Clinical assessment of the safety and efficacy of an inactivated hepatitis A vaccine: rationale and summary of findings. The objectives for the clinical testing of the inactivated hepatitis A vaccine developed by SmithKline Beecham Biologicals are reviewed and the results obtained are summarized. The first studies were carried out in healthy young adult volunteers using pilot vaccine lots prepared from the CLF and HM175 strains of hepatitis A virus (HAV). It was established that the candidate vaccines were well-tolerated, caused no hypersensitivity reactions and elicited a strong antibody response. As the yield in production with the HM175 strain was higher it was preferred over the CLF strain for further development of a vaccine. A dose-range study with HM175 vaccine in adults showed that an antigen dose of 720 ELISA units (El.U) produced almost 100% seroconversion after one injection. This dose was therefore chosen as appropriate for adults. A dose of 360 El.U was used in children. To date, a total of 67 studies in 18 countries involving 47,145 subjects, including 20,586 control subjects, have been initiated. In these studies, 55,259 doses of HM175-derived hepatitis A vaccine have so far been administered. This extensive experience has shown that the vaccine is well-tolerated, causing essentially only mild, transient local reactions. Vaccine reactogenicity was assessed using symptom checklists, filled in by the volunteers or their parents, for a period of 4 days following vaccination. Blood samples were obtained at different times after vaccination to evaluate the immune response. Clinical studies with six different lots, manufactured using commercial-scale production methods, have been carried out on 2901 adults. These studies have shown that a seroconversion rate of 95.7% (1225/1280) is obtained one month after the first vaccine dose.(ABSTRACT TRUNCATED AT 250 WORDS)


Properties and classification of hepatitis A virus. Hepatitis A virus (HAV) is a member of the picornavirus family. It was first provisionally classified as enterovirus 72, but subsequent determinations of its nucleotide and amino acid sequences showed them to be sufficiently distinct to assign the virus to a new genus. Heparna-virus (Hep-A-RNA-virus) has been suggested as the genus name. HAV shares the key properties of the picornavirus family: an icosahedral particle 28 nm in diameter with cubic symmetry, composed of 30% RNA and 70% protein. The genome is single-stranded 7.48 kb RNA, linear and positive-sense. Like other picornaviruses, HAV possesses four major polypeptides cleaved from a large precursor polyprotein. The surface proteins VP1 and VP3 are major antibody-binding sites. The internal protein VP4 is much smaller than the VP4s of other picornaviruses. As other picornaviruses, HAV has no envelope and replicates in the cytoplasm. HAV is stable to treatment with either and acid, and is much more heat-resistant than other picornaviruses. It withstands 60 degrees C for 1 h. MgCl2 stabilizes the virus to withstand temperatures up to 80 degrees C. The relative resistance of HAV to disinfection indicates a need for extra precautions in dealing with hepatitis patients and their products. Only one serotype is known. There is no antigenic cross-reactivity with other hepatitis viruses. HAV initially was identified in stool and liver preparations by employing immune electron microscopy as the detection system. Chimpanzees and marmoset monkeys are susceptible to HAV. HAV has been cultivated serially in primary explant cultures of adult marmoset livers and in cell lines of primate origin.(ABSTRACT TRUNCATED AT 250 WORDS)


Natural hosts of hepatitis A virus.

Natural hosts of hepatitis A virus. The host range for hepatitis A virus (HAV) is limited to man and several species of non-human primates, and involvement of vertebrates other than primates in HAV circulation is unlikely. Spontaneous hepatitis A infection has been reported to occur in captive non-human primates including the great apes (chimpanzee) as well as Old World (cynomolgus, African vervet, stump-tailed) and New World (aotus) monkeys. The presence of anti-HAV antibody in the sera of newly captured monkeys of these species shows that infection may also spread in their natural habitat. HAVs isolated from spontaneously infected monkeys, although antigenically closely related to human HAV, exhibit a significant degree of genomic heterogeneity. There are at least four distinct simian HAVs differing from each other and from all human HAV strains. It is suggested that each virus is native to a given species reflecting evolutionary relationships among HAVs and their hosts in the order of Primates.


Replication of hepatitis A virus and processing of proteins. Isolation and propagation of hepatitis A virus (HAV) in cell culture is routinely possible. All primary HAV isolates and most established virus strains, however, show a protracted replication behaviour and tend to establish a persistent infection. Rapidly replicating, cytolytic variant viruses can be selected from persistently infected cultures under distinct conditions. Factors critical for the outcome of HAV infection include the genetics of the virus, the physiological state of the infected cell, the presence of defective interfering particles, synthesis and encapsidation of viral RNA and possibly synthesis and processing of viral proteins. Analysis of the latter events have proved to be difficult. Only seven of the 11 peptides coded for by the HAV genome have been experimentally identified and the sequence of cleavages by which they are released from the precursor polyprotein is still a matter of discussion.


Molecular basis of virulence and growth of hepatitis A virus in cell culture. The ability of engineering variants of hepatitis A virus (strain HM175) to replicate in cell culture or to cause disease in marmosets was evaluated. Virus variants were encoded by chimeric genomes constructed from infectious cDNA clones of two viruses (wild type and cell-culture-adapted) which differed in their ability to grow in vitro and to cause acute hepatitis in marmosets. Transfection and infectivity assays indicated that virus growth in vitro could be enhanced by subcloning the cell substrate prior to infection or by introducing multiple combinations of two or more mutations into the wild type genome. Various chimeric viruses induced liver enzyme elevations in marmosets, indicating that attenuation of virulence also required multiple mutations.


Genetic, antigenic and biological differences between strains of hepatitis A virus. Recent studies have documented a considerable degree of genetic divergence among wild-type hepatitis A virus (HAV) strains recovered from different geographical locations. Human HAV strains can be grouped into four genotypes (I, II, III and VII) and unique simian strains belong to three additional genotypes (IV, V and VI). Between each of these genotypes, the nucleotide sequence varies at 15-25% of base positions in the P1 region. Despite this, there is good evidence that most, if not all, human strains of HAV are closely related antigenically. In contrast, although simian strains recovered from Old World monkeys are cross-reactive in immunoassays employing polyclonal antibodies, these strains have significant antigenic differences from human HAV strains. Nonetheless, because biological differences in the host range of these strains apparently preclude significant human infection, this is unlikely to pose a problem in controlling HAV infections with active immunization. Inactivated and attenuated vaccines produced from genotype I human strains (HM175 or CR326) are likely to provide protection against all relevant human HAV strains.


Passive immunization against hepatitis A. Administration of human serum immune globulin (Ig) is an effective means of protecting individuals against hepatitis A virus (HAV) infection and disease. Several large field studies have demonstrated that if given before exposure, Ig will prevent infection with HAV. Furthermore, if Ig is given during the incubation period of hepatitis A, the severity of infection may be reduced and potentially clinical infections may be converted into subclinical ones. Although uncommon, infection which occurs in the presence of circulating antibody may occasionally lead to passive-active immunity. Unfortunately, the duration of Ig protection is dose dependent, and high dose administration provides less than six months protection. Ig preparations contain HAV antibodies at levels detectable by commercial immunoassays; however, recipients of Ig do not have detectable levels of HAV antibodies when tested by the same method. Using more sensitive immunoassays and neutralization assays, low titres of HAV antibody can be detected in Ig recipients. Since Ig provides approximately 90% efficacy in preventing hepatitis A, it would appear that very low levels of HAV antibody are needed to prevent infection. Consequently, measurement of HAV antibodies elicited by HAV vaccines should provide a reasonable method to evaluate their immunogenicity and predict their efficacy.


Possible approaches to develop vaccines against hepatitis A. More than a decade ago, successful replication of hepatitis A virus (HAV) in cell culture opened the way to the development of live attenuated and inactivated vaccine candidates. Serial passages of HAV in cell culture led to attenuation as demonstrated by experiments in non-human primates. Several live vaccine candidates obtained through serial passages have been evaluated in volunteers. Significant improvements in the yield of viral antigen from infected cell cultures stimulated the development of killed vaccine candidates. These formalin-inactivated vaccines contain the viral capsid antigens assembled into viral particles. The immunogenic potential of the vaccine candidates depends strongly on the preservation of the configuration of the capsid proteins. Synthetic peptides covering immunogenic sequences of VP1 as well as soluble capsid proteins expressed as fusion proteins in Escherichia coli were therefore only weakly immunogenic when injected at high concentrations in rabbits. On the other hand, tamarin monkeys immunized with a live recombinant vaccinia expressing P1 were protected against virulent challenge. There are, however, considerable drawbacks related to the use of live vaccinia as a carrier virus. Chimeric polio-HAV VP1 viruses have been constructed. These hybrid viruses were not able to induce an immune response, probably because of configurational constraints of poliovirus on the inserted HAV epitopes. More recently, encouraging data on empty virus particles expressed in baculovirus and vaccinia virus systems have been reported.


Epidemiological patterns of hepatitis A in different parts of the world. Serological surveys in many communities show a high prevalence of antibodies to hepatitis A virus (HAV) in people over the age of 50 years. However, few of that age can recall a previous episode of hepatitis, indicating that subclinical infections are common. The outcome of infection with HAV depends on the age at which infection occurs and, perhaps, the infectious dose. Fulminant disease is well recorded, with the frequency varying from one to eight per 1000 cases. Information on the frequency of hepatitis A can be obtained by analysing hospital records and notifications to health authorities or by serological surveys. In many countries, these data are limited and seriously underestimate the true frequency of the disease. At a conservative estimate, the incidence of disease in most developed countries is probably four to five times higher than the number of notifications. HAV appears to circulate in most parts of the world and to be responsible for both epidemic and sporadic disease. Three major patterns of infection are known which reflect different epidemiological situations. These are demonstrated by different patterns of the age-specific prevalence of antibodies to HAV which reflect standards of hygiene and sanitation, the degree of crowding of the population and opportunities for the virus to survive and spread.


Epidemiology of hepatitis A in Mediterranean countries. Infection with hepatitis A virus (HAV) is still endemic in some Mediterranean areas. In most Northern Mediterranean countries, the incidence of acute icteric hepatitis in adults is increasing. This is due to the shifting of HAV infection to adulthood as a result of the decline of its overall prevalence due to improvements in socioeconomic, sanitary and hygienic conditions. The majority of adults remain susceptible and develop overt disease when infected, since the severity of disease is highly associated with age. Epidemics are now rare, but are more extensive when they do occur. They may sometimes be caused by accidental contamination of the water supply, but are usually due to contamination of food by diseased food-handlers or result from contaminated frozen foods. Outbreaks still may occur in day-care centres and in schools. Thus travelling to endemic areas is becoming the main source of HAV infection. Intrafamilial person-to-person spread also is an important source of infection. Transmission from children to parents and other adults may occur due to lack of immunity in the adult population. Selective immunization would further reduce the incidence of the disease. However, only inclusion of the vaccine in the routine programme of childhood immunization would guarantee the disappearance of hepatitis A.


