Sequence of the gene encoding flavocytochrome c from Shewanella putrefaciens: a tetraheme flavoenzyme that is a soluble fumarate reductase related to the membrane-bound enzymes from other bacteria. Flavocytochrome c from the Gram-negative, food-spoiling bacterium Shewanella putrefaciens is a soluble, periplasmic fumarate reductase. We have isolated the gene encoding flavocytochrome c and determined the complete DNA sequence. The predicted amino acid sequence indicates that flavocytochrome c is synthesized with an N-terminal secretory signal sequence of 25 amino acid residues. The mature protein contains 571 amino acid residues and consists of an N-terminal cytochrome domain, of about 117 residues, with four heme attachment sites typical of c-type cytochromes and a C-terminal flavoprotein domain of about 454 residues that is clearly related to the flavoprotein subunits of fumarate reductases and succinate dehydrogenases from bacterial and other sources. A second reading frame that may be cotranscribed with the flavocytochrome c gene exhibits some similarity with the 13-kDa membrane anchor subunit of Escherichia coli fumarate reductase. The sequence of the flavoprotein domain demonstrates an even closer relationship with the product of the yeast OSM1 gene, mutations in which result in sensitivity to high osmolarity. These findings are discussed in relation to the function of flavocytochrome c.


Kinetics of Bacillus cereus phosphatidylinositol-specific phospholipase C with thiophosphate and fluorescent analogs of phosphatidylinositol. Thiophosphate analogs (C-S-P bond) of phosphatidylinositol (Cn-thio-PI: racemic hexadecyl-, dodecyl-, and octylthiophosphoryl-1-myo-inositol) and a fluorescent analog (pyrene-PI: rac-4-(1-pyreno)-butylphosphoryl-1-myo-inositol) were all substrates for phosphatidylinositol-specific phospholipase C from Bacillus cereus. Hydrolysis of thio-PI was followed by coupling the production of alkylthiol to a disulfide interchange reaction with dithiobispyridine. Hydrolysis of pyrene-PI was followed using a HPLC-based assay with fluorescence detection. The activity of PI-PLC with thio-PI analogs showed an interfacial effect. C16-Thio-PI, which had a critical micelle concentration (CMC) of 7 microM, gave a hyperbolic activity versus concentration curve between 0 and 2 mM, while C8-thio-PI, which had a CMC above 10 mM, showed very low activity which increased greatly upon introduction of an interface in mixed micelles with hexadecylphosphocholine (HDPC). Pyrene-PI, which aggregates above 0.3 mM, gave a sigmoidal activity curve with much higher activity above the CMC. All three thio-PI homologs as mixed micelles with HDPC gave hyperbolic activity curves with PI-PLC that were a function of bulk concentration of substrate at constant surface concentration and surface concentration of substrate at constant bulk concentration. The maximal activity of PI-PLC with pure C16-thio-PI micelles was 6.25 mumol min-1 mg-1, while that with pyrene-PI was estimated to be 68 mumol min-1 mg-1. With pure C16-thio-PI micelles, 0.022 mM substrate gave half Vmax, similar to that in mixed micelles with HDPC.


Gene synthesis, bacterial expression, and 1H NMR spectroscopic studies of the rat outer mitochondrial membrane cytochrome b5. The gene coding for the water-soluble domain of the outer mitochondrial membrane cytochrome b5 (OM cytochrome b5) from rat liver has been synthetized and expressed in Escherichia coli. The DNA sequence was obtained by back-translating the known amino acid sequence [Lederer, F., Ghrir, R., Guiard, B., Cortial, S., & Ito, A. (1983) Eur. J. Biochem. 132, 95-102]. The recombinant OM cytochrome b5 was characterized by UV-visible, EPR, and 1H NMR spectroscopy. The UV-visible and EPR spectra of the OM cytochrome b5 are almost identical to the ones obtained from the overexpressed rat microsomal cytochrome b5 [Bodman, S. B. V., Schyler, M. A., Jollie, D. R., & Sligar, S. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 9443-9447]. The one-dimensional 1H NMR spectrum of the OM cytochrome b5 indicates that the rhombic perturbation of the ferric center is essentially identical to that in the microsomal beef, rabbit, chicken, and rat cytochromes b5. Two-dimensional 1H NMR spectroscopy (NOESY) and one-dimensional NOE difference spectroscopy were used to assign the contact-shifted resonances that correspond to each of the two isomers that result from the rotation of the heme around its alpha-gamma-meso axis. The assignment of the resonances allowed the determination of the heme orientation ratio in the OM cytochrome b5, which was found to be 1.0 +/- 0.1. It is noteworthy that the two cytochromes b5 that have similar populations of the two heme isomers (large heme disorder) originate from the rat liver.


Chemical modification of Torpedo acetylcholinesterase by disulfides: appearance of a "molten globule" state. Modification of Torpedo californica acetylcholinesterase (AChE) both by bis(1-oxy-2,2,5,5-tetramethyl-3-imidazolin-4-yl)disulfide (biradical) and by 4,4'-dithiopyridine, via a thiol-disulfide exchange reaction, was monitored by EPR and optical spectroscopy, respectively. Incubation with these reagents caused complete loss of enzymic activity. Treatment with glutathione of AChE modified by either of the two disulfides led to rapid release of the bound reagent with simultaneous regeneration of the single free thiol group of the enzyme. However, no concomitant recovery of catalytic activity was observed. SDS-PAGE showed that both the modified and demodified enzymes retained their structure as a disulfide-linked dimer. Circular dichroism revealed that modification of AChE by the disulfide agents with or without demodification by glutathione led to a complete disappearance of the ellipticity in the near-UV and to a much smaller decrease in ellipticity in the far-UV. The CD spectra observed are typical of the "molten globule" state of proteins. 1-Anilino-8-naphthalenesulfonate binding measurements and an enhanced susceptibility to trypsinolysis supported the supposition that chemical modification had transformed native AChE to a "molten globule".


Simultaneous measurements of the intra- and extra-cellular oxygen concentration in viable cells. An EPR method that can measure the intra- and extra-cellular oxygen concentration [O2] simultaneously in vitro has been developed using specially designed nitroxides. In the presence of Fe(CN)6(3-) in the medium, intracellular [O2] is measured by a neutral 15N-nitroxide and extracellular [O2] is measured by a negatively charged 14N-nitroxide, since charged species do not enter cells and the EPR spectrum of a 15N-nitroxide does not overlap with that of a 14N-nitroxide. The method is based in part on the minimal broadening of negatively charged nitroxides by Fe(CN)6(3-) and the very effective broadening of neutral nitroxides by the same paramagnetic ions. Results with this method confirm the existence of gradients in [O2] between the extracellular and intracellular compartments in CHO cells and M5076 tumor cells, even without stimulation of cellular respiration by CCCP. The nature of the barrier that needs to be involved to account for the experimental results raises some significant questions.


Reactivation of rat cytomegalovirus in lung allografts: an experimental and immunohistochemical study in rats. Reactivation of latent rat cytomegalovirus (RCMV) from a lung allograft or from a recipient was studied in RCMV-mismatched combinations (donor [D]-/recipient [R]+, D+/R-, and D+/R+) with latently infected lung grafts and chronically infected rats in an inbred rat model. Nineteen transplants in a major histocompatibility complex different strain combination (Brown-Norway/Lewis) were immunosuppressed daily (cyclosporine, azathioprine, and prednisolone) from day 3 after orthotopic left lung transplantation and killed on days 3, 6, and 21. Control groups consisted of nine chronically RCMV-infected rats with immunosuppression without transplantation and six allografts with immunosuppression without RCMV infection. Reactivation of latent RCMV was tested by immunohistochemical staining with monoclonal antibodies against RCMV-induced antigens and by plaque assays of the virus in the salivary glands. The following results were obtained: (1) All allotransplants developed acute ongoing rejection on days 3 and 6, and the rejection was resolved on day 21 by immunosuppression. (2) Reactivation was observed in allotransplanted groups, but not in the control rats. (3) In the D+/R+ and D-/R+ groups on days 3 and 6, the number of RCMV-related antigen-positive cells increased in the recipient spleen and lymph nodes and in the bronchus-associated lymphoid tissue of the donor lung in the D-/R+ group, but not in the chronically RCMV-infected controls. (4) In the D+/R- group on day 6, RCMV-induced antigen-positive cells were observed in the spleen and lymph nodes of the recipient and also around the vessels in the recipient lung. (5) In the D-/R+ group, vascular endothelial cells or mildly infiltrated mononuclear cell subpopulations around the vessels in the lung allograft showed weakly positive staining against RCMV-related antigens on day 6. (6) After the initial acute rejection on days 3 and 6 was treated by immunosuppressive drugs, reactivated acute RCMV infection became chronic or latent again on day 21. We conclude that RCMV infection could be transferred with latently infected lung allografts by reactivation of latent RCMV. In rats, as in man, alloimmune responses seemed to have a definite influence on the reactivation of latent RCMV after lung transplantation.


Vasopressin elevation of Na+/H+ exchange is inhibited by genistein in human blood platelets. The regulation of intracellular Na+ and pHi in human blood platelets is known to be controlled by the function of the Na+/H+ exchanger. The phosphorylation state of the Na+/H+ exchanger which determines the exchanger activity in human blood platelets is regulated by the activities of protein kinases and protein phosphatases. Observations in this study indicate that arginine vasopressin (AVP) that interacts with a V1 receptor, activates the Na+/H+ exchange in human blood platelets through a genistein-inhibited mechanism. The AVP-activated Na+/H+ exchange is probably not regulated by protein kinase C (PKC), since this activation is not inhibited by staurosporine. The multiple ways in which platelet Na+/H+ exchange can be modulated may indicate the critical role played by this exchanger in the homeostasis control of pHi in human blood platelets.


Analysis of thiol-topography in Na,K-ATPase using labelling with different maleimide nitroxide derivatives. Spin-label EPR spectroscopy of shark rectal gland Na,K-ATPase modified at cysteine residues with a variety of maleimide-nitroxide derivatives is used to characterize the different classes of sulphydryl groups. The spin-labelled derivatives vary with respect to charge and lipophilicity, and the chemical reactivity towards modification and inactivation of the Na,K-ATPase is dependent on these properties. Ascorbate is used to reduce the spin-labels in situ, and the kinetics of reduction of the protein-bound spin-labels are found also to depend on the nature of the maleimide-nitroxide derivative. The Na,K-ATPase is labelled either at Class I groups (with retention of enzymatic activity) or at Class II groups (where the enzymatic activity is lost). Although Class I groups are labelled more readily than are Class II groups they are only slightly more susceptible to reduction by ascorbate than the Class II groups, indicating no major difference in environment. The spectral difference observed between immobilized and mobile spin-labels with both Class I and Class II groups labelling is not reflected in widely different reduction kinetics for these two spectral components. Solubilization of the enzyme in an active form does not change the protein structure in terms of increased accessibility of the SH-groups to reduction by ascorbate. The results are discussed in terms of the location of the different SH-groups and the origins of the differences in mobility evident in the EPR spectra of the spin-labelled SH-groups.


