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B S Mitchell

Publications and source records attributed to B S Mitchell.

At least 55 records · Page 3Linked to original sources
PubMed1996

Inosine monophosphate dehydrogenase expression: transcriptional regulation of the type I and type II genes.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential rate-limiting enzyme in the de novo guanine nucleotide synthetic pathway that catalyzes the conversion of IMP to XMP. Enzyme activity is accounted for by the expression of two distinct but closely related genes termed IMPDH I and II. Increased IMPDH activity has been linked to both cellular proliferation and neoplastic transformation and generally ascribed to an increase in the expression of the type II gene. We have characterized the type I and type II genes and identified elements important in the transcriptional regulation of both genes. The type II IMPDH gene contains a 466 bp 5' flanking region spanning the translation start site that contains several transcription factor binding sites and mediates increased transcription of a CAT reporter gene in peripheral blood T lymphocytes when these cells are induced to proliferate. The single functional IMPDH type I gene contains exon-intron boundaries and exon structures that are nearly identical to those in the type II gene. In contrast to the type II gene, however, it contains two putative promoter sites, each with the potential for transcriptional regulation. We conclude that these two genes most probably arose from an early gene duplication event and that their highly conserved structures and differential regulation at the transcriptional level argue strongly for a significant role for each gene in cellular metabolism, growth, and differentiation.

Chloramphenicol O-Acetyltransferase↗
PubMed1996

Does the lectin Helix pomatia agglutinin bind to hyaluronic acid in breast and colon cancer?

The lectin Helix pomatia agglutinin (HPA) has been successfully used as an indicator of metastatic spread in a number of epithelial neoplasms including breast, colorectal, gastric, prostate and oesophageal cancers. Despite its use, the binding partners of HPA in tissue sections have not been defined at the molecular level. HPA has two main binding specificities: 1) N-acetylgalactosamine (GalNac) and 2) N-acetylglucosamine (GlucNac). In order to determine whether HPA binds to hyaluronic acid (a GlucNac-containing glycosaminoglycan) a dot blot assay was performed which confirmed hyaluronic acid as a binding partner for HPA. In the next step, experiments were performed using hyaluronate lyase predigestion before HPA application on clinical and experimental human tumours grown in severe combined immunodeficient (SCID) mice to assess whether the HPA binding in the dot blot system also occurred in tissue sections. The results from all samples indicate that hyaluronate lyase pretreatment does not alter HPA binding to tumour cells both in the patients' samples and in the human cancer cell lines grown in SCID mice. This also indicates that HPA binds to similar carbohydrate residues in patients' samples and in SCID mouse-grown human tumour cells. It seems, therefore, unlikely that HPA recognises hyaluronic acid as a binding partner in tissue sections of human tumours, and hence GalNac-containing glycoproteins seem to be the more likely ligands for HPA in cancer cells.

Animals↗
PubMed1996

Viral vector transduction of the human deoxycytidine kinase cDNA sensitizes glioma cells to the cytotoxic effects of cytosine arabinoside in vitro and in vivo.

Cytosine arabinoside (ara-C) is a cytidine analog that incorporates into replicating DNA and induces lethal DNA strand breaks. Although ara-C is a potent antitumor agent for hematologic malignancies, it has only minimal activity against most solid tumors. The rate-limiting step in intracellular ara-C activation is phosphorylation of the prodrug by deoxycytidine kinase (dCK). The present results demonstrate that both retroviral and adenoviral vector-mediated transduction of the dCK cDNA results in marked sensitization of glioma cells lines to the cytotoxic effects of ara-C in vitro. We also demonstrate that ara-C treatment of established intradermal and intracerebral gliomas transduced with dCK results in significant antitumor effects in vivo. These data suggest that viral vector transduction of the dCK gene followed by treatment with ara-C represents a new chemosensitization strategy for cancer gene therapy.

Adenoviridae↗
PubMed1995

Recombinant human inosine monophosphate dehydrogenase type I and type II proteins. Purification and characterization of inhibitor binding.