Ecology and prevention of a shellfish-associated hepatitis A epidemic in Shanghai, China. During a shellfish-borne hepatitis A outbreak in Shanghai during the first quarter of 1988, 300,000 cases were reported in two months. Using cell culture and experimental infection of marmosets, hepatitis A virus (HAV) was isolated from clams collected from the market and the sea bed during the epidemic. A dose-response curve correlating the quantity of clams consumed to the attack rate of hepatitis A was well documented. The occurrence of the epidemic was associated with a good harvest of clams in a new area, serious pollution of this area with sewage and importation of the clams in large quantities into Shanghai where most young adults were susceptible. Clams can apparently be decontaminated by using a continuous water flow. In this way, HAV titres can be reduced by 90% in one day and by 99.9% in two weeks. An attenuated live HAV vaccine which has been developed in China has been shown to be safe and immunogenic and may be used for prevention of such epidemics in the future.


Risk of hepatitis A in travellers.

Risk of hepatitis A in travellers. A review of the literature shows that 30-35 million travellers from industrialized nations annually visit a developing country where their incidence rate of symptomatic hepatitis A is 3 to 6 per 1000 per month of stay if they remain unprotected. The risk is 20 per 1000 for persons eating and drinking under poor hygienic conditions. Thus hepatitis A is now the most frequent vaccine-preventable disease in such travellers. Antibodies to hepatitis A virus are rarely found in potential travellers in many industrialized countries, except in those born before 1944, in those with a history of jaundice, or in those with a stay > 1 year in a developing country.


Hepatitis A in the US Army: epidemiology and vaccine development. Control of hepatitis A has been an important concern for US military forces in war and peace. Immune serum globulin, although effective, is exceedingly cumbersome to use. The prevalence of antibody against hepatitis A is decreasing in young American soldiers, putting them at risk of hepatitis A during deployment. The US Army has been an active participant in development of hepatitis A vaccine. The first successful cell-culture-derived, formalin-inactivated hepatitis A vaccine was developed at the Walter Reed Army Institute of Research. This prototype vaccine was shown, in 1986, to be safe and immunogenic for humans. Since then we have evaluated the following issues related to the use of inactivated hepatitis A vaccines in military populations. Immunogenicity of vaccine derived from the CLF and HM175 strains; immunogenicity of hepatitis A vaccine given by jet injector; immunogenicity of hepatitis A vaccine when given with hepatitis B vaccine; immunogenicity when given in shortened schedules; safety and immunogenicity in Thai children; and efficacy under field conditions in the tropics. The hepatitis A vaccines which we tested are safe and highly immunogenic. Immunization by jet gun confers immunity equivalent to immunization by needle. Hepatitis A vaccine is equally potent when given with hepatitis B vaccine. Data on rapid immunization schedules and efficacy are under evaluation. We conclude that hepatitis A vaccine is a major improvement in our ability to prevent hepatitis A in soldiers.


Hepatitis A as an occupational hazard.

Hepatitis A as an occupational hazard. Few studies have been carried out to evaluate the role of hepatitis A virus (HAV) as an occupational hazard. Our analysis of data on occupational diseases in Germany showed that hepatitis A ranks as third among infectious occupational diseases. Morbidity based on the frequency of compensation (15.2%) was in the same range as that observed for hepatitis B (19.7%). In another study, data were collected on anti-HAV prevalence among 2293 hospital workers in southwest Germany. Anti-HAV prevalence of hospital staff responsible for patient care and that of the general population were comparable, while food-handlers under the age of 30 years had a higher degree of anti-HAV prevalence. When an evaluation of anti-HAV prevalence data was carried out on persons younger than 30 years who comprised subsets of the medical staff, the relative risk was: charwomen 4.2, food-handlers 2.49, and paediatric nurses 1.84, showing that they had higher prevalence rates than nurses 1.25, physicians 1.09 and laboratory assistants 0.93. Vaccinations for the prevention of hepatitis A should therefore reach individuals that have an increased occupational risk: food-handlers, health care workers in infectious diseases and paediatrics, medical staff in laboratories handling stool samples, medical charwomen and, according to previously published work, staff of day care centres and sewerage workers.


Who should receive hepatitis A vaccine? Considerations for the development of an immunization strategy. The availability of efficacious hepatitis A vaccines should greatly facilitate the prevention of hepatitis A virus (HAV) infection. Groups at high risk of HAV infection have been identified from epidemiological studies and include both children and adults. While certain high-risk adults, such as travellers, could be a convenient target for vaccination, selective immunization of high-risk adults would not be expected to lower the overall rates of infection in most countries. Because a significant proportion of HAV infections occur in children, the eventual objective should be the integration of hepatitis A vaccine into routine childhood immunization schedules. This would reduce disease incidence by preventing infections in children and by preventing infections in adults that are acquired from children. The cyclical patterns of hepatitis A observed in many countries are related to levels of immunity in the population. The elimination of the susceptibility of a population to HAV infection through immunization could eliminate this well known human disease. This suggests that eradication of HAV infection should be attainable with effective hepatitis A vaccines.


Cost-effectiveness analysis of hepatitis A prevention in travellers. The advent of new vaccines and the changing epidemiology of hepatitis A call for an update of the economic evaluation of costs and benefits associated with the various alternative preventative strategies. A decision-tree-based model has been developed which enables the calculation of expected costs and expected numbers of hepatitis A virus HAV infections based on different intervention strategies. The model is sufficiently generic to allow for the evaluation of both population-wide strategies and strategies targeted at particular risk groups. An economic analysis focusing on travellers from Europe to high-endemic countries compared a non-intervention strategy to the following three strategies: active immunization with HAV vaccine; screening for HAV antibodies and vaccinating only susceptibles; passive immunization by means of immunoglobulin. The net cost per HAV infection prevented proved very sensitive to a number of important input parameters of the model. These included epidemiological characteristics such as HAV attack rate and prevalence of immunity, behavioural characteristics such as compliance with the vaccination scheme and vaccine characteristics such as rate and duration of protection. Our estimated expected cost per HAV infection prevented among Belgian travellers to high-endemic countries for three weeks per year over ten years amounts to approximately US$4880 for active immunization, US$5621 for screening followed by vaccination of susceptibles and US$29932 for passive immunization. Although these estimates are clearly sensitive to a number of crucial assumptions pertaining to the input parameters of the model, it seems safe to conclude that vaccination is more cost-effective than the currently recommended passive immunization with immunoglobulin.(ABSTRACT TRUNCATED AT 250 WORDS)


Perspectives on the control of hepatitis A by vaccination. The availability of an inactivated hepatitis A virus (HAV) vaccine, and the development of live attenuated virus vaccines against hepatitis represent great advances in the effort to control an important cause of viral hepatitis. There are a number of ways hepatitis A vaccines could be used, depending on the epidemiology of HAV infection in the country concerned, the cost of the product, the duration of protection that the vaccine affords and its effectiveness for postexposure prophylaxis. Expert groups could recommend vaccine to individuals who are at higher risk of exposure to hepatitis A as a result of behaviour, lifestyle or occupation, or to all infants and/or adolescents. The major public health problem with hepatitis A occurs in developing countries and regions with 'transitional economies' such as Eastern Europe and certain Middle Eastern countries. These countries have high levels of viral circulation, large cohorts of susceptible older children and adults, and high rates of HAV morbidity. Given the experience with hepatitis B vaccine and the economic and political realities of global immunization policy, it is unlikely that the Expanded Programme on Immunization and traditional vaccine donors will take a great interest in HAV vaccine in the near future. Individual countries may, however, decide to use hepatitis A vaccine on a widespread basis. Model cost effectiveness studies are needed for both developing and developed countries to help decide the feasibility of such widespread use. WHO has been involved in activities relevant to hepatitis A vaccine for many years.(ABSTRACT TRUNCATED AT 250 WORDS)


Production, quality control and characterization of an inactivated hepatitis A vaccine. The isolation and adaptation of hepatitis A virus to cell culture opened the way to the development of vaccines. Based on experience with inactivated poliovaccines, a similar approach was chosen for the development of an inactivated hepatitis A vaccine. Strain HM175, adapted to MRC-5 human diploid cells, was used as the virus strain. Vaccine production starts with growth and multiplication of the seed virus in MRC-5 cells. The harvests are clarified, purified and concentrated. Inactivation by formaldehyde is carried out on a pool of purified harvests. Close control of all process parameters results in consistent production of completely inactivated and highly immunogenic vaccine lots. Quality control testing is based on the general requirements for biologicals of WHO and National Control Authorities. Tests have been developed and validated to show the purity of the cell substrate used for each production cycle, the quality of the virus harvest, the adequacy of the purification and inactivation processes, and the conformity to stringent specifications for purity, safety and potency of the final bulk vaccine filled in final containers. The vaccine is characterized by adequate identity tests, by its reaction with polyclonal and monoclonal antibodies, by its immunogenicity in laboratory animals and by the detailed study of the immune response in primates and human volunteers. The final result of the development of adequate production and testing methods, confirmed by extensive characterization studies, is the availability of a consistent, safe and potent hepatitis A vaccine.


Modifications of the 5' untranslated region of foot-and-mouth disease virus after prolonged persistence in cell culture. The nucleotide sequence of the 5'-untranslated region (5'UTR) of the genome of foot-and-mouth disease virus (FMDV) R100, rescued after 100 passages of persistently infected BHK-21 cells, has been compared with that of the parental FMDV C-S8c1. The nucleotide sequence divergence between the two viruses in heteropolymeric regions is 1%. The few mutations located at the 5'-most terminal region (S fragment) and at the internal ribosome entry site (IRES) do not appear to affect significantly the tight secondary structure predicted for these RNA segments. Comparison of the 5'UTR of C-S8c1 or R100 RNA with that of other FMDV serotypes and subtypes indicates the presence of block deletions (or insertions) which do not correlate with the serological classification of FMDV. Remarkably, FMDV R100, a virus highly attenuated for mice and cattle, contains a polyribocytidylate (poly C) tract of about 420 nucleotides, 145 residues longer than its parental, virulent FMDV C-S8c1. This long poly C of R100 RNA includes a few uridine residues interspersed at fairly regular intervals. This is the longest highly homopolymeric tract described in a viral genome and, to our knowledge, in any informational biomolecule.


