Cholesterol promotes hemifusion and pore widening in membrane fusion induced by influenza hemagglutinin

Cholesterol-specific interactions that affect membrane fusion were tested for using insect cells; cells that have naturally low cholesterol levels (< 4 mol %). Sf9 cells were engineered (HAS cells) to express the hemagglutinin (HA) of the influenza virus X-31 strain. Enrichment of HAS cells with cholesterol reduced the delay between triggering and lipid dye transfer between HAS cells and human red blood cells (RBC), indicating that cholesterol facilitates membrane lipid mixing prior to fusion pore opening. Increased cholesterol also increased aqueous content transfer between HAS cells and RBC over a broad range of HA expression levels, suggesting that cholesterol also favors fusion pore expansion. This interpretation was tested using both trans-cell dye diffusion and fusion pore conductivity measurements in cholesterol-enriched cells. The results of this study support the hypothesis that host cell cholesterol acts at two stages in membrane fusion: (1) early, prior to fusion pore opening, and (2) late, during fusion pore expansion.     

Renal cell carcinoma tumors induce T cell apoptosis through receptor-dependent and receptor-independent pathways

Tumors can promote their own progressive growth by inducing T cell apoptosis. Though previous studies suggested that tumor-mediated T cell killing is receptor dependent, we recently showed that tumor gangliosides also participate, a notion consistent with reports indicating that, in some cell types, gangliosides can activate the intrinsic apoptotic pathway by stimulating reactive oxygen species production, cytochrome c release, and caspase-9 activation. In this study, we used normal peripheral blood T cells, as well as caspase-8-, caspase-9-, and Fas-associated death domain protein-deficient Jurkat cells, to assess whether the death ligands and gangliosides overexpressed by the renal cell carcinoma (RCC) cell line SK-RC-45 can independently stimulate T cell apoptosis as a mechanism of immune escape. Anti-FasL Abs and the glycosylceramide synthase inhibitor 1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP) each partially inhibited the ability of SK-RC-45 to kill cocultured activated T cells; together, as purified molecules, RCC gangliosides and rFasL induced a more extensive mitochondrial permeability transition and greater levels of apoptosis than either agent alone, equivalent to that induced by the FasL- and ganglioside-expressing RCC line itself. rFasL-mediated apoptosis was completely inhibited in caspase-8- and Fas-associated death domain protein-negative Jurkat cells, though apoptosis induced by purified gangliosides remained intact, findings that correlate with the observed partial inhibition of SK-RC-45-induced apoptosis in the Jurkat lines with defective death receptor signaling. Western blot analysis performed on lysates made from wild-type and mutant Jurkat cells cocultured with SK-RC-45 revealed caspase activation patterns and other biochemical correlates which additionally supported the concept that tumor-associated gangliosides and FasL independently activate the caspase cascade in T cells through the intrinsic and extrinsic pathways, respectively.     

The specific amino acid sequence between helices 7 and 8 influences the binding specificity of human apolipoprotein A-I for high density lipoprotein (HDL) subclasses: a potential for HDL preferential generation

Humans have two major high density lipoprotein (HDL) sub-fractions, HDL(2) and HDL(3), whereas mice have a monodisperse HDL profile. Epidemiological evidence has suggested that HDL(2) is more atheroprotective; however, currently there is no direct experimental evidence to support this postulate. The amino acid sequence of apoA-I is a primary determinant of HDL subclass formation. The majority of the alpha-helical repeats in human apoA-I are proline-punctuated. A notable exception is the boundary between helices 7 and 8, which is located in the transitional segment between the stable N-terminal domain and the C-terminal hydrophobic domain. In this study we ask whether the substitution of a proline-containing sequence (PCS) separating other helices in human apoA-I for the non-proline-containing sequence (NPCS) between helices 7 and 8 (residues 184-190) influences HDL subclass association. The human apoA-I mutant with PCS2 replacing NPCS preferentially bound to HDL(2). In contrast, the mutant where PCS3 replaced NPCS preferentially associated with HDL(3). Thus, the specific amino acid sequence between helices 7 and 8 influences HDL subclass association. The wild-type and mutant proteins exhibited similar physicochemical properties except that the two mutants displayed greater lipid-associated stability versus wild-type human apoA-I. These results focus new attention on the influence of the boundary between helices 7 and 8 on the properties of apoA-I. The expression of these mutants in mice may result in the preferential generation of HDL(2) or HDL(3) and allow us to examine experimentally the anti-atherogenicity of the HDL subclasses.     

The GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase (PDE5) is a sensor and a sink for cGMP

We describe here a novel sensor for cGMP based on the GAF domain of the cGMP-binding, cGMP-specific phosphodiesterase 5 (PDE5) using bioluminescence resonance energy transfer (BRET). The wild type GAFa domain, capable of binding cGMP with high affinity, and a mutant (GAFa F163A) unable to bind cGMP were cloned as fusions between GFP and Rluc for BRET (2) assays. BRET (2) ratios of the wild type GAFa fusion protein, but not GAFa F163A, increased in the presence of cGMP but not cAMP. Higher basal BRET (2) ratios were observed in cells expressing the wild type GAFa domain than in cells expressing GAFa F163A. This was correlated with elevated basal intracellular levels of cGMP, indicating that the GAF domain could act as a sink for cGMP. The tandem GAF domains in full length PDE5 could also sequester cGMP when the catalytic activity of PDE5 was inhibited. Therefore, these results describe a cGMP sensor utilizing BRET (2) technology and experimentally demonstrate the reservoir of cGMP that can be present in cells that express cGMP-binding GAF domain-containing proteins. PDE5 is the target for the anti-impotence drug sildenafil citrate; therefore, this GAF-BRET (2) sensor could be used for the identification of novel compounds that inhibit cGMP binding to the GAF domain, thereby regulating PDE5 catalytic activity.     

Zn2+ enhances the molecular chaperone function and stability of alpha-crystallin

Alpha-crystallin, the major eye lens protein, is a molecular chaperone that plays a crucial role in the suppression of protein aggregation and thus in the long-term maintenance of lens transparency. Zinc is a micronutrient of the eye, but its molecular interaction with alpha-crystallin has not been studied in detail. In this paper, we present results of in vitro experiments that show bivalent zinc specifically interacts with alpha-crystallin with a dissociation constant in the submillimolar range (Kd approximately 0.2-0.4 mM). We compared the effect of Zn2+ with those of Ca2+, Cu2+, Mg2+, Cd2+, Pb2+, Ni2+, Fe2+, and Co2+ at 1 mM on the structure and chaperoning ability of alpha-crystallin. An insulin aggregation assay showed that among the bivalent metal ions, only 1 mM Zn2+ improved the chaperone function of alpha-crystallin by 30% compared to that in the absence of bivalent metal ions. Addition of 1 mM Zn2+ increased the yield of alpha-crystallin-assisted refolding of urea-treated LDH to its native state from 33 to 38%, but other bivalent ions had little effect. The surface hydrophobicity of alpha-crystallin was increased by 50% due to the binding of Zn2+. In the presence of 1 mM Zn2+, the stability of alpha-crystallin was enhanced by 36 kJ/mol, and it became more resistant to tryptic cleavage. The implications of enhanced stability and molecular chaperone activity of alpha-crystallin in the presence of Zn2+ are discussed in terms of its role in the long-term maintenance of lens transparency and cataract formation.     

