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B S Mitchell

Publications and source records attributed to B S Mitchell.

At least 73 records · Page 4Linked to original sources
PubMed1993

Type I inosine monophosphate dehydrogenase: evidence for a single functional gene in mammalian species.

Human inosine monophosphate dehydrogenase activity is the result of the expression of two independent but closely related genes, termed IMPDH type I and type II. We have documented the presence of multiple, processed pseudogenes of type I IMPDH in human and Rhesus monkey genomic DNA, as well as a single functional gene encoding low levels of type I mRNA in human brain, heart, kidney and placenta. Single copy genes for each IMPDH isoenzyme were also found in rat, mouse, dog, cow, and chicken DNA and distinct mRNA species for type I and type II were identified by Northern blots in mouse and hamster RNA. Northern blot analysis of chicken RNA revealed a single mRNA species that hybridized to human IMPDH type I and II probes. These data document the high degree of evolutionary conservation of these two genes among mammals.

Animals↗
PubMed1993

IMP dehydrogenase inhibitors as immunomodulators.

IMP dehydrogenase is a key enzyme in the de novo pathway of purine biosynthesis and is responsible for catalyzing the first step in the formation of guanine ribonucleotides from inosine monophosphate. Mizoribine, an immunosuppressive agent in wide-spread clinical use in Japan, has been demonstrated to inhibit this enzyme. We have investigated the effects of mizoribine on human T cell activation. Stimulation of purified human peripheral blood T lymphocytes with phorbol ester and ionomycin leads to a five-fold increase in guanine ribonucleotide levels over 72 hours. The addition of mizoribine to these cultures at concentrations that are achieved in vivo leads to a dose-dependent inhibition of proliferation and concomitant 50% decrease in guanine ribonucleotide levels, an effect that is reversible with the addition of guanosine, which repletes the GTP pool. Similar effects are seen with direct stimulation via the CD3/T cell receptor complex. Inhibition of proliferation occurs at the G1/S interface of the cell cycle and is additive to that produced by cyclosporine. In order to determine whether inhibition of IMP dehydrogenase is a common mechanism of immunosuppression for drugs such as azathioprine and 6-mercaptopurine that interfere with purine biosynthesis, we compared the effects of these agents on the metabolism of purified T lymphocytes. The results of these studies demonstrate that mizoribine and mycophenolic acid, a highly specific inhibitor of IMP dehydrogenase, inhibit proliferation directly by the depletion of guanine ribonucleotides; 6-mercaptopurine, on the other hand, has a mixed effect on adenine and guanine ribonucleotide pools, whereas azathioprine inhibits proliferation by a mechanism completely independent of its effects on the purine metabolic pathway. We conclude from these studies that inhibitors of IMP dehydrogenase have potential as specific immunosuppressive agents.

Azathioprine↗
PubMed1993

Genomic structure and chromosomal localization of the human deoxycytidine kinase gene.

Deoxycytidine kinase (NTP:deoxycytidine 5'-phosphotransferase, EC 2.7.1.74) is an enzyme that catalyzes phosphorylation of deoxyribonucleosides and a number of nucleoside analogs that are important in antiviral and cancer chemotherapy. Deficiency of this enzyme activity is associated with resistance to these agents, whereas increased enzyme activity is associated with increased activation of such compounds to cytotoxic nucleoside triphosphate derivatives. To characterize the regulation of expression of this gene, we have isolated genomic clones encompassing its entire coding and 5' flanking regions and delineated all the exon/intron boundaries. The gene extends over more than 34 kilobases on chromosome 4 and the coding region is composed of 7 exons ranging in size from 90 to 1544 base pairs (bp). The 5' flanking region is highly G+C-rich and contains four regions that are potential Sp1 binding sites. A 697-bp fragment encompassing 386 bp of 5' upstream region, the 250-bp first exon, and 61 bp of the first intron was demonstrated to promote chloramphenicol acetyltransferase activity in a T-lymphoblast cell line and to have > 6-fold greater activity in a Jurkat T-lymphoblast than in a Raji B-lymphoblast cell line. Our data suggest that these 5' sequences may contain elements that are important for the tissue-specific differences in deoxycytidine kinase expression.