The herpes simplex virus type 1 ICP6 gene is regulated by a 'leaky' early promoter. Expression from the promoter for the large subunit (ICP6) of the ribonucleotide reductase encoded by herpes simplex virus type 1 (HSV-1) has been examined. Using the lacZ reporter gene fused in-frame with ICP6 regulatory sequences to assay expression quantitatively, we showed that the ICP6 promoter responded very weakly to the alpha-transinducing factor (TIF) in the absence of all other viral gene products, but much more strongly to immediate early proteins. Similar patterns of regulation were observed when the reporter gene construct was located at two different positions within the the viral genome or in a stably transfected Vero cell line. Infection of the stably transfected cells with various HSV-1 mutants identified ICP0 as the major transactivator of the ICP6 promoter.


Transcriptional expression of the viral genome in the Epstein-Barr virus-induced tamarin lymphoma and the corresponding lymphoblastoid tumour lines. Inoculation of the cottontop tamarin with Epstein-Barr virus (EBV) invariably gives rise to mono- or oligoclonal large cell lymphoma occurring at multiple sites, and which resembles to a certain extent B cell lymphoma that occurs in the immunodeficient patient. The viral transcriptional pattern in tamarin tumour biopsies and in the corresponding tumour cell lines was investigated by means of the synthesis of radioactive single-stranded cDNA. It was found that the EBV transcripts came mainly from the fragments BamH1-H, BamH1-S, BamH1-A and EcoR1-Dhet. Transcripts from a few other early or late genes, namely BARF1, BSLF1/BMLF1, BBLF-4, BLLF1 and BXLF2, were also detected in one of the three biopsies tested. It would be important to characterize the transcripts that originate from the region where viral latent expression has not previously been observed. Our results also revealed that there is a sharp increase in EBV transcription in the tumour cell lines derived from the tamarin lymphomas. Simultaneously, the copy number of the viral genome was found to be amplified. Such a significant change in viral activity might be indicative of a close virus-host cell interaction in vivo.


Mutations in the UL53 gene of HSV-1 abolish virus neurovirulence to mice by the intracerebral route of infection. The cell fusion protein, the product of the UL53 gene, is responsible for intracerebral (IC) pathogenicity of HSV-1. Recombinant HSV-1 R15 is apathogenic to mice by the IC route of inoculation, while intratypic recombinants, in which the UL53 gene in R15 was replaced by an analogous sequence from the pathogenic strain R19, regained IC pathogenicity. The nucleotide sequence of the UL53 gene of HSV-1 strains R15 (apathogenic) and R19 (pathogenic) was determined and compared to that of other pathogenic strains. Four mutations were found which are thought to be responsible for the apathogenic phenotype of HSV-1 strain R15. Northern blot hybridization of RNA extracted from BSC-1 cells infected with several HSV-1 strains indicated that all of the virus strains tested expressed equal amounts of UL53 mRNA in infected cell cultures. Demonstration of the expression of UL53 mRNA in brains of mice infected with HSV-1 strains was made possible by the combined use of a rapid method for mRNA extraction (Oligo dT-linked magnetic beads) and a highly sensitive technique for detection of the existence of the UL53-specific mRNA (cDNA synthesis followed by PCR). It was shown that both pathogenic (KOS and P42) and apathogenic (R15) HSV-1 strains expressed the UL53 gene in brains of IC infected mice.


[Enhancer-of-white-apricot and its effects on the copia insertion allele white-apricot in Drosophila melanogaster]. In Drosophila melanogaster, the w(a) allele differs from the red-eye gene (w+) by the insertion of the retrovirus-like transposable element copia within the transcription unit. The w(a) flies have the apricot eye color. Most RNAs derived from w(a) have 3' termini within the 3' LTR of copia, and only small amounts of structurally normal RNA are produced. The eye color of flies with w(a) is affected by the gene Enhancer-of-white-apricot [E(w(a))] and become lighter. This effect on pigmentation is correlated with a corresponding decrease in white RNA having wild-type structure which was determined by the Northern blot. Three revertant alleles of E(w(a)) was generated by the irradiation with gamma-ray. These alleles are recessive lethals with death occurring during the larval stage. The original E(w(a)) allele is an antimorph and produces a product that interferes with the activity of the wild-type gene which have the product expected to either have a positive role in the splicing of w(a) RNAs or a negative role in polyadenylation.


Restraint-review committee: a working model. Initially, the goal of this committee was to reduce the number of unnecessary physical restraints (see Table 4). This was achieved by a team approach and a systematic process of assessment, monitoring, and evaluation. In addition, this process provided a means by which restraint use could be thoroughly documented and functionally monitored. Efforts are continuing in order to provide a safe environment while at the same time promoting quality of life through reduction of restraints.


[Modulation of the adenylate cyclase-cAMP pathway by protein kinase C in chick retinal pigment epithelium]. I investigated stimulation of cyclic adenosine 3':5'-monophosphate (cAMP) production by isoproterenol and forskolin in cultured chick embryo retinal pigment epithelium. In addition, in order to study the interaction between signal transduction systems, I studied the effects of phorbol 12-myristate, 13-acetate (PMA), a protein kinase C (PKC) activator, on isoproterenol- or forskolin-stimulated cAMP production. After stimulation with 10(-9)-10(-5) M isoproterenol and forskolin, a marked and concentration-dependent increase in cAMP level was observed. However, addition of 10(-10)-10(-5) M PMA had no effect on the cAMP level. Preincubation of isoproterenol and forskolin with 100 nM PMA suppressed the increase of cAMP. The suppressive effect of PMA was stronger on isoproterenol-stimulated production than on forskolin-stimulated production. 4 alpha-phorbol 12, 13, didecanoate, a weak activator of PKC, had a weak suppressive effect on cAMP-production stimulated by isoproterenol. Both staurosporin and H-7, 1-(5-isoquinolinylsulfonyl)-methylpiperazine, inhibitors of PKC, decreased the suppression of cAMP production by PMA. It is speculated that in chick retinal pigment epithelium PKC stimulators, such as PMA, may inhibit the adenylate cyclase pathway.


Histoenzymic effects of thiophenate and fenbendazole on the absorptive surfaces of Haemonchus contortus. In vitro alterations induced by a 10 micrograms/ml and 50 micrograms/ml dose each of thiophenate and fenbendazole on the absorptive surfaces of Haemonchus contortus (Nematoda: Trichostrongylidae) were studied. The most significant changes were induced in the gut epithelium. Alkaline phosphatase and adenosine triphosphatase activities were decreased, succinic dehydrogenase activity was increased, while acid phosphatase and glucose-6-phosphatase were completely lost from the intestinal epithelium after treatment with either of the drugs. A stimulatory effect of these two anthelmintics was observe on lactic dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase distribution. Thiophenate caused an increase in the activities of glutamate dehydrogenase (GDH), glucose-6-phosphate dehydrogenase (G-6-PD) and nonspecific esterases and a decrease in reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-D) activity. Fenbendazole treatment led to the inhibition of GDH, while G-6-PD, NADPH-D, cytochrome oxidase, monoamine oxidase and nonspecific esterase activity remained unaltered in the epithelium.


A case of sudden infant death due to massive hemorrhage in primitive neuroectodermal tumor. A case of the sudden death of a 14-month-old girl due to massive hemorrhage in a primitive neuroectodermal tumor (PNET) is presented along with a review of the relevant literature. PNET is a rare, malignant brain neoplasm occurring predominantly in children.


Epidermodysplasia verruciformis as a model of human papillomavirus-induced genetic cancers: the role of local immunosurveillance. Epidermodysplasia verruciformis (EV) presents a genetically determined, unusual susceptibility to infection with EV-specific human papillomaviruses (HPVs) related to abrogation of immunosurveillance exclusively against these viruses. The cutaneous viral carcinogenesis depends upon potentially oncogenic HPVs, the cocarcinogenic effect of ultraviolet irradiation, and genetic host factors, presumably a defect of anti-oncogenes or alleles of major histocompatibility complex and tumor necrosis factor locus involved in antigen presentation.


Retinoids--"differentiation agents" for cancer treatment and prevention. The ability of vitamin A and its derivatives to induce differentiation in certain target tissues has been appreciated for nearly a century. Recently, oral all-trans retinoic acid (ATRA), a vitamin A metabolite, has been shown to induce terminal differentiation of leukemic cells in patients with acute promyelocytic leukemia (APL). Complete remissions are obtained and normal hematopoiesis is established in an outpatient setting with minimal side effects in the majority of cases. Although remissions are not durable, disseminated intravascular coagulation, a frequent complication of remission induction in APL, is avoided by oral ATRA prior to definitive chemotherapy. The molecular basis for the efficacy of ATRA in APL appears to be the involvement of the retinoic acid receptor alpha locus in the t(15;17) translocation breakpoint characteristic of APL.


Modulation of Na-H exchange activity by angiotensin II in opossum kidney cells. Angiotensin II (ANG II) was shown to modulate transport in the renal proximal tubule through both inhibition of adenylate cyclase and protein kinase C (PKC) activation. We evaluated the effects of ANG II on adenosine 3',5'-cyclic monophosphate (cAMP) content and Na-H exchange activity (amiloride-sensitive Na influx) in two strains of opossum kidney (OK) cells originating from different sources, OK-VD and OK-RR cells. In OK-VD cells, ANG II inhibited basal and parathyroid hormone (PTH)-induced cAMP generation in a pertussis toxin-sensitive manner and reversed PTH inhibition of Na-H exchange. These effects of ANG II were prevented by PD 123319, a selective nonpeptide antagonist of AT2 receptors. In contrast, DuP 753, which antagonizes selectively AT1 receptors, had no effect. In OK-RR cells, ANG II had no effect on cAMP content and decreased Na-H exchange activity. The effect of ANG II persisted in the presence of PTH but was abolished by PKC downregulation and by DuP 753, but not by PD 123319. In conclusion, two types of ANG II receptors, coupled to distinct signaling pathways, were expressed independently in OK cells originating from two different sources and mediated opposite effects of ANG II on Na-H exchange activity. Those models provide a powerful tool for studying the intracellular steps involved in the tubular effects of ANG II and to evaluate the effect of pharmacological inhibitors of ANG II binding to its receptors.


