Biomedical subjects

V Natarajan

Publications and source records attributed to V Natarajan.

At least 127 records · Page 7Linked to original sources

PubMed1984

JC virus enhancer-promoter active in human brain cells.

A human papovavirus, JCV, is the etiologic agent of the fatal demyelinating disease, progressive multifocal leukoencephalopathy. The JCV 98-base-pair tandem repeats, located to the late side of the viral replication origin, were shown to be a transcriptional regulatory element with enhancer-like activity in human fetal glial cells. These tandem repeats share significant homology with the 82-nucleotide rat brain-specific identifier RNA sequence.

Base Sequence↗

PubMed1984

Properties of canine myocardial phosphatidylethanolamine N-acyltransferase.

Dog heart contains a membrane bound N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of membrane phospholipids to the amino group of ethanolamine phospholipids in the presence of millimolar Ca2+ concentrations. Using crude membrane preparations, we found this N-acyltransferase activity to be heat sensitive and inhibited by sulfhydryl reagents. Pretreatment of a membrane fraction with trypsin reduced N-acyltransferase activity to 60% while pretreatment with trypsin and Triton X-100 together reduced it to 30% of the control value. At pH 8.0 both Sr2+ and Mn2+ could fully substitute for Ca2+ with respect to optimum ion concentration and molecular species of the product formed in dog heart membranes from endogenous substrates. Ba2+ was equally effective in achieving N-acylation of ethanolamine phospholipids while other divalent cations were less effective or ineffective. The reaction exhibited a pH optimum of 8.5 to 9.0 with both Ca2+ and Sr2+ while Mn2+ precipitated above pH 8.0 resulting in decreased N-acylation activity. Both phosphatidylcholine and 1-acyl lysophosphatidylcholine could serve as acyl donors. Triton X-100 at a concentration of 0.1% stimulated acyl transfer from exogenous phosphatidylcholine but inhibited acyl transfer from lysophosphatidylcholine.

Acyltransferases↗

PubMed1984

Possible regulation of phospholipase C activity in human platelets by phosphatidylinositol 4',5'-bisphosphate.

Phospholipase C from human platelets was found to catalyze the Ca2+-dependent degradation of phosphatidylinositol (PI), phosphatidylinositol 4'-phosphate (DPI), and phosphatidylinositol 4',5'-bisphosphate (TPI) at Ca2+ concentrations from 150 microM to 5 mM. Both DPI and TPI inhibited the hydrolysis of [2-3H]inositol-labeled PI (250 microM) in a concentration-dependent manner. The use of DPI and TPI from beef brain, both of which have fatty acid compositions different from that of soybean PI, permitted an assessment of the inhibitory effect of polyphosphoinositides on the hydrolysis of PI by phospholipase C. Fatty acid analysis of the diacylglycerols formed demonstrated that DPI and TPI, when incubated in mixture with PI, were competitive substrates for PI hydrolysis. Increasing the DPI/PI ratio from 0 to 0.3 caused a shift in the degradation of PI to DPI without greatly affecting the formation of 1,2-diacylglycerol. TPI alone, or in mixture with PI, was a poor substrate for phospholipase C. Increasing the TPI/PI ratio from 0 to 0.21, on the other hand, inhibited both PI degradation (greater than or equal to 95%) and overall formation of 1,2-diacylglycerol (greater than or equal to 82%). Kinetic analysis revealed that TPI acts as a mixed-type inhibitor with a Ki of about 10 microM. The Ka for Ca2+ in PI hydrolysis was profoundly increased from 5 to 180 microM when TPI (36 microM) was included with PI (250 microM). Optimum PI degradation under these conditions was only attained when the calcium concentration approached 4 mM. Analysis of phospholipids from unstimulated human platelets from five different donors revealed DPI/PI and TPI/PI ratios of 0.42 and 0.16, respectively. These findings, combined with the observed inhibition of PI hydrolysis by TPI at a TPI/PI ratio of 0.16, would suggest that in unstimulated platelets phospholipase C activity may be inhibited by greater than or equal to 75%. Changes in 33P-prelabeled phospholipids of intact platelets upon stimulation with thrombin indicated a transient decline in 33P label of both TPI and DPI (15 s) followed by an increase in [33P]phosphatidic acid but no change in [33P]PI. The finding that DPI is selectively degraded by phospholipase C in mixture with PI at DPI/PI ratios determined to be present in unstimulated platelets indicates that DPI may be more important than PI in the formation of 1,2-diacylglycerol which is believed to serve as precursor of arachidonic acid for thromboxane biosynthesis. Furthermore, the results suggest that in human platelets TPI may serve as modulator for the formation of 1,2-diacylglycerol from inositol phospholipids.

