In vivo evaluation of photoprotection against chronic ultraviolet-A irradiation by a new sunscreen Mexoryl SX. In a previous study on the hairless mouse it was shown that sub-erythemal doses of pure UV-A enhanced the numerous changes normally observed during chronological aging. A new sunscreen (a bis-benzylidene campho sulfonic acid derivative) has been synthesized in our research laboratory (lambda max: 345 nm, epsilon: 47,000). Its photoprotective properties against UV-A induced damages were assessed in our mouse model. Three month old albino hairless mice were exposed for 1 y to suberythemal doses (35 J/cm2) of UV-A obtained from a xenon source filtered through a WG 345 filter. One group of animals was exposed untreated, the other received a formulation containing 5% of the sunscreen prior to irradiation. At the end of the study the cutaneous properties of protected mice were compared to those of unprotected animals and to 3 and 15 month old unirradiated controls. We found that the visible changes induced by UV-A irradiation were mainly sagging and wrinkling. Histological and electron microscopic alterations consisted of hyperkeratosis, increased density of elastic fibers with alteration of fiber orientation and increased glycosaminoglycan deposits. Biochemical changes consisted of decreases in total collagen and collagen hydroxylation and increases in both collagen III/I + III ratio and fibronectin biosynthesis. All these changes were reduced or abolished by the sunscreen.


Breast carcinoma in women previously treated for Hodgkin disease: mammographic evaluation. Although the risk of second malignancies occurring after curative therapy for Hodgkin disease (HD) is well known, few cases of breast carcinoma developing in this setting have been reported. The authors performed a retrospective review of institutional records and identified 27 women with 29 breast carcinomas who had previously undergone treatment of HD and for whom mammograms were available. Two of the 29 carcinomas were synchronous, bilateral tumors. Although the patients' ages ranged from 33 to 75 years, most were young; 16 patients (55%) were younger than 45 years, and nine (31%) were younger than 40 years. Time from treatment for HD to development of breast cancer ranged from 8 to 34 years (mean, 18 years). All women developed ductal carcinoma. Mammography demonstrated 26 of the 29 cancers (90%); 11 of the 29 cancers (38%) were detected only with mammography. In 26 tumors for which clinical findings were known, the results of physical examination were positive in 18 (69%). Women previously treated for HD may be at increased risk of developing breast cancer, which may be bilateral and develop at a young age. This increased risk may be due to scattered radiation to the breast during mantle irradiation. Mammographic screening of these women is indicated. In women who were treated for HD at a young age, routine screening before the age of 35 years is recommended.


Psychosocial modulation of antibody to Epstein-Barr viral capsid antigen and human herpesvirus type-6 in HIV-1-infected and at-risk gay men. We investigated the effects of two behavioral interventions--aerobic exercise and cognitive behavioral stress management (CBSM)--on Epstein-Barr virus viral capsid antigen (EBV-VCA) and human herpesvirus type-6 (HHV-6) antibody modulation in 65 asymptomatic gay men measured at several time points in the 5 weeks preceding and following notification of their human immunodeficiency virus-type 1 (HIV-1) serostatus. After accounting for potential immunomodulatory confounds, we found that HIV-1 seropositive men had higher EBV-VCA antibody titers than those diagnosed as seronegative at every time point during the study; however, no significant differences were found with respect to HHV-6. Among HIV-1 seropositive and seronegative subjects, respectively, those randomized to either behavioral intervention had significant decreases in both EBV-VCA and HHV-6 antibody titers over the course of the intervention as compared with assessment-only controls (of HIV-1 seropositive and seronegative status) whose antibody titers did not significantly change and which remained consistently higher than either serostatus-matched intervention group over subsequent time points, independent of total immunoglobulin G levels and degree of polyclonal B cell activation. In attempting to explain serostatus differences in EBV and HHV-6 values, it was found that HIV-1 seropositive men had significantly lower CD4 cells, CD4:CD8 ratio, and blastogenic response to phytohemagglutinin (PHA), as well as significantly higher CD8 cells at baseline. No significant differences were found between the HIV-1 seropositive and seronegative men with respect to anxiety and depression at baseline. Since the greatest changes in EBV and HHV-6 occurred between baseline and week 10, we correlated changes in immune (CD4, CD8, CD4:CD8 ratio, PHA stimulation) and distress-related markers (state depression and anxiety) with EBV and HHV-6 change scores over this time period. No significant correlations were found between any of these immune- or distress-related variable and the antibody change scores suggesting that the mechanisms by which EBV and HHV-6 antibodies are being modulated by these interventions possibly involve other, yet to be determined, immune, neuroendocrine, and/or psychologic variables.


Detection of rabbit haemorrhagic disease virus antigen in tissues by immunohistochemistry. Formalin fixed liver, spleen, kidney, heart, lung, duodenum and appendix tissues from nine rabbits, experimentally infected with rabbit haemorrhagic disease virus (RHDV), were investigated for evidence of RHDV antigen by the direct avidin-biotin peroxidase complex immunohistochemical method. In all the rabbits examined, RHDV antigen was detected in degenerative and necrotic hepatocytes of the liver tissues. The area involved coincided with histopathological lesions on serial liver sections. The RHDV antigen was expressed in the cytoplasm of the hepatocytes, suggesting that RHDV replicated in these cells. RHDV antigen was also detected in the spleen. The results of immunohistochemistry were supported by the demonstration of RHDV protein by Western blot analysis and of RHDV particles by protein A-gold immunoelectron microscopy in the liver homogenate from all the rabbits that were examined.


Preservation of feline anti-Toxoplasma gondii antibody activity using blood absorbed on filter paper stored under different conditions. The present study deals with the successful long-term preservation of feline anti-Toxoplasma gondii antibody activity on filter paper strips stored with silica gel. When filter paper strips were kept at 25 degrees C with silica gel, antibody activity in blood samples was preserved for at least six months.


Long-term treatment with sodium cromoglycate, nedocromil sodium and beclomethasone dipropionate reduces bronchial hyperresponsiveness in asthmatic subjects. 165 patients (106 males, 59 females) entered an open group comparative study of a 12-week test treatment on bronchial hyperresponsiveness (BHR) determined by methacholine challenge. Patients were randomly allocated to receive nedocromil sodium (4 mg q.i.d.), sodium cromoglycate (10 micrograms q.i.d.) and beclomethasone dipropionate (500 micrograms t.i.d.). At the end of the study, an 2.25-fold increase of the PD20FEV1 was noted in all the treated patients. No significant difference was noted among the treatments.


Expression of a ryanodine receptor-Ca2+ channel that is regulated by TGF-beta. Ryanodine receptors (RyRs) are intracellular channels that release calcium ions from the sarcoplasmic reticulum (SR) in response to either plasma membrane depolarization (in skeletal muscle) or increases in the concentration of intracellular free Ca2+ (in the heart). A gene (beta 4) encoding a ryanodine receptor (similar to, but distinct from, the muscle RyRs) was identified. The beta 4 gene was expressed in all tissues investigated, with the exception of heart. Treatment of mink lung epithelial cells (Mv1Lu) with transforming growth factor beta (TGF-beta) induced expression of the beta 4 gene together with the release of Ca2+ in response to ryanodine (but not in response to caffeine, the other drug active on muscle RyRs). This ryanodine receptor may be important in the regulation of intracellular Ca2+ homeostasis.


[Alpha-receptor mediated effect of injection of norepinephrine into hippocampus on plasma cortisol level in cats]. In the present experiment, the effect of injection of NE into the different areas of hippocampus on the plasma cortisol level and the kind of NE receptor involved were studied in 83 cats anesthetized with Nembutal. The plasma cortisol level was increased following injection of NE into the ventral hippocampus (VHIP), however, there was no significant change when injection was made into dorsal hippocampus. The NE-VHIP effect can be blocked by injection of phentolamine, yohimbine or prazosin but not by propranolol. Thus, these results show that, in the regulation of the plasma cortisol level, the alpha-receptor system of VHIP is more specifically involved.


[Effects of beta-agkistrodotoxin on synaptic transmission in toad sympathetic ganglia]. The effects of beta-agkistrodotoxin (beta-AgTX) on synaptic transmission of the toad sympathetic ganglia were investigated by intracellular recording techniques. Superfusion of beta-AgTX (30 microgram/ml, 5-15 min) reversibly inhibited the cholinergic fast excitatory postsynaptic potential (f-EPSP, n = 16) and the fast components of acetylcholine (ACh) potential induced by micropressure administration of ACh (n = 24). Comparison of beta-AgTX effect in the same cell group showed significantly different inhibition rates on f-EPSP (77.2 +/- 27.7%) and ACh potential (25.5 +/- 17.5%) (n = 6, P less than 0.01, F test). During application of beta-AgTX (30 or 50 micrograms/ml) for 15-30 min, no detectable change was found in non-cholinergic late slow EPSPs (n = 22). The results suggest that beta-AgTX selectively inhibits the cholinergic transmission of the toad sympathetic ganglia by both presynaptic and postsynaptic mechanism.


[Effects of prolonged darkness and background illumination on cone horizontal cells in carp retina: a correlative study of morphology and physiology]. Using intact, immobilized carp preparations, changes in light responsiveness of cone horizontal cells and ultrastructures of their terminals in cone pedicles (HCTs) were correlatively examined in prolonged (greater than 2 h) darkness and after the presentation of a dim background light. Following background illumination cone horizontal cells exhibit high light responsiveness and HCTs give rise to a lot of long, fingerlike or ball-like extensions, called slender or round spinules. When the retina is left in the dark for more than 2 h, the light responsiveness of these cells is depressed, which is accompanied by a dramatic decrease of spinules. Thus light responsiveness of the cone horizontal cells seems to be well correlated with the number of spinules. The results suggest that spinules may play an important role in regulating light responsiveness of cone horizontal cells following background illumination by altering the efficacy of signal transfer across the synapses between cone photoreceptors and cone horizontal cells.


[Results of photon therapy of malignant tumors of the parotid gland]. Between 1971 and 1982 86 patients have been treated because of a malignant tumor of the parotid gland. 64 patients have been irradiated after complete (n = 49) or incomplete (n = 15) first resection. 12/64 (19%) relapsed locally. The loco-regional tumor control rate five and ten years after postoperative radiotherapy is 72%, 85% after complete resection, and 22% after incomplete resection (p less than 0.01). Tumor size and nodal disease are of prognostic value. Disease-free survival in patients without lymph nodes is 53%, with lymph node metastases 31% after five years (p less than 0.05). Small tumors (T1, 2) have a better local control rate compared to locally advanced tumors (five years: 83% vs. 53%, p less than 0.05). No difference was found neither for the total dose nor the histology of the tumor. Distant metastases became apparent after median eleven months.


