Geranoyl-CoA carboxylase: a novel biotin-containing enzyme in plants

Geranoyl-CoA carboxylase (EC 6.4.1.4) is a biotin-containing enzyme previously described in two genera of bacteria. Here we report the presence of geranoyl-CoA carboxylase in kingdom Plantae. Geranoyl-CoA carboxylase was purified 180-fold from maize leaves. The enzyme has a biotin-containing subunit of 122 kDa. The pH optimum for activity is 8.3. The apparent Km values for the substrates geranoyl-CoA, bicarbonate, and ATP are 64 +/- 5 microM, 0. 58 +/- 0.04 mM, and 8.4 +/- 0.4 microM, respectively. Subcellular fractionations indicate that geranoyl-CoA carboxylase is located in plastids. Geranoyl-CoA carboxylase activity is ubiquitous in organs of monocots and dicots and varies with development. We postulate that geranoyl-CoA carboxylase plays an important role in isoprenoid catabolism in plants, in a pathway analogous to that shown in Psuedomonas sp. In plants, this catabolic pathway would require the interaction of at least three subcellular compartments (plastids, microbodies, and mitochondria) and two biotin-containing enzymes, geranoyl-CoA carboxylase and 3-methylcrotonyl-CoA carboxylase.     

Precursor Influence on the Electrical Properties of Textured Bi-2212 Superconductors

Several synthetic methods, solid-state, sol-gel and polymer solution methods, have been used to prepare prereacted precursors, as well as a vitreous material obtained by melt quenching. The influence of the starting powder characteristics on the phase formation, microstructure, T _c and J _c of Bi-2212 textured rods prepared by directional crystallization from the melt has been analyzed. In all the cases, high transport critical current values (higher than 3000 A/cm² at 77 K) have been obtained, independently of the precursor type. Samples obtained by the polymer route show improved T _c values, associated to a lower oxygen content.     

Comparison of hindquarter metabolite uptakes in Belgian Blue double-muscled bulls at maintenance or during fattening

Metabolism of muscle growth in the hindquarter was investigated by the arterio-venous difference (AVD) technique in Belgian Blue double-muscled type bulls at maintenance or at fattening. The bulls were fitted with an aortic ultrasonic blood flow probe and with catheters in the aorta and vena cava. They were offered a diet allowing for maintenance (MP) during a period of 15 d, at the end of which measurements were made over 3 d. Bulls were then given a fattening diet (FP) and the measurements were repeated. Arterial blood flow was approximately 1 L/min greater when the bulls were standing than when lying. Blood flow was 2 L/min higher during FP than during MP. The AVD and uptake of glucose were maximal at 1400 and 1600. Uptake of alpha-amino nitrogen decreased immediately after a meal. The increase in glucose from MP to FP fitted very well with the calculated energy needs for muscle growth. The AVD and uptake of alpha-amino nitrogen, total amino acids, and total nonessential amino acids were negative during MP and positive and significantly higher during FP. There was also a significant increase in AVD and uptake of essential and branched-chain amino acids when the bulls were changed from MP to FP. When changing from maintenance to fattening, the incremental glucose and amino acid hindquarter uptake provided energy and supply for muscle protein accretion, respectively. The level of alanine transamination was also sharply reduced.     

Thiazolium‐Based Catalysts for the Etherification of Benzylic Alcohols under Solvent‐Free Conditions

Thiazolium and imidazolium hybrid materials were prepared by radical reactions between a mercaptopropyl‐modified SBA‐15 mesoporous silica and bis‐vinylthiazolium or bis‐vinylimidazolium dibromide salts. These hybrid materials were characterized by several techniques and were employed in the etherification reaction of 1‐phenylethanol. Solvent‐free conditions at 160 °C under different gas phases (oxygen, air, nitrogen and argon) were used. The thiazolium‐based material displayed excellent performances. Further studies were carried out using unsupported thiazolium salts, with or without a methyl group at the C‐2 position of the thiazolium moiety. These studies allowed us to propose a reaction mechanism. The supported thiazolium‐based material was successfully used in the etherification reaction of two other benzylic alcohols and also in seven consecutive cycles. This work represents the first use of thiazolium‐based compounds as catalysts for the etherification reaction of alcohols.

     

A quantitative ELISA for antigen-specific IgG subclasses using equivalence dilutions of anti-kappa and anti-subclass specific secondary reagents. Application to the study of the murine immune response against the capsular polysaccharide of Neisseria meningitidis serogroup B

We have developed an enzyme-linked immunosorbent assay (ELISA) to measure murine antigen-specific IgG antibodies of defined subclass using precalibrated equivalence dilutions of anti-kappa (in the standard) and each anti-IgG subclass-specific polyclonal secondary antibody (in the test sample). The calibration of secondary reagents could be carried out easily with a set of monoclonal antibodies (MoAbs) specific for all IgG subclasses. These MoAbs do not require purification or standardization. In addition the MoAbs can be of different antigenic specificity. Once the equivalence dilutions have been determined, they can be applied in a quantitative ELISA using the same antigen in the standard and sample, and using only one IgG subclass standard for the determination of all the IgG subclasses. The method is easy to standardize for many antigenic systems. It is particularly useful when the only standard available is one standardized MoAb of the appropriate specificity, and it could be adapted to use with standard polyclonal antibodies having a known content of total antigen-specific IgG bearing kappa chains but unknown IgG subclass composition. The use of this method to quantitate IgG specific for the capsular polysaccharide of Neisseria meningitidis serogroup B (CpsB) gave highly reproducible measures with an interbatch CV of 5-6% similar for all IgG subclasses and low detection limits ranging from 0.3 ng/well for IgG3 to 0.8 ng/well for IgG2a. The IgG subclass response observed after immunization with live meningococci was mainly IgG2a (74%) and IgG2b (18%). Hyperimmunization modified this IgG distribution to one of mainly IgG3 (62%) and IgG1 (28%) which was maintained in the response to a single immunization 4 weeks later, possibly indicating the generation of resting B cells during continuous stimulation.     

