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Objectives: To study the clinical and demographic profile of HIV/AIDS patients diagnosed at a tertiary care centre., Methods: The study was conducted on a group of 1141 patients suspected of having HIV/AIDS on clinical grounds. Screening was done using different Elisa's as advised by NACO and those confirmed as HIV positive were studied for their clinical spectrum and different demographic parameters., Results: Out of 1141 patients tested, 26 proved to have HIV 1 infection with no case of HIV 2 detected. Mean age of presentation was 40.04 +/- 7 years, main age group affected 31-40 years and a male: female ratio of 4.2:1 was observed. More than 42% were non Kashmiris with armed forces outnumbering all other occupational classes. Heterosexual transmission was the commonest with married out numbering unmarried. Fever, asthenia and weight loss were the predominant symptoms and pulmonary tuberculosis and oropharnygeal candidiasis commonest opportunistic infections., Conclusion: The clinical and demographic profile of HIV/AIDS patients in Kashmir is largely similar to the rest of India. Kashmir no longer stands immune to the menace of HIV/AIDS. With increasing globalization, frequent travel and change in social values the state is likely to witness an alarming rise in new cases unless a multipronged approach is undertaken to control the spread.

The effect of biotin on the induction (and possible requirement for uptake into mitochondria) of apopyruvate carboxylase has been examined in 3T3-L adipocytes. Cells fed biotin-sufficient medium contained only holoenzyme in mitochondria and no apoenzyme was detected. The amount of apoenzyme elaborated in biotin-deficient 3T3-L adipocytes was comparable to the holopyruvate carboxylase protein found in cells maintained on biotin-sufficient medium. Like the holoenzyme, the apoenzyme was detected exclusively in the mitochondrial fraction of 3T3-L adipocytes. This indicates that the synthesis of apopyruvate carboxylase and its translocation into mitochondria occur independently of the cofactor, biotin.

3T3-L fibroblasts differentiate into adipose cells when maintained in a non-growing state. The specific activity of fatty acid synthetase of differentiated cells was 25--30-fold higher than that present in 3T3-L fibroblasts or in 3T3-C2 cells that possess an extremely low incidence of differentiation to adipocytes. The results of immunochemical analysis indicate that the increased specific activity of fatty acid synthetase in the differentiated cells is due to an increase in the cellular content of this enzyme. The rate of conversion of adipose cells was accelerated by brief exposure of confluent non-growing cultures of 3T3-L cells to 3-isobutyl-1-methylxanthine. This was accompanied by an increase in the specificity activity of fatty acid synthetase, which was also shown to be due to an increase in the cellular content of this enzyme. The continuous presence of 3-isobutyl-1-methylxanthine in the culture medium was not required to elicit the morphological and biochemical changes in 3T3-L cells that occurred many days after the removal of the inducer but earlier than the onset of spontanous differentiation.

The effect of citrate on the different reactions catalyzed by rat mammary gland acetyl CoA carboxylase has been investigated. Citrate showed modest effect on the ATP-orthophosphate and ATP-ADP exchange reactions. In contrast, this tricarboxylic acid caused marked concentration-dependent stimulation of the acetyl CoA-malonyl CoA exchange reaction which was concomitant with the activation of acetyl CoA carboxylation. The data obtained are consistent with the suggestion that activation by citrate of the overall forward reaction (malonyl CoA synthesis) primarily reflects enhancement of the carboxyltransferase half-reaction catalyzed by rat mammary gland acetyl CoA carboxylase.

An acyl coenzyme A (CoA) carboxylase, which catalyzes the adenosine triphosphate-dependent fixation of CO2 into acetyl-, propionyl-, and butyryl-CoA, was detected in fractionated cell extracts of Propionibacterium shermanii. Catalytic activity was inhibited by avidin but was unaffected by avidin pretreated with excess biotin. The carboxylase levels detected were relatively small and were related to cellular growth. Maximal carboxylase activity was detected in cells grown for about 96 h. Thereafter, the activity declined rapidly. Optimal CO2 fixation occurred at pH 7.5. Other parameters of the assay system were optimized, and the apparent Km values for substrates were determined. The end product of the reaction (with acetyl-CoA as the substrate) was identified as malonyl-CoA. The stoichiometry of the reaction was such that, for every mole of acetyl-CoA and adenosine triphosphate consumed, 1 mol each of malonyl-CoA, adenosine diphosphate, and orthophosphate was formed. These data provide the first evidence for the presence of another biotin-containing enzyme, an acyl-CoA carboxylase, in these bacteria in addition to the well-characterized methylmalonyl-CoA carboxyltransferase.

