Fluidity,permeability and antioxidant behaviour of model membranes incorporated with α-tocopherol and vitamin E acetate

Magnetic resonance studies reveal a marked difference between the binding of α-tocopherol and that of the corresponding acetate (vitamin E acetate) with dipalmitoylphosphatidylcholine (DPPC) vesicles. This is reflected in differences in the phase-transition curves of the DPPC vesicles incorporated with the two compounds, as well as in the ¹³C relaxation times and line widths. A model for the incorporation of these molecules in lipid bilayers has been suggested. α-Tocopherol binds strongly with the lipids, possibly through a hydrogen bond formation between the hydroxyl group of the former and one of the oxygen atoms of the latter. The possibility of such a hydrogen bond formation is excluded in vitamin E acetate, which binds loosely through the normal hydrophobic interaction. The model for lipid-vitamin interaction explains the in vitro decomposition of H₂O₂ by α-tocopherol. α-Tocopherol in conjuction with H₂O₂ can also act as a free-radical scavenger in the lipid phase. The incorporation of α-tocopherol and vitamin E acetate in DPPC vesicles enhances the permeability of lipid bilayers for small molecules such as sodium ascorbate.     

Suppression of Oxygen Evolving System in Spinach Chloroplast Membranes due to Release of Manganese on Exposure to Osmotic Stress in vitro in Presence of Magnesium

When spinach chloroplast membranes were exposed to osmotic stress in vitro, by incubation in 1.0 M sorbitol + 10 mM MgCl₂ their oxygen evolving system was suppressed. The possible reasons for such inactivation of PS II mediated oxygen evolution were examined. There were conformational changes in the chloroplast membranes, as indicated by their absorption spectra. The pattern of sensitivity to DCMU was not altered. The sensitivity of PS II to water stress remained, even after a pre-wash treatment with NaCI (which removed 18 and 24 kD proteins) but not when the thylakoids were pretreated with NH₂0H or CaCl₂ (removed manganese and 33 kD). The manganese content of thylakoid membranes was markedly reduced under osmotic stress in presence of magnesium. We suggest that exposure of chloroplasts to 1.0 M sorbitol in presence of Mg₂₊ released manganese from thylakoid membranes, thereby leading to a suppression in oxygen evolution.     

Improved ethanol tolerance and production in strains of Clostridium thermocellum

Two Clostridium thermocellum strains were improved for ethanol tolerance, to 5% (v/v), by gradual adaptation and mutation. The best mutant gave an ethanol yield of 0.37 g/g substrate, with a growth yield 1.5 times more than its parent. Accumulation of acids and reducing sugars by the mutant strain with 5% (v/v) ethanol was lower than that of the parent strain with 1.5% (v/v) ethanol.     

Epididymal histoplasmosis diagnosed by isolation ofHistoplasma capsulatum from semen

An autochthonous case of epididymal histoplasmosis masquerading as tuberculosis in a 55-year-old male patient is reported from India. It was diagnosed by culture ofHistoplasma capsulatum from semen and by demonstration of the fungus upon re-examination of epididymal biopsy sections previously misinterpreted as tuberculous granuloma. The patient's main complaints were painful epididymal swelling, occasional fever and cough. He was treated successfully by excision of epididymis and vas deferens combined with amphotericin B therapy. This is believed to be the first case of epididymal histoplasmosis to be reported outside the American continent and the fourth of its type reported in the English literature. The case is also noteworthy in thatH. capsulatum was isolated for the first time from semen, and it underlines the importance of mycological culture of semen specimens for diagnosis of genitourinary infections of obscure etiology.Presented at the XII Congress of the International Society for Human and Animal Mycology, Adelaide, Australia, March 13–18, 1994.     

A general synthesis of 4-alkyl/aryl substituted saccharins

A new method for the synthesis of 4-alky/aryl substituted saccharins (2) has been developed. The key steps include conjugate addition/acylation of cyclohexenone (3) and reaction of the resulting β-ketoesters 4 with benzyl mercaptan to give the vinyl sulfides 5 and 6. The latter were converted to the saccharins 2 in high yield.     

Inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl during greening of groundnut leaves

The site of inhibition of chlorophyll biosynthesis by α′,α′-dipyridyl was found to be at the level of conversion of chlorophyllide (672 nm) to chlorophyll (678 nm) during greening of groundnut leaves. This inhibition was partially reversed by certain divalent cations.     

