The frozen shoulder: diagnosis and treatment. Prospective study of 50 cases of adhesive capsulitis. A prospective study of randomized analysis treatment of 50 cases of frozen shoulder was carried out in 3 Swiss medical centres. Three separate aetiological groups were studied: post-traumatic (40%), neurological (14%) and idiopathic (46%). An increased radioisotope bone scan (99 mTc diphosphonate) was found in 96% of cases, regardless of aetiology. The so-called idiopathic frozen shoulder showed a scapulo-humeral increase in radioisotope uptake in several areas (in 82% of cases) without involvement of the ipsilateral carpus. Clinically, the neurological type was associated with a shoulder-hand syndrome with positive bone scan of the shoulder and the wrist in all cases. The post-traumatic type showed a diffuse (in 50% of the cases) or at several circumscribed areas (also in 50%) increase in radioisotope uptake in the shoulder. In 45% of the post-traumatic type, there was also a shoulder-hand syndrome with uptake in the wrist also. A physical treatment and early mobilization, associated with the administration of subcutaneous salmon calcitonin for 21 days (100 U Calcitonin Sandoz) had a statistically significant increased effect on pain compared to treatment with physiotherapy alone by patients with post-traumatic frozen shoulders (p < 0.02). There was no significant difference, however, in the speed of recovery of function between the two treatment groups. These observations strengthen the hypothesis that adhesive capsulitis behave like an algoneurodystrophic process.


Monoclonal antibodies against partially deglycosylated colon cancer mucin that recognize Tn antigen. In order to develop reagents that can detect the exposed core carbohydrate antigens of mucins, we have prepared monoclonal antibodies against partially deglycosylated LS174T human colon cancer mucin. The three monoclonal antibodies, 10F4, 15D3a, and 91S8, stained cancers of the colon, pancreas, stomach, breast, prostate, and lung to a greater extent than corresponding normal tissues. There was no staining of normal pancreas or breast, suggesting that these antibodies may be particularly useful for detecting cancers in these two organs. In homogenates of cultured cancer cells, antigen was detectable in three colon cancer cell lines, but not in a variety of other epithelial cancers. The epitope specificity of all three monoclonal antibodies appears to be for Tn antigen, i.e. GalNAc-alpha-Ser/Thr, based on their recognition of alpha-linked GalNAc, but not T antigen, sialyl Tn, or a range of other structures. However, the three anti-Tn antibodies differed in tissue staining specificity and in relative binding to different mucins. These monoclonal antibodies, prepared against deglycosylated colon cancer mucin, appear to be useful reagents for the immunohistochemical detection of epithelial cancers, especially pancreatic cancer and breast cancer.


[Therapy of bone metastases of the lower extremity with the KMFTR modular tumor endoprosthesis system]. During the years 1975 through 1989, 189 patients have been operated because of metastases within bones of the lower extremity. Thereof, 34 patients received an endoprosthesis of the KMFTR (Kotz Modular Femur-Tibia reconstruction system) type. In detail, the following reconstruction elements had been used: proximal femur (n = 20), distal femur (n = 7), complete femur (n = 2), proximal tibia (n = 5). At the time of follow-up examination 30 patients had died. Mean survival time after resection of metastases was 18.6 months (range 8-44 months). According to histologic judgement, in 23 cases wide or marginal resections had been performed whereas 11 patients had been operated intralesionally. In no case local recurrence as relevant to therapy was observed. At the same time-point, survival periods after detection of secondary manifestations of the malignant diseases within the remaining 4 patients were 29, 61, 73 and 125 months, respectively. The following complications were observed: luxation of the prosthesis (n = 6), transient paresis of the peroneus (n = 3), deep vein thrombosis (n = 2), pulmonary infarction (n = 1), wound healing disturbances (n = 2) and hematoma (n = 3).


Reticulocyte quantification by flow cytometry, image analysis, and manual counting. Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis >> manual counting.


Suppression of interferon response of bovine leukocytes during clinical and subclinical ketosis in lactating cows. The influence of spontaneous ketosis on interferon alpha and gamma production in blood leucocytes and on PHA induced lymphocyte blastogenic response was investigated. Twenty three cows 4.13 +/- 2.8 weeks after calving were divided into three experimental groups on the basis of blood ketone bodies, glucose and free fatty acids concentrations. The leukocytes of cows with clinical symptoms and the highest concentration of ketones and free fatty acids in blood responded with the lowest levels of interferons alpha and gamma to three interferon inducers: Newcastle Disease Virus (NDV), phytohemagglutinin (PHA) and concanavalin A (ConA). Depression in interferon PHA stimulated synthesis correlated with a very low mitogenic response of blood lymphocytes. Blood leukocytes of cows with subclinical ketosis, characterized by mild clinical symptoms and a lower concentration of ketones in blood in comparison to cows with clinical ketosis, responded better to interferon and mitogenic stimulation; however, the interferon titer and blastogenesis were still lower than in leukocytes of healthy cows. Correlation between the stage of ketosis and the level of interferon production in milk leukocytes was also observed. A possible relationship between the suppression of interferon production in blood leukocytes and the increased concentration of ketone bodies in blood is discussed.


Diabetic gastroparesis. A critical reappraisal of new treatment strategies. Delayed gastric emptying, gastroparesis, is one of the sequelae of diabetes mellitus. Symptoms may include postprandial nausea, epigastric pain, bloating, vomiting, early satiety and unpredictable blood sugar fluctuations. Nowadays diagnosis is made by the measurement of gastric emptying with a radionuclide test meal. Using this technique some 50% of diabetic patients show signs of disordered gastric emptying. Relief is best delivered by agents promoting gastric emptying. In phase II single-dose studies metoclopramide, domperidone, cisapride, erythromycin and renzapride were all able to enhance gastric evacuation of solid and liquid meals in patients with diabetic gastroparesis. A few short term studies support the efficacy of domperidone and renzapride, but long term trials are lacking. Erythromycin, mimicking the potent gastrokinetic effect of motilin, may hold considerable promise for the future. Experience with erythromycin in diabetic gastroparesis is nonetheless very limited. To some extent the therapeutic effectiveness of metoclopramide and cisapride has been established in placebo-controlled trials. In trials with a placebo-controlled crossover design, however, only metoclopramide showed a sustained positive effect. Metoclopramide, which combines gastrokinetic and antiemetic properties seems, so far, the best therapeutic option in diabetic gastroparesis. Cisapride may be considered as a good alternative in cases where limited efficacy or side effects preclude the use of metoclopramide.


Immunosuppressive drugs in renal transplantation. A review of the regimens. Currently, expected 10-year first graft survival rates for kidneys from HLA-identical sibling, 1-haplotype-matched relative, and cadaver donors are 74, 51, and 40%, respectively. Histocompatibility, immunological conditioning with blood products, and immunosuppression with glucocorticoids, azathioprine, cyclosporin, and the antithymocyte (antilymphocyte) antibody preparations have been significant factors in the gradual improvement of kidney graft survival rates. Nearly all immunosuppression regimens are cyclosporin-based. Antithymocyte antibody induction therapy with delayed administration of cyclosporin is widely practised to avoid cyclosporin nephrotoxicity while the kidney graft is recovering from preservation injury. Late cyclosporin withdrawal results in inferior cadaver kidney transplant survival rates. Rejection crises usually respond to high dose glucocorticoid therapy. Glucocorticoid-resistant rejection usually responds to treatment with antithymocyte antibody. FK-506 is a promising new immunosuppressant that has properties similar to cyclosporin. Prophylaxis against viral, bacterial and fungal infections is necessary to reduce the morbidity of immunosuppression. The incidence of malignant conditions associated with viral infections is significantly increased with immunosuppression. New immunopharmacological agents and advances in genetic procedures may allow the induction of specific transplantation tolerance and successful xenotransplantation within the next decade.


Treatment considerations in acute renal failure. Acute renal failure (ARF) is characterised by progressive azotaemia, and for therapeutic purposes consideration of prerenal, intrinsic renal and postrenal types still holds good. Prerenal azotaemia is generally caused by loss of body fluids or blood, whereas postrenal azotaemia is effected by acute or chronic urinary tract obstruction. Provided these conditions are recognised on time and treated, they are reversible. However, delay in recognition or treatment could result in renal parenchymal damage and sustained ARF. Therefore utmost attention should be focused on identifying reversible factor(s) in the setting of ARF. Once reversible factors have been excluded, and ARF becomes sustained, a diagnosis of acute intrinsic renal failure is almost certain. Lack of natriuretic and diuretic responses to fluid challenge or infusion of furosemide (frusemide) and dopamine are further indications of this possibility. Management of acute intrinsic renal failure essentially consists of dietary control and dialysis therapy. The latter facilitates fluid and electrolyte management, but does not reduce the overall mortality. The potential benefit of parenteral hyperalimentation to promote renal function recovery must be carefully weighed against the risk of severe infectious complications.


Management of dysfunctional uterine bleeding. Dysfunctional uterine bleeding, although not usually life-threatening, can cause disruption and discomfort for many women. It has often been poorly researched in the past, possibly because of the difficulty in measuring menstrual blood loss. Several different therapies are available and individual women can choose from a number of options. Nonsteroidal anti-inflammatory drugs such as mefenamic acid or indomethacin will be the first choice for many women as they have few side effects and it is only necessary to take them when menstrual bleeding occurs. When contraception is also required, combined oral contraceptives are helpful. Progestogen and danazol therapy are also effective, although side effects do occur. A new development has been the levonorgestrel-containing intrauterine contraceptive device which has been shown to result in large decreases in menstrual blood loss. For those women who would like a surgical approach but do not want to undergo hysterectomy, the relatively new technique of endometrial resection results either in amenorrhoea or reduced menstrual blood loss in the majority of women.