Blood Platelets↗

PubMed1984

Proximal and distal domains that control in vitro transcription of the adenovirus IVa2 gene.

The adenovirus IVa2 gene, which is expressed at an intermediate time in the viral infectious cycle, is separated from the adenovirus major late promoter (MLP) 5' start site by 210 base pairs and is transcribed from the opposite strand. In contrast to the MLP, the IVa2 gene does not contain a "TATA" box upstream from its 5' start sites. By using a series of deletion mutants, two upstream control regions that are rich in cytidine residues, one proximal to the cap site at nucleotide positions -39 to -48 and a distal domain between nucleotide positions -152 and -242 have been identified as essential for IVa2 transcription (IVa2 cap site is nucleotide position + 1). Transcription efficiency is decreased by 70-90% after the deletion of a proximal C-rich domain when either linear or supercoiled DNAs were used as template. However, distal sequences functioned as transcriptional control domains only with covalently closed DNA templates. The deletion of both the proximal and distal regions from covalently closed DNA templates reduces the levels of IVa2 transcription by a factor of 100-150. When the plasmid pAd242 that contains the 5' start sites of adenovirus MLP and IVa2 is transcribed, there is essentially a complete suppression of transcription of the adenovirus IVa2 gene. The transcription efficiency of IVa2 is increased 10-fold after deletion of the MLP cap site. A model based on a shared entry site for RNA polymerase II and competition between major late and IVa2 promoters is proposed to explain the in vitro transcriptional results.

Adenoviridae↗

PubMed1984

Biosynthesis of long-chain alcohols by developing and regenerating rat sciatic nerve.

Cell-free preparations of rat sciatic nerve were found to catalyze the reduction of fatty acid to alcohol in the presence of NADPH as reducing cofactor. The reductase was membrane-bound and associated primarily with the microsomal fraction. When fatty acid was the substrate, ATP, coenzyme A (CoA), and Mg2+ were required, indicating the formation of acyl CoA prior to reduction. When acyl CoA was used as substrate, the presence of albumin was required to inhibit acyl CoA hydrolase activity. Fatty acid reductase activity was highest with palmitic and stearic acids, and somewhat lower with lauric and myristic acids. It was inhibited by sulfhydryl reagents, indicating the participation of thiol groups in the reduction. Only traces of long-chain aldehyde could be detected or trapped as semicarbazone. Fatty acid reductase activity in rat sciatic nerve was highest between the second and tenth days after birth and decreased substantially thereafter. Microsomal preparations of sciatic nerve from 10-day-old rats exhibited about four times higher fatty acid reductase activity than brain or spinal cord microsomes from the same animals. Wallerian degeneration and regeneration of adult rat sciatic nerve resulted in enhanced fatty acid reductase activity, which reached a maximum at about 12 days after crush injury.

Aging↗

PubMed1984

Catabolism of N-acylethanolamine phospholipids by dog brain preparations.

N- Acylphosphatidylethanolamine , incubated with dog brain homogenate or microsomes, was hydrolyzed to phosphatidic acid and N-acylethanolamine by a phosphodiesterase of the phospholipase D type. In the absence of F-, phosphatidic acid was further hydrolyzed to diacylglycerol and Pi while N-acylethanolamine was hydrolyzed by an amidase to fatty acid and ethanolamine. The phosphodiesterase showed an alkaline pH optimum and was also active towards N- acetylphosphatidylethanolamine , N-acyl-lysophosphatidylethanolamine, and glycerophospho (N-acyl)ethanolamine but showed little activity toward phosphatidylethanolamine and phosphatidylcholine. Ca2+ stimulated slightly at low concentrations but inhibited at higher concentrations. Triton X-100 stimulated the hydrolysis of N- acylphosphatidylethanolamine , inhibited that of N-acyl-lysophosphatidylethanolamine and glycerophospho (N-acyl)ethanolamine, and had no effect on phosphatidylethanolamine or phosphatidylcholine hydrolysis. The N-acylethanolamine hydrolase (amidase) was also present in the microsomal fraction and exhibited a pH optimum of 10.0. In addition to hydrolysis by the phosphodiesterase, N- acylphosphatidylethanolamine was also catabolized by microsomal phospholipases A1 and/or A2 to N-acyl-lysophosphatidylethanolamine, some of which was further hydrolyzed to glycerophospho (N-acyl)ethanolamine.

