Biomedical subjects

S M Morris

Publications and source records attributed to S M Morris.

At least 73 records · Page 4Linked to original sources

PubMed1998

Lactogenic hormone signal transduction.

The peptide hormone prolactin (PRL) is known to regulate numerous target tissues. Among the less well-known targets are cells of the immune system, including T cells, B cells, and macrophages. Our laboratory has cloned a panel of PRL-inducible T-cell activation genes for use in studies investigating how PRL modulates the biology of cells of the immune system. This article focuses on two such PRL-inducible genes. One is a transcription factor called interferon regulatory factor-1, whose expression is regulated by signaling molecules along the PRL-inducible JAK/Stat signaling pathway. These signaling molecules include Stat1 and CBP as positive mediators and, unexpectedly, Stat5b as a negative mediator. A second PRL-inducible gene is c15/RNUDC, a novel nuclear movement protein, which may provide a link between PRL signaling and signaling via the lipid second messenger, platelet activating factor.

Animals↗

PubMed1998

Differential regulation of arginases and inducible nitric oxide synthase in murine macrophage cells.

Activated macrophages avidly consume arginine via the action of inducible nitric oxide synthase (iNOS) and/or arginase. In contrast to our knowledge regarding macrophage iNOS expression, the stimuli and mechanisms that regulate expression of the cytosolic type I (arginase I) or mitochondrial type II (arginase II) isoforms of arginase in macrophages are poorly defined. We show that one or both arginase isoforms may be induced in the RAW 264.7 murine macrophage cell line and that arginase expression is regulated independently of iNOS expression. For example, 8-bromo-cAMP strongly induced both arginase I and II mRNAs but not iNOS. Whereas interferon-gamma induced iNOS but not arginase, 8-bromo-cAMP and interferon-gamma mutually antagonized induction of iNOS and arginase I mRNAs. Dexamethasone, which did not induce either arginase or iNOS, almost completely abolished induction of arginase I mRNA by 8-bromo-cAMP but enhanced induction of arginase II mRNA. Lipopolysaccharide (LPS) induced arginase II mRNA, but 8-bromo-cAMP plus LPS resulted in synergistic induction of both arginase I and II mRNAs. In all cases, increases in arginase mRNAs were sufficient to account for the increases in arginase activity. These complex patterns of expression suggest that the arginase isoforms may play distinct, although partially overlapping, functional roles in macrophage arginine metabolism.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Human type II arginase: sequence analysis and tissue-specific expression.

A full-length cDNA encoding type II arginase was isolated from a human kidney cDNA library and its sequence compared to those of vertebrate type I arginases as well as to arginases of bacteria, fungi and plants. The predicted sequence of human type II arginase is 58% identical to the sequence of human type I arginase but is 71% identical to the sequence of Xenopus type II arginase, suggesting that duplication of the arginase gene occurred before mammals and amphibians diverged. Seven residues known to be essential for activity were found to be conserved in all arginases. Type II arginase mRNA was detected in virtually all human and mouse RNA samples tested whereas type I arginase mRNA was found only in liver. At least five mRNA species hybridizing to type II arginase cDNA were found in the human RNA samples whereas only a single type II arginase mRNA species was found in the mouse. This raises the possibility that the multiple type II arginase mRNAs in humans arise from differential RNA processing or usage of alternative promoters.

Amino Acid Sequence↗

PubMed1997

Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols.

The aim of this study was to compare and improve standard methods to determine nitrite (NO2-), nitrate (NO3-) and S-nitrosothiol (RSNO) levels in cell culture supernatants, sera, and urine. We modified the conventional Griess reaction by replacing sulfanilamide with dapsone (4,4'-diamino-diphenylsulfone) and compared the NO2- levels in our study samples with a commercially available NO2- assay kit. Our modification, along with ultrafiltration of the samples, resulted in an enhanced sensitivity to measure NO2- down to 0.2 microM. The detection limit was further improved to 0.02 microM when NO2- was identified by the fluorochrome 2,3-diaminonaphthalene (DAN). To measure the stable end product NO3- by the Griess reaction or the DAN method, this anion must be reduced to NO2-. We compared the capacity of bacterial nitrate reductase with the reducing metal cadmium to convert NO3- to NO2-. After reduction, NO2- levels were determined either by the DAN method or by our modified Griess reaction. We found that there was a high correlation (r2 = 0.998) in total NO2- concentrations in the study samples using both methods for reducing NO3- to NO2-. The simultaneous determination of NO2- and NO3- was achieved by using anion-exchange chromatography (HPLC; Polyspher IC AN-1 column). The detection limit of this assay for each anion is 0.5 microM, and it can be applied equally well to sera, urine, and culture media. We also adapted the DAN method to determine RSNO levels in our study samples. Using this approach, we were able to measure RSNO levels down to 0.15 microM. As result we discovered that RSNO levels were markedly increased in urine from septic patients and in supernatants from cytokine-stimulated human tumor cell lines. L-Citrulline, a coproduct of NO biosynthesis, was measured using a colorimetric assay with a sensitivity limit of 3.0 microM. Increased L-citrulline levels in media from cultured cells, but not in sera or urine, correlated with increased NO production. Although all methods studied were suitable for quantifying end products of NO in biological fluids and media, the use of bacterial reductase and the modified Griess reaction proved successful to provide the greatest sensitivity and linear range for routine measurements of NO2- and NO3-.