Base Sequence↗
PubMed1993

Localization of substance P and leucine enkephalin in the nerve terminals of the guinea pig paracervical ganglion.

Immunoreactivities (IR) of substance P and leucine enkephalin have been demonstrated in the guinea-pig paracervical ganglion by an immunogold electron microscope method. Both substance P-IR and leucine enkephalin-IR were detected in large synaptic vesicles with electron-dense cores. The former neuropeptide was detected in nerve terminals and varicosities comprised mainly of large vesicles with electron-dense cores; the latter was detected in nerve terminals and varicosities that also included small, clear synaptic vesicles. In a minority of nerve terminals and varicosities coexistence of both immunoreactivities could be demonstrated within vesicles with an electron-dense core. Also present in these nerve terminals and varicosities were small, clear synaptic vesicles, though these were unreactive.

Animals↗
PubMed1993

An immunohistochemical and electron microscopic study of the peri-ureteric ganglia of the guinea-pig.

The neuropeptide- and catecholamine-synthesizing enzyme content and ultrastructure of the peri-ureteric ganglia of guinea-pigs were investigated. Small numbers of neuronal perikarya were present at frequent intervals forming ganglia close to, and along the entire length of, the ureter. Each of these ganglia was surrounded by a connective tissue capsule, and was located in the peri-ureteric connective tissues. Within each ganglion were typical nerve terminals and varicosities containing small, clear synaptic vesicles or synaptic vesicles with an electron-dense core, or a mixture of the two. In the ganglia, immunoreactivity to tyrosine hydroxylase, dopamine beta hydroxylase, neuropeptide tyrosine, or vasoactive intestinal peptide was present in neuronal perikarya; immunoreactivity to substance P or leucine enkephalin was present in nerve terminals and varicosities. Electron-microscopic immunogold studies indicated that there was no coexistence of substance P and enkephalin in the nerve terminals, unlike related ganglia in the pelvis of guinea-pigs.

Animals↗
PubMed1993

Projections of the guinea-pig paracervical ganglion to pelvic viscera.

The uterine cervix, urinary bladder and rectum of guinea pigs were injected with Fast Blue dye for retrograde transport studies. Dye-laden neuronal perikarya were detected for each viscus in the paracervical ganglion. These same perikarya also exhibited immunoreactivities for tyrosine hydroxylase, aromatic amino acid decarboxylase, dopamine beta-hydroxylase, neuropeptide Y, or vasoactive intestinal peptide, though the perikarya projecting to the urinary bladder did not exhibit immunoreactivity for aromatic amino acid decarboxylase. The results of this study indicate that the guinea-pig paracervical ganglion projects to viscera in addition to the uterus, and that the ganglion contains a range of immunoreactivities related to adrenergic and non-adrenergic neurotransmitters.

Amidines↗
PubMed1993

Regulation of human deoxycytidine kinase expression.

The human deoxycytidine kinase gene is a single copy gene and is comprised of seven exons that are spread over more than 34 kb of the genome. The 5'-flanking region is highly G/C rich and does not contain CAAT or TATA boxes. This region, when cloned into a recorder gene construct containing the chloramphenicol acetyltransferase gene, is capable of mediating CAT activity in human lymphoid cell lines and appears to have greater activity in human T, as compared to B, lymphoblast cell lines. The expression of the gene at the mRNA level does not appear to be cell-cycle regulated in that the levels of mRNA in human peripheral blood T lymphocytes remain constant as the cells progress from a resting to a proliferating state. Since this enzyme catalyzes the conversion of a number of chemotherapeutic agents to their corresponding monophosphate form and is thus essential for their activation, it will be important to define further the genetic elements which regulate the expression of this gene.

Cell Division↗
PubMed1993

Perforating vessel(s) of the orbital floor: a cadaveric study.