Motifs Q and I are required for ATP hydrolysis but not for ATP binding in SWI2/SNF2 proteins

Active DNA-dependent ATPase A Domain (ADAAD) is a SWI2/SNF2 protein that hydrolyzes ATP in the presence of stem-loop DNA that contains both double-stranded and single-stranded regions. ADAAD possesses the seven helicase motifs that are a characteristic feature of all the SWI2/SNF2 proteins present in yeast as well as mammalian cells. In addition, these proteins also possess the Q motif ~17 nucleotides upstream of motif I. Using site-directed mutagenesis, we have sought to define the role of motifs Q and I in ATP hydrolysis mediated by ADAAD. We show that in ADAAD both motifs Q and I are required for ATP catalysis but not for ATP binding. In addition, the conserved glutamine present in motif Q also dictates the catalytic rate. The ability of the conserved glutamine present in motif Q to dictate the catalytic rate has not been observed in helicases. Further, the SWI2/SNF2 proteins contain a conserved glutamine, one amino acid residue downstream of motif I. This conserved glutamine, Q244 in ADAAD, also directs the rate of catalysis but is not required either for hydrolysis or for ligand binding. Finally, we show that the adenine moiety of ATP is sufficient for interaction with SWI2/SNF2 proteins. The γ-phosphate of ATP is required for inducing the conformational change that leads to ATPase activity. Thus, the SWI2/SNF2 proteins despite sequence conservation with helicases appear to behave in a manner distinct from that of the helicases.     

Riboswitch Detection Using Profile Hidden Markov Models

Background  

Riboswitches are a type of noncoding RNA that regulate gene expression by switching from one structural conformation to another on ligand binding. The various classes of riboswitches discovered so far are differentiated by the ligand, which on binding induces a conformational switch. Every class of riboswitch is characterized by an aptamer domain, which provides the site for ligand binding, and an expression platform that undergoes conformational change on ligand binding. The sequence and structure of the aptamer domain is highly conserved in riboswitches belonging to the same class. We propose a method for fast and accurate identification of riboswitches using profile Hidden Markov Models (pHMM). Our method exploits the high degree of sequence conservation that characterizes the aptamer domain.     

Production of Recombinant Human Bile Salt Stimulated Lipase and Its Variant inPichia pastoris

hBSSL and its truncated variant hBSSL-C cDNA clones were expressed inPichia pastorisusing two different signal peptides, native signal peptide and invertase signal peptide, respectively, to facilitate secretion of the recombinant proteins into the culture medium. Both recombinant proteins were secreted into the culture medium to a level of 45–50 mg/liter in shake flask cultures. Native signal peptide of hBSSL was recognized inP. pastorisand was cleaved at the same site as in humans. The level of expression of the hBSSL gene was found to be dependent on the number of its copies integrated into the host chromosome. The multicopy transformant clone was found to be very stable. When grown and induced in a fermentor, the level of accumulation of the recombinant hBSSL in the culture medium improved from 50 mg/liter in shake flask cultures to 300 mg/liter. The recombinant hBSSL purified from the culture supernatant was found to be similar to the native hBSSL in its biochemical properties except for the lectin-binding profile.     

The interaction site of Flap Endonuclease-1 with WRN helicase suggests a coordination of WRN and PCNA

Werner and Bloom syndromes are genetic RecQ helicase disorders characterized by genomic instability. Biochemical and genetic data indicate that an important protein interaction of WRN and Bloom syndrome (BLM) helicases is with the structure-specific nuclease Flap Endonuclease 1 (FEN-1), an enzyme that is implicated in the processing of DNA intermediates that arise during cellular DNA replication, repair and recombination. To acquire a better understanding of the interaction of WRN and BLM with FEN-1, we have mapped the FEN-1 binding site on the two RecQ helicases. Both WRN and BLM bind to the extreme C-terminal 18 amino acid tail of FEN-1 that is adjacent to the PCNA binding site of FEN-1. The importance of the WRN/BLM physical interaction with the FEN-1 C-terminal tail was confirmed by functional interaction studies with catalytically active purified recombinant FEN-1 deletion mutant proteins that lack either the WRN/BLM binding site or the PCNA interaction site. The distinct binding sites of WRN and PCNA and their combined effect on FEN-1 nuclease activity suggest that they may coordinately act with FEN-1. WRN was shown to facilitate FEN-1 binding to its preferred double-flap substrate through its protein interaction with the FEN-1 C-terminal binding site. WRN retained its ability to physically bind and stimulate acetylated FEN-1 cleavage activity to the same extent as unacetylated FEN-1. These studies provide new insights to the interaction of WRN and BLM helicases with FEN-1, and how these interactions might be regulated with the PCNA–FEN-1 interaction during DNA replication and repair.