Ankylosing spondylitis. Current drug treatment. The administration of drugs constitutes an important component of the therapeutic programme in ankylosing spondylitis (AS). The main objective of initiating such therapy is to reduce pain, stiffness and discomfort. There are at present 3 groups of drugs available for the management of AS. The first group is represented by drugs thought to influence the disease process itself. In this group, sulfasalazine is the only drug which is controlled trials has been shown to suppress disease activity in AS. We recommend the use of sulfasalazine in patients with high disease activity, with peripheral arthritis and in those with AS of short duration. The second group of drugs includes nonsteroidal anti-inflammatory drugs (NSAIDs), which suppress inflammation without influencing the disease process. These drugs should be administered selectively during periods of high disease activity. Moreover, 1 drug should be used in appropriate dosage before it is assumed to be inefficient. High doses of NSAIDs may be prescribed before bedtime in patients suffering from severe pain and stiffness during the night. The toxicity profile of NSAIDs includes gastrointestinal and renal side effects. The third group comprises analgesics and muscle relaxants. Such drugs should be used rather frequently in patients with longstanding AS refractory to treatment with NSAIDs. Peripheral arthritis and enthesopathy are generally managed by local injections of corticosteroids, while AS complicated by psoriasis or inflammatory bowel disease is treated as primary AS. AS occurring in juveniles is best treated with aspirin and an NSAID, although careful observation is necessary for the development of Reye's syndrome (with aspirin) and gastric irritation (with NSAIDs). When patients with AS undergo surgery, the possibility of silent gastrointestinal bleeding due to the use of NSAIDs and salicylates should not be ignored. Patients treated with oral corticosteroids should receive a bolus injection of soluble corticosteroid prior to surgical intervention. NSAIDs may be administered pre- and postoperatively to relieve stiffness induced by immobility. Rapid treatment of intervening infections and use of NSAIDs is recommended in AS complicated by renal amyloidosis. During pregnancy and lactation, ibuprofen may be the preferred drug in AS.


Sertraline. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in depression and obsessive-compulsive disorder. Sertraline is a selective inhibitor of central serotonin reuptake. Thus, it enhances serotoninergic transmission--a property which appears to explain its antidepressant activity. Its elimination half-life (approximately 26 hours) makes it suitable for once daily administration. Although clinical experience with sertraline is limited, it appears to possess antidepressant efficacy similar to that of amitriptyline and dothiepin, marginally better than imipramine, and significantly better than placebo. Additionally, sertraline is the only antidepressant licensed in the UK for the prevention of recurrence of depression, and preliminary findings suggest that the drug may also be effective in the treatment of obsessive-compulsive disorder. Sertraline and other serotonin reuptake inhibitors possess tolerability advantages over tricyclic antidepressants. Sertraline has minimal anticholinergic activity, is essentially devoid of cardiovascular effects, has a wide therapeutic index and may be administered to elderly patients or those with underlying cardiovascular disorders. However, as with other serotonin reuptake inhibitors, sertraline has been associated with gastrointestinal disturbances (nausea, diarrhoea/loose stools) and male sexual dysfunction (primarily ejaculatory disturbance), although each of these effects is usually mild and transient, decreasing in frequency with continued treatment. As a drug class, serotonin reuptake inhibitors such as sertraline appear to provide significant advantages compared with the more established antidepressant agents, particularly in terms of tolerability. Although much broader clinical experience is required before sertraline's full therapeutic potential can be realised, if future studies confirm the encouraging initial findings, sertraline will undoubtedly become an important option in the treatment of depression.


Nicorandil. A review of its pharmacology and therapeutic efficacy in angina pectoris. Nicorandil belongs to the class of compounds known as potassium channel activators which are characterised by their arterial vasodilator properties. In addition, nicorandil has venodilating properties which are attributable to a nitrate group in its chemical structure. Therefore, by combining these two vasodilator mechanisms, nicorandil represents a novel type of compound for use in the treatment of angina pectoris. Furthermore, increasing experimental evidence suggests that potassium channel activation may also exert a direct cytoprotective effect by augmenting normal physiological processes which protect the heart against ischaemic events. Comparative studies of up to 3 months' duration suggest that nicorandil is equivalent in efficacy to isosorbide dinitrate, propranolol, atenolol, nifedipine or diltiazem in the treatment of stable angina. Preliminary evidence suggests that an improvement of anginal and ischaemic symptoms is maintained for up to 1 year. Whilst the efficacy of nicorandil in other types of angina has not been extensively studied, preliminary results indicate that intravenous nicorandil is as effective as isosorbide dinitrate in the treatment of unstable angina and is also effective in patients with variant angina. In addition, the limited data available indicate that nicorandil may be effective in patients with unstable and variant angina who are refractory to therapy with conventional antianginal agents, a potentially important area for further study. Headache, mostly of mild to moderate intensity was the most commonly reported adverse event, occurring in one-third of patients receiving the recommended therapeutic regimen of nicorandil 10 to 20mg twice daily. In comparative trials involving a total of 84 patients who received nicorandil, the incidence of headache was similar to that produced by isosorbide mononitrate and isosorbide dinitrate. Headache was most frequent on initiating therapy but declined with continued treatment. To date, approximately 5% of patients participating in European trials have withdrawn due to headache, although this rate may be reduced by using a lower starting dose of nicorandil (5 mg twice daily). In summary, clinical experience thus far indicates that nicorandil, with its novel combination of two distinct vasodilator mechanisms, offers an effective alternative to established vasodilator therapy with conventional nitrates and calcium antagonists in the long term treatment of stable angina pectoris. Further studies are warranted to establish whether the unique pharmacodynamic profile of nicorandil is advantageous for the treatment of other types of angina and/or the ischaemic myocardium.


Zalcitabine. A review of its pharmacology and clinical potential in acquired immunodeficiency syndrome (AIDS). Zalcitabine is an analogue of the nucleoside deoxycytidine which, when intracellularly converted to an active triphosphate metabolite, inhibits replication of human immunodeficiency virus (HIV). Zalcitabine is thought to act in the early phase of HIV replication by inhibiting reverse transcriptase and terminating the viral DNA chain. In vitro, zalcitabine is one of the more effective nucleoside analogues currently in clinical use for HIV infection, with 0.5 mumol/L concentrations completely inhibiting HIV replication in human T lymphocyte cell lines. In clinical trials, p24 antigen levels decreased and CD4 cell counts increased in patients with acquired immunodeficiency syndrome (AIDS) receiving zalcitabine > or = 0.03 mg/kg/day as monotherapy. Dose-dependent adverse effects that include peripheral neuropathy, stomatitis and rash, restrict long term use at higher dosages, and it is unclear whether zalcitabine monotherapy is as effective as zidovudine in extending survival in HIV-infected patients. Alternating or concomitant therapy with zalcitabine and zidovudine provides effective inhibition of viral replication and disease progression (as measured by improvements in CD4 cell counts) with lower and less toxic dosage regimens. At present, therefore, zalcitabine has a place in AIDS therapy both in combination with zidovudine, and as monotherapy for patients unable to tolerate zidovudine.


Anterior "cheek" electrodes are comparable to sphenoidal electrodes for the identification of ictal activity. Sphenoidal electrodes are used to localize epileptiform activity originating in the temporal lobe during complex partial seizures. Sphenoidal electrodes, however, are semi-invasive and uncomfortable to the patient. We compared skin electrodes placed on the cheek ("cheek electrodes") with sphenoidal electrodes for the detection of the side and site of complex partial seizure onset. In a masked, randomized comparison of single ictal recordings in 22 patients, there were no significant differences between sphenoidal and cheek electrode montages in detecting the side or site of ictal onset (P < 0.01). Signal/noise ratios for interictal spikes were a mean 16.5% greater at sphenoidal sites compared to cheek sites (paired t test, t = 2.4, P < 0.05). This difference, however, did not influence the detection of rhythmical ictal activity in cheek and sphenoidal montages in our study, nor the assignment of side, site or time of seizure onset by unbiased readers. Recordings from cheek electrodes are comparable to those from sphenoidal electrodes and are useful for localizing ictal activity.


Sleep deprivation in narcoleptic subjects: effect on sleep stages and EEG power density. Sleep of 8 narcoleptic and 8 control subjects was recorded under baseline (i.e., prior wakefulness 16 h) and after 24 h without sleep. During both baseline and recovery total sleep time and stage 2 non-REM sleep were significantly decreased in narcoleptic subjects. Slow wave activity (i.e., EEG power density in the range of 0.75-4.5 Hz) decayed exponentially during baseline and after sleep deprivation in both narcoleptic and control subjects. During both baseline and recovery EEG power density in delta and sigma frequencies in non-REM sleep was enhanced in narcoleptic subjects relative to controls. In REM sleep differences in the same direction were present in delta and beta frequencies. After sleep deprivation EEG power density in non-REM sleep was elevated in delta and some higher frequencies in both patients and controls, but the response to sleep deprivation was stronger in narcoleptic subjects. These data show that in narcoleptic subjects regulatory processes underlying non-REM sleep homeostasis are operative and indicate that the response to sleep deprivation is stronger than in control subjects.


Maturation of the coherence of EEG activity in normal and learning-disabled children. The age effect on coherence has been studied in control (98) and learning-disabled (LD, 54) school-aged children (from 6.0 to 16.8 years old). The EEG recordings were made at rest in 15 leads, and 105 pairwise combinations for coherence were calculated (each lead was compared with all the rest) for delta, theta, alpha, beta and total frequency bands. A significant increase of coherence with age was found in both groups, with a different pattern of maturation. In the control group, a significant increase with age was found in the coherences between posterior regions and vertex (Cz). A significant decrease with age in the coherence between frontal areas was observed, especially in the theta band. The LD group showed a different pattern: no significant relation with age was found in the coherence between any lead and vertex. A high effect of age on coherence between temporal regions was observed with a predominance of the left side in comparison with the contralateral and the ipsilateral. No decrease in frontal coherence was found: in the same region where the control group showed negative values with age, the LD groups had no age effect. The results obtained are discussed as differences in brain organization, in myelogenesis and synaptogenesis and an explanation of the etiology of LD is proposed.


A comparison of quantitative EEG frequency analysis and conventional EEG in patients with focal brain lesions. The sensitivity and specificity of quantified EEG frequency analysis (EEGFA) were assessed in a group of patients with CT or MRI verified unilateral cerebral lesions and compared with the results of conventional EEG interpretations. Digital EEG recordings were obtained in 25 patients and 25 normal control subjects during performance of an alerting task. Recording artifacts were carefully eliminated. The results of EEGFA were then statistically compared with those of 75 additional normal subjects in the same age range. Complete conventional EEGs were blindly interpreted by two independent electroencephalographers using a structured reporting procedure. We observed similar overall sensitivities for the two methods. Optimal yield for EEGFA was associated with the use of longer edited EEG lengths, longitudinal bipolar montage, and normative data based on total EEG power. In those recordings with normal or mildly abnormal EEG backgrounds the two techniques were to an extent complimentary, each detecting abnormalities missed by the other. In such circumstances EEGFA may be useful as an extension of conventional EEG interpretation.


EEG in liver transplantation: visual and computerized analysis. We prospectively evaluated 40 liver transplant candidates (including 28 recipients) with visually scored or computerized EEG. As expected, EEG frequencies rose significantly after successful transplant (P < 0.01). For all subjects, but especially for the transplant recipients, higher frequencies on computerized EEG at baseline had a strong association with survival at 18 months (P < 0.001). A logistic regression model allowed estimation of the odds for survival and indicated less than 50% long-term survival with central-occipital mean frequencies below 7.6 c/sec. Visual EEG scores closely paralleled the quantitative results, but only the latter were amenable to formal statistical analysis. EEG had much stronger predictive value for survival than serum albumin, although albumin improved more significantly in the months after transplantation.