A simple procedure was devised which allows purification of rat lactating-mammary-gland fatty acid synthase to a high degree of purity, with recoveries of activity exceeding 50%. Over 50 mg of enzyme was isolated from 60 g of mammary tissue. The specific activity of the purified enzyme was about 2.5 mumol of NADPH oxidized/min per mg of protein at 37 degrees. The enzyme appeared homogeneous by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by immunodiffusion analysis. Each mol (Mr 480 000) of the enzyme bound 3 mol of acetyl and 3-4 mol of malonyl groups when the binding experiments were performed at 0 degrees for 30 s. The presence of NADPH did not influence the binding stoicheiometry for these acyl-CoA derivatives. Approx. 2 mol of taurine was found per mol of the performic acid-oxidized enzyme, suggesting that there were 2 mol of 4'-phosphopantetheine in the native enzyme. Rat mammary-gland fatty acid synthase required free CoA for activity.

The activities of two lipogenic enzymes, acetyl-CoA carboxylase and fatty acid synthase, were determined in two transplantable mammary adenocarcinomas (13762 and R3230AC) carried by non-pregnant, pregnant and lactating rats, and in mammary tissue of control animals (non-tumour-carrying) of comparable physiological states. During mammary-gland differentiation of control or tumour-carrying animals, the activities of acetyl-CoA carboxylase and fatty acid synthase in the lactating gland increased by about 40--50-fold over the values found in non-pregnant animals. On the other hand, in tumours carried by lactating dams there were only modest increases (1.5--2-fold) in acetyl-CoA carboxylase and fatty acid synthase compared with the neoplasms carried by non-pregnant animals. On the basis of the Km values for different substrates and immunodiffusion and immunotitration data, the fatty acid synthase of neoplastic tissues appeared to be indistinguishable from the control mammary-gland enzyme. However, a comparison of the immunotitration and immunodiffusion experiments indicated that the mammary-gland acetyl-CoA carboxylase might differ from the enzyme present in mammary neoplasms.

A simple scheme for the purification of pyruvate carboxylase from rat liver mitochondria is described. It is rapid and provides high-purity pyruvate carboxylase with excellent yield and reproducibility. The final enzyme preparations appear to be homogeneous by the following criteria: elution behaviour on molecular-sizing matrix, SDS/polyacrylamide-gel electrophoresis, Ouchterlony double-diffusion analysis and Western blotting. Detection and quantification of nanogram amounts of pyruvate carboxylase (apo and holo forms) in total tissue homogenates by immuno-blotting and by enzyme-linked immunosorbent assay are described. The data provided suggest that under normal physiological conditions (both in vivo and in vitro) essentially all the pyruvate carboxylase molecules are biotinylated.

We have employed a flow cytometric method of monitoring DNA synthesis in order to study the stimulation of DNA replication during the induction of adipocyte differentiation in the preadipocyte cell line 3T3-L1. When confluent monolayers of this cell line are treated with a mixture of methyl isobutyl xanthine, dexamethasone, and insulin (MDI), they undergo at least one round of cell division, which is followed by the de novo synthesis of many enzymes that are involved in the formation of lipid. If the MDI-treated cells are incubated in iododeoxyuridine (IdUrd)- or bromodeoxyuridine (BrdUrd)-containing medium and subsequently stained with antibodies specific for these base analogues and with propidium iodide (PI), the kinetics of the induction of replication can be monitored. No differences in the patterns of IdUrd incorporation versus PI staining were observed between exponentially growing 3T3-L1 cells and those that had been stimulated to replicate DNA with MDI. In addition, we developed a flow cytometric (FCM) method for staining fatty acid synthetase and localizing the antigen in the G1 phase. We have thus demonstrated the feasibility of this methodology for correlating by FCM the production of enzymes such as fatty acid synthetase with IdUrd and BrUrd incorporation. The technique should permit studies of the inhibition of differentiation of adipocytes by halogenated pyrimidines.

Fatty acid synthase from lactating rat mammary gland is rapidly and irreversibly inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. Of the seven partial reactions catalysed by the enzyme, the inhibition of the overall catalytic activity is closely paralleled only by inhibition of the beta-oxoacyl synthase (condensing) partial reaction. Three partial reactions. Beta-oxoacyl reductase, beta-hydroxyacyl dehydratase and enoyl reductase, are inhibited to a modest degree. The three partial reactions known to involve an acyl-CoA/CoA-binding site, acetyl acyltransferase, malonyl acyltransferase and palmitoyl thioesterase, are not inhibited by S-(4-bromo-2,3-dioxobutyl)-CoA. The modification process does not cause the enzyme to dissociate into catalytically incompetent monomers. Stoichiometric studies suggest that approx. 6 mol of reagent are incorporated per mol of totally inhibited enzyme (dimer). The formation of acylated enzyme from either acetyl-CoA or malonyl-CoA protects the enzyme equally well against S-(4-bromo-2,3-dioxobutyl)-CoA. Also, pretreatment of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid), a thiol-specific reagent reported to block essential thiol groups in the condensing partial reaction, protects against inhibition by the reagent. On the other hand, the presence of up to 770 microM-S-acetonyl-CoA or dethio-CoA does not protect the enzyme from irreversible inhibition. Together, the results suggest that the primary inhibitory process is a bimolecular reaction resulting in alkylation of essential thiol groups in the condensing partial reaction: this process does not require the obligatory formation of a Michaelis-Menten complex of enzyme and reagent before the alkylation reaction.