Body Fluids↗

PubMed1997

Development and utilization of the rat lymphocyte hprt mutation assay.

Much of the recent progress in the field of genetic toxicology has come from an increased understanding of the molecular and cellular biology of the mammalian organism. Most prominent has been the ability to detect and quantify somatic mutation and relate the nature of the mutation to the specific type of chemical damage. Building upon the foundation of the human lymphocyte hypoxanthine guanine phosphoribosyl transferase (hprt) system, and later, the mouse hprt system, methods for the detection and quantification of hprt mutations in rat lymphocytes were developed. These methods are described in this report as is the ongoing validation of the assay. Additionally, the characterization of the recovered mutants and a comparison of the mutation spectrum in the rat lymphocyte system to the spectrum in cancer genes, such as H-ras and p53, and the spectrum in transgenic systems, such as lacI, are included. The development of the rat lymphocyte hprt system and validation of the assay at the molecular level, provide an effective and reliable measure of genetic damage in an in vivo system which is readily comparable to measurement of genetic damage in the human.

Animals↗

PubMed1997

Nitrite-induced photo-oxidation of thiol and its implications in smog toxicity to the eye: prevention by ascorbate.

Studies have been conducted on nitrite-induced oxidation of corneal thiols and reduced glutathione (GSH). Oxidation of GSH in the presence of nitrite (NaNO2) was minimal in the dark. Exposure of GSH to UV (365 nm) in the presence of nitrite substantially accelerated this oxidation; only < 10% of the original GSH remained at the end of 20 minutes. A similar Thiol depletion was observed in the case of corneal epithelial extracts irradiated with UV in the presence of the nitrite. Nitrite is therefore considered to be a potent phototoxicant with possible pathophysiological implications to the external eye tissues. Ascorbate was found to be effective in preventing thiol oxidation, suggesting the possibility of preventing nitrogen oxide-based smog irritation to the eye by this physiologically compatible antioxidant.

Animals↗

PubMed1997

Repair of elastase-digested elastic fibers in acellular matrices by replating with neonatal rat-lung lipid interstitial fibroblasts or other elastogenic cell types.

Disruption of elastic fibers is a major factor in the pathogenesis of pulmonary emphysema. Elastic fibers in culture, injured by exposure to elastase, undergo repair in the presence of elastogenic cells that restores the fibers toward normal as determined by biochemical and ultrastructural methods. The repair appears to be the result of both salvage and de novo repair mechanisms. The evidence for salvage repair is that hot-alkali resistance, lost as a result of elastase treatment, is restored to previously radiolabeled elastic fibers. This repair mechanism has been shown in aortic smooth muscle cell cultures. In order to determine the potential relevance of this mechanism for elastic fiber repair in the lungs, experiments were carried out using neonatal rat lung lipid interstitial fibroblasts (LIF). Treatment of the LIF cultures with elastase, in the absence of serum, caused solubilization of 12% of elastin; however, 81% of the elastin protein and 80% of the elastin-associated radioactivity (EAR) were solubilized by subsequent hot-alkali treatment, indicating that most of the elastin was retained in the matrix but was damaged. Ultrastructurally, the elastic fibers were frayed. After 6 additional wk in culture, hot-alkali resistant elastin protein and EAR were restored to 88 and 62% of control values, respectively, and the ultrastructural appearance of elastic fibers was restored to normal. We calculate that about 42% of the restored elastin represented salvage repair; the remainder was new elastin. No repair occurred in matrices rendered acellular by azide treatment; however, when acellular matrices were replated with LIF, repair was complete at 6 wk. No repair was seen when acellular matrices were replated with a transformed mouse macrophage cell line. We conclude that lung LIF are capable of mounting a robust repair process after elastolytic injury of elastin and that the repair is the result of both salvage and de novo repair mechanisms.