Animals↗

PubMed1983

Preferential stimulation of transcription from simian virus 40 late and adeno IVa2 promoters in a HeLa cell extract.

Transcription of certain SV40 and adenovirus genes can be selectively enhanced under suitable in vitro assay conditions. The major SV40 late promoters and the adeno IVa2 promoter do not have TATA box sequences upstream from the start sites of transcription. Analyses of transcripts synthesized in vitro from the SV40 late promoters by "run-off" and S1 nuclease mapping methods show that when transcription takes place in the presence of 15 mM (NH4)2SO4, there is a 3- to 5-fold stimulation of RNA synthesis initiating from the late promoter at map position 185 and from the SV40 early promoters. RNA initiating from map position 325 is stimulated 40-fold under these conditions. When the (NH4)2SO4 concentration is increased to 50 mM (NH4)2SO4, there is a 90% suppression of transcription. Ammonium sulfate also enhances transcription from the adeno IVa2 promoter, which is not expressed in extracts that lack (NH4)2SO4 (Fire, A., Baker, C. C., Manley, J. L., Ziff, E. B., and Sharp, P. A. (1981) J. Virol. 40, 703-719; Lee, D. C., and Roeder, R. G. (1981) Mol. Cell. Biol. 1, 635-651). These modified assay conditions may allow detection of new classes of promoters.

Adenoviridae↗

PubMed1983

Metabolism of N-acylethanolamine phospholipids by a mammalian phosphodiesterase of the phospholipase D type.

Rat heart contains a membrane-bound phosphodiesterase of the phospholipase D type which catalyzes the hydrolysis of 1,2-diacyl-sn-glycero-3-phospho(N-acyl)ethanolamine to N-acylethanolamine and phosphatidic acid. The enzyme also hydrolyzes the corresponding alkenylacylglycerophospho(N-acyl)ethanolamine and N-acylethanolamine lysophospholipids but not phosphatidylcholine or phosphatidylethanolamine. The activity is highest in the microsomal fraction, does not require Ca2+ or Mg2+, and is stimulated by Triton X-100. Bile salts, other ionic detergents, and Zn2+ are inhibitory. Hydrolysis occurs over a wide pH range, with the activity at acid pH being stimulated by freezing and thawing. Other rat tissues also release N-acylethanolamine from N-acylethanolamine phospholipids.

Animals↗

PubMed1983

The alkylacyl and alkenylacyl glycerophospholipids of human platelets.

The phospholipids of human platelets were isolated and analysed for their constituent 1-0-alkyl- and 1-0-(1'-alkenyl)glycerol species. The serine and inositol phospholipids contained only small amounts (0.5-1.6%) of both alkylacyl and alkenylacyl species; the ethanolamine phospholipids contained little alkylacyl (1.7%) but much more alkenylacyl (45.3%) species, while the choline phospholipids contained 4.5% and 1.4% of these compounds, respectively. Because choline phospholipids amount to 41% of the total phospholipids of human platelets, it appears that they could provide a sufficient supply of alkylacyl glycerophosphocholine for the generation of the alkylacetyl analog (platelet activating factor) by a deacylation-acetylation pathway.

Blood Platelets↗

PubMed1983

The role of cardiolipin as an acyl donor in dog heart N-acylethanolamine phospholipid biosynthesis.

N-Acylethanolamine phospholipids were produced from endogenous substrates with dog heart mitochondrial and microsomal preparations. With mitochondria the N-acyl group contained 13.8% linoleate, with microsomes only 3.6%. Cardiolipin comprised 18.5% of mitochondrial and 3.3% of microsomal lipid P and contained 93.7 and 72.4% linoleic acid, respectively. Incubation of dog heart subcellular fractions with [1-14C]linoleoyl cardiolipin in the presence of Ca2+ resulted in the formation of N-acylethanolamine phospholipids labeled primarily in the N-acyl and 1-O-acyl moieties. The data indicate that cardiolipin is the major source of linoleic acid used in the N-acylation of ethanolamine phospholipids by transacylase activity.