Carcinoma of the paranasal sinuses: results of treatment and some prognostic factors. Between 1979 and 1984, 46 patients, 23 males and 23 females, with carcinoma of the paranasal sinuses were treated at the Institute of Oncology in Ljubljana. A great majority of tumours were in an advanced stage of disease. 21 patients were operated on and all except one were postoperatively irradiated. Radiotherapy was the main treatment in the remaining 25 patients. 14 patients (30%) survived more than five years with no evidence of disease. Patients treated by a combination of surgery and postoperative radiotherapy survived significantly better (12/20-60%) than those treated by irradiation alone (2/25-8%), p less than 0.001. Patient's age and radicality of surgery also influenced the treatment results.


Computer simulation and modelling of tumor spheroid growth and their relevance for optimization of fractionated radiotherapy. As previous papers show, our group developed computer models simulating spatial (3D) tumor growth of an in vitro tumor spheroid. These models were extended by implementing irradiation models based on the linear-quadratic survival function in order to simulate and optimize radiation therapy schemes. The key idea in this study is to simulate different fractionation schemes (standard-, super-, hyperfractionation, irradiation with a weekly high dose) and to compare the model results with regard to their tumor effectiveness. After introducing simplified model assumptions the following treatment plans, as a result, represent an optimal scheduling: 1. the hyperfractionation (3 x 1...1.5 Gy per day) in the case of rapidly growing tumors; 2. the hyperfractionation (3 x 1...1.5 Gy per day) for moderately fast growing tumors; and 3. the treatment with a weekly high single dose (1 x 6 Gy per week) in the case of slowly growing tumors. The transfer of the results gained by simulating in vitro-experiments to clinical tumors are discussed. Single observations in clinical practice concerning the therapeutical benefit indicate a rather good agreement with the simulation results. Thereby, the possibility is given to comprehend in vitro tumor growth and clinical therapy schemes in a model and to successfully simulate optimal treatment schedules by computer experiments. This method enables a reduction of time-consuming studies prior to clinical therapy.


Major liver resection without a blood transfusion: is it a realistic objective? Observations from 75 patients undergoing resection of the liver during a recent period have been analyzed with respect to the use of perioperative blood transfusions and operative outcome. Twenty-six patients were operated on for benign disease and 49 patients for malignant disease. Twenty-one patients underwent a right or left hepatectomy, 49 patients underwent removal of one or more anatomic segments of the liver, and five patients underwent wedge resection of a lesion in the liver. There was one postoperative death. The median blood loss was 0 units (range, 0 to 15 units). Sixty-three percent of the patients underwent operation without the need for a blood transfusion. The median postoperative stay was 10 days (range, 4 to 88 days). In the group of patients who did not undergo a blood transfusion the median postoperative stay was 9 days (range, 4 to 28 days), and in the patients who had a blood transfusion it was 14 days (range, 6 to 84 days) (p less than 0.0003). The performance of major liver resection without a blood transfusion is a realistic objective.


The neutrophil respiratory burst and tissue injury in septic acute lung injury: the effect of cyclooxygenase inhibition in swine. Cyclooxygenase inhibition has been proposed as treatment for sepsis-induced acute lung injury. However, the mechanism of protection offered by the cyclooxygenase inhibitor ibuprofen is not well understood. To elucidate this mechanism, the effects of ibuprofen on the neutrophil respiratory burst and alveolar-capillary membrane leak were studied. Anesthetized swine (15 to 25 kg) were intubated and mechanically ventilated (fraction of inspired oxygen, 0.5). Control animals (n = 5) received a sham infusion of 0.9% NaCl, animals with sepsis (n = 10) received a 1-hour infusion of live Pseudomonas aeruginosa (5 x 10(8) colony-forming units/ml at 0.3 ml/20 kg/hr), and treated animals (ibuprofen-treated control animals [n = 4] or ibuprofen-treated animals with sepsis [n = 9]) received ibuprofen (12.5 mg/kg at 0 and 120 minutes). All animals were studied for 300 minutes. Neutrophils were isolated at 0, 60, and 300 minutes. Neutrophil superoxide anion production (O2-) was assessed in a kinetic fashion (in nanomoles per minute) by superoxide dismutase-inhibitable cytochrome C reduction (phorbol myristate acetate stimulation). Bronchoalveolar lavage protein estimation (0 and 300 minutes) and extravascular lung water (double indicator dilution) were performed to assess alveolar-capillary membrane leak. Ibuprofen significantly attenuated sepsis-enhanced maximum neutrophil generation of O2- (6.0 +/- 0.5 nmol/min for animals with sepsis, 300 minutes, vs 4.1 +/- 0.5 nmol/min for ibuprofen-treated animals, with sepsis, 300 minutes; p less than 0.05), indicating an in vivo down-regulatory effect on neutrophil oxidant generation. Ibuprofen also prevented increased airspace bronchoalveolar lavage protein and extravascular lung water accumulation, suggesting a protective effect on the alveolar-capillary membrane. This protective effect of ibuprofen in acute lung injury may be through a decreased neutrophil respiratory burst.


Partial characterization of an autoantibody recognizing the secondary binding site(s) of thrombin in a patient with recurrent spontaneous arterial thrombosis. A patient with an 18 year history of recurrent arterial thrombosis and no evidence of atherosclerosis or embolism of cardiac origin presented with a prolonged thrombin clotting time when performed with human thrombin. The bovine thrombin clotting time was only slightly prolonged. During 30 months of follow-up, the thrombin time fluctuated, but remained prolonged. The patient has been treated with an oral anticoagulant for the past 8 years, with no thrombotic recurrence. The inhibitor activity was due to the presence of polyclonal IgGs which bound to thrombin-Sepharose. The influence of IgGs purified from the patient's serum was compared to the influence of normal IgGs in several systems exploring the catalytic activity of thrombin and the binding of the enzyme to macromolecular substrates through secondary binding site(s). We found that the IgGs did not impair the catalytic activity toward small synthetic substrates, but inhibited the binding of thrombin to fibrinogen, thrombomodulin and heparin cofactor II. Such proteins are known to require a secondary binding site of thrombin to interact with the enzyme. The anti-thrombin antibody might have resulted from an abnormal generation of thrombin. This would be the consequence of the process favouring thrombosis. Alternatively, the autoantibody might have favoured thrombosis primarily, by impairing natural antithrombotic mechanisms triggered by thrombin.


Thrombomodulin is a cofactor for thrombin degradation of recombinant single-chain urokinase plasminogen activator "in vitro" and in a perfused rabbit heart model. Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) to a two-chain derivative (tcu-PA) fibrinolytically inactive. This reaction was accelerated in vitro by purified rabbit lung thrombomodulin in equimolar complex with thrombin. Polyclonal antibodies to rabbit thrombomodulin prevented this effect. We also observed that heparin and other sulfated polysaccharides had an accelerating effect on thrombin cleavage of recombinant scu-PA. Their effect was concentration-dependent and then reversed at high levels. The effect of heparin and heparan sulfate was independent and synergic with respect to thrombomodulin. All observations except the effect of heparin, could be confirmed in a Langendorff isolated rabbit heart model. From competition experiments carried out with scu-PA derivatives and mutants, we postulate that the amino-terminal sequence of rscu-PA, containing the epidermal growth factor (EGF)-like and the kringle domains is involved in the cofactor effect of thrombomodulin on scu-PA inactivation by thrombin. We conclude that a regulatory mechanism of scu-PA inactivation is present at the cell surface.


Human papillomavirus associated with bladder cancer. Recently published data have suggested a link between active human papillomavirus (HPV) infection and the development of bladder cancer. This study was undertaken to test for HPV genomic material in the tumors of patients without evidence of ongoing viral infection. Twenty-three consecutive patients with clinical evidence of intravesical neoplasia and no history of HPV infection or clinical evidence of intercurrent disease, underwent cystopanendoscopy and biopsy as part of the routine evaluation and treatment of their tumor. Routine pathologic evaluation and southern blot analysis of biopsy material were done to establish the presence or absence of HPV DNA in the bladder tumors. Twenty-one tumors were identified by routine histology: 20 were low-to-moderate grade transitional cell carcinomas; 1 was found to be squamous cell carcinoma; 1 patient had moderate dysplasia; and 1 patient had evidence of inflammation. Four of the 20 transitional cell tumors (20%) were found to contain HPV DNA. In addition, the patients with dysplasia and cystitis were also shown to have HPV genomic material in their biopsy specimens. Viral types 6/11, 16/18, and 31/33 were found. The 20 percent incidence of HPV genomic material in bladder tumors from patients without clinical evidence of viral infection is in keeping with the observations of other investigators. We present the implication of these findings within the context of our current understanding of viral oncogenesis in the urinary bladder.


Predictors of residual mass requiring surgical resection after chemotherapy of stage III testicular cancer. A prospective study. In a prospective study, 63 patients with histopathologically proved Stage III nonseminomatous testicular cancer (NSTC) were analyzed to predict the need for surgical resection of residual masses after cis-platinum-based chemotherapy. Of these 63 patients, 23 (37%) had residual masses after cis-platinum-based chemotherapy requiring surgical resection. Of the 23 patients undergoing surgical resections for their residual masses, 18 patients (78%) had matured teratoma, 3 (13%) had fibrosis with necrosis, and 2 (9%) had residual tumors. Twenty of the 23 (91%) patients with residual disease had either teratomatous elements in primary tumor or bulky metastatic disease at the time of initial chemotherapy. Two patients had incomplete resection of the metastatic disease containing teratoma and required additional resection of recurrent growing matured teratomas. We conclude that teratomatous elements in primary tumor having also bulky metastatic disease are strong predictors of residual disease after initial chemotherapy requiring surgery (21 of 23 or 91%).


[Lowering cholesterol with Anticholest--a high fiber guar-apple pectin drink]. In order to determine the efficacy of Anticholest, an apple-pectin-guar soft-drink in reducing elevated cholesterol (c) levels, 33 participants (aged from 8 to 73 years) were divided into three groups of comparable age, body-mass index, total c, LDL-c, HDL-c, and triglycerides. They received this combined fiber product either (group 1) at dosages of 1 cup (17 g) every second day, or (group 2) of 1 cup a day or (group 3) of 2 cups a day. Anticholest significantly reduced total c, LDL-c and the total c/HDL-c ratio. In group 3 HDL-c was increased significantly (p less than 0.01). The average percentage decrease in total c was 10.0% for group 1, 10.7% for group 2, and 15.7% for group 3. LDL-c was lowered 14.4% in group 1, 13.8% in group 2 and 19.1% in group 3. The highest individual reduction amounted to more than 30% for total-c and LDL-c. A significant decrease was also detected in the triglycerides (tg) in group 3 (p less than 0.05). 7 patients were non-responders. Anticholest appears to be an appropriate treatment for patients at risk of coronary heart disease with insufficient or inadequate response to dietary measures, but where long-term pharmacotherapy is not yet indicated. The lack of side effects and its form of administration as a viscous drink are important determinants for long-term compliance, which is essential in the management of a chronic progressive disease such as atherosclerosis.


