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Biomedical subjects

P S Bora

Publications and source records attributed to P S Bora.

At least 19 recordsLinked to original sources
PubMed2001

Functional properties of raw and heat processed cashew nut (Anacardium occidentale, L.) kernel protein isolates.

The functional properties viz. solubility, water and oil absorption, emulsifying and foaming capacities of the protein isolates prepared from raw and heat processed cashew nut kernels were evaluated. Protein solubility vs. pH profile showed the isoelectric point at pH 5 for both isolates. The isolate prepared from raw cashew nuts showed superior solubility at and above isoelectric point pH. The water and oil absorption capacities of the proteins were slightly improved by heat treatment of cashew nut kernels. The emulsifying capacity of the isolates showed solubility dependent behavior and was better for raw cashew nut protein isolate at pH 5 and above. However, heat treated cashew nut protein isolate presented better foaming capacity at pH 7 and 8 but both isolates showed extremely low foam stability as compared to that of egg albumin.

Absorption↗
PubMed2000

Chronic low level complement activation within the eye is controlled by intraocular complement regulatory proteins.

PURPOSE: To explore the role of the complement system and complement regulatory proteins in an immune-privileged organ, the eye. METHODS: Eyes of normal Lewis rats were analyzed for the expression of complement regulatory proteins, membrane cofactor protein (MCP), decay-acceleration factor (DAF), membrane inhibitor of reactive lysis (MIRL, CD59), and cell surface regulator of complement (Crry), using immunohistochemistry, Western blot analysis, and reverse transcription-polymerase chain reaction (RT-PCR). Zymosan, a known activator of the alternative pathway of complement system was injected into the anterior chamber of the eye of Lewis rats. Animals were also injected intracamerally with 5 microl (25 microg) of neutralizing monoclonal antibody (mAb) against rat Crry (5I2) or CD59 (6D1) in an attempt to develop antibody induced anterior uveitis; control animals received 5 microl of sterile phosphate-buffered saline (PBS), OX-18 (25 microg), G-16-510E3 (25 microg), or MOPC-21 (25 microg). The role of complement system in antibody-induced uveitis was explored by intraperitoneal injection of 35 U cobra venom factor (CVF), 24 hours before antibody injection. Immunohistochemical staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Western blot analysis were used to detect the presence of membrane attack complex (MAC) and C3 activation products, respectively, in normal and antibody-injected rat eyes. RESULTS: Complement activation product MAC was present in the normal rat eye, and intraocular injection of zymosan induced severe anterior uveitis. The complement regulatory proteins, MCP, DAF, CD59, and Crry, were identified in the normal rat eye. Soluble forms of Crry and CD59 were also detected in normal rat aqueous humor. Severe anterior uveitis developed in Lewis rats injected with a neutralizing mAb against Crry, with increased formation of C3 split products. Systemic complement depletion by CVF prevented the induction of anterior uveitis by anti-Crry mAb. Intracameral injection of anti-rat CD59 (6D1), anti-rat MHC class I antigen (OX-18), anti-rat Ig (G-16-510E3), or MOPC-21 caused no inflammatory reaction. CONCLUSIONS: The results suggest that the complement system is continuously active at a low level in the normal eye and is tightly regulated by intraocular complement regulatory proteins.

Animals↗
PubMed2000

Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59.

PURPOSE: To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system. METHODS: Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH(50) and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity. RESULTS: Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH(50)). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an M(r) >/= 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (>/=19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M(r) >/= 3 kDa also had an inhibitory effect on the alternative pathway activity (AH(50)). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid. CONCLUSIONS: Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59.

Antigens, CD↗
PubMed1998

Fatty acid ethyl esters: potentially toxic products of myocardial ethanol metabolism.

The chronic consumption of alcohol has proven detrimental to heart tissue and can lead to alcohol-induced heart muscle disease, a condition which may result in arrhythmias, cardiomegaly, and congestive heart failure. A search for the molecular mechanism underlying observed alcohol-induced end-organ damage, such as that seen in heart, has lead to the discovery of a nonoxidative pathway for the metabolism of alcohol in several human tissues including heart, brain, pancreas, and liver. It has been revealed that nonesterified fatty acids are esterified with ethanol to produce fatty acid ethyl esters (FAEE), neutral molecules which can accumulate in mitochondria and impair cell function. The observation that FAEEs are synthesized at high rates in the heart, and other organs that lack oxidative ethanol metabolism, provides a plausible link between the observed tissue damage, the ingestion of alcohol, and the subsequent development of alcohol-induced heart muscle disease. The synthesis of FAEEs are catalyzed by FAEE synthase enzyme, four of which have been characterized and purified to homogeneity from the human myocardium. Further analysis of these FAEE synthase enzymes opens up a new possibility to characterize and map a gene for alcohol-induced end-organ damage, such as that observed in heart and other organs. FAEEs have been found to be important metabolites of alcohol and are most commonly accumulated in those organs which are damaged by alcohol abuse, i.e. heart. It may now be important to establish a genetic link between alcohol abuse and alcohol-induced heart muscle disease in order to understand the mechanism of alcohol-induced cardiomyopathy.