Inosine monophosphate dehydrogenase (IMPDH) activity results from the expression of two separate genes, and the resulting proteins (type I and type II) are 84% identical at the amino acid level. Although the type II mRNA is expressed at higher levels in proliferating cells, both mRNAs, and by extrapolation both proteins, are present in normal and malignant cells. Since IMPDH is an important target for the development of drugs with both chemotherapeutic and immunosuppressive activity, we have compared the kinetic and physical properties of the two human enzymes expressed in and purified from Escherichia coli. Type I and II IMPDH had kcat values of 1.8 and 1.4 sec-1, respectively, with Km values for IMP of 14 and 9 microM and Km values for NAD of 42 and 32 microM. The two enzymes were inhibited competitively by the immunosuppressive agent mizoribine 5'-monophosphate (MMP) with Ki values of 8 and 4 nM and inhibited uncompetitively by mycophenolic acid with Ki values of 11 and 6 nM. The association of MMP to either isozyme, as monitored by fluorescence quenching, was relatively slow with kon values of 3-8 x 10(4) M-1 sec-1 and koff values of 3 x 10(-4) sec-1 (half-lives of 36-43 min). Thus, MMP is a potent, tight-binding competitive inhibitor that does not discriminate between the two IMPDH isozymes.

Binding, Competitive↗
PubMed1995

Characterization of the human inosine-5'-monophosphate dehydrogenase type II gene.

Inosine-5'-monophosphate dehydrogenase (IMPDH) activity and mRNA levels are induced up to 15-fold upon mitogenic or antigenic stimulation of human peripheral blood T lymphocytes. This increase in IMPDH activity is required for cellular proliferation and has been associated with malignant transformation. We have cloned the human IMPDH type II gene and show that it contains 14 exons and is approximately 5.8 kilobases in length. Exons vary in size from 49 to 207 base pairs and introns from 73 to 1065 base pairs. The transcription start site was mapped to a position 50 nucleotides upstream of the translation initiation site. The 5'-flanking region consisting of 463 base pairs upstream of the translation initiation site confers induced transcription and differential regulation upon a chloramphenicol acetyltransferase reporter gene when transfected into Jurkat T cells and human peripheral blood T lymphocytes, respectively. DNase I footprinting analysis using Jurkat T cell nuclear extract identified four protected regions in the promoter which coincide with consensus transcription factor binding sites for the nuclear factors AP2, ATF, CREB, Egr-1, Nm23, and Sp1. These findings suggest that several of these nuclear factors may play a critical role in the regulation of IMPDH type II gene expression during T lymphocyte activation.

Base Sequence↗
PubMed1995

Plastination for gross anatomy teaching using low cost equipment.

Plastination offers a means of keeping anatomical specimens without the usual problems associated with wet specimens ie desiccation, mould and specific storage requirements. Plastinated specimens are clean and odourless, require minimal aftercare and can be stored on shelves or in display cases. These specimens are more durable and robust than wet specimens showing similar features. The techniques described in this paper for plastination are cost effective, and produce good quality, robust specimens using low cost equipment which is readily available in most Anatomy departments. The procedures described are easy to follow used in conjunction with von Hagens [4] plastination technical notes.

Anatomy↗
PubMed1995

Characterization of the deoxycytidine kinase promoter in human lymphoblast cell lines.

Deoxycytidine kinase (dCK) phosphorylates 2'-deoxycytidine, as well as the purine deoxyribonucleosides and a number of nucleoside analogues that are important in the chemotherapy of leukemias. The enzyme is highly expressed in the thymus relative to other tissues and may play an important role in the T cell depletion associated with adenosine deaminase and purine nucleoside phosphorylase deficiencies. To characterize the dCK promoter region and to determine whether it mediates higher levels of gene expression in T lymphoblasts, we have analyzed a 700-bp genomic fragment encompassing 548 bp of 5' flanking region for functional activity and for transcription factor binding using T and B lymphoblast cell lines and nuclear extracts. The regions of the promoter that were defined as important to its function include a 5' GC box, and E box, a 3' GC box, and an E2F site. The transcription factor Sp1 binds to both GC boxes, activating at the 5' site but repressing at the 3' site. MLTF/USF activates transcription through the E box, whereas E2F activates through the E2F site, but binds weakly to this site in vitro and does not appear to mediate cell cycle-specific expression of dCK in vivo. No significant differences in promoter activity or transcription factor binding were observed between Jurkat T and Raji B lymphoblasts. The promoter of the dCK gene is thus regulated by a number of ubiquitously expressed transcription factors. DCK expression in cultured lymphoblast cell lines is not solely a function of the T or B lineage derivation.

B-Lymphocytes↗
PubMed1995

GTP depletion induced by IMP dehydrogenase inhibitors blocks RNA-primed DNA synthesis.