Immunogenicity, reactogenicity and consistency of a new, inactivated hepatitis A vaccine--a randomized multicentre study with three consecutive vaccine lots. We investigated immunogenicity, reactogenicity and consistency of three consecutive lots of an inactivated hepatitis A vaccine in 204 seronegative volunteers. Each volunteer received a total of three doses of vaccine (720 EIU) according to a 0, 1 month primary vaccination schedule with a booster dose given at month 6. Mild, moderate and mostly local side effects were reported in 49.7% after the first and in less than 30% after the third injection. Seroconversion rate after one vaccine dose was as high as 91%. All subjects but one had already seroconverted by 1 month after the second injection, corresponding to a seroconversion rate of 99%. The geometric mean titres (GMT) increased with each dose of vaccine administered. Our results show that this inactivated hepatitis A vaccine is highly immunogenic, safe and well tolerated. Furthermore, there were no significant differences between the three vaccine lots in respect to seroconversion rate, size of antibody titre or reactogenicity.


Ulnar nerve inoculation of poliovirus in bonnet monkey: a new primate model to investigate neurovirulence. A new monkey model of poliovirus neurovirulence has been developed avoiding the currently used intraspinal injection route which traumatizes the spinal cord. Poliovirus type 1 (0.1 ml) was inoculated into the ulnar nerve of bonnet monkeys (Macaca radiata) at the right elbow. Five monkeys were inoculated with 10(7) TCID of LSc/2ab (Sabin vaccine strain); none developed any illness. Limb paralysis, clinically resembling spinal poliomyelitis in children, developed in all four monkeys given greater than or equal to 10(5) TCID50 of Mahoney strain, and in three of four monkeys given 10(4) or 10(3) TCID50. Higher functions and cranial nerves were not affected. Paralysis occurred more frequently in the lower limbs (11 limbs in seven monkeys) than in upper limbs (six limbs in seven monkeys). The incubation period, from inoculation to onset of paralysis, ranged from 5 to 12 days. Further progression of paralysis to other limbs occurred within 2 to 6 days. No illness developed in two monkeys given 10(2) TCID50 of Mahoney virus. All monkeys given LSc/2ab and those given greater than 10(2) TCID50 Mahoney virus developed humoral antibody response; however, infection of the gastrointestinal tract was detected by virus isolation from throat swabs and stools only in monkeys given Mahoney virus, but not in those given LSc/2ab. Thus, intraneural spread of Mahoney virus to the spinal cord, neurovirulence of Mahoney but not of LSc/2ab and retrograde gastrointestinal infection with Mahoney but not with LSc/2ab are the features of this experimental model.


Antigen dependent adjuvant activity of a polydispersed beta-(1,4)-linked acetylated mannan (acemannan). The adjuvant properties of a polydispersed beta-(1,4)-linked acetylated mannan, acemannan (ACE-M), were evaluated. Day-old broiler chicks were randomly selected and allocated to four flocks (Vac 1-4). The Vac 1 flock was sham vaccinated with saline. The Vac 2 flock was vaccinated with an oil-based vaccine (Breedervac III; Newcastle disease virus (NDV), infectious bursal disease virus (IBDV) and infectious bronchitis virus). The Vac 3 flock was vaccinated with a vaccine-ACE-M mixture, and the Vac 4 flock was vaccinated with vaccine and ACE-M at separate anatomical sites. ELISA titres to NDV and IBDV were determined. The immune response to NDV at 21 days postvaccination (PV) was significantly enhanced (P less than or equal to 0.05) by the addition of ACE-M to the vaccine, compared with vaccination without ACE-M. Subsequently, the vaccine-ACE-M mixture appeared to suppress the immune response to NDV. However, at day 35 PV, 95% of the Vac 3 chicks compared with 90% of the Vac 2 and 89% of the Vac 4 chicks exhibited protective titres. The response to IBDV differed from that to NDV. At day 21 PV the immune response to IBDV was essentially the same for all flocks that received vaccine, i.e. addition of ACE-M to the vaccine did not significantly enhance the immune response; however, it did significantly (P less than or equal to 0.05) sustain the immune response at days 28 and 35. In addition to the observed effect on titres to NDV and IBDV, ACE-M also had an effect on flock immunity.(ABSTRACT TRUNCATED AT 250 WORDS)


GABA receptor-mediated modification of reticulo-ruminal myoelectrical activity in sheep. The involvement of central and peripheral GABA receptors in the control of the forestomach periodic motor activity was examined in five conscious ewes at rest, chronically fitted with electrodes implanted in the reticular and ruminal wall and a cannula placed in a cerebral lateral ventricle. Intravenous (IV) administration of the GABAA receptor agonist muscimol (5-50 micrograms/kg) did not affect reticulo-ruminal myoelectrical activity, while its intracerebroventricular (ICV) injection (0.05-0.10 micrograms/kg) dose-dependently increased the frequency of reticular and ruminal motor cycles and provoked rumination in 11 trials out of 15. Forestomach responses to ICV muscimol were abolished by a previous ICV treatment of the animals with the GABAA receptor antagonist bicuculline (0.5-4 micrograms/kg), which did not in itself influence reticular or ruminal motility. The GABAB receptor agonist baclofen, given either IV (300-800 micrograms/kg) or ICV (1-3 micrograms/kg), inhibited in a dose-dependent manner both reticular and ruminal activity, with the effects of its ICV administration appearing earlier and lasting longer. The GABAB receptor blocker baclofen given ICV alone (50-300 micrograms/kg) was ineffective, but such a treatment before IV or ICV administration of baclofen in part antagonized the inhibition of the forestomach myoelectrical cyclic activity. These pharmacological data suggest a possible excitatory role of strictly central GABAA and an inhibitory one of mainly central GABAB receptors in the regulation of reticulo-ruminal extrinsic motility in sheep. In this regulation, however, a probable peripheral component of inhibitory GABAB receptors should not be excluded.


Influenza A viruses: epidemiologic study in fatteners in Spain (1987-89). 2,979 sera were collected from slaughtered swine in two geographic areas of Spain from 1987 to 1989. They were tested for antibodies against an H1N1- and H3N2-influenza virus by haemagglutination-inhibition tests (HI). The percentage of positive sera was higher in area I (78%-69.2%) than in area II (63.1%-60.4%) for both viruses respectively. The coexistence of high titres to both H1N1- and H3N2-influenza virus became apparent in cold months simultaneously in each area, although influenza viruses circulated in the Spanish swine population for two years. Also this study suggests the possible circulation of A/Texas/1/77-like strains in Spain, results which have not been reported before.


Comparison of virus isolation, immunofluorescence and DNA probe hybridization for detection of pseudorabies virus in experimentally infected pigs. Virus isolation, immunofluorescent staining and DNA probe hybridization, three techniques for the detection of pseudorabies virus (PRV) have been compared in pigs experimentally infected with the Thailand CB-1 strain PRV. The virus isolation and DNA hybridization detection demonstrated a good correlation in detecting infection in live animals by nasal swabbing. In white blood cells an earlier detection was seen with the DNA-hybridization techniques. Consistent results with all the three techniques tested were found in organ materials as nasal mucosa and tonsils as well as in the olfactory bulb.


Effects of wheat bran and energy restriction on onset of puberty, cell proliferation and development of mammary tissue in female rats. Delayed onset of puberty and mammary development is assumed to reduce the risk of mammary cancer. An animal experiment was performed to investigate the influence of dietary fiber, which is known to affect hormonal balance, on these characteristics. Forty-five immature female rats were randomized into three groups, which were fed ad libitum a low-fiber diet (less than 0.5% dietary fiber based on white wheat flour), a high-fiber diet (9.2% dietary fiber based on wheat bran), or an energy-restricted low-fiber diet providing 90% of the energy of the ad libitum low-fiber diet. Energy intake in the second and third groups was similar. Wheat bran slightly delayed onset of puberty, whereas restricted energy intake delayed onset of puberty by about six days. At 48-58 days of age, 14 rats of the low-fiber group, 10 of the high-fiber group and 7 of the restricted group were in cycle. Development of mammary tissue was rudimentary in rats of the energy-restricted low-fiber group, stronger in the high-fiber group and strongest in the ad libitum low-fiber group. Cell proliferation in mammary tissue was similar for both groups fed ad libitum, but significantly lower in the restricted group. Peroxidase activity, a biomarker for estrogenicity, was lower in the high-fiber group than in the two low-fiber groups. It is concluded that wheat bran and, even more effectively, an imposed restricted energy intake delays onset of puberty and mammary development. This shortens the time for mammary cells to be initiated to tumor cells and hence reduces the risk of mammary cancer development.(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of serum thyrotropin levels on the concentration of messenger RNA for thyroid peroxidase in the rat. The effect of serum TSH on rat thyroid peroxidase mRNA levels was studied in order to investigate the regulation of thyroid peroxidase gene expression in vivo. A nearly full-length rat thyroid peroxidase cDNA clone was isolated from a bacteriophage cDNA library synthesized using poly A+ RNA isolated from the thyroids of propylthiouracil-treated rats. cDNA probes derived from this clone were used to study rat thyroid peroxidase mRNA levels in response to the level of serum TSH. Two major rat thyroid peroxidase mRNA bands were detected on Northern blots of total cellular RNA (at 3.2 kb and at 3.7 kb). Injection of thyroxine, which lowered the levels of serum TSH, also lowered the steady-state levels of both rat thyroid peroxidase mRNAs, whereas treatment with methimazole, which increased serum TSH, increased both rat thyroid peroxidase mRNA levels. In hypophysectomized rats 10 days postoperative, very low levels of thyroid peroxidase mRNA were observed. Injection of bovine TSH (1 IU/day) increased rat thyroid peroxidase mRNA expression, preferentially in the 3.2 kb band. These results clearly demonstrate that TSH regulates rat thyroid peroxidase mRNA levels in vivo.


Implementation of a tool to improve quality of care in psychogeriatric units. The examples of change that occurred in the units show that in most instances the impetus came from the top and middle levels, but then became total staff projects. The most important tactic introduced to cause change was re-education and ensuing discovery, using the tool as a guide. In at least one unit, much thought and effort were devoted to developing trust and confidence, as proposed by Bennis et al. (1976). Acceptance by the peer group (Mauksch and Miller, 1981) was achieved through the group decision-making process in the units and the interagency meetings of the head nurses and supervisors. Termination of the relationship between the units and project will probably take place at a later date, to be replaced by a special interest group made up of practice, education, and research psychogeriatric nurses. Only a few of the experiences of the units in utilizing the instrument to improve quality of care have been presented. Some were very successful; others had little impact. The overall impression is that of awakened desire by staff and administration to improve care; increased valuing of patients, their families, and staff; readiness to utilize the help available in the instrument and the project team; and recognition of the professional challenge and status inherent in psychogeriatric nursing.


The role of the geriatric consultation team in an acute care hospital. One of the most important health issues of this decade is the provision of effective health care for the elderly. The overwhelming needs of this increasing population segment add to growing hospital expenditures. Hospitals must develop new ways to provide quality health care to the acutely ill elderly. The special needs of this group are too complex to be addressed by a single discipline. A geriatric team approach can provide a comprehensive, cost-effective means of improving the quality of care provided. This care delivery model can result in positive outcomes for the geriatric patient.


