Diversity of Yeasts from Puddles in the Vicinity of Midre Lovénbreen Glacier, Arctic and Bioprospecting for Enzymes and Fatty Acids

A total of 132 yeast strains were characterised from 4 sediment samples collected from small puddles in the vicinity of Midre Lovénbreen glacier, Arctic. Based on the D1/D2 domain sequence similarity, the isolates could be categorised into 6 groups. The nearest phylogenetic neighbour of groups I to VI were identified as Cryptococcus gastricus, Cryptococcus terricolus, Rhodotorula muscorum, Mrakia psychrophila, Mrakia gelida and Rhodotorula glacialis, respectively. Strains representative of the six groups were psychrophilic and salt tolerant but varied in their ability to produce cold-active extracellular enzymes such as lipase, protease, pectinase, cellulase and amylase. C_{18:1 (w9C)} and C_{18:2 (w9,12C)} were the only two fatty acids common to all the yeasts and branched and (or) unsaturated fatty acids increased in yeasts growing at 8°C compared to 22°C, probably as an adaptation to low temperature. The present study establishes that psychrophilic yeasts are predominant in Arctic and could be used as work horses to produce cold-active enzymes and poly unsaturated fatty acids which have been implicated in low temperature adaptation and also for their use in biotechnology.     

Identification of a sulfonoquinovosyldiacylglyceride from Azadirachta indica and studies on its cytotoxic activity and DNA binding properties

Chromatographic separation of the methanolic extract of the leaves of Azadirachta indica led to the isolation of a sulfonoglycolipid characterized as a sulfonoquinovosyldiacylglyceride (SQDG), by extensive 2D NMR and mass spectral analysis. SQDG induces apoptosis in a dose dependent manner with IC(50) 8.3 μM against acute lymphoblastic leukemia (ALL) MOLT-4 cell lines. The compound showed significant DNA binding properties as evidenced by the enhancement of melting temperature and perturbation of the characteristic B-form in CD evidence of calf thymus DNA. The DNA binding was also characterized by isothermal calorimetry where a predominantly enthalpy driven binding to CT DNA was revealed.     

Quinazolino linked 4β-amidopodophyllotoxin conjugates regulate angiogenic pathway and control breast cancer cell proliferation

A series of new conjugates of quinazolino linked 4β-amidopodophyllotoxins 10aa–af and 10ba–bf were synthesized and evaluated for their anticancer activity against human pancreatic carcinoma (Panc-1) as well as breast cancer cell lines such as MCF-7 and MDA-MB-231 by employing MTT assay. Among these conjugates, some of them like 10bc, 10bd, 10be and 10bf exhibited high potency of cytotoxicity. Flow cytometric analysis showed that these conjugates arrested the cell cycle in the G2/M phase and caused the increase in expression of p53 and cyclin B1 protein with concomitant decrease in Cdk1 thereby suggesting the inhibitory action of these conjugates on mitosis. Interestingly, we observed a decrease in expression of proteins that control the tumor micro environment such as VEGF-A, STAT-3, ERK1/2, ERK-p, AKT-1 ser 473 phosphorylation in compounds treated breast cancer cells. Further, these effective conjugates have exhibited inhibitory action on integrin (αVβIII). Furthermore, the MCF-7 cells that were arrested and lost the proliferative capacity undergo mitochondrial mediated apoptosis by activation of caspases-9. Thus these conjugates have the potential to control breast cancer cell growth by effecting tumor angiogenesis and invasion.     

Chemical communication in Ropalidia marginata: Dufour's gland contains queen signal that is perceived across colonies and does not contain colony signal

Queens of the primitively eusocial wasp Ropalidia marginata appear to maintain reproductive monopoly through pheromone rather than through physical aggression. Upon queen removal, one of the workers (potential queen, PQ) becomes extremely aggressive but drops her aggression immediately upon returning the queen. If the queen is not returned, the PQ gradually drops her aggression and becomes the next queen of the colony. In a previous study, the Dufour's gland was found to be at least one source of the queen pheromone. Queen-worker classification could be done with 100% accuracy in a discriminant analysis, using the compositions of their respective Dufour's glands. In a bioassay, the PQ dropped her aggression in response to the queen's Dufour's gland macerate, suggesting that the queen's Dufour's gland contents mimicked the queen herself. In the present study, we found that the PQ also dropped her aggression in response to the macerate of a foreign queen's Dufour's gland. This suggests that the queen signal is perceived across colonies. This also suggests that the Dufour's gland in R. marginata does not contain information about nestmateship, because queens are attacked when introduced into foreign colonies, and hence PQ is not expected to reduce her aggression in response to a foreign queen's signal. The latter conclusion is especially significant because the Dufour's gland chemicals are adequate to classify individuals correctly not only on the basis of fertility status (queen versus worker) but also according to their colony membership, using discriminant analysis. This leads to the additional conclusion (and precaution) that the ability to statistically discriminate organisms using their chemical profiles does not necessarily imply that the organisms themselves can make such discrimination.     