Histamine-induced Cl- secretion in human nasal epithelium: responses of apical and basolateral membranes. The mechanism by which receptors coupled to phospholipase C (PLC) induce Cl- secretion in amiloride-pretreated cultures of human nasal epithelial (HNE) cultures was investigated. Histamine (10(-4) M, basolateral administration) stimulated a rapid increase in equivalent short-circuit current, an index of Cl- secretion, that returned to baseline within 5 min. Intracellular recordings with double-barreled Cl(-)-selective microelectrodes showed that the apical and basolateral membrane potentials rapidly hyperpolarized, the fractional resistance of the apical membrane increased, and the transepithelial resistance decreased in response to histamine. Intracellular Cl- activity remained constant. Equivalent circuit analysis revealed that the early portion (< 0.9 min) of the Cl- secretory response was driven by an activation of a hyperpolarizing basolateral conductance, likely K+, whereas the later (> 0.9 min) phase of Cl- secretion reflects activation of the apical membrane Cl- conductance. Histamine raised intracellular Ca2+ (Ca2+i) measured by fura-2 in HNE with a potency similar to that observed for induction of Cl- secretion. Both intracellular release and plasma membrane influx pathways were identified, typical of receptor-mediated activation of PLC. The intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (15 microM), coupled with reduced bathing solution Ca2+, blunted the rise in Ca2+i and the net transepithelial Cl- secretory response to histamine. We conclude that 1) histamine induced Cl- secretion in HNE by a sequential mechanism: the rapid initial component reflects activation of the basolateral K+ conductance, and the later component reflects activation of an apical Cl- conductance; and 2) the level of Ca2+i may participate in the activation of both the basolateral and apical conductances.


PGE2 regulates cAMP production in cultured rabbit CCD cells: evidence for dual inhibitory mechanisms. In cultured cortical collecting duct (CCD) cells, exogenous prostaglandin E2 (PGE2) inhibited arginine vasopressin (AVP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) production in a concentration-dependent manner. Although pertussis toxin (PT, 500 ng/ml) alone did not reverse the PGE2-dependent inhibition, PT and staurosporine, a protein kinase C inhibitor, together partially reversed the effect of exogenous PGE2. In contrast, PT completely reversed the inhibition of AVP-dependent cAMP production by sulprostone. These data suggest that exogenous PGE2 can inhibit AVP-stimulated cAMP production and that the inhibitory effects of PGE2 are mediated by staurosporine- and PT-sensitive component(s). Short-term (15-240 min) incubation with phorbol 12-myristate 13-acetate (PMA, 10(-7) M) inhibited PGE2-stimulated cAMP production. Long-term (20 h) incubation with PMA augmented PGE2-stimulated cAMP production. These data provide evidence for the maintenance of a PT-sensitive PGE2-dependent inhibitory pathway of cAMP production in cultured CCD cells. In addition, data are presented that support an inhibitory role for protein kinase C in the effects of PGE2 on the metabolism of cAMP in these cells.


Na channel kinetics remain stable during perforated-patch recordings. The results of studies on modulation of Na channel function are often difficult to interpret due to time-dependent changes in channel kinetics. Although the "tight-seal" whole cell voltage-clamp technique has proved very useful in studying the properties of the cardiac Na current, the spontaneous shift of parameters of inactivation and activation gating to more negative potential is a serious limitation to the use of the technique. The shifts are believed to result from changes in the intracellular milieu effected by dialysis; moreover, use of a variety of different anions and cations in the internal micropipette solution has not obviated the problem. The perforated-patch technique permits low-resistance intracellular access without free dialysis between the intracellular solution and the recording micropipette. We have compared steady-state inactivation and peak current-voltage relationship of whole cell Na currents measured with the conventional whole cell and perforated-patch techniques in rabbit atrial myocytes at 17 degrees C. Although gating parameters shifted to more negative potentials when recorded with the conventional technique, stable kinetics could be observed for up to 150 min with the perforated-patch technique. The potential for one-half Na channel inactivation was -73 +/- 5.1 mV and is consistent with measurements made using indirect techniques such as upstroke velocity measurements. The fact that the intracellular milieu is left relatively intact makes the approach attractive for studying modulation of the Na current by neurotransmitters and hormones.


Muscarinic receptors in MDCK cells are coupled to multiple messenger systems. Our studies on Madin-Darby canine kidney (MDCK) cells have demonstrated that high-affinity specific muscarinic receptors coupled to the phosphoinositide system are present in these cells. To determine whether muscarinic receptors in MDCK cells are linked negatively to the adenylate cyclase system, we measured the effect of muscarinic agonists and antagonists on vasopressin-, isoproterenol-, and forskolin-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation. Vasopressin produced a maximum stimulation of cAMP formation of 13 pmol.10(6) cells-1.2 min-1 at 10(-7) M. Isoproterenol and forskolin stimulated cAMP formation production to 21 pmol.10(6) cells-1.2 min-1 and 64 pmol.10(6) cells-1.10 min-1, respectively, at 10(-4) M. The effects of vasopressin, isoproterenol, and forskolin were blocked by arecoline, a cholinergic agonist, in a concentration-dependent manner. The arecoline response was blocked by treatment of the cells with pertussis toxin. The inhibition by arecoline of forskolin-stimulated cAMP formation was reversed by various muscarinic antagonists in the following order of potency: 4-diphenyl-acetoxy-N-methylpiperidine > p-fluorohexahydrosiladifenidol > pirenzepine > methoctramine. This order of potency of muscarinic antagonists is similar to that observed in our radioligand binding studies and is consistent with the M3 subtype of muscarinic receptors. Our results indicate that muscarinic receptors in MDCK cells are coupled negatively to the adenylate cyclase system via pertussis toxin-sensitive G protein. It is concluded that this intracellular system may at least be partially responsible for the action of cholinergic agonists in these cells and in the kidney.


Plasma membrane domain localization and transcytosis of the glucagon-induced hepatic system A carrier. We have utilized canalicular (cLPM) and basolateral (blLPM) liver plasma membrane vesicles to investigate the domain localization of several Na(+)-dependent amino acid transporters. Neutral amino acid transport by systems N and ASC was measurable in both domains but was greater in blLPM vesicles. Sodium-dependent glutamate uptake (system X-) was preferentially localized to cLPM. The absolute activity and domain distribution of these three carriers remained unchanged after treatment of rats with a combination of glucagon and dexamethasone. A low level of basal system A activity was present in both the blLPM and cLPM. Glucagon-induced system A activity was first observed in blLPM vesicles approximately 60 min posthormone treatment and, in cLPM vesicles, approximately 30 min later. In situ perfusion of glucagon-treated rat liver with the membrane-impermeant protein modification reagent glutathione maleimide specifically inactivated blLPM system A activity and abolished the delayed arrival of hormone-induced activity to the cLPM. Transient perfusion of the liver with glutathione maleimide followed by a recovery period in vivo showed that the reagent did not irreversibly inactivate the transcytotic process and also provided an independent demonstration of the time delay between arrival of the carrier activity at the two membrane surfaces. These results support the concept of a transcytotic process in which the blLPM is the sorting site for the hormone-induced system A carrier proteins that eventually reach the cLPM.


Chief cells possess a receptor with high affinity for PACAP and VIP that stimulates pepsinogen release. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a 38-amino acid peptide of the secretin-vasoactive intestinal peptide (VIP) family. To investigate whether PACAP alters chief cell function, we prepared isolated chief cells (> 90% pure) from guinea pig stomach. PACAP-38, PACAP-27, VIP, and secretin all caused a threefold increase in pepsinogen release. The dose-response curves of PACAP-38, PACAP-27, and VIP were biphasic, whereas with secretin it was not. The first phase comprised 40% of maximal release, and each of the three peptides (PACAP-38, PACAP-27, and VIP) were equipotent (EC50 0.1-0.3 nM). For the second phase, comprising 60% of maximal release, the relative potencies were PACAP-38 > PACAP-27 = VIP. 125I-labeled secretin, 125I-VIP, and 125I-PACAP-27 all demonstrated saturable binding to chief cells. Binding of both 125I-PACAP-27 and 125I-VIP was inhibited completely and with similar potencies by PACAP-38, PACAP-27, and VIP. Secretin had a > 500-fold lower affinity than PACAP-38 for displacing both 125I-PACAP-27 and 125I-VIP. With 125I-secretin, secretin was the most potent, and was 197 times more potent than PACAP-38, which was 6-8 times more potent than both PACAP-27 and VIP. We conclude that both PACAP-38 and PACAP-27 stimulate pepsinogen secretion from dispersed chief cells. In contrast to a number of other tissues, no evidence for a high-affinity receptor that interacted only with PACAP was found. PACAP and VIP interact with equal high affinity with a common receptor and with low affinity with the secretin receptor.


Differential expression of early response genes, c-jun, c-fos, and jun B, in A5 cells. We examined the expression of c-fos, c-jun, and jun B after activation of different signal transduction pathways in the A5 rat salivary epithelial cell line. Stimulation of beta-adrenergic receptors by isoproterenol, or addition of 8-bromoadenosine 3',5'-cyclic monophosphate, induces the expression of c-fos and jun B by a protein kinase A-mediated pathway. Phorbol 12-myristate 13-acetate (PMA) induces the expression of all three genes, but with different kinetics. While c-fos and jun B mRNA levels increase early (1 h) after stimulation and transiently, those of c-jun remain higher than control even after stimulation for 8 h and return to basal levels by 24 h. Inhibitors of protein kinase C block the effect of PMA on c-fos, c-jun, and jun B expression, indicating that these genes are also regulated by a protein kinase C-mediated mechanism in A5 cells. Increases in cytosolic Ca2+ by A23187 or ionomycin induce only the expression of c-fos gene. This induction is abolished when A5 cells are loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid before treatment with the ionophores, or when serum is excluded from the incubation medium. Exclusion of serum from the medium does not change the effects of isoproterenol or PMA on c-fos, c-jun, or jun B. These results strongly suggest that serum factors act synergistically with Ca2+ to induce c-fos expression in A5 cells. The studies presented here indicate that different signal transduction pathways operate in A5 cells for the induction of c-fos, c-jun, and jun B genes.


Organic cation transport by rat hepatocyte basolateral membrane vesicles. Hepatocyte basolateral membrane possesses transport systems for mediated uptake of organic cations, the first step in the subsequent biliary excretion and/or metabolism of these compounds. The purpose of these studies was to evaluate potential mechanisms for transport of this class of solutes across this membrane by measuring 3H-labeled tetraethylammonium ([3H]TEA) transport into rat hepatocyte basolateral membrane vesicles. [3H]TEA uptake was stimulated by an outwardly directed proton gradient consistent with TEA-proton exchange. Proton gradient-stimulated [3H]TEA uptake was inhibited by quinidine and by the combination of valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) but not by CCCP alone or by N1-methylnicotinamide (NMN). An outwardly directed TEA gradient also stimulated uptake of [3H]TEA with values at early time points exceeding those at equilibrium. This trans-stimulation or countertransport was saturable with an apparent Michaelis constant of 106 microM and maximal velocity of 434 pmol.mg-1.15 s-1. TEA countertransport was cis-inhibited by quinidine, cimetidine, and thiamine and by low temperature, but not by NMN. Thiamine was also capable of trans-stimulating [3H]TEA uptake. An outwardly directed potassium gradient enhanced and an inwardly directed potassium gradient reduced TEA countertransport but had no effect on [3H]TEA uptake occurring in the absence of other electrochemical driving forces. These studies indicate that there are at least two potential mechanisms in the hepatocyte basolateral membrane for transport of organic cations; organic cation-organic cation exchange (countertransport) and organic cation-proton exchange. Furthermore, the results are consistent with the existence of more than one transporter with different substrate affinities in each of these categories.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of H2 histamine receptor: linkage to both adenylate cyclase and [Ca2+]i signaling systems. We undertook these studies to examine the intracellular mechanisms of histamine action using a rat hepatoma-derived cell line that had been transfected to express the H2 histamine receptor cDNA. Transfected cells demonstrated increased adenosine 3',5'-cyclic monophosphate production, membrane inositol phospholipid turnover, and intracellular Ca2+ concentration ([Ca2+]i) in response to histamine. All of the effects could be inhibited with the H2 histamine receptor antagonist cimetidine, and the increased membrane inositol phospholipid turnover and [Ca2+]i were abolished by cholera toxin pretreatment of cells. These data support the notion that a single histamine H2 receptor can be linked to two stimulatory intracellular signaling systems.


