New monoclonal antibodies for the detection of immediate early antigens of cytomegalovirus. Two new monoclonal antibodies, CIE-1 and CIE-2, were developed for the rapid detection of human cytomegalovirus (HCMV) infection. They were found to be reactive with immediate early protein of HCMV in the nuclei of infected fibroblasts, as early as 3 hours post-infection. By radioimmunoprecipitation, CIE-1 was found to react with a protein with an apparent molecular weight of 70,000, whereas CIE-2 precipitated 2 proteins of 70,000 and 72,000 daltons, respectively. Both monoclonal antibodies recognized three prototype strains of HCMV: AD-169, Towne, and Davis, and did not cross-react with other human herpesviruses. CIE-1 and CIE-2 were compared with four commercial anti-HCMV monoclonal antibodies (Clonab, Dupont, Sera-Lab and Syva) by testing 88 clinical isolates. Culture confirmation tests and shell vial assays showed that CIE-1 and CIE-2 were more sensitive than several of these reagents and equally sensitive to the Dupont reagent. Moreover, CIE-1 and CIE-2 produced a bright, sharp staining of the nuclei of infected cells. These monoclonal antibodies should thus be valuable in rapid diagnosis of HCMV.


Immunization of cats against feline infectious peritonitis with anti-idiotypic antibodies. Anti-idiotypic antibodies (Ab2s) generated against neutralizing antibodies (Ab1s) specific for feline infectious peritonitis virus (FIPV) were shown to be specific for paratope-associated idiotopes of the Ab1s and not against isotypic determinants. In a study to determine the efficacy of an anti-idiotypic vaccine against feline infectious peritonitis (FIP), cats that were immunized with a pool of monoclonal Ab2s developed Ab3s that recognized the variable regions of the Ab2s as well as the natural antigen. In cats challenged with a lethal dose of virus the control group followed a predictable course of infection ultimately succumbing to FIP. Two immunized cats survived virus challenge and a third cat lived twice as long as the controls. The fourth immunized cat showed no evidence of protection. The ability to induce levels of protection against FIP lends support to the concept of using anti-idiotypic antibodies as a prophylactic vaccine.


How do CTL control virus infections? Evidence for prelytic halt of herpes simplex. Cytotoxic T lymphocytes (CTL) induce in target cells a rapid, prelytic fragmentation of target cell DNA, accompanied by apoptosis. In contrast, complement and (with a few exceptions) chemical and physical means of inducing cytolysis induce necrosis, without DNA fragmentation. The function of the unusual DNA fragmentation induced by CTL remains to be elucidated. The major recognized function of CTL is in halting virus infections. Earlier, we proposed that CTL might halt virus infections prelytically, by fragmenting viral and cellular nucleic acids, and that in this case, cytolysis per se might be a less important function of CTL. We report here experiments designed to detect prelytic halt of virus replication. We employed in vivo-like conditions: fibroblast targets (difficult to lyse) were infected with herpes simplex virus (HSV), then incubated at low E/T cell ratios overnight. At the highest E/T ratios which produced less than 10% CTL-induced lysis, plaque-forming unit yield was reduced by about 50%. At higher E/T ratios which lysed 1/6 to 1/3 of the infected target cells, 3/4 to 9/10 of the virus production was prevented. The discrepancy between the level of lysis and the reduction in virus yield is evidence for significant CTL-induced prelytic halt of HSV replication. At present, it is unclear whether the antiviral effect observed involves an activity of CTL distinct from their lytic ability, such as their DNA fragmenting ability.


Dietary effects of barley fibre, wheat bran and rye bran on bile composition and gallstone formation in hamsters. The effects of brewer's spent grain (BSG), wheat bran and rye bran on bile composition, gallstone formation and serum cholesterol were studied in Syrian golden hamsters. The frequency of gallstone formation in the animals fed diets supplemented with low (10%) and high (20%) concentrations of BSG or wheat bran was significantly lower than that of the animals fed a stone-provoking, fibre-free diet. The ratios of secondary to primary bile acids were lower in the animals fed the diets supplemented with a high dose of BSG, wheat bran and rye bran than in the controls. The ratio of LCA to DCA was reduced only in the animals fed the diet supplemented with rye bran as compared with controls. No significant changes in bile and serum cholesterol levels were observed in the experiment.


Tumours classified as "malignant histiocytosis" in children are T-cell neoplasms. During the last five years increasing evidence has accumulated that many tumours classified as 'histiocytic' in the past do not originate from macrophages, but from transformed (or anaplastic) large lymphoid cells. Most of these studies have focused upon adult neoplasms. Knowledge concerning the lineage of 'histiocytic' tumours in the paediatric age group is more limited. In this study we have examined the clinical, morphological and immunophenotypical features of six childhood malignancies originally diagnosed as being of histiocytic origin. Three patients showed an aggressive course with involvement of internal organs and very short survival times. Two patients were brought into remission: one is alive without active disease after seven years; the other died after seven years due to treatment-related cardiomyopathy. The remaining patient had a protracted course for two and a half years, but subsequently deteriorated and died three years after diagnosis. The histomorphological features in five cases were those of anaplastic large cell lymphomas. The remaining case consisted of pleomorphic (rather than anaplastic) large lymphoid cells. In all cases the immunophenotypical examination showed features characteristic of activated T lymphocytes. All cases were positive for Ki-1 (CD30), and three were positive for epithelial membrane antigen (EMA). Histiocyte-associated markers were positive in residual reactive macrophages, but nowhere could unequivocal positivity for macrophage-associated markers be seen in the neoplastic cells. It is concluded that most childhood malignancies in the past classified as 'histiocytic' are examples of anaplastic large cell (Ki-1) lymphomas of T-cell type and that true histiocytic malignancies are exceedingly rare in the paediatric age group.


Genetic characterization of Sabin types 1 and 3 poliovaccine virus following serial passage in the human intestinal tract. Poliovirus isolates types 1 and 3 were obtained from five and seven successive passages respectively, in infants who had been fed monovalent OPV in two separate clinical trials conducted in 1960. The purpose of these trials was to answer the question how much the vaccine virus would revert to its original neurovirulent phenotype following multiplication in the intestinal tract. Human passages were performed either by contact exposure or by feeding the excreted virus while the infants were maintained in isolation. Several virus isolates were obtained at each passage level. Infants participating in both studies showed no symptoms of disease. Antigenic studies (McBride, van Wezel) and protein analysis (PAGE) of the isolates, reported earlier from this laboratory, had shown that the isolates remained vaccine-like, although isolates from the later passages revealed some differences. Monkey neurovirulence test results showed that for both types 1 and 3 viruses the loss of attenuation of the vaccine strain upon passage was gradual, although the loss was faster for type 3. Examination of the oligonucleotide maps demonstrated that the oligonucleotide configuration of the isolates remained the same as for the vaccine strain but there was an increase of individual spot differences with increasing passage. The nucleotide sequence analysis of selected regions of the virus genomes revealed that there was no change from a G to A in nucleotide 480 of type 1 isolates; however, nucleotide 476 changed from a U to an A in type 1 passages 3, 4 and 5. Conversely, for type 3 the change of nucleotide 472 from a U to a C changed at the early first passage (4 days following administration of OPV), and remained a C in the six following passages; type 3 nucleotide 2034 did not change in the first passage from a U to a C, but it became a C in all further passages tested. The nucleotide changes mentioned for both virus types remained stable in successive passages. However, there was another nucleotide change for type 3 from a U to a C at position 1973 only for passages 5 and 6 which reverted to a U for passages 7L and 7LL. Study of selected human passage virus strains could further contribute to the identification of the critical nucleotides that are responsible for the attenuation of these two polio types of vaccine viruses.


Functional alterations of murine peritoneal macrophages during pregnancy. We have studied some functional characteristics of murine peritoneal macrophages (MOp) related to hormonal changes produced during pregnancy. Maximal expression of Ia antigen and release of interleukin 1 (IL1) were observed during the first week of pregnancy (implantation), when the highest peak of estradiol was produced. Both Ia antigen expression and IL1 levels progressively decreased as gestation advanced. Inversely, MOp capability to phagocyte and reduce nitro blue tetrazolium (NBT) was diminished at the beginning of pregnancy but returned to normal values in the last week. Sexual steroid levels (estradiol and progesterone) were inversely related, and the release of prostaglandin E2 (PGE2) by MOp decreased when progesterone levels increased. Although PGE2 production had no evident relation with Ia antigen expression and IL1 activity during the first and second weeks of pregnancy, the increment in PGE2 values and percentages of NBT+ cells could indicate a different stage of macrophage activation. These results suggest a possible hormonal regulation of macrophage functionality.


Possible influence of measles virus infection of cynomolgus monkeys on the outcome of the neurovirulence test for oral poliovirus vaccine. Macaque monkeys are susceptible to measles infection which triggers temporary immuno-depression similar to the well known phenomenon in humans. It is known that feral monkeys become infected with measles virus when they are exposed to humans. Since Macaca mulatta and M. fascicularis are species used to assay the neurovirulence of oral poliovirus vaccine, the immunodepression caused by measles infection of the test monkeys could significantly alter the results of the neurovirulence test. The serum titers of measles-neutralizing antibodies were studied in over 1500 monkeys used for neurovirulence tests. A high proportion of the feral monkeys had measles antibodies (51-100%); in contrast, none of 493 M. fascicularis monkeys which had been bred in a primate colony under strict isolation measures was found positive for measles antibodies. An increase in the prevalence of measles in the population of Ontario and Quebec provinces was accompanied with an increase in the proportion of measles-positive monkey and their serum antibody titers were found higher. It was observed that monkeys used in tests that had been performed during high measles prevalence presented with a poliomyelitis of more pronounced severity clinically and histologically. The analysis of 29 tests conducted on type 1 vaccines over several years showed a positive correlation (correlation coefficient = 0.5141, P less than 0.0022) between severity of poliomyelitis and the presence of measles serum antibodies in test monkeys (some animals seroconverted during the test). A similar observation, when type 3 Sabin vaccines were tested in M. fascicularis, was recently reported from another laboratory in Ontario.


Comparison of EBV-infectivity measured by EBNA-induction and immortalization of human B-cells. Epstein-Barr virus (EBV) infectivity was measured by two different systems. One was EBV-specific antigen (EBNA) induction in EBV-negative BJAB cells, the other was immortalization of human peripheral blood B-lymphocytes upon EBV infection. Because the sensitivity of the EBNA-induction assay, which takes 2-3 days, was similar with that of B-cell immortalization, which takes 6-8 weeks, the former can be used for the rapid and convenient determination of EBV-infectivity.