Serial changes in myocardial beta-adrenergic receptor after experimental brain death in dogs. Early donor heart failure is one of the serious problems in heart transplantation. This heart failure may be related to myocardial change during brain death before implantation of the heart. Actually, a gradual increment in catecholamine requirement to maintain hemodynamics is not infrequently seen in patients with brain death. This may presumably be related to alterations in the myocardial beta-adrenergic receptor system. In this experimental study, we investigated the changes in myocardial beta-adrenergic receptors after brain death induced by intracranial hypertension in dogs. To maintain the mean arterial pressure higher than 60 mm Hg after brain death, massive crystalloid infusion (group A) or 0.4 to 1.3 micrograms/kg/min of adrenaline infusion (group B) was applied. Blood adrenaline levels and myocardial beta-adrenergic receptor (receptor number and affinity with [125I]iodocyanopindolol binding) were measured at 3 hours (group A-3H or group B-3H, n = 5, respectively) and 6 hours (group A-6H or group B-6H, n = 5, respectively) after brain death. Blood adrenaline levels reduced in group A-6H and elevated in group B-3H and group B-6H. Beta-adrenergic receptor affinity was not different among each group. Beta-adrenergic receptor number decreased in group B-6H. Myocardial beta-adrenergic receptor density decreased in the adrenaline group during 6 hours of hemodynamic maintenance after brain death, whereas myocardial beta-adrenergic receptors did not change in the crystalloid group. Our data suggest that the large doses of catecholamines used to maintain hemodynamics in donors with brain death may depress myocardial beta-adrenergic receptors.


Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. In quiescent Balb-c mouse 3T3 fibroblasts, the application of whole or dialyzed 10% foetal calf serum elicits a biphasic electrical response, consisting of a transient outward current, flowing through Ca(2+)-activated K+ channels, followed by an inward one, lasting up to 15 min. On the basis of experiments with ion substitutions and blockers, the inward current can be attributed to the opening of cationic channels permeable to Na+ and Ca2+ ions. This current could mediate the calcium influx involved in the sustained elevation of [Ca2+]i that has been observed in many preparations in response to mitogen stimulation and that is involved in triggering cell proliferation.


Development of myocardial beta-adrenergic receptor density and adenylate cyclase activity after human heart transplantation. An increase in basal heart rate caused by a lack of vagal control and chronotropic supersensitivity to epinephrine has been shown in transplanted human hearts. Prejunctional and/or postjunctional origins for this supersensitivity have been suggested, the latter involving changes in the number of myocardial beta-adrenergic receptors or in the receptor adenylate cyclase system. To directly determine the time course of change, serial determinations were performed during the first 3 months after heart transplantation. The beta-adrenergic receptor density measured by iodine 125-labelled iodocyanopindolol binding in 61 endomyocardial biopsy specimens (a mean of 6.1 +/- 0.58 biopsies from each of 10 patients) showed great intraindividual and interindividual variability (56.6 +/- 6.8 fmol/mg protein) with no mean trend toward gradually changing receptor densities. Isoproterenol-stimulated adenylate cyclase activity measured in 33 biopsy specimens (a mean of 5.5 +/- 0.67 biopsy specimens from each of six patients) varied considerably (112.5 +/- 13.8 pmol cyclic adenosine monophosphate/mg protein/min), again with no definite tendency with regard to the development over time. The beta-adrenergic receptor densities showed no statistical correlation with the degree of rejection as assessed by histologic criteria and antimyosin ration. These results suggest that in the first 3 months after heart transplantation beta-adrenergic receptor density and adenylate cyclase responses to 10 mumol/L isoproterenol do not change and that beta-adrenergic receptor density in the transplanted myocardium does not seem to be affected by the degree of rejection.


Determination of the sidedness of the C-terminal region of the gastric H,K-ATPase alpha subunit. It cannot be predicted from hydropathy analysis whether the C-terminal end of the alpha subunit of the gastric H,K-ATPase is cytoplasmic or extracytoplasmic. The sideness of the C-terminal amino acids was determined by taking advantage of the two C-terminal tyrosines in the primary sequence of the enzyme. Intact, cytoplasmic side out vesicles derived from hog gastric mucosa or detergent solubilized vesicles were iodinated by the lactoperoxidase method and then the C-terminal amino acids hydrolyzed by carboxypeptidase Y. The alpha and beta subunits were separated by SDS gel electrophoresis. The level of iodination of the alpha subunit following solubilization was about three fold greater than when intact vesicles were iodinated, and the beta subunit was iodinated only when solubilized enzyme was used. Carboxypeptidase Y removed 28 +/- 4% of the radioactivity from the alpha subunit iodinated in intact vesicles. These data are consistent with a cytoplasmic location of the C-terminal amino acids of the alpha subunit and with a mostly extracytoplasmic location of the amino acids of the beta subunit.


Thermodilution measures of right ventricular ejection fraction and volumes in heart transplant recipients: a comparison with radionuclide angiography. A reliable, convenient measure of right ventricular ejection fraction may be a useful adjunct to evaluate cardiac allograft rejection. The purpose of this investigation was to compare two measures of right ventricular ejection fraction: (1) radionuclide angiography with the first-pass technique and (2) thermodilution with a balloon flotation catheter. The study was performed in 26 heart transplant recipients; hemodynamics, thermodilution cardiac output, and right ventricular ejection fraction were measured. First pass radionuclide angiography was performed either simultaneously (n = 11) or within 4 hours (n = 15) of the thermodilution study. Mean thermodilution right ventricular ejection fraction was 39% +/- 8%, and radionuclide angiography ejection fraction was 47% +/- 9%, which represents a highly significant difference (p < 0.001) in techniques. Linear regression showed no correlation between the two techniques (r = 0.3; p = NS). No differences in results were observed in those studied simultaneously versus less than 4 hours. We conclude that the thermodilution technique underestimates right ventricular ejection fraction in heart transplant recipients and that its usefulness as a tool to screen for systolic dysfunction related to rejection is limited.


Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana. Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana have been studied at the single-channel level using the patch-clamp technique. The Ca2+ channel in the plasma membrane opened for extracellular Ca2+ influx. The Ca2+ channel in the vacuolar membrane opened for cytoplasmic Ca2+ influx.


Glycogen synthase kinase-3: functions in oncogenesis and development. Study of GSK-3 had an inauspicious beginning rooted in intermediary metabolism. However, owing to the fortuitous convergence of several disparate areas of biology, the enzyme now offers unique opportunities for study of the control of a variety cellular processes. While at first sight a role in transcriptional regulation appears unlikely for a protein first identified as acting on glycogen synthase, it is even more surprising that the same protein should be functionally interchangeable with a fruit fly homeotic gene. Such understandable scepticism, however, is based on teleological bias. Glycogen synthase is a critical enzyme regulating glucose storage. The c-Jun oncoprotein may have the potential to transform cells but this does not excuse it from similar mechanisms of control to glycogen synthase. Likewise, homeotic genes play a crucial role in setting up the body plan of an embryo but must also be subject to control. The main difference is that when such control is lost, the result is rather graphic. It is, therefore, only to be expected that regulatory protein kinases will surface in superficially quite unrelated areas and that many of their targets will be 'housekeeping' proteins. Perhaps the most difficult aspect of protein phosphorylation research is the linking of physiological substrates with particular protein kinases, hence reconstructing pathways. No matter how compelling in vitro data appear, there must be demonstration that the protein is targeted by the specific protein kinase in cells, an extremely difficult process. Most progress in this respect has been made using genetic analysis in lower organisms, especially yeast. Here another problem arises: demonstration of biochemical linkages underlying genetic interactions which requires function to be ascribed to genes identified by a gross effect. The challenge is to co-ordinate these two approaches, a strategy currently being employed to further unravel the biological role of GSK-3.


[MR tomography of cystosarcoma phylloides. A case report]. This is the first time a report is being published in medical literature on the MR imaging of a cystosarcoma phylloides tumour after intravenous contrast agent administration. MR tomography enabled complete visualisation of the tumour even in the region close to the thoracic wall, as well as clear delineation against the non-tumorous glandular tissue. This means that the limits along which complete extirpation of the tumour could be performed, were determined accurately before the operation, such complete removal being imperative to avoid tumour recurrence.


Pathogenesis of human immunodeficiency virus-related Kaposi's sarcoma. Infection with the human immunodeficiency virus-1 is associated with a marked increase in the incidence of Kaposi's sarcoma. Recent studies suggest that the risk of Kaposi's sarcoma in human immunodeficiency virus infection is increased with oral-fecal contact and that a sexually transmitted agent possibly related to human papillomavirus-16 could be involved. Exposure to this or another sexually transmitted agent apparently alters both the morphology and growth regulation of the Kaposi's sarcoma progenitor cells. These changes include the expression of the alpha chain of the interleukin-6 receptor with the acquisition of an interleukin-6-dependent autocrine growth loop. Subsequent perturbation of multiple cytokines during human immunodeficiency virus infection, including Oncostatin-M, interleukin-1 beta and tumor necrosis factor-alpha alters the subsequent growth of Kaposi's sarcoma. These studies suggest that control of cytokine perturbations or the underlying human immunodeficiency virus-1 infection should result in a significant reduction in the rate of growth of acquired immunodeficiency syndrome-related Kaposi's sarcoma.


Pathology of preinvasive and excellent-prognosis cancers. Recent advances in breast cancer treatment have made recognition of different prognostic groups mandatory. In addition, further experience with screening-detected lesions has resulted in the definition of new associations. Therapeutic approaches to these frequently microscopic lesions are currently the source of much debate. This article reviews recent studies of excellent-prognosis carcinomas as well as premalignant and other histologically defined lesions that indicate an increased risk for the development or recurrence of carcinoma. Advances in fine-needle aspiration and in hormone receptor assays are also reviewed.


Radiation therapy for breast cancer.

Radiation therapy for breast cancer. Now that the radiation literature for breast cancer is well established in terms of both local control and survival endpoints, especially for invasive breast cancer, recent reports focus on refining selection criteria of patients for treatment and on minimizing complications and maximizing cosmetic effects. The optimal treatment of patients with ductal carcinoma in situ remains a controversial issue, and the optimal sequencing of adjuvant chemotherapy and primary radiation in patients with breast-conserving surgery is emerging as a topic requiring further study.


