Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. The membrane actions of three recently isolated polypeptide neurotoxins from the sea anemones Stichodactyla helianthus (toxin ShI), Condylactis gigantea (toxin CgII) and Calliactis parasitica (toxin CpI) were investigated on action potentials and voltage-clamp membrane currents of the giant axon of the crayfish Procambarus clarkii. The first two toxins were also tested on the cockroach (Periplaneta americana) giant axon. All three toxins were particularly lethal to crustaceans, moderately toxic to an insect (cockroach), and essentially non-toxic to a mammal (mouse). ShI and CgII were 50- to 100-fold more potent on crayfish than on cockroach axons; this difference in activity was correlated with the relative reversibility of their effects on these arthropod axons. The crustacean selectivity of these toxins is therefore due largely to their greater affinity for crustacean sodium channels. All three toxins prolonged crayfish giant axon action potentials by selectively slowing Na channel inactivation without greatly affecting activation. Before toxin treatment, inactivation was nearly exponential, with a time constant less than 1 msec. After treatment, the inactivation time course could be described as the sum of two exponentially decaying components, plus a large steady-state component. The major component possessed the slower (10-20 msec) time constant. The steady-state component increased with depolarization, causing the sodium channel steady-state inactivation curve to reach a minimum between -60 and -20 mV and then increase at more positive potentials. All three toxins shifted the peak sodium current-voltage relation to the left. This voltage shift was greater at 20 degrees C than at 10 degrees C. Maintained membrane depolarization during toxin wash-in delayed the appearance of modified Na channels. Also, prolonged depolarization of toxin-treated axons converted modified sodium channels back to normal ones. The toxins did not affect potassium and leakage currents. Our results indicate that the three crustacean-active sea anemone toxins share a common electrophysiological action on arthropod sodium channels, at least at the macroscopic level.


Broad substrate specificity of snake venom fibrinolytic enzymes: possible role in haemorrhage. We found previously that two fibrinolytic enzymes (jararafibrases I and II) purified from Bothrops jararaca venom displayed a haemorrhagic activity. To elucidate the mechanisms involved and the role of the enzymatic activity in haemorrhage, the enzymatic properties of the purified enzymes were examined. The substrate specificity of the enzymes was determined using type I collagen, type IV collagen, gelatin, laminin and fibronectin as substrates. The enzymes degraded type IV collagen, gelatin, laminin and fibronectin into smaller fragments, but degraded type I collagen only partially in a non-specific manner. The specific activities of jararafibrase I for type IV collagen and gelatin were 172 +/- 5 units/mg protein and 1315 +/- 177 units/mg protein, respectively. The specific activities of jararafibrase II for type IV collagen and gelatin were 9.2 +/- 0.6 units/mg protein and 143 +/- 15 units/mg protein, respectively. It was evident that the enzymes had rather broad substrate specificities and degraded basement membrane components including type IV collagen. The number of type IV collagen units of bacterial collagenase which gave the minimal haemorrhagic dose was 191.4, while the numbers of type IV collagenase units of jararafibrases I and II which gave the minimal haemorrhagic dose were 1.5 and 0.25, respectively. It is suggested that the broad substrate specificity of the enzymes is essential for inducing haemorrhage with a single enzyme.


Short-time cytotoxicity of mussel extracts: a new bioassay for okadaic acid detection. Okadaic acid (OA), the main toxin responsible for diarrhoeic shellfish poisoning (DSP) has high cytotoxicity for KB cell cultures (apparent after 3 hr of contact), facilitating rapid detection in contaminated mussels. We developed a method to determine the minimal active concentration (MAC) based on direct microscopic study of toxin-induced changes in cell morphology. A high correlation was found between the MAC of tested extracts and corresponding OA concentrations in mussel hepatopancreas as measured by high performance liquid chromatography. This technique is rapid and reproducible and does not require the use of living animals.


Tetrodotoxin and its analogues in extracts from the toad Atelopus oxyrhynchus (family: Bufonidae). Tetrodotoxin and its analogues, 4-epitetrodotoxin and 4,9-anhydrotetrodotoxin, were detected in the toad Atelopus oxyrhynchus by HPLC analysis. The toxin and its analogues were still present in a specimen which lived 3.5 years in captivity.


[The determination of the antiviral activity of the sorbent Evirkhip (in vitro studies)]. It was found that "Evirkhip" possesses a marked antiviral activity confirmed on a model of enteroviruses (coxsackie). The authors also studied the resistance to the drug in the chain: eukaryotic cell-viral particle-prokaryotic cell. The resistance decreased with the shift to right.


The participation of ribosomes in protein glycosylation. Interaction of the ribosome-UDP-N-acetyl-glucosamine complex with dolichol phosphate. UDP-N-acetylglucosamine can be bound by pure ribosomes. The part of N-acetylglucosamine-1-P can be transferred from the complex ribosome-UDP-N-acetylglucosamine onto dolichol phosphate. Evidence is presented that N-acetylglucosamine bound to dolichol phosphate can be transferred to the nascent peptide synthesized on the ribosome.


The desipramine-induced growth hormone response and the dexamethasone suppression test in obsessive-compulsive disorder. Ten patients with DSM-III-R obsessive-compulsive disorder (OCD) underwent the desipramine (DMI) growth hormone (GH) stimulation test as well as the dexamethasone suppression test (DST). The results were compared with the responses in a group of matched healthy controls. The GH response to DMI did not differ between patients and controls and 9 of 10 patients showed cortisol suppression in response to dexamethasone. The data suggest that neither alpha 2 adrenergic dysfunction nor DST non-suppression are features of primary OCD.


Search for evidence of herpes simplex virus, type 1, or varicella-zoster virus infection in postmortem brain tissue from schizophrenic patients. The highly sensitive polymerase chain reaction (PCR) was used to search for herpes simplex virus type 1 (HSV-I) or varicella-zoster virus (VZV) in the DNA extracted from postmortem temporal cortex samples of 8 schizophrenic subjects, 8 nonschizophrenic suicide victims and 8 normal controls. HSV-I or VZV-specific DNA amplification was not detected in any of the samples studied.


Detection and analyses by gel electrophoresis of cisplatin-mediated DNA damage. DNA damage induced by cis-diamminedichloroplatinum (II) (cisplatin: cis-DDP), an anticancer drug, was studied in vitro by monitoring the drug-induced conformational change of pUC18 plasmid DNA, the sensitivity to some restriction enzymes of the damaged DNA and the sequence-dependent termination of DNA synthesis caused by cisplatin. Closed circular, superhelical pUC18 DNA was treated at 37 degrees C for 16 h with various concentrations of cisplatin. Cisplatin-dose-dependent conformational change due to unwinding of the treated DNA was detected by agarose gel electrophoresis. To analyze the base-specificity of the cisplatin damage, the measurement for sensitivity of cisplatin-treated DNA to various types of restriction enzyme and sequence gel analysis of the treated DNA were conducted. The results suggested that cisplatin attacked preferentially the sequence of GG > AG > GNG in the order. In the present assay condition, the cisplatin/DNA nucleotide ratios required for the DNA damage detection were roughly 0.025 for the conformational analysis, 0.001 or more for the restriction enzyme analysis, and less than 0.001 for the sequence gel analysis. By using the present method, it was demonstrated that the cisplatin-mediated DNA damage was inhibited by NaCl, KCl, CaCl2 or MgCl2 at their nearly physiological concentrations, and by reducing agents such as thiourea and 2-mercaptoethanol in the reaction mixture.


[Annual changes of bacteria isolated from urinary tract infections and their susceptibility to new quinolone agents]. Statistical analyses were conducted on bacterial strains isolated from the urine of patients with urinary tract infections (UTI) in the Department of Urology at Aichi Medical University from January 1988 through December 1991. The drug sensitivity of the bacterial strains isolated from patients with complicated UTI between 1988 and 1991 was analyzed and compared. Among the outpatients with uncomplicated UTI, a total of 352 strains were isolated. E. coli was isolated the most frequently (90.1%). Among the outpatients with complicated UTI, a total of 829 strains were isolated. Enterococcus sp. was isolated the most frequently (21.2%). P. aeruginosa and S. aureus showed a tendency to increase. Among the inpatients with complicated UTI, a total of 671 strains were isolated. Enterococcus sp. was isolated the most frequently (22.5%). P. aeruginosa and S. aureus showed a tendency to increase, while E. coli and Enterobacter sp. showed a tendency to decrease. E. coli was highly sensitive to all drugs except ampicillin (ABPC). Proteus sp. and Klebsiella sp. were highly sensitive to norfloxacin (NFLX). P. aeruginosa was highly sensitive to gentamicin (GM). The frequency of NFLX-sensitive P. aeruginosa significantly dropped from 84.6% to 40.9% among the outpatients (P < 0.01), and from 75.0% to 44.4% among inpatients (P < 0.05). S. aureus was highly sensitive only to minocycline (MINO). The frequency of NFLX-sensitive S. aureus significantly dropped from 66.6% to 23.3% among inpatients (P < 0.05). Enterococcus sp. was highly sensitive to ABPC and MINO. Of the new quinolones, tosufloxacin (TFLX) had the strongest antimicrobial activity followed by (ciprofloxacin) CPFX on all the bacterial strains tested.


Predictive value of human papillomavirus DNA detection by filter hybridization and polymerase chain reaction in women with negative results of colposcopic examination. This study focused on women who had an abnormal Papanicolaou smear and a subsequent colposcopic examination negative for a cervical squamous intraepithelial lesion. Cervical swabs obtained during the initial colposcopic examination were analyzed for human papillomavirus (HPV) DNA, and these results were correlated with the follow-up examinations. Human papillomavirus DNA was detected by dot-blot hybridization in 31 of 109 (28%) cases. The rate of subsequent biopsy-proved squamous intraepithelial lesion was 17 of 31 (55%) for the HPV-positive group compared with 9 of 78 (11%) for the HPV-negative group. The HPV-DNA detection rate by polymerase chain reaction was 46 of 109 (42%), with a rate of subsequent squamous intraepithelial lesion of 18 of 46 (39%) for the HPV-positive group, compared with 8 of 63 (13%) for the HPV-negative group. Most women had no evidence of squamous intraepithelial lesions on follow-up if the concurrent Papanicolaou smear was normal, even when HPV DNA was detected, suggesting that HPV detection per se does not necessarily correlate with subsequent squamous intraepithelial lesion.