Half-field sinusoidally modulated light stimulation at subject's alpha frequency. Interhemispheric responses of alpha activity were investigated by use of half-field sinusoidally modulated light (SML) stimulation at the subject's alpha frequency. The left and right visual half-fields as well as full-field were separately stimulated by the SML. The power spectrum of SML responses, averaged with Wiener filtering, was obtained at the left and the right occipital area (O1 and O2) in 11 normal subjects. Power in each hemisphere, phase difference and coherence between O1 and O2 were estimated at an alpha frequency. A laterality index of power was defined as (P(l)-P(r))/(P(l) + P(r)) where P(l) is a power value at O1, and P(r) at O2. In most of the subjects, laterality index decreased in the order: left half-field, full-field and right half-field stimulation. This relationship revealed greater SML responses on the ipsilateral occipital area. There were significant differences in laterality index among 3 visual field conditions, thus showing that full-field responses ranged in lateralization between the left and right half-field responses in most of the subjects. This hemispheric distribution suggests that half-field SML stimulation affected alpha activity selectively in each hemisphere, mostly in the ipsilateral hemisphere.


Frontocentral DC-potential shifts predicting behavior with or without a motor task. This study was designed to investigate the predictive value of the event-related potentials (ERPs) preceding the initiation of a difficult perceptual-memory task and to investigate whether these ERPs require a motor movement on the part of the subject for their occurrence. Across 4 conditions the DC-potential shifts were recorded from 23 right-handed subjects using DC amplifiers. Although the start of each trial began with a ready signal, the conditions differed in that the subjects initiated the task by a button press in 2 conditions and the computer initiated it in 2 others without a press. The results showed that, especially in the frontocentral electrode sites, the DC-potential shifts which began those trials ending in correct performance were more negative relative to those trials ending in an incorrect response. Those conditions which required the subjects to self-initiate the trial and those which were initiated by the computer showed similar results indicating that the negative DC-potential shifts preceding correct performance are neither produced by nor depend on a task initiating motor movement. The onset of the DC-potential shifts preceded task initiation by up to 4.1 sec indicating that they were more than the Bereitschaftspotential.


Alterations in brain electrical activity caused by magnetic fields: detecting the detection process. Static and 60 Hz magnetic fields, 0.78 gauss, were applied individually and combined to each of 20 human subjects during 2 sec epochs, and the effect on the EEG was determined by comparing the power spectrum obtained during field exposure with that from control epochs. All but one subject exhibited field-induced alterations in the EEG; most subjects exhibited increased EEG activity at 2 or more frequencies within 1-18.5 Hz. The field-induced changes were recorded more often at the central and parietal electrodes than at the occipital electrodes. The responses observed during application of combined static and alternating fields did not differ from the sum of the responses observed when the fields were applied individually, even though the exposure conditions were specifically chosen to favor the hypothesized ion-resonance mechanism of interaction involving Ca2+. The data support the view that detection loci for magnetic fields exist within the nervous system.


Development of data reconstruction system of paper-recorded EEG: method and its evaluation. A new system by which paper-recorded EEG can be converted to computer treatable digital data was developed. It consists simply of an image scanner and a microcomputer. The performance of the system was evaluated and found to perform well in data reconstruction. The system makes it possible to apply various computer analyses to EEGs recorded on paper.


Native horizontal ultrathin polyacrylamide gel electrophoresis of proteins under basic and acidic conditions. The preparation of homogeneous ultrathin native polyacrylamide gels, using a basic as well as an acidic buffer system is described. The basic buffer system consists of Tris-HC1/Tris-glycine, the same buffer as in sodium dodecyl sulfate (SDS)-gel electrophoresis but without SDS. The acidic system uses potassium acetate, pH 4.3, as gel buffer and beta-alanine, pH 4.6, acetic acid as electrolytes. The gels are covalently bound on glass plates. Binding of acidic gels requires a special pretreatment of glass plates. The whole procedure is simple and extraordinarily fast: 100-120 min from the start of gel preparation to the end of electrophoresis. Coomassie staining is done in 40 min and silver staining in 90 min. The native gels are excellently suited for diffusion blotting. Further attractive properties of these gels are easy handling, simple drying and dimensional stability.


Improving the detection of proteins after transfer to polyvinylidene difluoride membranes. N-Terminal sequence analysis of proteins separated by two-dimensional polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes has become the method for molecular characterization of proteins contained in biological samples. However, the proteins of lower abundance cannot be sequenced directly, without improving the technique. We have studied a drying method on several PVDF membranes including Trans-Blott, Immobilon P and Problott. Using Amido Black, Coomassie Brilliant Blue R-250 and Ponceau S, we have obtained, in comparison with the non-dried membranes, an enormous increase in the number of detectable proteins.


Detection of the hypusine-containing protein (HP = eIF-5A) in crude yeast extracts by two-dimensional western blots. The hypusine-containing protein (HP) with its unique modification of a specific lysine residue resulting in the amino acid hypusine is highly conserved among all eukaryotes and is also found in Archaebacteria. Studies of the protein function in translational processes showed a stimulatory effect in the methionyl puromycin assay, but not in in vitro translation of native mRNA. It was therefore also designated as eIF-5A. To further investigate the role of HP in cellular metabolism, we purified the protein from Saccharomyces cerevisiae and raised polyclonal antibodies in chicken. Immunoglobulin preparations from the eggs of the immunized hens were used for Western blot analysis of HP in crude yeast extracts. For those studies, the soluble protein fraction of the yeast was resolved on two-dimensional gels (first dimension: isoelectric focusing using an immobilized pH gradient (IPG), pH 4-7; second dimension: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 12% T) and subsequently blotted onto Fluorotrans membrane. Anodic versus cathodic application of the extracts of the IPG strips was compared.


Two-dimensional electrophoresis analysis of human serum proteins during the acute-phase response. The serum of patients with meningitis, due to infection by Haemophilus influenzae type b, was analyzed. Several known acute-phase proteins were separated by two-dimensional electrophoresis and estimated quantitatively. In addition, hitherto undescribed reactants were recognized. Gels were calibrated and relevant spots related to master spot numbers in the human serum protein database.


Comparison of the cellular protein composition of aortic smooth muscle cells in vivo and in vitro by two-dimensional gel electrophoresis. Vascular smooth muscle cells are of major interest for the evaluation of atherosclerotic processes. By the use of two-dimensional gel electrophoresis and subsequent silver staining we investigated the protein expression of smooth muscle cells in the native thoracic aorta, immediately after enzymatic disaggregation, in subconfluent proliferating and in postconfluent nonproliferating primary cell cultures. Compared to the native thoracic aorta the protein composition of smooth muscle cells in cell culture is changed dramatically. Furthermore, significant differences in protein expression between proliferating and nonproliferating smooth muscle cells in cell culture were found.


Tyrosine phosphorylation and the mechanism of signal transduction by the B-lymphocyte antigen receptor. Lymphocytes provide a powerful defense against infectious agents with their exquisite ability to distinguish between macromolecules of the host and macromolecules of foreign invaders. This ability derives from the antigen receptors, which are created from precursor minigenes by a series of genetic-recombination reactions [1, 2] and from cellular mechanisms that inactivate lymphocytes expressing self-reactive antigen receptors [3, 4]. Central to the problem of distinguishing self from non-self is the means by which these antigen receptors recognize antigen and transmit the information of that recognition to the interior of the cell. This information ultimately leads to lymphocyte activation or inactivation, depending upon the context. In this review, I shall summarize recent advances in understanding the structural elements of the antigen receptor complex of B lymphocytes and in understanding the signal-transduction events initiated by this receptor.


Detection of polypeptides and amylase isoenzyme modifications related to malting quality during malting process of barley by two-dimensional electrophoresis and isoelectric focusing with immobilized pH gradients. Two cultivars ("Alexis" and "Lenka") of contrasting final attenuation values were malted, and the protein and amylase isoenzyme composition, as well as the change in protein and amylase isoenzyme composition during malting, was investigated by two-dimensional polyacrylamide gel electrophoresis of total proteins, and isoelectric focusing of amylase isoenzymes, respectively. Isoelectric focusing demonstrated that significant differences exist between the amylase isoenzyme patterns of the two cultivars, suggesting a correlation between the presence of certain amylase isoenzyme bands and final attenuation. This finding was confirmed by analysis of 36 barley cultivars with a wide range of quality. It was shown that all cultivars which are of low or, at best, moderate final attenuation values exhibit the amylase band "B" (isoelectric point approximately 6.8), whereas those cultivars which are predominantly of high malting grade do not possess this "B" isoenzyme band, but exhibit the pronounced "A" isoenzyme band (isoelectric point approximately 6.5) instead, suggesting that these isoenzymes (which we suppose to be beta-amylases) can be utilized to predict the final attenuation values of unknown barley samples or new lines. However, "final attenuation" is a complex function. Preliminary results of two-dimensional gel electrophoresis indicate that other factors, such as total amount of amylases, or a 19 kDa A hordein-like polypeptide, which was degraded faster in the low malting grade cultivar "Lenka", may also have a role in determining quality.


Characterization of S1 nuclease. Involvement of carboxylate groups in metal binding. Modification of the carboxylate groups of purified S1 nuclease resulted in a loss of its single-stranded DNAase, RNAase and phosphomonoesterase activities. The inactivation was due to the removal of zinc atoms from the enzyme and this in turn was dependent on the degree of modification. While the removal of one zinc atom resulted in the partial inactivation of the enzyme, removal of the remaining zinc atoms resulted in the complete inactivation of the enzyme. Similar results were obtained when the purified enzyme was incubated with various concentrations of the metal chelator, EDTA. The EDTA-(1 mM)-treated enzyme, depleted of one zinc atom, showing 40-45% residual activity, when incubated with 1 mM Zn2+ or 1 mM Co2+, regained a significant amount of its initial activity towards all the substrates. However, Woodward's-Reagent-K-modified enzyme depleted of one zinc atom and having the same level of activity (40-45%) could not regain its activity, indicating that the carboxylate groups are involved in the metal binding. Data obtained with carboxylate-group modification, EDTA-treatment, reconstitution with metal ions, zinc estimation and CD analysis of the enzyme suggests that, out of three zinc atoms present in S1 nuclease, zinc I is easily replaceable and is probably involved in the catalytic activity while zinc II and zinc III are involved in maintaining the enzyme structure.