Alcohol Drinking↗
PubMed1998

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: a gene for alcohol-induced cardiomyopathy.

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.

Acyltransferases↗
PubMed1997

Human fatty acid ethyl ester synthase-III gene: genomic organization, nucleotide sequencing and chromosomal localization.

The complete gene for human fatty acid ethyl ester Synthase-III (FAEES-III) was isolated from a human genomic lambda phage library for functional and structural determination. The gene spans approximately 3.3 kb which includes 791 base pairs of the 5' and 124 base pairs of the 3' flanking regions. The gene is comprised of seven exons and is interrupted by six introns. Several transcription regulatory sequences were identified in the promoter region. Primer extension experiments demonstrated the existence of two possible transcription initiation sites at nucleotide -29 and 32 position, 5' to the start of the translation. In addition to a TATA box at position-29 relative to the transcription initiation site and two Spl GGGCGG recognition sequences at nucleotide positions -42 to -37 and -50 to -45, the promoter contains a sequence motif matching the transcription activating factor AP-1. We also found an A + T rich region between nucleotide -505 and -390 which contained twenty-two AAAAT tandem repeats. The gene for FAEES-III was localized to human chromosome 11 by hybridizing the genomic fragment Xh01 to Chinese hamster/human somatic cell hybrid panels. These data extend our knowledge of non-oxidative alcohol metabolism and permit linkage analyses between this pathway and alcohol-related phenotypes.

Acyltransferases↗
PubMed1997

Moderate alcohol feeding attenuates postinjury vascular cell proliferation in rabbit angioplasty model.

Our studies in the cholesterol-fed rabbit model indicate that moderate alcohol consumption reduces the risk of restenosis by preventing low-density lipoprotein (LDL) oxidation. Eighteen hypercholesterolemic rabbits underwent arterial injury by Fogerty balloon endothelial denudation of iliac arteries. Two weeks later, balloon angioplasty of atherogenic or atherosclerotic arterial segments was performed. Nine rabbits (control) received water ad lib, whereas nine rabbits (moderate alcohol treated) received an average of 2.5 ml alcohol per 500 ml water daily, from the day of feeding hypercholesterolemic diet until they were killed, 10 weeks later. There was a 26% increase in lumen size of the moderate alcohol-treated group compared with the control group. The percentage neointima formation (NI) values of the moderate alcohol-treated and control groups were 77 +/- 2.1 and 61 +/- 1.9, respectively (p < 0.001). The lumen/neointima (L/NI) ratio of the moderate alcohol-treated group was 0.71 +/- 0.07 compared with the control group, 0.33 +/- 0.04 (p < 0.001). The number of foam cells in the moderate alcohol-treated group was threefold less than the control group [i.e., 1.4 +/- 0.4 and 3.9 +/- 0.8, respectively (p = 0.005)]. The arterial lesion malondialdehyde (MDA) values of the control and the moderate alcohol-treated groups were 13.6 +/- 2.8 and 4.4 +/- 0.5 (p = 0.004), respectively. By radioimmunoassay, the moderate alcohol-treated group had less macrophage chemotactic protein-1 (MCP-1; 3,277 cpm/microg protein) and platelet-derived growth factor (PDGF; 2,261 cpm/microg protein) compared with the controls (MCP-1, 4,529 cpm/microg protein; PDGF, 3,583 cpm/microg protein). Thus we conclude that low concentrations of alcohol reduce neointimal formation, and the extent of lipid oxidation, the number of foam cells in the neointimal area and may decrease the expression of MCP-1 and PDGF by reducing LDL oxidation in an animal model of postangioplasty restenosis.

Angioplasty, Balloon↗
PubMed1996

Purification and characterization of human heart fatty acid ethyl ester synthase/carboxylesterase.