Kinetics of urea exchange in air-filled and fluid-filled rat lungs. Urea has been used as an indicator for estimating 1) the dilution of epithelial lining fluid (ELF) that occurs during bronchoalveolar lavage (BAL) and 2) the permeability-surface area product (PS) of the pulmonary endothelium to this solute. Because relatively little is known about how urea equilibrates with fluid in the lung tissues and airspaces, we have undertaken a study of the kinetics of movement from the vasculature into the tissues of isolated, perfused rat lungs. Although instillation of 5 ml of 154 mM saline into the airspaces of this preparation increased the calculated extravascular volume of 3HOH from 0.64 +/- 0.23 to 2.10 +/- 0.58 ml (SE, n = 6) during a single transit through the pulmonary circulation, it did not have a detectable effect on the distribution of [14C]urea in the lung tissues. However, leakage of [14C]urea into saline within the airspaces was detected during constant infusions: concentrations in the airspace fluid reached 1.1 +/- 0.2% of those in the perfusate by 90 s and 1.90 +/- 0.2% at 120 s, levels that would significantly reduce estimates of the dilution of ELF by BAL. In contrast, concentrations of 99mTc-diethylenetriaminepentaacetic acetic acid (DTPA) in the airspaces remained < 0.2% of those in the perfusate, suggesting that 99mTc-DTPA may be a superior indicator for estimating dilution of ELF by BAL.(ABSTRACT TRUNCATED AT 250 WORDS)


Rabbit surfactant protein C: cDNA cloning and regulation of alternatively spliced surfactant protein C mRNAs. Surfactant protein C (SP-C), a hydrophobic protein of pulmonary surfactant is essential for surfactant function. Toward elucidating molecular mechanisms that mediate regulation of SP-C gene expression in rabbit lung, we isolated and characterized cDNAs encoding rabbit SP-C and studied the regulation of SP-C gene expression during fetal lung development and by adenosine 3',5'-cyclic monophosphate (cAMP) and dexamethasone in fetal lung tissues in vitro. We found that rabbit SP-C is highly homologous to SP-C of other species and is encoded by two mRNAs that differ by an insertion of 31 nucleotides in the 3' untranslated regions. SP-C mRNAs were classified into two types based on the nucleotide sequence; type I represents RNA without the 31 nucleotide insert and comprises approximately 80-90% of total SP-C mRNA content, whereas type II represents RNA containing the insert and comprises approximately 10-20% of total SP-C mRNA content. SP-C mRNAs were induced in a coordinate manner during fetal lung development and by cAMP and dexamethasone in fetal lung tissues in vitro. Southern hybridization analysis of genomic DNA suggested that SP-C mRNAs are encoded by a single gene. Polymerase [corrected] chain reaction-amplification of genomic DNA with oligonucleotide primers flanking the insertional sequence and sequence analysis of amplified DNA showed that SP-C mRNAs are produced by alternative use of 3' splice sites of intron 5 of SP-C gene.


Stimulation of bovine pulmonary artery endothelial cell ACE by dexamethasone: involvement of steroid receptors. After exposure of bovine pulmonary artery endothelial cells in culture to 1 microM dexamethasone for 24-48 h, angiotensin-converting enzyme (ACE) activity of these cells was elevated severalfold. The increase in ACE activity was preceded by an increase in ACE mRNA, which could be detected after treatment of cells with dexamethasone for 4 h. When the increase in ACE mRNA produced by dexamethasone at 4 h was blocked by alpha-amanitin, an RNA polymerase II inhibitor, the increase in ACE activity detected at 48 h was inhibited. RU 38486, a steroid receptor antagonist, inhibited the elevation of both ACE activity and mRNA produced by dexamethasone. Among other steroids tested, only hydrocortisone, aldosterone and corticosterone-21-acetate had a stimulatory effect on ACE activity. RU 38486 effectively blocked the elevation in ACE activity produced by both aldosterone and dexamethasone, but had no effect on the elevation of ACE activity produced by other agents (3-isobutyl-1-methylxanthine, A23187, and dibutyryl adenosine 3',5' cyclic monophosphate). From these data we conclude that dexamethasone and certain other steroids with an hydroxyl group in the 11th carbon position regulate ACE gene expression of bovine endothelial cells at the transcriptional level via a steroid receptor-mediated mechanism.


PMA-activated neutrophils decrease pulmonary endothelial ectoenzyme activities in perfused rabbit lungs. We have studied the effects of phorbol 12-myristate 13-acetate (PMA, 15 micrograms) on pulmonary endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] function in isolated rabbit lungs perfused in situ with platelet-poor (PPP) or platelet-rich (PRP) plasma in the presence or absence of neutrophils. Enzyme activities were estimated from the hydrolysis of the substrates [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) by ACE and 14C-labeled AMP by NCT during a single transpulmonary passage, using indicator-dilution techniques. In all treatment groups PMA produced a delayed increase in pulmonary vascular resistance to about three times the control value. PMA alone [in lungs perfused with PPP (n = 5 animals) or PRP (n = 6)] or neutrophils alone (in PPP-perfused lungs, n = 5) had no effect on enzyme activity. However, PMA-activated neutrophils (n = 5) decreased percent metabolism (%M) of [3H]BPAP from 87 +/- 3 to 77 +/- 4% (30 min after PMA), and the apparent first-order parameter [ratio of maximum activity to Michaelis constant (Amax/Km)] for ACE from 821 +/- 114 to 613 +/- 61 ml/min (30 min after PMA). At the same time, Km values of BPAP for ACE and AMP for NCT were elevated from 9.2 +/- 2.2 to 19.3 +/- 3 microM and 6.7 +/- 1.2 to 15.1 +/- 3.6 microM, respectively, whereas Amax (product of enzyme mass and rate of product formation, thus an index of perfused microvascular surface area) did not change.(ABSTRACT TRUNCATED AT 250 WORDS)


PMA-activated neutrophils decrease ectoenzyme activities in rabbit aortic endothelial cells in culture. We have studied the effects of phorbol 12-myristate 13-acetate (PMA)-activated neutrophils [polymorphonuclear leukocytes (PMN)] on endothelial ectoenzyme [angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT)] activities in cultured rabbit aortic endothelial cells (EC) with the use of [3H]benzoyl-Phe-Ala-Pro and 14C-labeled AMP as substrates, respectively, under first-order reaction conditions. PMA (1-1,000 ng/ml) or PMN alone had no effect on ACE activity. When PMA was incubated together with PMN (PMN/EC = 1.25:1 or 2.5 x 10(5) neutrophils/ml) for 4 h in Earle's salts, a PMA dose-dependent decrease in ACE activity was observed. Threshold PMA concentration was 2 ng/ml. At 8 ng PMA/ml, ACE activity was totally abolished, without any evidence of cytotoxicity, as inferred from release of 51Cr from prelabeled EC. The decrease in ACE activity was also dependent on PMN concentration and was detectable at PMN/EC values as low as 1.25:10 (0.25 x 10(5) PMN/ml). Inhibition of ACE occurred as early as 1 h after incubation (PMA 10 ng/ml, PMN/EC = 1.25:1). PMA alone caused a small but significant increase in NCT activity, whereas PMA coincubation with PMN produced a significant decrease in NCT activity (20-29%), which was PMA and PMN concentration independent. PMA increased PMN adherence to endothelial monolayers in a concentration-dependent manner. Pretreating PMN with monoclonal antibody 60.3 (raised against the adhesion glycoprotein CD18) or placing a 2-microns filter between PMN and EC, protected the decrease in ACE activity.(ABSTRACT TRUNCATED AT 250 WORDS)


Histamine, actin-gelsolin binding, and polyphosphoinositides in human umbilical vein endothelial cells. Histamine activates inositol phospholipid metabolism, increases calcium, and causes a change in shape of human umbilical vein endothelial (HUVE) cells. Changes in endothelial cell shape are determined, in part, by changes in the actin cytoskeleton. Gelsolin is an actin-binding protein with the potential to alter the actin cytoskeleton in response to changes in cell calcium and/or changes in polyphosphoinositides. Therefore, we examined the interactions of actin and gelsolin in HUVE cells in which inositol phospholipid metabolism was activated with histamine. In HUVE cells exposed to histamine we estimated actin-gelsolin binding by quantitating actin and gelsolin, immunoprecipitated with anti-gelsolin Sepharose. We estimated the relative amount of filamentous actin in the histamine-exposed HUVE cells by quantitating the amount of actin that was Triton soluble. We also measured the amount of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in the HUVE cells before and after exposure to histamine. We found that histamine decreased the amount of actin that was immunoprecipitated with gelsolin, decreased the fraction of cell actin that was Triton soluble, and increased PIP and PIP2. These results demonstrate that histamine promotes actin filament formation in HUVE cells and that histamine-mediated changes in actin-gelsolin binding in these cells are better predicted by changes in polyphosphoinositides than by increases in cell calcium.