Morphologic and immunoperoxidase study of neurologic lesions in naturally acquired rabies of raccoons. Histopathologic (hematoxylin and eosin [HE]) and immunoperoxidase (streptavidin-biotin complex) methods were used for examination of formalin-fixed tissues of rabid raccoons from an enzootic area of Pennsylvania. Extensive morphologic lesions of rabies encephalitis were present in the cerebrum and the brain stem regions. Negri bodies were detected by both methods and were present in the brain (cerebral cortex, hippocampus, brain stem, cerebellum, and cervical spinal cord) and in the ganglia of the trigeminal nerves. The viral inclusions were also seen in ganglion cells in the tongue, parotid salivary glands, pancreas, intestines, and adrenal glands. These sites were not associated with any inflammatory cellular infiltrate. The immunoperoxidase method was superior to HE for the detection of Negri bodies. Because lesions of rabies encephalitis were consistently observed in the cerebrum, brain stem, and cervical spinal cord regions, these areas of the brain should be included when raccoons are examined by the fluorescent antibody test for rabies.


Viral agents and associated lesions detected in a 10-year study of bovine abortions and stillbirths. In a 10-year survey started in 1980, specimens from 8,995 bovine abortions and stillbirths were submitted to the South Dakota Animal Disease Research and Diagnostic Laboratory. Of these, 8,962 were suitable for some type of examination. Viruses were associated with 948 (10.58%). Bovine herpesvirus-1 (IBR) was detected in 485 (5.41%), and bovine viral diarrhea virus (BVDV) was detected in 407 (4.54%). In 1 year of the survey, BVDV was detected in 8/32 fetuses that had lesions of passive congestion. Bovine herpesvirus-4 was isolated from 47 specimens (0.52%), parvovirus and enterovirus were each isolated from 2, and adenovirus, parainfluenza virus, and pseudorabies virus were each isolated from 1. Malignant lymphoid neoplasia was present in 2 fetuses, and their abortion was assumed to have been caused by the bovine leukosis virus.


Antigenic variant of swine influenza virus causing proliferative and necrotizing pneumonia in pigs. A new antigenic variant of swine influenza virus was isolated from the lungs of pigs experiencing respiratory problems in 7 different swine herds in Quebec. Pigs of different ages were affected, and the main clinical signs were fever, dyspnea, and abdominal respiration. Coughing was not a constant finding of the syndrome. At necropsy, macroscopic lesions included the overall appearance of pale animals, general lymphadenopathy, hepatic congestion, and consolidation of the lungs. Histopathologic findings were mainly proliferative pneumonia with a significant macrophage invasion, necrotic inflammatory cells in the alveoli and the airways, a marked proliferation of type II pneumocytes, and thickening of the alveolar septae. Fluorescent antibody examination of lungs of sick piglets did not demonstrate porcine parvovirus, transmissible gastroenteritis virus, or encephalomyocarditis virus. However, evidence of the presence of an influenza type A infection was demonstrated by indirect immunofluorescence (IIF) staining using monoclonal antibody directed to nucleocapsid protein (NP) of human type A influenza virus. The virus was isolated either by intra-allantoic inoculation of specific-pathogen-free embryonating hens' eggs or propagation in canine kidney (MDCK) cells in the presence of trypsin. By hemagglutination inhibition tests, no cross-reactivity was demonstrated with human influenza H1N1, H2N2, and H3N2 strains, and infected MDCK cells did not react by IIF with monoclonal antibodies to NP protein of type B influenza virus. The hemagglutination activity of plaque-purified isolates was only partly inhibited by hyperimmune serum produced to subtypes A/Wisconsin/76/H1N1 and A/New Jersey/76/H1N1 of swine influenza virus. Gnotobiotic piglets that were infected intranasally with egg-adapted isolates of this new antigenic variant of swine influenza virus developed the very same type of lesions observed in field cases.


Monoclonal and polyclonal antibodies for immunohistochemical detection of bovine parainfluenza type 3 virus in frozen and formalin-fixed paraffin-embedded tissues. Accurate identification of bovine parainfluenza type 3 virus in bovine respiratory disease requires dependable, sensitive, and specific techniques for detection in affected animals. Immunohistochemical testing can be a rapid and reliable means of demonstration of virus in tissues from suspect cases; however, this procedure is dependent upon the quality of the antisera directed against the viral antigens. The production of rabbit polyclonal and murine monoclonal antibodies directed against bovine parainfluenza type 3 virus and techniques for their use in fresh-frozen and formalin-fixed paraffin-embedded tissues in immunofluorescence and immunoperoxidase-based immunohistochemical tests are described.


Subclinical infectious bursal disease in an integrated broiler production operation. A field study was designed to determine the prevalence of subclinical infectious bursal disease (IBD) in broiler chickens from a commercial poultry company. Bursae of Fabricius (BF) from two vaccinated and three nonvaccinated broiler flocks were evaluated histologically, and antibody profiles of these broiler and matched parent breeder flocks were established. Lesions of IBD, including lymphoid necrosis, stromal edema, and infiltrates of heterophils and macrophages, were first detected in BF at 24 days of age in both vaccinated and nonvaccinated chickens. At 41 days, all BF had lesions characteristic of IBD, including severe lymphoid depletion, proliferation of epithelial cells, and mild fibroplasia. Although mean maternal antibody levels (measured by enzyme-linked immunosorbent assay) in broilers were apparently protective through day 12, IBD antibodies decreased to nonprotective levels (below 1,000) by day 16 or 20. Titers began to increase by day 28 or 32 because of field exposure. Sentinel birds, placed with broiler flocks, also developed IBD antibody titers. Broiler breeders had low and nonuniform antibody titers. Prevalence of field IBD exposure was high, and existing vaccination programs were not effective.


Studies on transmissibility of a tissue-culture-modified laryngotracheitis virus. The spreading characteristic of a tissue-culture-modified laryngotracheitis virus (LTV) was demonstrated in chickens under both laboratory and field conditions. An enzyme-linked immunosorbent assay was used to measure serum antibodies to LTV in experimental chickens. Contact controls developed immunity comparable to that of vaccinates and were able to resist challenge with a virulent LTV inoculated into the infraorbital sinus.


[Study of intermediate N using mutant forms of bacteriorhodopsin at Asp-96]. It has been found that the N(P, R)-type intermediate of the photocycle is formed in the Asp-96-->Asn mutant at acidic pH. Azide, which strongly activates the M decay in this mutant, allows the N intermediate to be shown also at neutral pH. Under these conditions mutant N decays in a pH-independent fashion. In the presence of azide, the H+ uptake by Asp-96-->Asn mutant bacteriorhodopsin follows the M decay, whereas the N decay occurs at a much slower rate. Two electrogenic stages have been shown to be associated with the M--->bR step in the Asp-96--->Asn mutant photocycle. The faster and slower stages correlate with the M--->N and N--->bR transitions, respectively. In the Asp-96--->Asn mutant, high concentrations of azide are found to increase the M decay rate up to the values higher than those in the wild-type protein, both with or without azide. Such an effect is absent for the Asp-96-->Glu mutant. The activation energies for M--->N and N--->bR transitions in the wild-type protein are equal to 18 and 19 kcal x mole-1, respectively. In the Asp-96-->Asn mutant without azide, the activation energy of the M decay is only 5 kcal x mole-1, whereas in the presence of azide in this mutant the activation energies for M and N decays are 8 and 9 kcal x mole-1, respectively. A scheme of events accompanying the Schiff base reprotonation during the photocycle is discussed.


Apoptotic cell death triggered by camptothecin or teniposide. The cell cycle specificity and effects of ionizing radiation. We have previously observed that the DNA topoisomerase I inhibitor camptothecin (CAM), or DNA topoisomerase II inhibitors teniposide (TEN) and amsacrine (m-AMSA) trigger endonucleolytic activity in myelogenous (HL-60 or KG1), but not lymphocytic (MOLT-4) leukaemic cell lines. DNA degradation and other signs of apoptotic death were seen as early as 2-4 h after cell exposure to these inhibitors. Cells replicating DNA (S phase) were selectively sensitive whereas cells in G1 were resistant; the sensitivity of G2 or M cells could not be assessed in these studies. The present studies were aimed at revealing whether DNA repair replication induced by ionizing radiation can sensitize the cells, and to probe the sensitivity of cells arrested in G2 or M, to these inhibitors. The data show that gamma-irradiation (0.5-15 Gy) of HL-60 cells does not alter their pattern of sensitivity, i.e. G1 cells, although engaged in DNA repair replication, still remain resistant to CAM compared with the S phase cells. Likewise, irradiation of MOLT-4 cells also does not render them sensitive to either CAM or TEN, regardless of their position in the cell cycle. Irradiation, however, by slowing the rate of cell progression through S, increased the proportion of S phase cells, and thus made the whole cell population more sensitive to CAM. HL-60 cells arrested in G2 either by irradiation or treatments with Hoechst 33342 or doxorubicin appear to be more resistant to CAM relative to S phase cells. Also resistant are cells arrested in M by vinblastine. The data suggest that some factor(s) exist exclusively in S phase cells, which precondition them to respond to the inhibitors of DNA topoisomerases by rapid activation of endogenous nuclease(s) and subsequent death by apoptosis. HL-60 cells in G1, G2 or M, or MOLT-4 cells, regardless of the phase of the cycle, appear to be protected from such a mechanism, and even induction of DNA repair replication cannot initiate DNA degradation in response to DNA topoisomerase inhibitors. These data, together with the evidence in the literature that topoisomerase I may be involved in DNA repair, suggest that a combination of these inhibitors with treatments that synchronize cells in the S phase and/or recruit quiescent cells to proliferation, including radiation, may be of value in the clinic.


cAMP effect on extracellular matrix synthesis in human breast cancer cells. The effect of a cAMP derivative (N6, O2-dibutyryl cyclic adenosine 3',5'-monophosphate: dBcAMP) on the cell cycle and on the synthesis of typical extracellular matrix (ECM) components, i.e. collagen and glycosaminoglycans (GAG), was studied in two hormone-responsive human breast cancer cell lines VHB-1 and MCF-7. The data showed that dBcAMP induced a decrease in the proportion of cells in S + G2 + M phases due to an increase of the non-cycling (G0 phase) cell number as revealed by the Ki-67 antigen immunocytochemical study. The collagen synthesis, estimated by [3H] proline incorporation into the cellular proteins followed by an enzymatic digestion with highly purified bacterial collagenase, was not modified by dBcAMP. In contrast, the GAG synthesis, analysed by [3H] glucosamine incorporation, was increased two-fold in the dBcAMP treated cells. As a comparison we also tested 4-hydroxy-Tamoxifen (4-OH-Tam) since it induces similar cell cycle perturbations as dBcAMP. However, we did not observe a stimulation of the GAG synthesis following 4-OH-Tam treatment. These data demonstrated that the increased GAG synthesis is due to cAMP and is not a consequence of perturbations in the cell cycle. We can therefore assume that the ECM modifications induced by dBcAMP may contribute to the growth inhibition of the hormone-responsive human breast cancer cells.