Brisk haemorrhage is occasionally encountered in subperiosteal orbital floor exploration. Personal experience of the authors of a vessel perforating the orbital floor in a roughly similar position in a number of cases prompted further study. Twelve fresh cadavers (24 orbits) were dissected specifically to identify any vessel(s) perforating the orbital floor. In this study a vessel was identified in 83% of the dissections.

Adult↗
PubMed1993

Prevalence of hepatitis C viral antibody in transfused and nontransfused Egyptian children.

Hepatitis C (HCV) virus is recognized as the major cause of what was previously referred to as parenterally acquired (blood-mediated) non-A, non-B hepatitis. A study involving 252 transfused and nontransfused Egyptian children was conducted from November 1990 through February 1991 to determine the prevalence of HCV and the role of blood and blood and blood product transfusions in the spread of the virus. Serum specimens were assayed by a second generation enzyme immunoassay and were considered reactive only after supplemental testing using the second generation recombinant immunoblot assay. Prevalence among 84 young study subjects with hematologic disorders was 55% (46 of 84), while no HCV antibodies were detected among the two nonhematologic pediatric populations studied: 84 hospital admissions and 84 acutely ill but otherwise healthy outpatients (seeking treatment for symptoms associated with a new condition less than three weeks old in the absence of any chronic health problem). Ninety-two percent (77 of 84) of the hematology-related cases had medical histories of multiple transfusions. Positive antibody responses (46) were significantly associated with increased duration of illness (P < 0.001) and the volume and number of transfusions (P < 0.01) when compared with negative ones (38). However, prior hospitalization and/or surgery were not related to HCV antibody status. The high prevalence of HCV antibody among multiply transfused infants and children suggests that blood and blood product supplies should be regularly screened for HCV antibody.

Adolescent↗
PubMed1993

Morphology and neurochemistry of the pelvic, and paracervical ganglia.

Autonomic ganglia are relays in the distribution of nerve fibres in the peripheral nervous system. In the pelvis there are local ganglion formations in the pathway of nerve fibres to and from pelvic viscera and vasculature: in rodents these are the male anterior major pelvic ganglion and the female paracervical ganglion (Frankenhäuser's ganglion). They are unusual in that they contain both sympathetic and parasympathetic ganglion cells. Homologous formations occur in humans. Since the best studied examples of these ganglionic formations are rodent ganglia the latter are reviewed in terms of their gross anatomy, cell morphology and immunohistochemistry. The synaptology, and neurotransmitter and neuropeptide contents of the neuronal perikarya and nerve terminals, of the ganglia are discussed in relation to the concepts of coexistence and chemical coding in autonomic ganglia in general. The neuropeptide content of the nerve fibres projecting to their visceral targets is described and discussed in functional terms. Conclusions are drawn with respect to the contributions made by study of these ganglia to further understanding of the organisation of the autonomic nervous system in general. The possible link between the autonomic nervous system and the endocrine system is discussed with respect to control of pelvic visceral activities.

Animals↗
PubMed1993

Hereditary overexpression of adenosine deaminase in erythrocytes: evidence for a cis-acting mutation.

Overexpression of adenosine deaminase (ADA) in red blood cells is inherited as an autosomal dominant trait and causes hemolytic anemia. The increased ADA activity in erythrocytes is due to an increase in steady-state levels of ADA mRNA of normal sequence. Increased ADA mRNA may be due to a cis-acting mutation which results in increased transcription or a loss of down-regulation during erythroid differentiation. Alternatively, it is possible that the mutation is in a trans-acting factor which interacts with normal ADA transcriptional elements to cause overexpression in red blood cells. To discriminate between a cis-acting and a trans-acting mutation, we took advantage of a highly polymorphic TAAA repeat located at the tail end of an Alu repeat approximately 1.1 kb upstream of the ADA gene. Using PCR to amplify this region, we identified five different alleles in 19 members of the family. All 11 affected individuals had an ADA allele with 12 TAAA repeats, whereas none of the 8 normal individuals did. We conclude that this disorder results from a cis-acting mutation in the vicinity of the ADA gene.

Adenosine Deaminase↗
PubMed1992

The effects of decentralization on substance P-immunoreactivity in the anterior major pelvic ganglion of the male guinea pig.

