Leishmania donovani: intracellular ATP level regulates apoptosis-like death in luteolin induced dyskinetoplastid cells

Leishmaniasis presents a spectrum of diseases ranging from benign cutaneous lesions to the often-fatal visceralizing form. Luteolin, a dietary flavone induces apoptosis-like death in both promastigote and amastigote forms of Leishmania, the causative agent of the diseases. Here, we have elucidated the mechanism of action of luteolin by analyzing the mitochondrial and cytosolic changes associated with apoptosis-like death of leishmanial cells. In Leishmania donovani, treatment with luteolin induces the loss of both maxicircles and minicircles which resulted in the formation of dyskinetoplastid cells. The loss of mitochondrial DNA causes reduction in the activities of complex I, II, III, and IV of electron transport chain. However, the mitochondrial ATPase activity of complex V remains almost unaltered during treatment with luteolin but the sensitivity to oligomycin is lost. The inactivation of ETC complex is associated with decrease in mitochondrial as well as glycolytic ATP production, which is responsible for depolarization of Deltapsi(m) and alteration in mitochondrial structure. This event is followed by the release of cytochrome c from mitochondria in mt-DNA depleted leishmanial cells and causes an activation of caspase like proteases. Collectively our results provide the first insight into the mechanistic pathway of apoptosis-like death where inhibition of glycolytic ATP production is an essential event responsible for depolarization of Deltapsi(m) in mt-DNA depleted cells to propagate apoptosis-like death in leishmanial cells.     

Rheology and composition of a multi-utility exopolymer from a desert borne cyanobacterium Anabaena variabilis

The desert cyanobacterium Anabaena variabilis produces an exopolymer during the stationary growth phase in batch culture. Optimal polymer production was observed at pH?10 under phosphorus limitation. Chemical analysis showed it to be composed of 49% carbohydrate and 19% protein. Monosaccharide analysis revealed a heteropolysaccharidic nature with glucose, mannose, and galactose as the main neutral sugars. Infrared (IR) spectrum of the exopolymer showed absorption bands at 1,645 and 1,421?cm^?1 characteristic of C=O in the carboxylate group. Strong band was observed at 1,072?cm^?1 due to C–O–C or C–O–P stretching vibrations. A band at 2,363?cm^?1 corresponding to C–H stretch of protein was also observed. IR spectrum suggested that the exopolymer is nonsulfated. Rheological properties of the polymer showed marked shear thinning non-Newtonian behavior in the concentration range of 0.1–0.4%. However, it appeared to undergo change in the internal structure on shearing thereby exhibiting thixotropic behavior. The polymer possessed 75% flocculating ability vis a vis alum, 71% emulsification of hexadecane, and good thermal stability making it a potent candidate for multiple industrial applications. The exopolymer bound 156?g H₂O g^?1 and exhibited antibacterial activity against Staphylococcus aureus suggesting a potential for application in wound management as well.     

Spectroscopic investigations to reveal the nature of interactions between the haem protein myoglobin and the dye rhodamine 6G

In the present investigation, steady‐state and time‐resolved fluorescence with the combination of circular dichroism (CD) spectroscopic techniques were applied to study the interactions of the well‐known dye rhodamine 6 G (R6G) with the haem protein human myoglobin (Mb). From the analysis of the results it appears that the static type of fluorescence quenching mechanism is primarily involved, due to ground‐state interactions. Although considerable overlapping of fluorescence emission of the dye R6G with the absorption of Mb in the Q‐band region exists, the possibility of occurrences of the excitational singlet–singlet non‐radiative energy transfer process from R6G to Mb appears to be unlikely, according to time‐resolved fluorescence measurements. From the determinations of the thermodynamic parameters, it was apparent that the combined effect of van der Waals' interactions and hydrogen bonding plays a vital role in Mb–R6G interactions. Induced circular dichroism (ICD) studies demonstrate the possibility of interactions between R6G and Mb. The binding constants, number of binding sites and thermodynamic parameters have been computed. From CD measurements it is apparent that the binding of the dye R6G with the haem protein Mb induces negligible conformational changes in the protein and Mb retains its secondary structure and helicity when it interacts with R6G. The present detailed studies on the interactions with Mb should be helpful in further advancement of medical diagnostics and biotechnology. Copyright © 2011 John Wiley & Sons, Ltd.     

Overexpression of a delayed early gene hlg1 of temperate mycobacteriophage L1 is lethal to both M. smegmatis and E. coli

Two genes of temperate mycobacteriophage L5, namely, gp63 and gp64, were hypothesized to be toxic to M. smegmatis. An identical L5 gp64 ortholog (designated hlg1) was cloned from homoimmune mycobacteriophage L1 and characterized at length here. As expected, hlg1 affected the growth of M. smegmatis when overexpressed from a resident plasmid. HLG1 (the protein encoded by hlg1) in fact caused growth retardation of M. smegmatis and the region encompassing its 57-114 C-terminal amino acid residues was found indispensable for its growth-retardation activity. Both nucleic acid and protein biosynthesis were severely impaired in M. smegmatis expressing HLG1. Interestingly, HLG1 also affected E. coli almost similarly. This putative delayed early lipoprotein did not participate in the lytic growth of L1.     

