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J M Cunningham

Publications and source records attributed to J M Cunningham.

At least 109 records · Page 6Linked to original sources
PubMed1995

Comparison of inhibition of glucose-stimulated insulin secretion in rat islets of Langerhans by streptozotocin and methyl and ethyl nitrosoureas and methanesulphonates. Lack of correlation with nitric oxide-releasing or O6-alkylating ability.

We have studied inhibition of glucose-stimulated insulin secretion in islets of Langerhans isolated from adult Sprague-Dawley rats and treated with different alkylating agents. Streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-ethyl-N-nitrosourea (ENU) all released nitric oxide, as demonstrated by an increase in medium nitrite and cellular cyclic GMP. Methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), which do not possess a nitroso group, did not show evidence of nitric oxide release. All five compounds, however, decreased glucose-stimulated insulin release, suggesting that nitric oxide release was not necessary for the inhibition of secretion. Lack of involvement of nitric oxide was further suggested by the failure of oxyhaemoglobin to reverse STZ and MNU inhibition of insulin secretion. Since ENU was at least as effective as MNU in inhibiting insulin secretion, it appears that alkylation of DNA at the O6 position of guanine may not be involved in this process.

Alkylating Agents↗
PubMed1995

Identification of two novel regulatory elements within the 5'-untranslated region of the human A gamma-globin gene.

Interaction between the stage selector element (SSE) in the proximal gamma-globin promoter and hypersensitivity site 2 in the locus control region partly mediates the competitive silencing of the beta-globin promoter in the fetal developmental stage. We have now demonstrated that a second SSE-like element in the 5'-untranslated region of the gamma-gene also contributes to this competitive silencing of the beta-gene. Utilizing transient transfection assays in the fetal erythroid cell line, K562, we have shown that the core enhancer of hypersensitivity site 2 can preferentially interact with the proximal gamma-promoter in the absence of the SSE, completely silencing a linked beta-promoter. Mutation of a 20-base pair sequence of the gamma-gene 5'-untranslated region (UTR) led to derepression of beta-promoter activity. A marked activation of gamma-promoter activity was also observed with this mutation, suggesting the presence of a repressor. Fine mutagenesis dissected these activities to different regions of the 5'-UTR. The stage selector activity was localized to a region centered on nucleotides +13 to +15. Electromobility shift assays utilizing this sequence demonstrated binding of a fetal and erythroid-specific protein. The repressor activity of the 5'-UTR was localized to tandem GATA-like sites, which appear to bind a complex of two proteins, one of which is the erythroid transcription factor GATA-1. These results indicate that the 5'-UTR of the gamma-gene contains sequences that may be important for its transcriptional and developmental regulation.

Base Sequence↗
PubMed1995

Hemoglobin switching in man and chicken is mediated by a heteromeric complex between the ubiquitous transcription factor CP2 and a developmentally specific protein.

The human stage selector protein (SSP) has been implicated in the developmental regulation of the globin genes. Binding of SSP to the stage selector element (SSE) in the proximal gamma-globin promoter is integral to the competitive silencing of a linked beta-promoter in embryonic/fetal stage erythroleukemia (K562) cells. We now report the biochemical purification of SSP from K562 cell nuclear extract and demonstrate that the ubiquitously expressed transcription factor CP2 is pivotal to, but not sufficient for, SSP binding activity. Although addition of anti-CP2 antiserum disrupts the formation of the SSP-SSE complex in the electrophoretic mobility shift assay (EMSA), recombinant CP2 fails to bind to the SSE. Binding of CP2 to the SSE requires a heterodimeric partner present in K562 cells. We have defined the molecular weight of the partner protein as 40-45 kDa in UV and protein cross-linking experiments. An element analogous to the human SSE has previously been demonstrated in the chicken beta A-gene-promoter. The effects of this element are dependent on the binding of the chicken stage selector protein, NF-E4. Comparative studies between human CP2 and chicken NF-E4 demonstrate homology between the protein complexes. SSP binds to the chicken SSE and formation of this complex is ablated by the addition of anti-CP2 antiserum or a monoclonal antibody to NF-E4. Western analysis of partially purified NF-E4 using anti-CP2 antiserum or the NF-E4 monoclonal antibody both demonstrate a dominant band at 66 kDa. Similarly, the NF-E4 antibody recognizes the 66 kDa human CP2 protein in Western analysis of the SSP-SSE complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The renal glomerulus of mice lacking s-laminin/laminin beta 2: nephrosis despite molecular compensation by laminin beta 1.

