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Biomedical subjects

R Subramaniam

Publications and source records attributed to R Subramaniam.

At least 19 recordsLinked to original sources
PubMed1998

Structural and functional changes in proteins induced by free radical-mediated oxidative stress and protective action of the antioxidants N-tert-butyl-alpha-phenylnitrone and vitamin E.

The free radical theory of aging proposes that reactive oxygen species (ROS) cause oxidative damage over the lifetime of the subject. It is the cumulative and potentially increasing amount of accumulated damage that accounts for the dysfunctions and pathologies seen in normal aging. We have previously demonstrated that both normal rodent brain aging and normal human brain aging are associated with an increase in oxidative modification of proteins and in changes in plasma membrane lipids. Several lines of investigation indicate that one of the likely sources of ROS is the mitochondria. There is an increase in oxidative damage to the mitochondrial genome in aging and a decreased expression of mitochondrial mRNA in aging. We have used a multidisciplinary approach to the characterization of the changes that occur in aging and in the modeling of brain aging, both in vitro and in vivo. Exposure of rodents to acute normobaric hyperoxia for up to 24 h results in oxidative modifications in cytosolic proteins and loss of activity for the oxidation-sensitive enzymes glutamine synthetase and creatine kinase. Cytoskeletal protein spin labeling also reveals synaptosomal membrane protein oxidation following hyperoxia. These changes are similar to the changes seen in senescent brains, compared to young adult controls. The antioxidant spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) was effective in preventing all of these changes. In a related study, we characterized the changes in brain protein spin labeling and cytosolic enzyme activity in a series of phenotypically selected senescence-accelerated mice (SAMP), compared to a resistant line (SAMR1) that was derived from the same original parents. In general, the SAM mice demonstrated greater oxidative changes in brain proteins. In a sequel study, a group of mice from the SAMP8-sensitive line were compared to the SAMR1-resistant mice following 14 days of daily PBN treatment at a dose of 30 mg/kg. PBN treatment resulted in an improvement in the cytoskeletal protein labeling toward that of the normal control line (SAMR1). The results of these and related studies indicate that the changes in brain function seen in several different studies may be related to the progressive oxidation of critical brain proteins and lipids. These components may be critical targets for the beneficial effects of gerontotherapeutics both in normal aging and in disease of aging.

Adult↗
PubMed1998

Effects of mitochondrial respiratory stimulation on membrane lipids and proteins: an electron paramagnetic resonance investigation.

Previous studies have implicated mitochondria-derived reactive oxygen species (ROS) in both the aging process and age-related diseases such as Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease etc. The current study, utilizing electron paramagnetic resonance (EPR) spectrometry, was designed to determine if mitochondrial respiratory stimulation, under state 4 conditions, caused extensive oxidative modifications to membrane cytoskeletal proteins and lipids in the brain. A mixed population of cortical synaptosomes and mitochondria, prepared by centrifugation techniques using rat brain cortex from adult (4-6 months) female Brown Norway rat brains, were labeled with the lipid-specific spin probe, 5-nitroxyl stearate (5-NS). Stimulation of the mitochondrial electron transport chain was accomplished using 20 mM succinate at 25 degrees C for 3 h. Mitochondrially derived free radicals, when reacted with the paramagnetic center of the spin probe, result in a loss of paramagnetism resulting in loss of intensity. A significant lowering (23%, P<0.0001) in the signal amplitude (B0) of 5-NS, indicative of generation of oxyradicals, was found. The order parameter, an inverse EPR-measure of membrane fluidity of the 5-NS spin labeled mitochondrial and synaptosomal membranes, also decreased following mitochondrial respiratory stimulation (P<0.005). Changes in the physical state of cytoskeletal and transmembrane proteins due to succinate oxidation were measured using MAL-6 (2,2,6, 6,-tetramethyl-4 maleimidopiperdin-1-oxyl), a thiol-specific nitroxide spin label. The ratio of the amplitudes of the weakly to strongly immobilized spin label reaction sites (W/S ratio) in the low-field region of the spectrum was used to determine any alteration in protein conformation. Previous studies in our laboratory have established that increased protein oxidation is associated with a decreased W/S ratio. In the current study, our results indicated significant lowering of the W/S ratio in cortex (30%, P<0.0001) upon stimulation of the mitochondria with 20 mM succinate. Thus, we conclude that respiratory stimulation of mitochondria, due to a hypermetabolic stress with succinate, caused significant oxidative modifications of cortical membrane lipids and proteins.

