The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose

The enzyme UDP-galactose-4-epimerase (GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDPglucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of beta-D-galactose to alpha-D-galactose and the hitter for epimerization of UDP-galactose to UDP-glucose. Absence of C albicans GAL10 (CaGAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H2O2. While the S. cerevsiae GAL10 (ScGAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose. (c) 2006 Elsevier Inc. All rights reserved.

Non-segmented negative sense RNA viruses as vectors for vaccine development

This article intends to cover two aspects of non-segmented negative sense RNA viruses. In the initial section, the strategy employed by these viruses to replicate their genomes is discussed. This would help in understanding the later section in which the use of these viruses as vaccine vectors has been discussed. For the description of the replication strategy which encompasses virus genome transcription and genome replication carried out by the same RNA dependent RNA polymerase complex, a member of the prototype rhabdovirus family - Chandipura virus has been chosen as an example to illustrate the complex nature of the two processes and their regulation. In the discussion on these viruses serving as vectors for carrying vaccine antigen genes, emphasis has been laid on describing the progress made in using the attenuated viruses as vectors and a description of the systems in which the efficiency of immune responses has been tested.

Role of an RNA polymerase interacting protein, MsRbpA, from Mycobacterium smegmatis in phenotypic tolerance to rifampicin

Rifampicin and its derivatives are at the forefront of the current standard chemotherapeutic regimen for active tuberculosis; they act by inhibiting the transcription activity of prokaryotic RNA polymerase. Rifampicin is believed to interact with the beta subunit of RNA polymerase. However, it has been observed that protein-protein interactions with RNA polymerase core enzyme lead to its reduced susceptibility to rifampicin. This mechanism became more diversified with the discovery of RbpA, a novel RNA polymerase-binding protein, in Streptomyces coelicolor that could mitigate the effect of rifampicin on RNA polymerase activity. MsRbpA is a homologue of RbpA in Mycobacterium smegmatis. On deciphering the role of MsRbpA in M. smegmatis we found that it interacts with RNA polymerase and increases the rifampicin tolerance levels, both in vitro and in vivo. It interacts with the beta subunit of RNA polymerase. However, it was found to be incapable of rescuing rifampicin-resistant RNA polymerases in the presence of rifampicin at the respective IC50.

Identification of protein interaction regions of VINC/NEAT1/Men epsilon RNA

The virus inducible non-coding RNA (VINC) was detected initially in the brain of mice infected with Japanese encephalitis virus (JEV) and rabies virus. VINC is also known as NEAT1 or Men epsilon RNA. It is localized in the nuclear paraspeckles of several murine as well as human cell lines and is essential for paraspeckle formation. We demonstrate that VINC interacts with the paraspeckle protein, P54nrb through three different protein interaction regions (PIRs) one of which (PIR-1) is localized near the 50 end while the other two (PIR-2, PIR-3) are localized near the 30 region of VINC. Our studies suggest that VINC may interact with P54nrb through a novel mechanism which is different from that reported for protein coding RNAs. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Metabolism of ubiquinone in relation to vitamin a status in the rat

1. 1. Biosynthetic experiments in vitro with slices of livers from normal and vitamin A-deficient rats confirmed that synthesis of ubiquinone did not increase in vitamin A deficiency. 2. 2. During development of deficiency of vitamin A in the rat, there was a definite increase in the synthesis of ubiquinone at the 10-days stage but this reverted to low, initial level by 20 days and after. 3. 3. Vitamin A analogues, 3-dehydroretinal, 5,6-monoepoxyretinal and retinoic acid, which supported growth have restored ubiquinone concentration to the normal levels and relieved the lowering in its catabolism. The biologically inert 5,8-monoepoxyretinal was the least active of the analogues tested. 4. 4. The concentration and synthesis of ubiquinone in the liver decreased under conditions of hypervitaminosis A. 5. 5. The experimental evidence does not support the hypothesis of inverse relationship between vitamin A and ubiquinone synthesis.

Effect of cold exposure on the metabolism of ubiquinone in the rat

1. Accumulation of ubiquinone in the livers of rats exposed to a cold environment was shown to be due to both decreased catabolism during the entire experimental period and increased synthesis during an intermediate stage (10–20 days). 2. The increased endogenous synthesis in the cold-exposed rats was eliminated when ubiquinone accumulated in the liver after exposure for 40 days (coinciding with cclimatization), or by absorption of the exogenous dietary supply, possibly by the mechanism of end-product regulation.

Metabolism of Plasmodium berghei. I. Krebs cycle

The free parasites of Plasmodium berghei, obtained from infected cells of rats using an antiserum method, were investigated to study the operation of Krebs cycle. P. berghei was found to respire only with succinate; pyruvate, and other substrates of the Krebs cycle were not oxidized. The presence of a succinate dehydrogenase and a functioning cytochrome oxidase system was demonstrated. Cell-free extracts of free parasites showed the presence of enzymes for the utilization of C4 dicarboxylic acids; other enzymes of the Krebs cycle could not be detected. P. berghei differs from other species of Plasmodium in this respect.