The anterior major pelvic ganglion (AMPG) of the male guinea pig possesses a substance P (SP)-immunoreactive peri-neuronal plexus. Selective nerve transections involving the principal inputs of the AMPG, the hypogastric and pelvic nerves, indicate that the SP-immunoreactive peri-neuronal plexus is derived from multiple sources: an extrinsic source involving both the hypogastric and pelvic nerves, and another source (possibly the projections of small intensely fluorescent cells). SP-immunoreactivity (IR) is not normally present in the neuronal perikarya of the AMPG. Evidence is presented that suggests the absence of SP-IR is due to an active suppression of SP-synthesis. This seems to be achieved by a trans-synaptic mechanism involving the hypogastric nerve which, after transection, leads to the appearance of neuronal perikarya exhibiting SP-IR (less than 1% of the total neuronal population of the AMPG). Up to 65% of the neuronal perikarya of the AMPG have the ability to synthesize SP, as demonstrated by SP-IR after 24 h in vitro. A more potent factor in the down-regulation of SP synthesis seems to be exerted by the pelvic genito-urinary organs, especially the prostate.

Animals↗
PubMed1992

Resistance to 1-beta-D-arabinofuranosylcytosine in human T-lymphoblasts mediated by mutations within the deoxycytidine kinase gene.

We have recently identified a complementary DNA clone which encodes the complete amino acid sequence for 2'-deoxycytidine kinase (dCK), the enzyme required for the initial phosphorylation of several deoxyribonucleosides and their analogues that are widely used as chemotherapeutic and antiviral agents. In order to identify the molecular basis for dCK deficiency in two clonal T-lymphoblast cell lines generated by virtue of their resistance to 1-beta-D-arabinofuranosylcytosine (ara-C-8D) or to 2',3'-dideoxycytidine (ddC50), we have cloned and sequenced their dCK complementary DNAs. The ara-C-8D cell line contained two identifiable mutations: (a) a 115-base pair deletion within the coding region, corresponding to the fifth exon of the gene and presumably resulting from a splice site mutation; and (b) a G to A point mutation that substitutes glutamic acid for glycine within the ATP-binding domain of the protein. Expression of each protein in Escherichia coli demonstrated a complete loss of catalytic activity and, in the case of the deletion, a proteolytic degradation product of the altered protein. The substitution of a negatively charged amino acid within the ATP-binding domain resulted in loss of enzyme activity with all nucleoside triphosphates tested. The ddC50 cell line contained a single identifiable structural gene mutation in all clones sequenced resulting in the substitution of arginine for glutamine at amino acid 156 of the protein. This mutation markedly diminished the catalytic activity of the expressed protein with the three substrates, deoxycytosine, deoxyadenosine, and deoxyguanosine. On the basis of the presence of a single point mutation and a marked reduction in dCK mRNA in this cell line, we postulate that the second allele either is not expressed or is expressed at extremely low levels. We conclude that cellular resistance to the toxicity of 1-beta-D-arabinofuranosylcytosine and dideoxycytidine in these cell lines is mediated by specific mutations within the dCK gene. Further elucidation of structural genes alterations in dCK-deficient cells will facilitate a more detailed understanding of the functional domains of this complex enzyme.

Base Sequence↗
PubMed1992

An immunohistochemical study of the catecholamine synthesizing enzymes and neuropeptides in the female guinea-pig uterus and vagina.