Inhibitors of IMP dehydrogenase (EC 1.2.1.14), including mizoribine (Bredinin) and mycophenolic acid, have significant antitumor and immunosuppressive activities. Studies were aimed at determining the mechanism by which intracellular GTP depletion induced by these agents results in inhibition of DNA synthesis. Incubation of human CEM leukemia cells for 2 hr with IC50 concentrations of either mizoribine (4 microM) or mycophenolic acid (0.5 microM) reduced cellular GTP levels an average of 68% or 58%, respectively, compared with the levels in control cells. Under similar conditions, mizoribine and mycophenolic acid decreased the amount of [3H]adenosine incorporated into primer RNA by 75% and 70%, respectively, relative to the untreated controls, but had no significant effect on total RNA synthesis. Repletion of the guanine nucleotide pools by coincubation of CEM cells with guanosine plus 8-aminoguanosine prevented both the inhibition of primer RNA synthesis and the inhibition of tumor cell growth induced by these agents. Additional studies demonstrated that GTP depletion alone was capable of directly inducing inhibition of primer RNA synthesis. Primer RNA synthesis was inhibited an average of 84% in whole-cell lysates that lacked GTP but contained all remaining ribo- and deoxyribonucleoside triphosphates. On an M13 DNA template, RNA-primed DNA synthesis catalyzed by the purified complex of DNA primase (EC 2.7.7.6) and DNA polymerase alpha (EC 2.7.7.7) was decreased an average of 70% in the absence of GTP, compared with synthesis in the presence of 0.5 mM GTP. These results provide evidence that mizoribine and mycophenolic acid inhibit DNA replication by inducing GTP depletion, which suppresses the synthesis of RNA-primed DNA intermediates.

Adenosine Triphosphate↗
PubMed1994

Assignment of the human type I IMP dehydrogenase gene (IMPDH1) to chromosome 7q31.3-q32).

Two phage lambda clones that contain the 5' portion of the human type I inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) gene were isolated. Both polymerase chain reaction analysis of a panel of human-mouse and human-hamster cell somatic hybrids using primers specific for the type I IMPDH gene and fluorescence in situ hybridization with metaphase human chromosomes using type I IMPDH genomic DNA as probes indicate that the type I IMPDH gene (symbol IMPDH1) is located on chromosome 7. Sequential GTG-banding was performed to assign the band location of the type I IMPDH gene to chromosome 7q31.3-q32.

Animals↗
PubMed1994

Hereditary overexpression of adenosine deaminase in erythrocytes: studies in erythroid cell lines and transgenic mice.

Overexpression of adenosine deaminase (ADA) in red blood cells is characterized by a marked, tissue-specific increase in levels of structurally normal ADA mRNA and enzymatic activity in the erythrocytes of affected individuals, leading to adenosine triphosphate (ATP) depletion and hemolytic anemia. This autosomal dominant trait is linked to the ADA gene. To investigate the molecular mechanism responsible for this disorder, we examined relative reporter gene activity using constructs containing 10.6 kb of 5' flanking sequence and 12.3 kb of the first intron of the ADA gene from the normal and mutant alleles. No differences in chloramphenicol acetyltransferase (CAT) activity were found in transient transfection experiments using erythroleukemia cell lines. Transgenic mice containing the ADA constructs expressed CAT in the appropriate tissue-specific fashion, with 10(2)- to 10(4)-fold higher activity in the thymus. However, CAT activities in erythrocytes and bone marrow of mice containing high transgene copy numbers did not differ between the normal and mutant alleles. These results indicate that the mutation responsible for ADA overexpression is unlikely to reside in the 5' and promoter regions or in the regulatory regions of the first intron. It is possible that the erythroid-specific overexpression of ADA results from a mutation at some distance from the gene or requires an interaction of a proximal mutation with more distal DNA elements.

Adenosine Deaminase↗
PubMed1994

Pneumonia hospitalizations in the US Navy and Marine Corps: rates and risk factors for 6,522 admissions, 1981-1991.

The authors identified hospitalizations for pneumonia (n = 6,522) in active-duty Navy and Marine Corps personnel during 1981-1991 from computerized inpatient records. The crude mean annual rate of pneumonia hospitalization was 77.6 per 100,000 active-duty personnel; 65% of pneumonia hospitalizations had no etiologic agent identified. The most commonly reported agents to cause pneumonia hospitalization were Streptococcus pneumoniae (12.3%), Mycoplasma pneumoniae (10.8%), other streptococcal species (2.1%), and Haemophilus influenzae (1.9%). The median age at hospitalization was 22 years. The median duration of hospital stay was 4 days and the case fatality rate was 0.4%. The authors used a 2% sample of the entire population and by means of stepwise unconditional multivariate logistic regression modeling for pneumonia found that, independent of age, the most junior Navy and Marine Corps personnel were at highest risk. Whites were at higher risk than blacks, Hispanics, or Filipinos. These results indicate that among this generally healthy US young adult military population, pneumonia hospitalization is common, often brief, and frequently without specifically identified pathogens.