Application of sequential extraction procedures and glycoprotein blotting for the characterization of the 2-D polypeptide patterns of barley seed proteins. Barley (Hordeum vulgare L.) proteins were sequentially extracted from ground seeds with Tris-HCl buffer, 55% 2-propanol, 55% 2-propanol containing 1% dithiothreitol, and 6 M urea containing 2% Nonidet P-40 and 1% dithiothreitol. The protein composition of these solubility fractions was then analyzed by high resolution two-dimensional gel electrophoresis with immobilized pH gradient 4-9 in the first dimension, followed by silver staining and glycoprotein blotting, respectively, for a more detailed characterization of the two-dimensional polypeptide pattern of barley seed proteins.


The structure of the O-antigenic polysaccharide from lipopolysaccharide of Vibrio cholerae strain H11 (non-O1). After acid degradation of the lipopolysaccharide (LPS) of Vibrio cholerae strain H11 (non-O1), a tetrasaccharide was obtained, the structure of which was determined by quantitative and methylation analyses, periodate oxidation, one- and two-dimensional NMR spectroscopy, and fast-atom-bombardment and four-sector tandem mass spectrometry as beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-alpha-D-GalANGr o-(1-4)-NeuAc, in which GalANGro is N-galacturonoyl-2-aminoglycerol and QuiN 2-amino-2,6-dideoxy-glucopyranose. In addition, the trisaccharide beta-D-GalANGro-(1-3)-beta-D-QuiNAc-(1-4)-D-altro-hept ulose and the disaccharide alpha-D-GalANGro-(1-4)-NeuAc were isolated from acid-degraded lipopolysaccharide; the occurrence of sedoheptulose in lipopolysaccharide has not been described before. Based on the result of methylation analysis showing that galacturonic acid was the terminal sugar of the polysaccharide chain, and on the assumption that the tri- and the disaccharide represented the reducing and the non-reducing ends of the polysaccharide, respectively, the chemical structure of the O-specific chain of V. cholerae H11 is proposed as alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro-(1- 3)-beta-D-QuiNAc- (1-[4)-alpha-D-GalANGro-(1-4)-alpha-NeuAc-(2-3)-beta-D-GalANGro -(1-3)-beta-D- QuiNAc-(1-]n-(1-4)-D-altro-heptulose. However, other possible structures can not be ruled out since the tri- and the disaccharide could be localised at different positions.


Purification and characterization of a pore-forming protein from the marine sponge Tethya lyncurium. A pore-forming protein was detected and purified for the first time from a marine sponge (Tethya lyncurium). The purified protein has a polypeptide molecular mass of 21 kDa and a pI of 6.4. Tethya pore-forming protein (also called Tethya hemolysin) rapidly lysed erythrocytes from a variety of organisms. After binding to target membranes, the hemolysin resisted elution with EDTA, salt or solutions of low ionic strength and hence resembled an integral membrane protein. Erythrocytes could be protected from hemolysis induced by Tethya hemolysin by addition of 30 mM dextran 4 (4-6 kDa; equivalent hydrodynamic diffusion radius, 1.75-2.3 nm) to the extracellular medium, but not by addition of uncharged molecules of smaller size [sucrose, raffinose and poly(ethylene glycol) 1550; equivalent hydrodynamic diffusion radii, 0.46, 0.57 and 1.2 nm, respectively]. This result indicates that hemolysin is able to form stable transmembrane pores with an effective diameter of about 2-3 nm. Treatment of osmotically protected erythrocytes with Tethya hemolysin caused a rapid efflux of intracellular K+ and ATP, and a rapid influx of extracellularly added Ca2+ and sucrose. In negative-staining electron microscopy, target erythrocyte membranes exposed to purified Tethya hemolysin displayed ultrastructural lesions but without visible pores.


The alpha subunit of the stimulatory guanine-nucleotide-binding regulatory protein forms high-molecular-mass aggregates, concomitant with iloprost-induced desensitization of human platelet adenylyl cyclase. Prolonged treatment of human platelets with the prostacyclin analog iloprost led to desensitization of the response to various prostaglandin derivatives. However, basal adenylyl cyclase activity and stimulation by agents acting directly via Gs, the stimulatory guanine-nucleotide-binding regulatory protein of adenylyl cyclase, were likewise decreased. Reconstitution of desensitized membranes with purified Gs from turkey erythrocytes indicated no alteration in the catalyst itself. However, the function of Gs (in cholate extracts) appeared to be severely impaired when reconstituted with adenylyl cyclase catalyst. Modification of Gs was also indicated by its altered sedimentation in sucrose density gradients. From Western blots, the alpha subunit of Gs, alpha s, from control platelets sedimented as a 5.6S species, while that from desensitized cells appeared at higher S values (in a polydisperse distribution). Activation by guanosine 5'-[gamma-thio]triphosphate of Gs from control platelets shifted alpha s to 3.5-3.7S, while activation of Gs from desensitized platelets induced such shift only for a minor portion of alpha s. This small fraction alone appeared to be susceptible to ADP-ribosylation by cholera toxin/[32P]NAD. Furthermore, an antibody directed against the C-terminal hexadecapeptide of alpha s precipitated much less alpha s from cholate extracts derived from desensitized platelets. Modification of alpha s during desensitization was also suggested from cross-linking experiments using the homobifunctional agent bismaleimidohexane: alpha s from desensitized platelets formed a single product of 80 kDa, while that from untreated platelets yielded a doublet (100 kDa and 110 kDa).


Measuring the quality of life of patients with benign prostatic hyperplasia. Assessment of the usefulness of a new quality of life questionnaire specially adapted to benign prostatic hyperplasia patients. A questionnaire consisting of 36 questions based on a visual analogue scale for measuring the quality of life (QOL) of benign prostatic hyperplasia (BPH) patients is presented and assessed. The sensitivity of the questionnaire is demonstrated by its ability to register a statistically significant (p less than 0.01) improvement of QOL after prostate surgery (transurethral resection of the prostate). Its reliability is shown by its ability to give reproducible results in a nonoperated BPH patient group. Patient compliance has been excellent. It is discussed to what extent questions directly concerning symptoms from prostatism should be included in QOL questionnaires for BPH patients. Nonparametric statistics are applied. It is concluded that questionnaires like this are useful tools in the assessment of BPH patients, irrespective of the treatment modality. They will probably be of particular value in studies of drug therapy of BPH, complementing methods of assessment like symptom score and urodynamic parameters.


Prostate-specific antigen and prostatic acid phosphatase in the detection of early prostate cancer and in the prediction of regional lymph node metastases. Serum values of prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) were determined in 180 patients prior to pelvic lymphadenectomy and radical prostatectomy and in 40 patients prior to pelvic lymphadenectomy alone. In all tumor stages, PSA was superior to PAP in detecting cancer of the prostate. By PSA determination using a cutoff level of 4 ng/ml (Tandem assay), 28.8% of the patients with prostate cancer, stage pT2pN0M0, and 17.8% of the cases with a stage pT3pN0M0 tumor could not be detected. All these tumors had been noticed, however, by digital rectal examination. This indicates that PSA determination cannot replace digital rectal examination as a screening method for prostate cancer. In this study, it was possible neither by PSA nor by PAP to define a practicable cutoff level for patients with and without lymph node metastases. A clear differentiation between the stages pT2pN0M0 and pT3pN0M0 was not possible by either PSA or PAP alone.


Evidence that Serenoa repens extract displays an antiestrogenic activity in prostatic tissue of benign prostatic hypertrophy patients. A double-blind placebo-controlled study was performed in 35 benign prostatic hypertrophy (BPH) patients never treated before. The patients were randomized into two groups, the 1st (18 cases) receiving Serenoa repens extract (160 mg t.d.) for 3 months up to the day before the operation of transvesical adenomectomy and the 2nd (17 cases) receiving placebo. Steroid receptors were evaluated in the nuclear (n) and cytosolic (c) fraction using the saturation analysis technique (Scatchard analysis or single saturating-dose assay) for androgen (AR) and estrogen (ER) receptors and the enzyme immunoassay (EIA) for ER and progesterone receptors (PgR). Scatchard analysis of ERc and ERn revealed the presence of two classes of binding sites, one with high-affinity low-capacity binding and the other with low-affinity high-capacity binding. In the untreated BPH group, ER were higher in the n than in the c fraction: ERn were positive in 14 cases and ERc in 12 of 17 cases. In the BPH group treated with S. repens extract on the contrary, ERn were negative for both binding classes in 17 cases and ERc in 6 of 18 cases. Using EIA, ERn and ERc were detected in all 15 samples examined, but in the treated group, ERn were significantly (p less than 0.01) lower than in the untreated group, whilst ERc remained almost unchanged. Similar results were obtained measuring PgR: the n fraction of the treated group prostatic samples was significantly (p less than 0.01) lower than that of the untreated group.(ABSTRACT TRUNCATED AT 250 WORDS)


Noradrenergic and peptidergic innervation of secondary lymphoid organs: role in experimental rheumatoid arthritis. Noradrenergic (NA) and peptidergic nerve fibres are present in both primary and secondary lymphoid organs, distributing with the vasculature, trabecular and capsular smooth muscle, and within the parenchyma among cells of the immune system. NA nerve terminals directly abut lymphocytes and macrophages in spleen and lymph nodes. In these organs, norepinephrine has fulfilled the basic criteria for neurotransmission with cells of the immune system as targets. In vitro and in vivo studies have demonstrated NA modulation of primary and secondary antibody responses, cytotoxic T cell responses, natural killer cell activity, and proliferation and differentiation of both T and B lymphocytes. Substance P (SP) has been shown to modulate inflammatory responses, lymphocyte proliferation, and other immunologic reactivity. We investigated the role of NA and SP nerve fibres within lymph nodes in experimental allergic auto-immune arthritis in Lewis rats. Denervation of NA nerve fibres in popliteal and inguinal lymph nodes with 6-hydroxy-dopamine resulted in earlier onset and enhanced severity of arthritic changes as well as inflammation in bilaterally induced experimental arthritis, while denervation of SP nerve fibres in popliteal and inguinal lymph nodes with capsaicin resulted in delayed onset and diminished severity of the inflammatory changes ipsilaterally, and prevention of contralateral arthritic changes in unilaterally induced experimental arthritis. These findings suggest that NA and SP nerve fibres in lymph nodes can modulate the time course of onset and the severity of experimental arthritis in Lewis rats. These modulatory effects are distinctly different from the effects of NA and SP nerve fibres in the joints themselves.


