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J M Cunningham

Publications and source records attributed to J M Cunningham.

At least 127 records · Page 7Linked to original sources
PubMed1993

Identification of a low affinity, high capacity transporter of cationic amino acids in mouse liver.

The liver regulates the supply of amino acids required for protein synthesis and intermediary metabolism between feeding and fasting in mammals. The flux of amino acids between the liver and other tissues is determined, in part, by the activity of specific carrier proteins. We have identified a carrier of the cationic amino acids arginine, lysine, and ornithine in mouse liver that is closely related to a previously identified transporter with the same substrate specificity expressed in nonhepatic tissues. Uptake studies were performed in Xenopus oocytes injected with cRNA encoding these proteins. The comparison of the two transporters in these studies demonstrated that, unlike the widely-expressed transporter, arginine uptake mediated by the liver carrier is significant only at substrate concentrations that exceed systemic plasma levels and is less dependent on the intracellular concentration of cationic amino acids. These properties enable hepatocytes expressing this carrier to remove excess cationic amino acids from the blood without interfering with their uptake by nonhepatic tissues that express the related transporter.

Amino Acid Sequence↗
PubMed1993

Interleukin-1 beta effects on cyclic GMP and cyclic AMP in cultured rat islets of Langerhans-arginine-dependence and relationship to insulin secretion.

When islets were cultured with interleukin-1 beta (1 or 100 pmol/l) for 12 h in arginine-containing medium, cyclic GMP levels were increased 1.6- and 4.5-fold respectively. The arginine analogue, N-omega-nitro-L-arginine methyl ester, which blocks nitric oxide formation and partially reverses inhibition of insulin secretion by 100 pmol/l interleukin-1 beta, largely, but not completely, blocked generation of cyclic GMP. Treatment of islets with 100 pmol/l interleukin-1 beta for 12 h significantly decreased islet cyclic AMP generation in the absence of isobutylmethylxanthine (from 13.1 +/- 0.7 to 9.3 +/- 0.8 fmol/micrograms islet protein), this fall was arginine-dependent and may have resulted from an effect on a cyclic AMP phosphodiesterase, since it was masked if isobutylmethylxanthine was present. Isobutylmethylxanthine (0.4 mmol/l) reduced the inhibitory potency of interleukin-1 beta in 15 h slightly but significantly from 80.5 to 59.0%. The morpholinosydnonimine SIN-1, which is a nitric oxide donor, inhibited insulin secretion, raised islet cyclic GMP and lowered cyclic AMP; its effects were similar to those of interleukin-1 beta. However, 6-anilinoquinoline-5,8-quinone, [LY83583 (1-10 mumol/l)], inhibited insulin secretion, and significantly decreased cyclic GMP while 8-bromocyclic GMP stimulated insulin secretion. Both low- and high-dose interleukin-1 beta treatment give a large arginine-dependent and a small, yet significant, arginine-independent increase in cyclic GMP. The inhibitory effect of SIN-1 or interleukin-1 beta on insulin secretion seems to depend to a small extent on decreased islet cyclic AMP, though sustained increases in nitric oxide or depleted islet GTP may directly affect the secretory process.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Envelope-binding domain in the cationic amino acid transporter determines the host range of ecotropic murine retroviruses.

Infection of rodent cells by ecotropic type C retroviruses requires the expression of a cationic amino acid transporter composed of multiple membrane-spanning domains. By exchanging portions of cDNAs encoding the permissive mouse and nonpermissive human transporters and examining their abilities to specify virus infection upon expression in human 293 cells, we have identified the amino acid residues in the extracellular loop connecting the fifth and sixth membrane-spanning segments of the mouse transporter that are required for both envelope gp70 binding and infection. These findings strongly suggest that the role of the mouse transporter in determining infection is to provide an envelope-binding site. This role is analogous to those of host membrane proteins composed of a single membrane-spanning domain that serve as binding proteins or receptors for other enveloped viruses such as human immunodeficiency virus, Epstein-Barr virus, and murine and human coronaviruses.

3T3 Cells↗
PubMed1993

Retroviral infection and expression of cationic amino acid transporters in rodent hepatocytes.

