Biomedical subjects

James Haorah

Publications and source records attributed to James Haorah.

11 recordsLinked to original sources

PubMed2006

Partial purification from hot dogs of N-nitroso compound precursors and their mutagenicity after nitrosation.

Hot dogs contain apparent N-nitroso compounds (ANC) and ANC precursors (ANCP). ANCP purification was followed by nitrosation, sulfamic acid treatment, and analysis for ANC. Aqueous hot dog extracts were adsorbed on silica gel, which was eluted with MeCN and MeOH. The MeOH eluate was adsorbed on cation exchange resin (H+ form) and eluted with NH4OH. Eluted ANCP traveled at moderate speeds in high-performance liquid chromatography (HPLC) on amino and Pb2+ columns. Gas chromatography-mass spectrometry (GC-MS) of trimethylsilyl (TMS) derivatives of crude water extract indicated the presence of glycerol, phosphate, lactic acid, and two monosaccharides. GC-MS of TMS derivatives of Pb2+ column HPLC eluates indicated that ANCP included 1-deoxy-N-1-glucosyl glycine. The nitrosated NH4OH eluate showed 4x background mutagenic activity for Salmonella typhimurium TA-100. Un-nitrosated fractions showed 2x background activity. Although tryptophan nitrosation gave 88% ANC yield, tryptophan is probably not a major ANCP in hot dogs. Hot dog patties prepared with or without sucrose or glucose showed similar ANC and ANCP levels. We discuss possible implications of these findings for the etiology of colon cancer.

Amino Acids↗

PubMed2006

Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE).

The blood-brain barrier (BBB) is compromised during progressive HIV-1 infection, but how this occurs is incompletely understood. We studied the integrity of tight junctions (TJs) of brain microvascular endothelial cells (BMVECs) in an in vitro BBB system and in human brain tissues with HIV-1 encephalitis (HIVE). A downregulation of TJ proteins, claudin-5 and occludin, paralleled monocyte migration into the brain during HIVE. Because small G proteins (such as Rho) can play a role in BMVEC TJ assembly, an artificial BBB system explored the relationship among TJs, Rho/Rho kinase (RhoK) activation, and transendothelial monocyte migration. Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. BMVEC transfection with a constitutively active mutant of RhoK led to dislocation of occludin from the membrane and loss of BMVEC cell contacts. When dominant-negative RhoK-transfected BMVECs were used in BBB constructs, monocyte migration was reduced by 84%. Thus, loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVECs could be an underlying cause of BBB impairment during HIVE.

Blood-Brain Barrier↗

PubMed2006

Alcohol abuse enhances neuroinflammation and impairs immune responses in an animal model of human immunodeficiency virus-1 encephalitis.

Neuroinflammatory disorders (including human immunodeficiency virus-1 encephalitis, HIVE) are associated with oxidative stress and inflammatory brain injury, and excessive alcohol use can exacerbate tissue damage. Using a murine model of HIVE, we investigated the effects of alcohol abuse on the clearance of virus-infected macrophages and neuroinflammation. Severe combined immunodeficient mice were reconstituted with human lymphocytes, and encephalitis was induced by intracranial injection of HIV-1-infected monocyte-derived macrophages (HIV-1(+) MDM). Animals were fed an ethanol-containing diet beginning 2 weeks before lymphocyte engraftment and for the entire duration of the experiment. Lymphocyte engraftment was not altered by ethanol exposure. Alcohol-mediated immunosuppression in ethanol-fed mice was manifested by a significant decrease in CD8(+)/interferon-gamma(+) T lymphocytes, a fivefold increase in viremia, and diminished expression of immunoproteasomes in the spleen. Although both groups showed similar amounts of CD8(+) T-lymphocyte infiltration in brain areas containing HIV-1(+) MDMs, ethanol-fed mice featured double the amounts of HIV-1(+) MDMs in the brain compared to controls. Ethanol-exposed mice demonstrated higher microglial reaction and enhanced oxidative stress. Alcohol exposure impaired immune responses (increased viremia, decreased immunoproteasome levels, and prevented efficient elimination of HIV-1(+) MDMs) and enhanced neuroinflammation in HIVE mice. Thus, alcohol abuse could be a co-factor in progression of HIV-1 infection of the brain.