Polysialic acid influences specific pathfinding by avian motoneurons. The influence of polysialic acid (PSA) on the neural cell adhesion molecule on motoneuron outgrowth and pathway formation was investigated by determining its temporal and spatial pattern of expression and by the effect that its removal had on motoneuron projection patterns. Motoneurons first expressed PSA as their growth cones began to segregate into motoneuron pool-specific groups in the plexus region; furthermore, PSA levels differed between motoneurons projecting to different targets. When PSA was removed during the period of axonal segregation in the plexus region projection errors were common. However, later removal during the process of muscle nerve formation did not result in projection errors. These results suggest that PSA modulates interactions between motoneuron axons and guidance molecules in the plexus region during axonal pathfinding.


RNA splicing regulates agrin-mediated acetylcholine receptor clustering activity on cultured myotubes. Agrin is a component of the synaptic basal lamina that induces the clustering of acetylcholine receptors (AChRs) on muscle fibers. A region near the carboxyl terminus of the protein exists in four forms that are generated by alternative RNA splicing. All four alternatively spliced forms of agrin are active in inducing AChR clusters on rat primary and C2-derived muscle fibers. In contrast, only two forms of the protein, each containing an 8 amino acid insert, are capable of inducing clusters on myotubes of S27 cells, a C2 variant that has defective proteoglycans. These two forms are also most active in inducing clusters on chick myotubes. This pattern of differential activity suggests that RNA splicing of agrin transcripts and interactions with proteoglycans or other components of basal lamina have important roles in regulating the localization of neurotransmitter receptors at synaptic sites.


Functional modulation of brain sodium channels by cAMP-dependent phosphorylation. Voltage-gated Na+ channels, which are responsible for the generation of action potentials in brain, are phosphorylated by cAMP-dependent protein kinase in vitro and in intact neurons. Phosphorylation by cAMP-dependent protein kinase reduces peak Na+ currents 40%--50% in membrane patches excised from rat brain neurons or from CHO cells expressing type IIA Na+ channels. Inhibition of basal cAMP-dependent protein kinase activity by transfection with a plasmid encoding a dominant negative mutant regulatory subunit increases Na+ channel number and activity, indicating that even the basal level of kinase activity is sufficient to reduce Na+ channel activity significantly. Na+ currents in membrane patches from kinase-deficient cells were reduced up to 80% by phosphorylation by cAMP-dependent protein kinase. These effects could be blocked by a specific peptide inhibitor of cAMP-dependent protein kinase and reversed by phosphoprotein phosphatases. Convergent modulation of brain Na+ channels by neurotransmitters acting through the cAMP and protein kinase C signaling pathways may result in associative regulation of electrical activity by different synaptic inputs.


Primary structure and alternative splice variants of gephyrin, a putative glycine receptor-tubulin linker protein. A 93 kd polypeptide associated with the mammalian inhibitory glycine receptor (GlyR) is localized at central synapses and binds with high affinity to polymerized tubulin. This protein, named gephyrin (from the Greek gamma epsilon phi upsilon rho alpha, bridge), is thought to anchor the GlyR to subsynaptic microtubules. Here we report its primary structure deduced from cDNA and show that corresponding transcripts are found in all rat tissues examined. In brain, at least five different gephyrin mRNAs are generated by alternative splicing. Expression of gephyrin cDNAs in 293 kidney cells yields polypeptides reactive with a gephyrin-specific antibody, which coprecipitate with polymerized tubulin. Thus, gephyrin may define a novel type of microtubule-associated protein involved in membrane protein-cytoskeleton interactions.


Predictability of FEV1 after pulmonary resection for bronchogenic carcinoma. The aim of this study was to review the reliability of prediction of postoperative FEV1 in patients with bronchogenic carcinoma using a Tc-99m perfusion scan and simple spirometry. Over a 27-month period, 40 patients without known recurrent disease had their FEV1 measured. One quarter of the postoperative values for FEV1 differed from predicted values by less than 5% (2/11 pneumonectomies, 5/23 lobectomies, 3/6 segmental resections) and half differed by no more than 10% of predicted FEV1 (4/11 pneumonectomies, 12/23 lobectomies, 3/6 segmentectomies). One tenth of the predicted values differed by more than 30% and up to 760 mls (1/11 pneumonectomies, 2/23 lobectomies, 1/6 segmentectomy). Disease recurrence, phrenic nerve paralysis, exacerbation of obstructive pulmonary disease and poor collaboration during spirometry explained the most severe erroneous results. Age, preoperative smoking, tumour stage and histology, absence of symptoms at the time of diagnosis and adjuvant radiotherapy showed no statistically significant effect on predictability. Twenty-one patients had a postoperative Tc-99m pulmonary scan simultaneous to the spirometric control. Overestimation of postoperative FEV1 was associated with heterogeneous distribution of ventilation and perfusion.


The antioxidant potential of propofol (2,6-diisopropylphenol). We have examined in vitro the antioxidant properties of 2,6-diisopropylphenol. In studies using electron spin resonance spectroscopy we have demonstrated that 2,6-diisopropylphenol acts as an antioxidant by reacting with free radicals to form a phenoxyl radical--a property common to all phenol-based free radical scavengers. In additional experiments, the antioxidant properties of the clinical formulation of 2,6-diisopropylphenol (propofol) have been measured using an assay of antioxidant potential. In these experiments, propofol, but not Intralipid, was found to exhibit significant antioxidant activity, such that in the range of propofol concentrations examined (10(-6)-10(-5) mol litre-1), each molecule of 2,6-diisopropylphenol was able to scavenge two radical species. We conclude that the free radical scavenging properties of 2,6-diisopropylphenol resemble those of the endogenous antioxidant alpha-tocopherol (vitamin E).


Management of pituitary adenomas.

Management of pituitary adenomas. Pituitary adenomas arise from the anterior lobe of the pituitary gland and may secrete, in poorly controlled fashion, one or more of the hormones normally produced at this site, leading to specific endocrine syndromes. Pituitary tumors may also compress or invade adjacent structures, and the function of the normal pituitary may be compromised when a sellar mass exerts pressure on the stalk or on the gland itself. Advances in radiologic imaging and in microsurgical apparatus have made pituitary tumors more amenable to treatment, with surgery the treatment of choice. Radiotherapy is used mainly to control residual tumor after incomplete excision. Remission can be obtained in up to 90% of patients with microadenomas and in about 50% to 60% of those with macroadenomas.


Temporal correlation of succinylcholine-induced fasciculations to loss of twitch response at different stimulating frequencies. The present study was undertaken to determine the time courses of succinylcholine-induced fasciculations and adductor pollicis single-twitch responses at two stimulating frequencies. A prospective, randomized study. The main operating room of a university teaching hospital. Forty-four patients undergoing general anesthesia and requiring tracheal intubation. In 22 patients, anesthesia was induced with thiopental sodium, fentanyl, and midazolam and maintained with 70% nitrous oxide in oxygen prior to the administration of succinylcholine 1 mg/kg. In another 22 patients, end-tidal isoflurane 0.75% to 1% was included in the induction regimen. Patients in each group were randomly assigned to receive ulnar nerve stimulation at either 0.1 Hz or 1.0 Hz. The times at which fasciculations were first noticed and no longer visible and the times to 25%, 50%, 75%, 90%, and 100% twitch depression were recorded. These times were compared for the two rates of neurostimulation. With single-twitch nerve stimulation at 1.0 Hz, 100% twitch depression occurred 6 +/- 16 seconds following the end of fasciculations, while at a stimulating frequency of 0.1 Hz, it occurred 52 +/- 32 seconds later (p less than 0.05). At the end of fasciculations, single-twitch depression of 50% or more was noted in only 19% of patients in the 0.1 Hz groups and in all patients in the 1.0 Hz groups (p less than 0.05). In the 0.1 Hz groups, 50% twitch depression occurred 19 +/- 27 seconds after the end of fasciculations, with more than three-quarters of the patients having achieved 50% twitch depression 30 seconds following the disappearance of fasciculations. The addition of isoflurane did not significantly alter any of these times. The data reveal that cessation of fasciculations may be an inaccurate clinical sign of the readiness for intubation and confirm that standardized methods of neurostimulation are necessary in the pharmacodynamic evaluation of neuromuscular blocking drugs. In settings where profound neuromuscular relaxation is not required, waiting at least 30 seconds beyond the disappearance of fasciculations should provide good intubating conditions.


The effects of hormonal and other stimuli on cell-surface Ro/SSA antigen expression by human keratinocytes in vitro: their possible role in the induction of cutaneous lupus lesions. Ultraviolet B light (UVB) has previously been shown to induce the expression of the extractable nuclear antigens (e.g. Ro/SSA) on the surfaces of human keratinocytes in vitro. This study assessed whether injurious, metabolic, inflammatory, immunological or hormonal stimuli would also induce this expression or modulate that produced by UVB. No stimulus initiated expression alone, but 17-beta oestradiol doubled that found in response to UVB. These findings confirm the potential role of UVB in the initiation and potentiation of cutaneous lupus lesions and may help to explain the female preponderance of the disease.


Genetic aspects of the Klippel-Trenaunay syndrome. An extensive search for a genetic pattern in Klippel-Trenaunay syndrome (KTS) revealed two other cases of KTS in the families of two of the 86 patients with this vascular syndrome who were questioned. Patients with KTS also had family members with other malformations: e.g. hemihypertrophy in one family, and a prevalence of 7/400 of naevi flammei in first-degree relatives of KTS patients was observed. We suggest that KTS can be inherited in a multifactorial way and a range of vascular malformations can be observed in the family members of patients with this syndrome.


Intrahepatic cholangiocarcinoma: clinical aspects, pathology and treatment. Intrahepatic cholangiocarcinoma (ICC) is the second most common primary tumor of the liver. To further define its clinicopathology and surgical management, we reviewed our experience. Clinical presentations of 32 patients with ICC was similar to that with hepatocellular carcinoma. Jaundice occurred in only 27 percent. ICC was unresectable due to advanced disease stage in 81 percent. Six patients had curative resections with two 5 year disease free survivors. Underlying liver disease was associated with ICC in 34 percent of patients.


A discrete mode of the antipyretic action of AVP, alpha-MSH and ACTH. The antipyretic effect of AVP, alpha-MSH and ACTH consists in lowering the thermoregulatory threshold and in shortening the time span of the fever. Thus, neuropeptides influence activity of hypothalamic neurones regulating body temperature. This was confirmed by recent experiments of Moravec (this volume) which indicate that spontaneous activity and thermosensitivity of neurones in hypothalamic slices can be influenced, by AVP. Why neuropeptides of different chemical structure such as AVT, on one hand, and alpha-MSH and ACTH, on the other hand, induce the same effect on thermoregulation remains to be elucidated.