Rat mammary gland acetyl-CoA carboxylase (acetyl-CoA:carbon dioxide ligase (ADP forming), EC 6.4.1.2) is rapidly and irreversibly inactivated by micromolar concentrations of S-(4-bromo-2,3-dioxobutyl)-CoA (BDB-CoA) or p-hydroxymercuribenzoate (PHMB). Inhibition of both half reactions (i.e., the biotin carboxylation and the carboxyltransferase) catalyzed by acetyl-CoA carboxylase closely parallels loss in overall activity (malonyl-CoA synthesis). The presence of a substrate or product (acetyl-CoA, ATP, ADP, Pi) or inhibitor (palmitoyl-CoA) does not protect the enzyme from inhibition caused by BDB-CoA or PHMB. On the other hand, citrate, an activator of acetyl-CoA carboxylase, affords substantial protection against inhibition by BDB-CoA and PHMB. Covalent modification by BDB-CoA or PHMB appears to lock acetyl-CoA carboxylase in an inactive conformation (15-30 S) that is unable to undergo citrate-induced self-association into the catalytically competent polymeric form.

Three enzymes involved in the conversion of 3T3-L2 fibroblasts into fat cells, acetyl CoA carboxylase (ACC), fatty acid synthetase (FAS) and pyruvate carboxylase (PC) have been localized by immunofluorescence techniques. The method enables the identification of cells undergoing the conversion while they are still fibroblastic in appearance, often before the obvious appearance of fat droplets. Specific fluorescence for each enzyme can be seen in "clones" of cells derived from single cells, which may undergo an event during logarithmic growth, which programs the cells to differentiate subsequent to confluence of and addition of induction medium.

1. Acetyl coenzyme A carboxylase from lactating rat mammary gland has been purified to apparent homogeneity. 2. The purified enzyme has the following characteristics: (a) its specific activity approaches 15 units/mg of protein, (b) the sedimentation constants of the protomeric and polymeric forms of the enzyme are 12 to 13 S and greater than or equal to 40 S, respectively, (c) the polymeric form of the enzyme shows filamentous structures in the electron microscope, and (d) the polypeptide(s) arising from its dissociation reveals a single major component of Mr = 240,000 to 260,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 3. The enzyme contains 1 mol of biotin and approximately 6 mol of phosphate/240,000 g of protein.

Biotin-binding antibodies were raised in rabbits by injecting biotin-bovine serum albumin conjugate. Neither the protomer nor the polymer of rat mammary-gland acetyl-CoA carboxylase formed precipitin bands with the anti-biotin. By virtue of its ability to bind biotin (apparent binding constant for free biotin about 1mum), the anti-biotin inhibited the carboxylase activity under certain conditions. This property of the antibody was employed to detect the ligand-induced changes affecting the biotinyl group in different conformational states of mammalian carboxylase. Depending on the ligand present, the biotinyl group in the protomeric form was either accessible or inaccessible to the antibody. The biotinyl group of the protomer generated by a relatively high concentration of NaCl (0.5m) reacted with the antibody, and the antibody-carboxylase complex could not be converted into active enzyme by citrate. Further experiments showed that citrate failed to induce polymerization in this protomer-antibody complex and that anti-biotin could be displaced rapidly from this complex with excess of biotin. The resulting protomer was converted into the polymeric state on citrate addition, with parallel regain of enzyme activity. In the presence of ADP+Mg(2+), ATP+Mg(2+) or ATP+Mg(2+)+HCO(3) (-), however, the enzyme remained as a protomer, but its configuration was such that the biotinyl group was essentially inaccessible to the antibody. Likewise, the biotinyl group of the different polymeric forms of the carboxylase (s approximately 30-45S) engendered by phosphate, malonyl-CoA, acetyl-CoA or citrate remained essentially inaccessible, since their activity was minimally affected by the anti-biotin. In the presence of 0.15m-NaCl, the phosphate-induced polymer reverted to a approximately 19S form with concomitant appearance of anti-biotin-sensitivity, whereas the other polymeric forms remained unaffected under similar experimental conditions.