The susceptibility of rodent hepatocytes to infection by mouse type C retroviruses was examined in vivo and in vitro and compared with the expression of two membrane proteins that function as transporters for the cationic amino acids CAT-1 and CAT-2. CAT-1 expression in rodents determines susceptibility to ecotropic retrovirus infection by serving as the virus receptor. Recently, it has been suggested that CAT-2 may be a receptor for amphotropic murine leukemia virus. In the present study, CAT-1 expression was observed in Hepa1, a cell line derived from a murine hepatoma, and in rat hepatocytes propagated on collagen monolayers in vitro but not in intact or regenerating rat liver in vivo. The expression of CAT-1 correlated with susceptibility to infection by an ecotropic retrovirus encoding beta-galactosidase. CAT-2 expression was observed in hepatocytes in vitro and in vivo, consistent with reports of infection of regenerating and cultured hepatocytes by amphotropic retroviruses. However, introduction of murine CAT-2 into nonpermissive Chinese hamster cells was not sufficient to confer susceptibility to amphotropic retrovirus infection, using a protocol that could easily demonstrate CAT-1-dependent infection by an ecotropic virus. Our data establish CAT-1 as a major determinant of ecotropic retrovirus infection in rodent hepatocytes and suggest that CAT-2 is not a receptor for viruses in the amphotropic subgroup.

Animals↗
PubMed1993

Minus-strand DNA is present within murine type C ecotropic retroviruses prior to infection.

Viral minus-strand DNA has been identified within ecotropic murine retroviruses prior to infection. The abundance of minus-strand DNA is inversely proportional to the distance from the primer binding site, suggesting that viral DNA is synthesized by reverse transcriptase with the genomic RNA as template. These findings demonstrate that replication of the retroviral genome is not initiated by infection and may begin after activation of reverse transcriptase by gag-pol cleavage during virus assembly.

Animals↗
PubMed1993

Methylation-enhanced binding of Sp1 to the stage selector element of the human gamma-globin gene promoter may regulate development specificity of expression.

The human gamma-globin gene promoter contains a stage selector element (SSE) responsible for preferential interaction of the promoter with a powerful erythroid-specific enhancer in the fetal developmental stage (S.M. Jane, P.A. Ney, E.F. Vanin, D.L. Gumucio, and A.W. Nienhuis. EMBO J. 11:2691-2699, 1992). The element binds two proteins, the ubiquitous activator Sp1 and a protein previously known as -50 gamma and now named the stage selector protein (SSP). Binding of the second protein correlates with SSE activity in transient-transfection assays. We now report that a de novo binding site for the SSP is created by the -202(C-->G) mutation that causes hereditary persistence of fetal hemoglobin (HPFH). This site functions in an analogous manner to the SSE in hybrid beta-promoter/reporter gene constructs transfected into K562 cells. In contrast, the wild-type -202 sequence, which fails to bind the SSP, is incapable of activating the beta-gene promoter. Both the -50 and -202 HPFH sites for SSP binding overlap a consensus sequence for the transcriptional regulator Sp1. In addition, both sites contain CpG dinucleotides that are contact bases for SSP. Since the gamma promoter is known to be hypomethylated in fetal cells but fully methylated at CpG residues in adult erythroid cells, we examined the effects of this DNA modification on protein binding to the two regions. Gel mobility shift assays with nuclear extract from K562 cells (which contain both Sp1 and SSP) demonstrate preferential binding of SSP to the SSE and HPFH sites under conditions in which probe was limiting. Methylation of the CpG residues reverses this preference only in the SSE site, with a marked increase in the binding of Sp1 at the expense of the SSP. Purified Sp1 binds with 10-fold higher affinity to the methylated than to the nonmethylated -50 probe but with the same affinity to the -202 HPFH probe. The methylation-induced preferential binding of Sp1 to the SSE at the expense of SSP may be part of the mechanism by which the gamma genes are repressed in normal adult erythroid cells. In cells containing the -202 HPFH mutation, the inability of Sp1 to displace SSP in the methylated state may explain the persistence of gamma-promoter activity and gamma-gene expression observed in adults with this mutation.