Interaction of aristololactam-β-D-glucoside and daunomycin with poly(A): spectroscopic and calorimetric studies

The binding of two sugar containing antibiotics viz. aristololactam-β-D-glucoside and daunomycin with single and double stranded poly(A) was investigated by spectroscopic and calorimetric studies. The binding affinity of daunomycin to ss poly(A) was of the order of 10⁶ M^− 1 and that to ds poly(A) was of the order of 10⁵ M^− 1. Aristololactam-β-D-glucoside showed a relatively weaker binding with an affinity of the order of 10⁴ M^− 1 with both the conformations of poly(A). Fluorescence studies showed maximum quenching for daunomycin-ss poly(A) complexes. The binding constants calculated from fluorescence spectroscopy were in good agreement with that obtained from UV spectroscopy. Moderate perturbation of circular dichroic spectra of both the conformations of poly(A) in presence of these molecules with concomitant formation of prominent extrinsic CD bands in the 300-450 nm region further revealed the association. Isothermal titration calorimetry results showed an overall entropy driven binding in all the four systems though the entropy change was maximum in daunomycin-ss poly(A) binding. The binding affinity was also maximum for daunomycin-ss poly(A) and varied as daunomycin-ds poly(A)>aristololactam-β-D-glucoside-ds poly(A)>aristololactam-β-D-glucoside-ss poly(A). A 1:1 binding stoichiometry was observed in all the cases, as confirmed by Job plot analysis, indicating the interaction to consist of a single binding mode. Ferrocyanide quenching studies showed good stacking interaction in all cases but was best for daunomycin-ss poly(A) interaction. No self-structure formation was observed in poly(A) with both daunomycin and aristololactam-β-D-glucoside suggesting the hindrance of the sugar moiety for such structural organization.     

IL-7 and IL-15 do not synergize during CD8 T cell recall response against an obligate intracellular parasite

Long-term protection against Toxoplasma gondii is dependent on robust CD8⁺ T cell immunity. In the absence of this response, the host is unable to maintain chronicity, which results in recrudescence of infection and possible death. Factors needed for the persistence of protective CD8⁺ T cells against the parasite need to be evaluated. Previous studies from our laboratory have reported that synergism between γ chain cytokines like IL-7 and IL-15 is critical for the generation of CD8⁺ T cell response needed for protection during acute infection. In this study we report that the situation is different during the recall response where CD8⁺ T cell response is almost entirely dependent on IL-15, with IL-7 at best playing a minor role. In the absence of IL-15, CD8⁺ T cells fail to respond optimally to parasitic re-challenge and hosts are unable to control their replication, which leads to their death. Thus T. gondii infection may represent a unique situation where CD8⁺ T cell response during secondary challenge is primarily dependent on IL-15 with other γ chain cytokines having nominal effect. These findings provide important information regarding factors involved in the generation of protective immunity against T. gondii with strong implications in developing immunotherapeutic agents against the pathogen.     

Interaction of Isoquinoline Alkaloids with Polymorphic DNA Structures

The interaction of berberine, palmatine, and coralyne with the B, Z, and H^L form of poly[d(G‐C)] was studied. Berberine and palmatine showed moderate binding to the B form, while coralyne showed higher binding, as revealed from spectroscopic and thermodynamic data. Berberine and coralyne binding to the B form was exothermic and enthalpy‐driven, while palmatine showed exothermic binding which was favored by both negative enthalpy and negative entropy changes. Berberine and palmatine neither bind nor converted the Z‐form structure to B form. Coralyne, on the other hand, exhibited a strong binding affinity to Z DNA structure that was enthalpy‐driven. Berberine binding to the H^L form was cooperative, exothermic, and favored by both negative enthalpy and negative entropy changes with the formation of an induced CD band. Palmatine showed weak binding, while coralyne showed a strong binding with the H^L form. The structural differences in the isoquinoline alkaloids appear to influence the affinity and mode of interactions with these polymorphic DNA structures.