Acylation↗

PubMed1983

N-Acylation of dog heart ethanolamine phospholipids by transacylase activity.

Radioactive N-acylethanolamine phospholipids were produced in dog heart homogenates incubated with acyl-labeled phosphatidylcholine in the presence of 5 mM Ca2+ and Triton X-100. 70-80% of the label in the N-acylethanolamine phospholipids was recovered in the N-acyl groups and most of the remainder was in the 1-O-acyl groups. Incubations with 1,2-dipalmitoylPC and 1-palmitoyl-2-linoleoylPC labeled in either the 1-O-acyl or 2-O-acyl moiety showed the predominant utilization of the acyl groups at the sn-1 position, indicating transacylation by phospholipase A1 (or lysophospholipase) activity. It is suggested that intramolecular transacylation from 1-O-acyl to N-acyl groups of phosphatidylethanolamine also occurred to some extent, thus providing a free primary hydroxy group as an additional acyl acceptor for the transacylation reaction.

Acylation↗

PubMed1983

Biosynthesis of N-acylethanolamine phospholipids by dog brain preparations.

Dog brain homogenates and subcellular preparations incubated in the presence of Ca2+ produced a new phospholipid that was isolated and identified by its infrared spectrum and by chemical degradation as a mixture of 1,2-diacyl, alkenylacyl, and alkylacyl sn-glycero-3-phospho(N-acyl)ethanolamines, 50, 45, and 5%, respectively. The N-acyl groups consisted almost exclusively of 16:0, 18:0, and 18:1 fatty acids. Formation of N-acylethanolamine phospholipids from endogenous substrates was linear for about 90 min at approximately 4.5 nmol/h/mg protein and exhibited a pH optimum of 10. Biosynthetic activity was associated with particulate fractions, primarily microsomes, synaptosomes, and mitochondria, but not with myelin. In each case, small amounts (approximately 0.5 nmol/h/mg protein) of long-chain N-acylethanolamines were also produced. Incubation of dog brain microsomes with 1,2-di[1'-14C]palmitoyl glycerophosphocholine yielded N-acylethanolamine phospholipids labeled at both N-acyl (55%) and O-acyl (45%) moieties. It appears that dog brain organelles may contain a phosphatidylethanolamine N-acyl transferase (transacylase) analogous to that recently demonstrated in the myocardial tissue.

Animals↗

PubMed1983

Art of teaching.

Explore the source record for details and available documents.

Education, Medical↗

PubMed1982

N-Acylation of ethanolamine phospholipids in canine myocardium.

N-Acylethanolamine phospholipids, which are found in infarcted but not in normal canine myocardium, were produced by preparations of normal myocardial tissue incubated in the presence of millimolar concentrations of Ca2+. Biosynthetic activity from endogenous substrates was associated with both microsomal and mitochondrial fractions and exhibited an alkaline pH optimum. The time course of N-acylethanolamine phospholipid synthesis and degradation was followed after pulse labeling of myocardial ethanolamine phospholipids with [1,2-14C]ethanolamine. Enzymic N-acylation of both phosphatidylethanolamine and lysophosphatidylethanolamine was demonstrated by incubating these substrates with homogenates of myocardial tissue. Neither free fatty acids nor acyl-CoA derivatives served as acyl donor but some of the constituent fatty acids of exogenous phosphatidylethanolamine were recovered among the amide-linked fatty acids of the N-acylethanolamine phospholipids. N-Acylation may thus occur by the transfer of O-acyl groups catalyzed by a Ca2+ -dependent transacylase. Since N-acylethanolamine phospholipids are precursors of the biologically active N-acylethanolamines, their biosynthesis may constitute an important injury-induced metabolic event aimed at the protection of ischemic myocardial tissue.

Animals↗

PubMed1982

Site-specific base substitution and deletion mutations that enhance or suppress transcription of the SV40 major late RNA.