Role of the endothelial system in Bay K 8644 enantiomer and nifedipine vasomodulator action in rat aorta. The potential importance of the endothelial system in regulating the effects of (-)-Bay K 8644 (0.1 microM), (+)-Bay K 8644 (0.1 microM) and nifedipine (10 nM) on resting tension, on contractile responses to noradrenaline (NA) and Ca2+ (in a Ca(2+)-free high-K+ solution), and on basal, NA-induced and K(+)-induced 45Ca2+ uptake, was investigated in rat aorta rings. Mechanical removal of endothelium considerably potentiated the contractile response induced by NA in standard medium and by Ca2+ in Ca(2+)-free high-K+ (15 mM) medium, but did not modify the response induced by Ca2+ in Ca(2+)-free high-K+ (55 mM) medium or by NA in Ca(2+)-free medium. Furthermore, the basal 45Ca2+ uptake and that induced by NA (10 microM) or KCl (15 and 55 mM) were similar in endothelium-rubbed and intact rings. (-)-Bay K 8644 (0.1 microM) shifted the NA and Ca2+ concentration-response curves to the left with potentiation of the maximal contraction. However, (+)-Bay K 8644 (0.1 microM) and nifedipine (10 nM) caused a shift to the right, with depression of the maximal contraction. The NA concentration-response curves, and those of Ca2+ in Ca(2+)-free high-K+ (55 mM) medium, were affected by the drugs to similar extents, and were not modified by the presence or absence of endothelial cells. The drugs tested did not affect resting tension. Basal 45Ca2+ uptake was not modified by either nifedipine or the Bay K 8644 enantiomers.(ABSTRACT TRUNCATED AT 250 WORDS)


Alpha 2-adrenoceptor-mediated contractions in the rabbit aorta treated with BAY K 8644. We tested the effect of the selective alpha 2-adrenoceptor agonists B-HT 920 and B-HT 933 on the rabbit aorta. These drugs had weak contractile effects in the tissue, which were inhibited by the selective alpha 1-adrenoceptor antagonist prazosin (10(-5) M). Their contractile effects were potentiated by the Ca2+ channel facilitator BAY K 8644 (10(-6) M). The selective alpha 2-adrenoceptor antagonist yohimbine (10(-5) M) reduced the contractions elicited by B-HT 920 and B-HT 933 in the presence of BAY K 8644 (10(-6) M), but did not alter the control effect of these drugs. In the rabbit aorta, the contractile effect of B-HT 920 and B-HT 933 in the presence of BAY K 8644 (10(-6) M) was partly caused by alpha 1-adrenoceptor stimulation, because prazosin (10(-5) M) relaxed the contractions elicited under these conditions. In the aorta preincubated with BAY K 8644 (10(-6) M) and prazosin (10(-5) M), B-HT 920 (3 x 10(-4) M) elicited non-sustained phasic contractions (1-5 g), which were probably due to alpha 2-adrenoceptor stimulation, as they were inhibited by yohimbine (10(-5) M). In similar experiments B-HT 933 (3 x 10(-4) M) caused inconsistent and slight contractions (< 0.5 g developed tension).


Rat hepatocytes in primary culture synthesize and secrete cellular fibronectin. Fibronectins, involved in cell-matrix interactions and cell attachment, are glycoproteins which show a remarkable heterogeneity, due to alternative splicing. The type III-related domains, ED-A and ED-B, are present in cellular fibronectin in a variety of ratios whereas they are absent in circulating plasma fibronectin. Fibronectin synthesis by hepatocytes which are accepted as suppliers of plasma fibronectin was studied in primary cultures during a 6-day culture period. Using site-specific antibodies we demonstrate that rat hepatocytes are also able to synthesize and secrete fibronectin bearing the ED-A domain from Day 3 on after inoculation. By immunocytological characterization of the hepatocyte monolayer with antibodies directed against desmin, laminin, collagen IV, alpha-SM-actin, or ED-1 or factor VIII-related antigen, contaminating mesenchymal hepatic cell-types as a source for cellular fibronectin production could be ruled out. Dexamethasone treatment caused enhanced fibronectin synthesis and cellular fibronectin was already detectable at Day 1 after plating. Elevation of cellular fibronectin synthesis after prolonged culture-terms and by dexamethasone could also be demonstrated on mRNA steady-state level, using ED-A cDNA as a probe in hybridization analysis. Dot blot hybridisation proved a prominent response of cellular fibronectin mRNA level to dexamethasone at Day 1 when dexamethasone treatment resulted in an increased contribution of ED-A-positive fibronectin transcripts to total fibronectin mRNA level.


Synthesis of undulin by rat liver fat-storing cells: comparison with fibronectin and tenascin. Fat-storing cells (FSCs) are known to synthesize various components of the hepatic extracellular matrix and thereby play an important role during liver fibrogenesis. The aim of our study was to investigate the synthesis of undulin, a recently described connective tissue protein belonging to the fibronectin-tenascin superfamily of glycoproteins, by fat-storing cells in primary culture. SDS-PAGE analysis of immunoprecipitates from cell layer lysates or media pulse-labeled with radioactive methionine revealed undulin-specific bands A (270 kDa), B1 (190 kDa), and B2 (180 kDa) after reduction. A single undulin-specific transcript was detected at about 7 kb. Undulin synthesized by cell-free translation revealed two polypeptides migrating about 5000 Da below the B1 and B2 subunits. Treatment of FSCs with tunicamycin created two novel bands slightly below the B2 chain. Since the electrophoretic patterns of undulin chains recovered by cell-free translation and tunicamycin treatment of cells were very similar we suggest that N-glycosylation is the major post-translational processing event. Newly synthesized undulin was detected after 30 min of pulse labeling in the cell layer fraction and was secreted into the medium at a slower rate than fibronectin. In contrast to fibronectin and tenascin, undulin was already synthesized by freshly isolated FSCs and during the early stage of primary FSC culture ("resting" cells), supporting the hypothesis that undulin is associated with a differentiated mesenchyma. However, in analogy to fibronectin and tenascin, undulin was also synthesized by "activated" FSCs, indicating that undulin might also be of importance in dedifferentiated tissues.


A role of retinoic acid in the regulation of the morphology and the levels of intermediate filament proteins and mRNAs in PC12 cells. An adrenal tumor-derived cell line (PC12W) cultured in the presence of nerve growth factor exhibited a spindle-shaped cell morphology resembling neuronal cells. The shape of these cells can be specifically changed in vitamin A-depleted medium supplemented with retinoic acid. Retinoic acid promoted an epithelial-like cell morphology except for occasional neuronal processes. These morphological results were correlated with differential expression of intermediate filaments at the mRNA and protein levels in these cells. Retinoic acid suppressed the synthesis of peripherin, an intermediate filament protein predominantly found in peripheral nerve cells, but a high level of simple keratins, normally found in simple epithelial cells, was present in retinoic acid-treated PC12 cells. The neurofilaments typically expressed in neurons remained virtually unaffected under the same conditions. In contrast, nerve growth factor induced the production of neurofilaments, but suppressed the synthesis of simple keratins. Since intermediate filament expression is known to be tissue-specific, these changes in expression together with the cell morphology changes are consistent with PC12 cells undergoing an epithelial-like differentiation in the presence of retinoic acid and a neuronal-like differentiation in the presence of nerve growth factor. These results suggest that retinoic acid and nerve growth factor are both effective regulators of PC12 cell differentiation but stimulate opposing pathways.


Expression of decorin by sprouting bovine aortic endothelial cells exhibiting angiogenesis in vitro. In our recent studies, we have demonstrated that monolayer cultures of bovine aortic endothelial (BAE) cells that do not express type I collagen also fail to express and synthesize decorin, a small chondroitin/dermatan sulfate proteoglycan that interacts with type I collagen and regulates collagen fibrillogenesis in vitro. However, BAE cells exhibiting a spontaneous sprouting phenotype and a predisposition toward the formation of cords and tube-like structures (an in vitro model for angiogenesis) initiate the synthesis of type I collagen during their morphological transition from a polygonal monolayer to an angiogenic phenotype. In the present study, we examined whether BAE cells also initiate the synthesis of the proteoglycan decorin during this morphological transition. We show by Northern blot analysis and by immunochemical methods that BAE cell cultures containing sprouting cells and cords, but not monolayer cultures of these cells, express and synthesize decorin (M(r) approximately 100,000). We also show that type I collagen expression by BAE cell cultures is initiated concomitantly. However, the localization of decorin and type I collagen in cord and tube-forming BAE cell cultures is not completely identical. Type I collagen is detected only in sprouting BAE cells and in endothelial cords, whereas decorin is also apparent in BAE cells surrounding the cords and tubes. Our results indicate that the synthesis of decorin as well as type I collagen is associated with endothelial cord and tube formation in vitro.


An in vitro model of rodent nongenotoxic hepatocarcinogenesis. An in vitro model of liver in which rat hepatocytes are maintained as cocultures with nonparenchymal epithelial cells (NPC) derived from liver has been developed and characterized with respect to maintenance of hepatocyte viability and differentiated function. The system was then evaluated as a model for studying peroxisome proliferator-induced rodent liver nongenotoxic carcinogenesis. Within the coculture model, hepatocyte viability and morphology were maintained for 1 month or more within a system that is both easily accessible for microscopic examination and is free of any additives that may lead to artifacts. Even after 1 month or more, hepatocyte cocultures retained expression of the constitutive liver marker albumin. In addition, they maintained the ability to show induction of the peroxisome proliferator-inducible enzymes peroxisomal bifunctional enzyme (PBE) and cytochrome P450IVA1 in response to the peroxisome proliferator nafenopin. After 4 weeks, NPC cocultures showed a six- and a fourfold induction of PBE and cytochrome P450IVA1 expression, respectively, which compared well with the three- and fivefold induction seen in freshly isolated cells. This was paralleled by an increase in the cytoplasmic volume fraction of peroxisomes averaging eightfold. Interestingly, great heterogeneity was exhibited between adjacent hepatocytes in terms of the degree of peroxisome proliferation, a finding reflected by immunocytochemical staining which indicated heterogeneity in the level of expression of the peroxisome proliferator-inducible enzymes. Other cell lines representing different tissue types, morphologies, and species were also examined for their ability to support hepatocyte survival but were found to be ineffective, with the exception of a bovine corneal endothelial cell line. This line supported hepatocyte survival and maintenance of differentiated function but to a lesser extent than that observed with NPC. Ultrastructural examination of NPC cocultures revealed extensive interhepatocyte junctional complexes and interdigitation of adjacent membranes together with the presence of bile canalicular structures. There were no junctional complexes between the hepatocytes and the supporting feeder cells with any contact being limited to a close association of the hepatocytes with the extracellular matrix presumably produced by the NPC. The data demonstrate that hepatocytes maintained in vitro within an NPC coculture system retain differentiated function and the ability to respond to the peroxisome proliferator class of nongenotoxic carcinogens. Cocultures will provide us with a model system for the study of changes in hepatocyte growth regulation during rodent liver nongenotoxic carcinogenesis.