Effects of temperature and neuroactive substances on hypothalamic neurones in vitro: possible implications for the induction of fever. This paper reviews some of our findings which have shown the usefulness of in vitro methods in the study of hypothalamic neurones. (1) Membrane current analyses of dispersed neurones of the rat preoptic and anterior hypothalamus (POA) during thermal stimulation have revealed that warm-sensitive neurones are endowed with a non-inactivating Na+ channel having a high Q10 in the hyperthermic range (35-41 degrees C). (2) A brain slice study has shown that neurones in the organum vasculosum lamina terminalis (OVLT) region have much higher sensitivity to PGE2 than POA neurones. This provides further evidence of a critical role of the OVLT in translation of blood-borne cytokine signals into brain signals for fever induction. (3) Local application of IL-1 beta and IFN alpha altered the activity of thermosensitive (TS) neurones and glucose responsive (GR) neurones in vitro in an appropriate way to produce fever and anorexia. While the responses to IL-1 beta required the local release of prostaglandins, the responses to IFN alpha were found to be mediated by opioid receptor mechanisms. (4) The responses of POA TS neurones and VMH GR neurones to IL-1 beta but not those to IFN alpha, were reversibly blocked by alpha MSH, an endogenous antipyretic peptide. Thus, immune cytokines and their related neuroactive substances may affect hypothalamic TS and GR neurones thereby producing elaborately regulated changes in homeostatic functions such as thermoregulation (fever) and feeding (anorexia), which are considered as host defence responses.


In vitro receptor autoradiography: a map for exploring the hypothalamus. In rabbits and guinea pigs, hypothalamic sites for prostaglandin E2 (PGE2) action were studied by means of in vitro receptor autoradiography. The density of PGE2 binding sites (probably PGE2 receptors) was the highest in the anterior wall of the third ventricle (A3V). This result is consistent in all mammalian species ever studied, suggesting a fundamental role of the A3V in the hypothalamic action of PGE2, such as fever.


Binding of a mitochondrial presequence to natural and artificial membranes: role of surface potential. The binding of a synthetic mitochondrial presequence to large, negatively charged, unilamellar vesicles and to unenergized yeast mitochondria has been measured. The presequence, which corresponds to the amino-terminal 25 residues of the yeast cytochrome oxidase subunit IV precursor, was labeled with a fluorescent probe and used to examine the importance of the surface potentials of membranes on the interactions with the presequence. Binding of the fluorescent presequence to the membranes was determined by measuring a decrease in the fluorescence emission of the bound presequence. Binding both to the vesicles and to the mitochondria could be described as a simple partitioning of the presequence between the aqueous and lipid phases. The partitioning was found to depend on the ionic strength of the medium, and the Gouy-Chapman theory could be used to describe the partitioning at various ionic strengths. Application of the theory allowed the determination of an apparent charge on the presequence (+2.31 +/- 0.25), salt-independent apparent partition coefficients for vesicles (99 +/- 84 M-1) and for unenergized mitochondria (14.5 +/- 3.6 L g-1), and an estimated charge density for the mitochondrial outer membrane (-0.0124 +/- 0.0016 C m-2). This study shows that electrostatic effects are significant for the binding of a mitochondrial presequence both to lipid vesicles and to mitochondria, the natural target membrane of the presequence. The accumulation of positively charged presequences at the negative mitochondrial surface and the subsequent partitioning of the presequences directly into the mitochondrial outer membrane probably represent early steps in the translocation of precursor proteins into mitochondria.


A kinetic and allosteric model for the acetylcholine transporter-vesamicol receptor in synaptic vesicles. The ligand binding relationship between the acetylcholine transporter (AcChT) and the vesamicol receptor (VR) and the kinetics of active transport were studied in synaptic vesicles purified from the Torpedo electric organ using analogues of AcCh and vesamicol. Methoxyvesamicol, which should exhibit better equilibration properties for kinetics measurements than the more potent parent, inhibits active transport in a nonlinear noncompetitive manner. AcCh analogues competitively inhibit binding of [3H]vesamicol with higher affinity in hyposmotically lysed vesicle ghosts than in intact vesicles, apparently due to removal of a competing internal, osmotically active factor. AcCh and actively transported analogues of AcCh that are up to 57% larger in van der Waals volume exhibit up to a 200-fold ratio for the dissociation constant measured by inhibition of vesamicol binding to ghosts (KIAg) compared to the Michaelis constant for transport (KM) or the IC50 value for inhibition of [3H]AcCh active transport. In contrast, two AcCh analogues that are about 120% larger and that almost surely are not transported exhibit a KIAg/IC50 ratio of about 1. The data demonstrate that the vesamicol family of compounds binds to an allosteric site in the AcChT. Initiation of active transport has no apparent effect on the affinities of vesamicol and AcCh analogues, which suggests that most of the AcChT-VR in purified vesicles is transport incompetent. Vesicle ghosts actively transport [3H]AcCh nearly as well as intact vesicles, which suggests that internal factor does not affect transport-competent AcChT-VR. A kinetics model is proposed that predicts that AcCh analogues exhibiting a KIAg/IC50 ratio significantly greater than 1 are actively transported. Some of the microscopic constants in the model are estimated. The AcChT binds AcCh very weakly with a dissociation constant of about 20-50 mM, but it transports substrates rapidly in a process exhibiting remarkably little selectivity for the detailed shape and volume of the transported ion.


Comparison of new iron chelating agents in the prevention of ischemia/reperfusion injury: a swine model of heart-lung transplantation. The preservation of the heart and lung for transplantation remains a major concern in extended ischemic intervals. This experimental endeavor evaluates and compares the efficacy of iron chelating agents such as high molecular weight deferoxamine and 21-aminosteroid (U74006F) in a swine model of heart-lung transplantation. Heat-lung blocks were exposed to 4 h and 45 min of ischemia and 2 h of reperfusion. Animals were divided into three groups. Group A was a control without pharmacological intervention. In groups B and C, 21-aminosteroid (U74006F), 10 mg/kg, and high molecular weight deferoxamine, 50 mg/kg, were used, respectively. The results of functional parameters (cardiac index, stroke index, lung water, PO2, PCO2, alveolar-arterial gradient, and alveolar-arterial ratio) demonstrated superior heart and lung function for group C, where high molecular weight deferoxamine was used. Alterations of heart and lung function were significantly more (p less than .001) for control animals and for group B where U74006F was used. This study suggests that formation of hydroxyl radicals was affected by chelation of iron with high molecular weight deferoxamine, which reflects better heart and lung function and consequently less damage to this group of animals. The compound 21-aminosteroid U74006F failed to protect the heart and lung from ischemic-reperfusion injury in this model of heart-lung transplantation.


Photoaffinity labeling of the acetylcholine transporter. The acetylcholine (AcCh) binding site in the AcCh transporter-vesamicol receptor (AcChT-VR) present in synaptic vesicles isolated from the electric organ of Torpedo was characterized. A high-affinity analogue of AcCh containing an aryl azido group, namely, cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide (AzidoAcCh), was synthesized in nonradioactive and highly tritiated forms. AzidoAcCh was shown to be a competitive inhibitor of [3H]AcCh active transport and binding of [3H]-vesamicol to the allosteric site. The [3H]AzidoAcCh saturation curve was determined. In all cases the AcChT.AzidoAcCh complex exhibited an inhibition or dissociation constant of about 0.3 microM. Binding of [3H]AzidoAcCh was inhibited by vesamicol and AcCh. AzidoAcCh irreversibly blocked greater than 90% of the [3H]vesamicol binding sites after multiple rounds of photolysis and reequilibration with fresh ligand. Autofluorographs of synaptic vesicles photoaffinity-labeled with [3H]AzidoAcCh showed specific labeling of material exhibiting a continuous distribution from 50 to 250 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The result demonstrates that the AcChT has an unexpected structure highly suggestive of the synaptic vesicle proteoglycan.


Linkage of the acetylcholine transporter-vesamicol receptor to proteoglycan in synaptic vesicles. The relationship of the acetylcholine transporter-vesamicol receptor (AcChT-VR) to proteoglycan in Torpedo electric organ synaptic vesicles was investigated. The cholate-solubilized VR was immunoprecipitated by a monoclonal antibody directed against the SV1 epitope located in the glycosaminoglycan portion of the proteoglycan. AcChT that was photoaffinity-labeled with a tritiated high-affinity analogue of AcCh [cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate] and then denatured in sodium dodecyl sulfate also immunoprecipitated. The labeled AcChT exhibited a M(r) range of 100,000-200,000. Proteoglycan did not engage in detectable nonspecific reversible aggregation that might mask the presence of another subunit during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In vesicles permeabilized with cholate, the enzymes keratanase and testicular hyaluronidase inactivated binding of vesamicol and destroyed the SV1 epitope without detectable proteolysis. Other glycosaminoglycan-degrading enzymes were without effect. The results demonstrate that the AcChT-VR and proteoglycan are very strongly linked and that glycosaminoglycan-like polysaccharide controls the conformation of the VR. The unexpected linkage to proteoglycan suggests that AcChT-VR in intact terminals might communicate with extracellular matrix and participate in stabilization and operation of the synapse.


Differential nucleotide binding to catalytic and noncatalytic sites and related conformational changes involving alpha/beta-subunit interactions as monitored by sensitive intrinsic fluorescence in Schizosaccharomyces pombe mitochondrial F1. Mitochondrial F1 from the yeast Schizosaccharomyces pombe exhibits an intrinsic tryptophan fluorescence sensitive to adenine nucleotides and inorganic phosphate [Divita, G., Di Pietro, A., Deléage, G., Roux, B., & Gautheron, D.C. (1991) Biochemistry 30, 3256-3262]. The present results indicate that the intrinsic fluorescence is differentially modified by nucleotide binding to either catalytic or noncatalytic sites. Guanine or hypoxanthine nucleotides, which selectively bind to the catalytic site, produce a hyperbolic saturation monitored by fluorescence quenching at 332 nm, the maximal emission wavelength. On the contrary, adenine nucleotides, which bind to both catalytic and noncatalytic sites, exhibit a biphasic saturation. High-affinity ATP binding produces a marked quenching as opposed to the lower-affinity one. In contrast, ADP exhibits a sigmoidal saturation, with high-affinity binding producing no quenching but responsible for positive cooperativity of binding to the lower-affinity site. The catalytic-site affinity for GDP is almost 20-fold higher at pH 5.0 as compared to pH 9.0, and the high sensitivity of the method allows detection of the 10-fold lower-affinity GMP binding. In contrast, high-affinity binding of ADP, or AMP, is not pH-dependent. The selective catalytic-site saturation induces a F1 conformational change decreasing the Stern-Volmer constant for acrylamide and the tryptophan fraction accessible to iodide. ATP saturation of both catalytic and noncatalytic sites produces an additional reduction of the accessible fraction to acrylamide.