The uterus and vagina of the guinea pig have been examined, region by region, for acetylcholinesterase, tyrosine hydroxylase, dopamine beta-hydroxylase and aromatic amino acid decarboxylase activity, as well as for the neuropeptides, neuropeptide Y, vasoactive intestinal peptide, substance P, enkephalin and somatostatin. No acetylcholinesterase activity was localized in the uterus, though it was present in associated paracervical ganglion tissues. Of the catecholamine-synthesizing enzymes, tyrosine hydroxylase and dopamine beta-hydroxylase activity was found virtually throughout the reproductive tract, whereas aromatic amino acid decarboxylase activity was restricted in its distribution. Neuropeptide distribution was quite varied. Neuropeptide Y was found throughout the endometrium/submucosa but only in the muscularis of the vagina and not in the myometrium. Substance P was localized in the vagina and uterine horn, though not the body of the uterus. Vasoactive intestinal peptide was present in all regions of the endometrium/submucosa, but not in the myometrium of the uterine horn. Enkephalin and somatostatin were not localized in any part of the reproductive tract examined, apart from paracervical ganglion tissues. The types and significance of the nerves supplying the reproductive tract are discussed.

Acetylcholinesterase↗
PubMed1992

In situ hybridisation: a review of methodologies and applications in the biomedical sciences.

In situ hybridisation methodology is reviewed with particular emphasis on the latest modifications of technique. Methodology is evaluated, with suggestions on how to maximise its successful application. Emphasis is placed upon non-radioactive techniques which can be used by morphologically orientated laboratories, and details of the use of digoxigenin-labelled oligonucleotide probes are given special mention. Recent applications in morphology, and neurobiology, virology, pathology and cytogenetics are detailed. Developments where significant advances have been made in the biomedical sciences are highlighted.

DNA↗
PubMed1992

Macrophages and other endocytic cells in the mouse uterus during the second half of pregnancy and into the postpartum period.

The spatiotemporal distribution of macrophages in the uterine wall of mice in the second half of pregnancy and the early postpartum period has been studied. Macrophages were identified using a combination of morphological criteria, the capacity to endocytose horseradish peroxidase and the expression of the Mac-1 antigen. Macrophages were a consistent feature of the myometrium. Numerous endocytic cells were found in the decidua basalis and metrial gland of pregnant mice and in the 'scar' regions of postpartum mice. However, few of the endocytic cells expressed the Mac-1 antigen. It is concluded that stromal/decidual cells, and not monocyte-derived macrophages, are primarily responsible for the removal of cellular debris in the decidua basalis and metrial gland during the last week of pregnancy and in repair of the uterine wall in the postpartum period.

Animals↗
PubMed1992

Comparison of the effects of mizoribine with those of azathioprine, 6-mercaptopurine, and mycophenolic acid on T lymphocyte proliferation and purine ribonucleotide metabolism.

The immunosuppressive drug mizoribine has been demonstrated to inhibit T lymphocyte proliferation by depleting these cells of guanine ribonucleotides as a consequence of inhibiting the enzyme inosine monophosphate (IMP) dehydrogenase. Because the immunosuppressive agents azathioprine and 6-mercaptopurine (6MP) are both converted to the IMP analog 6-thio-IMP, we postulated that these drugs might inhibit T cell activation and/or proliferation by a similar mechanism. Incubation of isolated peripheral blood T cells with either mizoribine or the selective IMP dehydrogenase inhibitor mycophenolic acid caused a dose-dependent inhibition of T cell proliferation, which was reversible with the addition of 50 microM guanosine to replete guanine ribonucleotide pools. In contrast, guanosine exacerbated the inhibition of proliferation induced by azathioprine and restored proliferation at IC50 concentrations of 6MP by only 10%. Complete restoration of proliferation in the presence of 6MP, but not azathioprine, was achieved with the addition of adenine. The inhibitory effects of azathioprine, as well as those of mizoribine, 6MP, and mycophenolic acid, were identical in cells stimulated with antibody to the T cell receptor and in cells stimulated with phorbol ester and ionomycin. We conclude from these studies that mizoribine selectively inhibits guanine ribonucleotide formation in purified T cells, whereas the effect of 6MP appears to be more dependent on adenine ribonucleotide depletion. Azathioprine, on the other hand, inhibits proliferation by a mechanism independent of purine ribonucleotide depletion. None of these agents inhibits T cell proliferation by interfering with signal transduction mediated by the T cell receptor. Inhibition of guanine ribonucleotide biosynthesis appears to be a novel and perhaps more selective mechanism of inhibiting T cell proliferative responses after T cell activation.

Azathioprine↗