3T3 Cells↗

PubMed1997

A prolactin-inducible T cell gene product is structurally similar to the Aspergillus nidulans nuclear movement protein NUDC.

Clone 15 (c15) was originally identified as a PRL-inducible gene in activated T cells. Sequence analysis of c15 revealed that the last 94 amino acids of c15 are 68% identical and 78% similar to the filamentous fungus Aspergillus nidulans nuclear movement protein NUDC. The identification of the mammalian (rat) c15 protein suggests that the carboxy-terminal NUDC-like region has been conserved over evolution for an important structure and/or function. To determine whether c15 is functionally analogous to NUDC, complementation studies were performed using the inducible/repressible pAL5 vector system. The results of the complementation experiments show that the full-length mammalian c15 protein is not only capable of rescuing the nuclear movement defect of the nudC3 mutants, but is also able to restore the expression of the downstream endogenous NUDF protein to near wild type levels. These results indicate that rat c15 and fungal NUDC not only share similar structures, but also serve similar functions. Taken together, the structural and functional conservation between c15 and NUDC is consistent with the notion that c15 is the rat homolog of nudC and has therefore been given the name RnudC.

Animals↗

PubMed1997

Vasoactive intestinal peptide stimulation of human trabecular meshwork cell growth.

PURPOSE: To demonstrate that vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, is a growth factor of human trabecular meshwork (TM) cells in culture and in a corneoscleral explant organ culture treated with laser trabeculoplasty (LTP). METHODS: Proliferating human TM cells in cell cultures were incubated with VIP for 20 hours, followed by total cell number determination, using a Coulter counter. The percentage of proliferating TM cells was assessed, using an antibody against the proliferating cell nuclear antigen (PCNA). To test the growth effect of VIP on TM cells in situ, corneoscleral explants in organ cultures were first treated with argon LTP to initiate TM-cell proliferation and then were exposed to VIP for 48 hours. The mitotic TM cells were demonstrated immunocytochemically, using anti-PCNA in paraffin sections of the explants; and the total number of TM cells was determined after paraffin sections were counterstained by hematoxylin. RESULTS: Vasoactive intestinal peptide dose-dependently stimulated the proliferation of TM cells in cell culture. Treatment with 5 x 10(-10) M VIP resulted in a maximal increase of 40% in cell number. The effect of VIP was blocked by a VIP antagonist. The number of PCNA-stained TM cells and the total cell number in the TM in LTP-treated corneoscleral explants were increased by VIP. CONCLUSIONS: Exogenously applied VIP stimulated the proliferation of human TM cells in subconfluent cultures and in LTP-treated corneoscleral explants. In that LTP has been shown to increase the number of TM cells in situ, the growth stimulatory effect of VIP may help enhance this therapy.

Adolescent↗

PubMed1996

A role for apoptosis in the toxicity and mutagenicity of bleomycin in AHH-1 tk+/- human lymphoblastoid cells.

The chromosomal mutagen, bleomycin, is also noted for its toxic properties, although the mechanism of cell death is not fully understood. In order to determine if cell death occurred by apoptosis or necrosis, AHH-1 tk+/- cells were exposed to bleomycin and the percentage of viable, apoptotic and necrotic cells quantified by flow cytometry. Logistic regression analysis indicated that the primary manner of cell death was through the apoptosis pathways, that apoptosis was delayed, and that apoptosis was accompanied by an arrest in the G2 phase of the cell cycle. Once apoptosis was established as a mechanism for cell death, the efficiency with which these pathways removed damaged cells from the population was evaluated with the use of specific-locus mutation assays (tk and hprt) as indicators of cells with DNA damage that maintained viability and clonogenicity. Linear regression analysis detected a significant, concentration-dependent increase in the numbers of TFTr clones with the slow-growth phenotype. This suggests that a proportion of cells with bleomycin-induced DNA damage did not undergo cell death by apoptosis and that apoptosis, a mechanism for the destruction of damaged cells, is not fully efficient in the AHH-1 tk +/- cell line.

Apoptosis↗

PubMed1996

A mutation in the p53 tumor suppressor gene of AHH-1 tk+/- human lymphoblastoid cells.

