The Saccharomyces cerevisiae YLL012/YEH1, YLR020/YEH2, and TGL1 genes encode a novel family of membrane-anchored lipases that are required for steryl ester hydrolysis

Sterol homeostasis in eukaryotic cells relies on the reciprocal interconversion of free sterols and steryl esters. The formation of steryl esters is well characterized, but the mechanisms that control steryl ester mobilization upon cellular demand are less well understood. We have identified a family of three lipases of Saccharomyces cerevisiae that are required for efficient steryl ester mobilization. These lipases, encoded by YLL012/YEH1, YLR020/YEH2, and TGL1, are paralogues of the mammalian acid lipase family, which is composed of the lysosomal acid lipase, the gastric lipase, and four novel as yet uncharacterized human open reading frames. Lipase triple-mutant yeast cells are completely blocked in steryl ester hydrolysis but do not affect the mobilization of triacylglycerols, indicating that the three lipases are required for steryl ester mobilization in vivo. Lipase single mutants mobilize steryl esters to various degrees, indicating partial functional redundancy of the three gene products. Lipase double-mutant cells in which the third lipase is expressed from the inducible GAL1 promoter have greatly reduced steady-state levels of steryl esters, indicating that overexpression of any of the three lipases is sufficient for steryl ester mobilization in vivo. The three yeast enzymes constitute a novel class of membrane-anchored lipases that differ in topology and subcellular localization.     

Role of Salmonella surface components in immunomodulation of inflammatory mediators

Salmonella enterica serovar Typhimurium and its surface components were assessed for their inflammatory potential by footpad oedema test using plethysmometer. Inflammation was found to be the highest when outer membrane proteins (OMPs) were used as inflammagen followed by lipid associated protein-lipopolysaccharide complex (LAP-LPS) and lipopolysaccharides (LPS). Inflammation produced by OMPs was found to be comparable to that by carrageenan (a known positive inflammagen). However, injection of L-histidine (an antioxidant) prior to administration of carrageenan or Salmonella enterica serovar Typhimurium inhibited the inflammation, which indicated the involvement of oxidants during inflammatory response. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and nitric oxide (NO) production by peritoneal macrophages from infected mice exhibited a significant increase as compared to those of the immunized mice. In contrast, glutathione production was found to be the maximum in the macrophages taken from OMPs-immunized mice followed by LAP-LPS and LPS alone. The biochemical studies correlated well with histopathological studies of intestinal tissue of animals from various groups. Based upon these parameters, inflammation seems to be modulated by OMPs and LAP-LPS, which may be because of the protein moieties present in the components. Hence, immunization with protein moieties having L-histidine or L-histidine-like structures may suggest an alternative to the potential therapeutic values of anti-inflammatory drugs. Thus the results of this study form the basis for evaluating these antigens (either alone or in combination with polysaccharides) for preventive intervention rather than therapeutic. (Mol Cell Biochem 270: 167–175, 2005)     

Carbonic anhydrase in relation to higher plants

The review incorporates recent information on carbonic anhydrase (CA, EC: 4.2.1.1) pertaining to types, homology, regulation, purification, in vitro stability, and biological functions with special reference to higher plants. CA, a ubiquitous enzyme in prokaryotes and higher organisms represented by four distinct families, is involved in diverse biological processes, including pH regulation, CO₂ transfer, ion exchange, respiration, and photosynthetic CO₂ fixation. CA from higher plants traces its origin with prokaryotes and exhibits compartmentalization among their organs, tissues, and cellular organelles commensurate with specific functions. In leaves, CA represents 1–20 % of total soluble protein and abundance next only to ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) in chloroplast, facilitating CO₂ supply to phosphoenol pyruvate carboxylase in C₄ and CAM plants and RuBPCO in C₃ plants. It confers special significance to CA as an efficient biochemical marker for carbon sequestration and environmental amelioration in the current global warming scenario linked with elevated CO₂ concentrations.     

Rab11 is required during Drosophila eye development

In an effort to identify the role of Rab11, a small GTP binding protein, during Drosophila differentiation, phenotypic manifestations associated with different alleles of Rab11 were studied. The phenotypes ranged from eye-defects, bristle abnormalities and sterility to lethality during various developmental stages. In this paper, our focus is targeted on eye defects caused by Rab11 mutations. A novel P-element insertion in the Rab11 locus, Rab11mo, displayed characteristic retinal anomalies, which could be reverted by P-element excision and expression of Rab11+ transgenes. During larval development, Rab11 is widely synthesized in photoreceptor cells and localizes to the rhabdomeres and lamina neuropil in adult eyes. Photoreceptors and associated bristles failed to be formed in homozygous clones generated in Rab11EP(3)3017 eyes. Decreased levels of Rab11 protein and increased cell death in Rab11mo third-instar larval eye-antennal discs suggest that the retinal defects originate during larval development. Our data indicate a requirement for Rab11 in ommatidial differentiation during Drosophila eye development.     