The maximizing urinary functioning program: an innovative approach to managing incontinence in long term care. Urinary incontinence is a common occurrence in long term care and, given the client population, continence is often an unrealistic expectation. However, it was recognized that nursing plays a pivotal role in the maintenance and enhancement of clients' abilities with respect to their urinary functioning. In our facility, it was found that a need existed not only to assist clients to maximize their urinary functioning, but also to encourage the independent decision-making of our professional staff. The MUF Program developed to meet these needs was based upon the use of a systematic method for decision-making in nursing. The Maximizing Urinary Functioning Program was implemented on a pilot unit and evaluated using a pre-test/post-test design. Based upon the results of this evaluation and projections for the benefit to new admissions, the program is now being implemented hospital-wide. This project has also served to raise several research questions of interest to those working in long term care and levelled practice: how will the management of new admissions with urinary incontinence change over several years with a program of systematic decision-making in place? will there be a decline in the number of incontinent clients at our facility over a two-year period? is there a relationship between independent decision-making and job satisfaction for professional nursing staff working in levelled practice? and what is the effect of enhancing the independent decision-making skills of professional nursing staff on the job satisfaction of the nonprofessional staff?


Central motor conduction to upper and lower limbs after magnetic stimulation of the brain and peripheral nerve abnormalities in 20 patients with Friedreich's ataxia. Central motor conduction time (CMCT) to thenar and soleus muscles was measured after magnetic stimulation of the cortex in 20 cases of Friedreich's ataxia (FA) and was abnormal in all. CMCT values were related to disease duration and disability. The amplitude of CMAP after cortex stimulation was severely reduced in the most disabled patients. Reduction in amplitude of the nerve evoked potentials was related neither to disease duration nor grade of disability. These results suggest that clinical worsening in FA is mainly due to progressive central motor pathway involvement. CMCT study is a better index of disease progression than peripheral nerve examination. Abnormalities in CMCT may be the third electrophysiological diagnostic criterion in FA, after reduced amplitude of nerve action potentials and absent H reflex.


Newest human herpesvirus (HHV-6) in the Guillain-Barré syndrome and other neurological diseases. To investigate the presence of human herpesvirus-6 (HHV-6) in patients affected by Guillain-Barré syndrome (GBS) and by other neurological diseases (OND), we examined by indirect immunofluorescence analysis (IFA) the sera and cerebrospinal fluid (CSF) from 28 GBS and 63 OND. Moreover, we tested 150 blood donors (BD) to appreciate the diffusion of HHV-6 infection in the Italian adult healthy population. We found a significantly higher titre of antibody to HHV-6 in the GBS patients compared with OND and BD, although the pathogenicity of the virus is not known.


Pain-related somatosensory evoked potentials following CO2 laser stimulation in peripheral neuropathies. Pain-related somatosensory evoked potentials (pain SEPs) following CO2 laser stimulation were examined in 30 patients with peripheral neuropathies, and the results were compared with clinical sensory findings. Pain SEP findings showed a significant correlation with the clinical impairment of pain sensation, but not with the impairment of deep sensations. In contrast, conventional electrically-stimulated SEPs (electric SEPs) showed a significant correlation with deep sensations, but not with the impairment of pain sensation. Examinations of both pain SEPs and electric SEPs, therefore, are considered to be very useful to evaluate physiological functions of sensory nerves in patients with peripheral neuropathies.


Recurrent herpes zoster myelitis treated with human interferon alpha: a case report. Recurrent herpes zoster myelitis is very rare. However, a case was recently observed in our hospital. A 43-year-old woman developed myelitis 2 weeks after development of shingles. Her condition was improved by methylprednisolone. Seven months later, she developed myelitis after development of shingles again. Antibody against varicella-zoster (VZV), increased in the serum, but was negative in the cerebrospinal fluid. Methylprednisolone was not sufficiently effective against this attack. The refractory sensory disturbance was improved by human interferon alpha (IFN-alpha). Natural killer cell activity, the helper T-cell/suppressor T-cell ratio and the kappa/lambda ratio of B-cells increased with clinical improvement. In this case, delayed-type hypersensitivity after VZV infection played a role in the occurrence of myelopathy and clinical improvement resulted from the immunosuppressive effects of IFN-alpha.


Immunocytochemical and ultrastructural studies of neuronal and oligodendroglial cytoplasmic inclusions in multiple system atrophy. 1. Neuronal cytoplasmic inclusions. Neuronal alterations in five cases of multiple system atrophy (MSA) were investigated histologically, immunocytochemically and ultrastructurally. Argentophilic neuronal cytoplasmic inclusions (NCIs) were observed in all cases. They were distributed, in order of decreasing frequency, in the pontine nucleus, striatum, subiculum, amygdala, hippocampus, dentate fascia, substantia nigra and inferior olivary nucleus. Anti-ubiquitin antibodies visualized many thickened neurites in the degenerating gray matter as well as NCIs. Some NCIs were also recognized by anti-phosphorylated neurofilament antibodies. Ultrastructurally, NCIs consisted of a meshwork of granule-associated filaments, the diameter ranging from 18 to 28 nm, that were mixed with neurofilaments. The granule-associated filaments were also present in the axoplasm of myelinated fibers. Our studies demonstrate widespread distribution of NCIs in the central nervous system of MSA. The same pathological process that forms the granule-associated filaments in axons may also be responsible for the formation of ubiquitin-positive thickened neurites. These axonal alterations, as well as neuronal perikaryal changes, may play an important role in the impaired neuronal function in MSA.


Immunocytochemical and ultrastructural studies of upper motor neurons in amyotrophic lateral sclerosis. The pathological alterations in upper motor neurons were investigated in 27 cases of adult-onset sporadic amyotrophic lateral sclerosis (ALS). No significant cytoskeletal alterations were found in the Betz cells of any of the cases except one, although cytoskeletal pathology was consistently present in lower motor neurons. The one case had severe circumscribed atrophy of the precentral gyrus and, microscopically, had argentophilic intracytoplasmic inclusions in Betz cells and other pyramidal neurons in the primary motor area as eell as in the lower motor neurons. Immunocytochemically these inclusions contained the epitope of phosphorylated neurofilament and ubiquitin and ultrastructurally consisted of granule-associated filaments with neurofilaments. This is the first demonstration of alterations of cytoskeleton and ubiquitination in the giant cells of Betz, an established subset of upper motor neurons in ALS. Thus, although uncommon, cytoskeletal changes can be found in upper motor neurons in some ALS cases.


Relationships between Lewy bodies and pale bodies in Parkinson's disease. The prevalence of pale bodies and Lewy bodies was studied in the substantia nigra of 12 patients with typical Parkinson's disease (PD), in 5 patients with diffuse Lewy body disease (DLBD), and in a group of neurologically normal controls. Anti-ubiquitin antibodies labelled pale bodies and Lewy bodies in typical PD and DLBD, and there was a strong positive correlation between numbers of ubiquitin-immunoreactive pale bodies and Lewy bodies. BF10, a monoclonal antibody against a phosphate-dependent epitope of neurofilament 155-kDa polypeptide subunit, immunolabelled 57% of Lewy bodies and 15% of pale bodies in typical PD. Some pale bodies and Lewy bodies were seen in the substantia nigra of 2 of 5 neurologically normal, aged controls, probably representing "incidental PD". We conclude that there is a close relationship between pale bodies and typical Lewy bodies in the substantia nigra in clinical varieties of PD, and that these inclusions share antigenic determinants. If pale bodies and Lewy bodies reflect separate aspects of the cellular pathology in PD, their formation probably occurs in parallel. Alternatively, these observations may suggest that pale bodies represent a stage in the formation of Lewy bodies.


Sural nerve immunoreactivity for nerve growth factor receptor in a case of localized hypertrophic neuropathy. Immunoreactivity for nerve growth factor receptor (NGFR) was examined using a monoclonal antibody against human NGFR in the sural nerve of a 24-year-old woman, affected by localized hypertrophic neuropathy (LHN). NGFR expression was correlated with electron microscopy and with immunoreactivity for S-100 protein, laminin, HLA-DR, HNK-1, P0 glycoprotein and neurofilament peptides. Our results indicate that in LHN most of whorl-forming cells are NGFR positive and S-100 protein or HLA-DR negative. These data along with the ultrastructural features suggest their origin from perineurium.


[Non-chromaffin non-functioning aortosympathetic paraganglioma]. The paraganglia system is the largest component in the construction of the Disseminated Neuroendocrine System, comprising cells derived from the neural crest, with ability to synthesize and secrete catecholamines, hormones and peptides. A general explanation on the subject with a personal contribution is made.


In vitro activity of silicon carbide whiskers in comparison to other industrial fibers using four cell culture systems. Silicon carbide whiskers (SiCW) and continuous glass filaments are important components of composite materials having potentially widespread use in the automotive, aerospace, and power generation industries. We determined the in vitro activity of three well-characterized samples of silicon carbide whiskers and a continuous glass filament sample in four different cellular assays and compared this to the activities of UICC crocidolite, JM Code 100 glass microfiber, and erionite in the same assay systems. The SiCW had a diameter range of 0.32-0.75 microns and a length range of 4.5-20.1 microns. The SiCW was significantly toxic; on a mass basis, one SiCW sample was more toxic than crocidolite; however, JM Code 100 glass microfiber, which is not toxic in vivo (i.e., it does not cause fibrogenesis or carcinogenesis when inhaled), was also more toxic than crocidolite. The glass filament sample was the least cytotoxic of all the samples tested. On a fiber number basis, all three SiCW samples were more toxic than crocidolite. The results of our study showed that SiCW exhibits significant in vitro biological reactivity. Thus, despite the caution that must be exercised in extrpolating the results of in vitro studies to conclusions about in vivo health effects, SiCW should be considered toxic until further toxicological data are available.


Adverse effects of neutrophils on the lung. Studies of both emphysema and adult respiratory distress syndrome (ARDS) support the premise that lung injury is due to unregulated host defense mechanisms. A major mediator of host defense and injury is the neutrophil, which is relatively incapable of regulating its own function. Accordingly, defects in regulatory mechanisms allow neutrophils to damage the lungs. Emphysema serves as a prime example of this link between host defense and injury. Hereditary emphysema is caused by a deficiency in alpha 1-antitrypsin (alpha 1-AT), a protease inhibitor. The decreased levels of this enzyme in affected individuals result in inadequate protection against neutrophil elastase and other proteolytic enzymes, leading to lung damage. Patients with acquired emphysema, associated with cigarette smoking, have normal levels of alpha 1-AT in their lungs. However, the alpha 1-AT in these patients has a reduced ability to associate with and inhibit the action of neutrophil elastase. Thus, both types of emphysema involve an alteration in the balance between proteases and antiproteases. The lung damage observed in patients with ARDS also appears to involve neutrophils, but in this case elastase may not be the culprit. In these patients, neutrophil elastase appears to be inactivated by high levels of alpha 1-AT, thus preventing excess protease action. It is hoped that a more complete understanding of the mechanisms involved in host defense and injury will enable the development of specific therapeutic interventions, such as the alpha 1-AT replacement therapy that is being used to treat patients with hereditary emphysema.