Loss-of-function mutations in the p53 tumor suppressor gene result in an altered response to DNA-damaging agents. Included in the mutant p53 phenotype are the loss of the G1 checkpoint and delayed apoptotic cell death, characteristics we have consistently observed in the AHH-1 tk+/- cell line following exposure to DNA-damaging agents. In order to determine the functional status of p53 in the AHH-1 tk+/- cell line, molecular analysis (single-strand conformational polymorphism [SSCP] and sequence analysis) was performed on exons 5-9 of the p53 gene. In addition, the status of the p53 gene in the closely related lymphoblast line, MCL-5, which, in our hands, has a much higher spontaneous rate of apoptosis than AHH-1 tk+/-, was also determined by molecular analysis. Initial SSCP analysis of AHH-1 tk+/- revealed an abnormal migration pattern of exon 8 when compared to a wild-type control. Subsequent sequence analysis indicated that a base-pair substitution (CGG-->TGG) mutation had occurred at codon 282, a reported "hot spot' for 5-methylcytosine mutations in the human p53 gene. Neither SSCP nor sequence analysis of exons 5-9 of MCL-5 indicated any differences from wild-type DNA. These results suggest that the lack of a G1 arrest and the delayed entrance into apoptosis observed in chemically-exposed AHH-1 tk+/- cells are, at least partially, accounted for by a loss-of-function mutation in the p53 gene.

Apoptosis↗

PubMed1996

A cohort of supporting metabolic enzymes is coinduced with nitric oxide synthase in human tumor cell lines.

Although nitric oxide (NO) has cytotoxic activity against certain tumor cell lines, some human tumor cell lines can themselves produce NO by expressing the inducible isoform of NO synthase (iNOS). As rates of cellular NO synthesis play a major role in determining whether NO has cytotoxic or cytoprotective effects at anatomic sites of NO production, identification of cellular processes which regulate rates of NO synthesis by iNOS is important for understanding the role of NO in tumor cell biology. This study demonstrates that argininosuccinate synthetase and GTP-cyclohydrolase-I, which catalyze rate-limiting steps in the synthesis of iNOS substrate (arginine) and cofactor (tetrahydrobiopterin), respectively, are coinduced with iNOS expression in two human tumor cell lines. These results indicate that coinduction of these supporting metabolic pathways helps to maximize cellular NO synthesis by iNOS in human tumor cells, suggesting that these pathways might be useful targets for pharmacologic intervention in NO-producing human tumor cells.

Adenocarcinoma↗

PubMed1996

Neutrophil deactivation by influenza A viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies.

Bacterial superinfections are a major cause of morbidity and mortality during influenza A virus (IAV) epidemics. Depression of phagocyte functions resulting from attachment of the IAV hemagglutinin (HA) to cell surface sialo-glycoproteins is a likely contributory cause of these infections. We have proposed that the group of collagenous lectins (termed collectins) present in blood and pulmonary surfactant play a role in initial host defense against IAV. We used here several recombinant human surfactant protein D (RhSP-D) preparations to determine the mechanism through which opsonization of IAV with collectins protects neutrophils against the deactivating effects of IAV on cellular respiratory burst responses in vitro. RhSP-D was markedly more potent than antibodies that inhibited viral hemagglutination activity (anti-HA antibodies) at protecting neutrophils in this assay. Unlike the anti-HA antibodies, RhSP-D was protective at concentrations that minimally inhibited viral hemagglutination activity. Two related features of SP-D--the degree of multimerization and the ability to cause aggregation of IAV particles--were critical determinants of the ability of SP-D to protect neutrophils against deactivation. Similarly SP-D-induced viral aggregate formation resulted in enhanced IAV binding to neutrophils and potentiated the ability of the virus itself to trigger neutrophil respiratory burst responses. In contrast to the case of IAV-antibody complexes, SP-D-IAV complexes attached to and activated neutrophils through a neuraminidase-sensitive mechanism (ie, similar to unopsonized IAV). These results indicate that collectin-mediated viral aggregation per se may be an important host defense mechanism not only by virtue of reducing the number of infectious viral particles, but also by promoting phagocyte responsiveness.

Animals↗

PubMed1996

Oxyradical scavenging effects of dehydroascorbate.

Dehydroascorbate (DHA) has been shown to possess vitamin C like activities as well as to protect the lens against oxidative stress and cataract formation. The results presented here suggest that some of the beneficial effects of DHA can be attributed to its property of undergoing peroxidative decarboxylation and of O2-. scavenging. Incubation of 1-14C-DHA with peroxide at physiological pH has been found to liberate 14CO2 in quantitative yields, with recovery of 79-94%. The recovery increases with the increase in the amount of DHA used. Its O2-. scavenging activity was apparent by inhibition of O2-. dependent reduction of ferricytochrome c and nitroblue tetrazolium. The authenticity of the latter mechanism was proved by inhibition of the O2-. dependent reactions also by superoxide dismutase.

Decarboxylation↗

PubMed1996

Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

Blotting, Northern↗

PubMed1996

Cell cycle traverse in AHH-1 tk +/- human lymphoblastoid cells exposed to the chromosomal mutagen, m-amsa.