S-laminin/laminin beta 2, a homologue of the widely distributed laminin B1/beta 1 chain, is a major component of adult renal glomerular basement membrane (GBM). Immature GBM bears beta 1, which is replaced by beta 2 as development proceeds. In mutant mice that lack beta 2, the GBM remains rich in beta 1, suggesting that a feedback mechanism normally regulates GBM maturation. The beta 2-deficient GBM is structurally intact and contains normal complements of several collagenous and noncollagenous glycoproteins. However, mutant mice develop massive proteinuria due to failure of the glomerular filtration barrier. These results support the idea that laminin beta chains are functionally distinct although they assemble to form similar structures. Laminin beta 2-deficient mice may provide a model for human congenital or idiopathic nephrotic syndromes.

Animals↗
PubMed1995

Gene transfer into human hemopoietic progenitor cells.

Considerable progress is being made in the transfer of genetic material to hematopoietic stem cells. In this chapter we describe how gene transfer is being used to: mark marrow and peripheral blood progenitor cells prior to autologous transplantation, to track their fate on reinfusion and to detect contaminating tumorigenic cells; modulate immunocyte function--important in immunologic disorders and perhaps in cancer therapy; generate tumor vaccines from tumor cells isolated from marrow; correct single gene defects--the 'classical' concept of gene therapy; and finally to modify the drug sensitivity of progenitor cells--enabling them to resist the suppressive effects of cytotoxic drugs during cancer therapy and perhaps providing a mechanism for in vivo selection of gene modified cells.

Animals↗
PubMed1995

Developmental regulation of the human beta-globin cluster.

Expression of the genes of the human beta-globin cluster is subject to stringent developmental regulation. An integral component of this control is competition between the gamma- and beta-promoters for the upstream regulatory sequences of the Locus Control Region (LCR). We have defined two stage selector elements (SSE and SSE-2), located in the proximal gamma-promoter and gamma-genes 5'UTR which mediate the preferential interaction of the gamma-gene with the LCR in the foetal developmental stage. The activity of both elements is dependent upon the binding of foetal and erythroid-specific proteins. We have purified the protein binding the proximal SSE and characterised it as a complex between the ubiquitous transcription factor CP2 and a 40 hD partner protein. Comparative studies between human CP2 and the chicken stage selector protein, NF-E4, demonstrate homology between the protein complexes. These findings demonstrate that competitive silencing of globin genes mediated by developmentally-specific protein complexes is conserved in evolution.

Animals↗
PubMed1995

Novel pattern of P53 mutation in breast cancers from Austrian women.

Since mutagens produce an extraordinary diversity of mutational patterns, differential mutational exposures among populations are expected to produce different patterns of mutation. Classical epidemiological methods have been successful in implicating specific mutagens in cancers such as those of lung and skin in which one mutagen predominates. In breast cancer, however, no mutagens have been implicated in an unequivocal manner. In an attempt to facilitate epidemiological studies, we have been studying the pattern of p53 gene mutations in breast cancers from multiple populations with high and low breast cancer incidences. We previously reported that breast cancers from Midwest United States, predominantly rural Caucasian women, have a different pattern of p53 gene mutation from populations of Western European women. Herein, we analyze patterns of p53 mutations from Graz, Austria, another population with a high incidence of breast cancer. Among the 60 Austrian breast cancers analyzed, 14 (23%) have a p53 gene mutation in exons 5-9 or in adjacent splice junctions. Analysis of the patterns of mutation shows differences between the "Western European" profile and the Austrian and Midwest United States groups (P = 0.027 and 0.024, respectively). The Austrian pattern is characterized by a high frequency of A:T-->T:A transversions (P = 0.006). The presence of distinct patterns of mutation among the limited number of analyzed populations of Western European origin supports the idea that differential mutagenic exposure and/or genetic differences contribute to breast cancer mutagenesis among geographically distinct Caucasians of Western European origin.