Towards an understanding of the mode of action of Bacillus Calmette-Guérin-therapy in bladder cancer treatment, especially with regard to the role of fibronectin. The interaction between Bacillus Calmette-Guérin (BCG) and the host was investigated after repeated intravesicular BCG-therapy for superficial bladder cancer. Studies were performed on (a) the local reaction in the bladder, (b) the systemic reaction, and (c) short and long term interactions in both the bladder and the serum/plasma. The analytes measured included anti-BCG IgA and IgG, fibronectin, lactoferrin, elastase-alpha 1-proteinase inhibitor, myeloperoxidase and alpha 2-proteinase inhibitor. All analytes, with the exception of alpha 2-proteinase inhibitor, were measured in both serum/plasma and urine. An additional group of 94 patients undergoing bronchoalveolar lavage was used for comparison with other diseases affecting mucous membranes. In vitro studies on human bladder in culture were also carried out to study the relationship between BCG, elastase and fibronectin. The results revealed a normal defence reaction, in which IgA and IgG antibodies specific to BCG were produced by the host. Maximal concentrations of all analytes in urine were found about 4 h after BCG instillation. Immunoglobulins, soluble fibronectin, and granulocyte markers all appeared in urine after instillation and all showed a similar time course. The in vitro study showed the synergistic effect of elastase and BCG in stimulating the host defence reaction. The relationship between BCG and fibronectin can be seen as fortuitous but not indicative of the efficacy of BCG-therapy in patients with superficial bladder cancer.


CSF diazepam-binding inhibitor concentrations in panic disorder. Diazepam-binding inhibitor (DBI) is a neuropeptide that has been detected in the brain and cerebrospinal fluid (CSF). Previous studies have suggested the possible role of DBI as a potential endogenous anxiogenic ligand modulating GABAergic transmission at the benzodiazepine-GABA receptor complex. The measurement of DBI immunoreactivity (DBI-IR) in CSF of panic-disorder patients and normal controls was undertaken to assess whether there were differences in the CSF concentration of this peptide to assess possible relationships with other monoamines and peptides. Lumbar CSF was obtained from 18 panic patients (4 men, 14 women) and 9 controls (5 men, 4 women). As a group, no significant differences were found between panic patients' CSF concentration of DBI-IR (1.12 +/- 0.27 pmol/mL) and normal volunteers (1.23 +/- 0.27 pmol/mL). No gender differences were demonstrated. However, we did find a positive correlation between CSF levels of DBI and CSF corticotropin releasing hormone (CRH) in our panic patients.


Dementia, myoclonus, peripheral neuropathy, and lipid-like material in skin biopsy during psychotropic drug treatment. Chronic treatment of humans with several drugs is associated with lesions resembling lipidosis in different tissues. Recently, a Creutzfeldt-Jacob-like syndrome has been observed during tricyclic antidepressant therapy, but no evidence of interaction of these drugs with lysosomal function has been reported during such treatment. We report a case of dementia, myoclonus, peripheral neuropathy, and lipid storage in the skin due to antidepressant drug therapy, in which the discontinuation of drugs resulted in an improvement of clinical and electrophysiologic signs together with reduction of morphological evidence of lipid lysosomal storage.


The role of retinoids in normal and abnormal embryonic craniofacial morphogenesis. The objective of this article is to evaluate the role of retinoids in the developing head and face. This article covers two lines of evidence that strongly support a role for retinoids in craniofacial development. First, the specific effects of exogenous retinoids on the head and face are covered and mechanisms for the specificity discussed. Second, the function of endogenous retinoids in facial development is discussed in relation to the distribution of retinoid-binding substances in the face. Finally, the interaction of retinoids with other genes known to be expressed in the face as well as other factors required for facial growth is discussed.


Hemophan versus hemophan--a philippic against the suggestion of constant membrane quality. All statements referring to biocompatibility of dialyzers depend on the suggestion of a constant and reproducible structure of the investigated membrane material. The data presented here strike this concepts in case of hemophane. Two commercially available hemophane dialyzers, the GF MC 120 (Gambro) and the MO 450 (SMAD) membranes, were compared. Significant differences could be seen in the induction of leukopenia and leukocyte sequestration reaction within patients lungs, but also in capacity to influence granulocyte oxidative metabolism and release of granular enzymes. According to these data it seems questionable whether hemophane is a membrane material with constant structure and quality normally expected for products bearing trade names. Excluding other differences (like surface-area or sterilization method) variable amounts of DEAE substituents and/or an inconstant distribution of the DEAE groups within the membranes may be the most possible reason responsible for the differences found. The study underlines the urgent need of an exact physico-chemical characterization of dialyzer membranes and is therefore a challenge for free publication of such basic data by the producers.


Synthesis and biological activity of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine deoxyuridine diastereomers. The synthesis of the two diastereomers (9 and 10) of 5-(2,2-difluorocyclopropyl)-2'-deoxyuridine are described. Their antiviral and cytotoxic activities were determined, in comparison with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 5-fluoro-2'-deoxyuridine (FDU), respectively. 5-[(1R)-2,2-Difluorocyclopropyl]-2'-deoxyuridine (10) was the most active antiviral agent against HSV-1 (IC50 = 5 micrograms/ml) relative to BVDU (IC50 = 0.082 micrograms/ml), and cytotoxic agent in the CCRF-CEM (IC50 = 230 microM) screen relative to FDU (IC50 = 4.7 x 10(-3) microM). The 5-[(1S)-2,2-difluorocyclopropyl] diastereomer was inactive in both screens. Partition coefficients (P) and affinity for the mouse erythrocyte nucleoside transporter (Ki) were not determinants of antiviral or cytotoxic activities. However, the (1R)-diastereomer (10) was more resistant to glycosidic bond cleavage by thymidine phosphorylase than the (1S)-diastereomer (9).


Miniature urea sensor based on H(+)-ion sensitive field effect transistor and its application in clinical analysis. An urea-ENFET (Enzyme field effect transistor) probe was made by covering one of the grids of the dual ISFET (Ion sensitive field effect transistor) with a membrane of cross-linked bovine serum albumin (BSA)-urease and the other with cross-linked BSA, and the response characteristics of the probe was then tested through differential measurements. In different concentrations of phosphate buffer, the sensor responded to various concentrations of urea solution within 10-60s. From the calibration curve plotted on logarithmic scales a linear concentration range of 1.0-8.0 mg/dl was acquired, and the correlation coefficient and response sensitivity were 0.997 and 50mV/dec. (mg/dl), respectively. However, in dilute urea solution, the sensor responded linearly to the contents of urea over the range of concentration of 0.1-1.0 mg/dl with a correlation coefficient of 0.998 and a response sensitivity of 12-15mV/mg/dl. The standard deviation and the variation coefficient for 20 performances responding to 100mg/dl urea in 0.01M pH7.0 phosphate buffer were found to be 1.39mV and 1.44%, respectively. The urea-ENFET was used for the determination of BUN (Blood urea nitrogen) and the BUN values were compared with those determined by enzymatic method, the repression equation and correlation coefficient for 50 assays were y = -0.1272 + 0.9695x and r = 0.9912, respectively. When the urea-ENFET was used for determining urea either in buffer solution or in serum for 250 runs over a period of 1.5 months (the enzyme FET was stored at 4 degrees C between measurements during this period), the observed decrease of response sensitivity was only about 10%.(ABSTRACT TRUNCATED AT 250 WORDS)


The utility of side-chain cyclization in determining the receptor-bound conformation of peptides: cyclic tripeptides and angiotensin II. The effect of side-chain cyclization on accessible backbone conformations of tripeptides, X-Ala-Y (X and/or Y = Cys, Hcy (Hcy: homocysteine), cis 4-mercaptoproline (MPc), and trans 4-mercaptoproline (MPt)), was elucidated using two variants of systematic conformational search. In addition to cyclization through a disulfide bond, the thioether (-S-CH2-) and amide (-CO-NH-) side-chain analogues of Cys-Ala-Cys and Hcy-Ala-Hcy were evaluated. The number of valid backbone conformations and the allowed phi, psi space were evaluated for each compound, and the ability of the cyclic tripeptides to accommodate beta-turn conformations was examined in order to assess the value of cyclization in limiting conformational freedom. Based on the number of conformations, cyclization was highly effective in reducing the backbone degree of freedom: in order of decreasing number of conformations, Ala-Ala-Ala 1 >> Hcy-Ala-Hcy 2 >> Cys-Ala-Hcy 3 approximately equal to Hcy-Ala-Cys 4 >> MPc-Ala-Hcy 5, 7 > Cys-Ala-Cys 6 > MPc-Ala-Cys 8 > Hcy-Ala-MPt 9 > Cys-Ala-MPt 10 approximately equal to MPc-Ala-MPt 11. Although Hcy-Ala-Hcy 2 had the greatest number of conformations of the cyclic peptides studied, it was still greatly constrained relative to its linear analogue 1. The bicyclic ring system introduced by MP was even more effective in constraining the cycle, having greater impact at position 3 than at position 1. Under the conditions of the study, cyclization of MP-containing analogues could be effected only with the cis isomer (MPc) at position 1 and/or the trans isomer (MPt) at position 3. Sterically allowed conformations of Ala2 for the cyclic tripeptides 2-4 were generally similar to those of the linear tripeptide 1, while those of Cys-Ala-Cys 6 and MPc-Ala-Hcy 7 were restricted to a smaller region of phi 2, psi 2 space: the right- and left-handed alpha-helical conformation and the beta-conformation. This trend was even more pronounced for Hcy-Ala-MPt 9, Cys-Ala-MPt 10, and MPc-Ala-MPt 11, in which Ala2 was severely restricted to a very small region of phi, psi space: the left-handed alpha-helical conformation for 9-11, plus the beta conformation for 9. This suggests that MP at the 3-position is incompatible with a right-handed alpha-helical conformation at position 2.(ABSTRACT TRUNCATED AT 400 WORDS)


Abnormalities in Na+/H+ antiporter activity in diabetic nephropathy. In hypertensive humans and the spontaneously hypertensive rat, increased cellular Na+/H+ antiport activity has been demonstrated in leukocytes, platelets, skeletal muscle, and vascular smooth muscle cells. This membrane abnormality may be associated with medial thickening of resistance vessels. A similar membrane transport abnormality has also been demonstrated in leukocytes and fibroblasts from type 1 diabetic patients with nephropathy. This membrane transport marker of hypertension may indicate a predisposition to essential hypertension in such patients and may lead to diabetic nephropathy, possibly from mesangial expansion.