Alcoholism↗

PubMed2005

Ethanol-induced activation of myosin light chain kinase leads to dysfunction of tight junctions and blood-brain barrier compromise.

BACKGROUND: Brain endothelial cells form the blood-brain barrier (BBB) that regulates solute and macromolecule flux in and out of the brain, leukocyte migration, and maintains the homeostasis of the central nervous system. BBB dysfunction is associated with disruption of tight junctions (TJ) in the brain endothelium. We propose that alcohol abuse may impair BBB permeability through TJ modification. METHODS: Primary cultured bovine brain microvascular endothelial cells (BBMEC) were treated with 50 mM ethanol (EtOH), and monolayer tightness was assessed by measurement of transendothelial electrical resistance (TEER). Changes in TEER were correlated with alterations in TJ protein distribution [occludin, zonula occludens-1 (ZO-1), claudin-5] using immunofluorescence (IF). Expression of myosin light chain (MLC) kinase (MLCK), ZO-1, claudin-5, and phosphorylated MLC, occludin and claudin-5 were determined by immunoprecipitation and Western blot. EtOH-induced changes in monocyte migration across in vitro BBB constructs were also examined. RESULTS: EtOH induced a decrease in TEER of BBMEC monolayers that was reversed by EtOH withdrawal. Treatment of BBMEC with EtOH or its metabolite, acetaldehyde, prior to monocyte application resulted in a 2-fold increase in monocyte migration across the BBB. IF demonstrated decrease in claudin-5 staining, occludin translocation from cell borders to cytoplasm and gap formation in EtOH-treated BBMEC monolayer. These changes paralleled significant increase in phosphorylation of MLC, occludin and claudin-5. EtOH-treated BBMEC showed reduction of total occludin and claudin-5 without changes in ZO-1 or MLC. TEER decrease, changes in occludin/claudin staining, increase in MLC, occludin and claudin-5 phosphorylation and enhanced monocyte migration across the BBB were all reversed by inhibition of MLCK. Inhibition of EtOH metabolism in BBMEC also reversed these events. CONCLUSION: These results suggest that EtOH activates MLCK leading to phosphorylation of MLC, occludin and claudin-5. Cytoskeletal alterations (MLC) and TJ changes (occludin and claudin-5 phosphorylation) result in BBB impairment (decrease in TEER). TJ compromise is associated with increased monocyte migration across the BBB.

Animals↗

PubMed2004

Peroxynitrite alters the catalytic activity of rodent liver proteasome in vitro and in vivo.

The proteasome is an important multicatalytic enzyme complex that degrades misfolded and oxidized proteins, signal transduction factors, and antigenic peptides for presentation. We investigated the in vitro effects of peroxynitrite (PN) on the peptidase activity of both crude 20S and 26S and purified 20S proteasome preparations from rat liver as well as proteasome activity in Hep G2 cells and in mouse liver. Crude and purified proteasome preparations were exposed to PN or to the PN donor, 3-morpholinosydnonimine hydrochloride (SIN-1), and then assayed for chymotrypsin-like activity. For in vivo experiments, mice were treated with molsidomine, which is metabolized to SIN-1 in liver. PN and SIN-1 dose-dependently modulated the chymotrypsin-like activity of the 20S proteasome: lower concentrations enhanced proteasome activity, and higher concentrations caused its decline. The NO donor S-nitroso-N-acetylpenicillamine (SNAP), at all concentrations, suppressed 20S proteasome activity. We observed similar results when liver soluble fractions (S-100) were treated with PN, SIN-1, or SNAP, except that enzyme activity in S-100 fractions was less sensitive than the purified enzymes to these agents. Treatment of Hep G2 cells with 0.01 or 0.1 mmol/L SIN-1 stimulated in situ proteasome activity in these cells, while 1 mmol/L SIN-1 suppressed it. SNAP treatment did not affect proteasome activity in Hep G2 cells. Mice treated with molsidomine had enhanced liver proteasome activity 6 hours after treatment, but after 24 hours enzyme activity declined below control levels. In conclusion, PN dose-dependently modulated proteasome activity, regulating protein degradation by the proteasome in liver cells.