Role of CO2 in proton activation by histidine decarboxylase (pyruvoyl). The amino acid decarboxylases that use an intrinsic pyruvoyl cofactor have been viewed in terms of the pyridoxal-P paradigm whereby a Schiff base is formed between the enzyme-bound cofactor and the substrate, setting up a cation sink for electrons of the C alpha-CO2- bond, ejecting CO2, and the reversal of these steps with a proton with overall retention stereochemistry. With histidine decarboxylase (pyruvoyl) it is found that the presence of CO2 is required for T-exchange between histamine and water. Since the forward reaction including formation of the C-H bond does not require added CO2, it might be assumed that the CO2 that is formed in the fragmentation step is retained by the enzyme perhaps to assist in proton transfer. No such requirement for CO2 has been reported for the pyridoxal-P-dependent decarboxylases which are generally thought to liberate CO2 prior to proton transfer. In seeking a connection between bound CO2 and proton transfer in the histidine decarboxylase reaction, one is reminded of the carboxybiotin enzymes also known for an invariant stereochemistry of retention and for the requirement that the biotin be in the carboxylated form for H-exchange to occur. Perhaps the bound CO2 of histidine decarboxylase forms a carbamate by addition to Lys155 or to an amide group of the active site. The new carboxy group could then be the vehicle for protonating the carbon from which it originated, giving overall retention of the stereochemistry at the alpha-C.(ABSTRACT TRUNCATED AT 250 WORDS)


Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy. In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits. Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system. In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence. Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta. Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C. Unlike trypsin, the latter enzyme did not remove PDE's ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly. The results indicate that mutations in many regions of gamma affect its binding to alpha beta. A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity. However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)


Spectrophotometric detection of the interaction between cytochrome c and heparin. Heparin inhibits transport of electrons from reduced cytochrome c to cytochrome c oxidase. The effect is due to the interaction of heparin with cytochrome c. It has been observed that binding of heparin to the reduced or oxidized cytochrome c changes the spectrum of cytochrome c at the Soret region. Affinity chromatography of heparin in cytochrome c immobilized to thiol-Sepharose shows that commercial heparin is eluted in the low-affinity and high-affinity fractions. Both participate in the interaction with cytochrome c. Polylysine induces decay of the cytochrome c-heparin complex.


Pleiotropic effects of pufX gene deletion on the structure and function of the photosynthetic apparatus of Rhodobacter capsulatus. By deletion of the pufX gene of Rhodobacter capsulatus from a plasmid carrying the puf operon and complementation of a chromosomal puf operon deletion, we created pufX mutants and used them to characterize possible functions of the pufX gene product. The pufX mutants were incapable of photosynthetic growth in a minimal medium, or in a rich medium at low light intensities, although second-site mutations suppressed this phenotype. Measurements made in vitro with intact and solubilized chromatophore preparations indicated that the individual complexes of the photosynthetic unit seemed to function normally, but electron transfer from the reaction center to the cytochrome b/c1 complex was impaired. The structures of the photosynthetic apparatus of pseudo-wild type and mutant strains were evaluated using absorption spectroscopy and electron microscopy. The pufX mutants had intracytoplasmic membrane invaginations about 50% larger in diameter than those of the pseudo-wild type and higher levels of B870 light-harvesting complex. It is concluded that the PufX protein plays an important role in the structure of the functional photosynthetic unit, and its absence results in loss of efficient electron transfer from the QB site of the reaction center to the Qz site of the cytochrome b/c1 complex.


Spectroscopic studies of Rhodobacter capsulatus cytochrome c' in the isolated state and in intact cells. Ferricytochrome c' from Rhodobacter capsulatus was investigated by 1H-NMR, EPR and optical spectroscopies. A haem-linked ionisation, occurring with a pKa of 8.4 at 25 degrees C, was observed and assigned to the ionisation of the axial histidine ligand by comparison with data for related proteins. At pH values below this pKa the spin-state of the haem Fe3+ is shown to be a quantum mechanically admixed S = 3/2, 5/2 state. Above the pKa the Fe3+ is high-spin. EPR studies of intact cells grown photoheterotrophically reveal that in situ cytochrome c' exists largely in the ferrous state. Upon the addition of [Fe(CN)6]3- the protein becomes oxidised and EPR spectra reveal that the Fe3+ spin-state is a quantum mechanically admixed S = 3/2, 5/2 state. These data indicate that the unusual spin-state of ferricytochrome c' is not a consequence of changes to the protein on its isolation, as had been suggested previously. They also indicate that in situ cytochrome c' is located in an environment with a pH less than 7.


Pyrroloquinoline quinone, a method for its isolation and identification by mass spectrometry. Procedures for the unambiguous detection and for the isolation and mass spectrometric identification of pyrroloquinoline quinone (PQQ) are presented. The procedure involved acid hydrolysis of protein in the presence of phenylhydrazine and successive isolation and identification of the formed adduct using mass spectrometry. In HPLC the phenylhydrazone of PQQ gave many methylated products, of which the predominant compound was the pentamethylated derivative. After reaction of the phenylhydrazone derivative of PQQ (PHPQQ) with ammonia, a product was obtained which did not contain phenylhydrazine and which formed a pentamethylated derivative as the main methylation product. The HPLC profiles of the methylated products of PHPQQ and of its ammonia derivative were very characteristic and could be used for identification in addition to mass spectrometry. However, prolonged treatment of proteins with phenylhydrazine during hydrolysis can result in the formation of a material that resembles PQQ in some aspects of its behaviour. Thus, analysis by MS is essential for unambiguous identification. This analytical procedure was applied to pig plasma benzylamine oxidase, pig aorta lysyl oxidase, pig kidney diamine oxidase and bovine serum albumin with negative results. However, samples of pronase contained variable quantities of non-covalently bound PQQ: this can lead to erroneous identification of PQQ in enzyme after pronase digestion.


The Ku complex is modulated in response to viral infection and other cellular changes. The complex of Ku with DNA is demonstrated to have multiple forms as assayed by gel retardation analysis. In CV1 cells this variation of complex can be modulated in response to viral infection with SV40. By Western blot analysis, a correlation can be made between modification of the complex formed on DNA in response to viral infection with variation of the 85 kDa subunit of Ku. Modification of the 85 kDa subunit can also be seen when cells are exposed to various extracellular stimuli including variation in serum levels, PMA and CaPO4.


Purification of proteinase-like and Na+/K(+)-ATPase stimulating substance from plasma of insulin-dependent diabetics and its identification as alpha 1-antitrypsin. The purpose of this study was to purify and identify the proteinase-like substance previously recognized as responsible for the Na+/K(+)-ATPase stimulating property of plasma from insulin-dependent diabetic subjects. Anion-exchange chromatography followed by two-step heparin affinity chromatography resulted in a fraction highly enriched in both potent Na+/K(+)-ATPase stimulating activity and potent proteolytic activity. Approx. 400 micrograms of purified protein was isolated from 62 mg of starting plasma proteins. When analyzed on sodium dodecyl sulfate gels the active fraction consisted mainly of one polypeptide band with an apparent molecular mass of 66 kDa under either reducing or nonreducing conditions. The proteinase-like properties of the purified fraction were further revealed by its ability to clot plasma, split fibrinogen with production of fibrinopeptide A and induce shape change in human platelets and irreversible platelet aggregation in the presence of the stable analogue of endoperoxides U46619. Its additional capacity to affect platelet phosphoinositol metabolism was shown by the stimulation of protein kinase C-dependent phosphorylation of 47 kDa platelet membrane protein. In designing an identification protocol for the purified fraction, it was postulated that plasma proteinases are probably bound to their inhibitors, to form a stable covalently linked complex. The possibility that a proteinase-proteinase inhibitor complex was purified instead of single proteinase(s) was investigated. Neither trypsin nor neutrophil elastase were present in the active fraction whereas, among the possible plasma proteinase inhibitors tested, immunoreactivity was observed only in the presence of alpha 1-antitrypsin (alpha 1 AT) antiserum. Double immunodiffusion showed that control human alpha 1 AT and the plasma-purified fraction shared common antigens. Furthermore, both isoelectric focusing and amino acid composition analysis showed that the two substances were similar. The results obtained indicate that alpha 1 AT is apparently the only active component of the purified fraction from the plasma of insulin-dependent diabetics, thus suggesting that an altered form of the inhibitor is responsible for the broad range of proteinase-like effects elicited by the plasma-purified fraction.


Steady-state transcript levels of cytochrome c oxidase genes during human myogenesis indicate subunit switching of subunit VIa and co-expression of subunit VIIa isoforms. Steady-state levels of the mitochondrial rRNAs, of mRNAs for mitochondrially and nuclear-encoded subunits of cytochrome c oxidase and for the beta subunit of ATP synthase were assessed by Northern blot hybridizations during the in vitro differentiation of human myoblasts. Transcript levels of the so-called liver-type form of subunit VIa of cytochrome c oxidase diminished during the course of differentiation, while transcription of the so-called heart-type form was induced. Transcripts for the liver-type form and for the heart-type form of subunit VIIa of cytochrome c oxidase were detected in all myogenic cultures; the levels of the heart-type form progressively increased during the course of differentiation. The levels of the other transcripts studied did not change substantially. The results suggest subunit switching of subunit VIa and co-expression of subunit VIIa isoforms during myogenesis. The differential changes in mRNA levels of the heart-type subunits VIa and VIIa and the differential changes in mRNA levels of the liver-type subunits VIa and VIIa demonstrate that different transcriptional regulation mechanisms are present for both heart-type genes as well as for both liver-type genes.