Induction of alpha v beta 3 integrin-mediated attachment to extracellular matrix in beta 1 integrin (CD29)-negative B cell lines. beta 1 integrin containing complexes have been implicated as the primary adhesion structures in many lymphocyte extracellular matrix (ECM) interactions. However, many B lymphocytes lack surface expression of the beta 1 subunit, implying that this subpopulation of lymphoid cells must employ alternate adhesion structures if they are to maintain an interactive capacity with ECM. An examination of the adherence properties of the beta 1 integrin-negative B cell line JY indicated that these cells exhibit little or no basal adherence to any of the ECM components examined. However, these cells could be induced to adhere to the ECM components fibronectin, laminin, and vitronectin following treatment with PMA. Blocking studies with monoclonal antibodies indicated the alpha v beta 3 integrin complex was involved in the attachment to each of these ligands. However, the adherence to fibronectin displayed a complex pattern of inhibition suggesting the involvement of other ECM receptors. The utilization of the alpha v beta 3 complex was not unique to the JY cell line. Other B cell lines were observed to employ alpha v beta 3, and these lines similarly lacked expression of beta 1 integrin. These results indicate that alpha v beta 3 can act as a lymphoid ECM-adhesion structure which may provide an alternative means for lymphocytes to interact with ECM. Furthermore, these studies provide evidence for the presence of lymphoid-associated alpha v beta 3 integrins with regulatable activity, which contrasts with the constitutive adhesive potential of these complexes when present on other cell types.


Induction of nuclear lamins A/C during in vitro-induced differentiation of F9 and P19 embryonal carcinoma cells. Lamin B is the major constituent of the nuclear lamina of undifferentiated mouse embryonal carcinoma cells. The full complement of the three major lamins A, B, and C, found in somatic mammalian cells, is acquired after induction of differentiation in vitro by certain drugs. In this study we have examined the time course of lamin A/C expression in the two embryonal carcinoma cell lines F9 and P19. We show here that lamins A/C are detectable in these cell lines, at the mRNA level and at the protein level, after 3 days of growth in media containing retinoic acid or retinoic acid + 3-isobutyl-1-methylxanthine. The data reported here indicate that the expression of lamins A/C is mainly regulated at the transcriptional level and occurs when the cells, by morphological and functional criteria, have differentiated along their developmental pathway.


Irreversible oxidative inactivation of protein kinase C by photosensitive inhibitor calphostin C. Isolated protein kinase C (PKC) was irreversibly inactivated by a brief (min) incubation with calphostin C in the presence of light. This inactivation required Ca2+ either in a millimolar range in the absence of lipid activators or in a submicromolar range in the presence of lipid activators. In addition, an oxygen atmosphere was required suggesting the involvement of oxidation(s) in this inactivation process. Furthermore, PKC inactivation might involve a site-specific oxidative modification of the enzyme at the Ca(2+)-induced hydrophobic region. Physical quenchers of singlet oxygen such as lycopene, beta-carotene, and alpha-tocopherol all reduced the calphostin C-induced inactivation of PKC. In intact cells treated with calphostin C, the inactivation of PKC was rapid in the membrane fraction compared to cytosol. This intracellular PKC inactivation was also found to be irreversible. Therefore, calphostin C can bring prolonged effects for several hours in cells treated for a short time. Taken together these results suggest that the calphostin C-mediated inactivation of PKC involves a site-specific and a 'cage' type oxidative modification of PKC.


Sperm nuclear chromatin normality: relationship with sperm morphology, sperm-zona pellucida binding, and fertilization rates in vitro. To study whether the results of tests of sperm chromatin and deoxyribonucleic acid (DNA) normality are related to fertilization rates in vitro. Normal morphology, nuclear maturity determined by acidic aniline blue stain, and DNA normality determined by acridine orange fluorescence of sperm in insemination medium and the number of sperm bound to the zona pellucida (ZP) of the oocytes that had failed to fertilize in vitro were determined. The relationship between sperm test results and fertilization rates were analyzed by logistic regression. Samples were obtained from patients undergoing in vitro fertilization (IVF) treatment. The number of sperm bound to the ZP, the percentage of sperm with normal morphology, and the percentage of sperm with normal DNA were the most significant factors related to fertilization rates in vitro. In patients with normal morphology > or = 15% or with > 10 sperm bound per ZP, the percentage of sperm with normal DNA, the number of sperm bound to the ZP, and motility grade were significantly related to IVF rates. In patients with normal morphology > or = 15%, failure of fertilization may be because of defects of sperm-ZP binding or abnormal DNA. Assessment of DNA normality of motile sperm in the insemination medium may aid prediction of fertilization rates in addition to normal morphology and sperm-ZP binding.


Treatment of interstitial pregnancy with methotrexate via hysteroscopy. We present a case in which treatment of interstitial pregnancy with local MTX administration was performed successfully through hysteroscopic vision, without the need to operate. Decreased gestational sac dimension and increased or low beta-hCG level ( < 1,400 mIU/mL) facilitates the success rate. The follow-up showed disappearance of the gestational sac and decrease of beta-hCG levels to < 10 mIU/mL. We conclude that local MTX administration via hysteroscopy after tubal ostium visualization is feasible. The procedure should be considered in women during the reproductive age, especially in rare cases of interstitial pregnancy.


The evaluation of various culture media in combination with dimethylsulfoxide for ultrarapid freezing of murine embryos. Bicarbonate-buffered HTF medium, Medicult, and T6 are as effective as PB1 medium when used in combination with DMSO in ultrarapid freezing of two-cell mouse embryos. However, the use of phosphate-buffered T6 results in reduced in vitro development and inner cell mass size as compared with bicarbonate- and Hepes-buffered T6 when used with 3.5 M of DMSO. Hence, the use of this media for ultrarapid freezing should be avoided when this concentration of DMSO is used.


[Serum growth hormone binding protein (GHBP) and insulin-like growth factor binding protein (IGFBP)]. Insulin--like growth factors bind to specific binding proteins (IGFBPs) in serum and tissues. At present, six different IGFBPs have been characterized. Recent studies suggest that IGFBPs act as a reservoir for IGFs but also modulate the bioavailability of IGFs. Binding protein for growth hormone (GH) in serum has been recognized recently. Interestingly, the high affinity GH binding protein (GHBP) is identical with the extracellular domain of GH receptor and is absent in patients with Laron-type dwarfism, suggesting that serum GHBP might serve as a marker for the GH receptor in tissue. In this short review, updated information on serum GHBP and IGFBP is presented.


[Change in serum G-CSF levels in patients with Graves' disease by treatment with methimazole]. We evaluated the determination of serum G-CSF in the diagnosis of granulocytopenia due to methimazole (MMI) in 54 patients with Graves' disease, while they were being treated with MMI, by way of measuring WBC counts and serum levels of G-CSF, thyroid hormones, IgE, and interleukin-2. Serum TSH was measured by immunoradiometric assay, serum G-CSF was done by enzyme immunoassay, thyroid hormones and IgE were done by radioimmunoassay, and serum Interleukin-2 was done by enzyme-linked immunosorbent assay. The population whose G-CSF levels were higher than the minimum detectable level (30pg/ml) was 6 (30%) in normal subjects, 4 (22%) in patients with untreated Graves' disease, 2 (12%) in patients with treated euthyroid Graves' disease, 3 (23%) in patients with Graves' disease who had gone through agranulocytosis, and 2 (33%) in patients with Graves' disease complicated with granulocytopenia. There was no significant change in WBC counts for 4 weeks, but there was a significant difference between WBC counts before treatment and those at 8 weeks after treatment. We observed no significant change of serum G-CSF levels in patients with Graves' disease under treatment. However, there were significantly high levels of serum G-CSF and significantly low counts of WBC in patients with Graves' disease complicated with granulocytopenia induced by MMI, compared with those in normal subjects, patients with untreated Graves' disease, patients with treated euthyroid Graves' disease, and patients with euthyroid Graves' disease who had gone through agranulocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)


Prostacyclin rather than nitric oxide lowers human umbilical artery tone in vitro. This study was designed to determine vasodilator activities of two endothelium-derived relaxing factors: prostacyclin (PGI2) and nitric oxide (NO) in human umbilical arteries. Isolated vessel segments were contracted by submaximal concentrations of serotonin and bradykinin. These contractions were enhanced after inhibition of prostaglandin formation by the cyclooxygenase inhibitor indomethacin and after removal of the endothelium, both resulting in a pronounced decrease in PGI2 formation. Contractions remained unchanged after treatment of the vessels with nitro-L-arginine, a selective inhibitor of endogenous NO biosynthesis. The efficacy of inhibition of NO biosynthesis was established by a more than 60% reduction in cyclic GMP accumulation. Even inhibition of stimulated NO formation by histamine did not change vascular tone. These data suggest that PGI2 rather than NO is an endothelium-derived relaxing factor in human umbilical arteries.


Galanin in sensory neurons in the spinal cord. The distribution and physiological effects of the neuropeptide galanin (GAL) have been examined in the somatosensory system. GAL is normally present in a few sensory neurons that terminate in the dorsal horn of the spinal cord and it is colocalized with substance P and calcitonin gene-related peptide. After peripheral nerve section, but not dorsal root section, the amount of GAL produced and present in sensory fibers proximal to the section is dramatically upregulated. In parallel functional studies, we could demonstrate that exogenous GAL has a complex effect on the spinal cord reflex excitability, facilitatory at low doses and inhibitory at high doses. Furthermore, GAL inhibits the effect of excitatory neuropeptides physiologically released at the peripheral and central terminals of small diameter afferents that subserve a nociceptive function. After axotomy, the inhibitory effect of GAL is increased. We conclude that GAL may have an important role in the control of nervous impulses that underlie pain states that can occur after peripheral nerve injury.