Evaluation of the shell vial pre-CPE method using monoclonal antibodies for the diagnosis of human cytomegalovirus infection in the field of pediatrics. A method of pre-cytopathological effects (CPE) detection in shell vial cultures using monoclonal antibodies (mAb) was evaluated for detection of cytomegalovirus (CMV) in pediatric specimens. The diagnostic value of antibody detection by enzyme immunoassay (EIA) was also evaluated. CMV was isolated from 47 of 1179 specimens tested by the routine tissue culture method. Positive results were obtained in 51 of these 1179 specimens using the pre-CPE method. The pre-CPE shell vial assay was demonstrated to be a rapid and sensitive method of detecting CMV. Detection of the virus by the pre-CPE method was considered much more reliable than by EIA IgM assays for pediatric patients.


Infectivity determinants encoded in a conserved gene block of human herpesvirus-6. The nucleotide sequence was determined for a 9.3 kb BamHI DNA fragment derived from a cosmid clone (Lorist 6) library of the 160 kb human herpesvirus-6 (HHV-6) strain U1102 genome. Analysis of the sequence showed two different sources for the DNA; 8.0 kb was derived from HHV-6, while 1.3 kb was derived from the right repeat of transposon Tn10, the insertion sequence (IS) element IS10R. The IS element sequence is shown to be derived from the host bacteria of the plasmid. The HHV-6 sequence represents a highly conserved part of the genome encoding 15% of the genes conserved among the other human herpesviruses in only 5% of the genome. Six genes were identified, five encoding products with amino acid sequence similarity to homologues in herpes simplex virus (HSV), Varicella Zoster Virus (VZV), human cytomegalovirus (CMV) and Epstein-Barr Virus (EBV). All had closest amino acid similarity to CMV proteins. Three clustered structural genes, included glycoprotein H, a major conserved determinant of infectivity, were jointed to a putative dUTPase homologue in an arrangement distinct to CMV and HHV-6. In the other herpes viruses these genes are separated by over 50 kb. The gene at this point of genetic rearrangement had no sequence similarity to proteins of other herpesviruses. However, there is a protein at this locus in CMV with similar composition and character. Both appear to be highly glycosylated, secreted glycoproteins with repetitive elements similar to those of human mucins. Comparison of sequence available in the HHV-6 GS strain also shows this to be a variable region (5% nucleotide differences) in an overall conserved DNA sequence (0.5%).


Cell biology of viruses that assemble along the biosynthetic pathway. In this review we discuss five groups of viruses that bud into, or assemble from, different compartments along the biosynthetic pathway. These are herpes-, rota-, corona-, bunya- and pox-viruses. Our main emphasis will be on the virally-encoded membrane glycoproteins that are responsible for determining the site of virus assembly. In a number of cases these proteins have been well characterized and appear to serve as resident markers of the budding compartments. The assembly and dissemination of these viruses raises many questions of cell biological interest.


Neurotensin stimulates the growth and secretion of rat adrenal zona glomerulosa. Within 2 days neurotensin (NT) and ACTH administrations markedly enhanced the average volume of zona glomerulosa (ZG) cells and plasma aldosterone concentration (PAC) in intact rats. In dexamethasone-treated rats, both NT and ACTH evoked a clearcut ZG-cell hypertrophy, but only NT was able to raise PAC. In conclusion, our findings indicate that NT is a potent stimulator of the growth and secretion of rat ZG in vivo, and suggest that the mechanism underlying this action of NT does not involve the well-known NT-induced stimulation of ACTH release.


Sequence of bovine interleukin 7.

Sequence of bovine interleukin 7. We report the cloning and the sequencing of a cDNA coding for the mature bovine interleukin 7 (IL-7). The clone was isolated from a bovine leukemia virus (BLV)-induced B cell-lymphosarcoma cDNA library. The 5' non-coding sequence and the sequence of the signal peptide were obtained from a clone isolated from a bovine genomic library. The entire bovine IL-7 protein is 176 amino acids long and shows 75 and 65% homology to published sequences of human and murine IL-7, respectively.


Characterization of plasmids with antimicrobial resistant genes in Pasteurella haemolytica A1. Two R plasmids, pYFC1 and pYFC2, from Pasteurella haemolytica A1 encoding sulfonamide, streptomycin (pYFC1), and ampicillin (pYFC2) resistances have been characterized by restriction endonuclease digestions, subcloning or DNA sequencing. pYFC1 consists of 4225 bp and is 51.9% in AT content. Physical mapping indicated a highly conserved region of restriction sites among pYFC1, RSF1010, pGS05, pFM739, pHD148 and pGS03B. pYFC1 encoded a dihydropteroate synthase (29.8 kDa), and streptomycin kinase (29.6 kDa) which is homologous in nucleotide sequences or deduced amino acid sequence to that encoded by a broad-host range IncQ plasmid RSF1010. Based on the primary structure of pYFC1, the sulfonamide and streptomycin genes are derived from the same ancestor of RSF1010. pYFC2 is similar to the plasmid from P. haemolytica LNPB51 isolated in France by partial restriction enzyme mapping. pYFC1 and pYFC2 have the same size of 4.2 kbp.


cAMP signaling in neurons: patterns of neuronal expression and intracellular localization for a novel protein, AKAP 150, that anchors the regulatory subunit of cAMP-dependent protein kinase II beta. In mammalian brain, physiological signals carried by cyclic AMP (cAMP) seem to be targeted to effector sites via the tethering of cAMP-dependent protein kinase II beta (PKAII beta) to intracellular structures. Recently characterized A kinase anchor proteins (AKAPs) are probable mediators of the sequestration of PKAII beta because they contain a high-affinity binding site for the regulatory subunit (RII beta) of the kinase and a distinct intracellular targeting domain. To establish a cellular basis for this targeting mechanism, we have employed immunocytochemistry to 1) identify the types of neurons that are enriched in AKAPs, 2) determine the primary intracellular location of the anchor protein, and 3) demonstrate that an AKAP and RII beta are coenriched and colocalized in neurons that utilize the adenylate cyclase-cyclic AMP-dependent protein kinase (PKA) signaling pathway. Antibodies directed against rat brain AKAP 150 were used to elucidate the regional, cellular and intracellular distribution of a prototypic anchor protein in the CNS. AKAP 150 is abundant in Purkinje cells and in neurons of the olfactory bulb, basal ganglia, cerebral cortex, and other forebrain regions. In contrast, little AKAP 150 is detected in neurons of the thalamus, hypothalamus, midbrain, and hindbrain. A high proportion of total AKAP 150 is concentrated in primary branches of dendrites, where it is associated with microtubules. We also discovered that the patterns of accumulation and localization of RII beta (and PKAII beta) in brain are similar to those of AKAP 150. The results suggest that bifunctional AKAP 150 tethers PKAII beta to the dendritic cytoskeleton, thereby creating a discrete target site for the reception and propagation of signals carried by cAMP.


Multiple cyclic AMP receptors are linked to adenylyl cyclase in Dictyostelium. cAMP receptor 1 and G-protein alpha-subunit 2 null cell lines (car1- and g alpha 2-) were examined to assess the roles that these two proteins play in cAMP stimulated adenylyl cyclase activation in Dictyostelium. In intact wild-type cells, cAMP stimulation elicited a rapid activation of adenylyl cyclase that peaked in 1-2 min and subsided within 5 min; in g alpha 2- cells, this activation did not occur; in car1- cells an activation occurred but it rose and subsided more slowly. cAMP also induced a persistent activation of adenylyl cyclase in growth stage cells that contain only low levels of cAMP receptor 1 (cAR1). In lysates of untreated wild-type, car1-, or g alpha 2- cells, guanosine 5'-O-'(3-thiotriphosphate) (GTP gamma S) produced a similar 20-fold increase in adenylyl cyclase activity. Brief treatment of intact cells with cAMP reduced this activity by 75% in control and g alpha 2- cells but by only 8% in the car1- cells. These observations suggest several conclusions regarding the cAMP signal transduction system. 1) cAR1 and another cAMP receptor are linked to activation of adenylyl cyclase in intact cells. Both excitation signals require G alpha 2. 2) cAR1 is required for normal adaptation of adenylyl cyclase. The adaptation reaction caused by cAR1 is not mediated via G alpha 2. 3) Neither cAR1 nor G alpha 2 is required for GTP gamma S-stimulation of adenylyl cyclase in cell lysates. The adenylyl cyclase is directly coupled to an as yet unidentified G-protein.


An integrative approach to the proteolytic control of the cell-mediated cytotoxicity. The possible role of intracellular proteases in the control of the cell-mediated cytotoxicity of NK or CTL type, as components of the sequential molecular events leading to the target cell lysis, is emphasized by an integrative structural and functional approach. Starting from the own cytochemical researches and based on recent data, the effector cell proteases are analysed concerning their cellular and ultrastructural compartmentalization and their involvement in the sequential stages of the cytotoxic cycle. Membranous, granular, lysosomal and cytosolic compartments of the proteolytic activity are differentially elicited to interfere in the stimulus-secretion pattern of the cytotoxic function. Serine proteases (trypsin and chymotrypsin-like) and thiol proteases (calpain, cathepsins B and L) are specifically involved in the receptor-mediated signal transduction by the phosphatidyl inositol pathway, in the programming of the secretory machinery, in the exocytosis of the cytotoxic factors and in the final lytic phase. An integrative model of the cell-mediated cytotoxicity is proposed including the protease compartments of the effector cell and their specific involvement in the triggering, the modulation and the control of the cellular and molecular events, as main components of the informational networks of the cytotoxic cells.


Identification of the domains in cyclin A required for binding to, and activation of, p34cdc2 and p32cdk2 protein kinase subunits. The binding of cyclin A to p34cdc2 and p32cdk2 and the protein kinase activity of the complexes has been measured by cell-free translation of the corresponding mRNA in extracts of frog eggs, followed by immunoprecipitation. A variety of mutant cyclin A molecules have been constructed and tested in this assay. Small deletions and point mutations of highly conserved residues in the 100-residue "cyclin box" abolish binding and activation of both p34cdc2 and p32cdk2. By contrast, large deletions at the N-terminus have no effect on kinase binding and activation, until they remove residues beyond 161, where the first conserved amino acids are found in all known examples of cyclin A. At the C-terminus, removal of 14 or more amino acids abolishes activity. We also demonstrate that deletion of, or point mutations, in the cyclin A homologue of the 10-residue "destruction box," previously described in cyclin B (Glotzer et al., 1991) abolish cyclin proteolysis at the transition from M-phase to interphase.