An EPR investigation of spin-labelled erythrocytes as a diagnostic technique for malignant hyperthermia. Spin labelled (7-, 12- and 16- doxylsteric (DS) acid) erythrocyte (red blood cell) membranes isolated from six malignant hyperthermia susceptible (MHS) and six malignant hyperthermia normal (MHN) volunteer donors were characterized using the order parameter (S) and rotational correlation time (tau r) determined from 37 degrees C, 9 GHz electron paramagnetic resonance spectra. These parameters were found to decrease due to the increasing fluidization of the membrane as the halothane and benzyl alcohol sample concentration is increased and that significant differences exist in the values of S and tau r between the MHS and MHN sample groups, but that the differences in these parameters between the presence and absence of an external fluidizing agent are not significant for both sample groups. In the absence of these fluidizing agents, the mean values and standard errors of S at 37 degrees C for MHS and MHN are 0.643(2) and 0.652(3) for 7-DS, 0.554(2) and 0.563(3) for 12-DS, respectively, and of tau r are 2.139(12) and 2.223(13) ns for 16-DS, respectively. The values of S and tau r are significantly smaller for the MHS group than for the MHN group contrary to some previous reports. These observations revise and extend previous reports and show that the observed values of S and tau r in the absence of a fluidizing agent depend on the erythrocyte membrane structure in MHS and MHN subjects. They also suggest that the potential to use spin-labelled red blood cells as a screening protocol for MH deserves further investigation.


Differences in the repertoires of basement membrane degrading enzymes in two carcinoma sublines with distinct patterns of site-selective metastasis. Basement membrane-degrading enzymes of two clonal sublines of the murine Lewis lung carcinoma with distinct patterns of organ-selective metastasis were analyzed. Subline M-27 is highly metastatic to the lung and does not form liver metastases, while subline H-59 is highly metastatic to lymph nodes and liver, but not to lung. Qualitative and quantitative differences in the enzymatic profiles were found. H-59 cells which were significantly more invasive in vitro in the Matrigel invasion assay were found by zymogram analysis to secrete high levels of a 72 kDa gelatinase, while M-27 cells produced low levels of this gelatinase and of a higher molecular weight species which migrated in the 107 kDa region. On the other hand, M-27 cells produced significantly higher levels of urokinase type plasminogen activator (uPA) as indicated by a fibrinolysis assay and by Western blot analysis. Northern blot assays revealed an increase of approx. 3-fold in mRNA for cathepsin B in tumor M-27 which was reflected in a quantitative difference in plasma membrane cathepsin B levels as detected by Western blot analysis. H-59 cells on the other hand expressed approx. 8.5-fold more mRNA for cathepsin L. The quantitative differences in the levels of basement membrane degrading proteinases released by these tumor cells suggest that invasion by these cells is differentially regulated--a possible factor in their distinct patterns of dissemination.


[Superselective interventional angiography]. The authors report on the clinical application of superselective interventional angiography within the framework of revascularisation and occlusion. A coaxial catheter system is used for probing, consisting of an F 5 angiography catheter as guiding catheter and an F 3 catheter as internal catheter. The internal catheter is equipped with a shaft with segments of different flexibility and can take up a guide wire of 0.018" that is sufficiently stable to be rotated and guided. The interplay between the guide wire, which can be manipulated, and the flexible internal catheter enables superselective probing even of peripheral vascular areas. As may be required by the basic disease, the necessary interventional measures can be taken via the superselectively placed microcatheter. Superselective interventional angiography is indicated as an occlusive measure in preoperative vascular occlusion followed by palliative tumour resection, embolisation in haemangioma, chemoembolization in tumours of the liver. Superselective angiography is used for revascularisation in the local lysis of peripheral vessels. Due to the on-target superselective approach, side effects are markedly less than those observed with the interventions performed to date.


Postnatal development of pyruvate oxidation in quadriceps muscle of the rat. In order to evaluate the age dependency of enzymes involved in the energy-generating system, skeletal muscle specimens from rats of different ages were investigated for several mitochondrial enzymes. [1-14C]pyruvate (+/- ADP) oxidation rates and pyruvate dehydrogenase complex (PDHC) activity increased significantly from low early values during the neonatal period to nearly adult values at the end of the suckling period. Other enzymes of the pyruvate oxidation route such as citrate synthase and cytochrome c oxidase showed similar patterns of development. Immunoblot studies of PDHC detected a clear increase in the intensity of the bands of the alpha subunits of E1 (pyruvate dehydrogenase) and E2 (dihydrolipoyl transacetylase) within the first 3 weeks of life. The ratio between the individual PDHC proteins indicated that E1 alpha, the regulatory subunit of the multienzyme complex, is the most rapidly increasing protein with age.


Increased superoxide anion production in polymorphonuclear leucocytes on exposure to isobutyl-2-cyanoacrylate. Alkyl-2-cyanoacrylates have been employed in a variety of surgical procedures, although the pathophysiological basis for their cytotoxicity has not been resolved. Previous reports indicated that leucocytes infiltrate the site of cyanoacrylate application in situ. Consequently, human polymorphonuclear leucocytes were treated with 0-200 mM cyanoacrylate isobutyl-2- and superoxide anion radical production, lactate dehydrogenase release, and intracellular reduced glutathione content were quantitated following this treatment. In addition, some cells were also treated with 0-20 mM ascorbic acid before exposure to the adhesive. Pretreatment with ascorbic acid resulted in a statistically significant decrease in superoxide production (up to 111%), decrease in lactate dehydrogenase release (up to 26.4%) and a like increase in glutathione content (up to 26.6%). These results indicated that the cytotoxic properties of isobutyl-2-cyanoacrylate were diminished in a dose-dependent manner with addition of ascorbic acid, a free radical trapping agent. Moreover, isobutyl-2-cyanoacrylate appeared to increase specifically the production of superoxide anion. These findings suggested that the cytotoxicity of this class of adhesives may be associated with the formation of reactive oxygen intermediates.


Trichomonas vaginalis (TV) and human papillomavirus (HPV) infection and the incidence of cervical intraepithelial neoplasia (CIN) grade III. The temporal relationship between cervical infection with Trichomonas vaginalis (TV) or human papillomavirus (HPV) and the incidence rate of cervical intraepithelial neoplasia grade three (CIN III) was examined in a cohort of 43,016 Norwegian women. From 1980 to 1989, a cervico-vaginal infection from TV and HPV was diagnosed cytologically in 988 and 678 women, respectively. During the 181,240 person-years of observation, 440 cases of CIN III/cervical cancer developed. The age-adjusted incidence rates (IR) of CIN III were 225 per 100,000 person-years among women with no cytologic evidence of infection, 459 among women with TV infection, and 729 among women with HPV infection. A multiple regression model yielded a relative rate (RR) of CIN III of 2.1 (95 percent confidence interval [CI] = 1.3-3.4) among women with TV infection and 3.5 (CI = 1.9-6.6) among women with HPV infection, compared with women with neither infection. As CIN can be misclassified as HPV infection, the entry Pap-smears of 10 women with HPV infection who later developed CIN III were re-examined. Excluding the four discordant cases with the corresponding person-years decreased the RR of CIN III to 2.1 (CI = 0.9-4.8). Our report demonstrates the limitations of studies that rely only on cytologic detection of HPV infection. Nevertheless, the results support the hypothesis that HPV is a causal factor for CIN III lesions, and also display an association between TV infection and cervical neoplasia.


Urinary eicosanoids and the assessment of glomerular inflammation. Nephrotoxic nephritis, a model system for glomerulonephritis, is characterized by glomerular inflammation, proteinuria, and a marked increase in ex vivo glomerular eicosanoid production. This study addressed whether urinary eicosanoids might serve as noninvasive markers for glomerular inflammation and damage with nephrotoxic nephritis and its accelerated variant. Accelerated nephritis, relative to simple nephritis, was characterized by more substantial glomerular inflammation, particularly that due to neutrophils. Correspondingly, accelerated nephritis was accompanied by greater proteinuria and more marked elevations in glomerular eicosanoids generated ex vivo. With respect to urinary eicosanoids, thromboxane, but not leukotriene B4, was detected in the urine of normal animals. After the induction of nephrotoxic nephritis, urinary thromboxane was moderately elevated (twofold) and urinary leukotriene B4 was variably present (three of seven animals). In accelerated nephritis, urinary thromboxane was more markedly elevated (sixfold) and leukotriene B4 was consistently present. The presence of urinary leukotriene B4 was confirmed by gas chromatography/mass spectrometry. Urinary eicosanoids together correlated with glomerular leukocyte numbers and proteinuria by linear regression. Urinary leukotriene B4 individually correlated with glomerular neutrophil numbers. Renal metabolism of leukotriene B4 to omega oxidation products by the rat kidney was not apparent. These data validate that the enhanced glomerular eicosanoid metabolism seen in nephrotoxic nephritis takes place in vivo and additionally suggest that both urinary thromboxane and leukotriene B4 may serve as noninvasive markers for glomerular inflammation and damage. In light of these and prior studies, urinary thromboxane may be a general marker of glomerular inflammation and leukotriene B4 may be a more specific index of acute inflammation.


Evidence against increased hydroxyl radical production during oxygen deprivation-reoxygenation proximal tubular injury. The purpose of this study was to assess whether proximal renal tubules generate excess hydroxyl radical (.OH) during hypoxia/reoxygenation or ischemia/reperfusion injury, thereby supporting the hypothesis that reactive oxygen species contribute to the pathogenesis of postischemic acute renal failure. In the first phase of the study, rat isolated proximal tubular segments (PTS) were subjected to hypoxia (95% N2- 5% CO2) for 15, 30, or 45 min, followed by 15 to 30 min of reoxygenation in the presence of sodium salicylate, a stable .OH trap. Cellular injury after hypoxia and reoxygenation was assessed by lactate dehydrogenase release; .OH production was gauged by hydroxylated salicylate by-product generation (2,3-, 2,5-dihydroxybenzoic acids (DHBA); quantified by HPLC/electrochemical detection). Continuously oxygenated PTS served as controls. Despite substantial lactate dehydrogenase release during hypoxia (8 to 46%) and reoxygenation (8 to 11%), DHBA production did not exceed that of the coincubated, continuously oxygenated control PTS. In the second phase of the study, salicylate-treated rats were subjected to 25 or 40 min of renal arterial occlusion +/- 15 min of reperfusion. No increase in renal DHBA concentrations occurred during ischemia or reperfusion, compared with that in sham-operated controls. To validate the salicylate trap method, PTS were incubated with a known .OH-generating system (Fe2+/Fe3+); in addition, rats were treated with antioxidant interventions (oxypurinol plus dimethylthiourea). Fe caused marked DHBA production, and the antioxidants halved in vivo DHBA generation. In conclusion, these results suggest that exaggerated .OH production is not a consequence of O2 deprivation/reoxygenation tubular injury.