Animals↗
PubMed1998

The free radical antioxidant vitamin E protects cortical synaptosomal membranes from amyloid beta-peptide(25-35) toxicity but not from hydroxynonenal toxicity: relevance to the free radical hypothesis of Alzheimer's disease.

Amyloid beta-peptide (Abeta) is a key factor in the neurotoxicity of Alzheimer's disease (AD). Recent research has shown that Abeta-mediated neurotoxicity involves free radicals and that Abeta peptides can initiate multiple membrane alterations, including protein oxidation and lipid peroxidation, eventually leading to neuronal cell death. Research also has emphasized the role of 4-hydroxynonenal (HNE), a downstream product of lipid peroxidation, in being able to mimic some of the effects of Abeta peptides. In the current investigation, electron paramagnetic resonance (EPR) studies of spin labeled cortical synaptosomal membrane proteins has been employed to study conformational changes in proteins, spectrophotometric methods have been used to measure protein carbonyl content, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for mitochondrial function has been used to study the effect of vitamin E on samples that were treated with Abeta or HNE. The free radical dependence of beta-amyloid-associated toxicity was confirmed by the ability of the free radical scavenger vitamin E to prevent the toxic effects of Abeta. In contrast, HNE was still toxic in the presence of vitamin E. These results support our Abeta-associated free radical model for neurotoxicity in AD brain and are discussed with reference to potential therapeutic strategies for AD.

Aldehydes↗
PubMed1998

Delleman syndrome: anesthetic implications.

An infant with oculocerebrocutaneous (Delleman) syndrome (1), only 26 cases of which have been reported (2), presented with focal alopecia of scalp, periorbital skin appendages, hypertrophy of the skin (Fig. 1A), left-sided orbital cyst, lid coloboma, cleft palate (Fig. 1B), neonatal seizures, cerebral hemiatrophy, multiple intracranial cystic spaces, and enlarged lateral ventricles. The anomalies often require multiple anesthetics for examination of the eye, drainage of the orbital cyst, repair of lid coloboma, enucleation of the eye, excision of skin tags, and repair of cleft palate. Although this infant's perioperative course was uneventful, he had significant preoperative problems, such as neonatal seizures and an episode of aspiration pneumonia. Because the Delleman syndrome is rare, this case is presented to illustrate possible anesthetic implications of the disease.

Abnormalities, Multiple↗
PubMed1997

Oxidatively induced structural alteration of glutamine synthetase assessed by analysis of spin label incorporation kinetics: relevance to Alzheimer's disease.

The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken. Ovine GS was oxidized by exposure to iron/hydrogen peroxide and subsequently labeled with the thiol-specific nitroxide probe MTS [(1-oxyl-2,2,5,5-tetramethyl-pyrroline-3-methyl)methanethiosulfonate]. The reaction of MTS with cysteine residues within GS was monitored in real time by electron paramagnetic resonance spectrometry. Structural perturbation of GS, manifested as decreased thiol accessibility, was inferred from an apparent decrease in the rate constant for the second-order reaction of MTS with protein thiols. A subsequent spin-labeling study was undertaken to compare the structural integrity of GS purified and isolated from Alzheimer's disease-afflicted brain (AD-GS) with that of GS isolated from nondemented, age-matched control brain (C-GS). The rate constant for reaction of MTS with AD-GS was markedly decreased relative to that for the reaction of spin label with C-GS. The kinetic data were partially corroborated by spectroscopic data obtained from circular dichroism analysis of control and peroxide-treated ovine GS. In an adjunct experiment, the interaction of GS with a synthetic analogue of the Alzheimer's-associated beta-amyloid peptide, known to induce free radical oxidative stress, indicated strong interaction of the enzyme with the peptide as reflected by a decrease in the rate constant for MTS binding to reactive protein thiols.

Alzheimer Disease↗
PubMed1997

The lipid peroxidation product, 4-hydroxy-2-trans-nonenal, alters the conformation of cortical synaptosomal membrane proteins.