Fatty acid ethyl ester synthase metabolizes ethanol non-oxidatively in those extrahepatic organs most commonly damaged by alcohol abuse. This study was designed to purify human myocardial fatty acid ethyl ester synthase (FAEES)/carboxylesterase from human heart. The enzyme was purified to homogeneity after chromatography over DEAE-cellulose, Sephadex G-100 and hydroxylapatite. The homogenous enzyme, 62 kDa, has both synthase and carboxylesterase activities. The N-terminal amino acid sequence of the first 17 residues of the purified enzymes were 88% homologous to that of the carboxylesterase from rat liver and adipose tissue. Antibody was raised against pure synthase/carboxylesterase cross-reacted with human cytosolic and microsomal fractions. With a constant oleic acid concentration of 0.25 mM, a calculated apparent Km and Vmax for ethanol were 0.30 M and 3700 nmol/mg protein/h., respectively. With constant ethanol concentrations of 1.2 M, the activity increased with the concentration of oleic acid to 0.17 mM, plateau to 0.25 mM. Because synthase/carboxylesterase esterifies free fatty acids with ethanol to produce its esters with potentially toxic effects, it may now be feasible to establish a link between alcohol consumption and end-organ damage.

Acyltransferases↗
PubMed1996

Myocardial cell damage by fatty acid ethyl esters.

Fatty acid ethyl ester (FAEE), a myocardial metabolite of ethanol, causes mitochondrial dysfunction in vitro in rabbits. We investigated the effect of these esters on rat heart mitochondria in vitro and in vivo. In vitro studies were conducted to investigate the binding of ethyl oleate (FAEE) to mitochondria and their capacity to hydrolyze these FAEE. In vivo effects of ethyl esters were studied by the direct transfer of [3H]oleate into the myocardium. Mitochondria were prepared from the myocardium of injected rats, and the amount of [3H]oleate bound to them was determined. In another in vivo study, 50 microliters of 50 microM cold oleic acid ethyl ester was injected into the rat myocardium and the histopathological changes induced by oleic acid ethyl ester were examined by light microscopy. Our results show that fatty acid ethyl ester can bind to myocardial mitochondria in vitro as well as in vivo and the mitochondria can hydrolyze FAEE to fatty acid, which is a known uncoupler of oxidative phosphorylation. Of the total ethyl [3H] oleate injected, 8 microM [3H]oleate and 1 microM ethyl [3H]oleate was bound to the mitochondria. Significant myocardial cell damage was first observed on day 4 and markedly increased on day 30 after ethyl ester injection, with cells showing gross deformation and enlargement. However, no significant histopathological changes were observed in the myocardial tissue on day 2 after injection. Our results suggest that the FAEE may damage the myocardial cells as well as the mitochondria and may provide a metabolic link between ethanol abuse and myocardial dysfunction.

Animals↗
PubMed1996

Identification, quantitation, and purification of a 36 kDa circulating protein associated with active pars planitis.

PURPOSE: To establish a correlation between the presence of a 36 kDa protein in the blood of patients with pars planitis and to characterize and purify this protein. METHODS: Blood samples were obtained from patients with pars planitis and other types of uveitis and from various controls. Samples were treated with polyethelene glycol and protein A and were analyzed on 10% SDS-PAGE for the presence of a 36 kDa protein. Quantitative estimation of the level of this protein was determined by densitometric tracing of the stained gels. Polyclonal antibodies were raised by immunizing New Zealand White rabbits with a mixture of the gel fragment containing the 36 kDa protein (p-36) and complete Freund's adjuvant. These antibodies were used in the immunoaffinity purification of this protein. RESULTS: The levels of p-36 were sixfold to eightfold higher in 81% of the patients with active pars planitis than in controls (P < 0.05). Furthermore, the levels of this protein correlated with disease activity. A partial amino terminal sequence analysis revealed that p-36 may be a novel protein. It has been purified from the patient's blood using affinity chromatography. CONCLUSIONS: A 36 kDa protein (p-36) is found in elevated concentrations in the blood of many patients with active pars planitis. Its putative role in the etiopathogenesis of pars planitis is unknown.

Amino Acid Sequence↗
PubMed1996

Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein.

PURPOSE: Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein. METHODS: A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells. RESULTS: The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein. CONCLUSIONS: The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.

Amino Acid Sequence↗
PubMed1995

Genetic and radiation-reduced somatic cell hybrid sublocalization of the human GSTP1 gene.