Mechanism of H2O2-induced modulation of airway smooth muscle. We investigated the effects of H2O2 generated by glucose (G) and glucose oxidase (GO) on the isolated rabbit tracheal smooth muscle suspended in Krebs-Ringer solution. H2O2 generated by G+GO was measured with luminol-dependent chemiluminescence. G+GO in the concentrations of 1x (1.80 microM G, 0.075 U/ml GO) and 2, 4, and 8x generated 1.35, 3.2, 6.10, and 6.00 microM of H2O2, respectively. H2O2 produced relaxation of rabbit tracheal smooth muscle, relaxed acetylcholine (ACh)-precontracted muscle, and reduced muscle responsiveness to ACh. These effects were concentration dependent. H2O2, however, produced contraction of guinea pig tracheal smooth muscle. Catalase completely inhibited the H2O2-induced relaxation of ACh-precontracted tracheal smooth muscle. H2O2-induced relaxation was greater in preparations with intact epithelium (65%) than in those denuded of epithelium (40%). The relaxant effects of H2O2 in the presence of an inhibitor of nitric oxide synthesis (NG-monomethyl-L-arginine), an inhibitor of guanylate cyclase (methylene blue), an inhibitor of cyclooxygenase (indomethacin), and an ATP-sensitive K+ channel blocker (glipizide) were 44, 44, 39, and 48%, respectively. H2O2-induced relaxation in the presence of indomethacin in preparations with denuded epithelium was 29%. These results suggest that H2O2-induced relaxation of tracheal smooth muscle is partly epithelium dependent and is mediated by inhibitory arachidonic acid metabolites, epithelium-derived relaxing factor (nitric oxide), ATP-sensitive K+ channels, and the synthesis and release of prostaglandins from epithelium and the underlying smooth muscle.


Digoxin-like immunoreactive substance in urine of patients with mucocutaneous lymph node syndrome (MCLS). Levels of digoxin-like immunoreactive substance (DLIS) and dehydroepiandrosterone sulfate (DHEA) in urine from patients with mucocutaneous lymph node syndrome (MCLS) were measured by radioimmunoassay. Because DLIS of stored samples was often below the level of detection by conventional immunoassay, the authors used 80% ammonium sulfate and extraction with phosphate buffer and then 80% hot ethanol. To study the origin of raised levels of DLIS in urine, the synthesis of DLIS by cultured human umbilical vein endothelial cells (HUVEC) was tested in vitro. The correlation between DLIS and DHEA levels was not significant. Mean levels of urinary DLIS corrected for creatinine in the patients with MCLS were significantly higher than in both normal and diseased controls. The culture medium of HUVEC was found to contain DLIS activity. These results show that MCLS should be added to the clinical states associated with an increased urinary concentration of DLIS and that the endothelial cells are one source of DLIS in man.


[Thrombocytopenia induced by low molecular weight heparin]. Two cases of thrombocytopenia due to a low molecular weight heparin (Fraxiparine) are reported. The first case was a 35-year-old alcoholic man with acute mild pancreatitis. After having been treated with Fraxiparine for 12 days to prevent venous thrombosis, routine laboratory studies revealed a thrombocytopenia (49 G.l-1). At the same time, a minor haemorrhage occurred in the nasogastric tube. Prothrombin time, partial thromboplastin time, fibrin degradation products and D-dimers remained normal. There were no soluble fibrin monomers. Fraxiparine was discontinued. The thrombocyte count continued to decrease (12 G.l-1) up to the thirteenth day, it raised 3 days later to 110 G.l-1, and returned to normal after 9 days more (395 G.l-1). The second patient was a 58-year-old man given prophylactic Fraxiparine between the 5th and 16th days after admission for a severe asthma attack. Here again, after 12 days of treatment, the thrombocyte count decreased to 74 G.l-1. There were no other abnormalities, neither clinically nor in laboratory findings. Heparin administration was discontinued and the thrombocytopenia had resolved 3 days later. In both patients, the diagnosis of thrombocytopenia elicited by low molecular weight heparin was confirmed by finding, in vitro, a platelet aggregating factor in the presence of Fraxiparine. The literature concerning this topic is reviewed and discussed.


Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection. A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.


Analysis of glycoprotein I (gI) negative and aberrant pseudorabies viral diagnostic isolates. Glycoprotein I (gI) phenotypes and genotypes of 4 pseudorabies viral diagnostic isolates were evaluated by use of in vitro DNA amplification, monoclonal antibody binding, gI-specific serodiagnostic responses, and in vivo virulence approaches. Three viruses were avirulent and did not elicit gI-specific serologic responses, react with gI-specific monoclonal antibodies, or contain gI epitope-encoding DNA sequences. The fourth virus was virulent and did elicit a gI-specific serodiagnostic response. Compared with reference virulent pseudorabies viruses, however, the fourth isolate had reduced reactivity with a group of gI monoclonal antibodies and had a single nucleotide sequence substitution with a corresponding putative amino acid change in the epitopically dominant portion of the gI molecule. Presumably, the first 3 isolates represented diagnostic recoveries of viruses derived from gI-deleted modified-live pseudorabies viral vaccines, whereas the fourth isolate was a virulent but gI-aberrant wild-type virus. Thoroughly assessing the gI status of pseudorabies viral diagnostic isolates was considered to be essential in evaluating the epidemiologic importance of these viruses and in monitoring the validity of gI-based vaccine companion tests now used worldwide in pseudorabies control and eradication programs.


Pharmacokinetic evaluation of enrofloxacin administered orally to healthy dogs. Enrofloxacin was administered orally to 6 healthy dogs at dosages of approximately 2.75, 5.5, and 11 mg/kg of body weight, every 12 hours for 4 days, with a 4-week interval between dosage regimens. Serum and tissue cage fluid (TCF) concentrations of enrofloxacin were measured after the first and seventh treatments. The mean peak serum concentration occurred between 1 and 2.5 hours after dosing. Peak serum concentrations increased with increases in dosage. For each dosage regimen, there was an accumulation of enrofloxacin between the first and seventh treatment, as demonstrated by a significant (P = 0.001) increase in peak serum concentrations. The serum elimination half-life increased from 3.39 hours for the 2.75 mg/kg dosage to 4.94 hours for the 11 mg/kg dosage. Enrofloxacin accumulated slowly into TCF, with peak concentrations being approximately 58% of those of serum. The time of peak TCF concentrations occurred between 3.8 hours and 5.9 hours after drug administration, depending on the dosage and whether it was after single or multiple administrations. Compared with serum concentrations (area under the curve TCF/area under the curve serum), the percentage of enrofloxacin penetration into TCF was 85% at a dosage of 2.75 mg/kg, 83% at a dosage of 5.5 mg/kg, and 88% at a dosage of 11 mg/kg. All 3 dosage regimens of enrofloxacin induced continuous serum and TCF concentrations greater than the minimal concentration required to inhibit 90% (MIC90) of the aerobic and facultative anaerobic clinical isolates tested, except Pseudomonas aeruginosa.(ABSTRACT TRUNCATED AT 250 WORDS)


Regulation of adrenocorticotropin secretion from cultured canine anterior pituitary cells. Pituitary cells, collected from five healthy dogs, were cultured and treated with various doses of ovine corticotropin-releasing hormone (CRH), arginine vasopressin (AVP), oxytocin (OT), or angiotensin II (AII) to determine which of these hypothalamic peptides affected adrenocorticotropin (ACTH) secretion. Of the 4 peptides, only CRH significantly increased ACTH secretion from cultured canine anterior pituitary cells. The lowest dose of CRH tested, 0.01 nM, significantly stimulated ACTH release. Co-addition of AVP, OT, or AII with CRH did not increase ACTH secretion beyond that caused by addition of CRH alone. Similarly, neither co-addition of AVP with OT, AVP with AII, or OT with AII significantly stimulated ACTH secretion. These results support a role for CRH in the physiologic regulation of ACTH secretion from the canine anterior pituitary, but do not support regulatory roles for AVP, OT, or AII.


Cohort study of natural transmission and two methods for control of caprine arthritis-encephalitis virus infection in goats on a California dairy. A prospective observational cohort study of 361 dairy goat kids was conducted to compare 2 methods of controlling caprine arthritis-encephalitis virus infection under commercial dairy conditions. To compare effectiveness of feeding kids pasteurized milk vs serologic testing and segregation in addition to pasteurized milk feeding, goats were monitored up to the age of 30 months by use of monthly agar gel immunodiffusion testing. Survival analysis methods were used to determine whether age at seroconversion differed between the 2 groups. Significantly lower rates of seroconversion were observed in the segregated group (P < 0.001), compared with the nonsegregated group. Of 193 goats in the pasteurized milk-only group, 146 (75.6%) seroconverted within the 30-month study period, whereas infection was detected in 39 (23.2%) of 168 goats in the test/segregated group. Nonsegregated goats were 3.37 times more likely to seroconvert by 24 months of age, and 70.3% of seroconversions by 24 months of age could be attributed to nonsegregation. For age-specific intervals beyond 180 days of age, 70 to 100% of seroconversions could be attributed to lack of segregation. Cohort life tables for age at seroconversion were reported for each group. Type of colostrum fed, sex, and weaning group (season) were not significantly associated with age at seroconversion. Saanen goats had lower age-specific risk of seroconversion in the nonsegregated group alone and overall. Non-Saanen goats wee 1.5 times more likely to seroconvert than were Saanen goats, when adjusted for a possible confounding effect of weaning group. Results indicate that pasteurized milk feeding and routine test and segregation would be a substantially more effective means of control of the disease in dairy goat herds than would pasteurized milk feeding alone.


Risk factors associated with the incidence of seroconversion to caprine arthritis-encephalitis virus in goats on California dairies. Incidence of seroconversion to caprine arthritis-encephalitis virus (CAEV) was determined for 1,194 goats on 11 dairies, using 2 repeated annual herd tests for CAEV. Current life table methods were used to compare age-specific incidence of seroconversion for pasteurized milk-raised and unpasteurized milk-raised goats. Logistic regression models were used to determine the risk factors associated with CAEV seroconversion, and to estimate odds ratios for seroconversion for various factor levels. Goats raised by unpasteurized milk-feeding methods were 2.5 to 6.7 times more likely to seroconvert than were goats raised by pasteurized milk-feeding methods, depending on the method of comparison. Similarly, 61.6 to 85.0% of seroconversions in yearling goats possibly were attributable to unpasteurized milk feeding. Among yearling goats, CAEV seroconversion was associated with feeding method, breed, and source of goat (herd of origin) when the effect of dairy was considered. In addition to the 6.7 times greater risk of seroconversion for unpasteurized milk-raised goats, yearling goats of the Saanen and Toggenburg breeds were 2.2 and 3.3 times, respectively, more likely to seroconvert than were Alpine yearling goats. Yearling goats purchased from another source were less likely to seroconvert than were yearlings raised on the dairy where they were studied. Among goats > 1 year old, age was associated with risk of seroconversion. Goats that were 3 years old or were > or = 4 years old were 1.7 and 3.2 times, respectively, more likely to seroconvert than were 2-year-old goats, when adjusted for effect of dairy. The effects of dairy were significant (P < or = 0.001) in yearling and older goats.