Alzheimer's disease (AD) is widely held to be a disorder associated with oxidative stress due, in part, to the membrane action of amyloid beta-peptide (A beta). A beta-associated free radicals cause lipid peroxidation, a major product of which is 4-hydroxy-2-trans-nonenal (HNE). We determined whether HNE would alter the conformation of synaptosomal membrane proteins, which might be related to the known neurotoxicity of A beta and HNE. Electron paramagnetic resonance spectroscopy, using a protein-specific spin label, MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl), was used to probe conformational changes in gerbil cortical synaptosomal membrane proteins, and a lipid-specific stearic acid label, 5-nitroxide stearate, was used to probe for HNE-induced alterations in the fluidity of the bilayer domain of these membranes. Synaptosomal membranes, incubated with low concentrations of HNE, exhibited changes in protein conformation and bilayer order and motion (fluidity). The changes in protein conformation were found to be concentration- and time-dependent. Significant protein conformational changes were observed at physiologically relevant concentrations of 1-10 microM HNE, reminiscent of similar changes in synaptosomal membrane proteins from senile plaque- and A beta-rich AD hippocampal and inferior parietal brain regions. HNE-induced modifications in the physical state of gerbil synaptosomal membrane proteins were prevented completely by using excess glutathione ethyl ester, known to protect neurons from HNE-caused neurotoxicity. Membrane fluidity was found to increase at higher concentrations of HNE (50 microM). The results obtained are discussed with relevance to the hypothesis of A beta-induced free radical-mediated lipid peroxidation, leading to subsequent HNE-induced alterations in the structure and function of key membrane proteins with consequent neurotoxicity in AD brain.

Aldehydes↗
PubMed1997

A functional homolog of mammalian protein kinase C participates in the elicitor-induced defense response in potato.

The elicitor-induced activation of the potato pathogenesis-related gene PR-10a is positively controlled by a protein kinase(s) that affects the binding of the nuclear factors PBF-1 (for PR-10a binding factor-1) and PBR-2 to an elicitor response element (ERE). In this study, we have identified a kinase that has properties similar to the conventional isoenzymes of the mammalian protein kinase C (PKC) family. the treatment of potato tuber discs with specific inhibitors of PKC abolished the elicitor-induced binding of the nuclear factor PBF-2 to the ERE. This correlated with a reduction in the accumulation of the PR-10a protein. In contrast, treatment of the tuber discs with 12-O-tetradecanoylphorbol 13-acetate (TPA), an activator of PKC, led to an increase in binding of PBF-2 to the ERE and the corresponding increase in the level of the PR-10a protein, mimicking the effect seen with the elicitor arachidonic acid. Biochemical characterization of proteins extracted from the particulate fraction of potato tubers demonstrated that a kinase belonging to the conventional isoforms of PKC is present. This was confirmed by immunoprecipitation with antibodies specific to the conventional isoforms of human PKC and in-gel kinase assays. The ability of the immunoprecipitates to phosphorylate the alpha-peptide (a PKC specific substrate) in the presence of the coactivators calcium, phosphatidylserine, and TPA strongly suggested that the immunoprecipitated kinase is similar to the kinase characterized biochemically. Finally, the similar effects of the various modulators of PKC activity on the elicitor-induced resistance against a compatible race of Phytophthora infestans implicate this kinase in the overall defense response in potato.

Animals↗
PubMed1995

Brain regional correspondence between Alzheimer's disease histopathology and biomarkers of protein oxidation.

Four biomarkers of neuronal protein oxidation [W/S ratio of MAL-6 spin-labeled synaptosomes, phenylhydrazine-reactive protein carbonyl content, glutamine synthetase (GS) activity, creatine kinase (CK) activity] in three brain regions [cerebellum, inferior parietal lobule (IPL), and hippocampus (HIP)] of Alzheimer's disease (AD)-demented and age-matched control subjects were assessed. These endpoints indicate that AD brain protein may be more oxidized than that of control subjects. The W/S ratios of AD hippocampal and inferior parietal synaptosomes are 30 and 46% lower, respectively, than corresponding values of tissue isolated from control brain; however, the difference between the W/S ratios of AD and control cerebellar synaptosomes is not significant. Protein carbonyl content is increased 42 and 37% in the Alzheimer's HIP and IPL regions, respectively, relative to AD cerebellum, whereas carbonyl content in control HIP and IPL is similar to that of control cerebellum. GS activity decreases an average of 27% in the AD brain; CK activity declines by 80%. The brain regional variation of these oxidation-sensitive biomarkers corresponds to established histopathological features of AD (senile plaque and neurofibrillary tangle densities) and is paralleled by an increase in immunoreactive microglia. These data indicate that senile plaque-dense regions of the AD brain may represent environments of elevated oxidative stress.