A number of related enzymes like glutathione S-transferases (GSTs) and fatty acid ethyl ester synthases (FAEESs) have been implicated in detoxification and drug resistance. The anionic class of GSTs, pi, and closely related FAEES-III exhibit tissue-specific and developmentally regulated expression, and the former has been shown to be overexpressed or amplified in a variety of tumors. The GSTP1 gene has previously been cloned and cytogenetically localized to human 11q13 by in situ hybridization. Using a series of previously described radiation-reduced somatic cell hybrids, we have sublocalized GSTP1 to 11q13. We isolated a genomic clone containing the entire GSTP1 gene and sequenced it. Analysis of the 5'region revealed 23 (TAAAA) tandem repeats interrupted by a single TA and TAA insertion. This repeat number differs among individuals. Eleven alleles in a mostly Caucasian sample were observed. This repeat has a polymorphism information content of 0.74. Linkage analysis of the Venezuelan reference pedigree places GSTP1 5 cM distal to PYGM and 4 cM proximal to FGF3 thereby providing a genetic marker half-way between these two loci. The sublocalization and genetic characterization of GSTP1 facilitates linkage analysis of several disease genes mapped to this chromosome band as well as the correlation of genetic and physical markers in the region.

Animals↗
PubMed1994

Cardioprotective effects of alcohol: mediation by human vascular alcohol dehydrogenase.

Numerous studies have shown that moderate drinking protects against coronary disease, but no mechanism for this effect has been established. In the present study we show that the B1 isoenzyme of alcohol dehydrogenase (ADH) is expressed in human blood vessels. Polymerase chain reaction (PCR) employing total human aortic cDNA as a template detected a 0.6 kb band, the nucleotide sequence of which is an identical match to the low Km (50 microM) B1 ADH isoenzyme nucleotide sequence. Immunoblot of vascular homogenates shows a 40 KDa band, i.e., the size of the B1 ADH subunit, and immunohistochemical studies of vessel sections demonstrate high density staining with anti-human ADH (Class I) but not control sera. These studies identify within blood vessels the existence of a metabolic pathway sensitive to low substrate concentrations and capable of producing a reductive (NADH) environment that could antagonize lipoprotein oxidation and hence could account for a protective effect of ethanol on atherosclerosis.

Alcohol Dehydrogenase↗
PubMed1993

Molecular mechanism of ethanol metabolism by human brain to fatty acid ethyl esters.

Ethanol metabolism in the human brain has been documented to occur with the formation of fatty acid ethyl esters. These neutral lipids can disorder membranes and interrupt mitochondrial function. Their formation is under the control of three synthases, localized to grey matter and purified to homogeneity. cDNA cloning demonstrates two of these enzymes to be GSH S-transferases and has enabled initiation of genetic studies of alcohol-induced CNS injury.

Acyltransferases↗
PubMed1992

Purification and characterization of fatty acid ethyl ester synthase-II from human myocardium.

Fatty acid ethyl ester synthases metabolize ethanol nonoxidatively in those extrahepatic organs most commonly damaged by alcohol abuse. This study was designed to isolate and purify human myocardial synthase-II, one of the enzymes responsible for catalyzing the formation of fatty acid ethyl esters. DEAE-cellulose chromatography of human myocardial cytosol at pH 8.0 separated synthase-I, synthase-II, and synthase-III activities, eluting at conductivities of 5, 7, and 11 mS, respectively. From this elution profile, fatty acid ethyl ester synthase-II accounts for up to 50% of total synthesis in the human heart. This enzyme species was purified over 2200-fold to homogeneity after chromatography over hydroxylapatite, CM-cellulose, and hydroxylapatite. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this homogeneous species showed a single band at 65 kDa which corresponded to its molecular weight determined by gel filtration. This molecular weight and its lack of glutathione transferase activity indicate that this species is not related to synthase-I and -III. Homogeneous synthase-II has a Vmax for palmitate, stearate, oleate, and linoleate of 70, 80, 140, and 120 nmol/mg/h, respectively. The Km for palmitate, stearate, oleate, and linoleate is 0.19, 0.12, 0.10, and 0.18 mM, respectively. The substrate specificity with respect to alcohol chain length was also investigated in the presence of 0.65 mM [14C]oleic acid. The Vmax for methanol, ethanol, propanol, and butanol was 180, 100, 280, and 410 nmol/mg/h, respectively. The Km for methanol, ethanol, propanol, and butanol was 1.16, 1.04, 0.58, and 0.33 M, respectively. The N-terminal 17-amino acid sequence of human synthase-II does not correspond to any known N-terminal amino acid sequence, indicating that this may be a novel protein. However, it has over 70% homology to a sequence close to the C terminus of rabbit cytochrome P-450IIC1 and over 50% homology to a sequence of human hemopexin starting at residue 16. Synthase-II does not cross-react with human hemopexin antibody and rat cytochrome P-450C antibody. Thus, this study provides evidence that synthase-II is a novel protein, distinct from synthase-I and -III, and it also provides a foundation for subsequent cloning and genetic studies of fatty acid ethyl ester synthase-II in man.

Acyltransferases↗