Adult↗
PubMed1993

Immunotherapy with interleukin 2 after ABMT in AML.

Myeloablative chemo (+/- radio) therapy and rescue with ABMT has been used as final consolidation therapy in 18 patients with AML in first remission. In seven (6 autologous, 1 syngeneic) marrow reinfusion was followed by iv IL-2. One patient, who commenced IL-2 4 days after BMT, died from pulmonary oedema due to the capillary leak syndrome. Thereafter, treatment with IL-2 was delayed until the platelet count reached 30 x 10(9)/l. All patients developed reversible hypotension (treated with infusion of colloid), but treatment was otherwise well tolerated. With 21-58 months (median 32 months) from the time of ABMT there has been one relapse (actuarial risk 17%, 95% confidence intervals (CI) 3-31%). The disease-free survival is 71% (95% CI 38-100%). Eleven patients with comparable remission induction and consolidation therapy, and an identical interval between diagnosis and ABMT (5-11 months, median 6 months) received an autograft without immunotherapy. With 24-45 months (median 29 months) follow-up the actuarial disease-free survival is 36% (95% CI 8-64%), the actuarial relapse risk is 54% (95% CI 18-90%). We conclude that immunotherapy given after ABMT to patients with AML in first remission when the platelet count exceeds 30 x 10(9)/l is safe and may induce an immunological environment which results in the elimination of residual leukaemia.

Adolescent↗
PubMed1992

Roles for the integrin VLA-4 and its counter receptor VCAM-1 in myogenesis.

Mammalian myogenesis is biphasic: primary myoblasts fuse to form primary myotubes, then secondary myoblasts align along the primary myotubes and form secondary myotubes, which comprise most of adult muscle. We provide evidence that an integrin (VLA-4) and its counter receptor (VCAM-1) have a role in secondary myogenesis. Both receptors are synthesized by cultured muscle cells: VLA-4 is induced as myotubes form, whereas VCAM-1 is present on myoblasts and myotubes. In vivo, both molecules are expressed at sites of secondary myogenesis, VLA-4 on primary and secondary myotubes, and VCAM-1 on secondary myoblasts and on regions of secondary myotubes apposed to primary myotubes. These patterns suggest that VLA-4-VCAM-1 interactions influence alignment of secondary myoblasts along primary myotubes and/or the fusion of secondary myoblasts. In support of the latter possibility, antibodies to VLA-4 or VCAM-1 inhibit myotube formation in culture.

Animals↗
PubMed1992

Molecular cloning and functional expression of an inducible nitric oxide synthase from a murine macrophage cell line.

Macrophages activated by exposure to cytokines and/or to endotoxin produce nitric oxide (NO.), a free radical that is a mediator of the host response to infection. Activation induces the expression of nitric oxide synthase, the enzyme that catalyzes formation of NO. from L-arginine and molecular oxygen. We report the cloning of a cDNA encoding the inducible nitric oxide synthase from a murine macrophage cell line, RAW264.7, exposed to interferon-gamma and lipopolysaccharide. Oocytes injected with mRNA transcribed from this cDNA demonstrate arginine-dependent production of nitrite, a stable metabolite of NO.. Nitric production is blocked by the enzyme inhibitor, NG-monomethylarginine, and is independent of calcium/calmodulin. RAW264.7 cells demonstrate rapid accumulation of the nitric oxide synthase-encoding mRNAs upon activation. Comparison of the deduced amino acid sequence to the calcium/calmodulin-dependent nitric oxide synthase previously purified (Bredt, D. S., and Synder, S.H. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 682-685) and cloned (Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R., and Synder, S. H. (1991) nature 351, 714-718) from rat brain identifies shared binding sites for the cofactors NADPH and flavins in the C-terminal half of both proteins and an additional conserved region near the N terminus that may recognize L-arginine and/or contribute to the active site.

Amino Acid Oxidoreductases↗
PubMed1992

Considerations in the skeletonization technique of internal thoracic artery dissection.