Aged↗
PubMed1994

Tropical enterocolitis in children.

Besides classical necrotizing enterocolitis (in neonates), which is seen in India as elsewhere in the world, we observe sporadic cases of tropical enterocolitis, i.e. segmental jejunitis, ileitis or colitis and rarely duodenitis. This is a distinct clinico-pathological entity presenting as "acute abdomen", with pain, bilious vomiting, constipation or bloody diarrhoea. The clinical course is not as fulminating as neonatal necrotizing enterocolitis. Most cases are salvaged by conservative treatment especially after the confidence brought by laparoscopic vision of the abdomen, thus excluding perforation or gangrene of the bowel involved. Without laparoscopy, most of the cases end up in laparotomy. The pathology appears to be a kind of local hyperimmune reaction in the segment of bowel involved, ranging from punctate haemorrhages in the seromuscular layer of the bowel to a generalized red fiery look or perforation due to mucosal ulceration. Whatever the causative agent, the pathogenesis is of local vasculitis leading to ischemia and various patterns of disease.

Child, Preschool↗
PubMed1993

Combinatorial interactions between positive and negative cis-acting elements control spatial patterns of 4CL-1 expression in transgenic tobacco.

The phenylpropanoid enzyme 4-coumarate:coenzyme A ligase (4CL) plays a key role in linking general phenylpropanoid metabolism to end-product specific biosynthetic pathways. During vascular system and floral organ differentiation, the parsley 4CL-1 gene is expressed in a restricted set of tissues and cell types where 4CL activity is required to supply precursors for the synthesis of diverse phenylpropanoid-derived products such as lignin and flavonoids. In order to localize cis-acting elements which specify complex patterns of 4CL-1 expression, we analyzed the expression of internally deleted promoter fragment-GUS fusions in tobacco plants and parsley protoplasts. Elements located between -244 and -78 were required for most aspects of developmentally regulated expression. Within this region, three separate promotor domains containing partially redundant cis-elements directed vascular-specific expression when combined with a TATA-proximal domain. A negative cis-acting element which represses phloem expression was revealed in one of the domains and appears to be responsible for restricting vascular expression to the xylem. Distinct but overlapping promoter domain combinations were required for expression in floral organs, suggesting that different combinations of cis-acting elements may direct expression in different organs. Gel retardation assays were used to demonstrate the formation of DNA-protein complexes between factors present in nuclear extracts of parsley tissue culture cells and various tobacco organs and a 4CL-1 promoter fragment. Competition experiments showed that complex formation required the presence of a 42 bp promoter domain shown to be critical for 4CL-1 expression in vascular and floral tissues. The results are discussed in light of the coordinate expression of 4CL and other phenylpropanoid genes.

Base Sequence↗
PubMed1993

Structure, inheritance, and expression of hybrid poplar (Populus trichocarpa x Populus deltoides) phenylalanine ammonia-lyase genes.

A heterologous probe encoding phenylalanine ammonia-lyase (PAL) was used to identify PAL clones in cDNA libraries made with RNA from young leaf tissue of two Populus deltoides x P. trichocarpa F1 hybrid clones. Sequence analysis of a 2.4-kb cDNA confirmed its identity as a full-length PAl clone. The predicted amino acid sequence is conserved in comparison with that of PAL genes from several other plants. Southern blot analysis of popular genomic DNA from parental and hybrid individuals, restriction site polymorphism in PAL cDNA clones, and sequence heterogeneity in the 3' ends of several cDNA clones suggested that PAL is encoded by at least two genes that can be distinguished by HindIII restriction site polymorphisms. Clones containing each type of PAL gene were isolated from a poplar genomic library. Analysis of the segregation of PAL-specific HindIII restriction fragment-length polymorphisms demonstrated the existence of two independently segregating PAL loci, one of which was mapped to a linkage group of the poplar genetic map. Developmentally regulated PAL expression in poplar was analyzed using RNA blots. Highest expression was observed in young stems, apical buds, and young leaves. Expression was lower in older stems and undetectable in mature leaves. Cellular localization of PAL expression by in situ hybridization showed very high levels of expression in subepidermal cells of leaves early during leaf development. In stems and petioles, expression was associated with subepidermal cells and vascular tissues.