Animals↗

PubMed2004

Alcohol and HIV decrease proteasome and immunoproteasome function in macrophages: implications for impaired immune function during disease.

Proteasomes (proteinase complexes, PR) and immunoproteasomes (IPR) degrade damaged proteins and affect protein processing required for antigen presentation by mononuclear phagocytes. These critical immune processes are attenuated during progressive HIV-1 infection and are affected by alcohol abuse. To investigate the mechanisms underlying these functional changes, we measured PR and CYP2E1 activities [an ethanol (EtOH) metabolizing enzyme] and reactive oxygen species (ROS) in human monocyte-derived macrophages (MDM) following HIV-1 infection and EtOH treatment. We observed progressive declines of PR activity and PR/IPR contents in HIV-1-infected MDM. PR activity and IPR expression increased after IFN-gamma stimulation but reduced after HIV-1 infection. EtOH inhibited both IFN-gamma-induced PR and IPR. Paradoxically, EtOH attenuated PR catalytic activity in infected MDM and suppressed viral replication. Elevated ROS followed EtOH exposure and paralleled decreased PR activity. The latter was restored by anti-oxidant. The data support the notion that HIV-1 infection and EtOH may work in concert to affect immune function including antigen presentation and thereby affect disease progression.

Alcoholism↗

PubMed2004

Nitrosation of glycine ethyl ester and ethyl diazoacetate to give the alkylating agent and mutagen ethyl chloro(hydroximino)acetate.

Whereas nitrosation of secondary amines produces nitrosamines, amino acids with primary amino groups and glycine ethyl ester were reported to react with nitrite to give unidentified agents that alkylated 4-(p-nitrobenzyl)pyridine to produce purple dyes and be direct mutagens in the Ames test. We report here that treatment of glycine ethyl ester at 37 degrees C with excess nitrite acidified with HCl, followed by ether extraction, gave 30-40% yields of a product identified as ethyl chloro(hydroximino)acetate [ClC(=NOH)COOEt, ECHA] and a 9% yield of ethyl chloroacetate. The ECHA was identical to that synthesized by a known method from ethyl acetoacetate, strongly alkylated nitrobenzylpyridine, and may have arisen by N-nitrosation of glycine ethyl ester to give ethyl diazoacetate, which was C-nitrosated and reacted with chloride to give ECHA. Nitrosation of ethyl diazoacetate also yielded ECHA. Ethyl nitroacetate was not an intermediate as its nitrosation did not produce ECHA. ECHA reacted with aniline to give ethyl (hydroxamino)(phenylimino)acetate [PhN=C(NHOH)CO2Et]. This product was different from ethyl [(phenylamino)carbonyl]carbamate [PhNHC(=O)NHCO2Et], which was synthesized by reacting ethyl isocyanatoformate (OCN.CO2Et) with aniline. ECHA reacted with guanosine to give a derivative, which may have been a guanine-C(=NOH)CO2Et derivative. ECHA showed moderate toxicity and weak but significant mutagenicity without activation in Salmonella typhimurium TA-100 (mean, 1.31 x control value for 12-18 microg/plats) and for V79 mammalian cells (1.5-1.7 x control value for 60-100 microM). In conclusion, gastric nitrosation of glycine derivatives such as peptides with a N-terminal glycine might produce ECHA analogues that alkylate bases of gastric mucosal DNA and thereby initiate gastric cancer.