Aged↗
PubMed1994

The regulatory element 3' to the A gamma-globin gene binds to the nuclear matrix and interacts with special A-T-rich binding protein 1 (SATB1), an SAR/MAR-associating region DNA binding protein.

A cis-acting DNA regulatory element 3' to the A gamma-globin gene contains eight distinct regions of DNA-protein interaction distributed over 750 bp of DNA. The sequences of two foot-printed regions (sites I and IV) are A-T rich and generate a highly retarded complex on gel shift analysis with nuclear extract from human erythroleukemia (K562) cells. We have purified a 98-kD protein that reproduces this gel shift. Tryptic cleavage and peptide sequence analysis demonstrated that the 98-kD protein is identical to a recently cloned protein, special A-T-rich binding protein 1 (SATB1), that binds selectively to nuclear matrix/scaffold-associated regions of DNA (MARs/SARs). We have shown by functional analysis that the 3' A gamma regulatory element associates with the nuclear matrix. SATB1 mRNA was identified in K562 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated its transcript in several other hematopoietic lines. Antisera to SATB1 caused ablation of the gel shift complex generated by both the crude nuclear extract and the purified 98-kD protein with the site I oligonucleotide. Furthermore, oligonucleotides that bind SATB1 inhibited formation of the site I gel shift complex when added as excess unlabeled competitor. An immunoblot analysis of the site I gel shift complex documented the presence of SATB1. Binding of SATB1 to two sites within the 3' A gamma regulatory element and its MAR/SAR activity suggests that this element may influence gene expression through interaction with the nuclear matrix.

Amino Acid Sequence↗
PubMed1994

The effect of nitric oxide donors on insulin secretion, cyclic GMP and cyclic AMP in rat islets of Langerhans and the insulin-secreting cell lines HIT-T15 and RINm5F.

The aim of this study was to investigate whether short-term treatment with nitric oxide donors could mimic cytokine inhibition of insulin secretion. We tested the nitric oxide generating compounds 3-morpholinosydnonimine (SIN-1), S-nitroso-N-penicillamine (SNAP), S-nitrosoglutathione and hydroxylamine for their ability to inhibit insulin secretion, raise cyclic GMP and lower cyclic AMP levels in isolated rat islets of Langerhans and the insulin-secreting cell lines HIT-T15 and RINm5F. In islets, all nitric oxide donors inhibited glucose-induced insulin secretion and raised cyclic GMP levels. SIN-1 and S-nitrosoglutathione also reduced cyclic AMP, while SNAP and hydroxylamine had no effect. Insulin secretion in HIT-T15 cells was inhibited by SIN-1, SNAP and hydroxylamine and in RINm5F cells by hydroxylamine. Inhibition of HIT-T15 and RINm5F cell insulin secretion was not accompanied by an increase in cyclic GMP levels. The degree of inhibition of insulin secretion was unrelated to the extent of release of nitric oxide by the compounds as measured by nitrite and nitrate production. More effective inhibition by S-nitrosoglutathione and hydroxylamine versus SIN-1 and SNAP may be related to intracellular versus extracellular site of nitric oxide generation.

Animals↗
PubMed1994

Differential toxicity of alpha-keto hydroxypyridine iron chelators and desferrioxamine to human haemopoietic precursors in vitro.

Compliance with iron chelation therapy improves life expectancy in transfusion-dependent haematological disorders. However, failure of compliance with parenteral desferrioxamine (DF) therapy and the expense incurred makes this drug unavailable for most patients in the developing world. We have been evaluating the orally active iron chelator 1,2-dimethyl-3-hydroxypyrid-4-one (L1) in both preclinical and clinical trials. Five patients have developed reversible agranulocytosis during treatment with this agent. We have now studied the effects of L1, other alpha-ketohydroxypyridines and DF on bone marrow myeloid progenitors using the CFU-GM system. The results show that L1 is less toxic than DF to normal bone marrow myeloid progenitors (ID50:130 mumol/l versus 7.9 mumol/l). The L1 ID50 is within the previously reported range of peak plasma values (80-450 mumol/l). When saturating concentrations of iron were added to the cultures, the mean toxicity of all the chelators was significantly decreased over the range of doses tested, e.g. L1 ID50, 567 mumol/l; DF ID50, > 1000 mumol/l. The toxicity of L1 in vitro was similar for marrows from 3 normal donors and for the recovery marrow from a patient with thalassaemia major who had experienced agranulocytosis. Further studies are required to elucidate the mechanisms of L1-induced agranulocytosis.