Metabolism of Plasmodium bergheistar, open: III. Carbon dioxide fixation and role of pyruvate and dicarboxylic acids

Formation of C4 dicarboxylic acids in Plasmodium berghei by carbon dioxide fixation reaction has been demonstrated by the use of labeled NaH14CO3. The reactions require glucose, which may be required not only as an energy source but also to contribute to the formation of pyruvate in the process of carbon dioxide fixation. Intracellular concentration of pyruvate may play an important role in the metabolism of P. berghei; an increased intracellular level of pyruvate seems to be a prerequisite before some of these reactions could be detected. The distribution of the label indicates extensive randomization of amino acids and suggests an extensive cycling of the amino acid and organic acid pools of the parasites. This investigation formed part of the thesis submitted in 1965 for the doctoral degree at the Indian Institute of Science, Bangalore 12, India, and was supported in part by the Council of Scientific and Industrial Research, India.

Metabolism of Plasmodium bergheistar, open: II. 32Pi incorporation into high-energy phosphates

Free parasites of Plasmodium berghei were found to incorporate labeled inorganic phosphate into high-energy phosphates by substrate linked and oxidative hosphorylation. But the parasites also appear to utilize the reserve ATP of the host cells when they are within the host cells which may indicate the dependence of the parasite on the host cells for provision of energy. This investigation formed part of the thesis submitted in 1965 for the doctoral degree at the Indian Institute of Science, Bangalore 12, India, and was supported in part by the Council of Scientific and Industrial Research, India.

Studies on the metabolism of o-aminophenol purification and properties of isophenoxazine synthase from Bauhenia monandra

An enzyme which catalyzes the oxidative conversion of o-aminophenol to 2-amino-3-H-isophenoxazin-3-one has been purified 396-fold by using standard fractionation procedures. The enzyme is specific for o-aminophenol and has pH and temperature optima at 6.2 and 40 °, respectively. It is insensitive to metal chelating agents but is inhibited by several reducing substances. There is no cofactor or metal ion requirement for the reaction. A competitive type of inhibition was observed with structural analogs such as anthranilic acid and 3-hydroxyanthranilic acid. There are no free sulfhydryl groups in the enzyme, but preincubation of the enzyme with substrate or substrate analogs resulted in the liberation of titratable free sulfhydryl groups. The mechanism of biosynthesis of isophenoxazine ring is discussed.

Studies on iron metabolism in Neurospora crassa

Neurospora crassa Em 5297a secretes an ironbinding compound (X) when grown under conditions of iron deficiency. Decreasing the concentration of iron in the medium results in an increase of X and a corresponding fall in catalase activity. Under iron-deficient conditions the production of X precedes the fall in catalase activity. The iron complex of the iron-binding compound (XFe) can act as a good iron source to the organism to maintain normal growth and catalase activity, even though the iron is held very firmly in the chemical sense. While ferrichrome is as potent as XFe, as an iron source to N. crassa, ferrichrome A and ferric acethydroxamate are only partially beneficial. XFe, when provided as the sole iron source, also influences nonheme iron enzyme activities like succinic dehydrogenase and aconitase. XFe is permeable to N. crassa mycelia and is incorporated at a much faster rate compared with that from a simple chelate such as ferric citrate.

Primer-independent initiation of RNA synthesis by SeMV recombinant RNA-dependent RNA polymerase

Sesbania mosaic virus (SeMV),a single-strand positive-sense RNA plant virus, belongs to the genus Sobemoviruses. Mechanism of replication in Sobemoviruses is poorly understood. In the present study, SeMV RNA-dependent RNA polymerase (RdRp) was overexpressed and purified as a thioredoxin-tagged protein. The recombinant SeMV RdRp could synthesize RNA from genomic or subgenomic RNA templates, even in the absence ofthe protein primer, VPg. Analysis of the product indicated that it was double-stranded and that the mode of initiation was de novo. Mutational analysis of the 3' UTR of subgenomic RNA revealed that a stem-loop structure at the 3' end was important. Further, analysis of this stem-loop showed that the SeMV RdRp was capable of recognizing stem-loop structures of various lengths and forms. These results demonstrate that the SeMV RdRp is capable of primer-independent RNAsynthesis in vitro. (C) 2010 Elsevier Inc. All rights reserved.

Studies of the enzymes involved in nicotinamide adenine dinucleotide metabolism in Aspergillus niger

The enzyme nicotinamide amidase (nicotinamide amidohydrolase) was purified 57-fold from Aspergillus niger. The purified preparation was specific towards its substrate nicotinamide and did not deamidate NADP, NAD, NMN, N′-methyl nicotinamide, asparagine, glutamine, benzamide, α-naphthaleneamide and indoleacetamide. The asparagine, glutamine, benzamide, α-naphthaleneamide and indoleacetamide.vThe optimum pH was found to be 7.5. Temperature optimum was 40°. It had a Km value of 6.504 · 10−4 M towards nicotinamide. The enzyme exhibited Mg2+ ion requirement for its optimum activity. NAD-glycohydrolase (EC 3.2.2.5) was purified 109-fold from the mold. A. niger. The enzyme preparation was active only towards NAD and NADP and did not attack NMN, N′-methylnicotinamide and NADH. The Km value for NAD was found to be 7.693 · 10−6 M. The enzyme did not require any metal ion for its activity. It is suggested that A. niger will serve a better source for a large scale preparation of NAD-glycohydrolase than the Neurospora mold. The biological role of both NAD-glycohydrolase and nicotinamide amidase in the regulation of cellular NAD level has been discussed. It is, further, observed that NAD did not exert its feedback control on nicotinamide amidase at least in A. niger.