Altered sensitivity of alpha 2-adrenoceptors in the brain during aging in rats. The soporific and cardiovascular responses to the injection of clonidine into the locus coeruleus and nucleus tractus solitarius, respectively, were studied in rats of different ages. Clonidine (0.09, 0.19, 0.28, and 0.56 nmol) microinfused into the LC of young rats induced dose-dependent behavioral and ECoG SWS with a significant increase in total voltage in the lower frequency bands. In contrast to young rats, clonidine (0.19 and 0.28 nmol) given into the LC did not affect the behavior and ECoG spectrum power in old rats. However, after higher doses of clonidine (0.56 and 1.2 nmol) a small, short-lasting period of behavioral and ECoG SWS was still evident. In 3-month-old rats clonidine (0.25, 0.5 and 1 micrograms) injected into the nucleus tractus solitarius in anesthetized rats produced a significant fall in blood pressure and a significant decrease in heart rate. In contrast, in 12-month-old rats the maximum fall in blood pressure and heart rate was significantly less than in young animals. In addition, in older rats (24-month old) clonidine at the same or larger doses given into the nucleus tractus solitarius did not produce any significant changes in the cardiovascular parameters studied. In conclusion, the present experiments provide evidence that during aging there is a progressive decrease in the soporific and cardiovascular responses to alpha 2-adrenoceptor stimulation in the locus coeruleus and nucleus tractus solitarius, respectively.


Cell-matrix interactions in the genesis of arteriosclerosis and atheroma. Effect of aging. The progression of atheroarteriosclerosis was shown to be age dependent. This designation covers two separate entities: arteriosclerosis, the progressive and diffuse hardening of the walls of arteries with loss of elasticity, and atheromatous plaque formation, which can start early in life according to nutrition and genetic factors (LDL-receptor expression). Lipoprotein-receptor interactions play a crucial role in lipidic plaque formation. There is, however, no indication that the diffuse hardening of the vascular wall would also be influenced by these mechanisms. We described recently a high-affinity receptor for elastin peptides, present on smooth muscle cells, fibroblasts, and also on monocytes and PMNs. When activated, this receptor will increase intracellular calcium. Circulating elastin peptides were determined by a sensitive Elisa method and found to be between 0.1 and 20 micrograms/ml, in the range of activation of the elastin receptor. They increase in obliterative arteriopathies and type IIb hyperlipidemia. Elastolysis accompanies aging and vascular pathology; the sensitivity of this receptor changes with age, intracellular Ca++ increases, but the receptor appears to be uncoupled from its normal transmission mechanism. These results may well explain the increasing diffuse calcification of the vessel wall. The previously demonstrated potentiation of cholesterol deposition in elastic fibers by calcium is in agreement with simultaneous deposition of calcium and lipids. The recent demonstration of the efficient competition of fibronectin for LDL in proteoglycan-LDL complexes suggests that this reaction may be involved in foam cell formation by the opsonization of LDL for phagocytosis. Fibronectin was shown to accumulate in atherosclerotic plaques. Altogether these recent results confirm the importance of cell-matrix interactions in atherogenesis and lead to a better understanding of the age dependence of these disease processes.


Transposable elements as a factor in the aging of Drosophila melanogaster. We have considered the hypothesis that transposable elements may contribute to the aging process through somatic mutation. We have presented evidence to suggest that at least two elements, Copia and 412, are capable of somatic activity in adult Drosophila tissue. A strain harboring a third transposable element, P, was produced that showed eye color mosaicism and reversion to wild phenotype (red eyes) as a result of somatic and germ line transposition. A high-fat diet, known to accelerate aging, increased the frequency of eye color mosaicism and red eyes. We induced life span shortening by artificially activating somatic transposition of P elements, and the extent of reduction in life span was similar in both sexes. These data are consistent with the notion that some aspects of the age phenotype may be caused by mutational activity of transposable elements in somatic tissues. The hypothesis is readily tested in other organisms, including humans. It offers new dimensions in the understanding and management of age-associated changes.


The problem of ductal carcinoma in situ of the breast. Ductal carcinoma in situ (DCIS) of the breast is a problem seen with increasing frequency as a result of the more widespread use of mammography. Treatment options are discussed together with on-going clinical trials seeking to determine the role of radiotherapy and tamoxifen in breast conservation treatment for DCIS.


[Biological and developmental aspects of macronodular adrenal hyperplasia in Cushing's disease]. The pathophysiology of macronodular adrenocortical hyperplasia (MNH) associated with Cushing's diseases remains debatable. Some authors have claimed that MNH represents an autonomous condition that has to be treated by surgical adrenalectomy while others advocate the use of pituitary surgery. We have compared the biochemical features, before and after pituitary surgery, between 7 cases of MHN and 22 cases of diffuse hyperplasia (DH) associated with Cushing's disease. In addition, the diameter of the adrenal nodules after surgery was evaluated in 6 cases. The mean ACTH level in MNH and DH subjects was similar. Dexamethasone suppressibility and the stimulatory effect of metyrapone on ACTH secretion were less in MNH than in DH subjects, suggesting a greater degree of adrenal autonomy in the former. The cortisol responses to metyrapone and CRF tests suggested an increased responsiveness of the adrenals to endogenous ACTH in the MNH group. Only one MNH patient had biochemical features suggesting a primary adrenal disease. The use of a sensitive ACTH assay and the results of inferior petrosal sinus sampling identified the ACTH-dependency of the disease. A short to medium term remission of the disease was obtained in all cases of MNH and in 87% of cases of DH after surgery. The diameter of the adrenal nodules significantly decreased in only 43% of cases. Our results suggest that the adrenal autonomy in MNH is incomplete and that, despite the persistence of the adrenocortical nodules, the disease can be cured with pituitary microsurgery in most cases.


What is the contribution of occupational environmental factors to the occurrence of scleroderma in men? An occupational analysis of 56 men with scleroderma in the United Kingdom showed no evidence that silica exposure was implicated in the onset of the disease, in contrast with older published reports suggesting that such exposure explained an important proportion of the occurrence of the disease in men. Of the major occupational exposures suggested from case reports, only organic solvents were reported to any extent in this series. No significant increase in exposure to organic solvents was noted, however, in a case control analysis.


Interleukin 6 production by mononuclear phagocytes can be stimulated by leukotrienes. Leukotriene B4 is a potent lipid mediator of inflammation, and some of its bioactivities may involve inflammatory cytokines. Human monocytes, cultured in the presence of graded concentrations of LTB4, were significantly stimulated in their production of IL6. Nanomolar concentrations of the mediator were optimal for stimulation of IL6 production, which was already significant at 6 hours. LTC4 showed a similar, albeit lower activity. In addition to stimulating IL6 protein production, LTB4 also augmented IL6 mRNA accumulation, which was maximal at 1 hour. Furthermore, LTB4-treated monocytes contained increased amounts of nuclear protein capable of binding to potential transcriptional promoter regions of the IL6 gene. These data suggest that leukotrienes may modulate the production of IL6 and indicate some underlying mechanisms which may be involved.


Regulation of neutrophil functions by elastase-generated IgG fragments. IgG is split by neutrophil elastase into Fc and Fab fragments. These IgG fragments influence the functions of stimulated neutrophils such as chemotaxis, oxidative burst, and enzyme release. FMLP stimulated leukocyte chemotaxis is specifically inhibited by the elastase generated Fc fragments. Seven nmol Fc/10(6) PMN totally inhibit the chemotaxis stimulated by 16 to 125 nM FMLP. Native IgG and Fab fragments show no effect. FMLP-stimulated superoxide anion generation is specifically inhibited by Fc fragments with half maximal inhibition by 1.2 nmol/10(6) PMN. The generation of hydrogen peroxide is concomitantly stimulated, resulting in a superoxide dismutase-like effect. FMLP-stimulated elastase and myeloperoxidase release are enhanced by Fab fragments (10 nmol/10(6) PMN) to 206 and 155%, respectively, of reference values by 25 nM FMLP, while Fc and native IgG stimulate to a less extent. Consequently, elastase-generated Fc fragments have an inhibitory effect on inflammation by reducing chemotaxis and oxidative burst of stimulated neutrophils. The release stimulating activity of Fab fragments results in an up-regulation of elastase induced IgG degradation.


When should granulocyte function be checked? Behind many clinical cases with recurrent, severe infections, absesses, delayed wound healing and especially in antibiotic resistant sepsis some granulocyte function abnormalities can be detected. The abnormalities are of inherited and acquired origin. The inherited dysfunctions are discussed here in details, but the appearance of some failures in neutrophil functions should be taken into consideration when examining patients with other diseases (e.g. diabetes, infections, periodontal disease, zinc deficiency, malignancies, uremia etc.). The main clinical tools for the diagnosis of the qualitative abnormalities in neutrophil functions are chemotaxis with migration, and an NBT test with and without stimulation, as a first indication. Any deviation in the result of these function tests requires further determinations.


Further evidence that the nucleic acid of avian encephalomyelitis virus consists of RNA. The nucleic acid of the Van Roekel strain of avian encephalomyelitis virus (AEV) was determined to be RNA, according to the inability of the nucleoside analog 5-bromo 2'-deoxyuridine (BUdR) to inhibit its growth in chicken embryo kidney cell cultures. The test was carried out using known DNA and RNA viruses as controls, and the results are consistent with classification of AEV as a member of the family Picornaviridae within the genus Enterovirus.


Effect of route of administration on the efficacy of a recombinant fowlpox virus against H5N2 avian influenza. A recombinant fowlpox vaccine virus containing the H5 hemagglutinin gene of avian influenza virus was administered to susceptible chickens via wing-web puncture, eye drop, instillation into the nares, and drinking water. Even though there was a negligible hemagglutination-inhibition (HI) serologic response, all 10 chickens vaccinated by wing-web puncture remained without obvious signs of disease and survived challenge with a highly pathogenic strain of H5N2 avian influenza virus. All unvaccinated chickens and those vaccinated by nasal and drinking-water routes died following challenge. Eight of 10 chickens vaccinated with the recombinant by eyedrop died. All vaccinates were negative on the agar gel precipitin (AGP) test, and only one chicken had a positive HI titer before challenge. All chickens that survived challenge had high levels of HI antibody and were positive on the AGP test, indicating that they were infected by the challenge virus.


Hemorrhagic enteritis virus inclusions in turkey renal tubular epithelium. Commercial turkeys from four Iowa flocks, two Illinois flocks, and three California flocks were submitted to state diagnostic laboratories because of a variety of health problems. The turkeys ranged in age from 5 to 12 weeks, included both hens and toms, and were owned by five different companies. Some flocks had previously been immunized with live hemorrhagic enteritis vaccine, and other flocks were unvaccinated. In all accessions, basophilic intranuclear inclusion bodies were observed in renal tubular epithelium by light microscopy. Transmission electron microscopy showed that the inclusions consisted of densely packed virus particles. The virions were identified as adenoviruses based upon the icosahedral morphology and average particle diameters of 72 nm. Avidin-biotin immunoperoxidase staining of formalin-fixed, paraffin-embedded kidneys was used to identify this adenovirus as hemorrhagic enteritis virus.