Subcellular mechanism and site of action of ionic lanthanum at the motor nerve terminal. The mechanism by which ionic lanthanum (La3+) increases and subsequently decreases spontaneous transmitter release was investigated by recording miniature endplate potentials (MEPPs) at frog neuromuscular junctions. Addition of tetrodotoxin and Co2+ delayed the onset of MEPP frequency increase but did not otherwise prevent the response. Dinitrophenol substantially reduced but did not eliminate the increase, whereas 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) completely abolished it. Thus, La3+ does not act by depolarizing the terminal or by substituting for Ca2+ at transmitter release sites. Instead, it appears to enter the terminal through Na+ channels and promote Ca2+ release from intracellular organelles. The profound depletion of transmitter with time may be due to the high turnover of transmitter coupled with the inhibition of metabolic processes by La3+.


ORG 2766 fails to improve visual functions in rats with occipital lesions. Adult rats were trained on a white versus black card discrimination in a circular water tank. Three independent variables were manipulated: lesion (sham, lateral occipital, medial occipital), dose of ORG 2766 administered (0 or 25 micrograms in saline on alternate days for 18 days), and time of administration (during the post-surgical recovery interval or during post-operative testing). Both visual cortical lesions produced a prominent retention deficit and defective pattern vision. Neither post-surgical nor concurrent administration of ORG 2766 improved visual functions. These results, along with a growing body of evidence, challenge the generality of the positive influences of ORG 2766 upon behavioral recovery observed in animals with limbic lesions.


Anti-HCV immunoglobulin M antibody in patients with hepatitis C. Anti-hepatitis C virus (HCV) immunoglobulin M antibody titres were measured by an enzyme-linked immunosorbent assay method in 16 patients with non-A, non-B acute hepatitis (NANB AH), 13 with non-A, non-B fulminant hepatitis (NANB FH) and nine with type C chronic hepatitis. Anti-HCV IgM was positive in one of the 16 patients with NANB AH, six of the 13 with NANB FH, and five of the nine with type C chronic hepatitis. Anti-HCV IgG was positive in eight of the 16 patients with NANB AH and eight of the 13 with NANB FH. Either anti-HCV IgM or anti-HCV IgG were positive in 10 of the 13 patients with NANB FH. All of the five anti-HCV IgM positive patients with type C chronic hepatitis were undergoing an exacerbation of the diseases, while all of the anti-HCV IgM negative patients were in a remission stage which had lasted for more than 6 months. The findings of this study suggest that anti-HCV IgM is useful for the early diagnosis of type C FH and may be a useful marker of diseases activity in type C chronic hepatitis.


Hepatitis C virus infection in chronic liver disease and hepatocellular carcinoma in Saudi Arabia. The prevalence of antibody to hepatitis C virus (HCV) was determined in 139 patients with chronic liver disease (CLD) and 42 patients with hepatocellular carcinoma (HCC) during one year at the Riyadh Military Hospital, Saudi Arabia. The anti-HCV was detected in 36 of 96 (37.5%) HBsAg-negative patients with chronic liver disease and six of 43 (13.9%) HBsAg-positive patients with chronic liver disease. In addition, 11 (42.3%) HBsAg-negative hepatocellular carcinoma patients and two of 16 (12.5%) HBsAg-positive hepatocellular patients had antibody to HCV. The anti-HCV prevalence was 1.5% in 4818 healthy blood donors and 1% in 385 antenatal patients. The overall HCV seropositivity of 30.4% in 181 liver disease patients (CLD and HCC) in Saudi Arabia is lower than that reported from European countries.


Ubiquitin is associated with aggregates of arginine modified proteins in injured nerves. Crush injury to rat sciatic nerves results in a 10-fold increase in the post-translational arginylation of proteins. In other systems, N-terminal arginylation leads to ubiquitination and proteolysis of the arginylated proteins. In the present experiments, proteins obtained from the 150 kg supernatant of crushed sciatic nerves were posttranslationally modified by 3H-arginine. These arginine modified proteins formed aggregates (precipitated at 20 kg) which then partially separated by SDS-PAGE were immunoreactive to a monoclonal antibody to ubiquitin. The results indicate that following injury to sciatic nerves, certain proteins are arginylated and ubiquitinated, probably targeting them for degradation. It is likely that these reactions help to rid cells of proteins damaged by the crush which would otherwise be cytotoxic.


Selectivity of serum immunoreactive prolyl 4-hydroxylase as a marker for hepatic necrosis. To investigate the selectivity of serum immunoreactive prolyl 4-hydroxylase (S-IRPH) as a marker for hepatic necrosis, sera were taken from patients with hepatic inflammation and hepatic necrosis. The concentration of immunoreactive prolyl 4-hydroxylase in the sera was determined by radio-immunoassay, and S-IRPH and alanine aminotransferase (ALT) data were plotted. Patients with hepatic necrosis showed mainly prolyl 4-hydroxylase elevation while patients with inflammation had both prolyl 4-hydroxylase and alanine aminotransferase elevations. The addition of serum immunoreactive prolyl 4-hydroxylase to current serum markers would be useful in investigating the pathophysiology of hepatocellular damage, particularly in differentiating between hepatic necrosis and inflammation.


Complications of hepatolithiasis.

Complications of hepatolithiasis. Hepatolithiasis or intrahepatic stone is associated with a variety of complications of which biliary sepsis is one. Left untreated, infection results in formation of micro-abscesses, portal thrombophlebitis and fistulation into adjacent structures. With repeated infection, biliary strictures and severe destruction of liver parenchyma occur. Biliary cirrhosis, portal hypertension and bleeding varices are the terminal manifestations. Early recognition and proper treatment are essential for the prevention of severe complications and functional deterioration.


Interleukin-1 beta directly excites hypothalamic supraoptic neurons in rats in vitro. Responses of 42 neurosecretory neurons in the supraoptic nucleus (SON) to human recombinant interleukin-1 beta (IL-1 beta) were examined during intracellular recordings in rat brain slices. IL-1 beta (10(-9)-10(-8) M) depolarized the membrane and caused increased firing in 25 neurons (59.5%). In 11 other neurons (26.6%), depolarization was also seen, followed by membrane hyperpolarization. The IL-induced depolarizing effect remained in the presence of tetrodotoxin (TTX) but was abolished by sodium salicylate. The results suggest that IL-1 beta mainly exerts a direct excitatory effect on SON neurons and further, that prostaglandins may be involved in such an effect.


Protein tyrosine kinases in nervous system development. Protein tyrosine kinases are important mediators of intracellular signaling during nervous system development. Activation of receptor protein tyrosine kinases by neurotrophic factors are initial events in the development of discrete cell populations. The patterns of expression and characterization of substrates for nonreceptor protein tyrosine kinases indicates that they also play a crucial role in neuronal development. The observed functional redundancy among protein tyrosine kinases and their associated intracellular signaling pathways underscores the need for further characterization of these novel interactions to elucidate the mechanisms regulating nervous system development.


Retinoid and thyroid hormone receptors: ligand-regulated transcription factors as proto-oncogenes. The retroviral v-erb A locus is derived from a cellular gene, c-erb A, encoding a thyroid hormone receptor. The v-erb A and c-erb A proteins are, in turn, members of a larger family of structurally and functionally interrelated polypeptides that includes the steroid, retinoic acid, and vitamin D3 receptors. These nuclear hormone receptors act by binding to specific sites on the cell genome and, in response to cognate hormone, modulating the transcription of adjacent 'target' genes. The expression, properties, and mechanisms of action of the thyroid hormone receptors (c-erb A proteins) and the closely related retinoic acid receptors are discussed.


[Adrenergic receptors in the liver parenchyma in children with chronic hepatitis]. In the present study adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of extrahepatic portal hypertension children without parenchymal affection (control group, n-7) and the resection of children in parenchymal affection (group of chronic hepatitis children, n-6). It has been shown, that the binding of beta-adrenergic radioligand 3H-dihydroalprenolol (3H-DHA) in liver parenchyma membranes of both control and chronic hepatitis groups was saturable and showed high affinity. The Scatchard analysis of the binding data indicated that the binding site was characterized by Kd and Bmax of 1.2 +/- 0.5 nM, 261.2 +/- 50 fmol/mg, respectively, for the control group; and 0.9 +/- 0.15 nM, 68.5 +/- 18.8 fmol/mg, respectively, for the group of chronic hepatitis patients; (mean+SEM). The binding of alpha 1-adrenergic antagonist 3H-prazosin (3H-PRZ) in liver parenchyma was also saturable and showed high affinity. The binding site is characterized by Kd = 0.6 +/- 0.12 nM, Bmax = 92.8 +/- 8.0 fmol/mg, for the control group; and Kd = 0.8 +/- 0.15 nM, Bmax = 195.0 +/- 22.0 fmol/mg, for the group of chronic hepatitis. It has been found that the number of binding sites of 3H-DHA significantly decreased and the number of binding sites of 3H-PRZ did not change in chronic hepatitis liver parenchyma in comparison with the control group. The results obtained suggest the important role of beta-adrenergic receptors in the pathogenesis of chronic hepatitis and in liver regeneration in children.


[Increase of calcium-dependent activity of phospholipase C in the myocardium in adaptation of animals to short-term stress effects]. The effect of adaptation of rats to repeated short-term stress exposures was studied on the density and the affinity of alpha 1-adrenoceptors in the heart and on the phospholipase C activity and sensitivity to changes in Ca2+ concentration. It was found that adaptation to stress was accompanied by a desensitization of alpha 1-adrenoceptors and also by an increase in Ca(2+)-dependence of phospholipase C activity in the heart. The role of increased activity of phospholipase C and activated inositol triphosphate-diacylglycerol regulatory cascade is discussed as regard to the previously revealed accumulation of heat shock proteins in the myocardium and to the adaptive protection of the heart.


[3H-tert-butylbicyclo-ortho-benzoate--a new ligand for chloride ion channel of GABA-A receptor]. The study was made of the binding of 35S-t-butylbicyclophosphorothionate and 3H-t-butylbicycloorthobenzoate (TBOB) which label a GABAA receptor-regulated chloride ionophores to glass fiber filters GF/B and GF/C. The rate of 3H-TBOB binding was higher. GABA displayed a biphasic effect on 3H-TBOB binding to rat brain synaptic membrane; enhancement at low concentrations of the agent and inhibition at higher ones. The results suggest that GABA may modulate the 3H-TBOB binding.


[Danazol action on 5'-nucleotidase activity in mouse peritoneal macrophages]. The paper reports the results of danazol action in vivo and in vitro on 5'-nucleotidase activity of mouse peritoneal macrophages. The results showed, that danazol at a concentration of 10 M (the pharmacologic level in women taking 600 mg/day) significantly stimulated the enzyme activity.