Acetates↗

PubMed2003

Ethanol consumption decreases the synthesis of the mannose 6-phosphate/insulin-like growth factor II receptor but does not decrease its messenger RNA.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) is a protein that facilitates the transport of acid hydrolases into the lysosome. We have shown that chronic ethanol consumption lowers the M6P/IGF-IIR content in rat hepatocytes. Here, we determined the steady-state level of mRNA encoding M6P/IGF-IIR, as well as the rate of receptor synthesis, to ascertain whether the ethanol-elicited reduction in receptor protein content is related to changes in either or both of these parameters. Rats were pair-fed the normal carbohydrate (NC) or low carbohydrate high-fat (LC) liquid diets containing either ethanol or isocaloric maltose-dextrin for 7-8 weeks. RNA was isolated from hepatocytes and from whole livers of these animals and subjected to reverse transcription-polymerase chain reaction (RT-PCR) to determine the mRNA levels encoding M6P/IGF-IIR. Hepatocytes isolated from these animals were also radiolabeled with Pro-mix L-[35S] in vitro cell labeling mix to measure incorporation into total cellular protein and the immunoprecipitated M6P/IGF-IIR protein. The steady-state levels of M6P/IGF-IIR mRNA in both hepatocytes and whole livers from ethanol-fed rats were the same as those from their respective controls regardless of whether they were fed the NC or the LC diets. Hepatocytes from ethanol-fed rats showed a 36% lower rate of total protein synthesis and an even greater reduction (70%) in receptor synthesis. When the relative rate of receptor synthesis was calculated, hepatocytes from ethanol-fed rats had a 53% lower relative rate of receptor synthesis compared with controls. Autoradiographic analysis of the immunoprecipitated receptor protein from ethanol-fed rats also indicated a 79% decline in the total M6P/IGF-IIR protein synthetic rate compared with pair-fed controls. We conclude that the ethanol-elicited reduction of M6P/IGF-IIR content was, in part, related to a concomitant reduction of receptor protein synthesis but not to a decline in its mRNA level. Thus, the ethanol-elicited decline in receptor protein synthesis may be due to defective M6P/IGF-IIR mRNA translation.

Alcohol Drinking↗

PubMed2003

N-nitroso compounds in the gastrointestinal tract of rats and in the feces of mice with induced colitis or fed hot dogs or beef.

Because colonic N-nitroso compounds (NOC) may be a cause of colon cancer, we determined total NOC levels by Walters' method in the gastrointestinal tract and feces of rodents: (i) feces of C57BL mice fed chow and semi-purified diets contained 3.2 +/- 0.4 and 0.46 +/- 0.06 NOC/g, respectively (P < 0.01, mean +/- SD). (ii) NOC levels for gastrointestinal contents of three groups of Sprague-Dawley rats fed chow diet were 0.9 +/- 0.05 (diet), 0.2 +/- 0 (stomach), 0.3-0.4 (small intestine), 0.7-1.6 (cecum and colon) and 2.6 +/- 0.6 (feces) nmol/g. NOC precursor (NOCP) levels (measured as NOC after mild nitrosation) for two rat groups fed chow diet showed a 16-fold increase from stomach to proximal small intestine (mean, 6.2 micromol/g), and a 1.7-fold increase from distal colon to feces (mean, 11.6 micromol/g). (iii) Eight Min and five C57BL/6J mice received 4% dextran sulfate sodium in drinking water on days 1-4 to induce acute colitis. This increased fecal NOC levels 1.9-fold on day 5 in both strains (P < or = 0.04), probably due to NO synthase-derived nitrosating agents in the colon. (iv) Following studies on humans fed beef [Hughes et al. (2001) Carcinogenesis, 22, 199], Swiss mice received semi-purified diets mixed with 18% of beef plus pork hot dogs or sautéed beef for 7 days. On day 7, individual 24-h fecal NOC outputs were determined. In three hot dog and two beef groups with 5 mice/group, mean fecal NOC output/day was 3.7-5.0 (hot dog) and 2.0-2.9 (beef) times that for control groups fed semi-purified diet alone (P < 0.002 for each of combined groups). These groups showed little change in fecal NOCP output. (v) Initial purification of rat fecal NOCP by adsorption-desorption and HPLC is described. Results should help evaluate the view that colonic NOC causes colon cancer associated with colitis and ingestion of red and nitrite-preserved meat.