Structural proteins of classic and variant strains of infectious bursal disease viruses. Structural polypeptides of six tissue-culture-origin (BGM-70 continuous cell line) infectious bursal disease viruses representing classic and variant strains of serotype 1 and one serotype 2 strain were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Additionally, two of the variant strains were propagated in vivo in bursa of Fabricius and compared with those grown in cell culture. Differences among the structural proteins of serotype 1 viruses were minor and probably of no value in differentiating these viruses. However, distinct differences were observed between serotype 1 and 2 viruses. The bursa-derived viruses were different from those propagated in cell culture in molecular weights and in proportions of the proteins. The bursa-derived strains had protein migration patterns similar to those described for tissue-culture-incomplete virus particles.


Response of B congenic chickens to infection with infectious bursal disease virus. Six congenic lines of chickens that differ from the parental inbred line RPRL-15I5 for genes in the major histocompatibility (B) complex were used to study the influence of the B haplotypes on the response of chickens to infection with virulent infectious bursal disease virus (IBDV) at 1 day or 4 weeks of age, and on the antibody response to vaccination with live or inactivated oil-emulsion (OE) IBDV vaccines at 7 weeks of age. IBDV-induced immunodepression and lesions in the bursa, spleen, and thymus in chickens infected with virus at 1 day of age were of the same degree of severity, regardless of line of chickens used. The response of blood cells to the mitogens phytohemagglutinin-M and concanavalin A was elevated in chickens infected with IBDV at 1 day of age. In an experiment conducted to study the effect of the B haplotype on IBDV infection in 4-week-old chickens, B congenic line C-12 (B12B12) showed the highest susceptibility to clinical IBD, with mortality of 79%. No detectable difference in the serological response to vaccination with live or OE IBDV vaccines was noted among chickens of various congenic lines. We conclude that the B haplotypes may influence IBDV-induced mortality, but not immunodepression or severity of lesions in lymphoid organs, or the antibody response to live or OE IBDV vaccines.


Escherichia coli multiplication and lesions in the respiratory tract of chickens inoculated with infectious bronchitis virus and/or E. coli. Escherichia coli numbers and histopathological changes were studied in the respiratory tract of line 151 chickens intranasally inoculated with infectious bronchitis virus (IBV) and/or virulent E. coli; this line is highly susceptible to IBV. Chickens inoculated with IBV alone showed increased numbers of E. coli in the trachea and had tracheitis, airsacculitis, and bronchiolitis. One of 17 chickens inoculated with IBV alone died with fibrinopurulent serositis. Chickens inoculated with IBV and E. coli had more severe and persistent respiratory lesions than those inoculated with IBV alone. E. coli was isolated from tracheas of chickens inoculated with IBV and E. coli more frequently than from chickens inoculated with IBV alone. In this group, 14 of 27 chickens died with tracheal plugs or with fibrinopurulent serositis. There was neither increased numbers of E. coli nor significant lesions in the respiratory tract of the group inoculated with E. coli alone. These results suggest that IBV may facilitate E. coli invasion into the lower respiratory tract of the chicken.


Immunosuppressive effect of a highly virulent infectious bursal disease virus isolated in Japan. The immunosuppressive effect of infectious bursal disease virus (IBDV) on vaccination against Newcastle disease (ND) was compared among 2-, 3-, and 4-week-old chickens inoculated with the highly virulent IBDV field isolate 90-11 and the reference serotype 1 strain GBF-1. In all age groups, isolate 90-11 severely suppressed antibody response to ND vaccination and protective vaccinal immunity against ND. In contrast, chickens inoculated with strain GBF-1 and vaccinated with ND vaccine were well protected from the ND virus challenge. The mitogenic response to phytohemagglutinin of splenic lymphocytes from chickens inoculated with isolate 90-11 or strain GBF-1 was significantly lower than that of uninoculated controls. There was no difference between the two inoculated groups in responsiveness, although lymphocyte depletion in the thymus was more severe in chickens inoculated with isolate 90-11 than in chickens inoculated with strain GBF-1.


Cholangiocarcinomas in Japanese and Thai patients: difference in etiology and incidence of point mutation of the c-Ki-ras proto-oncogene. Point-mutational activation of the c-Ki-ras proto-oncogene has been shown to be rare in human hepatocellular carcinoma, the most common primary liver cancer and one usually associated with chronic viral infection. To reveal the association of c-Ki-ras activation with cholangiocarcinogenesis under different etiological backgrounds, the incidence of point mutation at codons 12 and 13 of the c-Ki-ras proto-oncogene was examined in three groups of human liver cancers with differentiation to biliary epithelial cells: Group 1, cholangiocellular carcinoma in Japanese with normal livers; Group 2, cholangiocellular carcinoma in Thais who had lived in an area where the liver fluke Opisthorchis viverrini is endemic; and Group 3, combined hepatocellular-cholangiocellular carcinoma, a rare type showing features of both hepatocellular and biliary epithelial differentiation, in Japanese with chronic viral hepatitis with or without cirrhosis. The polymerase chain reaction and direct sequencing of its product were used to detect the mutation. Point mutation at codon 12 of the c-Ki-ras gene was detected in five (56%) of nine cases in Group 1. In contrast, the mutation was not detected in any of the cases in Groups 2 and 3. Therefore, point-mutational activation of c-Ki-ras did not seem to be involved in the development of primary liver cancers associated with apparent chronic irritation of liver cells or biliary epithelial cells caused by exogenous liver-fluke or viral infection. On the other hand, point-mutational activation of the c-Ki-ras proto-oncogene may be involved in cholangiocarcinogenesis in liver without preexisting liver-fluke or viral infection.


[Presynaptic effects induced by pretreatment with formamide of the neuromuscular junction of the mouse]. Some techniques to block muscular nerve evoked contraction involve pharmacological approaches using synaptic blocking agents. Such methods interfere with normal synaptic transmission, and could introduce artifacts making difficult the experimental interpretation. The method based on the use of formamide pre-treatment should not interfere with synaptic physiology, indeed previous works suggest that the mechanism involved in block of muscle activity could depend on the decrease in specific postsynaptic membrane capacitance, and on the disruption of the morphology of the transverse tubule system. To prove this assumption we evaluated before and after formamide pre-treatment, some pre and postsynaptic parameters related to the spontaneous quantal release (MEPC). By means of the Loose patch clamp technique, we demonstrated, that formamide pre-treatment increases in an irreversible manner the frequency of spontaneous quantal release. Morphology of MEPC appear not modified by formamide pretreatment, which does not interfere with postsynaptic cholinergic receptors activity.


In vitro susceptibility of avian Escherichia coli and Pasteurella multocida to danofloxacin and five other antimicrobials. The in vitro susceptibility of Escherichia coli and Pasteurella multocida isolated from poultry was determined to danofloxacin, a novel fluoroquinolone, and five other commonly used antimicrobials. A total of 1737 E. coli field isolates and 107 P. multocida isolates were tested by veterinary diagnostic laboratories in Europe, Japan, South Africa, and North America during the period 1989-91. The antimicrobial susceptibility of these isolates was determined using the Sensititre broth microdilution technique. The minimum inhibitory concentrations (MIC) of danofloxacin, furaltadone, lincomycin, oxytetracycline, spectinomycin, and trimethoprim:sulfamethoxazole that prevented growth of 90% of the bacteria were 0.25 > 64, > 64, > 64, > 128, and > 16 micrograms/ml, respectively, against E. coli isolates and 0.25, 64, 64, 16, 128, and 8 micrograms/ml, respectively, against P. multocida isolates. Danofloxacin demonstrated considerable in vitro potency against these important poultry pathogens, many of which showed extensive resistance to the other antimicrobials tested.


Protein phosphatases in cell signalling. Protein phosphorylation is the most common post-translational modification and plays a role in all known pathways of signal transduction. Net phosphorylation is a result of the balance of activities of protein kinases and phosphatases. There is increasing evidence that the regulated removal of phosphate groups from proteins, catalyzed by protein phosphatases, is required for the downstream activation of other signalling proteins.


Ubiquitin and intracellular protein degradation. In eukaryotes, the ubiquitin-dependent protoelytic pathway is one of the major routes by which intracellular proteins are selectively destroyed. Recent work has shown that conjugation of ubiquitin to substrate proteins is mediated by a remarkably diverse array of enzymes. Proteolytic targeting may also be regulated at steps between ubiquitination of the substrate and its degradation to peptides by the multisubunit 26S protease. The complexity of the ubiquitin system suggests a central role for protein turnover in eukaryotic cell regulation.


Does the antiarrhythmic effect of DMPO originate from its oxygen radical trapping property or the structure of the molecule itself? Using the isolated perfused rat heart with transient (30 min) normothermic global ischemia, it was shown that DMPO (5,5-dimethyl-pyrroline-N-oxide), an organic spin trap agent designed specifically to trap free radicals, dramatically reduced the vulnerability of the myocardium to reperfusion-induced ventricular fibrillation (VF) and ventricular tachycardia (VT). DMPO (concentration range 30-500 mumol/l) infused in the heart at the moment and during the first 10 min of reperfusion exerted a dose-dependent antiarrhythmic effect. Thus, the doses of 30, 100, and 500 mumol/l of DMPO reduced the incidence of reperfusion-induced VF and VT from their control values of 100% and 100% to 83% and 91%, 50% (p < 0.05) and 67%, 25% (p < 0.01) and 50% (p < 0.05), respectively. Furthermore, the recovery of myocardial function was improved during postischemic reperfusion. A modification in the molecular structure of DMPO leading to HMIO (1,2,2,4,5,5-hexamethyl-3-imidazoline-oxide), so-called inactive DMPO which does not trap free radicals in the presence of a radical generating system or in the effluent of reperfused hearts, failed to reduce the incidence of reperfusion-induced arrhythmias or improve the recovery of postischemic reperfused myocardium. These findings suggest that the free radical trapping properties of DMPO or the effects of the formed DMPO-OH, a stable nitroxyl radical adduct, are responsible for the reduction of reperfusion-induced arrhythmias, and not the molecular structure of DMPO itself. Finally, it is of interest to note that the detection of free radicals was observed in fibrillating hearts, but not in nonfibrillating hearts. This consideration should be taken into account when making therapeutic interventions and risk assessments of a radical scavenger in this setting.


Chaperones and protein folding.