Reactivation of latent herpes simplex virus by excimer laser photokeratectomy. We tested whether excimer laser photorefractive and phototherapeutic keratectomy may reactivate latent herpes simplex and cause recurrent keratitis in mice. Two of ten latently infected mice that were treated with ten excimer laser pulses to the corneal epithelium shed herpes simplex virus type 1, as did four of ten mice that were treated with 50 excimer laser pulses. Ocular shedding of herpes simplex virus was detected in four of ten mice that were treated with ethylenediamine-tetraacetic acid (EDTA) scraping of the corneal epithelium without laser keratectomy, and in six of ten mice on which combined EDTA-facilitated epithelial removal was performed followed by the application of ten excimer laser pulses. In both EDTA-treated groups, viral shedding was prolonged and 18 of 20 mice developed marked corneal opacification or neovascularization, or both. Corneal photoablation with the excimer laser may induce reactivation of latent herpes simplex virus, even in mice with clear and smooth-appearing corneas, and should be considered in the differential diagnosis of humans with persistent corneal epithelial defects after refractive or therapeutic excimer procedures.


A cloned porcine renal calcitonin receptor couples to adenylyl cyclase and phospholipase C. The signal transduction pathways of the recently cloned porcine kidney calcitonin (CT) receptor were evaluated. This receptor, when stably transfected into MC-3T3 cells, avidly bound salmon CT (SCT) [dissociation constant (Kd) = 4 nM]. Incubation with SCT resulted in a dose-dependent accumulation of adenosine 3',5'-cyclic monophosphate (cAMP) [50% effective concentration (EC50) = 0.02 nM] in transfected cells (referred to as PC-1 cells). Binding kinetics and cAMP dose response relationships were similar to those of the native receptor in LLC-PK1 cells. PC-1 cells also responded to calcitonin gene-related peptide (CGRP), but the EC50 value for cAMP accumulation was more than three orders of magnitude higher than for SCT. Exposure of PC-1 cells to SCT (5 nM to 1 microM) produced a dose-dependent rise in cytosolic free Ca2+ concentration ([Ca2+]i), whereas CGRP did not. The initial rise in [Ca2+]i was not dependent on extracellular Ca2+, suggesting that SCT induced release of Ca2+ from intracellular stores. SCT also increased inositol trisphosphate production in PC-1 cells. In conclusion, the cloned, transfected porcine CT receptor functionally couples to and activates both adenylyl cyclase and phospholipase C. This dual coupling is also a characteristic of the parathyroid hormone receptor, which has significant homology in amino acid sequence with the CT receptor.


Cyclic 3',5'-nucleotide diesterases in dynamics of cAMP and cGMP in rat collecting duct cells. We studied cyclic 3',5'-nucleotide phosphodiesterase (PDE) isozymes and their role in adenosine 3',5'-cyclic monophosphate (cAMP) and cGMP metabolism in a rat inner medullary collecting duct (IMCD) cell line. The homogenized and fractionated IMCD cells of cAMP-PDE and all of cGMP-PDE activity were found in the cytosol. The majority of cytosolic cAMP-PDE (greater than 50%) was isozyme PDE-IV; the Ca(2+)-calmodulin-sensitive PDE-I was present only in cytosol. Preincubation of IMCD cells with PDE-IV inhibitor rolipram markedly (5x) enhanced levels of cAMP both basal and in the presence of [Arg8]vasopressin (AVP). Cilostamide (for PDE-III) or vinpocetine had no effect, whereas PDE-I inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MeoM-IBMX) enhanced AVP-dependent cAMP levels. Exposure of IMCD cells to 2 microM ionomycin decreased both basal and AVP-stimulated cAMP. Depletion of Ca2+ by preincubation of IMCD cells in the Ca(2+)-free medium with ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid markedly enhanced the stimulatory response of cAMP to AVP, and addition of 8-MeoM-IBMX further enhanced the AVP response. The levels of cGMP, basal or in response to atriopeptin (ANP), were not affected by PDE-V inhibitor zaprinast, but both inhibitors of PDE-I, 8-MeoM-IBMX and vinpocetine, increased basal cGMP, and 8-MeoM-IBMX also increased cGMP levels enhanced by ANP. The depletion of Ca2+ from IMCD cells alone had no effect on cGMP levels, but effects of 8-MeoM-IBMX and vinpocetine on the ANP-stimulated cGMP levels were enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)


Discordant aspects of aldosterone resistance in potassium depletion. Aldosterone resistance, defined as absent kaliuretic response to exogenous hormone, has been described in K depletion. It is not clear whether the absent kaliuresis is due to activation of K-conserving mechanisms or to failure of activation of the Na-K pump in cortical collecting tubules (CCT) by mineralocorticoids. Adrenalectomized male Sprague-Dawley rats were allocated to either a normal or low-K diet. Na-K pump activity (pmol.mm-1.h-1) in microdissected CCT and medullary collecting tubules (MCT, inner stripe of the outer medulla) was determined at 7 or 21 days after allocation to the dietary groups before and after exogenous aldosterone (50 micrograms twice daily, for 3 days). K depletion led to progressive hypertrophic changes in the CCT and MCT manifest in an increase in basal Na-K pump activity. In both K repletion and short-term K depletion (7 days), aldosterone led to the expected increase in CCT Na-K pump activity. With long-term K depletion, the CCT Na-K pump response to aldosterone was blunted. In the MCT where under normal conditions the Na-K pump is aldosterone unresponsive, an increasing aberrant responsiveness to the mineralocorticoid was observed with progressive K depletion. We conclude that apparent aldosterone resistance in short-term K depletion is likely due to activation of K-conserving mechanisms with early preservation of the CCT biochemical response to the hormone. With long-term K depletion, a blunted biochemical response to aldosterone may contribute to the absent kaliuretic response. In the MCT, K depletion led to the development of aberrant responsiveness to aldosterone.


Kinetics of urea and water permeability activation by vasopressin in rat terminal IMCD. The vasopressin-regulated urea carrier and the vasopressin-regulated water channel are distinct transporters present in the apical membrane of the inner medullary collecting duct (IMCD) cells. To assess whether these transporters may be activated by common mechanisms, we investigated the time course of increase of urea and water permeability in response to vasopressin in isolated perfused terminal IMCD segments. The permeability responses were determined through the use of a specially designed continuous-flow fluorometer for rapid analysis of collected tubule fluid samples. The time courses of activation of the two transporters by vasopressin were virtually identical. Both urea and water permeability displayed a rapid initial increase for the first 10 min followed by a slower secondary response lasting at least 30 additional min. The lag periods between vasopressin addition and the initial rise in permeability were the same for urea (34.2 +/- 8.8 s) and water (34.8 +/- 8.9 s) transport activation. Furthermore, the initial rate of permeability increase (normalized by the total increase) was not significantly different for the two transport processes. The lag periods for the increase in urea permeability in response to 8-bromoadenosine 3',5'-cyclic monophosphate and vasopressin were not significantly different. The results are consistent with the view that the rate-limiting step in vasopressin-induced activation is the same for both the urea carrier and water channel and may lie at a step beyond generation of adenosine 3',5'-cyclic monophosphate.


Characterization of Na(+)-H+ exchange in segments of rat mesenteric artery. To develop a technique for measuring Na(+)-H+ exchange activity and intracellular pH (pH(i)) "on line" in resistance vessels, we utilized strips of rat mesenteric arteries loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Strips were held at a fixed length within a 3-ml cuvette, and fluorescence emission was monitored at 530 nm. The spectrofluorimeter was monitored in the ratio mode, and the excitation wavelength was alternated between 440 and 505 nm. Tissues were maintained by perfusing with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid containing buffers. The introduction of ammonium chloride produced a rapid alkalinization. Washout of ammonium caused rapid acidification. Restoration of pH(i) was Na+ dependent and inhibited by dimethyl amiloride (concentration that produces half-maximal inhibition, K0.5 = 30 microM), features characteristic of Na(+)-H+ exchange. Further studies assessed the transport rate of the exchanger, which averaged 0.19 +/- 0.02 pH U/min (means +/- SE, n = 8). An estimate of the dependence of Na(+)-H+ exchange on external Na+ gave an apparent Michaelis constant for external Na+ of 10 mM and an apparent maximal velocity of 0.1 mM H+/s. Intracellular H+ was found to have a cooperative effect (Hill coefficient = 4) on Na(+)-H+ exchange.


Alterations in Na(+)-H+ exchange in mesenteric arteries from spontaneously hypertensive rats. Na(+)-H+ exchange was examined in segments of the superior mesenteric artery from spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats using the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Basal pH values were identical in the two strains (SHR = 7.18 +/- 0.02, WKY = 7.15 +/- 0.01) and exposure to 20-50 mM NH4Cl produced comparable changes in pH in both strains (tissues acidified to pH 6.8). The recovery of intracellular pH (pH(i)) from acid loading in bicarbonate-free buffer was dependent on extracellular Na+, inhibited by dimethyl amiloride, and thus was presumably mediated by Na(+)-H+ exchange. Exchange activity was markedly increased in SHR (0.308 vs. 0.182 pH U/min), and the increased activity could not be explained by differences in intracellular buffer capacity or in the Na+ affinity of the transporter. The rate of Na(+)-H+ exchange was more steeply dependent on pH(i) in SHR vessels Hill coefficient = 6.6 in SHR vs. 4.1 in WKY), suggesting an alteration in the transport protein. Young SHR (4-6 wk) exhibited kinetic characteristics resembling those of 12- to 14-wk-old WKY rats. Exposure to the Ca2+ ionophore bromo-A23187 significantly increased the Hill coefficient of WKY arteries but had no effect on vessels from SHR. Exposure of vessels from either strain to the phorbol ester phorbol 12-myristate 13 acetate (PMA) had no effect on Na(+)-H+ exchange.