Biodegradability of slaughterhouse wastewater with high blood content under anaerobic and aerobic conditions

In this work, the biodegradability of wastewater from a slaughterhouse located in Ke?an, Turkey, was studied under aerobic and anaerobic conditions. A very high total COD content of 7230 mg dm^?3 was found, due to an inefficient blood recovery system. Low BOD₅/COD ratio, high organic nitrogen and soluble COD contents, were in accordance with a high blood content. A respirometry test for COD fractionation showed a very low readily biodegradable fraction (S_S) of 2%, a rapidly hydrolysable fraction (S_H) of 51%, a slowly hydrolysable fraction (X_S) of 33% and an inert fraction of 6%. Kinetic analysis revealed that hydrolysis rates were much slower than these of domestic sewage. The results underlined the need for an anaerobic stage prior to aerobic treatment. Tests with an anaerobic batch reactor indicated efficient COD degradation, up to around 80% removal. Further anaerobic degradation of the remaining COD was much slower and resulted in the build up of inert COD compounds generated as part of the metabolic activities in the anaerobic reactor. Accordingly, it is suggested that an appropriate combination of anaerobic and aerobic reactors would have to limit anaerobic degradation to around 80% of the tCOD and an effluent concentration above 1000 mg dm^?3, for the optimum operation of the following aerobic stage. © 2003 Society of Chemical Industry     

LFA-3 co-stimulates cytokine secretion by cytotoxic T lymphocytes by providing a TCR-independent activation signal

T cell activation is known to depend not only on efficient antigen recognition and subsequent signaling through TCR, but also on interactions involving multiple adhesion and accessory molecules such as CD28/B7, LFA-1/ICAM-1 and LFA-3/CD2. The present study dissects the role of LFA-3/CD2 interactions in the activation of melanoma-specific CD8+ T cell clones. To this end we analyzed the influence of LFA-3 density on melanoma cells on lysis and cytokine production (TNF, IL-2, IFN-gamma) by T cells following activation by various amounts of antigenic peptides. Our results indicate that increasing LFA-3 density on melanoma cells variably affects their lysis susceptibility, but systematically and considerably enhances cytokine production by melanoma-specific cytotoxic T lymphocyte (CTL) clones. At any stimulatory antigen density, LFA-3 increased the fraction of responding cells and/or cytokine amounts produced by individual cells, without affecting TCR down-regulation. These results show that CD2 engagement increases cytokine gene activation essentially by providing to T cells a TCR-independent co-activation signal. From a practical point of view, our data demonstrate that the level of LFA-3 expressed on tumors critically affects cytokine production by specific CTL and thus the efficiency of specific immune reactions mediated by these cells.     

Activation of p70(S6) kinase by beta-adrenoceptor agonists on adult cardiomyocytes

Cardiac cellular hypertrophy plays an important role in cardiovascular diseases. Up until now, little has been known about the regulation of cellular growth on the level of intracellular signalling. Here, the implication of the p70(S6)-kinase (p70(S6K)) in the hypertrophic response after beta-adrenergic stimulation of cardiac myocytes from adult rats was investigated. Isoproterenol stimulation can activate p70(S6K) in adult cardiomyocytes analysed by direct kinase assays and retarded gel mobility. This signalling of beta-adrenoceptor stimulation is found only under conditions where the cardiomyocytes exhibit also a hypertrophic response to beta-adrenoceptor stimulation as measured by increase in protein content, RNA content and incorporation of radiolabelled amino acids. Rapamycin, a specific inhibitor of this kinase, reduces the trophic responses to control levels, suggesting an involvement of the p70(S6)-kinase in the development of cellular hypertrophy. An engagement of the MAP-kinase (ERK-1/2) pathway in the beta-adrenergic induced growth of cardiac myocytes from adult rats was excluded.     

Epstein Barr virus in Argentine pediatric Hodgkin''s disease

Pancreatic islets prelabelled with either 86Rb or 45Ca were exposed to a rise in D-glucose concentration from 2.8 to 16.7 mM whilst perifused in the presence of 2 microM glibenclamide, 30 mM extracellular K+ and both 30 mM K+ and 250 microM diazoxide. In all three situations, the rise in glucose concentration provoked a dramatic increase in insulin output, despite unchanged or even increased efflux of 86Rb from the prelabelled islets. Also in all three situations, glucose sharply decreased effluent radioactivity from islets prelabelled with 45Ca but perifused in the absence of extracellular Ca2+, while augmenting 45Ca efflux, to a variable extent, from islets perifused at normal extracellular Ca2+ concentration (1.0 mM). It is proposed, therefore, that the insulinotropic action of D-glucose in depolarized islets, and presumably also under normal conditions, may involve the gating of voltage-insensitive Ca2+ channels.