Chaperones and protein folding. Chaperones are centrally involved in the control of protein structure, function, localization and transport. A flurry of scientific activity continues to examine the molecular nature of chaperone-substrate recognition and the role of auxiliary chaperones (cohort proteins) and small molecules that expedite these processes. Chaperones have been implicated in processes as diverse as protein secretion, nuclear transport, thermotolerance, the steroid receptor signal transduction pathway, T-cell receptor and major histocompatibility complex class I and II multimeric assembly and bacterial virulence.


Production and characterization of monoclonal antibodies to three infectious bronchitis virus serotypes. Three panels of monoclonal antibodies (MAbs) were prepared against the spike (S) proteins of infectious bronchitis virus (IBV) strains Arkansas 99, Connecticut 46, and Massachusetts 41. Based on enzyme-linked immunosorbent assay (ELISA), the MAbs were grouped into three categories: 1) group-specific, which reacted with a broad spectrum of homologous and heterologous IBV serotypes; 2) serotype-specific, which reacted only with strains of the homologous serotype; and 3) strain-specific, which reacted "selectively" with only certain strains of homologous and heterologous serotypes. MAbs that displayed serotype specificity were all specific to S1 fractions of the homologous serotype, confirming that epitopes that determine virus serotype are associated with the S1 protein. An excellent correlation was found when the results of IBV serotyping by MAb-based indirect ELISA were compared with those from the conventional virus-neutralization test. This confirms that the MAbs described here will serve as valuable tools in epizootiological studies and serotype-specific diagnosis of IBV infection.


Fixation of femoral shaft osteotomy with intramedullary metallic or absorbable rod: an experimental study on growing dogs. The purpose of this study was to examine the effects of an intramedullary self-reinforced polyglycolic acid (SR-PGA), self-reinforced poly-L-lactic acid (SR-PLLA) and a metallic rod on growing bone and their applicability in the fixation of a femoral shaft osteotomy in growing dogs. In 5 dogs, 12 weeks of age, a SR-PGA rod and in another 5 dogs a SR-PLLA rod, both 4.7 mm in diameter and 60 mm in length, were introduced into the intramedullary cavity of the right femur to fix a femoral shaft osteotomy. In a third group of 5 dogs the femoral shaft osteotomy was fixed with an intramedullary metallic rod of equal size. The follow-up intervals were 1, 3, 6, 12, 24 and 48 weeks. Solid union of the osteotomy without secondary displacement was seen radiographically 6 weeks after the operation in all dogs. Neither an intramedullary SR-PGA-, SR-PLLA- nor metallic rod caused any significant disturbance to the longitudinal growth of the operated femur. Narrowing of the femoral neck and a slight valgization of the angle between the femoral neck and shaft without any functional disability was seen 48 weeks after the operation.


Brain, lung, and cardiovascular interactions with cocaine and cocaine-induced catecholamine effects. The pathophysiologic effects of cocaine on neuronal, pulmonary, and cardiovascular tissue are related to the drug's interaction with select catecholamine and neuroendocrine systems. Cocaine has been shown to alter circulating levels of the neurotransmitters, dopamine, norepinephrine, epinephrine, as well as the hypothalamic-pituitary-adrenal axis hormones corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH), and cortisol. Furthermore, brain and lung tissue have been identified as primary sites of cocaine sequestration and metabolism. This paper reviews evidence suggesting that steroid-potentiated actions of catecholamines on vascular tissues contributes to the etiology of cocaine-related medical complications, including ischemic stroke, coronary ischemia, and ischemia-based renal failure.


Effect of sumatriptan, a new selective 5HT1-like agonist, on liquid gastric emptying in man. Paired studies were carried out on 12 healthy male subjects to compare the effect of intravenous doses of the 5-HT1-like agonist sumatriptan (GR43175; 3 mg), 10 mg metoclopramide and saline control on the rate of gastric emptying of a radiolabelled liquid test-meal. Intravenous administration of metoclopramide accelerated gastric emptying by decreasing the lag period, while intravenous administration of sumatriptan delayed gastric emptying by increasing the lag period. The observation that sumatriptan causes a delay in gastric emptying in normal healthy volunteers, but relieves nausea and vomiting during migraine attacks, suggests that sumatriptan may be acting via a central mechanism to relieve symptoms of nausea and vomiting associated with migraine.


Human papillomavirus detection in cervical cells by in situ hybridization with biotinylated probes. We have investigated the applicability of human papillomavirus (HPV) DNA detection by in situ hybridization with biotinylated probes in epithelial cells obtained from the cervix using a cotton tip swab. We describe a simple procedure for obtaining homogeneous cell samples and good preservation of cellular structure. This is achieved by pretreatment of cells with L-cysteine before hybridization. Separate denaturation of cellular DNA and probe DNA is also necessary for satisfactory results. Both benign HPV DNA 6/11 and potentially oncogenic HPV DNA 16/18 could be identified in our series. In situ hybridization on cervical scrapes is a rapid, simple and very specific method for detecting patients infected with oncogenic HPV types.


Mouse mammary tumor proviral gene expression in cells of the B lineage. Mouse mammary tumor proviruses (MMTV) use a common enhancer/promoter region to accommodate their transcription in two different cellular environments. In mammary tissue, transcription is regulated through the hormone response element located in the 5' LTR. In B cells, transcription is hormone independent, can be stimulated following B cell activation, and is distinct from the transcription of other known inducible genes, including immunoglobulin. The open reading frame (ORF) in the viral 3' LTR has at least two functions. Its gene product(s) acts as a viral superantigen, but also has autoregulatory properties, leading to MMTV transactivation. We propose a scheme suggesting that MMTV evolved to use the B cell as an intermediary in its viral life cycle.


Three-dimensional structure of Na,K-ATPase determined from membrane crystals induced by cobalt-tetrammine-ATP. The three-dimensional structure of Na,K-ATPase has been analyzed with electron microscopy and image processing. The enzyme, purified from pig kidney outer medulla, was arranged in a new form of tetragonal two-dimensional membrane crystals after incubation with cobalt-tetrammine-ATP, a stable MgATP complex analogue. Each continuous protein domain, as delineated by negative stain, consists of two alpha beta-protomers related by a dyad axis. The two rod-like regions are connected by a bridge displaced about 20 A away from the center of the structure toward the lipid bilayer. The domain connecting the two promoters is more constricted and closer to the center of the structure in the Co(NH3)4ATP-induced crystals than in the vanadate-induced p21 crystals. These observations suggest that the difference between previously analyzed dimers of two-dimensional p21 crystals induced with vanadate/magnesium and dimers of p4 crystals induced with Co(NH3)4ATP reflects two different conformational states of the enzyme.


Infectious minor lymphocyte stimulating (Mls) antigens. Minor lymphocyte stimulating (Mls) antigens have profound effects on the murine immune system and have been very important for our current understanding of immune tolerance. It has recently been discovered that these Mls antigens are encoded in an open reading frame located in the 3' long terminal repeat of endogenous and infectious mouse mammary tumor viruses (MMTV). In this review we will discuss the effects of a novel infectious MMTV with properties of Mls-1a on the neonatal and adult immune system in comparison to the effects of endogenous Mtv-7 (Mls-1a).


Mls superantigens: how retroviruses influence the expressed T cell receptor repertoire. The murine Mls antigens were originally discovered because they have an enormous T cell stimulatory capacity, exceeding in strength any other antigen. The finding that Mls interacts directly with the V beta segment of the T cell receptor, leading to in vivo deletion and in vitro stimulation of T cells expressing particular V beta genes, provided an explanation for this phenomenon and led to the formulation of the new term of superantigen. Recently it was discovered by several groups that these superantigens are encoded by type B retroviruses, the mammary tumor viruses which are transmitted vertically as infectious particles or exist as proviruses in the murine genome.


T cell recognition of Mls-like superantigens: analysis of TCR requirements, superantigenic ligands, and signal transduction. Mls-like endogenous superantigens have been reported to be unique, differing from conventional antigens in the T cell receptor (TCR) requirements for their recognition, in the signal transduction events which they elicit in T cells, and in the identity of the stimulatory ligand. This review describes recent findings indicating that TCR V alpha as well as V beta products play a role in Mls recognition. Data are also summarized which indicate that T cell recognition of Mls leads to increased intracellular [Ca2+] and phosphatidyl inosotol hydrolysis. Finally, genetic linkage between mouse mammary tumor virus (MMTV) proviruses and endogenous superantigens is analyzed, and the possible role of non-MMTV products is examined.


16,16-dimethyl prostaglandin E2 modulates aflatoxin B1-induced injury of rat hepatocytes in primary culture: possible role of cAMP. The hepatocellular cytoprotective effects of 16,16-dimethyl prostaglandin E2 (dmPGE2), an analogue of PGE2, were investigated using primary cultures of rat hepatocytes and aflatoxin B1 as the hepatotoxin. Lactic dehydrogenase (LDH) release by hepatocytes was used as an index of hepatotoxicity. When aflatoxin-treated hepatocytes were co-cultured with 16,16-dmPGE2 (0.01-0.5 micrograms/mL) LDH release was significantly reduced and ultrastructural changes of hepatocellular injury were markedly diminished. The magnitude of the cytoprotective effect was not dependent on the concentration of the prostaglandin over the range tested. A significant cytoprotective effect was also induced when hepatocellular cyclic AMP (cAMP) levels were increased by the addition of dibutyl-cAMP. In contrast to 16,16-dmPGE2, PGF2 alpha Tromethamine, an analogue of PGF2 alpha, which does not stimulate cAMP, induced insignificant changes in cytoprotection. These findings indicate that only a low concentration of 16,16-dmPGE2 (> or = 0.01 micrograms/mL) is necessary to induce a maximal hepatocellular cytoprotective effect and suggest that this effect may be dependent on activation of cAMP.


Regulation of T cell function by Mtv-7 gene products. Mls-1, a superantigen encoded by the endogenous mouse mammary tumor virus Mtv-7 induces immunological tolerance through deletion of antigen-reactive T cells. A remarkable difference between this self-antigen and self-MHC antigens is that while the mouse establishes tolerance against self MHC antigens by the time of birth it does not begin to delete T cells specific for the self-superantigen until they had mounted an immuneresponse against it. An immune response occurs normally several days after birth and may be delayed experimentally for weeks before the deletion process ensues. However, for effective deletion of Mls-1 reactive T cells the mouse must be exposed to Mtv-7 positive lymphoid cells within hours after birth. In reviewing here data obtained in this and other laboratories regarding experimental induction of Mls-1 tolerance in neonatal mice we are trying to make a case for the involvement of Mtv-7 encoded antigens distinct from the superantigen. We propose that T cells reactive with non superantigenic Mtv-7 determinants pose a threat to the establishment of chimaerism between Mls-1- neonates and Mls-1+ inocula, as they may cause the rejection of Mls-1 superantigen bearing lymphocytes. Chimaerism is essential for the establishment of lasting Mls-1 tolerance.