Adolescent↗
PubMed1994

Effects of human T lymphocyte activation on inosine monophosphate dehydrogenase expression.

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the first step in the formation of guanine ribonucleotides from inosine monophosphate and the activity of this enzyme appears to be essential for cell proliferation. Inhibitors of IMPDH have been demonstrated to be effective immunosuppressive agents and to inhibit T cell activation in vitro. IMPDH activity results from the expression of two different genes (types I and II) that encode protein subunits of identical size with 84% amino acid identity. To determine the relative contribution of the expression of these two genes to T cell activation, we have examined the effects of T cell stimulation on IMPDH activity, mRNA levels, and protein. The stimulation of isolated peripheral blood CD28+ T cells with PMA and ionomycin causes a 15-fold increase in IMPDH activity over a 72-h period. This is associated with a 10-fold increase in type II mRNA levels at 48 h. Type I mRNA is expressed at very low levels in resting T cells, but increases 10-fold by 24 h after stimulation. The type I cDNA probe also detects a second larger mRNA species of 4.0 kb that is not detectable in a variety of normal tissues or in a panel of leukemic cell lines. RNase protection assays using RNA probes corresponding to the entire coding region of the type I enzyme reveal a single protected fragment, demonstrating that the 4.0-kb message is the result of alternate splicing in the 5' or 3' untranslated regions or the use of an alternative polyadenylation site. Western blot analysis demonstrates a concomitant increase in total IMPDH protein on T cell activation, although posttranslational modifications do not allow the distinction between type I and type II on isoelectric focusing gels. We conclude that the induction of both type I and type II IMPDH contribute significantly to the T cell proliferative response. Both enzymes therefore should be considered important targets for immunosuppressive therapy.

Base Sequence↗
PubMed1994

Are breast tumours innervated? Immunohistological investigations using antibodies against the neuronal marker protein gene product 9.5 (PGP 9.5) in benign and malignant breast lesions.

The aim of the present study was to assess the innervation pattern of benign and malignant breast lesions using the neuronal marker protein gene product (PGP) 9.5. An unlabelled antibody technique (using streptavidin biotin complex formation) was used on paraffin wax sections of tissues fixed in neutral buffered formalin. In 2/4 cases of chronic mastopathy, PGP 9.5 immunoreactivity was seen in relation to blood vessels and the ductal system. No immunoreactivity for PGP 9.5 was seen in the affected tissues of 9/10 cases of fibroadenomata. In 9/16 breast cancers, PGP 9.5-labelled perivascular nerve fibres were detected in connective tissue stroma supporting carcinoma tissue, though not in the immediate vicinity of such tumour tissue. Labelled nerve fibres were detected in large bundles at the periphery of tumours, possibly unrelated to the latter. Our results indicate that the newly formed blood vessels within a tumour are not innervated, though major blood vessels which supply the tumour are innervated.

Breast↗
PubMed1994

The neuronal marker protein gene product 9.5 (PGP 9.5) is phenotypically expressed in human breast epithelium, in milk, and in benign and malignant breast tumors.

The aim of this study was to examine the expression of the well-established neuronal marker protein gene product 9.5 (PGP 9.5) in nonneuroendocrine tissues of the human breast. Using antibodies directed against PGP 9.5 in an immunohistological technique, a positive staining of nerves and, surprisingly, a positive cytoplasmic reaction were obtained in normal breast epithelium, in all cases of fibroadenomata (n = 7) and in carcinoma cells in 5 out of 16 cases of breast cancers. In screening several human breast cancer cell lines, a positive immunoreaction was observed in 4 out of 6 of the cell lines. To exclude the possibility that this immunoreactivity was false, positive lysates from the same cell lines and preparations from human breast milk were subjected to NaDodSO4-polyacrylamide gel electrophoresis and Western blotting. This study confirmed the presence of PGP 9.5 in both milk and breast cancer cell lines. Because any contamination with nerve fibers can be excluded having used cell culture material or milk, it can be concluded that the presence of PGP 9.5 in the normal epithelium and in breast cancer cells is genuine. PGP 9.5 expression is, therefore, a feature of normal breast epithelia and breast cancer cells and cannot be regarded as 'neuron' specific.