Transcriptional analysis of SV40 late promoter mutants indicates that the DNA sequence 5'-GGTACCTAACC-3' (map positions 294-304) is important in the control of SV40 late RNA expression. C to T base substitutions at map positions 298, 299 and 304 increased initiation of late RNA synthesis at map position 325 five to ten fold. G to A base substitutions at map positions 294 and 295 decreased RNA initiation by a factor of 2 to 3. Deletion of nucleotides 295-298 reduced RNA initiation by a factor of 4 to 5. S1 analysis of in vivo RNA, isolated 24-36 hr after infection, demonstrated that the four base deletion not only decreased RNA initiation at nucleotide 325, but also increased RNA initiation at three alternate sites located approximately 125 nucleotides upstream from the major late RNA initiation site.

Base Sequence↗

PubMed1982

Early stimulation of phosphatidylcholine biosynthesis during Wallerian degeneration of rat sciatic nerve.

Phospholipid metabolism was studied in rat sciatic nerve during Wallerian degeneration induced by crush injury. Portions of crushed sciatic nerve, incubated with labeled substrates, showed significantly higher phosphatidylcholine synthesis than normal nerve, prior to any measurable alterations of phospholipid composition. Maximum synthesis occurred 3 days after crush injury, at which time the metabolism of other phospholipids was unchanged. After a rapid decrease in biosynthetic activity, a second phase of enhanced phosphatidylcholine synthesis occurred, beginning 6 days after crush injury. Increased incorporation of [33P]phosphate, [2-3H]glycerol, and [Me-14C]choline indicated stimulation of de novo synthesis of phosphatidylcholine 3 days after injury. Neither base exchange reactions nor sequential methylation of ethanolamine phospholipids contributed significantly to phosphatidylcholine synthesis. Assay of certain key enzymes under optimal conditions in subcellular fractions of sciatic nerve revealed higher activities of cholinephosphate cytidyltransferase, choline phosphotransferase, and acyl-CoA:lysophosphatidylcholine acyltransferase in injured nerve, while choline kinase activity remained unchanged. This indicates that stimulation of phosphatidylcholine synthesis occurs via the cytidine nucleotide pathway, as well as by increased acylation of lysophosphatidylcholine. Although the cause of stimulated phosphatidylcholine synthesis remains unexplained, it is possible that trace amounts of lysophospholipids or other metabolites produced by injury-enhanced phospholipase activity may be responsible.

Animals↗

PubMed1981

Mechanism of aminoacylation of tRNA. Influence of spermine on the kinetics of aminoacyl-tRNA synthetases by isoleucyl- and valyl-tRNA synthetases from Mycobacterium smegmatis.

Isoleucyl-tRNA synthetase has been purified to homogeneity from Mycobacterium smegmatis. The influence of spermine on the kinetics of valyl-tRNA and isoleucyl-tRNA formation has been investigated by Cleland's method (Cleland, W.W. (1963) Biochim. Biophys. Acta 67, 104-137, 173-187, 188-196). The results suggest that in the presence of spermine and suboptimal concentration of Mg2+, the formation of valyl-tRNA and isoleucyl-tRNA follows a sequential mechanism. In the presence of an optimal concentration of Mg2+, both valyl-tRNA and isoleucyl-tRNA formation proceeds by a ping-pong mechanism. However, in the presence of spermine and optimal concentrations of Mg2+, valyl-tRNA formation follows the ping-pong mechanism while isoleucyl-tRNA formation follows the sequential mechanism.

Amino Acyl-tRNA Synthetases↗

PubMed1981

On the biosynthesis and metabolism of N-acylethanolamine phospholipids in infarcted dog heart.

Minced tissues from both infarcted and apparently normal areas of canine myocardium 6 h after ligation of the left descending branch of the coronary artery were incubated with [1,2-(14)C]ethanolamine. In each case substantial amounts of radioactivity were incorporated into both phosphatidylethanolamine and N-acylphosphatidylethanolamine. Incubation of homogenates from the same tissues with N-[1-(14)C]palmitoyl phosphatidylethanolamine yielded labeled N-palmitoyl ethanolamine. The data support the concept that N-acylethanolamines, pharmacologically active compounds which accumulate in infarcted myocardium, are produced by N-acylation of ethanolamine phospholipids followed by phosphodiesterase action.

Acylation↗