The role of hepatitis B virus in the development of primary hepatocellular carcinoma: Part I. Chronic infections with hepatitis B virus (HBV) of humans and animal hepadnavirus infections in their natural hosts are strongly associated with primary hepatocellular carcinoma (HCC). Although viral integrations are found in cells of many HCC, no general viral-specific hepatocarcinogenic mechanism for hepadnaviruses has been identified. In approximately one half of HCC in woodchuck hepatitis virus (WHV) infected woodchucks, viral integrations near the c-myc or N-myc genes have been reported which result in enhanced expression of the respective gene. Such host gene-specific insertional mutagenesis has not been found in HCC of other hepadnavirus infected hosts. Thus in humans, ground squirrels and ducks hepadnaviral integrations appear to be at different host chromosomal DNA sites in each HCC and few integrations have been found within or near any cellular gene. Other possible hepadnavirus-specific carcinogenic mechanisms that are being investigated include transactivation of cellular gene expression by an hepadnavirus gene product (e.g. the X-gene), and mutation of host genes by unknown hepadnavirus-specific mechanisms. It should be noted, however, that chronic hepadnavirus infection is associated with chronic necroinflammatory liver disease with hepatocellular necrosis and regeneration (sometimes leading to cirrhosis in humans), a pathological process that is common to numerous other risk factors for HCC. This suggests the possibility that this pathological process is hepatocarcinogenic irrespective of the inciting agent and the role of hepadnavirus infection is no different from that of other risk factors in causing chronic necroinflammatory liver disease.


Cytogenetics and molecular genetics of nervous system tumors. Cytogenic and molecular genetic analyses of the major histological subtypes of nervous system tumors, gliomas, meningiomas, and neurinomas, have provided interesting information on the mechanisms responsible for or contributing to their origin and development. Regarding malignant gliomas, a complex pattern of chromosomal involvement has been documented at the cytogenetic level: gains of chromosome 7 and losses of chromosome 10, 9p, 17p, and 22; further molecular characterization of these abnormalities has shown that mutational alterations of the p53 gene, together with the loss of alleles at 17p, seem to be the earliest abnormalities occurring during the genesis and progression of these neoplasms. The losses of regions on chromosomes 22 and 13 might also be relatively early events, perhaps characterizing subgroups of low grade gliomas. The mutations of the p53 gene in low grade tumors leads to a selective advantage in vivo and seems to be a critical step in the transformation from low grade to high grade gliomas. The loss of sequences on chromosome 10 and the deletions of 9p (that is loss of tumor suppressor genes on these locations), and epidermal growth factor receptor gene amplification, have been proposed as sequential abnormalities participating in glioblastoma tumorigenesis. The available data on meningiomas and neurinomas show that loss of regions on chromosome 22 is the main characteristic feature. Thus, tumor suppressor genes located in this chromosome are non-randomly involved in both neoplasms, and may present as solitary, sporadic tumors or as multiple associated lesions in neurofibromatosis type 2 (NF-2). The molecular analysis of a large series of meningiomas to determine the common chromosome 22 region lost has revealed that a putative meningioma tumor suppressor gene should be located at the distal 22q12.3-qter region. In parallel, the linkage data on the mapping of the NF-2 gene suggest that the NF-2 and meningioma loci are separate entities. However, some evidence exists on a possible participation of the NF-2 locus in the genesis of some meningiomas. The efforts to identify and isolate the genes involved, as well as their functional analysis, will contribute to a better understanding of the mechanisms of oncogenesis in these neoplasms and will doubtless have a clinical impact in the diagnosis, treatment and prognosis of nervous system tumors in patients.


Chalcogenapyrylium dyes as dual-action sensitizers for photodynamic therapy. Cytochrome c oxidase activity in isolated tumor mitochondria was inhibited by exposure to 570-800 nm of light after addition of cationic dye 1. The alteration in enzyme activity is a consequence of the formation of the highly reactive oxygen species, singlet oxygen, a photochemical product resulting from the irradiation (770 nm of light) of dye 1 in the presence of oxygen. The oxidation of dye 1 by singlet oxygen produces dye 2. Irradiation of mitochondrial suspensions treated with 10(-5) M solutions of dye 2 with 489 +/- 5 nm of light also caused inhibition of cytochrome c oxidase activity, but the addition of scavengers of singlet oxygen, hydrogen peroxide, superoxide, and hydroxyl radical did not prevent the observed inhibition. Similarly, lowering the atmospheric oxygen concentration to 3% had no effect on mitochondrial inhibition. In solution, photoreduction of dye 2 occurred at an increased rate in the presence of 0.01 M tryptophan. ESR studies of dye 2 with 5,5 dimethyl-1-pyrroline-N-oxide (DMPO) (0.1 M) and added tryptophan (0.01 M) suggested the photoproduction of hydroxyl radicals as a possible mechanism for its photodynamic action. We interpret the data as indicating the possibility that after the photooxidative transformation of dye 1 to dye 2, a second mechanism becomes operative for further photodynamic inhibition of enzyme activity. The in vivo or in vitro conversion of dye 1 to dye 2 would allow dye 1 to function as a novel, dual-action photosensitizer.


[Autoimmunity and cancer: paraneoplastic neurological syndromes associated with small cell cancer]. Paraneoplastic neurological syndromes are mostly associated with small cell lung cancer. Lambert-Eaton myasthenic syndrome appears to be caused by anti-presynaptic calcium channel antibodies. Calcium channels are also present in the cell membrane of small cell lung cancer, which may trigger the formation of anti-calcium channel antibodies. It is the most convincing argument in support of the auto-immune paraneoplastic theory, which refers to cross-antigenicity. Serum of patients with small cell carcinoma and cancer-associated retinopathy contains immunoglobulins against several antigens in the retinal and tumor cells. Patients with chronic intestinal pseudoobstruction (gastrointestinal neuropathy) associated with small cell lung cancer displayed circulating IgG antibodies reactive with neurons of myenteric plexus (anti-enteric neuronal antibodies). On the other hand, high levels of anti-neuronal antibodies (anti-Hu) have been found in the serum and cerebrospinal fluid of patients suffering from subacute encephalomyelitis (limbic encephalitis, cerebellar degeneration, sensory neuronopathy) associated with small cell lung cancer. The pathogenic role of the anti-neuronal antibody is not well established. Nevertheless, the finding of high titer antineuronal antibody in patients with a suggestive clinical syndrome is of great interest since it confirms the paraneoplastic syndrome and suggests the location of the primary tumor when the cancer is unknown.


Non-absorbable antibiotics in the treatment of diverticular disease of the colon. Diverticular disease of the colon is a common health problem in western societies. Most patients with colonic diverticula are asymptomatic; it has been estimated that only 20% of individuals harboring diverticula will develop symptoms and signs of illness and a minority will develop major complications. Medical treatment of diverticular disease is aimed to the relief of symptoms and to prevent inflammatory complications. High fiber diets and antispasmodics are widely used in the treatment of uncomplicated diverticular disease although their real efficacy has not been fully elucidated. Antibiotics are used to treat major inflammatory complications of diverticular disease but apparently there is no rationale for their use in uncomplicated disease where an inflammatory component is, by definition, excluded. However two recent papers suggest a possible role of rifaximin, a broad-spectrum poorly absorbable antibiotic, in the management of mild acute diverticulitis and in obtaining symptomatic relief in patients with uncomplicated disease. Prospective studies with an adequate sample size per group of treatment are needed to assess the efficacy of cyclic long term administration of poorly absorbable antibiotics in preventing major complications of diverticular disease.


Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications. A complete protocol for the expression of recombinant cytochrome c genes from yeast, Drosophila melanogaster, and rat in a yeast strain, GM-3C-2, which does not express its own cytochromes c is described. The construction of the expression vectors, transformation and large-scale growth of the yeast, and preparation and purification of the recombinant cytochromes c are described. It was found that, contrary to the way yeast modifies its own cytochromes c, the recombinant proteins were partially acetylated at their N-terminus, except for the drosophila protein, which remained entirely unblocked. Furthermore, the yeast and rat proteins were close to fully trimethylated at lysine 72, while the drosophila protein could be separated chromatographically into forms containing tri-, di-, mono-, and unmethylated lysine 72 showing corresponding resonances in the NMR spectrum. These observations emphasize that, in employing expression procedures to obtain native or mutant forms of cytochrome c, it is essential to identify the variety and extent of post-translational modifications and to separate the preparation into pure monomolecular species. Otherwise, it may become impossible to distinguish between the influence of a site-directed mutation and unexamined post-translational modifications.


Isolation and characterization of pro-barley lectin expressed in Escherichia coli. Lectins are a class of proteins with specific carbohydrate-binding properties found in a wide variety of plants and animals. Gramineae lectins are presumably defense-related proteins in plants that exert their effect by binding to N-acetylglucosamine. Barley lectin is a vacuolar protein synthesized with an amino-terminal signal sequence for entering the secretory pathway and a carboxyl-terminal propeptide necessary for proper targeting to the vacuole. To analyze the three-dimensional structure of barley lectin with the carboxyl-terminal extension and to investigate whether the conversion of the prolectin into the mature molecule leads to a conformational change, the precursor and the mature forms of barley lectin were expressed in Escherichia coli. Both proteins accumulated in denatured form in inclusion bodies were solubilized in 8 M urea and renatured in a redox buffer system. Active pro- and mature barley lectins were purified to homogeneity by affinity chromatography.


Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: recovery and renaturation from bacterial inclusion bodies. A synthetic gene encoding the Group II phospholipase A2 (PLA2) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA2's were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA2. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA2 demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.


Long-term feeding of corn oil, beef tallow, or menhaden oil and eicosanoid levels in BHE/cdb rats. The effects of long-term feeding of 2 or 10% fat diets containing corn oil, beef tallow, or menhaden oil on the levels of eicosanoids in brain, plasma, and kidney medulla were studied. Male BHE/cdb rats, which carry a genetic trait for non-insulin-dependent diabetes mellitus, were fed these diets for 9 mo, at which time their glucose tolerance levels were determined, as were brain, kidney medulla, and plasma levels of PGE2, 6-keto-PGF1 alpha, and LTB4. Glucose tolerance was abnormal in the 2 and 10% corn oil groups and normal in the 10% menhaden oil groups. The glucose tolerance levels of the other groups were only slightly disturbed. The levels of LTB4 in kidney and plasma were not affected by dietary fat type or amount. LTB4 levels were significantly higher in the brains of rats fed a low level of beef tallow than in the brains of rats fed a higher level of beef tallow or a low level of menhaden or corn oil. LTB4 values for the brains of rats fed the other diets were intermediate and not different from the aforementioned values. The levels of 6-keto-PGF1 alpha were highest in rats fed the 10% corn oil diet regardless of the tissue assayed. Rats fed the 2 or 10% beef tallow or menhaden oil were not different but had lower levels of this eicosanoid metabolite than rats fed the corn oil diet. PGE2 levels followed the same pattern, suggesting that the impaired glucose tolerance of the corn oil-fed rats had a strong influence on the tissue levels of these eicosanoids.


