The alpha helix dipole: screened out?

Aligned alpha helix peptide dipoles sum to a "macroscopic" dipole parallel to the helix axis that has been implicated in protein folding and function. However, in aqueous solution the dipole is counteracted by an electrostatic reaction field generated by the solvent, and the strength of the helix dipole may reduce drastically from its value in vacuum. Here, using atomic-detail helix models and Poisson-Boltzmann continuum electrostatics calculations, the net effective dipole moment, mu(eff), is calculated. Some initially surprising results are found. Whereas in vacuum mu(eff) increases with helix length, the opposite is found to be the case for transmembrane helices. In soluble proteins, mu(eff) is found to vary strongly with the orientation and position of the helix relative to the aqueous medium. A set of rules is established to estimate of the strength of mu(eff) from graphical inspection of protein structures.     

Identification and molecular characterization of the rainbow trout calpains (Capn1 and Capn2): their expression in muscle wasting during starvation

Calpains are calcium regulated proteases involved in cellular functions that include muscle proteolysis both ante- and postmortem. Here, we describe the molecular characterization of the rainbow trout catalytic subunits of the mu- and m-calpains, respectively. The cDNA sequence for Capn1 encodes a protein of 704 amino acids with a calculated molecular mass of 79.9 kDa. The amino acid sequence shows 66% and 86% identity with the mouse and zebrafish Capn1, respectively. The Capn2 cDNA codes for a protein consisting of 701 amino acid residues with a calculated molecular mass of 78.2 kDa. The protein shows 65% amino acid sequence identity with the mouse and chicken Capn2. The two isozymes of rainbow trout have the characteristic domains: I (propeptide), II (cysteine catalytic site), III (electrostatic switch), and IV (contains five EF-hands). Because starvation induces muscle wasting, the hypothesis of this study was that starvation could affect regulation of the calpain system in muscle. Starvation of rainbow trout fingerlings (15-20 g) for 35 days stimulated the expression of Capn1 (2.2-fold increase, P < 0.01), Capn2 (6.0-fold increase, P < 0.01), and calpastatins (1.6-fold increase, P < 0.05) as measured by quantitative real-time RT-PCR. The mRNA changes led to a 1.23-fold increase in the calpain catalytic activity. The results suggest a potential role of calpains in protein mobilization as a source of energy under fasting condition.     

Cysteine and serine protease-mediated proteolysis in body homogenate of a zooplankter, Moina macrocopa, is inhibited by the toxic cyanobacterium, Microcystis aeruginosa PCC7806

The paper describes the characterization of proteases in the whole body homogenate of Moina macrocopa, which can possibly be inhibited by the extracts of Microcystis aeruginosa PCC7806. With the use of oligopeptide substrates and specific inhibitors, we detected the activities of trypsin, chymotrypsin, elastase and cysteine protease. Cysteine protease, the predominant enzyme behind proteolysis of a natural substrate, casein, was partially purified by gel filtration. The substrate SDS-polyacrylamide gel electrophoresis of body homogenate revealed the presence of nine bands of proteases (17-72 kDa). The apparent molecular mass of an exclusive cysteine protease was 60 kDa, whereas of trypsin, it was 17-24 kDa. An extract of M. aeruginosa PCC7806 significantly inhibited the activities of trypsin, chymotrypsin and cysteine protease in M. macrocopa body homogenate at estimated IC(50) of 6- to 79-microg dry mass mL(-1). Upon fractionation by C-18 solid-phase extraction, 60% methanolic elute contained all the protease inhibitors, and these metabolites could be further separated by reverse-phase liquid chromatography. The metabolites inhibitory to M. macrocopa proteases also inhibited the corresponding class of proteases of mammalian/plant origin. The study suggests that protease inhibition may contribute to chemical interaction of cyanobacteria and crustacean zooplankton.     

Seasonal effects of melatonin on ovary and plasma gonadotropin and vitellogenin levels in intact and pinealectomized catfish, Clarias batrachus (Linn)

Effects of daily administration of melatonin for 15 days were evaluated with respect to ovarian activities and plasma gonadotropin (GtH II) and vitellogenin (Vg) levels in intact (INT) and pinealectomized (Px) female catfish, C. batrachus, during preparatory (April), prespawning (May and June), spawning (July) and post-spawning (September) periods. Px (saline control groups) caused a stimulatory effect during preparatory (with respect to Vg synthesis and incorporation) and prespawning (with respect to Vg synthesis) periods whereas no effect was observed during spawning and post-spawning periods with respect to the reproductive parameters studied. During April, melatonin-treatment significantly decreased plasma GtH II levels and percentage of vitellogenic oocytes without any significant changes in plasma Vg levels and gonadosomatic index (GSI). During early prespawning period, in May, 50microg melatonin brought about a significant reduction in plasma GtH II levels in INT group, whereas 100microg caused a decrease in all parameters; on the other hand, in Px groups both dose levels proved to be inhibitory. In June (late prespawning period) melatonin-treatment could not bring about any change in GSI and plasma Vg levels compared to the control groups regardless of Px but plasma GtH II and mean number of yolky oocytes were significantly reduced in melatonin-treated INT group. During spawning period (July) melatonin inhibited the GSI, mean number of yolky oocytes and plasma GtH II levels without affecting plasma Vg levels. In September (post-spawning period), melatonin did inhibit both GSI and plasma GtH II levels. The results, thus, indicate that melatonin showed variable effects (inhibitory and/or no effect) to GSI, mean number of yolky oocytes and plasma Vg levels but a consistent inhibiton of plasma GtH II levels indicating that melatonin may control the reproduction by blocking the GtH II release from the pituitary via affecting the hypothalamo-hypophysial axis.     

Studies on Trypsin Inhibitor in Bean (Phaseolus vulgaris L) Cultivars of Himalayan Region

Eight Phaseolus vulgaris L cultivars of Himalayan region were analyzed for trypsin inhibitor activity and inhibition of gut trypsin enzyme extracted from Spodoptera littoralis larvae. Trypsin unit inhibited per gram seed weight was maximum in local yellow cultivar. The trypsin inhibitor was purified to 65.9-fold with 55.6% recovery from seeds of selected cultivar. The purified protein had a molecular weight of 14,130 Daltons and was found to be a monomer by SIDS-PAGE. It was heat stable at 100°C for 10 minutes and had a pH optimum of 7.5. Hence, the purified inhibitor appears to be of Bowman-Birk type. It lost its activity on exposure to 0.2M 2-mercaptoethanol. The inhibition pattern was of non-competitive type and the Ki value was 0.8μM. The KM value of trypsin enzyme for the substrate BApMA was 2.2mM.     

Identification of an antimicrobial entity from the cyanobacterium Fischerella sp. isolated from bark of Azadirachta indica (Neem) tree

The active principle in a methanolic extract of the laboratory-grown cyanobacterium, Fischerella sp. isolated from Neem (Azadirachta indica) tree bark was active against Mycobacterium tuberculosis, Enterobacter aerogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhi, Escherichia coli as well as three multi-drug resistant E. coli strains in in vitro assays. Based on MS, UV, IR ¹H NMR analyses the active principle is proposed to be hapalindole T having the empirical formula C₂₁H₂₃N₂ClSO and a molecular weight of 386 with the melting point range 179–182 ^°C. The estimated production of Hapalindole T from the cyanobacterium is 1.25 mg g⁻¹ lyophilized biomass. It is suggested that cyanobacteria colonizing specialized niches such as tree bark could be an antibacterial drug resource.