Blotting, Western↗
PubMed1994

Are tumours innervated? Immunohistological investigations using antibodies against the neuronal marker protein gene product 9.5 (PGP 9.5) in benign, malignant and experimental tumours.

The aim of the present study was to assess the innervation pattern in benign haemangiomata, malignant colo-rectal carcinomata and experimental malignant tumours transplanted into severe combined immunodeficient (SCID) mice using the general nerve fibre marker protein gene product (PGP) 9.5. An indirect immunohistological technique (using streptavidin-biotin complex formation) was used on paraffin wax sections of tumour tissues fixed in neutral buffered formalin. In 5/7 haemangiomata nerve fibres were detected in the vicinity of some tumorous blood vessels, though the majority of such vessels were without immunoreactive nerve fibres. In some cases the reaction product for PGP 9.5 appeared to be distributed in a diffuse perivascular manner. In the colo-rectal carcinomata, no intra-tumorous blood vessels received a nerve supply, though nerves were observed in the stroma supporting the tumour tissues. In the experimental tumours derived from either human colo-rectal carcinomata or breast carcinomata cell lines, no nerve fibres were detected within the tumours themselves. The conclusions reached were that tumour blood vessels are not innervated. Angiogenesis, which is a prerequisite for tumour growth, must, therefore, be regulated by a means other than neural.

Animals↗
PubMed1994

Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside.

Deficiency of deoxycytidine kinase (dCK) activity represents one possible cause of resistance to cytosine arabinoside (ara-C). Mutations of the dCK gene have recently been shown to be responsible for dCK deficiency and increased resistance in vitro. In order to define the relevance of this mechanism in vivo, we analyzed the dCK gene in 16 adult patients with relapsed/refractory acute myeloid leukemia (AML) and clinical resistance to standard-dose and/or high-dose ara-C. Southern blot analysis using genomic DNA from peripheral blood or bone marrow samples containing > or = 70% leukemic blasts and agarose gel electrophoresis of cDNA obtained by RT-PCR did not reveal gross rearrangements of the dCK gene. Sequencing of the dCK coding region showed point mutations in seven patients. Besides two silent mutations (or RFLPs) in codon 42 and 86, base pair mutations resulting in amino acid replacements were found in five patients affecting codon 20, 93, 98, 99, and 154, respectively. dCK cDNA clones from three patients with > or = 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E. coli and analyzed for dCK activity. Normal enzyme activity was found in two patients (codon 20 and 98), and a complete loss of activity in one patient (codon 99). We conclude that structural alteration of the coding region of the dCK gene represents one possible mechanism for ara-C resistance in vivo, but, considering the frequency of this event, other mechanisms may play a more important role for clinical resistance to ara-C in patients with AML.

Adult↗
PubMed1994

Chemical coding of neurons projecting to pelvic viscera in the male guinea pig: a study by retrograde transport and immunohistochemistry.

Retrograde transport studies using Fast Blue dye demonstrated that the ductus deferens, seminal vesicle, prostate and rectum, but not the urinary bladder of the male guinea pig are at least in part innervated by the anterior major pelvic ganglion. In the ductus deferens, seminal vesicle and prostate innervation is derived from ipsilateral and contralateral ganglia. In addition to retrograde studies, dye-filled neurons were analysed immunohistochemically for neuronal markers and associations with specifically identified neuronal projections. Neurons of the ganglion projecting to the ductus deferens either contained tyrosine hydroxylase alone, tyrosine hydroxylase and neuropeptide Y, neuropeptide Y alone, neuropeptide Y and vasoactive intestinal peptide, or vasoactive intestinal peptide alone. These neurons were associated with three classes of neuronal projections, substance P-, leucine-enkephalin-, and methionine-enkephalin-immunoreactive. Neurons projecting to the seminal vesicles were similar to the neurons supplying the ductus deferens, except none of the seminal vesicle-specific neurons exhibited vasoactive intestinal peptide immunoreactivity. Neurons supplying the prostate were immunoreactive for either tyrosine hydroxylase or neuropeptide Y; these neurons were infrequently associated with the three classes of varicose neuronal projections. Neurons projecting to the rectum contained neuropeptide Y and were only associated with methionine-enkephalin immunoreactive neuronal projections in one animal.

Amidines↗