Tumour and tumour-like conditions of the soft tissue: magnetic resonance imaging features differentiating benign from malignant masses. 49 primary soft-tissue masses were studied by magnetic resonance imaging (MRI) in an effort to differentiate benign from malignant lesions. There were 24 benign and 25 malignant tumours. Images were evaluated for the presence of low-signal septation, changes in pattern of homogeneity, signal intensity, and margin definition and shape. 20 of the 25 malignant tumours (80%) demonstrated low-signal septation, and two of the 24 benign tumours (8%) also demonstrated this feature. Malignant tumours tended to change pattern from being homogeneous on T1-weighted images to being heterogeneous on T2-weighted images (72%). Benign tumours maintained their pattern on both sequences (67%). Only three of the 24 benign tumours (12.5%) changed from being homogeneous to heterogeneous. Our findings suggest that tumours which change pattern, from homogeneous on T1-weighted images to heterogeneous on T2-weighted images, and tumours which have low-signal septations are likely to be malignant. Moreover benign lesions tend to have well defined margins, and some benign masses have characteristic appearances that aid in their differentiation from malignant processes.


99Tcm-mercapto acetyl triglycine in paediatric renal tract disease. The use of 99Tcm-mercapto acetyl triglycine (99Tcm-MAG3), a new hippuran substitute, has been reported widely in adults but not in children. Our experience of its use in 100 infants and children for renography and indirect micturating cystography (IMC) is reported. The average age was 5.6 years. 65 patients completed IMC studies and nine patients had 99Tcm-dimercaptosuccinic acid (99Tcm-DMSA) scans performed on the same day. The majority of patients were referred for the investigation of urinary tract infection. 32 kidneys were found to be scarred on 99Tcm-MAG3 scans, 17 kidneys and ureters refluxed on IMC and 14 kidneys were obstructed. The results of 99Tcm-MAG3 scans were compared with those of other urinary tract investigations including ultrasound, micturating cystography (MCUG), intravenous urography (IVU) and 99Tcm-DMSA scintigraphy. No kidney which was scarred had a normal 99Tcm-MAG3 scan. All significant degrees of dilatation or obstruction on IVU or ultrasound were also detected by 99Tcm-MAG3. 99Tcm-MAG3 gave more information than any other single imaging modality and we believe it represents an ideal initial screening test in the investigation of urinary tract infection in older toilet-trained children.


Choledochal cyst and congenital anomalies of the pancreatico-biliary junction: the clinical findings, radiology and outcome in nine cases. Nine patients with choledochal cyst have been reviewed. There was a 6:3 preponderance of females. Four patients presented in adulthood. Cholangiocarcinoma developed in three patients with an interval ranging from 2 to 35 years following surgery. The predominant complaint was of abdominal pain and symptoms had been present from weeks up to 28 years. Imaging by sonography may be all that is required in the paediatric age group, but in adults further confirmation of the choledochal cyst and the associated anomalous junction of the pancreatico-biliary duct (AJPB) by direct cholangiography is recommended.


Magnetization transfer contrast (MTC) imaging of skeletal muscle at 0.26 Tesla--changes in signal intensity following exercise. Magnetization transfer contrast (MTC) imaging of skeletal muscle before and after exercise has been studied in normal volunteers at 0.26 Tesla. A saturation pulse was applied over a range of offset frequencies immediately before a gradient recall echo sequence. Substantial signal loss was observed in all muscles. After exercise, selective saturation resulted in a significant increase of contrast between active and less active muscle, a phenomenon attributable to an increase in extracellular water content. MTC imaging provides a more sensitive method to detect changes in water distribution in human skeletal muscle.


Biological intercomparisons of neutron beams used for radiotherapy generated by p(+)-->Be in hospital-based cyclotrons. The new generation of hospital-based neutron therapy facilities involve cyclotrons using protons on beryllium. The spectrum of neutrons produced includes a large and variable proportion of low-energy neutrons that are poorly penetrating but biologically effective. Cells cultured in vitro were used to compare the three US facilities at Seattle, M.D. Anderson and UCLA, together with the UK facility at Clatterbridge. Cyclotrons were compared within a given experiment on the same day using cells from a common suspension. Among the three US facilities, the relative potency factor at a depth of 25 mm differs by about 11%, with Seattle the least and UCLA the most biologically effective. Clatterbridge was compared directly with M.D. Anderson and found to be less effective by about 5%; it has a slightly lower biological effectiveness than any of the US facilities. There is evidence for an increased biological effectiveness in the build-up region, which reduces the effective skin sparing potential. There is not much difference in build-up between the three US facilities. Using the proton-on-beryllium neutron production process results in a wide spectrum of neutrons with a large but variable low-energy component. The biological effectiveness of the beam depends on target design and thickness as well as the design of the collimating system. Consequently the biological effectiveness of neutron beams generated by this process must be assessed on an individual basis. It cannot be assumed that because cyclotrons have similar accelerating energies that the relative biological effectiveness will be the same.


Wilms' tumour in New Zealand 1960-1986.

Wilms' tumour in New Zealand 1960-1986. Between 1960 and 1986, 177 cases of Wilms' tumour were reported to the New Zealand National Cancer Registry. There was an equal sex distribution and 93.2% of patients were in the paediatric age group with a mean age of 3.4 years. The mean symptom interval was 2.0 months and the majority of patients presented with localised disease, although the incidence of advanced disease was greater than that reported from other series. The 5-year survival rate for all cases was 52.5%. Data were divided into patients presenting before and after the publication of the first National Wilms' Tumour Study (NWTS) in the United States in 1976. In patients presenting after 1976 the diagnosis was based on histology in 100%, compared to 58.6% in patients before 1977. Treatment was considered curative in a greater proportion of the post-1976 group. Treatment modalities reflected the recommendations of the NWTS and the 5-year survival rate of this group was 78.3%, this being similar to that reported from the NWTS. Multivariate analysis showed patient age, symptom interval, tumour stage and treatment (pre- and post-NWTS) to be independent predictors of survival.


Subcutaneous mastectomy with immediate reconstruction as treatment for early breast cancer. Subcutaneous mastectomy and axillary dissection combined with immediate reconstruction was used to treat primary breast cancer in a consecutive series of 111 patients. Local recurrence occurred in 19 patients (17 per cent) during a mean follow-up of 30 (range 6-60) months. Complete control of local disease was achieved in all patients except one, who had terminal disease. Prophylactic radiotherapy was avoided for 83 per cent of patients, and good cosmetic results were obtained in 89 per cent. These results support the use of this technique in the treatment of early breast cancer.


Regional variation in the surgical treatment of early breast cancer. A questionnaire survey of consultant surgeons in England was undertaken to establish the preferred surgical management of early breast cancer. The Regional Health Authority in which each surgeon worked was recorded to document regional variation. The 14 Regional Health Authorities were divided into three geographical areas: north, central and south. The main finding was that proportionately more surgeons in the north would recommend mastectomy (P < 0.0001) rather than conservative surgery compared with those in the central and southern areas. The nature of the study does not allow explanations for such variation.


Incidence and prognosis of Krukenberg tumour in Northern Ireland. The case notes of 15 patients with Krukenberg tumours were studied to determine the incidence, presentation, clinical findings and prognosis of the disease in Northern Ireland. Patients tended to be young and premenopausal. A short history of abdominal pain was the most frequent presenting complaint, abdominal swelling being second. Menstrual irregularities occurred infrequently and no patient was virilized. Tumours tended to be large, bilateral and associated with ascites. The stomach was the commonest primary site. The overall prognosis was poor, especially if the primary tumour remained covert at the time of diagnosis. The prognosis was unaffected by subsequent discovery of the primary source; extensive investigation is inconvenient for the patient and wasteful of resources. The incidence of the tumour in Northern Ireland was 0.16 per 100,000 per annum: it will be rarely encountered by general surgeons.


Rectal cancer risk in patients treated for familial adenomatous polyposis. The Leeds Castle Polyposis Group. Total colectomy with ileorectal anastomosis (IRA) in familial adenomatous polyposis (FAP) leaves patients at risk for rectal cancer. To assess this risk, the rectal cancer incidence in 297 patients with FAP undergoing IRA since 1951 was determined in the population-based registers of Denmark, Finland and Sweden. At the same time, detailed data on 50 patients with FAP and invasive rectal cancer were obtained from 11 international polyposis registries. The cumulative incidence of rectal cancer was 13.1 per cent at 25 years. The 5-year survival rate of patients with FAP developing rectal cancer was 71 per cent. Combining both studies, the risk of dying from rectal cancer after IRA was 2.0 per cent at 15 years of follow-up. These results justify IRA as primary treatment for most patients; restorative proctocolectomy is preferred for some subgroups. The high all-cause mortality rate observed in this relatively young population necessitates lifelong surveillance of patients with FAP.


Cholinergic 'blockade' as a model of the cognitive deficits in Alzheimer's disease. The performance of 44 Alzheimer patients and 33 controls was examined on tests previously found to be differentially affected by scopolamine administration. Tests of secondary memory, performance intelligence, primary memory, semantic retrieval, procedural memory and verbal intelligence were included. It was found that Alzheimer patients performed more poorly than controls on tests of secondary memory, as measured by selective reminding, recall and recognition. Procedural memory, as measured by stem completion, homophone spelling and transformed text reading, did not differ between Alzheimer patients and controls. Semantic memory, verbal intelligence and primary memory were impaired in moderate and severe cases. However, patients with 'mild' dementia, as measured by the Mini-Mental State Examination, did not differ from controls on tests of semantic memory, verbal intelligence and primary memory. It was concluded that the pattern of anterograde memory deficits and preserved abilities in mild dementia mimicked that previously observed in scopolamine administration in young subjects.