Characterization of osteoblastic properties of 7F2 and UMR-106 cultures after acclimation to reduced levels of fetal bovine serum

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated the development of a culturing environment reduced in estrogenic compounds. The selected medium (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red, a suspected estrogenic agent. This protocol reduced the concentration of FBS supplementation to 0% through successive 24 h incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast-like phenotype of 7F2 and UMR-106 cell lines when compared with control cells grown in various concentrations of FBS. Although the rate of mitotic divisions declined, the 7F2 and UMR-106 cultures continued to express osteoblast-specific markers and exhibited estrogen responsiveness. These experimental findings demonstrate that the culture protocol developed did not alter the osteoblast nature of the cell lines and provides a model system to study estrogen's antiresorptive role on skeletal turnover.     

Immunomodulatory effect of Tylophora indica on Con A induced lymphoproliferation.

Our preliminary studies with tylophora alkaloids had shown that they inhibit cellular immune responses like contact sensitivity to dinitro-flurobenzene and delayed hypersensitivity to sheep red blood cells, in vivo. Investigations were hence carried out to determine the cellular targets of tylophora alkaloids in in vitro systems. Con A induced proliferation of splenocytes was used as a model system to study the effect of the alkaloids on cellular immune responses. The alkaloid mixture was found to inhibit proliferation of splenocytes at higher concentrations and augment the same at lower concentrations. Both macrophages and T cells were found to be vulnerable to tylophora alkaloids. The alkaloid mixture suppressed IL-2 production in Con A stimulated splenocytes at the inhibitory or higher concentrations and enhanced production at the lower concentrations. IL-1 production by activated macrophages on the contrary was doubled in the presence of inhibitory concentrations of tylophora. These studies indicate that tylophora alkaloids have a concentration dependent biphasic effect on Con A induced mitogenesis. At lower concentrations they augment Con A induced lymphoproliferation by enhancing IL-2 production. Inhibition of proliferation at higher concentrations of the alkaloid is due to inhibition of IL-2 production and activation of macrophages, which have a cytostatic effect.     

High herkogamy but low reciprocity characterizes isoplethic populations of Jasminum malabaricum,a species with stigma‐height dimorphism

• Studies of floral polymorphisms have focused on heterostyly, while stigma‐height dimorphism has received considerably less attention. Few studies have examined the reproductive biology of species with stigma‐height dimorphism to understand how factors influencing mate availability and pollen transfer are related to morph ratios in populations.
• Floral morphological traits, especially herkogamy and reciprocity, pollinator visitation, breeding system and spatiotemporal mate availability, are known to affect inter‐morph pollination and morph ratios in species with stigma‐height dimorphism. In this study, we investigated the presence of stigma‐height dimorphism and estimated morph ratios in four naturally occurring populations of Jasminum malabaricum. We quantified morph‐ and population‐specific differences in the abovementioned factors in these populations to understand the observed morph ratios.
• The positions of anthers and stigmas were characteristic of stigma‐height dimorphism, the first report of this polymorphism in the genus. All study populations were isoplethic, implying equal fitness of both morphs. Herkogamy was higher in the short‐styled morph, while reciprocity was higher between the long‐styled stigma and short‐styled anthers. Long‐ and short‐tongued pollinators were common floral visitors, and we observed no differences between morphs in spatiotemporal mate availability or pollinator visitation. Neither morph exhibited self‐ or heteromorphic incompatibility.
• The short‐styled stigma had lower reciprocity but likely receives sufficient inter‐morph pollen from long‐tongued pollinators, and also by avoiding self‐pollination due to higher herkogamy. These results highlight the importance of sufficient effective pollinators and floral morphological features, particularly herkogamy, in maintaining isoplethy in species with stigma‐height dimorphism.

     

Studies of the distribution of vitamin A as ester and alcohol and of carotenoids in plasma proteins of several species

The distribution of vitamin A ester, alcohol, and lutein was studied in fractions of chicken plasma proteins obtained by ammonium sulfate fractionation or dialysis, 6–8 hr. after oral administration of vitamin A and lutein in an oily medium. The losses of vitamin A and carotenoids during fractionation were considerable in some cases, losses of vitamin A being as large as 50% at times. Although this made the interpretation of the distribution between precipitate and supernatant difficult, the following conclusions seem to be possible. Vitamin A ester was found to be associated with the least soluble, and vitamin A alcohol and lutein with the more soluble protein fractions. No mono- or diesters of lutein could be demonstrated in the plasma.     

Purification of a retinol binding protein from the hen-oviduct cytosol and its immunological cross-reactivity with those from the nucleus

Cellular retinol-binding protein was purified from the cytosol of the oviducts of laying hens by ammonium sulphate fractionation and chromatography on Sephadex G-75 and DEAE-Sephadex A-50 columns. Analysis of the purified retinol-binding protein on 10% SDS-polyacrylamide gel revealed the presence of a doublet representing very similar molecular sizes. Antiserum was prepared against the purified cellular retinol-binding protein, and on the basis of (a) immunodiffusion test and (b) immunoneutralization of 3H-labelled retinol-cellular retinol-binding protein complex on a column of Sephadex G-75, the antiserum appeared to be specific. The antiserum showed cross-reactivity with the nucleosol and a 0.4 M NaCl extract of the chromatin of the oviduct nuclei, while it did not react with the major egg-white proteins such as ovalbumin, conalbumin and ovomucoid.