Characterization of transforming growth factor-beta (TGF-beta) receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein. Transforming growth factor-beta (TGF-beta) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-beta 1 and TGF-beta 2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGF-beta 1 and TGF-beta 2. The Kd values obtained from Scatchard analyses were approximately 65 pM for 125I-TGF-beta 1 and approximately 40 pM for 125I-TGF-beta 2, with 70,000 and 85,000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-beta 1 and TGF-beta 2 were equipotent (apparent half maximal inhibition [IC50] approximately 70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 125I-TGF-beta 1 and 125I-TGF-beta 2 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-beta binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-beta 1 than for -beta 2 yet exhibits a 5- to 10-fold higher affinity for TGF-beta 2 than for -beta 1. The 38-kDa TGF-beta binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-beta 2 than for -beta 1 that is strikingly similar to that of the type III/betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the BeWo cells.


Haemopoietic growth factors in acute myeloblastic and lymphoblastic leukaemia. Acute leukaemia blast cells fail to mature into terminally differentiated cells and accumulate in the haemopoietic tissues. In analogy with normal haemopoiesis, the leukaemic mass is largely non-dividing and descends from a small pool of leukaemic progenitor cells with high proliferative activity. In vitro culture methods have become powerful instruments to analyse human leukaemia progenitor cells. These techniques have in recent years been considerably improved as the result of the availability of the recombinant HGFs. Here we have summarized the current insights in the growth properties of acute leukaemia progenitor cells, derived from the application of fully defined in vitro assays. We have discussed the proliferation and maturation abilities in relation to cytogenetic abnormalities, status of growth factor receptors and the property of autocrine growth stimulation and evaluated the significance of these findings for the understanding of the pathogenesis of acute myeloblastic leukaemia and acute lymphoblastic leukaemia.


Cellular signalling events stimulated by myeloid haemopoietic growth factors. In haemopoietic cells, proliferation, commitment to development, lineage restriction and survival via suppression of apoptosis can all be controlled by haemopoietic growth factors. The mechanisms underlying the regulation of these events can now be studied since recombinant forms of most of these haemopoietic growth factors are now available. Recent advances in cell purification techniques and the development of multipotent cell lines (see Spangrude et al, 1988; Whetton, 1990; Heyworth et al, 1988, 1990a; Jones et al, 1990) have provided suitable cell populations on which to study the cellular signalling events associated with differentiation and lineage restriction. This process has started with the elucidation of the structure and expression of many of the myeloid growth factor receptors, which should now facilitate progress in the study of the signal transduction mechanisms these growth factors employ. Another important facet of these studies will be to determine whether a single growth factor with multiple target cell types, ranging from multipotent cells to postmitotic cells (e.g. neutrophils), employs distinct signalling mechanisms depending on the target cell in question. The cellular signalling events elicited by each of these growth factors and the ways in which they can regulate the transcriptional activation of genes associated with specific developmental events are going to be key areas of haemopoietic research in the next few years.


Native cytokine antagonists.

Native cytokine antagonists. Cytokines orchestrate the complex homeostasis of cells and tissues by acting in both an autocrine and paracrine fashion. The processes responsible for regulation of cytokines is not well understood. This chapter has summarized what is known about antagonism and inhibition of the action of cytokines. Several concepts have emerged from work in this area. At least two cytokines (IL-1 alpha and IL-1 beta) have an endogenous receptor antagonist, the IL-1 receptor antagonist. This is the first example of one endogenous molecule directly blocking the binding of another molecule to its receptor: most forms of regulation occur through independent receptors. Several cytokines, including TNF, IFN-gamma, IL-2 and IL-4, are inhibited by soluble receptors. Several cytokines, including IL-10, TGF-beta and MDF, act to inhibit other cytokines. It is likely that these inhibitors will be found to have pleiotropic actions in vivo. Finally, we describe antibody inhibition of cytokines. Detailed studies will be required to understand the complex interplay of the aforementioned cytokine inhibitors and the processes they regulate.


Transforming growth factor beta and interleukin-1: a paradigm for opposing regulation of haemopoiesis. The polypeptide cytokines, IL-1 and TGF-beta affect nearly every tissue and cell type in the body. IL-1 is the prototype of the proinflammatory molecule while TGF-beta is essentially anti-inflammatory. IL-1 is part of the cascade of cytokines that are produced during microbial invasion or bodily injury and enhance a variety of host responses, particularly in the immunological and haemopoietic systems, while TGF-beta acts as an inhibitor of these responses. At several levels, IL-1 and TGF-beta act in opposition to one another. IL-1 stimulates the expression of many genes in lymphoid and marrow stromal cells that stimulate haemopoietic cell growth and differentiation, while TGF-beta inhibits these IL-1 mediated effects. Also, TGF-beta stimulates secretion of the IL-1Ra. In addition, IL-1 induces the cell surface expression of cytokine receptors on lymphoid and haemopoietic cells, while TGF-beta dramatically inhibits the cell surface expression of these receptors, including the IL-1 receptor. Finally, IL-1 augments lymphoid and haemopoietic cell growth and TGF-beta potently inhibits this proliferation. The interactions of these cytokines serve to illustrate that the net balance of stimulatory and inhibitory signals determines the fate of a given cell which may be responsible for regulating homeostatic cell growth (Figure 1). Thus, the regulation of cytokine production and/or antagonism of their effects continues to be a therapeutic goal in the treatment of many diseases.


The repair of tracheal defects using bioabsorbable mesh. This study compares the use of synthetic bioabsorbable mesh with that of non-absorbable mesh in repairing tracheal defects. Two patch windows, each 1 cm x 1 cm, were created on the ventral wall of the cervical trachea in 12 adult mongrel dogs. One window was repaired using a bioabsorbable polyglycolic acid (PGA) mesh graft, and the other using a nonabsorbable Marlex mesh (polypropylene mesh; PP mesh) (C. R. Bard, Inc., Billerica, MA) graft. The dogs were killed at intervals from 1 to 35 weeks after surgery. Macroscopically, there was no difference between the two mesh groups until 3 weeks after surgery. At 6 weeks in the PP mesh group, the connective tissue over the mesh was remarkably thick, and after 9 weeks, constriction by scarlike tissue was evident, as compared with the PGA mesh group, which showed no constriction. Microscopically, epithelium covered the whole mesh in both groups at 6 weeks. However, in the PP mesh group, flat and poorly differentiated cells occupied a large area, and after 9 weeks the height of the epithelial cells in the PP mesh group was less than in the PGA mesh group. Furthermore, in the PP mesh group, nonciliated cells occupied a large area even at 15 weeks, and the submucous connective tissue layer was thicker than in the PGA mesh group after 6 weeks. The results indicate that, from the point of view of wound-healing, bioabsorbable mesh may be more adequate for repairing tracheal defects.


Expression of intermediate filaments and other special markers by testicular germ cell tumors. With reference to embryogenesis. Distribution of intermediate filament proteins (IFs) and several special markers was studied in 39 testicular germ cell tumors and 8 embryos and foetuses. The similarity and difference between development of germ cell tumor and embryogenesis were immunohistochemically investigated. Seminoma and embryonal carcinoma, as tumoral counterparts of undifferentiated germ cells, were characterized by little IF expression. This study revealed that the maturing and differentiating process in germ cell tumor is different from normal embryonal development and the tumor cells showed leaping maturing steps in tumorigenesis. Immunostaining for IFs helped to discover the further differentiation occurring in embryonal carcinoma and to demonstrate heterogeneous elements in non-seminoma germ cell tumors, which sometimes might not be apparent by light microscopical observation of H&E staining section. According to the findings, two patterns in mixed germ cell tumors are suggested; i.e., combined and diffuse types. The mechanism of tumorigenesis of the two types is supposed to be different. Clinically, the prognosis of most patients with testicular germ cell tumor is fairly good because of the improved chemotherapies that are dependent on histological diagnosis.


Long-term use of low molecular weight heparin ameliorates hyperlipidemia in patients on hemodialysis. Hyperlipidemia is one of the major risk factors for cardiovascular death in long-term hemodialysis (HD) patients. To clarify whether unfractionated heparin (UFH) contributes to the pathogenesis of hyperlipidemia, nine Type IIb, seven Type IV, and 10 normolipidemic patients, who had been dialyzed with 80.7 IU/Kg heparin, were dialyzed with 40 anti-Xa U/kg of low molecular weight heparin (LMWH) (Logiparin, Novo-Nordisk, Gentfe, Denmark) for 6 months. Seven normolipidemic patients were also dialyzed with heparin as controls. Decreases in triglyceride (TG) during HD with LMWH were significantly less than those with heparin. However, lipoprotein lipase activities (LPL) during HD with LMWH and heparin, and those before and after 6 months on LMWH, were no different. During the 6 months on LMWH, serum total cholesterol, TG, and alpha lipoprotein significantly decreased in Type IIb patients but did not change in Type IV. In contrast, beta lipoprotein slightly increased in Types IIb, IV, and normolipidemic patients who were dialyzed with LMWH but was unchanged in the controls. These observations suggest that UFH aggravates hyperlipidemia in patients, but these effects cannot be attributed to depletion of endothelial LPL liberated by UFH.


Small cell carcinoma of the stomach: an immunohistochemical and electron microscopic study. A 4 x 6 cm ulcerative mass in the antrum was found to consist of papillary adenocarcinoma in the surrounding wall and the small round cell neoplasm at its base. Immunohistochemical staining revealed that elements of the papillary adenocarcinoma were positive for carcinoembryonic antigen, epithelial membrane antigen, keratin, endocrine granule constituent, and CA19-9, while components of the small cell carcinoma were weakly positive only for neuron-specific enolase. In one portion of the small cell carcinoma, particularly large cells with pleomorphic nuclei which were intensely positive for desmin were detected. Electron microscopic examination revealed dense-cored granules and intercellular junctions in the small neoplastic cells and bundles of intermediate filaments in the desmin-positive large cells. These findings suggest that ultrastructural examination is vital in diagnosis of small cell carcinoma and they reveal the capability of this carcinoma toward multidirectional differentiation.


Metanephric adenoma.

Metanephric adenoma. In a recent survey of more than one hundred childhood renal tumors in our Laboratory files, we identified a unique case characterized by an unusual degree of differentiation and cell maturity. Histologically this case was notable for an orderly array of small and uniformly-packed tubules with a rosette-like configuration. The nuclei were oval, smooth and of a bland appearance. Mitoses were absent. Many glomerular figures were intermingled. This renal tumor picture is somewhat different from that known as tubular Wilms' tumor because of the well-differentiated adenomatous pattern and the absence of any blastema. The term metanephric adenoma is suggested for this tumor, which may represent the benign counterpart of Wilms' tumor.