Activation of locus coeruleus neurons by nucleus paragigantocellularis or noxious sensory stimulation is mediated by intracoerulear excitatory amino acid neurotransmission. The nucleus paragigantocellularis (PGi), located in the rostral ventrolateral medulla, is one of two major afferents to the nucleus locus coeruleus (LC). Electrical stimulation of PGi exerts a robust, predominantly excitatory influence on LC neurons that is blocked by intracerebroventricular (i.c.v.) administration of the broad spectrum excitatory amino acid (EAA) antagonists kynurenic acid (KYN) or gamma-D-glutamylglycine (DGG), but not by the selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoate (AP7). I.c.v. injection of KYN or DGG also blocked activation of LC neurons evoked by noxious somatosensory stimuli. These results indicate that activation of LC neurons by PGi and noxious stimuli may be mediated by an EAA acting at a non-NMDA receptor in LC. In the present study, microiontophoretic techniques were used to determine the sensitivity of LC neurons in vivo to the selective EAA receptor agonists kainate (KA), NMDA and quisqualate (QUIS). Microinfusion and microiontophoresis were also used to determine whether direct application of KYN, the preferential non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX) or the selective NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP5) onto LC neurons blocked excitation elicited by stimulation of PGi or the sciatic nerve. The results demonstrated that individual LC neurons were robustly activated by direct application of KA, NMDA and QUIS. Iontophoretically applied KYN reduced or completely antagonized responses evoked by all 3 agonists. In contrast, iontophoretically applied AP5 strongly attenuated NMDA-evoked excitation, while KA-and QUIS-evoked responses were not affected by this agent. Furthermore, direct application of KYN or the specific non-NMDA receptor antagonist, CNQX, onto LC neurons substantially attenuated or completely blocked synaptic activation produced by PGi or sciatic nerve stimulation in nearly every LC neuron tested. Microinfusion of the selective NMDA receptor antagonist AP5 had no effect on sciatic nerve-evoked responses. These results confirm our hypothesis that activation of LC neurons from PGi is mediated by an EAA operating primarily at a non-NMDA receptor subtype on LC neurons. Furthermore, these findings provide additional support for the hypothesis that this pathway mediates at least some sensory-evoked responses of LC neurons.


Low-voltage-activated calcium channels in human retinoblastoma cells. Retinoblastoma cells represent pluripotent neural-progenitor cells which, if induced to differentiate, express many features of mature human retinal neurons. Ca channel currents were recorded from isolated, undifferentiated human retinoblastoma Y79 cells in a bath solution containing 20 mM BaCl2 using whole-cell patch-clamp pipettes containing CsCl. The transient, macroscopic currents inactivated with a time constant of about 20 ms at -20 mV and had other properties similar to low-voltage-activated calcium channels described in other cell types: Activation curves fit by the Boltzmann relation had a midpoint of -32 mV and a slope factor of 6.8 mV (-80 mV holding potential) and inactivation curves had a midpoint of -40 mV and a slope factor of 3.7 mV. Non-stationary fluctuation analysis of the currents performed over a 2 kHz bandwidth indicated that each channel contributed 0.44 pA to the macroscopic transient current at -20 mV, suggesting a unitary conductance of about 7 pS.


Sexual differences in the stimulatory effects of bombesin on tuberoinfundibular dopaminergic neurons. The purpose of this study was to (1) examine the effects of central administration of bombesin on the activity of tuberoinfundibular dopaminergic (DA) neurons in male and female rats, and (2) determine if sexual differences in the responsiveness of these neurons to bombesin were due to the presence of prolactin or gonadal steroids. The activity of tuberoinfundibular DA neurons was estimated by measuring the concentrations of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in terminals of these neurons in the median eminence. The effects of bombesin on the secretion of prolactin and alpha-melanocyte stimulating hormone (alpha MSH), and the activities of nigrostriatal, mesolimbic, and periventricular-hypophysial DA neurons were also determined in gonadally intact male and female rats. Central administration of bombesin (10 ng/rat; i.c.v.) decreased prolactin secretion in gonadally intact male and female rats, but only in males was this associated with an increase in the activity of tuberoinfundibular DA neurons. In contrast, bombesin increased the activity of periventricular-hypophysial DA neurons terminating in the intermediate lobe of both male and female rats, and this was associated with a decrease in alpha MSH secretion in both sexes. Bombesin had no effect on the activities of nigrostriatal, mesolimbic, or periventricular-hypophysial DA neurons terminating in the neural lobe in either sex. The loss of endogenous gonadal hormones following ovariectomy rendered tuberoinfundibular DA neurons responsive to the stimulatory effects of bombesin, whereas immunoneutralization of endogenous prolactin following administration of prolactin antiserum had no effect on the inability of bombesin to alter the activity of these neurons in female rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Hyperemia of injured peripheral nerve: sensitivity to CGRP antagonism. Intrinsic mechanisms of vasodilatation may prevent injury-related ischemia in peripheral nerve endoneurium. We examined local perfusion up to 10 days following local crush, partial injury or simple exposure of the rat sciatic nerve. By employing epineurial hCGRP(8-37), a receptor antagonist of CGRP, and serial hydrogen clearance measurements, we estimated the component of post-injury blood flow related to local CGRP action. Injury-related ischemia was not observed at any of the time points studied at or proximal to injury. Instead, endoneurial blood flow (EBF) increased at 24 h proximal to crush or partial injury, and at 48 h within the crush zone when compared to sham operated controls or to a pooled reference range of EBF. Composite blood flow (F) was also elevated at 48 h and 5 days within the crush zone suggesting hyperemia involving the epineurial plexus, perineurial vessels and AV shunts. hCGRP(8-37) constricted vasa nervorum at most time points but its effect on EBF was maximum and exceeded controls within the crush zone at 48 h. The findings indicate that certain types of nerve injury, including focal crush, are associated with hyperemia, not ischemia. CGRP vasodilatation may account for part of this response, implying a local peptidergic afferent fiber response to nerve trunk injury.


Chromosome abnormalities and oncogenesis in cat leukemias. Chromosome abnormalities are found in feline leukemia virus (FeLV)-infected tumor cells as well as in tumor cells free of the virus. Three cell lines derived from tumors in the domestic cat (Felis catus), two of thymic origin and one of multicentric lymphoma origin, were analyzed cytogenetically to determine whether the FeLV virus was associated with chromosomal abnormalities in these tumor cell lines. One thymic tumor and the multicentric lymphoma were FeLV infected. The other thymic tumor cell line was FeLV-free. The normal diploid number in the domestic cat is 38. All three cell lines had numerical chromosome abnormalities with modal numbers of 37, 38 (pseudodiploid), and 39, respectively and had consistent structural chromosome abnormalities. Three markers in the virus-free cell line (S markers) were shared with one or the other of the virus-positive cell lines. The two FeLV-positive cell lines did not have S markers in common. The finding of chromosome abnormalities in both the virus-infected and the virus-free cell lines suggests that these abnormalities may be important in oncogenesis. The FeLV virus could not be considered the only causative agent of the abnormalities observed.


A TP53 mutation detected in cells established from an osteosarcoma, but not in the retinoblastoma of a patient with bilateral retinoblastoma and multiple primary osteosarcomas. A patient with bilateral retinoblastoma and subsequent multiple primary osteosarcomas has been described previously. Osteosarcoma cell lines established from this patient were shown to express a shortened RB1 mRNA transcript and no detectable normal Rb protein. We now show that the osteosarcoma cell lines have lost one TP53 allele and contain a mutation in exon 8 codon 286 [GAA to AAA (Glu to Lys)] in the remaining allele. Consequently, the osteosarcoma cell lines have no normal Rb protein and no normal p53 protein. Neither constitutional DNA nor DNA extracted from a retinoblastoma of the left eye of the patient contained the TP53 mutation, suggesting that the TP53 mutation in the osteosarcoma cells may represent a tumor-promoting mutation, which confers a selective growth advantage. If both RB1 and TP53 are involved in the initiation of osteosarcoma, the mechanisms for development of the retinoblastoma and osteosarcoma tumors are different.


[The effect of alpha 1-adrenergic stimulation of the myocardium depends on the opposite actions of 2 receptor subtypes]. Two different myocardial alpha 1-adrenergic receptor subtypes have been recently described. Since their functional role is still unknown we examined the effect of alpha 1A and alpha 1B on contractile properties, cell Ca2+ homeostasis and myofilament sensitivity to Ca2+ in indo-1 loaded ventricular myocytes. Non selective alpha 1-adrenergic stimulation increases twitch amplitude, systolic Ca2+ and myofilament sensitivity. Same effects but amplified are associated with alpha 1A-receptors stimulation. In contrast, alpha 1B-receptors stimulation decreases twitch amplitude and systolic Ca2+ below control value. Myocardial alpha 1-adrenergic stimulation results from the opposite actions of the 2 subtypes.


[The arrhythmogenicity of alpha 1-adrenergic stimulation following myocardial acidosis]. Ischemia is associated with myocardial acidosis which recovers upon reperfusion. In such conditions, alpha 1-adrenergic stimulation is arrhythmogenic. We used single cardiac myocytes loaded with the pH fluorescent dye, SNARF-1, to determine if a modulation of pH could explain the effect of alpha 1-adrenergic stimulation. Cells were exposed to acidosis (CO2 15%) for 15 min and then normocapnia restored. During acidosis, alpha 1-adrenergic stimulation caused an increase in pH which was abolished by blocking Na+/H+ exchange with ethylisopropylamiloride (EIPA). After removal of acidosis aftercontractions were manifest in 8 out of 10 and 1 out of 5 cells in the presence of an alpha 1-adrenergic agonist and in control, respectively (p < 0.001). EIPA abolished the occurrence of after contractions. Thus, the arrhythmogenicity of alpha 1-adrenergic stimulation depends on activation of Na+/H+ exchanger.


Increase in functional activity rather than in amount of Gi-alpha in failing human heart with dilated cardiomyopathy. The aim was to investigate whether or not increased pertussis toxin catalysed ADP ribosylation correlates with increased amount of Gi-alpha in failing human heart. Antisera raised against unique synthetic peptides corresponding to alpha subunits of Gs and Gi 1-3 were used in immunoblotting and ELISA to determine amounts of various G proteins. Adenylyl cyclase activity, beta adrenoceptors, and muscarinic receptors were then measured in cardiomyopathic hearts (n = 6) obtained at transplant in order to study whether or not an altered expression of G proteins has relevance to the integrity and function of the receptor--adenylyl cyclase system. Six non-failing control hearts were also studied. No significant differences in the peptide equivalent amounts of either Gs or Gi were found in the failing human heart as compared to the non-failing heart. However, functional activity of Gi was shown to increase significantly since there was a decrease in basal (57%), isoprenaline stimulated (60%), and guanyliminodiphosphate stimulated (52%) adenylyl cyclase activity. In contrast the density of beta adrenoceptors was markedly decreased (51%) in failing human heart in comparison to non-failing hearts. Neither the density nor the affinity of muscarinic receptors changed in the failing human heart. These results suggest that in the failing human heart, there is an increase in functional activity rather than in amount of Gi, and an important part of functional expression of Gi-alpha may be regulated at the post-translational level.