Chemical and enzymatic synthesis of 4'-thio-beta-D-arabinofuranosylcytosine monophosphate and triphosphate

N4-Acetyl-1-(2, 3-di-O-acetyl-4-thio-beta-D-arabinofuranosyl) cytosine (2) was synthesized in three steps from 1-(4-thio-beta-D-arabinofuranosyl) cytosine (1). The reaction of this partially blocked 4'-thio-ara-C derivative 2 with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one gave the 5-phosphitylate derivative 3, which on reaction with pyrophosphate gave the 5'-nucleosidylcyclotriphosphite 4. Product 4 was then oxidized with iodine/pyridine/water and deblocked with concentrated ammonium hydroxide to provide the desired 4'-thio-ara-C-5'-triphosphate 5. This triphosphate 5 was converted to 4'-thio-ara-C -5'-monophosphate 6 by treatment with snake venom phosphodiesterase I. The details of the synthesis, purification, and characterization of both nucleotides are described.     

Synthesis of some 2'-fluoro-2'-deoxy-3'-C-ethynyl and 3'-C-vinyl-beta-D-lyxofuranosyl nucleosides

1-(2-Fluoro-2-deoxy-beta-D-drabinofuranosyl) uracil (5) and 1-(2-fluoro-2-deoxy-beta-D-arabinofuranosyl)cytosine (6) were synthesized as reported earlier. Both of these compounds were converted into 2'-fluoro-2'-deoxy-3'-C-ethynyl and 3'-C-vinyl-beta-D-lyxofuranosyl nuclearsides (16-19) by a multistep sequence. All these new nucleosides were evaluated against seven human tumor cell lines in vitro.     

Differential role of the alpha1C subunit tails in regulation of the Cav1.2 channel by membrane potential, beta subunits, and Ca2+ ions

Voltage-gated Ca(v)1.2 channels are composed of the pore-forming alpha1C and auxiliary beta and alpha2delta subunits. Voltage-dependent conformational rearrangements of the alpha1C subunit C-tail have been implicated in Ca2+ signal transduction. In contrast, the alpha1C N-tail demonstrates limited voltage-gated mobility. We have asked whether these properties are critical for the channel function. Here we report that transient anchoring of the alpha1C subunit C-tail in the plasma membrane inhibits Ca2+-dependent and slow voltage-dependent inactivation. Both alpha2delta and beta subunits remain essential for the functional channel. In contrast, if alpha1C subunits with are expressed alpha2delta but in the absence of a beta subunit, plasma membrane anchoring of the alpha1C N terminus or its deletion inhibit both voltage- and Ca2+-dependent inactivation of the current. The following findings all corroborate the importance of the alpha1C N-tail/beta interaction: (i) co-expression of beta restores inactivation properties, (ii) release of the alpha1C N terminus inhibits the beta-deficient channel, and (iii) voltage-gated mobility of the alpha1C N-tail vis a vis the plasma membrane is increased in the beta-deficient (silent) channel. Together, these data argue that both the alpha1C N- and C-tails have important but different roles in the voltage- and Ca2+-dependent inactivation, as well as beta subunit modulation of the channel. The alpha1C N-tail may have a role in the channel trafficking and is a target of the beta subunit modulation. The beta subunit facilitates voltage gating by competing with the N-tail and constraining its voltage-dependent rearrangements. Thus, cross-talk between the alpha1C C and N termini, beta subunit, and the cytoplasmic pore region confers the multifactorial regulation of Ca(v)1.2 channels.     

Inhibition of chaperone activity is a shared property of several Cu,Zn-superoxide dismutase mutants that cause amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron degeneration, paralysis, and death. Mutant Cu,Zn-superoxide dismutase (SOD1) causes a subset of ALS by an unidentified toxic property. Increasing evidence suggests that chaperone dysfunction plays a role in motor neuron degeneration in ALS. To investigate the relationship between mutant SOD1 expression and chaperone dysfunction, we measured chaperone function in central nervous system tissue lysates from normal mice and transgenic mice expressing human SOD1 variants. We observed a significant decrease in chaperone activity in tissues from mice expressing ALS-linked mutant SOD1 but not control mice expressing human wild type SOD1. This decrease was detected only in the spinal cord, became apparent by 60 days of age (before the onset of muscle weakness and significant motor neuron loss), and persisted throughout the late stages. In addition, this impairment of chaperone activity occurred only in cytosolic but not in mitochondrial and nuclear fractions. Furthermore, multiple recombinant human SOD1 mutants with differing biochemical and biophysical properties inhibited chaperone function in a cell-free extract of normal mouse spinal cords. Thus, mutant SOD1 proteins may impair chaperone function independent of gene expression in vivo, and this inhibition may be a shared property of ALS-linked mutant SOD1 proteins.