Skeletonization of the internal thoracic artery during myocardial revascularization procedures may provide some degree of protection from sternal wound infection in diabetic, obese, or pulmonary compromised patients when both internal thoracic arteries are used as conduits. Prior descriptions of the technique fail to provide specific details and possible pitfalls of the dissection. A method of skeletonization that has been used at the California Center for Cardiothoracic Surgery in more than 1,000 patients is presented in detail. Potential advantages and disadvantages are discussed.

Humans↗
PubMed1992

The human cationic amino acid transporter (ATRC1): physical and genetic mapping to 13q12-q14.

The product of the mouse Rec-1 locus is an integral membrane protein that determines susceptibility to infection by murine ecotropic retroviruses. Recently it has been determined that its role in normal cell metabolism is transport of the cationic amino acids, arginine, lysine, and ornithine across the plasma membrane. Southern blot analysis of genomic DNA from a panel of 48 mouse-human somatic cell hybrids assigned the human version of this gene, ATRC1, to chromosome 13. Chromosomal in situ hybridization localized the gene to 13q12-q14. A restriction fragment length polymorphism (RFLP) was detected with TaqI. There were two alleles with frequencies of 0.29 and 0.71. Pairwise linkage analysis established linkage between ATRC1 and ATP1AL1, D13S1, D13S6, D13S10, D13S11, D13S21, D13S22, D13S33, D13S36, and D13S37. Multilocus linkage analysis of five of the loci indicated that the most likely order of loci in this region was D13S11-ATP1AL1-ATRC1-D13S6-D13S33.

Amino Acid Transport Systems, Basic↗
PubMed1992

Microcarrier culture of hepatocytes in whole plasma for use in liver support bioreactors.

Contact activation of plasma clotting may limit the use of some microcarrier types for hepatocyte attachment in a liver assist device. Activation by seven microcarrier types was studied in plasma containing 2-500 units heparin/mL. Clotting was activated by dextran (Cytodex 1 and 2) and collagen-coated (Cytodex 3) microcarriers at 2-25 units/mL (Cytodex 1) and 2-100 units/mL (Cytodex 2 and 3). There was no activation by polystyrene, gelatin, glass or fibronectin-coated polystyrene microcarriers. Compared with culture medium, incubation of HepG2 cells in plasma did not affect cell viability but increased cell number (56.4 versus 65.1 x 10(4) cells; P less than 0.05) and incorporation of [3H]-amino acids into protein (204913 versus 279624 dpm; P less than 0.05). Polystyrene-attached cells demonstrated time-linear protein synthesis, glucose and 7-ethoxycoumarin metabolism. We conclude that polystyrene-attached hepatocytes maintain viability and metabolic activity in plasma and are of potential use in a liver support bioreactor.

Artificial Organs↗
PubMed1991

Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.

Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.

Animals↗
PubMed1991

Dimethyl sulphoxide induces a reduced growth rate, altered cell morphology and increased epidermal-growth-factor binding in Hep G2 cells.

Culture of Hep G2 cells in medium containing 2% (v/v) dimethyl sulphoxide (DMSO) resulted in a slowing of growth and a marked change in morphological appearance. By day 6, cultures containing DMSO had one-third the number of cells compared with parallel control cultures. Measurement of 125I-epidermal-growth-factor (EGF) binding to DMSO-treated cells revealed a striking time-dependent elevation in specific EGF binding to their cell surface. Increased binding was detectable within 24 h of the start of DMSO treatment, reaching, by 6 days, levels almost 25 times greater than those for control cells. Addition of EGF to DMSO-treated cells caused a rapid down-regulation of the EGF receptor, but did not alter their proliferation rate. Slowing of growth by other means, such as serum starvation, growth to confluence or culture in the presence of sodium butyrate, did not affect 125I-EGF binding, indicating a specific effect of DMSO on these cells.

Carcinoma, Hepatocellular↗
PubMed1991

A method for the purification of DNA/protein complexes applied to DNA topoisomerase II cleavage sites.