Effects of polyethylene glycol conjugated superoxide dismutase on coxsackievirus B3 myocarditis in mice. The aim was to examine the role of oxygen derived free radicals in the development of myocarditis by investigating the effects of polyethylene glycol conjugated superoxide dismutase (PEG-SOD), a potent scavenger of oxygen free radicals, upon coxsackievirus B3 (CB3) myocarditis. Two week old male C3H/He mice were inoculated intraperitoneally with 10(3) plaque forming units of CB3. PEG-SOD, 1 x 10(3), 5 x 10(3), 1 x 10(4), and 1 x 10(5) U.kg-1 x d-1, was given subcutaneously daily on days 0 to 14. Treated groups were compared to the infected control. The survival rate of the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group was lower than that of the infected control group (40% v 78%, p < 0.01). The survival rates of the other treated groups did not differ significantly from the infected control. The myocardial calcification score in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group was higher than in the infected control group on d 7, when myocardial virus titres did not differ significantly among the five groups. The scores for myocardial cellular infiltration and myocardial necrosis in the 1 x 10(3) and the 5 x 10(3) U.kg-1 x d-1 PEG-SOD groups were significantly lower than in the infected control on d 14, when myocardial viruses were not detected in the five groups. However, the myocardial necrosis and myocardial calcification scores in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group were higher than in the infected control. The improvement of cardiac pathology in the 1 x 10(3) and the 5 x 10(3) U.kg-1 x d-1 PEG-SOD groups seems to have resulted not from the reduction in myocardial virus titres but from inhibition of generation of oxygen free radicals. The mechanism of the impaired survival and aggravation of cardiac pathology in the 1 x 10(5) U.kg-1 x d-1 PEG-SOD group is unknown. The results suggest that oxygen free radicals may be involved in the pathogenesis and development of CB3 myocarditis and that appropriate dosages of PEG-SOD have therapeutic potential for clinical CB3 myocarditis, although caution must be paid to the treatment window.


Structural and enzymatic studies on the plasma membrane domains and sodium pump enzymes of absorptive epithelial cells in the avian lower intestine. The coprodaeum of the domestic hen maintained on a low-NaCl diet adapts by enhanced sodium transport. This study examines the adaptive response at the single cell and whole organ levels. Surface areas of apical (microvillous) and basolateral plasma membranes of columnar absorptive epithelial cells were estimated by use of ultrastructural stereology. The activities of succinic dehydrogenase (a mitochondrial enzyme) and ouabain-sensitive, potassium-dependent paranitrophenyl phosphatase (a sodium pump enzyme) were determined in tissue homogenates. Sodium, potassium-ATPase (pump enzyme) activity in cell membranes was localized by ultrastructural cytochemistry. Apical and basolateral membranes responded differently. In high-NaCl hens, the membrane signature of the average cell was 32 microns 2 (apical), 932 microns 2 (lateral) and 17 microns 2 (basal). Cells from low-NaCl hens had more apical membrane (49 microns 2 per cell) but essentially the same area of basolateral membrane. However, total surfaces per organ were greater for all membranes. Sodium pump enzymes were localized in basolateral membranes. Enzyme activities per unit mitochondrial volume and per unit basolateral membrane surface were higher in low-NaCl birds. These findings are discussed in the context of known mechanisms of transcellular sodium transport via apical ion channels and basolateral pumps.


[Acute hemorrhagic viral conjunctivitis]. Two cases of acute hemorrhagic conjunctivitis are described, in which the enterovirus Coxsackie 24 was found by serological examination to be the etiological agent. The virus was important from Nigeria. The patients suffered by the acute hemorrhagic keratoconjuntivitis with transient iritic irritation without the systemic symptoms. Since now this disease with serological verification was not diagnosed in our country. The question of the viral hemorrhagic conjunctivitis and their treatment is discussed. The necessity of virological investigation in inflammations of the anterior segment is stressed.


Location and dynamics of alpha-tocopherol in model phospholipid membranes with different charges. Studies were made on the position and dynamics of the OH-group of alpha-tocopherol in phospholipid membranes. There was no difference in the spin-lattice (T1) relaxation times at the 5a-position of alpha-tocopherol labeled with 13C- or C19F3-determined from the nuclear magnetic resonance (NMR) spectra of liposomes positively charged with stearylamine (SA) and negatively charged with dicetylphosphate (DCP). The zeta-potentials of egg yolk phosphatidylcholine (EYPC) liposomes with and without SA or DCP were not affected by incorporation of 20 mol% alpha-tocopherol, though incorporation of 10 mol% ascorbyl-palmitate decreased the zeta-potentials of EYPC and EYPC-SA liposomes. The P==O stretching band (1235 cm-1) of the phosphate group and C==O stretching band (1734 cm-1) of the acyl ester linkage in dimyristoylphosphatidylcholine (DMPC) liposomes, measured by Fourier transform-infrared (FT-IR) spectroscopy, were not changed by incorporation of alpha-tocopherol. These results suggest that no specific interaction occurred between the OH-group of alpha-tocopherol and the polar interfacial region of the bilayer. The dynamic quenching effects of n-(N-oxy-4,4'-dimethyloxazolidine-2-yl)stearic acids (n-NSs) on the intrinsic fluorescence of alpha-tocopherol were in the order 5-NS > 7-NS = 12-NS > 16-NS. Acrylamide, a water-soluble fluorescence quencher with a very low capacity to penetrate through phospholipid bilayers, had very low quenching efficiency. These results indicate that the bulk of the chromanol moiety of alpha-tocopherol is located in a position close to that occupied by the nitroxide group of 5-NS in the membranes and is poorly exposed at the membrane surface.(ABSTRACT TRUNCATED AT 250 WORDS)


Human papillomavirus in cervix carcinoma and condylomata acuminata--identification of HPV-DNA by improved dot-blot-hybridization. Using the benzoylated naphthoylated DEAE cellulose method (BND-method) we have designed a more efficient approach for the detection of human papillomavirus-DNA (HPV-DNA) via dot-blot and hybridization. Biopsy material from anogenital warts (40 patients), invasive carcinoma uteri (12 patients) and normal controls (20 patients) were studied for the presence of HPV-DNA. Phenol extracted DNA from representative lesions was loaded onto a pretreated nitrocellulose filter, was incubated under stringent conditions with 32-P-dCTP labelled HPV-DNA and exposed to a Kodak X-OMAT film. DNA of HPV types 11, 16, 18 were cloned into plasmid vectors. The common, time-consuming caesium-chloride density-gradient centrifugation used for purification of plasmid DNA (20-36 h), was substituted by the BND-method (15 min). Complete HPV genomes were excised using the restriction endonucleases Eco RI and Bam HI. The HPV-DNA fragments obtained were then electroeluted using the 'Bio-Trap' method and subsequently labelled with 32P-dCTP by nick translation. Without resorting to more complex and sensitive technology, such as the polymerase chain reaction, efficiency of specific analysis of large numbers of cervical samples and condylomata was achieved without loss of accuracy or increased costs. The time required for HPV identification from biopsy or sample receipt was shortened considerably (approximately 50%).


The inter-relation between inflammation and epidermal proliferation in normal skin following epicutaneous application of leukotriene-B4--an immunohistochemical study. Topical application of leukotriene-B4 (LTB4) on normal skin has been used as an in-vivo model to investigate cutaneous inflammation and epidermal proliferation, which are important phenomena in the pathogenesis of psoriasis. The aim of the present investigation is to further elucidate the interrelation between inflammation and epidermal proliferation, using specific monoclonal antibodies as markers for the different cell types involved. Aliquots of LTB4 were applied on the upperarms of eight healthy volunteers. After LTB4-application, biopsies were taken at consecutive time intervals. On frozen sections, epidermal proliferation was assessed by Ks8.12-(keratin 16) and Ki-67-binding (cycling cells), inflammation was characterized using anti-elastase (PMN), T11 (T-lymphocytes), pan-B (B-lymphocytes), WT 14 (CD14-positive cells) and OKT 6 (Langerhans cells). New observations were that the density of CD14-positive cells was increased even at 8 h and decreased slightly at 72 h. A striking rearrangement of Langerhans cells was seen in close vicinity to intra-epidermal accumulations of PMN. Remarkably an increased density of these cells in the dermis at 72 h was seen and a decrease in the epidermis. In line with previous studies, the accumulation of PMN reached a maximum 24 h after LTB4-challenge. The identity of the mononuclear infiltrate cells which have been reported 48-72 h after LTB4 proved to be T-lymphocytes. No B-lymphocytes were observed. Ki-67-positive nuclei were maximally increased 72 h after LTB4-application, which implies that recruitment of cycling cells is of relevance for the LTB4-induced proliferation in vivo. The hyperproliferation-related keratin 16 was expressed inconsistently in the suprabasal compartment.(ABSTRACT TRUNCATED AT 250 WORDS)


[Subdivision of Ia fibers of de-efferented cat muscle spindles into two groups]. The discharge frequency of primary muscle spindle afferents of the tibial anterior muscle of the cat was determined under a ramp-and-hold stretch of the host muscle. The rate of ramp stretch was increased stepwise from 1 to 100 mm/s. Its amplitude was kept constant and was 7 mm. The initial length of the host muscle was enhanced in steps of 3 mm to reach a maximal augmentation of the muscle length by 12 mm. At each discharge pattern obtained under a rate of ramp stretch of 10 mm/s the slow receptor adaptation was established. The slow receptor adaptation is the difference between the discharge frequency occurring at the beginning of static phase of a ramp-and-hold stretch, the static maximum, and at the end of the third second of static phase, the static value (Fig. 1). The amplitude of the slow receptor adaptation was plotted against the amplitude of static maximum (Fig. 2). The diagram showed two groups of Ia fibres. One of the two groups had a slow receptor adaptation of high amplitudes. Its primary afferents will be called bag1 Ia fibres. The other group had a slow receptor adaptation of low amplitudes. Its primary afferents will be named bag2-chain Ia fibres. Furthermore, the dynamic response and the static value were determined at the discharge patterns of the two groups of spindle fibres. The dynamic response of the bag1 Ia fibres was with p < 0.0001 significantly higher in its mean amplitude than that one of the bag2-chain Ia fibres (Fig. 4a).(ABSTRACT TRUNCATED AT 250 WORDS)