Participation of alpha 2-adrenergic receptors in neural vascular tone of canine skeletal muscle. Studies were carried out in anesthetized, paralyzed, and ventilated dogs to determine whether postsynaptic alpha 2-adrenergic receptors participated in neurally mediated vascular tone in skeletal muscle. Hindlimb skeletal muscle resistance (RL) and blood flow (QL) were determined before, during, and after reversible cold block of the sciatic nerve. This sequence of observations was repeated 30 min after blockade of alpha 1-adrenergic receptors with prazosin. Then the alpha 2-adrenergic receptors were blocked with yohimbine, and the nerve cold block was repeated. When the sciatic nerve was cold blocked before alpha 1-adrenergic blockade, RL decreased approximately 50% and QL increased 75% (P less than 0.05) and then returned to control when the nerve was rewarmed. After alpha 1-block 76% of neural tone remained as assessed by nerve cooling (P less than 0.05). This phenomenon occurred despite effective alpha 1-adrenergic blockade as assessed by the alpha 1-receptor agonist methoxamine. With alpha 1- plus alpha 2-block no change in RL or QL was seen with nerve cold block. The same protocol was repeated in a second series of animals, but mean arterial pressure, which fell after alpha 1-block in the group above, was maintained by dextran infusion at normotensive levels. In these animals, 40% of neural tone remained after alpha 1-block. Both alpha 1- and alpha 2-adrenergic blockers were again needed to abolish the QL and RL response to nerve cold block. In another series of animals, yohimbine was administered before prazosin. In this series, alpha 2-adrenergic blockade greatly reduced neural tone as assessed by nerve cooling.(ABSTRACT TRUNCATED AT 250 WORDS)


Intravenous infusion of trieicosapentaenoyl-glycerol and LTB4 and LTB5 production by leukocytes of rabbits. During myocardial infarction leukotriene B4 (LTB4) is probably a major determining factor of tissue damage because it can amplify the inflammatory reaction by recruiting leukocytes and degranulating them. Oral administration of eicosapentaenoic acid (EPA) is known to reduce LTB4 production by polymorphonuclear leukocytes (PMNL). However, it takes several weeks for EPA to take effect. In this study, we formulated a trieicosapentaenoyl-glycerol emulsion and infused it into rabbits (0.8 g EPA/kg). In the ex vivo study, the inhibition of LTB4 production by PMNL from EPA-infused rabbits was maximal (32-60% of preinfusion values, P less than 0.01) 6 h after the infusion. There was also a tendency toward reduced LTB4 production 1, 24, and 168 h after the infusion. A lower dose (0.2 g EPA/kg) also reduced LTB4 production (45% of preinfusion values, P less than 0.02) 6 h after the infusion. There was no significant change in LTB4 production in control groups in which soybean oil emulsion was infused instead of EPA. EPA infusion might be useful for reduction of tissue damage in the acute phase of LTB4-related diseases such as acute myocardial infarction.


Role of superoxide anion and endothelium in vasoconstrictor action of prostaglandin endoperoxide. The vasoconstrictor actions of prostaglandin (PG) endoperoxide, PGH2, were examined in isolated rabbit aortic rings suspended for measurement of isometric tension. In aortic rings with an intact endothelium, PGH2 caused concentration-dependent contractions which were blocked by SQ 29548 (a PGH2-thromboxane A2 receptor blocker) or superoxide dismutase (a superoxide anion scavenger) but not by carbethoxyhexyl imidazole (a thromboxane A2 synthase inhibitor) or catalase (a hydrogen peroxide scavenger). In contrast U 46619, a thromboxane A2 mimic, caused contractions, which were blocked by SQ 29548 but not by superoxide dismutase. PGH2 caused significantly greater contractions in aortic rings without endothelium or in those with intact endothelium treated with NG-nitro-L-arginine, a nitric oxide inhibitor; these contractions were inhibited by SQ 29548 but not by superoxide dismutase. In aortic rings with endothelium contracted with phenylephrine, a subthreshold concentration of PGH2, but not U 46619, impaired relaxations to acetylcholine; the inhibition was prevented by treatment with SQ 29548 or superoxide dismutase, indicating that the abnormality of endothelial cell function was specific for PGH2. These observations indicate that PGH2 causes contractions and inhibits endothelium-dependent relaxation by a mechanism involving formation of superoxide anion, which interacts with endothelium-derived nitric oxide.


Evidence of a K(+)-H(+)-ATPase in vascular smooth muscle cells. In earlier studies Na(+)-K(+)-adenosinetriphosphatase (ATPase) and Na(+)-K(+)-2Cl- cotransport partially accounted for vascular smooth muscle cell (VSMC) K+ (Rb+) uptake. In other cells Rb+ is taken up by a K(+)-H(+)-ATPase that is sensitive to NC-1300-B, SCH28080, omeprazole, and N-ethylmaleimide (NEM). This study examines the effects of K(+)-H(+)-ATPase inhibitors on VSMC. Rubidium uptake by primary cultures of canine coronary artery (CCA) VSMC or cultured rat aortic (CRA) VSMC line A7r5 was reduced 19-37% by NC-1300-B, SCH28080, or omeprazole. N-ethylmaleimide reduced CCA VSMC K+ content from 1.55 +/- 0.02 to 1.24 +/- 0.06 mu eq/mg protein. The NC-1300-B-sensitive portion of CRA VSMC Rb+ uptake was not blocked by ouabain (0.1 mM) or bumetanide (0.1 mM), but was reduced by alkalinization with 7.5 mM NH4Cl, and increased by acidification with 7.5 mM Na-acetate. Intracellular pH (pHi) of CRA VSMC was reduced 0.14 +/- 0.03 U by NC-1300-B and 0.22 +/- 0.03 U by NEM. pHi of CCA VSMC was reduced 0.20 +/- 0.03 U by omeprazole (1 mM) and 0.20 +/- 0.03 U or 0.20 +/- 0.05 U by amiloride in the absence or presence of omeprazole, respectively. Fluorescence of 2',7'-bis(carboxyethyl)-5-(6')- carboxyethyl)-5-(6')-carboxyfluorescein due to excitation at 500:441 nm in rat aortic strips was reduced by 0.21 +/- 0.02 U by omeprazole and 0.22 +/- 0.03 U by K+ removal and increased by 0.21 +/- 0.06 U by K+ repletion. We conclude that VSMC possess a previously unknown Rb+ uptake mechanism. This newly discovered mechanism helps to maintain K+ gradient and pHi by extruding H+ in exchange for K+, and is presumably a K(+)-H(+)-ATPase similar to those described in other tissues.


Changes in insulin sensitivity from stress during repetitive sampling in anesthetized rats. The effect of repetitive sampling on insulin sensitivity was studied in anesthetized rats. During glucose clamp studies, glucose disposal decreased from 9.3 +/- 0.9 (SE) to 6.5 +/- 1.1 mg.kg-1.min-1 (P less than 0.05), and hepatic glucose output (HGO) increased from 1.2 +/- 0.8 to 2.4 +/- 1.1 mg.kg-1.min-1 (P less than 0.05) after a cumulative blood loss of 9 ml/kg. After a loss of 15 ml/kg, HGO rose further to 4.7 +/- 1.6 mg.kg-1.min-1 (P less than 0.05). During repetitive sampling under identical conditions, plasma adrenocorticotropic hormone (ACTH) increased, despite simultaneous saline infusion, from 68 +/- 11 to 102 +/- 15 pg/ml (P less than 0.05) with a loss of 8 ml/kg, while plasma insulin increased from 39 +/- 7 to 124 +/- 20 mU/l (P less than 0.01) with a loss of 10 ml/kg. Thereafter, ACTH and insulin rose progressively. Plasma corticosterone closely followed the pattern of the ACTH response, indicating that the stress of cumulative blood loss had a significant effect on adrenal steroid production. Increases in ACTH were retarded by reduced volume loss and accelerated by increased loss. It is concluded that stress from blood loss greater than 7 ml/kg may be a source of error in the evaluation of glucose turnover and insulin sensitivity during clamp experiments in rats.


Mosquito blood feeding patterns as a factor in the epidemiology of Japanese encephalitis in southern India. Determinations were made of the source of 16,330 bloodmeals from 10 species of Culex mosquitoes, including recognized vectors of Japanese encephalitis (JE) virus, in two epidemiologically distinct areas in southern India. In Madurai, where cases occurred sporadically and pigs were reared only in some villages, Cx. tritaeniorhynchus, Cx. pseudovishnui, and Cx. vishnui had fed predominantly on cattle (89.2-91%), but less frequently on humans (2.1-6.2%) and on pigs and ardeid birds (0-0.1%). In Nallur, which was endemic for JE and had a large pig population, 4.4-5.4% of the feedings were on these hosts. Cattle feedings accounted for 84.6-88% of the total feedings, human feedings for 2.4-6.2%, but there were no ardeid-positive feedings. Culex tritaeniorhynchus and Cx. vishnui showed a marked increase in the proportion of human feedings during the hot season, due to increased availability of humans sleeping outdoors to mainly exophagic mosquitoes. Feeding indices were corrected for spatial and temporal concurrence of hosts in each season, but these factors were found to require further elucidation. Discrepancies in the relative abundance of vectors as monitored by two different methods are discussed in the light of these observations.


Persistence of eggs and hepatic fibrosis after treatment of Schistosoma mansoni-infected mice. In 1971 we estimated that Schistosoma mansoni eggs in the tissues of mice were destroyed with an approximate half-life of four weeks. Our present results of five experiments suggest that egg destruction is not as rapid, and no significant destruction of eggs was detected for up to 26 weeks after treatment. However, in these experiments, a mean of 60% of the eggs in intestinal tissues were found in the feces at the time of treatment. In previously reported experiments, only 15% of gut eggs were passed in the feces. We now believe that underestimation of the number of eggs passed in the feces led to an overestimation of the number of eggs destroyed in the tissues. We analyzed liver eggs separately because eggs lost from this site are unaffected by eggs passed in the feces. No significant decrease in liver eggs occurred in the present experiments, but reanalysis of previously published data showed significant egg destruction in the liver in several experiments, although at a much slower rate than previously estimated. However, inspection of the data in the previously published and present experiments does not show a convincing difference in the number of eggs in the liver after treatment. The persistence of egg shells is probably not important in the pathogenesis of disease, but is of concern in calculating worm fecundity. Hepatic collagen levels increased markedly two weeks after treatment and subsequently decreased significantly in some, but not all, experiments.


[Effects of craniocerebral hypothermia on the course of climacteric syndrome]. Craniocerebral hypothermia was used in the treatment of 43 women aged 48 to 55, suffering from the climacteric syndrome of varying severity. The treatment efficacy was confirmed by EEG data and changes in blood levels of ACTH, LH, prolactin, and hydrocortisone.


ACTH treatment in intractable seizures of childhood. Between 1980 and 1989, 21 children suffering from intractable seizures other than infantile spasms were treated with intramuscular ACTH at the Children's Hospital Camperdown. Five patients had two courses of ACTH therapy, 24% of patients had a good response (group A), 56% responded transiently (group B) and 20% did not respond (group C). Group A had normal development and no neurological deficits prior to seizures. A favourable response was not observed in patients with partial seizures, 90% of the patients who responded had a recurrence of seizures. Mean time to recurrence was 9.0 +/- 7.3 months in group A and 1.6 +/- 2.0 months in group B. Hypokalaemia, hypertension and infection were found in 42.9%, 33.3% and 19.1% respectively. ACTH also had effects on concurrent anti-epileptic drug levels.