Differential stability of mRNAs coding for alpha and gonadotropin beta subunits in cultured rat pituitary cells. Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) result from the assembly of a common subunit alpha and a unique subunit beta, expressed in the same cell by single, structurally-related genes. In order to compare the intrinsic stability of the alpha, LH beta and FSH beta mRNA transcripts, we used cultured rat pituitary cells incubated in presence of actinomycin D. Hybridization with 32P-labelled rat cDNA probes showed that the cell content of all three mRNAs decreased with time, but at different rates. Apparent half-lives, estimated as the time necessary to observe a 50% mRNA decay, were 1.0 +/- 0.13 h for FSH beta, 6.5 +/- 0.25 h for alpha and 44 +/- 0.5 h for LH beta, stability thus exhibiting an inverse relation to the sizes of the corresponding mRNAs (approximately 1700, 800 and 700 nucleotides, respectively). Northern analysis revealed that the decline in mRNA abundance was associated with a progressive decrease in the length of mRNAs, most clearly visible for alpha and LH beta. For the most stable LH beta mRNA, shortening was apparent as early as 2 h after exposure to actinomycin D thus preceding neatly the decrease in amount starting at about 10-12 h. In vitro RNase H digestion demonstrated that shortening resulted from a reduction of the length of the poly(A) tract. These data establish that the three mRNAs coding for gonadotropin subunits have different stabilities although they share substantial homology. Diversity in size and sequence essentially resides in untranslated regions in which, we suggest, specific motifs and protein factors may interact to determine mRNA stability.(ABSTRACT TRUNCATED AT 250 WORDS)


Tyrosine iodination and iodotyrosyl coupling of the N-terminal thyroid hormone forming site of human thyroglobulin modulate its binding to auto- and monoclonal antibodies. The present work was aimed at studying the interaction of autoantibodies (aAb) and monoclonal antibodies (mAb) with the N-terminal thyroid hormone forming site of human thyroglobulin (TG). Obtained by CNBr treatment of TG, the peptide (22 kDa) containing the complete major hormonogenic site of human TG was purified in three forms according to the degree of iodination and iodotyrosine coupling: the native, poorly iodinated form (n-22K), the iodinated form containing iodotyrosine but not hormone residues (i-22K) and the form containing thyroid hormone (t-22K). We report that aAb from some patients with autoimmune thyroid diseases showed significant binding to both iodinated 22 kDa forms. Furthermore, a detailed study using mAb evidenced that iodination and coupling induced changes in the antigenicity of the molecule, some occurring without direct implication of iodine or thyroid hormones. The 22 kDa peptide appears as an interesting model to study the antigenic changes induced by the structural modifications in the course of thyroid hormone synthesis. This observation could be relevant to the etiopathogenic process of thyroid autoimmune diseases.


Direct estradiol down-regulation of secretogranin II and chromogranin A mRNA levels in rat pituitary cells. Estradiol (E2) has been previously shown to negatively regulate, in vivo, the secretogranin (SgII) and chromogranin A (CgA) mRNA levels in the rat pituitary. Using cultured pituitary cell aggregates, experiments were undertaken to discriminate between direct or indirect effects of E2 on SgII and CgA levels. SgII, CgA and gonadotropin alpha- and beta-subunit mRNA levels were determined by Northern blotting. SgII and CgA protein levels were quantitated by Western blotting, and by immunoprecipitation of radioactive SgII after [35S]methionine labeling. Increasing concentrations of E2 (from 10(-12) M to 10(-8) M) in the culture medium promoted a decrease of SgII and CgA mRNA levels to 30% and 50% of the control, respectively, after 72.h treatment. By contrast, none of the gonadotropin subunit mRNAs exhibited changes in concentration. A 24 h treatment with 10(-8) M E2 was sufficient to promote such a decrease in SgII and CgA mRNAs. Quantitation of the proteins after Western blotting revealed that 10(-8) M E2 lowered by 30% in CgA content of aggregates (P < 0.05 vs. control) while SgII content remained unaffected. Moreover, quantitation of the newly synthesized SgII by immunoprecipitation of the 35S-labeled SgII gave evidence for a lack of E2 effect. These data demonstrate: (1) a direct effect of E2 on the pituitary cells to down-regulate SgII and CgA mRNA steady-state levels; (2) though contained within the same secretory granules, a distinct pathway for negative E2 regulation of the gonadotropins and both granins; and (3) a differential effect of E2 on cell SgII and CgA contents as was previously demonstrated in vivo.


Characterization of muscarinic receptor subtypes on rat pancreatic acini: pharmacological identification by secretory responses and binding studies. In order to identify subtypes of muscarinic receptor on the rat pancreas, the effects of new muscarinic receptor antagonists, [11-[[2-(diethylamino)-methyl]-1-piperidinyl]acetyl]-5, 11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) and 4-diphenylacetoxy-N-methylpiperadine-methiodide (4-DAMP), on amylase secretion stimulated by carbachol and binding of [3H]quinuclidinyl benzilate (QNB) were evaluated using isolated rat pancreatic acini. Atropine, pirenzepine, AF-DX 116 and 4-DAMP inhibited carbachol-stimulated amylase release in a dose-dependent manner. All these antagonists caused a concentration-dependent rightward shift of the dose-response curve for carbachol-stimulated amylase release without altering the maximal response. Schild plots revealed that pA2 values for atropine, pirenzepine, AF-DX 116 and 4-DAMP were 9.15, 6.78, 6.09 and 8.79, respectively. Every slope of Schild plots was not different from unity, suggesting that these antagonists act as competitive inhibitors. These antagonists also inhibited the binding of [3H]QNB in a dose-dependent manner. The inhibition constants were 1.21 x 10(-9) M (atropine), 1.26 x 10(-7) M (pirenzepine), 0.57 x 10(-6) M (AF-DX 116) and 2.75 x 10(-9) M (4-DAMP). Thus, the order of inhibitory potencies was atropine > or = 4-DAMP > pirenzepine > AF-DX 116. These findings suggest that 4-DAMP-sensitive M3 receptor may play an important role in the pancreatic exocrine functions.


Reciprocal expression of c-jun, proline-rich protein and amylase genes during rat parotid salivary gland development. We have investigated the temporal expression and cellular localization of the c-jun proto-oncogene and two major rat parotid gland secretory protein genes, PRP (proline-rich protein) and amylase, during postnatal development. c-jun mRNA steady-state levels increased at days 1, 7 and 14 after birth and decreased to basal levels at 21 days and older. PRP mRNA was first detected at 14 days and abruptly increased to adult levels at day 21. Amylase transcripts were first seen at day 7 and progressively increased to adult levels by 28 days. In situ hybridization demonstrated c-jun mRNA accumulation in the differentiating acinar cells and the ducts. The c-jun mRNA accumulation with time corresponds with the proliferative activity reported to occur in these two cellular populations. PRP transcripts were present exclusively in the well differentiated acinar cells while the accumulation of amylase mRNA corresponded to the progressive commitment of parotid cells to acinar differentiation. Our data suggest that during the postnatal development of the rat parotid gland: (a) c-jun expression associates with parotid gland proliferation and precedes the expression of PRP and amylase genes, and (b) activation of PRP and amylase genes is not concomitant and apparently occurs only in differentiating acinar cells.


Nucleotide sequence of the nfaA gene encoding the antigen 8786 adhesive factor of enterotoxigenic Escherichia coli. The nucleotide sequence of a 714-bp DNA fragment containing the enterotoxic Escherichia coli (ETEC) adhesive factor 8786 structural gene, designated nfaA, revealed an open reading frame of 498 bp encoding a polypeptide of 166 amino acids. Primer-extension experiments showed that the nfaA gene is within a single transcription unit. No homology was found with the ETEC adhesive factors already sequenced. In contrast, a homology with Salmonella enteritidis fimbrin SEF 14 was observed.


Transcription in vitro and in vivo of the 7S RNA gene associated with the ribosomal RNA operon in the hyperthermophilic archaeon Methanothermus fervidus. The gene encoding the archaeal 7S RNA in the hyperthermophile Methanothermus fervidus is linked to a tRNA(Ser) and rRNA operon in the arrangement 5'-7S RNA-14nt-tRNA(Ser)-196nt-16S rRNA and the promoter directing transcription of this 7S RNA gene has now been identified. Initiation of transcription of the 7S RNA gene has been shown to occur both in vivo in M. fervidus and in vitro, using a Methanococcus thermolithotrophicus derived cell-free transcription system, at the first G residue within the initiator sequence ATGG, located 6 bp upstream of the 5' end of the 7S RNA coding region. Cotranscription of the 7S RNA and tRNA(Ser) has been demonstrated in vitro.


The effect of superoxide dismutase in the myocardium during reperfusion in the dog. The aim of the study was to investigate the pathological role of free radicals during myocardial reperfusion. Low (0.5 mg/kg body weight) and high doses (5 mg/kg) of superoxide dismutase (SOD) were infused into the left atrium of mongrel dogs for 4 min starting 29 min after ligation and 1 min before reperfusion of the left anterior descending coronary artery (LAD). Arterial blood pressure, heart rate, electrocardiogram, and the regional contractile force of the left ventricle were monitored throughout the ligation (30 min) and reperfusion periods (20 min). Concentrations of creatine kinase (CK) and malondialdehyde (MDA) in the coronary sinus blood were determined before (0 min) and during ligation (15 and 25 min) and during reperfusion of the LAD (2, 7, and 20 min). In other groups of dogs, the effect of the two doses of SOD on epicardial blood flow was investigated during ligation and reperfusion by the measurement of epicardial temperature using a thermocardiograph. Experimental subjects were mongrel dogs of either sex (n = 25), weight 10-35 kg. Compared to controls (mean +/- SEM, 43.1 +/- 1.2; n = 7), the number of ventricular extrasystoles during the first 5 min of reperfusion was significantly (p < .001) decreased in dogs treated with the high dose (15.01 +/- 2.14; n = 5), but not in those receiving the low dose of the drug (34.6 +/- 5.66; n = 5). The concentrations of CK increased gradually until the end of reperfusion without differences among the different groups. Plasma MDA was the highest in control dogs 7 min after reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)


MspI polymorphism of the apolipoprotein A-II gene, serum lipids and apolipoproteins in Chinese from Singapore. The effect of a DNA polymorphism (MspI) of the apolipoprotein (apo) A-II gene on serum lipid and apo levels was studied in a group of 125 healthy Chinese of both sexes. The frequency of the 3.7-kb rarer allele (M2) was found to be significantly higher in the Chinese (0.30) than in Caucasians (0.16; p < 0.025). The distribution of apo A-II genotypes was in Hardy-Weinberg equilibrium in the Chinese population. The presence of a polymorphic site (MspI) within an Alu sequence at the 3' end of the gene, the 3.0 kb (M1) allele, was associated with significantly higher levels of serum apo A-I and A-II (p < 0.05 and < 0.01, respectively). Serum high-density Lipoprotein cholesterol levels were also correspondingly higher in individuals with M1, but did not reach a significant level. Male heterozygotes of the apo A-II polymorphism had significantly higher levels of serum triglycerides compared to homozygotes (p < 0.05). Thus the MspI polymorphism of the apo A-II gene appears to be associated with altered levels of lipids and apos in the Chinese population.