Mechanistic aspects of DNA transposition. The past year has seen a number of important advances in our understanding of the mechanisms of DNA transposition. The molecular details of the protein-protein, protein-DNA and chemical-reaction steps in several transposition systems have been revealed and have highlighted remarkable uniformity in some areas, ranging from bacterial to retroviral mechanisms.


Yeast retrotransposons.

Yeast retrotransposons. In the decade since Ty elements were discovered, advocates have argued they could be used as a genetic entrée to elusive host-type functions required by retroviruses. However, the advent of the polymerase chain reaction, coupled with a boom in funding for human immunodeficiency virus research have moved retroviral research apace, raising questions as to whether novel contributions would be realized. The past year, with the implication of the cell cycle and specific host proteins, such as the debranching enzyme and transcription initiation factors, in Ty retrotransposition has provided a positive answer and raised new questions.


RNA splicing in lower eukaryotes.

RNA splicing in lower eukaryotes. Recently, cis-acting elements and trans-acting RNA and protein factors necessary for splicing nuclear pre-mRNAs, group II introns or group III introns, have been discovered, and new roles for the splicing factors have been elucidated. Parallels among the pathways for splicing these different classes of introns have been identified.


Mechanistic insights relevant to protein secretion in yeast. During the past year, a powerful combination of genetic and biochemical approaches has yielded fascinating information with respect to the question of how proteins cross membranes and subsequently traffic between intracellular compartments of the yeast secretory pathway. Fundamental advances have been made in two specific areas. These include experiments that have provided new perspectives with respect to the nature of the soluble machinery involved in facilitating protein traffic from the cytoplasm to the lumen of the endoplasmic reticulum, and work that has provided a biochemical description of what may in effect represent a membranous ligand-gated channel that is required for protein translocation into the endoplasmic reticulum lumen.


The consequences of a change in formulation of methadone prescribed in a drug clinic. Methadone mixture DTF (1mg in 1ml) is a safe non-injectable alternative to methadone tablets (5mg). It also allows for a more gradual detoxification from opiate dependence. For these reasons it was decided to 'rationalise' our prescribing so that methadone in mixture form only would be dispensed. At the beginning of 1989, 66 opiate-dependent patients were receiving methadone tablets, 61 the methadone mixture. We report the consequences of instituting a policy change which was clearly very unpopular with patients. Of the 66 patients receiving methadone tablets prior to the change to mixture, 53 were represcribed tablets by the end of the 3-month follow-up period. In many cases this was because of an intense resistance to the change over, physical complications of methadone mixture occurred in very few. We were able to observe a decline in social stability and an increased use of non-prescribed drugs in some patients who changed to methadone mixture. More far-reaching consequences of the change included an increase in chemist break-ins, an increase in the street value of methadone tablets and greater hostility and threatening behaviour towards staff. These changes reflect psychological rather than pharmacological or pharmacokinetic effects.


Isolation of amine oxidase from bovine plasma by a two-step procedure. A novel method for isolation of amine oxidase from bovine plasma is reported; it involves a two-step procedure, namely ammonium sulfate fractionation and affinity chromatography with elution by aniline, which is a competitive inhibitor of the enzyme. A homogeneous enzyme, characterized by a specific activity of 0.44 U/mg, was obtained with a yield higher than 50%.


Purification methods for recombinant Lactobacillus casei thymidylate synthase and mutants: a general, automated procedure. General procedures for the rapid, large-scale purification of recombinant Lactobacillus casei thymidylate synthase and its mutants have been established. An effective method employs sequential phosphocellulose and hydroxylapatite chromatography. Crude cell extracts are directly applied to phosphocellulose, and the enzyme is obtained in a nearly pure state by stepwise elution with KCl. The eluate is directly applied to hydroxylapatite, and the homogeneous enzyme is eluted with a gradient of potassium phosphate. The method has been successful for the purification of recombinant wild-type enzyme and all mutants thus far examined. The entire purification procedure has been automated using a commonly available FPLC system and can be performed in several hours with minimal operator time.


Preparation and properties of an affinity support for purification of cyclic AMP receptor protein from Escherichia coli. Reaction of cyclic AMP with 1,1'-carbonyldiimidazole produces an intermediate which reacts with primary amines to provide a stable 2'-O-carbamyl derivative. This chemistry has been used to tether cyclic AMP to a Sepharose gel. The resulting affinity support has been used to effect a simple, nondenaturing purification of cyclic AMP receptor protein from crude cell extracts.


Reaction of bone to HA, carbonate-HA, hydroxyapatite + calcium orthophosphate and to hydroxyapatite + calcium ortho- and pyrophosphate. Slip cast conical implants of HA, carbonate-HA, calcium orthophosphate/hydroxyapatite and hydroxyapatite/calcium pyrophosphate/calcium orthophosphate with weight ratios 75/25 and 50/30/20 were implanted in rabbit tibia. The bone-implant interfaces were evaluated histologically, by means of SEM/EDX analysis and by push-out test. Thirty-six implanted samples were investigated after 2, 8, and 24 weeks. The triphasic calcium phosphate showed a moderate disintegration. This material showed 2 weeks after implantation a bonding between new bone and implant could be seen in parts of the operation site by SEM. Eight weeks after implantation an intimate relationship between the bone tissue and all specimens of each material was found. After 24 weeks the Ca/P ratio in the bone had reached the Ca/P ratio of mature bone determined by SEM/EDX analysis.


[The effect of ftorotan on the lipid and cAMP content of the liver in hypoxia]. A study was made of the content of cAMP, triglycerides and nonesterified fatty acids in the liver of rats anesthesized with halothane under conditions of spontaneous respiration depending on the oxygen content in the inhalation mixture. As the content of oxygen in the inhalation mixture was reduced, the content of cAMP in the liver was decreased, the intensity of lipolysis was disturbed with subsequent derangement of fatty acids oxidation. The raising of the oxygen content in the inhalation mixture did not disturb the activity of the adenylate cyclase system or provoked deep lipolysis disorders in the liver.


[The properties of the brain dopamine and GABA receptors in rats with chronic alcoholic intoxication with different ethanol withdrawal times]. Marked changes in dopamine and GABA receptors in the brain of rats with chronic alcohol intoxication were registered 48 h and 3 months after ethanol withdrawal. The changes in dopamine receptors characterized by the increased number of the sites of binding with ligand (Bmax) were most pronounced 48 h after ethanol withdrawal. In 3 months the properties of these receptors tended toward recovery. The changes in GABA receptors also manifested by the increase in Bmax become further pronounced 3 months after ethanol withdrawal.


[The neurochemical mechanisms underlying possible recurrences of alcoholism]. Experiments on chronic alcoholized rats revealed the similar changes in brain dopamine receptors, in brain and blood catecholamines as well as in blood cyclic adenosine monophosphate during both short- and long-term alcohol deprivation. It is concluded that such changes may form material basis for alcoholism relapses.


[The mechanism of the effect of reserpine on endocrine system function]. The experiments in rabbits and guinea-pigs showed that the use of reserpine, 0.1 and 0.25 mg/kg body weight a day, led to a 2-2.8-fold increase in thyroid hormones and 25-45% enhancement in cortisol in the blood. At the same time cAMP concentrations increased by 87% and almost by 5 times in the thyroid and adrenals, respectively. The pancreas exhibited a 5-fold increase in cAMP, 2.5- and 2.3-fold decrease in insulin in rabbit peripheral blood and in the activity of pancreatic aldolase and dehydrogenases, respectively. Blood levels of glucose reduced by 15-17%.


Effects of mediobasal hypothalamic lesion on immunoreactive ACTH/beta-endorphin levels in cerebrospinal fluid, in discrete brain regions, in plasma, and in pituitary of the rat. One week after complete destruction of the mediobasal hypothalamus, immunoreactive adrenocorticotropin (ACTH) and beta-endorphin levels were determined in cerebrospinal fluid, trunk blood, as well as in brain and pituitary tissue samples collected from anaesthetized and cisternally cannulated rats. Control rats were sham operated. In lesioned rats we observed: (a) 60% decrease in the immunoreactive beta-endorphin concentrations in the cerebrospinal fluid, (b) decreased immunoreactive ACTH and beta-endorphin levels in the hypothalamus, in the thalamus and in the amygdala, (c) unaffected immunoreactive ACTH/beta-endorphin levels in the septum and in the hippocampus, (d) decreased immunoreactive beta-endorphin levels both in the anterior and neurointermediate pituitary but unchanged immunoreactive ACTH contents in the anterior lobe, and (e) unaffected immunoreactive ACTH and beta-endorphin levels in the plasma under stressful conditions. From these findings the following conclusions can be drawn: (1) more than 50% of the beta-endorphin-like peptide content of the cerebrospinal fluid originates from the periventricular nuclei of the hypothalamus and thalamus in the rat; (2) the loss of the hypothalamic control probably enhances the intracellular proteolytic degradation of beta-endorphin both in the anterior and neurointermediate pituitary lobe; (3) rats with mediobasal hypothalamic lesion cannot react to the stressful stimuli of ether anaesthesia or cisternal cannulation with elevated plasma immunoreactive ACTH and beta-endorphin levels.


Zinc (Zn2+) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells. Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1-5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at -80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn2+) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5-15 min, depending upon the concentration used. The IC50 for inhibition of the peak current evoked by a step depolarization from -80 mV to 0 mV (N plus L channels) for 80 ms was 69 microM Zn2+ and the Hill slope about 1. The calcium current evoked by a voltage step from -80 mV to voltages between -40 mV and -15 mV (T-type current) was more sensitive (> 80% block with 20 microM Zn2+). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn2+ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.


Opioid receptors and awareness of the Greek alphabet. Several years ago a colleague, John Hugh Rees, set the following question for a final BSc Pharmacology exam: 'Describe current views about the classification of opioid receptors. Is the classification you describe of much relevance or has it merely served to make you more aware of the Greek alphabet?' This question is still relevant today.


Developmental changes in the levels of Ca2+/calmodulin-dependent protein kinase II alpha and beta proteins in soluble and particulate fractions of the rat brain. Developmental changes in Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been immunochemically examined in the forebrain, cerebellum and brainstem of the rat using antibodies against its alpha or beta protein. The concentration of alpha and beta proteins varied markedly in different brain regions at increasing postnatal ages. In early postnatal brain, the concentration of the alpha and beta proteins was low, and a large increase was observed between postnatal days 10 and 30. The maximum expression of the alpha protein was in the order of 6.01, 2.33 and 0.168 micrograms/mg of forebrain, brainstem and cerebellum proteins respectively, in the soluble or particulate fraction. On the other hand, that of the beta protein was in the order of 1.81, 0.495 and 0.291 micrograms/mg of forebrain, cerebellum or brainstem protein. The ratio of alpha and beta proteins also differed in the soluble and particulate fractions. The maximum expression of the alpha protein was observed at day 30 in soluble and particulate fractions of forebrain, and at day 20 in those of the brainstem. The major alpha protein peak was observed on or after day 30 in particulate and soluble fractions from cerebellum, respectively. The maximum expression of the beta protein was observed at day 20 in soluble and particulate fractions of the forebrain as well as in soluble fraction of the cerebellum, and was observed at day 30 in the particulate fraction of cerebellum. The expression of the alpha and beta proteins roughly correlated with the CaM kinase II activity from forebrain and brainstem.