Demyelinating peripheral neuropathy in Cockayne syndrome: a histopathologic and morphometric study. The clinical and histopathological features of Cockayne syndrome in a 2-year-old girl are reported. Sural nerve biopsy revealed segmental demyelination and remyelination. The density of myelinated fibers, especially small ones, was decreased in comparison with an age-matched control. Although the total number of unmyelinated fibers showed no difference from that in the control, the number of small unmyelinated fibers was slightly increased. A study of teased fibers from the patient's nerve revealed that 1% of the fibers had segmental demyelination, and 7% showed remyelination. Ultrastructurally, demyelinated fibers were present sporadically. No degeneration of axons was evident. Our pathological and morphometric data for the sural nerve suggest the presence of primary demyelination in early childhood.


Transient, unilateral, isolated hypoglossal nerve palsy. We report a boy with isolated hypoglossal nerve palsy that recovered within twelve weeks. Investigations of the cervix, base of the skull, medulla and hypoglossal nerve by CT-scan and MRI did not show any abnormal findings. There are a few reports concerning isolated unilateral hypoglossal nerve palsy with benign course. Although we could not establish the cause of our patient's disease, an awareness of this condition should save such patients from unnecessary invasive studies.


Quantitation of supercoiled DNA cleavage in nonradioactive DNA: application to ionizing radiation and synthetic endonuclease cleavage. Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to "normalize" the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.


Assay of reducing end-groups in oligosaccharide homologues with 2,2'-bicinchoninate. For the first time, reducing values of homologous series of oligosaccharides have been determined by the 2,2'-bicinchoninate assay. The extent of Cu2+ reduction was monitored by spectrophotometric measurement of Cu+/2,2'-bicinchoninate complexes. Conditions of the assay were optimized so that relatively uniform reducing values were obtained with oligosaccharides derived from starch, polygalacturonic acid, and chitin, regardless of degree of polymerization. The uniform values resulted because members of each oligosaccharide series were oxidized to similar levels beyond the aldonic acid stage, while oxidation was limited to the reducing-terminal residue. This conclusion was based on direct measurements of quantitative loss of paramagnetic copper (Cu2+) by electron paramagnetic resonance spectroscopy.


An enzymatic cycling procedure for beta-NADP+ generated by 3'-phosphodiesterase, 2':3'-cyclic nucleotide. An enzymatic cycling procedure for beta-NADP+ generated by the enzyme 3'-phosphodiesterase, 2':3'-cyclic nucleotide (EC 3.1.4.37) from its substrate 2':3'-cyclic NADP+ is described. The enzymes glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and diaphorase (EC 1.8.1.4) are used to cycle the cofactor between its oxidized and reduced forms in the presence of glucose-6-phosphate and p-iodonitrotetrazolium violet (INT) with the concomitant production of colored INT-formazan, monitored at 492 nm. The amplification is about 400-fold per hour and is sensitive enough to detect 6 x 10(-13) mol of NADP(H). A simple procedure for the optimization of this cycling assay is also described. Conjugates to 3'-phosphodiesterase, 2':3'-cyclic nucleotide may be used in heterogeneous enzyme immunoassays for the detection of small quantities of haptens or proteins in biological fluids.


Effect of direct control of electroosmosis on peptide and protein separations in capillary electrophoresis. The separations of peptide and protein mixtures in capillary zone electrophoresis (CZE) at various solution conditions were studied with the direct control of electroosmosis. The zeta potential at the aqueous/capillary interface and the resulted electroosmosis in the presence of an electric field were directly controlled by using an additional electric field applied from outside of the capillary. The controlled electroosmotic flow affected the migration time and zone resolution of peptide and protein mixtures. The changes in the magnitude and polarity of the zeta potential caused the various degrees of peptide and protein adsorption onto the capillary through the electrostatic interactions. The separation efficiencies of peptide and protein mixtures were enhanced due to the reduction in peptide and protein adsorption at the capillary wall. The direct manipulations of the separation efficiency and resolution of peptide and protein mixtures in CZE were demonstrated by simply controlling the zeta potential and the electroosmotic flow with the application of an external electric field.


Some new aryl-sydnones: effects on murine tumours. The effects of new aryl-sydnones: 3-[4-X-3-nitrophenyl]-1,2,3-oxadiazolium-5-olates, where X = Cl (SYD-1); pyrrolidino (SYD-2); piperidino (SYD-3) and morpholino (SYD-4) on the survival of mice bearing Sarcoma 180, Ehrlich carcinoma, B10MCII (Fibrous histiocytoma) and L1210 leukemia ascitic tumours, on the proliferation of cultured tumour cells and on the synthesis of DNA in L1210 leukemia were determined. SYD-1 and SYD-2 in vivo significantly enhanced the survival of S180, Ehrilich and B10MCII tumour-bearing mice. Furthermore, SYD-2 showed significant activity against L1210. SYD-3 and SYD-4 did not show antitumour activity. SYD-1, in vitro was the most cytotoxic against all the above tumour cells. All of the drugs tested inhibited thymidine uptake by L1210 cells, SYD-4 being the least active.


Treatment with antioxidant and other nutrients in combination with chemotherapy and irradiation in patients with small-cell lung cancer. Eighteen non-randomized patients with small cell lung cancer (4 women and 14 men, mean age 60.4, SD 7.8 years) received in addition to conservation small cell lung cancer treatment antioxidant treatment with vitamins, trace elements and fatty acids. All patients were out-patients who, except for one were also treated with chemotherapy and/or irradiation at regular intervals at a university of central hospital. Five patients (28%) were in an advanced stage of the disease. At the end of the follow-up period (31.7.90) the median survival time for the whole group was 505 days. Fourteen (77%) of the patients survived for more than 12 months and six patients (33%) for more than two years. One patient (5%) survived more than five years. Eight patients (44%) were still alive with a mean survival time of 32 months at the end of the study. Ten patients succumbed earlier from progression of the disease. Antioxidant treatment, in combination with chemotherapy and irradiation, prolonged the survival time of patients with small cell lung cancer compared to most published combination treatment regimens alone. We also noticed that the patients receiving antioxidants were able to tolerate chemotherapy and radiation treatment well. Surviving patients started antioxidant treatment in general earlier than those who succumbed.


Detection of human papillomavirus in primary hepatocellular carcinoma. Human papillomaviruses (HPV) have been linked causally to some human cancers such as cervical carcinoma. To determine whether any additional type of human malignancy contained HPV DNA, we examined 16 hepatocellular carcinoma (HCC) specimens by Southern blot technique and by polymerase chain reaction (PCR). One HCC contained HPV 16 DNA as demonstrated by both Southern blot and PCR. Two other HCC samples contained HPV 18-related nucleotide sequences by PCR but were negative by Southern blot of genomic DNA. HPV could have been carried via blood to the liver, thus indirectly supporting the presence of an HPV viremia. Our findings suggest that oncogenic HPV might constitute a cofactor acting synergistically with hepatitis B virus (HBV) in the development of the HCC in these patients. Alternatively, the presence of HPV in the tumor tissue might be the result of an opportunistic infection.


Suppression by a pine cone extract of Pinus parviflora Sieb et Zucc of mammary tumour virus in milk of mice. The intravenous or the oral administration of a pine cone extract of Pinus parviflora Sieb et Zucc (Fr VI) and the related synthetic agent (DHP-FA) to lactating SHN mice prevented an increase of milk levels of mouse mammary tumour virus (MMTV) from day 7 to day 14 of lactation. Furthermore, Fr VI decreased the MMTV level at the 2nd lactation compared to the 1st lactation. This is the first report on the inhibition of milk MMTV of mice in the in vivo system.


Establishment and characterization of two cell lines derived from human glioblastoma multiforme. We established and characterized two cell lines derived from glioblastoma multiforme. Both cell lines exhibited tumor cell morphology and growth kinetics and showed variable expression of glial fibrillary acidic protein (GFAP), S-100, fibronectin and vimentin. Cytofluorimetrical analysis of tumor samples showed a diploid DNA distribution, whereas permanent culture cells evolved to the hyperdiploid DNA content. Karyotype studies revealed cytogenetical abnormalities described in glial tumors including gain of chromosome 7, loss of chromosome 10 and presence of double minutes (DMs). Enhanced expression of Ha-ras and c-myc genes resulted from high p-21 and p-62 levels. The contemporary presence of TGF-alpha and EGF-Rc transcripts suggested an autocrine mechanism in the cell lines growth.


In vitro activity of sparfloxacin compared with those of five other quinolones. The in vitro activity of sparfloxacin, a new difluorinated quinolone, was evaluated against 857 gram-positive and gram-negative clinical isolates and compared with those of ciprofloxacin, norfloxacin, ofloxacin, fleroxacin, and lomefloxacin. The MIC of sparfloxacin for 90% of the members of the family Enterobacteriaceae tested was 0.5 microgram/ml (range, 0.06 to 4.0 micrograms/ml); only for members of the genera Serratia, Citrobacter, and Providencia were MICs above 1 microgram/ml. Some 90% of Pseudomonas aeruginosa isolates were inhibited by 8 micrograms of the drug per ml. The MICs for 90% of Staphylococcus spp. and Enterococcus faecalis were 0.12 and 2 micrograms/ml, respectively. All (100%) Streptococcus pneumoniae strains were inhibited by 0.5 microgram/ml. The inoculum size had little effect on either the MIC or the MBC of sparfloxacin. An increase in the magnesium concentration from 1.1 to 8.4 mM increased the MIC between 2 and 10 times, depending on the genus tested. Sparfloxacin was less active at pH 5. The antibacterial activity of sparfloxacin against gram-positive bacteria was generally higher than those of the quinolones with which it was compared; against Streptococcus pneumoniae, sparfloxacin was four- and eightfold more active than ofloxacin and ciprofloxacin, respectively. The activity of sparfloxacin against gram-negative rods was generally comparable to that of ciprofloxacin except against Enterobacter and Acinetobacter spp., Pseudomonas cepacia, Xanthomonas maltophilia, and Alcaligenes and Flavobacterium spp., against which sparfloxacin was the most active quinolone.


Non-random chromosome rearrangements in herpes simplex virus type 1 transformed diploid CHEF cells. The effects of Herpes simplex virus type 1 (HSV-1) on diploid, non-tumourigenic Chinese hamster embryo fibroblasts (CHEF/18-1D-3) were studied. Six independent lines transformed by HSV-1 alone or by HSV-1 in combination with acyclovir or aqueous tobacco extract were isolated. In contrast to uninfected CHEF/18-1D-3 cells, all transformants grew in soft agar and were tumourigenic in nude mice. Neither infectious virus nor viral antigens could be detected in any of the lines. Cytogenetic analysis revealed clonal chromosome abnormalities in all lines including trisomy for the long arm of chromosome 3 in five lines. In three of these the extra 3q was translocated onto 6p. All lines showed loss of the corresponding 3p arm. The remaining line had a hypodiploid stemline with loss of one chromosome 7. This line also showed a pronounced chromosomal instability with a multitude of mainly sporadic rearrangements. These results show that HSV-1 induced transformation and tumourigenesis in CHEF cells is associated with the induction of chromosome rearrangements, in particular trisomy for 3q.