Continuous production of 6-APA in an aqueous two-phase system. An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate provided a favorable environment for bioconversion of penicillin G to 6-aminopenicillanic acid (6-APA). The recombinant E. coli cells containing penicillin acylase were partitioned in the phosphate-rich bottom phase, and the product 6-APA in the PEG-rich top phase, which protected the enzyme from deactivation for a longer period of time. The continuous production of 6-APA by recycling the bottom phase showed a slight decrease in enzyme activity from initial 80 mM of 6-APA production to 61 mM during the eight days of operation at a space velocity of 0.12 hours-1. This work showed a possibility that the aqueous two-phase whole-cell enzyme bioconversion could serve as an alternative to immobilized cells on solid matrices.


Viral haemorrhagic disease of rabbit: purification and characterization of a strain isolated in France. The causative agent of rabbit viral haemorrhagic disease (VHD) was purified by CsCl density gradient centrifugation from liver homogenates of rabbits infected with a strain of VHD virus isolated in France. Electron microscopy observations revealed that the virions were icosahedral with a diameter of 33-35 nm, and with an average buoyant density of 1.33. Structural proteins were investigated by SDS-polyacrylamide gel electrophoresis and western blotting. The results indicated the presence of a major structural protein of 64 kDa and other minor proteins of molecular weight of 43, 36 and 32 kDa, the last presumably resulting from the proteolytic degradation of VP 64. These results correspond with the description made by the different authors who identify the VHD virus as a member of the Caliciviridae family.


The Entner-Doudoroff pathway in Escherichia coli is induced for oxidative glucose metabolism via pyrroloquinoline quinone-dependent glucose dehydrogenase. The Entner-Doudoroff pathway was shown to be induced for oxidative glucose metabolism when Escherichia coli was provided with the periplasmic glucose dehydrogenase cofactor pyrroloquinoline quinone (PQQ). Induction of the Entner-Doudoroff pathway by glucose plus PQQ was established both genetically and biochemically and was shown to occur in glucose transport mutants, as well as in wild-type E. coli. These data complete the body of evidence that proves the existence of a pathway for oxidative glucose metabolism in E. coli. PQQ-dependent oxidative glucose metabolism provides a metabolic branch point in the periplasm; the choices are either oxidation to gluconate followed by induction of the Entner-Doudoroff pathway or phosphotransferase-mediated transport. The oxidative glucose pathway might be important for survival of enteric bacteria in aerobic, low-phosphate, aquatic environments.


The membrane-induced proton motive force influences the metal binding ability of Bacillus subtilis cell walls. Bacillus subtilis 168 is a gram-positive bacterium whose cell wall contains the highly electronegative polymers peptidoglycan (chemotype A1 gamma) and glycerol-based teichoic acid to produce a surface with a net negative charge with high metal binding capacity. During metabolism, a membrane-induced proton motive force continuously pumps protons into the wall fabric. As a result, a competition between protons and metal ions for anionic wall sites occurs, and less metal is bound in living cells than in nonliving cells or those in which the plasma membrane has been uncoupled. This was shown by using two metallic ions, UO2(2+) and Sc3+, on control cells, cells uncoupled with either carbonyl cyanide m-chlorophenylhydrazone or NaN3, or cells killed by gamma radiation. Transmission electron microscopy, energy-dispersive X-ray spectroscopy, and inductively coupled plasma atomic-emission spectroscopy showed that more metal was retained in the walls of nonliving cells and those with deenergized membranes than in their living counterparts.


Mechanisms of strontium uptake by laboratory and brewing strains of Saccharomyces cerevisiae. Laboratory and brewing strains of Saccharomyces cerevisiae were compared for metabolism-independent and -dependent Sr2+ uptake. Cell surface adsorption of Sr2+ to live cells was greater in the brewing than in the laboratory strain examined. However, uptake levels were greater in denatured (dried and ground) S. cerevisiae, and the relative affinities of Sr2+ for the two strains were reversed. Results for the brewing S. cerevisiae strain were similar whether the organism was obtained fresh from brewery waste or after culturing under the same conditions as for the laboratory strain. Reciprocal Langmuir plots of uptake data for live biomass were not linear, whereas those for denatured biomass were. The more complex Sr2+ binding mechanism inferred for live S. cerevisiae was underlined by cation displacement experiments. Sr2+ adsorption to live cells resulted in release of Mg2+, Ca2+, and H+, suggesting a combination of ionic and covalent bonding of Sr2+. In contrast, Mg2+ was the predominant exchangeable cation on denatured biomass, indicating primarily electrostatic attraction of Sr2+. Incubation of live S. cerevisiae in the presence of glucose resulted in a stimulation of Sr2+ uptake. Cell fractionation revealed that this increased Sr2+ uptake was mostly due to sequestration of Sr2+ in the vacuole, although a small increase in cytoplasmic Sr2+ was also evident. No stimulation or inhibition of active H+ efflux resulted from metabolism-dependent Sr2+ accumulation. However, a decline in cytoplasmic, and particularly vacuolar, Mg2+, in comparison with that of cells incubated with Sr2+ in the absence of glucose, was apparent. This was most marked for the laboratory S. cerevisiae strain, which contained higher Mg2+ levels than the brewing strain.


Purification and properties of NADP-dependent glutamate dehydrogenase from Ruminococcus flavefaciens FD-1. Glutamate dehydrogenase (GDH) (L-glutamate:NADP+ oxidoreductase, deaminating, EC 1.4.1.4) from the cellulolytic ruminal bacterium Ruminococcus flavefaciens has been purified and characterized. The native enzyme and subunit are 280 and 48 kDa, respectively, suggesting that the native enzyme is a hexamer. The enzyme requires 0.5 M KCl for optimal activity and has a pH optimum of 6.9 to 7.0. The Kms for ammonia, alpha-ketoglutarate, and glutamate are 19, 0.41, and 62 mM, respectively. The sigmoidal NADPH saturation curve revealed positive cooperativity for the binding of this coenzyme. The first residue in the N-terminal amino acid sequence from R. flavefaciens GDH was alanine, suggesting that the protein may be modified posttranslationally. Comparison of the N-terminal sequence with those of Escherichia coli, Salmonella typhimurium, and Clostridium symbiosum revealed only 39% amino acid homologies. The GDH from R. flavefaciens was unique in that its specific activity was highest during ammonia-limited growth but was not affected by ammonia shock treatment (20 mM).


The clinical significance of the thyrotropin-releasing hormone test in alcoholic men. Sixty-six alcoholic men who had been abstinent from alcohol for at least four weeks were assessed clinically and then investigated in terms of Thyroid-Stimulating Hormone (TSH) and prolactin responses to a Thyrotropin-Releasing Hormone (TRH) challenge. Consistent with other studies, a third of the subjects had a blunted TSH response to TRH. This blunted response was not associated with a family history of alcoholism, or current depressive symptoms, past history of depression or family history of depression. However, subjects with a blunted TSH response were more likely to have had an earlier onset of alcoholism and to have had shorter alcoholic remissions in the past.


Plasma beta-endorphin, but not met-enkephalin levels are abnormal in chronic alcoholics. Plasma beta-endorphin, met-enkephalin and ACTH concentrations were measured in 20 male alcoholics (age: 32-60 yr; duration of ethanol addiction: 13.2 +/- 6.2 yr; mean +/- SE) immediately after admission to the hospital (at a time not exceeding 8 hr from the last ethanol consumption) and after 5 weeks of forced abstinence. The results were compared with those obtained in 12 age-matched normal controls. Plasma ACTH and met-enkephalin levels were normal in alcoholics on both occasions. In contrast, in samples taken at admission to the hospital, the circulating concentrations of beta-endorphin in alcoholics were half (17.1 +/- 5.3 pg/ml; mean +/- SE) of those observed in the normal controls (34.1 +/- 6.0). beta-endorphin levels rose significantly after 5 weeks of abstinence (30.1 +/- 4.9); at this time, they were not significantly different from those observed in normal controls. These data indicate that acute alcohol consumption induces significant alterations in plasma beta-endorphin, but not met-enkephalin levels, which are reversed after 5 weeks of abstinence.


Effect of ethanol on the in vivo kinetics of thiamine phosphorylation and dephosphorylation in different organs of rat--II. Acute effects. The effects of acute ethanol administration on different steps of thiamine (T), thiamine monophosphate (TMP) and pyrosphosphate (TPP) metabolism were determined in vivo in nervous tissues (cerebral cortex, cerebellum, brain stem and sciatic nerve) and in other tissues (small intestinal mucosa, kidney, heart, skeletal muscle and liver) of rats. The radioactivity of T and its phosphoesters in plasma and tissues was determined under steady-state conditions and at fixed time intervals (0.25-24 hr) after an i.p. injection of Thiazole-[2-14C]-thiamine (30 micrograms: 1.25 microCi) in the presence of a constant plasma ethanol concentration (37 mM; 1.75 g.l-1) produced by repeated intragastric administration of ethanol. Control animals received water intragastrically. Ethanol-treated rats and controls were starved, with water ad libitum during the 24 hr study period. Data were evaluated by using appropriate compartmental models, which allowed calculation of fractional rate constants, turnover rates and turnover times. In nervous tissues ethanol enhanced TMP entry (without affecting T entry or T and TMP release), reduced turnover time of total T and TPP, caused an almost general enhancement of TPP dephosphorylation without affecting T pyrophosphorylation, and increased markedly T content in the mixture released by tissues. Overall, ethanol appeared to enhance exchanges of T compounds in nervous tissues. In other tissues, the effects of ethanol were less consistent. Ethanol increased T uptake in kidney and liver and T release in liver and heart, but had no effect on T exchanges in the small intestinal mucosa and in skeletal muscle. In the presence of ethanol, TMP uptake increased in heart and skeletal muscle and decreased in the small intestinal mucosa, while TMP release decreased in heart and remained unchanged in all other organs. Turnover times tended to increase for total T and to decrease for TPP. T pyrophosphorylation was generally reduced, and T phosphates dephosphorylation generally enhanced. T became the most abundant component in the mixture released from all tissues.


Effects of dexamethasone on G protein levels and adenylyl cyclase activity in rat vascular smooth muscle cells. Dexamethasone administration in vitro has been shown to increase adenylyl cyclase activity in vascular smooth muscle cells (VSMC) from renal arteries and in non-vascular cell lines. To investigate whether G proteins are involved in this response, cultured VSMC from mesenteric arteries of Sprague-Dawley rats were incubated in the presence and absence of 10 nM dexamethasone for 24 and 48 h. Basal and stimulated adenylyl cyclase activities were increased by approximately 50% after treatment with dexamethasone. The changes were neither specifically associated with ligands which stimulate adenylyl cyclase catalytic unit via Gs (isoproterenol and prostaglandin E1) nor with guanylylimidodiphosphate (0.1 nM), which inhibits the catalytic unit via Gi. This suggests that dexamethasone enhances adenylyl cyclase activity through changes at the level of the catalytic unit, rather than through the G proteins which modulate its activity. No differences were seen in immunoblotting studies of the levels of Gi alpha 2, Gs alpha, Gi alpha 3 and beta subunits. Similarly, dexamethasone had no effect on the expression of mRNA for Gi alpha 2 and Gs alpha. The results indicate that glucocorticoid-induced increases of adenylyl cyclase activity are due to changes at the level of the adenylyl cyclase catalytic unit rather than alteration of the levels or turnover of Gs alpha, Gi alpha 2, Gi alpha 3 and beta subunits in the membranes of VSMC.