Regulation of ornithine decarboxylase by parathyroid hormone in osteoblastic cell systems. Parathyroid hormone (PTH) has been shown to induce osteoblastic activity via a complex signal transduction process which is mediated either by cAMP or cytosolic calcium ([Ca2+]i), or a combination thereof. One of the PTH functions in osteoblasts is the induction of ornithine decarboxylase (ODC) activity. We have analyzed the second messengers involved in this process. 8-Bromo cAMP, a cAMP derivative, enhanced ODC activity in UMR106-01 osteoblastic cell system. The calcium ionophore A23187 and the protein kinase stimulator phorbol-12-myristate 13-acetate did not alter ODC activity. ODC activity was increased by bPTH-(1-34), PGE1, and PGE2 which stimulated both cAMP and [Ca2+]i. In contrast, PTH-(2-34), propionyl bPTH-(2-34), bPTH-(3-34), bPTH-(7-34), and PGF2 alpha, which only enhanced [Ca2+]i but not cAMP, had no effect on ODC activity. Thus, the stimulation of ODC in UMR106 cells by PTH appeared to be mediated primarily via the cAMP signal transduction pathway, and the mere increase in intracellular calcium could not account for the stimulation of ODC activity. ODC mRNA level was found to be increased by PTH treatment. Therefore, translation of ODC may be stimulated by PTH. Moreover, PTH also stimulated ODC antizyme activity, suggesting that the ODC degradation rate was increased.


Solubilization of functional receptors for parathyroid hormone and parathyroid hormone-related peptide from clonal rat osteosarcoma cells, ROS17/2.8. ROS17/2.8 cells, a cell line derived from a rat osteosarcoma, have abundant receptors for parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP). A particulate membrane fraction was prepared from these cells and it was solubilized using relatively mild conditions with digitonin (0.25%), a nonionic detergent. When radioligands of both PTH and PTHrP were incubated with this membrane fraction in the absence of any protease inhibitor at 15 degrees C, approximately 75% of these radioligands were degraded within 2 hours. This degradative activity was inhibited more effectively by bacitracin than by any of several other protease inhibitors tested. The digitonin-solubilized PTH/PTHrP receptors were radiolabeled in the presence of bacitracin using radioiodinated [Tyr36]PTHrP(1-36) amide (PTHrP(1-36)) and N-hydroxysuccinimidyl-4-azidobenzoate (HSAB), as cross-linker. When an aliquot of the reaction solution was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography, a broad band was observed that had an apparent molecular size of 90,000 daltons (M(r) = 90 kD). This band was no longer seen when the binding was conducted in the presence of 10(-6) M of unlabeled PTHrP(1-36), and it was decreased in density when binding was conducted in the presence of 10(-6) M of unlabeled [Nle8,18, Tyr34] bovine PTH(1-34) amide (NlePTH). The solubilized receptors retained their capacity to bind the radioligand after partial purification by wheat-germ agglutinin affinity-chromatography. The use of relatively mild detergent conditions thus offers a means to solubilize receptors that retain their capacity to bind PTH and PTHrP.


Nutritional deficiencies and gastrointestinal disorders in the edentulous elderly: a literature review. Many factors influence the food selection and nutritional status of elderly individuals. In this brief review, it is hypothesized that the functional status of dental prostheses is a prime determinant in the food selection, dietary fibre intake and prevalence of gastrointestinal disorders in edentulous elderly subjects.


Facial neuralgias: a clinical review of 34 cases. This paper reviewed the clinical features of the facial neuralgias, and described the demographic data for 24 cases of atypical facial neuralgia, seven cases of trigeminal neuralgia, two cases of cluster headache and one of glossopharyngeal neuralgia, which were all referred for investigation and treatment over a three-month period to a teaching hospital dental department.


Clonal chromosome changes in renal carcinoma do not correlate with clinical stages and histopathologic grades. We analyzed the correlations between chromosome abnormalities and clinical and histopathologic characteristics in 77 cases of renal cell carcinoma (RCC). Chromosome changes such as +5,+7,+8,+10,+18,+X,+Y, and -Y have been excluded from the analysis because they also occur in nonneoplastic kidney tissue and cytogenetic analysis indicates that these anomalies are not involved in tumor progression. The most frequent specific chromosome abnormalities in this sample were 3p rearrangements, trisomy 17, and hyperdiploidy and were not related to tumor stage or grade or to development of distant metastases.


Cytogenetic findings in a case of pediatric glioblastoma. We report a patient with glioblastoma multiforme (GBM) which showed stable and unstable telomeric associations involving the short arms of chromosomes 4 and 7. The karyotype was hyperdiploid, with chromosome numbers ranging from 84 to 87 in all cells, and showed a single stemline with variations in the number of marker chromosomes, teleomeric associations, and double minutes (dmin). The karyotype designation is 83-86,XX,-X,rea(X),-4,tas(4;7)(p16;?p22),der(6)t(6;?)(p21;?), -8, -9, der(9)t(9;?)(?p11;?), dup(9)(p12p23), -10 x 2, del(10)(p11), -11,del(11)(p11), -12, der(12)t(12;?) (p13;?),-13, -14 x 2,der(14)t(14;?) (p11;?), -16 x 2, -19, -21 x 2, -22 x 2, + 9-13mar, + dmin. Loss of the short arm of chromosome 10, structural aberrations of the short arm of chromosome 9, and dmin are consistent findings in GBM, whereas the high chromosome number is less common. Chromosome instability associated with the phenomenon of telomeric association/fusion has not been reported in GBM.


Establishment and characteristics of a T-cell acute lymphoblastic leukemia cell line, JK-T1, with a chromosomal translocation between 8q24 and 14q13. A human leukemia cell line, JK-T1, was established from the bone marrow of a 10-year-old boy with T-cell acute lymphoblastic leukemia. The origin of the leukemic cell line, JK-T1, was demonstrated by its chromosomal and immunologic similarity to the patient's fresh leukemic cells. Karyotypic analysis revealed 46,XY,del(6)(q?),t(8;14)(q24;q13),der(9)t(9;?)(q34;?). In JK-T1, neither rearrangement nor amplification of the c-myc gene was observed apparently because the breakpoint of chromosome 14 was not q11 but q13. JK-T1 was independent of interleukin 2 (IL-2) because of little production of IL-2, little IL-2 receptor (CD25) on the surface, and no response to exogenous IL-2. JK-T1 had lymphocyte function associated antigen-1 (LFA-1) (CD11a, CD18) on its surface and could adhere to the hematologic stromal layer. These characteristics of JK-T1 cell line are considered to be useful not only for evaluating the role of t(8;14) but also in studying the adhesion molecules of leukemia.


Invasion of connective tissue by human carcinoma cell lines: requirement for urokinase, urokinase receptor, and interstitial collagenase. We have screened six human squamous carcinoma cell lines for their ability to invade connective tissue by using the experimentally modified chorioallantoic membrane of a chick embryo as an in vivo model of invasion. In confirmation of our earlier studies, all the invasive cell lines expressed high levels of surface-bound urokinase type plasminogen activator (uPA). However, some cell lines expressing this activity were not invasive, suggesting that surface uPA, although necessary, was not sufficient. Since in addition to fibronectin, that can be degraded by uPA or plasmin, chorioallantoic membrane connective tissue contains collagen, we examined the profile of collagenases secreted by the various cell lines in search for an activity that would coincide with the invasive phenotype. We found, using gelatin substrate gels, that type IV gelatinase was produced by all six cell types tested, three cell types produced the M(r) 92,000 gelatinase, and three a lower-molecular-weight activity, which we identified by immunoprecipitation with specific antibodies, and by a direct assay of activity, as interstitial collagenase. Only the latter cells were found to be highly invasive. We showed previously that continuous culture in vitro of one of the carcinoma cell lines, HEp3, led to a gradual extinction of their malignant phenotype. To confirm the correlation between invasion and the production of interstitial collagenase, we examined these two functions in cells freshly isolated from a HEp3 tumor and intermittently during passage in vitro. We found that, although the surface uPA activity was slightly diminished in the in vitro grown cultures, it was still within the range of values found in highly malignant cells, suggesting that it is not the reason for the decrease in invasiveness. In contrast, the reduction in interstitial collagenase closely followed the loss of the invasive phenotype; after 30 in vitro passages the cells were almost completely devoid of interstitial collagenase and unable to invade. The decrease in collagenase activity was not the result of an increased tissue inhibitor of metalloproteinases production.


Tamoxifen stimulates human papillomavirus type 16 gene expression and cell proliferation in a cervical cancer cell line. The widely adopted use of tamoxifen as a chemotherapeutic agent is primarily based on its inhibition of cancer cell growth. However, we report that tamoxifen at low concentrations (10(-9) and 10(-11) M) causes stimulation of cell proliferation in a cervical cancer cell line, SFR. The facts that SFR cells do not contain estrogen receptors and are estrogen nonresponsive imply the existence of an antiestrogen-specific binding protein and suggest that the effect of tamoxifen is possibly mediated through a pathway other than estrogen receptors. Tamoxifen at low concentrations stimulated human papillomavirus type 16 (HPV-16) gene transcription and E7 protein production. Levels of HPV-16 mRNA and E7 protein reached a peak at approximately 2-4 h after tamoxifen treatment, persisted for several hours, and subsequently decreased to their prestimulation levels by about 24 h after treatment. Our results indicate for the first time that tamoxifen stimulates cell proliferation of cervical cancer cells, and we suggest that the enhanced HPV-16 mRNA and E7 protein levels are probably responsible.


Developmental regulation of annexin II (Lipocortin 2) in human brain and expression in high grade glioma. In experiments to identify molecules that might be important in the pathogenesis of glioblastoma multiforme, the most common malignant brain tumor, we found that annexin II (Lipocortin 2, p36), a likely second messenger in several different mitogenic pathways, was highly expressed in tumor tissue of glioblastoma multiforme (9 of 9) and highly anaplastic astrocytoma (2 of 6), but not in astrocytomas of lower pathological grade (0 of 6). We also detected high levels of annexin II expression in fetal brain during the period when radial glia proliferate, although annexin II expression was not detected in normal adult brain. These data demonstrate that annexin II expression is developmentally regulated in the human central nervous system and suggest that the early progenitor radial glia share important characteristics with highly malignant glial tumors.