A novel human lymphoma cell line (Deglis) with dual B/T phenotype and gene rearrangements and containing Epstein-Barr virus genomes. We report a new and apparently unique human lymphoma cell line termed Deglis. The line was established from a polymorphic centroblastic lymphoma. The cell line and its source carry a dual B-cell and T-cell phenotype and Epstein-Barr virus (EBV) genomes. Simultaneous expression of B-cell (CD19+, CD20+, CD23+, CD37+) and T-cell (CD2+, CD3+/-, CD7+, CD43+) antigens, activation antigens (CD30+, CDw70+) as well as CD68+, a macrophage-associated antigen, was observed on the cell line and its source. Genotypic studies of the cell line showed dual gene rearrangements. JH (on both primary tumor and the cell line) and C kappa were rearranged without expression of cytoplasmic or surface immunoglobulins. T-cell receptor-alpha (TCR-alpha) and TCR-beta genes were rearranged, but TCR-delta and TCR-gamma genes were in germline configuration. Apparently, functional transcripts of TCR-alpha and truncated transcripts for TCR-beta and TCR-delta were observed. EBV-encoded proteins (LMP and EBNA2) were expressed by the parent tumor and the cell line. Southern blot analysis showed the same clonal EBV genomes in the primary tumor and the cell line. Karyotypic analysis of the cell line showed several chromosomal abnormalities but normal chromosomal number. The characteristics of this cell line suggest that neoplastic transformation has occurred in a precursor cell broadly committed to lymphoid lineage. Further studies on this cell line may help resolve some issues in the physiopathology of lymphoid tumors.


Effects of gamma-interferon on human neutrophils: protection from deterioration on storage. Human polymorphonuclear leukocytes (PMN) preincubated overnight with 100 U/mL gamma-interferon (IFN-gamma) had an increased metabolic response, as measured by iodination and/or superoxide production, to stimulation by tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor (GM-CSF), formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan, and lipopolysaccharide (LPS), as compared with cells comparably preincubated in the absence of IFN-gamma. The decline in the staphylocidal activity of the stored PMN was also prevented in part by IFN-gamma, as was the depressed adherence of PMN stimulated with phorbol myristate acetate (PMA), FMLP, TNF, GM-CSF, and LPS. This protective effect of IFN-gamma on PMN function was associated with the prolonged surface expression of the complement receptor three (CR3) alpha-chain (CD11b), CR3 beta-chain (CD18), FcRII (CD32), and FcRIII (CD16), and the appearance of surface FcRI (CD64). The polymerase chain reaction (PCR) was used to amplify neutrophil RNA-derived cDNA recognized by synthetic oliogonucleotides designed from published nucleotide sequences for specific proteins. Using this procedure, mRNA for gp91-phox, p67-phox, p47-phox, CD64, two forms of CD32, CD16, CD11b, CD18, and actin were found to be depressed after overnight storage of neutrophils, and this decrease in steady-state mRNA levels was in part or totally prevented by IFN-gamma. CD64 and gp91-phox mRNA were generally increased by IFN-gamma to a level greater than that of freshly isolated neutrophils. Northern analysis of CD64 and p47 phox mRNAs confirmed the findings with the PCR method. These findings suggest that storage of PMN in a functionally active state is favored by the presence of IFN-gamma.


Merocyanine 540-sensitized photoinactivation of enveloped viruses in blood products: site and mechanism of phototoxicity. The amphipathic dye, merocyanine 540 (MC540), which preferentially photosensitizes enveloped viruses and virus-infected cells, is currently being evaluated in preclinical models as a blood sterilizing agent. In this communication, we report on an initial analysis of the site and nature of MC540-mediated photodynamic damages to human herpes simplex virus type 1 and human cytomegalovirus. The comigration of dye molecules and virions on a gel filtration column, the red-shift of the fluorescence emission spectrum of virus-containing fractions, and the distribution of MC540-treated virions in an aqueous two-phase partition system were indicative of MC540 binding to the enveloped viruses and localizing in a lipophilic environment (most likely the viral envelope). Fluorescence quenching and fluorescence resonance energy transfer experiments suggested that both dye monomers and dimers were capable of partitioning into the lipid bilayer of the viral envelope. Adsorption and penetration assays and immunohistochemical analyses of viral antigen expression showed that MC540-sensitized irradiation interfered with early phases of the infectious process, the adhesion to the host cell, the penetration of the host cell, and the translocation of the virus into the nucleus of the host cell. The inactivation of viruses was inhibited if oxygen in the medium was displaced by argon, enhanced if air was displaced by pure oxygen or if water was replaced by deuterium oxide. This suggested that the MC540-sensitized photoinactivation of enveloped viruses is an oxygen-dependent process and that singlet oxygen is one but not necessarily the only mediator of the antiviral effects of MC540.


Spreading of HeLa cells on a collagen substratum requires a second messenger formed by the lipoxygenase metabolism of arachidonic acid released by collagen receptor clustering. HeLa cells attach to a variety of substrata but spread only on collagen or gelatin. Spreading is dependent on collagen-receptor upregulation, clustering, and binding to the cytoskeleton. This study examines whether second messengers are involved in initiating the spreading process on gelatin. The levels of cytosolic free calcium ([Ca++]i), cAMP, and cytoplasmic pH (pHi) do not change during cell attachment and spreading. However, a basal level of [Ca++]i and an alkaline pH(i) are required for spreading. There is an activation of protein kinase C (PKC) and a release of arachidonic acid (AA) on attachment and before cell spreading. Inhibition of PKC does not block cell spreading, indicating that PKC activation is not essential for spreading. Inhibition of phospholipase A2 blocks cell spreading, whereas addition of exogeneous AA overcomes this inhibitory effect. Among AA metabolic pathways, inhibitors of lipoxygenase (LOX) block cell spreading, suggesting that a LOX product(s) formed from AA initiates spreading. Clustering receptors for collagen with polyclonal antibodies, or with anti-collagen-receptor antigen-binding fragments (Fab) in combination with a secondary antibody, induce AA release. Also, AA is released when cells attach to either immobilized gelatin or immobilized Arg-Gly-Asp (RGD) peptide. Thus, AA is released whenever receptor clustering is observed. Receptor occupancy is not sufficient to release AA; when cells are treated with gelatin or RGD peptide in solution or anti-collagen-receptor Fab fragments without secondary antibody, conditions where receptor clustering is not observed, AA is not released. Thus, a LOX metabolite(s) of AA formed by collagen-receptor clustering is a second messenger(s) that initiates HeLa cell spreading. LOX inhibitors also block the spreading of bovine aortic endothelial cells, chicken embryo fibroblasts, and CV-1 fibroblasts on gelatin or fibronectin, indicating that other cells might use the same second messenger system in initiating cell-substratum adhesion.


Independent pathways regulate the cytosolic [Ca2+] initial transient and subsequent oscillations in individual cultured arterial smooth muscle cells responding to extracellular ATP. Stimulation with extracellular ATP causes a rapid initial transient rise followed by asynchronous periodic oscillations in cytosolic calcium ion activity ([Ca2+]i) in individual aortic smooth muscle cells in either HEPES-buffered or HCO3(-)-buffered saline. The dose at which one-half of the cells display an initial rise in cytosolic calcium is 0.11 microM ATP in the presence of external Ca2+ and 0.88 microM ATP in the absence of external Ca2+; the corresponding value for oscillations in the presence of external Ca2+ is 2.6 microM ATP. While the initial transient displays rapid desensitization, the oscillations persist for greater than 30 min in the continuous presence of ATP. The presence of the agonist ATP is also absolutely required for the maintenance of the oscillations, presumably to provide continuous activation of P2 purinoceptors. The average frequency of oscillation is approximately 0.9 min-1. The frequency depends only slightly on the concentration of ATP, and oscillations do not collapse into a prolonged elevated [Ca2+]i at high concentrations of ATP. Both Ca2+ influx and release from internal stores participate in the initial transient. Oscillations are not produced in the absence of external Ca2+ but are initiated upon the addition of external Ca2+ in the continued presence of ATP. Oscillations in progress are abolished by the removal of extracellular Ca2+ with one additional peak occurring after the Ca2+ removal. These data suggest that extracellular Ca2+ influx is required for the maintenance of the posttransient oscillations, presumably to provide the Ca2+ necessary for refilling intracellular Ca2+ pools that are the source of the oscillating [Ca2+]i. The Ca2+ influx is not regulated by voltage-gated Ca2+ channels. The data in this report are consistent with the view that the initial transient has contributions from two receptor-mediated pathways, and the oscillations are controlled either by a mechanism separate from the ones that control the initial transient or by steps whose control diverges before the point of desensitization.


The beta-PDGF receptor induces neuronal differentiation of PC12 cells. Expression of the mouse beta-PDGF receptor by gene transfer confers PDGF-dependent and reversible neuronal differentiation of PC12 pheochromocytoma cells similar to that observed in response to NGF and basic FGF. A common property of the PDGF, NGF, and basic FGF-induced differentiation response is the requirement for constant exposure of cells to the growth factor. To test the hypothesis that a persistent level of growth factor receptor signaling is required for the maintenance of the neuronal phenotype, we examined the regulation of the serine/threonine-specific MAP kinases after either short- (10 min) or long-term (24 h) stimulation with growth factors. Mono Q FPLC resolved two peaks of growth factor-stimulated MAP kinase activity that coeluted with tyrosine phosphorylated 41- and 43-kDa polypeptides. MAP kinase activity was markedly stimulated (approximately 30-fold) within 5 min of exposure to several growth factors (PDGF, NGF, basic FGF, EGF, and IGF-I), but was persistently maintained at 10-fold above basal activity after 24 h only by the growth factors that also induce PC12 cell differentiation (PDGF, NGF, and basic FGF). Thus the beta-PDGF receptor is in a subset of tyrosine kinase-encoded growth factor receptors that are capable of maintaining continuous signals required for differentiation of PC12 cells. These signals include the constitutive activation of cytoplasmic serine/threonine protein kinases.