Glucocorticoid-mediated responses of plasma ACTH and anterior pituitary pro-opiomelanocortin, growth hormone and prolactin mRNAs during adjuvant-induced arthritis in the rat. Adjuvant arthritis (AA) in the rat leads to chronic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis and the loss of its diurnal rhythmicity. We have investigated the effects of adrenalectomy (ADX) and different levels of corticosterone replacement upon plasma ACTH levels and anterior pituitary pro-opiomelanocortin (POMC), GH and prolactin mRNAs during the development of AA. In control ADX animals, we observed the negative feedback effects of exogenous corticosterone on plasma ACTH and anterior pituitary POMC mRNA. In the ADX animal with AA, however, the increased POMC mRNA which was observed was not reduced by exogenous corticosterone on day 7 of AA, although the negative feedback effect of corticosterone on plasma ACTH was intact. On day 14, however, even high dose corticosterone replacement failed to have a significant feedback effect on the raised levels of plasma ACTH. In control ADX animals, corticosterone replacement resulted in increased anterior pituitary GH mRNA and reduced prolactin mRNA. In contrast, in ADX animals with AA, GH mRNA was reduced and there was a further decrease in prolactin mRNA. In these animals, corticosterone replacement did not affect GH or prolactin mRNA expression. These data demonstrate a disruption of the normal mechanisms underlying feedback inhibition of the HPA axis by glucocorticoids during AA. Similarly, the glucocorticoid-dependent regulation of GH and prolactin mRNA expression is altered in AA.


Identification of exchange mechanisms for the regulation of intracellular pH in rat thyroid FRTL-5 cells. pH is maintained in cells by plasma membrane exchange mechanisms. In the absence of HCO3- ions, FRTL-5 cells regulate intracellular pH (pHi) by an Na+/H+ antiport but HCO3(-)-dependent exchangers cannot operate. We have investigated pHi regulation (by microfluorimetry and the pH sensitive dye 2',7'-bis(2-carboxyethyl)-5(6')-carboxyfluorescein) in small groups (five to six cells) of FRTL-5 thyroid cell monolayers held in Krebs-Ringer buffer (pH 7.4) with or without HCO3- ions. The exchangers were investigated with inhibitors (amiloride or its derivative dimethylamiloride for the Na+/H+ antiporter and the stilbene derivative disodium 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) for HCO3(-)-dependent mechanisms), ionic substitution and by NH4+/NH3 (10 mM) acid loading. Basal pHi was lower in the presence (7.3 +/- 0.058, mean +/- S.D., n = 14) than in the absence (7.59 +/- 0.078, n = 10) of HCO3- ions. In HCO3(-)-free media, cells recovered from acid load by 0.34 +/- 0.04 pH units in the first 2 min and finally reached a pHi of 7.35 +/- 0.06. This recovery was Na(+)-dependent and blocked by dimethylamiloride during the 15 min following intracellular acidification. In HCO3(-)-containing media, cells recovered from an acid load at a similar rate, but reached 99 +/- 10% (n = 9) of the baseline pH; this recovery was also dependent on Na+ ions. Moreover, although dimethylamiloride and DIDS reduced the rate of recovery to 0.06 +/- 0.02 and 0.18 +/- 0.04 pH units respectively during the 2-min period, the cells returned to the basal pHi within 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)


Studies of electron spin resonance on bilirubin free radicals. The nature of ESR signals derived from bilirubin-IX alpha has been studied by focusing on the samples treated with free radical generating and inhibiting systems, i.e. X-XOD, Fe/EDTA, SOD, mannitol/ascorbate, CO, KCN, etc. In all the cases, the stable signals comprise those originated from semiquinone radical (g = 2.0012) and superoxide free radical (g parallel = 2.041, g perpendicular = 2.0040). The superoxide is shown binding with certain metal ions chelated by bilirubin. The free radical scavengers are able to destroy these radicals. The kinetic curve of the regeneration of bilirubin radicals has been determined and the reaction follows a zero order mechanism. It is likely that both the physiological and toxic actions of bilirubin are related to the characters of its free radicals. Bilirubin is discussed as "active oxygen sink" in mammalians.


The influence of alpha- and beta-galactose residues and sialic acid O-acetyl groups of rat erythrocytes on the interaction with peritoneal macrophages. The significance of glycoconjugates on the surface of rat erythrocytes was studied in the interaction of these cells with homologous peritoneal macrophages. The erythrocytes exposing terminal alpha-galactose and thus of B blood group specificity, as well as sialic acid are not bound by the macrophages. beta-Galactose residues exposed by sialidase induced strong binding and additional alpha-galactosidase treatment enhanced the binding. beta-Galactose exposed on glycolipids after pronase and alpha-galactosidase treatment induced no binding. An intact protein core of the glycoproteins on the erythrocyte surface was necessary for interaction with macrophages. Partial de-O-acetylation of sialic acids prior to sialidase treatment stimulated subsequent binding of the erythrocytes.


Effects of cholesterol on viability and (n - 6) fatty acid metabolism in cultured human monocyte-like cells (U937). Effects of supplementation of growth-promoting cholesterol on metabolism of the cytotoxic (n - 6) polyunsaturated fatty acids in cultured human monocyte-like cells (U937) have been examined. U937 cells were incubated in 5% delipidated fetal bovine serum containing 0 or 38.7 microM cholesterol. The rate of uptake and the distribution of metabolites of (n - 6) fatty acids (such as 18:2(n - 6), 18:3(n - 6), and 20:3(n - 6), and 20:4(n - 6)) were examined by adding radiolabelled fatty acid at a level of 1 microgram/mL (3.3 microM for 20-carbon fatty acids and 3.6 microM for 18-carbon-fatty acids). For assessing the cytotoxicity, (n - 6) fatty acids were added to medium at a concentration of 5 micrograms/mL (16.4 microM for 20-carbon fatty acids and 17.9 microM for 18-carbon fatty acids). Cholesterol supplementation suppressed the uptake of all (n - 6) fatty acids and reduced the cytotoxic effects of 18:2(n - 6), 20:3(n - 6), and 20:4(n - 6), but not 18:3(n - 6). In addition, cholesterol supplementation increased peroxide production and metabolism of (n - 6) fatty acids in U937 cells. Thus, the differential suppressive effect of cholesterol on the cytotoxicity of different fatty acids could not be attributed to an inhibitory effect on fatty acid delta 6- and delta 5-desaturation, or to an antioxidant effect on peroxide formation.


Enhancement of differentiation of cultured adipogenic cells (TA1) by pertussis toxin. Differentiation of adipocytes is controlled by a variety of hormones and growth factors. To investigate the possible role of GTP-binding proteins (G proteins) in the process of adipose conversion, we studied the effect of pertussis toxin on differentiation of the fibroblast/adipocyte cell line (TA1). Pertussis toxin potentiated dexamethasone- and indomethacin-induced adipocyte differentiation in a time- and dose-dependent fashion. Addition of dibutyryl cAMP or forskolin inhibited adipose conversion, indicating that an abolishment of inhibitory control of adenylate cyclase is not responsible for the action of pertussis toxin. The B oligomer of the toxin did not mimic the effect of the holotoxin. Pertussis toxin catalyzed ADP-ribosylation of 40,000 molecular mass protein of the membrane fraction was dose-dependently inhibited by the pretreatment of the cells with the toxin. These results indicate the possible involvement of pertussis toxin-sensitive G proteins in adipogenesis.


Vibrio harveyi RNA polymerase: purification and resolution from gyrase A. RNA polymerase was purified from Vibrio harveyi and found to contain polypeptides (beta, beta', alpha and sigma) closely corresponding to those of the Escherichia coli enzyme. In vitro transcription studies using V. harveyi and E. coli RNA polymerase demonstrated that the purified V. harveyi RNA polymerase is functional and that the two enzymes have the same promoter specificity. Chromatography through a monoQ column was required to remove a 100-kilodalton protein that was present in large amounts and copurified with the RNA polymerase. N-terminal amino acid sequencing showed that the first 18 amino acids of the 100-kilodalton protein shares 78% sequence identity with the A subunit of gyrase or topoisomerase II. The abundance of the gyrase A protein is unprecedented and may be linked to bioluminescence.


Rapid affinity-purification and biotinylation of antibodies. A simple and rapid method to affinity-purify and biotinylate antibodies was developed. The method utilizes separation of antigens by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose and binding of the antibodies to the specific antigen. The antibodies are biotinylated, while still bound to the antigen, thus avoiding the conjugation of the active antigen-binding sites of the antibodies. These antibodies have been successfully used in double-label immunofluorescence studies, but they should be likewise applicable in other immunological protocols.


Gene transfer into established and primary fibroblast cell lines: comparison of transfection methods and promoters. The stable transfection of immortalized Rat-1 and rat skin primary fibroblast cell lines by calcium phosphate precipitation, lipofection and electroporation methods have been examined. The lipofection method was found to be better than the other methods in terms of higher transfection efficiency and convenient use. Expression of beta-galactosidase from two different viral promoters showed that the level of transgene expression depends on the promoter strength in a particular cell type. The results presented here show that the transgene expression is extremely variable among different colonies generated from individually transfected cells. Therefore, it is necessary to examine individual colonies of cells for the production of reporter gene to obtain cell lines expressing high amounts of gene products.


Preparation of uniform, intact DNA samples from resected tumor tissues for pulsed-field gel electrophoretic analyses. Preparation of high molecular weight DNA from resected tumor tissues suitable for pulsed-field gel electrophoresis (PFGE) can be complicated by the presence of nonviable cells and lymphocytes. We have developed a simple procedure to reduce the level of degraded DNA in PFGE DNA samples prepared from resected tumor tissues. The procedure employs a single, three component Percoll step gradient centrifugation and can be performed on several tumor samples simultaneously. Analyses of DNAs from 15 tumor specimens (7 solid tumors and 8 aspirated fluids) demonstrate that the technique enriches the integrity of PFGE DNA samples. Morphologic evaluation of 9 specimens suggested that both cellular debris and contaminating normal lymphocytes are removed from starting cell populations during the enrichment procedure. Fractionation of cells also reduced cell clumping, allowing for the formation of more uniform PFGE DNA samples.