Screening for antimitotic compounds using the cdc25 tyrosine phosphatase, an activator of the mitosis-inducing p34cdc2/cyclin Bcdc13 protein kinase. A universal intracellular factor, the "M phase-promoting factor" (MPF), triggers the G2/M transition of the cell cycle in all organisms. In late G2, it is present as an inactive complex of tyrosine-phosphorylated p34cdc2 and unphosphorylated cyclin Bcdc13. In M phase, its activation as an active MPF displaying histone H1 kinase activity originates from the specific tyrosine dephosphorylation of the p34cdc2 subunit by the tyrosine phosphatase p80cdc25. We describe here a colorimetric assay of recombinant human cdc25A tyrosine phosphatase used as a cell cycle-specific target to screen for antimitotic compounds. The glutathione-S-transferase/cdc25A tyrosine phosphatase fusion protein is produced in large amounts of Escherichia coli and easily purified by affinity chromatography on glutathione-agarose. Optimal purification, storage and assay conditions (concentrations of enzyme, p-nitrophenylphosphate and dithiothreitol; duration of assay) have been determined. Using this system we tested 15 compounds currently used in cancer treatment; none of them displayed any inhibitory activity. However, the assay detected the inhibitory activity of vanadate, a reported tyrosine phosphatase inhibitor. The simplicity, speed and possible extensive automation of this assay using an essential cell cycle-regulating component provide a highly specific mechanism-based screen for antimitotic drugs discovery.


Autolysis of methicillin-resistant and -susceptible Staphylococcus aureus. The autolytic activities, including unstimulated, Triton X-100-stimulated, and daptomycin-induced, of various sets of methicillin-resistant and related methicillin-susceptible strains were compared. Faster rates of autolysis were noted in two heterogeneous methicillin-resistant transductants than in their methicillin-susceptible parental recipients, in a heterogeneous resistant strain than in a susceptible derivative created by chemical mutagenesis, and in a homogeneous resistant strain than in a derivative that had decreased methicillin resistance and was created by transposon Tn551 mutagenesis. These results suggest that the presence of the methicillin resistance region, mec, either directly or indirectly through an interaction with other host genes, confers a faster rate of autolysis on strains. Various auxilliary genes are known to affect methicillin resistance expression, and one of these genes, femA, was necessary for the expression of this faster rate of autolysis. These differences in autolytic activities were not observed in isolated crude cell walls retaining autolytic activities, suggesting different modes of regulation of autolysins in intact cells and isolated walls. In contrast, one homogeneous, highly resistant strain, DU4916, had a lower autolytic activity than did derived heterogeneous resistant and susceptible strains created by chemical mutagenesis and a strain that had decreased resistance and was created by transposon mutagenesis. Our observations suggest that methicillin resistance expression is associated with an enhanced rate of autolysis, in heterogeneous resistant strains at least.


Decreasing expression of NM23 gene in metastatic murine mammary tumors of viral etiology (MMTV). Murine mammary tumors induced by the Murine Mammary Tumor Virus (MMTV) were chosen to study the expression of the NM23 gene during the metastatic process because of their viral etiology, different from that of the previously reported experimental tumor systems. NM23 mRNA levels are higher in non metastatic tumors than in metastatic ones. Moreover, the NM23 expression is higher in tumors induced by the C3H variant of the MMTV than in tumors induced by the RIII variant. These data are a further support to the hypothesis of a basic role of the NM23 gene in the down-regulation of tumor progression.


In vitro and in vivo antibacterial activities of a new quinolone, OPC-17116. The in vitro and in vivo antibacterial activities of OPC-17116 were compared with those of ofloxacin, enoxacin, ciprofloxacin, and tosufloxacin. The MICs of OPC-17116 for 90% of the strains tested were 0.125 to 8 micrograms/ml against gram-positive bacteria such as members of the genera Staphylococcus, Streptococcus, and Enterococcus: less than or equal to 0.063 to 16 micrograms/ml against members of the family Enterobacteriaceae; and less than or equal to 0.063 to 16 micrograms/ml against glucose-nonfermentative bacilli such as Pseudomonas aeruginosa. The activity of OPC-17116 against gram-positive organisms was comparable to that of tosufloxacin and higher than those of other reference drugs. The in vitro activity of OPC-17116 against gram-negative bacteria was similar to those of the reference drugs. In experimental systemic infections in mice with various organisms, the efficacy of OPC-17116 was similar to that of tosufloxacin and greater than those of ofloxacin, enoxacin, and ciprofloxacin. In a pyelonephritic model in mice with P. aeruginosa KU-1, OPC-17116 was as active as ciprofloxacin and more active than ofloxacin, enoxacin, and tosufloxacin. In respiratory tract infections in mice with Staphylococcus aureus Smith, Streptococcus pneumoniae TMS 3, and Klebsiella pneumoniae 3K25, the efficacy of OPC-17116 was generally greater than that of tosufloxacin. The peak level of OPC-17116 in the lungs of mice was 10 times higher than that in serum and was significantly greater than levels in lung achieved with an equivalent dose of the other quinolones. The therapeutic efficacy of OPC-17116 may depend not only on its in vitro activity but also on its high concentration in tissue.


Activities of the four optical isomers of 2',3'-dideoxy-3'-thiacytidine (BCH-189) against human immunodeficiency virus type 1 in human lymphocytes. Four different isomers of 2',3'-dideoxy-3'-thiacytidine [beta-DL-(+-)-BCH-189] were evaluated in primary human lymphocytes infected with human immunodeficiency virus type 1. The beta-L-(-) isomer was the most potent enantiomer, with a median effective concentration of 1.8 nM and no discernible cytotoxicity up to 100 microM. The relative order of potencies for the isomers was beta-L-(-) greater than beta-DL-(+-) racemic greater than beta-D-(+) greater than alpha-L-(+) greater than alpha-D-(-). The beta-L-(-) enantiomer was as potent as 3'-azido-3'-deoxythymidine.


In vitro activities of five quinolones against Chlamydia pneumoniae. The in vitro susceptibilities of 10 strains of Chlamydia pneumoniae were determined for five quinolones, including ciprofloxacin, ofloxacin, fleroxacin, temafloxacin, and sparfloxacin. Sparfloxacin was the most active compound tested, followed by ofloxacin and temafloxacin. Ciprofloxacin and fleroxacin were the least active. The use of HEp-2 cells for testing C. pneumoniae resulted in larger inclusions but essentially the same endpoints as were seen with use of HeLa 229 cells.


Inactivation of poliovirus type 1 in mixed human and swine wastes and by bacteria from swine manure. The persistence of poliovirus type 1 (PO1) in mixed septic tank effluent and swine manure slurry was determined, and the antiviral effects of several bacterial cultures isolated from swine manure slurry were demonstrated. In two field experiments, PO1 was consistently inactivated more rapidly in the mixed waste than in the control Dulbecco's phosphate-buffered saline (D-PBS). D values (time [in days] for a 90% reduction of virus titer) were 18.7 and 29.9 for the mixed waste and 56.5 and 51.8 for the D-PBS control, respectively. The virus inactivation in the mixed waste was temperature dependent. A comparison of PO1 inactivation in raw mixed waste, autoclaved mixed waste, and bacterium-free filtrate of raw mixed waste at the same pH and temperatures provided an initial demonstration that the virus inactivation in the mixed waste is related, at least in part, to microbial activity. At 25 degrees C, the D value was 6.8 for the mixed waste, 11.2 for the autoclaved mixed waste, and 10.5 for the bacterium-free filtrate of raw mixed waste. At 37 degrees C, D values were 1.3, 3.9, and 3.1 for these three suspending media, respectively. Three bacterial isolates which had shown antiviral effects in a screening test each caused virus inactivation in autoclaved mixed waste, in which the effect of other microorganisms was excluded. Inhibition of PO1 inactivation by protease inhibitors suggests that the virus inactivation in the mixed waste was due in part to proteolytic enzymes produced by bacteria in the waste.


Glucose toxicity and inability of Bacteroides ruminicola to regulate glucose transport and utilization. Ammonia-limited (3.5 mM ammonia) cultures of Bacteroides ruminicola B(1)4 had a high number of viable cells (greater than 10(9)/ml), but only when the concentration of glucose was not too high (10 mM or less). When the glucose concentration was increased from 10 to 50 mM, there was a marked decrease in viability (10(5)-fold or greater). Because there was little decline in pH and only a small increase in succinate and acetate as the glucose concentration was increased, it did not appear that end products were killing the cells. This conclusion was supported by the observation that reinoculated cultures grew in the spent medium which had been supplemented with ammonia. Unlabeled rhamnose did not inhibit [14C]-glucose uptake, and cultures which were selected with a low concentration of rhamnose tolerated high concentrations of glucose (50 mM). The glucose-resistant mutant transported glucose at a lower rate than the wild type, and the Vmax of glucose transport was fourfold lower. The wild type stored much more polysaccharide than the glucose-resistant mutant, but it is not clear if polysaccharide accumulation per se is responsible for the glucose toxicity. These results indicated that B. ruminicola B(1)4 is unable to regulate glucose transport and utilization when growth is limited by ammonia.


[Inhibitory effects of vasoactive intestinal peptide on superoxide anion generation from stimulated human inflammatory cells]. Vasoactive intestinal peptide (VIP) is suspected to be a neurotransmitter of nonadrenergic and noncholinergic inhibitory nerves in human respiratory tracts. Although VIP affects T lymphocytes through its specific receptor, the effects of VIP on inflammatory cells except T lymphocytes are obscure. We investigated the effects of VIP on superoxide anion (O2-) generation from five kinds of human cells; neutrophils, eosinophils and mononuclear cells isolated from peripheral blood, alveolar macrophages obtained from the bronchoalveolar lavage, and human monocyte cell line, U937, the capacity of which for O2- generation was induced by interferon-gamma. O2- generation from human cells stimulated by 10(-5) M fMLP was measured by the cypridina luciferin analog, MCLA, -dependent chemiluminescence method. VIP inhibited O2- generation from fMLP-stimulated neutrophils, mononuclear cells and U937 in a dose-dependent manner. 3 x 10(-6) M VIP inhibited O2- generation from fMLP-stimulated eosinophils and alveolar macrophages significantly. These results indicate that VIP might inhibit the activation of inflammatory cells and act as an antiinflammatory agent.


Protein kinase-dependent effects of okadaic acid on hepatocytic autophagy and cytoskeletal integrity. The protein phosphatase inhibitor okadaic acid suppressed autophagy completely in isolated rat hepatocytes, as measured by the sequestration of electroinjected [3H]raffinose into sedimentable autophagic vacuoles. Okadaic acid was effectively antagonized by the general protein kinase inhibitors K-252a and KT-5926, the calmodulin antagonist W-7, and by KN-62, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMK-II). These inhibitors also antagonized a cytoskeleton-disruptive effect of okadaic acid, manifested as the disintegration of cell corpses after breakage of the plasma membrane. CaMK-II, or a closely related enzyme, would thus seem to play a role in the control of autophagy as well as in the control of cytoskeletal organization.