Animals↗

PubMed2002

Chronic ethanol administration decreases the ligand binding properties and the cellular content of the mannose 6-phosphate/insulin-like growth factor II receptor in rat hepatocytes.

We have shown previously that chronic ethanol administration impairs the maturation of lysosomal enzymes in rat hepatocytes. The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) is a protein that facilitates the transport of lysosomal enzymes into the lysosome. Therefore, we examined whether ethanol consumption altered the ligand binding properties and the cellular content of M6P/IGF-IIR. Rats were pair-fed liquid diets containing either ethanol (36% of calories) or isocaloric maltose-dextrin for either 1 week or 5-7 weeks. Hepatocytes prepared from these animals were examined for receptor-ligand binding and receptor content. One week of ethanol feeding had no significant effect on ligand [radioiodinated pentamannose phosphate conjugated to bovine serum albumin ((125)I-PMP-BSA)] binding to hepatocytes, but cells from rats fed ethanol for 5-7 weeks bound less (125)I-PMP-BSA than pair-fed controls. Scatchard plot analysis revealed that the number of (125)I-PMP-BSA binding sites in hepatocytes from ethanol-fed rats was 49% lower than that of controls. (125)I-PMP-BSA binding by perivenular (PV) and periportal (PP) hepatocytes from ethanol-fed rats was, respectively, 40 and 48% lower than their controls, but there was no significant difference between these two types of hepatocytes. Ligand blot analysis using (125)I-insulin-like growth factor II ((125)I-IGF-II) also showed that the receptor in lysates of hepatocytes from ethanol-fed rats bound 26-27% less ligand than controls. Similarly, immunoblot analysis of cell lysates from ethanol-fed rats revealed 62% lower levels of immunoreactive M6P/IGF-IIR than controls. Feeding rats a low carbohydrate-ethanol diet did not exacerbate the reduction in M6P/IGF-IIR-ligand binding nor did it reduce the levels of immunoreactive receptor. Our findings indicate that chronic ethanol consumption lowers M6P/IGF-IIR activity and content in hepatocytes. This reduction may account, in part, for the impaired processing and delivery of acid hydrolases to lysosomes previously observed in ethanol-fed rats.

Animals↗

PubMed2002

Total N-nitroso compounds and their precursors in hot dogs and in the gastrointestinal tract and feces of rats and mice: possible etiologic agents for colon cancer.

We review evidence that red and processed meat are causes of colon cancer and that processed meat is a risk factor for childhood cancer and type 2 diabetes. Associations could be due to N-nitroso compounds (NOCs) derived from nitrosation of NOC precursors (NOCPs). We review our survey of total NOC and NOCP content of foods. Only rapidly nitrosated amines, including a glycosyl amino acid, were efficiently determined as NOCPs. NOCPs in hot dogs and rat feces were partly purified by adsorption-desorption and HPLC. After nitrosation, purified hot dog fractions were directly mutagenic in Ames test. The main NOCPs in these materials may be N-glycosyl amino acids and peptides. NOC levels in rat gastrointestinal tract rose steadily from stomach to feces. NOCP levels showed similar trend but with sharp increases from stomach to duodenum. One day after Min and C57BL/6J mice were fed 4% dextran sulfate sodium to induce acute colitis, fecal NOC levels increased 1.9-fold compared with untreated mice (P < 0.05). For 7 d Swiss mice received semipurified diet, 180 g beef-pork hot dogs mixed with 820 g diet or 180 g sautéed beef mixed with 820 g diet. Fecal NOC outputs on day 7 were 3.7-5.0 (hot dog) and 2.0-2.9 (beef) times those for control groups (P < 0.002 for combined groups), perhaps reflecting higher dietary NOC intakes. Feeding a similar hot dog mixture to mice did not affect normal 7-methyldeoxyguanosine level in colonic mucosal DNA. Overall, results support the hypothesis that colonic NOCs are a cause of colon cancer.

Animals↗