Use of monoclonal antibodies for discrimination between natural and recombinant human interferon-tau. Different monoclonal antibodies (MAbs) were raised against denaturated and native recombinant human Interferon-tau (IFN-tau). This approach gave us MAbs which recognized either N-term (prepared with SDS-denaturated IFN-tau) or C-terminal part of the antigen as well as MAbs which linked to non linear epitopes (obtained with native form of IFN-tau). Some of them inhibited or enhanced their respective binding to IFN-tau. After characterization, these antibodies were used as probes and some were selected to prepare two quantitative sensitive sandwich IRMAs able to discriminate between recombinant and natural IFN-tau.


Development of human monoclonal antibodies against human cytomegalovirus. Human monoclonal antibodies (HMAbs) against human cytomegalovirus (HCMV) have been developed by fusion of human spleen cells and human lymphoblastoid cell lines (NP101 and NP197). The cell line NP101 had great advantages in its high fusion frequency and the stability of the resultant hybridomas. The specificity of HMAbs was confirmed by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. Two of the six HMAbs obtained, which were IgG3 subclass, neutralized viral infectivity in the absence of complement. The neutralizing activity of one of these two HMAbs was enhanced in the presence of human complement, whereas the other was not. Another IgG1 subclass HMAb neutralized viral infection only in the presence of complement. The remaining three HMAbs showed no neutralizing activity. Those HMAbs may provide an important approach to studying human immune responses to HCMV. HMAbs having neutralizing activity may prove to be useful for passive immunotherapy of HCMV diseases.


Monoclonal antibodies against heavy and light chains of domestic mink IgG. A panel of 26 monoclonal antibodies (MAbs) specific to mink IgG was produced and analyzed by ELISA, immunodiffusion assay (IDA) and immunoblotting assay. All the raised MAbs were directed against the isotypic IgG epitopes. Immunoblotting assay demonstrated that 11 MAbs reacted only with the Fc-fragments of IgG and 7 only with the light chains. Four antibodies bound to the Fab-containing fragments and failed to react with the Fc-fragments or isolated L-chains. Three MAbs did not react with IgG in IDA. Based on the results of IDA and cross-blocking assays, the MAbs were divided into 10 groups, with the MAbs of each group recognizing the same epitope. In IDA some MAbs were able to react with the epitopes which are common to the IgGs of some other representatives of Mustelidae family and also to some mammalian species remote from mink (dog, horse, pig, fox and rabbit).


Influence of exposure to tobacco smoke on serum alpha fetoprotein levels of women in midtrimester pregnancy. The influence of exposure to tobacco smoke on maternal serum alpha-fetoprotein (AFP) levels at 16 wk gestation was examined. Urinary cotinine levels were used to quantify exposure to tobacco smoke. Significantly higher levels of maternal serum AFP were found in 101 women who had more than 1.0 microgram cotinine/mg urinary creatinine compared with 180 women whose urinary cotinine levels were below this level [(mean +/- SD) 1.23 +/- 0.64 and 1.06 +/- 0.54 respectively; 95 per cent CI of difference of means 0.01-0.31; P < 0.05]. There was a mild albeit statistically significant correlation between urinary cotinine levels and maternal serum AFB (r 0.099; P < 0.05). However, the difference in maternal serum AFP levels between the two groups was not found to be significant, when adjustments for maternal body mass index were made.


des-(1-3)-IGF-I, an insulin-like growth factor analog used to mimic a potential IGF-II autocrine loop, promotes the differentiation of human colon-carcinoma cells. HT29-D4 human colon-carcinoma cells have been shown to secrete insulin-like growth factor (IGF)-II and to simultaneously express type-I IGF receptors. However, the sequestration of IGF-II by several molecular forms of IGF-binding proteins (IGFBP) in the culture medium prevents the establishment of an operative IGF-II autocrine loop. IGFBPs secreted by HT29-D4 cells (HT29-D4 IGFBP) comprise isoforms of IGFBP-4 (25, 27 and 30 kDa) and 2 unidentified forms (34.5 and 32-34 kDa). This latter does not bind 125I-IGF-I. The net affinity of HT29-D4 IGFBP is about 12 times stronger for IGF-II (KD approx. 10(-10) M) than for IGF-I. All the HT29-D4 IGFBP molecular forms are unable to bind the N-terminally truncated IGF-I analog, des-(1-3)-IGF-I. In contrast, HT29-D4 cell-surface type-I IGF receptors bind IGF-I and des-(1-3)-IGF-I identically (KD approx. 5 x 10(-10) M). We have taken advantage of these particular binding properties to use des-(1-3)-IGF-I to mimic a potential IGF autocrine loop and to observe its biological consequences. Nanomolar concentrations of des-(1-3)-IGF-I induce HT29-D4 cells to develop into a differentiated phenotype, as judged by a substantial carcinoembryonic antigen release and the induction of numerous intercellular cysts with well-organized microvilli. In the same way, des-(1-3)-IGF-I early induces a slight inhibition of HT29-D4 cell proliferation. Based on these findings, we conclude that the type-I IGF receptor primarily controls the differentiation of these colonic cells, and that HT29-D4 cancer cells remain in an undifferentiated state because of their inability to use endogenous IGF-II as an autocrine regulatory factor.


VLA-4 integrin on sarcoma cell lines recognizes endothelial VCAM-1. Differential regulation of the VLA-4 avidity on various sarcoma cell lines. Osteosarcomas and rhabdomyosarcomas are vigorously invading tumors. Before they can extravasate to the parenchymal organs and form metastases, they have to adhere to the endothelial cells lining the blood vessels and then penetrate through the endothelium. We show that several human sarcoma cell lines, osteosarcomas HOS, MG-63, U2-OS, and a rhabdomyosarcoma RD, express VLA-4 molecule on their surface and bind to the VCAM-I-expressing activated endothelial cell line Ea.hy 926. The increased sarcoma-cell adhesion could be abolished by treating the sarcoma cells with monoclonal antibodies (MAbs) VLA4 (both alpha- and beta-chain, HP2/1 and 4B4 respectively) or treating endothelial cells with VCAM-I antibody (4B9). Furthermore, we show that sarcoma cells adhere to recombinant soluble VCAM-I protein. On the other hand, these sarcoma cell lines do not express marked amounts of other ligands (such as CDII/18 or sialyl-Lex) for other endothelial adhesion molecules (ICAM-I, ICAM-2, E- and P-selectin) indicating that the VLA-4-VCAM-I dependent pathway might be of major importance in sarcoma extravasation. VLA-4 is not always in an avid form and therefore the expression of VLA-4 does not directly predict adherence to VCAM-I. The avidity of VLA-4 (measured by adherence to soluble VCAM-I) of MG-63 and U2-OS cells could be increased by a 30-min PMA treatment, whereas the avidity of VLA-4 on HOS cells increased only after 48 hr of PMA induction. Our results show that sarcoma cell lines (HOS, MG-63, U2-OS and RD) adhere to stimulated endothelium via VLA-4-VCAM-I adhesion molecules and that VLA-4 avidity on sarcoma cells can be differentially modulated by PMA.


The usefulness of ethylephrine (Efortil-R) in the treatment of priapism and intraoperative penile erections. We present our experience in the treatment of 8 patients with priapism after intravenous injection of vasoactive drugs, and of 15 patients with persistent erections in the course of transurethral cystoscopy surgery. All of them were treated with intracavernous injection of 10 mg ethylephrine (1 ml Efortil-R). The results were satisfactory in all cases. In one patient we had to draw 75 ml blood and give another 10 mg dose of ethylephrine. We have not observed secondary effects of drug administration except two local haematomas with spontaneous resolution. We consider that this treatment is very useful in the management of patients with persistent erections or priapism because of the excellent results obtained without adverse effects.


The prognostic value of prostate-specific antigen and prostatic acid phosphatase in serum of patients with prostate cancer after orchidectomy. Correlations between the serum levels of PAP and PSA before and 1, 3 and 6 months after orchidectomy in 27 prostatic cancer patients (advanced clinical stages C and D according to Whitemore scale) were studied. The PSA values correlated more distinctly than PAP with the general clinical condition. PSA is a reliable tumour marker when used at regular intervals, especially for monitoring therapeutic results. A high preoperative PSA level correlates with a high postoperative level and progression of the disease.


Immunological detection of nitric oxide synthase(s) in human tissues using heterologous antibodies suggesting different isoforms. Nitric oxide (NO) is generated from L-arginine by NO synthases. Localization of the brain enzyme has been carried out in the rat; however, despite data suggesting that NO is a major regulator of vascular and neural functions in man, there is no information about the localization of NO synthase in human tissues. Rabbit antisera to NO synthase purified from rat brain (antisera A and B) were raised, tested by Western blotting, affinity purification and enzyme immunoprecipitation assay, and used to investigate the distribution of the enzyme in a variety of human tissues by immunohistochemistry. Antisera to two synthetic peptides from cloned neural NO synthase were used to aid specificity testing. Anti-sera A and B reacted with a approximately 160-kDa protein in Western blots of human brain extracts, gave immunostaining of nerves, and precipitated enzyme activity from rat brain homogenates. Antiserum B to NO synthase also reacted with proteins of M(r) between 125 and 140 kDa in extracts of well-vascularised tissues, and immunostained vascular endothelium; the neural and vascular immunoreactivity persisted after affinity purification of antiserum B with the approximately 160 kDa protein. Endothelial staining with antiserum B was seen in respiratory tract, liver, skin and umbilicus; syncytial trophoblasts stained in the placenta. Neural staining with antiserum A and B was seen in the myenteric and submucous plexus, and in nerve fibres in smooth muscle of the gut and in many areas of the central nervous system, particularly cortex, hippocampus, hypothalamus, cerebellum, brain stem and spinal cord.(ABSTRACT TRUNCATED AT 250 WORDS)


Single-base mutations at position 2661 of Escherichia coli 23S rRNA increase efficiency of translational proofreading. Two single-base substitutions were constructed in the 2660 loop of Escherichia coli 23S rRNA (G2661-->C or U) and were introduced into the rrnB operon cloned in plasmid pKK3535. Ribosomes were isolated from bacteria transformed with the mutated plasmids and assayed in vitro in a poly(U)-directed system for their response to the misreading effect of streptomycin, neomycin, and gentamicin, three aminoglycoside antibiotics known to impair the proofreading control of translational accuracy. Both mutations decreased the stimulation of misreading by these drugs, but neither interfered with their binding to the ribosome. The response of the mutant ribosomes to these drugs suggests that the 2660 loop, which belongs to the elongation factor Tu binding site, is involved in the proofreading step of the accuracy control. In vivo, both mutations reduced read-through of nonsense codons and frameshifting, which can also be related to the increased efficiency in proofreading control which they confer to ribosomes.