Sumatriptan: a selective 5-hydroxytryptamine receptor agonist for the acute treatment of migraine. The clinical pharmacology, pharmacokinetics, clinical efficacy, adverse effects, and associated drug interactions of the novel antimigraine drug sumatriptan are reviewed. English-language publications pertaining to sumatriptan were identified via a search of the MEDLINE computerized database. Open and controlled clinical studies were reviewed in assessing clinical efficacy, although only the results of controlled, randomized trials from the basis for the conclusions pertaining to the effectiveness of sumatriptan. The primary measure of drug effectiveness in all clinical studies was significant improvement in headache severity scores. Secondary measures included functional ability, time to relief, rescue medication use, associated symptoms of nausea/vomiting and photo/phonophobia, and, in some studies, headache recurrence rate. These data were obtained from each published clinical trial and used in the overall analysis of sumatriptan efficacy. Sumatriptan is a serotonin agonist that has been studied for the acute treatment of migraine and cluster headache. The drug appears to work via specific serotonin receptors to mediate selective vasoconstriction within the cranial vasculature and to prevent the release of inflammatory mediators from trigeminal nerve terminals. The recommended dose of sumatriptan is 6 mg given subcutaneously at the onset of headache; an oral formulation is under investigation. In the published clinical trials of the oral and subcutaneous dosage forms to date, sumatriptan was effective in reducing headache severity from moderate/severe to mild/absent in approximately 70-80 percent of patients treated with active drug, compared with only 20-30 percent in the placebo groups, and 48 percent in the oral ergotamine tartrate/caffeine (Cafergot)-treated group. Secondary measures of effectiveness also favored sumatriptan. There may be a higher rate of headache recurrence with sumatriptan compared with placebo or Cafergot, although further study is necessary to confirm this observation. Adverse effects associated with sumatriptan administration generally were mild and transient and included tingling, warm/hot sensations, and pressure and tightness in the chest and neck. No significant drug interactions have yet been identified. Sumatriptan appears to represent a safe and effective alternative to the ergot alkaloids for the abortive treatment of acute migraine. However, further clinical trials, especially those yielding comparative data with current antimigraine agents, are needed to determine the full therapeutic contribution of sumatriptan.


Studies on the relationship between chronic fatigue syndrome and Epstein-Barr virus in Japan. Among 1,153 consecutive patients, 22 patients (1.9%) who complained of chronic fatigue for a period of over 6 months without detectable causes were studied. Ten patients (0.86%) satisfied the criteria of chronic fatigue syndrome (CFS) and were classified to be definite cases of CFS. The other patients were classified as probable cases. In order to clarify the role of Epstein-Barr virus (EBV) as a cause of CFS, we measured various antibodies for EBV. The definite cases had significantly higher titers of early antigen complex (EA)-IgG than both the probable cases and controls. We proposed the EA-IgG/EBNA ratio as the indicator of activation of EBV and attempted to estimate the degree of fatigue by the EA-IgG/EBNA ratio. The highest ratio value (16.0) of the 22 patients ratios was the most serious case. In general, the ratio correlated with the degree of fatigue. Based on these results, it was concluded that a relationship does exist between CFS and EBV.


A case of adenoid cystic carcinoma of the bronchus producing cancer-associated antigen, CA19-9. A 73-year-old male was admitted to Hyogo College of Medicine Hospital for further evaluation of chest x-ray abnormalities. Chest roentgenogram taken at admission showed right lower lobe atelectasis and bronchoscopic examination revealed an endobronchial tumor obstructing the left lower lobe bronchus. The biopsy specimen showed cribriform adenoid cystic carcinoma. The serum CA19-9 level was markedly elevated at admission, leading to immunohistochemical analysis of the biopsy specimen. As a result, in the tumor, CA19-9 was positively stained. This is probably the first reported case of adenoid cystic carcinoma of the bronchus which produces CA19-9.


Cytomegalovirus infection associated with acute pancreatitis, rhabdomyolysis and renal failure. The occurrence of rhabdomyolysis and acute renal failure associated with cytomegaloviral infection is rare. A 27-year-old housewife was admitted to our hospital with complaints of thirst, muscle weakness, abdominal pain and oliguria. There was no past history of diabetes, drinking, fever or drug habituation and a negative family history. Laboratory tests revealed myoglobinuria, hyper-pancreatic type amylaseuria, hyperglycemia, azotemia and highly increased creatine phosphokinase in the plasma. She was treated with hemodialysis and insulin therapy. Serological studies showed a 4-fold increase in cytomegalovirus antibody titers 4 weeks after admission. Muscle biopsy specimens showed hyaline degeneration and infiltration of T cell lymphocytes in the muscle. Renal biopsy specimens showed acute tubular necrosis and some myoglobin casts. No cytomegalovirus antigen was found in renal specimens by immunofluorescence study. From these results, it was determined that a systemic cytomegalovirus infection triggered pancreatitis which caused diabetic ketoacidosis, rhabdomyolysis and acute renal failure.


Prevalence of antibodies to hepatitis viruses in blood donors with a clinical history of hepatitis. A comparison between the 1979, 1982 and 1989 findings indicates that the number of adults susceptible to HAV infections has increased. This fact should be given attention in view of the strongly altered travelling pattern of fairly large sections of the population. It should also be kept in mind that the group of adults born between 1951 and 1960 comprises those adults who have the most frequent contacts with younger children being at the greatest risk of acquiring HAV infection.


Rapid reversal of angiotensin converting enzyme inhibition by lisinopril in the perfused rabbit lung. Lisinopril is a potent competitive inhibitor of purified rabbit lung ACE (dissociation t1/2 = 105 min). To examine reversibility of binding and ACE functional activity in situ, the single-pass extraction (E) of an 125I-lisinopril analogue (351A) and the hydrolysis of an ACE substrate, benz-phe-ala-pro (BPAP) were studied. Lungs were perfused at 50 ml/min with a Krebs-albumin (3%) solution. A bolus containing [14C]dextran, [3H]BPAP, and 351A was injected and (E)351A measured by multiple indicator dilution technique. BPAP metabolism (M) was reflected by the appearance of its hydrolysis product [3H]benz-phe in lung effluent. Control (E)351A was 66 +/- 5% (mean +/- SD, n = 6) and (M)BPAP was 69 +/- 9% (n = 6). Unlabeled lisinopril (30 nmol) in the bolus significantly reduced E(351A) and M(BPAP) to 16 +/- 16% and 3 +/- 3%, respectively. Ten minutes later E(351A) and M(BPAP) had returned to control values. Reduction of E(351A) was partially reversible and M(BPAP) completely reversible after 1 min. After recirculation with 0.25 mM lisinopril for 30 min, however, significant depression of E(351A) was evident for 60 min after exposure to lisinopril was discontinued. Thus, rapid as well as slowly reversible components of inhibition of ACE inhibitor binding can be demonstrated in the perfused rabbit lung.


Regulation of matrix vesicle phospholipid metabolism is cell maturation-dependent. We have developed a chondrocyte culture model for assessing the regulation of matrix vesicles at two different stages of chondrogenic maturation. These chondrocytes, resting zone (RC) and growth zone (GC), retain their phenotypic markers in culture, including production of matrix vesicles with distinctive lipid compositions and enzyme activities. Isolated matrix vesicles incubated in vitro with 1,25-(OH)2D3 (1,25) or 24,25-(OH)2D3 (24,25) respond differentially. 1,25 stimulates phospholipase A2 (PA2) in GC vesicles, but not on those from RC. 24,25 inhibits PA2 in RC vesicles, but has no effect on GC. PA2 activity is required for fatty acid turnover and is the rate-limiting step in prostaglandin production. Plasma membrane phospholipids are more susceptible to the release of arachidonic acid by PA2 than are matrix vesicle phospholipids. Matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25 and 24,25 stimulate arachidonic acid turnover in their target cells, but by different mechanisms. 1,25 has no effect on arachidonic acid turnover in RC; however, 24,25 inhibits turnover in RC and GC. 1,25 and 24,25 also affect isolated matrix vesicle membrane fluidity. These results suggest that vitamin D metabolites modulate PA2 activity, change the composition of membrane phospholipids by altering fatty acid composition, and affect calcium transport. The effects are mediated by altering membrane fluidity and is dependent on the stage of cell differentiation.


The incidence and severity of hypoxia associated with 99Tcm Technegas ventilation scintigraphy and 99Tcm MAA perfusion scintigraphy. Inhalation of the ventilatory radiopharmaceutical 99Tcm Technegas leads, in some patients, to symptoms that may be attributed to temporary lowering of oxygen saturation. In order to evaluate this, oxygen saturation was measured by pulse oximetry in a series of patients undergoing Technegas ventilation scintigraphy. A decrease in oxygen saturation was recorded in 87% of the patient group. The mean change, as a percentage of the initial value, was 8.3% (range 1-24%). Hypoxia arising in association with Technegas administration may be reduced by pre-oxygenation. In patients who were pre-oxygenated, oxygen saturation did not fall below 85% (PaO2: 50 mmHg) but in 39% of those not pre-oxygenated the value fell below this level. Oxygen saturation was also monitored in a series of patients undergoing perfusion scintigraphy. In 17% of patients a decrease was recorded (range 2-11%). In view of the large number of perfusion scans performed annually in this department and elsewhere without untoward effect, such temporary decreases in oxygen saturation presumably present no hazard to the patient.


Prevention of venous thromboembolism after surgery: a review of enoxaparin. This review discusses the problem of deep vein thrombosis (DVT) after operation and identifies three levels of risk of DVT: low (less than 10 per cent), moderate (10-40 per cent) and high (40-80 per cent). Special emphasis is placed on the most recent prophylactic treatment, low molecular weight heparins (LMWHs), particularly enoxaparin. Several LMWHs are now available, but they differ slightly and each must be evaluated on its own merits. In general, however, LMWHs are both effective and safe in those patients at moderate or high risk of DVT. Thromboprophylaxis is cost effective when analysed using health-economic methodology.


Autoradiographic localization of binding sites for 2-[125I]iodomelatonin in the pars tuberalis of the western spotted skunk (Spilogale putorius latifrons). Pineal melatonin secretion mediates photoperiodic regulation of implantation of blastocysts in the female western spotted skunk. Autoradiography studies demonstrated that high affinity 2-[125I]iodomelatonin binding sites are present in the pars tuberalis but not in any other hypophyseal region or in the hypothalamus or thalamus of this species. This restricted localization of 2-[125I]iodomelatonin binding, which was characteristic of spotted skunks sacrificed at various times during their annual reproductive cycles, suggests that the pars tuberalis mediates photoperiodic responses in this species.


Enhancement of working memory performance in the rat by MDL 26,479, a novel compound with activity at the GABAA receptor complex. MDL 26,479, a novel triazole compound with action at the GABAA receptor complex, was examined in 2 models of working memory deficit in the rat. MDL 26,479 attenuated a scopolamine-induced acquisition deficit in a T-maze reinforced alternation task. MDL 26,479 also reversed a performance deficit induced by increased delay between sample and choice trials in the same task, suggesting that the compound may have memory enhancing potential. This finding further supports claims that drugs acting at the GABAA receptor complex are of potential use in the treatment of memory disorder.