Bone Marrow Cells↗
PubMed1994

Cytokines, nitric oxide and insulin secreting cells.

Cytokines are a group of regulatory and immunomodulatory proteins involved in a number of physiological processes. Various disease states are believed to involve alteration of normal cytokine activity, including insulin-dependent diabetes mellitus, an autoimmune disease in which insulin secreting beta cells within pancreatic islets of Langerhans are selectively destroyed. Glucose-induced insulin secretion is inhibited by the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 and tumour necrosis factor alpha (TNF) when combined with IL-1 beta in cultured rat islets, by IL-1 beta, TNF and interferon gamma in mouse islets, and by combined treatment of IL-1 beta, TNF and interferon gamma in human islets. Continued cytokine treatment in many cases leads to destruction of some, if not all, islet cells. A key factor in the inhibitory effect of IL-1 beta and TNF in rat islets is the generation of nitric oxide which inactivates enzymes such as aconitase and ribonucleotide reductase by formation of iron-nitrosyl complexes. This in turn may lead to reduced oxidation of glucose and synthesis of ATP and DNA respectively. The causes of cytokine-induced beta cell death are less well defined, but important factors may be nitric oxide-mediated DNA damage, depletion of NAD levels and toxic effects of oxygen free radicals and eicosanoids generated in addition to nitric oxide. Potentially important defence and repair responses induced by IL-1 beta treatment of rat islets are formation of heat shock protein, haem oxygenase, and superoxide dismutase. Other protective responses may be induction of cytokines and cytokine receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Characterization of the third member of the MCAT family of cationic amino acid transporters. Identification of a domain that determines the transport properties of the MCAT proteins.

We have identified the third member of a family of cationic amino acid transporters in lipopolysaccharide-stimulated murine macrophages. The deduced amino acid sequence of this transporter is the same as MCAT-2 (mouse cationic amino acid transporter-2), the low affinity transporter expressed in hepatocytes, except for a stretch of 41 amino acids that connects the eighth and ninth membrane-spanning domains. These transporters apparently result from differential splicing of transcripts from a single gene and therefore have been named MCAT-2A (hepatocyte) and MCAT-2B (macrophage). Despite their similarity, MCAT-2B is saturated at one-fifth the arginine concentration, has a lower apparent Vmax, and is more sensitive to trans-stimulation than MCAT-2. Introduction of the unique regions of MCAT-2A and MCAT-2B into the equivalent portion of the related protein, MCAT-1, created chimeric transporters with properties most like the donor of this region. Our findings suggest these 41 amino acids contain a domain that binds the amino acid substrate during its translocation across the membrane.

Amino Acid Sequence↗
PubMed1993

N-linked glycosylation of the receptor for murine ecotropic retroviruses is altered in virus-infected cells.

Pulse-chase labeling identified incomplete maturation of the cationic amino acid transporter that serves as the receptor for ecotropic retroviruses in infected NIH 3T3 fibroblasts. The molecular basis and functional consequences of this change were studied by expressing viral and receptor proteins in Xenopus oocytes. Expression of the ecotropic, but not polytropic, envelope surface protein, gp70, interfered with N-linked glycosylation of the permissive mouse, but not the nonpermissive, human transporter. Incomplete glycosylation may be a direct consequence of gp70 binding since the glycosylated residues, Asn223 and Asn229, are present in the envelope binding domain. Receptors bearing amino acid substitutions in Asn223 and/or Asn229 that prevent glycosylation function normally in virus infection and amino acid transport. However, gp70 expression sufficient to block binding of 125I-gp70 to the receptor in the plasma membrane decreased receptor-mediated arginine uptake by 50%. Taken together, these findings suggest that newly synthesized gp70 may bind to the receptor in the endoplasmic reticulum and prevent normal glycosylation during their transit to the plasma membrane.

3T3 Cells↗