The object of this study was to devise a purification method for DNA/topoisomerase II complexes, with which to examine the enzyme's cleavage site specificity in cellular differentiation. Retinoic acid-induced differentiation involves topoisomerase II-mediated transient changes in DNA supercoiling, but it is not known whether this occurs at specific sites in the genome. Topoisomerase II forms a covalent DNA enzyme complex as it acts, which can be recovered by the sodium dodecyl sulfate (SDS)/KCl precipitation method, but this method fails to recover significantly more DNA from cells induced to differentiate. This may in part reflect the low numbers of retinoic acid-induced protein-linked breaks in DNA and also the method's relative inefficiency for DNA with few attached topoisomerase molecules. This suggested that an additional purification method would be required to enrich sufficiently for cleavage site DNA to address the issue of site specificity. The principle of our method is to couple poly(ethylene glycol) (PEG) to topoisomerase while it is covalently attached to DNA and then to use phase partitioning in an aqueous two-phase system of PEG and phosphate to separate free DNA from DNA bound to PEG-modified topoisomerases (which have high affinities for the phosphate-rich and PEG-rich phases, respectively). The method can be used in conjunction with DNase protection and, unlike the SDS/KCl method, can fractionate short fragments of DNA to which single protein molecules are attached. Using the SDS/KCl precipitation and new method in series, we have recovered protein-linked DNA from HL60 cells induced to differentiate to the granulocyte lineage (by retinoic acid) or to the monocyte/macrophage lineage (by phorbol myristate acetate) and have demonstrated that specific sequences become protein linked, probably to topoisomerase II, during induced differentiation.

Chemical Fractionation↗
PubMed1991

Rostrocaudal variation of fiber type composition in rat intercostal muscles.

We used the histochemical stain for ATPase to compare the fiber-type composition of rat internal and external intercostal muscles from thoracic (T) segments 2-5, 8, and 11. At each level, type II fibers were more numerous than type I fibers, type II B fibers were more numerous than II A fibers, and type I fibers were more numerous in external than in internal intercostals. However, fiber type composition varied from segment to segment. For example, the proportion of type II A fibers increased in a rostrocaudal gradient in internal but not external intercostals, and type I fibers were more prevalent at rostral and caudal than at intermediate levels in both internal and external intercostals. These results provide a basis for interpreting previous physiological and molecular studies which have compared intercostal muscles from different segmental levels.

Adenosine Triphosphatases↗
PubMed1991

Aberrant DNA topoisomerase II activity, radioresistance and inherited susceptibility to cancer.

Inherited susceptibility to a wide variety of neoplasias (Li-Fraumeni syndrome), has been shown in studies of one cancer-prone family, to have an intriguing association with an aberrant c-raf-1 gene and inheritance of a radioresistant phenotype in their non-cancerous skin fibroblasts. This association together with observations that DNA topoisomerases, when defective, can introduce errors into DNA and that these enzymes are perturbed in vitro by serine/threonine kinases similar to raf encoded proteins, prompted investigation of DNA topoisomerase activity of the family's fibroblasts. Since radioresistance was transferred to murine cells (NIH-3T3) when the aberrant c-raf-1 gene from this family was transfected, we also examined transformants containing this and other oncogenes. V-raf/c-myc and EJ-ras transformants were examined, the former because the family's skin fibroblasts also have 3-8-fold elevated myc expression (not apparently relevant to radioresistance) and the latter because ras, like raf, conveys radioresistance. The family members' fibroblasts and the three transfected murine lines, showed a similar perturbation of a spermidine and ATP-dependent DNA catenation activity (typical of DNA topoisomerase II). There was a significant positive correlation (r = 0.93; P = 0.0026) between the degree of activation of topoisomerase II and one measure of radioresistance (the Dq value). Relaxation of DNA supercoiling (topoisomerase I activity and other DNA nicking enzymes) was not abnormal. Cytotoxicity assays and evaluation of the influence of topoisomerase II inhibitors on DNA/protein complex formation, corroborated the existence of a qualitative topoisomerase II defect in the family's cells and transfectants. Although the contention that the qualitative topoisomerase II abnormalities observed here may be associated with malfunction is highly speculative, these findings may be relevant to the mechanism of oncogenesis, not only in this family, but with raf and ras type oncogenes.

Amsacrine↗