Amino Acid Sequence↗
PubMed1991

Chlorophyll a/b binding (CAB) polypeptides of CP29, the internal chlorophyll a/b complex of PSII: characterization of the tomato gene encoding the 26 kDa (type I) polypeptide, and evidence for a second CP29 polypeptide.

CP29, the core chlorophyll a/b (CAB) antenna complex of Photosystem II (PSII), has two nuclear-encoded polypeptides of approximately 26 and 28 kDa in tomato (Lycopersicon esculentum). Cab9, the gene for the Type I (26 kDa) CP29 polypeptide was cloned by immunoscreening a tomato leaf cDNA library. Its identity was confirmed by sequencing tryptic peptides from the mature protein. Cab9 is a single-copy gene with five introns, the highest number found in a CAB protein. In vitro transcription-translation gave a 31 kDa precursor which was cleaved to about 26 kDa after import into isolated tomato chloroplasts. The Cab9 polypeptide has the two highly conserved regions common to all CAB polypeptides, which define the members of this extended gene family. Outside of the conserved regions, it is only slightly more closely related to other PSII CABs than to PSI CABs. Sequence analysis of tryptic peptides from the Type II (28 kDa) CP29 polypeptide showed that it is also a member of the CAB family and is very similar or identical to the CP29 polypeptide previously isolated from spinach. All members of the CAB family have absolutely conserved His, Gln and Asn residues which could ligate the Mg atoms of the chlorophylls, and a number of conserved Asp. Glu, Lys and Arg residues which could form H-bonds to the polar groups on the porphyrin rings. The two conserved regions comprise the first and third predicted trans-membrane helices and the stroma-exposed segments preceding them.

Amino Acid Sequence↗
PubMed1991

Trends in selected obstetric complications from University Hospital, Kuala Lumpur, Malaysia.

Trends in selected pregnancy complications from 1969 to 1987 in a tertiary hospital in Malaysia are presented. Complications reviewed were abortion, ectopic pregnancy, anemia, hypertension, hyperemesis, antepartum and postpartum hemorrhage. Possible explanations for the observed trends were discussed, including the role of improved obstetric care and changes in the characteristics of the childbearing population. The data presented give some indication of maternal morbidity in the childbearing population served by this tertiary center and should lead to improvements in provision of services as well as in health data collection in the future.

Abortion, Spontaneous↗
PubMed1990

The formation of covalent adducts between benzo[a]pyrenediol epoxide and RNA: structural analysis by mass spectrometry.

Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention times with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribonucleotides were poly(G) greater than yeast RNA greater than poly(A) greater than poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Several adducts exhibited molecular ions by both LSIMS and EIMS. Large fragments observed by EIMS usually resulted from the loss of CH3OH, CH3O., CH2O, CH3., and H. from the molecular ion. Major fragmentation pathways also resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions. Each of these major ions in turn resulted in further characteristic fragmentation patterns.

Animals↗
PubMed1990

Pattern of rheumatic diseases in south India. IV. Clinical profile of juvenile rheumatoid arthritis.

An analysis of 100 consecutive cases of juvenile rheumatoid arthritis from South India revealed a male preponderance (62%), a lower incidence of the systemic onset variety (10%) and equal incidence of systemic features when compared with the West. Knees and ankles were the joints commonly involved. The incidence of elevated erythrocyte sedimentation rate and C reactive protein, with haemoglobin levels below 10 g/dl was highest in the systemic onset variety. The polyarticular and systemic onset group responded well to aspirin, while the pauciarticular group responded well to indomethacin.

Arthritis, Juvenile↗