Wild edible plants of the Sikkim Himalaya: Nutritive values of selected species

The wild edible plants form an important constituent of traditional diets in the Himalaya. In the Sikkim Himalaya a total of 190 species have been screened as edible species out of which nearly 47 species come to the market. The present paper deals with nutritive values of 27 most commonly consumed wild edible plants in the Sikkim Himalaya. Of 27 plant species that were analyzed for their nutritive values, 22 were edible for their fruits and five for leaves/shoots. Among different plant parts, generally higher nutrient concentration was recorded for leaves, followed by new shoots and fruits. For different species the crude fiber content ranged between 2.15–39.90%, and the total soluble salts between 4.66–21.0%, and the vitamin C content from 6-286 mg/100 g. The fat content was determined high in the fruits of Castanopsis species, Machilus edulis, and Cinnamomum species, while the protein content was highest in Hippophae rhamnoides, Cuc-umis melo, and Eleagnus latifolia. The total carbohydrate content ranged from 32-88% in the fruits of various wild edibles, the reducing sugar from 1.25–12.42%, total sugar from 2.10–25.09%, the lignin content varied from 9.05–39.51%, the hemicellulose between 25.63-55.71% and cellulose content varied from 9.57–33.19% in different species. Among the various mac-ronutrients estimated in the plant samples of different wild edible species, nitrogen was present in highest quantity, followed by potassium, calcium, magnesium, phosphorus, and sodium. Mi-cronutrients, such as iron, zinc, magnesium, and copper contents were analyzed in different plant parts of various wild edible species. The iron content was higher in leaves and new shoots. The study shows that wild edible plants are good source of nutrient for rural population, and also well comparable with various commercial fruits. It is suggested that a few wild edible species need to be grown for commercial cultivation and adopted in the traditional agroforestry systems, which will lead to reduced pressure on them in natural forest stands as well as producing economic benefits for poor farmers.     

Study on the diuretic activity of Strychnos potatorum Linn. seed extract in albino rats.

Methanol extract of Strychnos potatorum Linn. seeds (SPSE) was evaluated for its diuretic activity in Wistar albino rats. The SPSE was administered at the graded doses of 200, 400, and 600 mg/kg body weight. The parameters which were taken into account during the experimental on each rat were: total urine volume (corrected for water intake during the test period), body weight before and after the experiment, and the concentration of sodium, potassium, and chloride ions in urine. The total urine volumes of the SPSE (600 mg/kg)-treated rats were evaluated nearly two and half fold then compared with the control (saline treated) group. Excretion of cations (sodium and potassium ions) and anions (chloride ions) also increased significantly with respect to the control group. The diuretic effect was comparable with that of the standard drug Furosemide. The increase of cations in the urine on treatment with Strychnos potatorum seed extract (SPSE) was dose-dependent. This effect supports the use of the Strychnos potatorum seeds as a diuretic in folk remedies.     

Antifungal activity of Syzygium samarangense leaf extracts against Candida

Candida species are opportunistic human fungal pathogens that cause acute and chronic infections against which only few antifungal agents are available. Here we have elucidated the antifungal effect of Syzygium samarangense leaf extracts (SSLE). Antifungal activity of SSLE was studied against Candida albicans, C. krusei, C. parapsilosis, C. glabrata, C. auris and C. tropicalis. Following experiments were performed: minimum fungicidal concentration (MFC) determination, agar well disc diffusion assays, fungal morphology analysis using scanning electron microscope (SEM), ex vivo fungal survival assays on porcine tongue and skin and in vivo fungal survival assays using Drosophila melanogaster fly model. Results demonstrated MFC of SSLE ranges between 100 and 125 mg ml⁻¹. SEM images showed cell wall degradation of C. albicans when treated with SSLE. Around 75% decrease in C. albicans viability was observed when infected porcine tongue and skin were treated using SSLE. The C. albicans infected D. melanogaster when fed with SSLE showed significant decrease (around 80%) of fungal count than the infected control. Furthermore, agar plate disc diffusion assays demonstrated that the antifungal activity of SSLE could be due to chalcone, which is one of the active constituents in SSLE. Our study demonstrated that SSLE could be used for the topical treatment of Candida infections.