AHH-1 tk +/- cells were exposed to the chemotherapeutic agent, m-amsa, both in complete medium and in medium without serum, subcultured in complete medium, and the effect on the traverse of the cell cycle determined by flow cytometric analysis of bromodeoxyuridine (BrdUrd)-labeled DNA. After exposure to m-amsa (day 0), the percentage of S-phase cells increased significantly (P < 0.0017) with increasing concentration. Cells also accumulated in G2/M as evidenced by the significant (P < 0.0026), concentration-dependent increase in the percentage of cells detected within this phase. Serum deprivation during exposure resulted in significantly (P = 0.024) more cells in S-phase than in cultures exposed to m-amsa in complete medium. After three days in culture, a significant (P = 0.0001) accumulation of cells in G2/M was present; the percentage of cells in G2/M did not differ significantly (P = 0.148) in cultures exposed to m-amsa in complete medium or in serum-free medium. However, a significant (P < 0.001) loss of S-phase cells was found in cultures exposed without serum. At day 7, no significant concentration effects were detected (GO/G1, P = 0.6026; S-phase, P = 0.9773; G2/M, P = 0.8401). These results demonstrate that exposure to m-amsa perturbs the traverse of the cell cycle, initially by inhibiting the completion of S-phase and followed by an accumulation of cells in G2/M. In addition, exposure to m-amsa under conditions of serum deprivation results in an increased percentage of cells in the initial S-phase after exposure, the loss of S-phase cells from the culture after three days, and the appearance of subdiploid peak, consistent with cells undergoing apoptosis.

Amsacrine↗

PubMed1996

Novel actions of aspirin and sodium salicylate: discordant effects on nitric oxide synthesis and induction of nitric oxide synthase mRNA in a murine macrophage cell line.

Aspirin and sodium salicylate each inhibit to a similar extent the production of nitric oxide (NO) in the RAW 264.7 murine macrophage cell line following stimulation by either lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma). The similar potencies of aspirin and sodium salicylate indicate that acetylation of cellular macromolecules is not essential for the observed effects. The failure of added prostaglandin E2 to overcome the effects of aspirin or sodium salicylate indicates that these effects are not simply the result of inhibition of prostaglandin synthesis. The inhibition of NO production occurs irrespective of the effect of these agents on induction of nitric oxide synthase (iNOS) mRNA by LPS or IFN-gamma. Aspirin and sodium salicylate inhibit iNOS mRNA induction in LPS-stimulated cells but enhance iNOS mRNA induction in IFN-gamma-stimulated cells. In contrast, these agents consistently inhibit induction of argininosuccinate synthetase mRNA in both LPS- and IFN-gamma-stimulated cells. Concentrations of aspirin in the 3-10 mM range inhibit induced NO production and expression of iNOS protein without inhibiting induction of iNOS mRNA. Discordances between effects on NO synthesis and induction of iNOS mRNA indicate that aspirin and sodium salicylate have multiple sites of action in their effects on pathways that are involved in the production of NO by stimulated RAW 264.7 cells.

Animals↗

PubMed1996

Role of the citrulline-nitric oxide cycle in the functional response of adult human and rodent pancreatic islets to cytokines.

The present study aims to characterize the role of the citrulline-nitric oxide cycle in the response of adult human and rat pancreatic islets to cytokines. Citrulline (0.1-1.0 mM) or arginine (0.1-1.0 mM) led to a similar dose dependent nitric oxide (NO) production by rat islets exposed to interleukin 1 beta (IL-1 beta) or human islets exposed to IL-1 beta + tumour necrosis factor alpha (TNF-alpha) + interferon gamma (IFN-gamma). In the absence of citrulline or arginine cytokines failed to induce NO production. Cytokines induced argininosuccinate synthetase activity in both species. Studies of IL-1 beta exposed rat islets revealed both NO-dependent and NO-independent effects: (1) IL-1 beta inhibits glucose-induced insulin release even in the absence of NO synthesis, but this inhibition is more severe when the presence of citrulline or arginine enables NO production; (2) NO formation in the presence of arginine or citrulline is necessary for cytokine-induced inhibition of protein biosynthesis. In conclusion, the citrulline-NO cycle enables rodent and human islet of Langerhans to regenerate arginine from citrulline and maintain NO production, thus contributing to islet functional inhibition. Considering that arginine availability may be limiting for NO production in vivo, the citrulline-NO cycle may be important for the regulation of NO production during insulitis in early insulin-dependent diabetes mellitus.

Adult↗