Photoinactivation of δ-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The -aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of -ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.Abbreviations ALAD -aminolevulinic acid dehydratase - FMN flavin mononucleotide - O₂ ^- superoxide - H₂O₂ hydrogen peroxide - 10₂ singlet oxygen - LA levulinic acid - PBG porphobilinogen - BSA bovine serum albumin - BME 2-mercaptoethanol - SOD superoxide dismutase - pHMB para-hydroxymercuribenzoate - DTT dithiothreitol - FAD flavin adenine dinucleotide - NADH nicotinamide adenine dinucleotide     

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.     

Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

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.     

π–π Interactions in Structural Stability: Role in RNA Binding Proteins

RNA binding proteins play significant roles in many bio-macromolecular systems. Aromatic amino acid residues are vital for several biological functions. In the present work, the influences of π–π interactions in RNA binding proteins are analyzed. There are a total of 3,396 π-residues in RNA binding proteins out of which 1,547, 1,241, and 608 are phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp), respectively. Among these 945, 634, and 356 Phe, Tyr, and Trp residues, respectively, are involved in π–π interactions. The observations indicate that majority of the aromatic residues in RNA binding proteins are involved in π–π interactions. Side chain–side chain π–π interactions are the predominant type of interactions in RNA binding proteins. These π–π interactions stabilize the core regions within RNA binding proteins. π–π interacting residues are evolutionary conserved. Residue-wise analysis indicates that π–π interacting residues have higher long-range contacts and hence they are important in the global conformational stability of these proteins.     

Cation–π Interactions in β-Lactamases: The Role in Structural Stability

β-lactam group of antibiotics is the most widely used therapeutic molecules for treating bacterial infections. The main mode of bacterial resistance to β-lactams is by β-lactamases. In the present study, we report our results on the role of cation–π interactions in β-lactamases and their environmental preferences. The number of interactions formed by arginine is higher than lysine in the cationic group, while tyrosine is comparatively higher than phenylalanine and tryptophan in the π group. Our results indicate that cation–π interactions might play an important role in the global conformational stability of β-lactamases.