Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

Even a mild spell of cold weather affects feeding in migrating redheaded buntings

We investigated whether a climatic change in temperature affected daily food intake in migrating male redheaded buntings. Groups of adult male birds (n = 18) were photoinduced into migratory phenotype under increasing spring daylengths (NDL); as the birds began to exhibit night restlessness, Zugunruhe, these were allocated into groups, either with ambient (NDL, variable daily temperature: maximum – 29–44 °C and minimum – 16–33 °C; for food intake (six birds) and activity recording, six birds) until 2 weeks after they concluded migration or with constant temperature (NDT, 22 ± 1 °C; for food intake (six birds)) conditions. As day length increased March onwards, daily food intake increased (hyperphagia) in NDL and NDT groups. However, hyperphagia was slower in NDT birds as compared to NDL birds, suggesting that altered ambient temperature affects daily food intake in migrating buntings. Another group of 12 birds were held under constant daylengths (12L:12D; EDT and constant temperature 22 ± 1 °C). Although the onset of Zugunruhe was delayed under EDT, the day of onset of Zugunruhe was taken as day 0. Daily food intake and body weight before and during migration of EDT birds were compared with that of NDT and NDL groups. Daily food intake and body weight increased in all migrating birds, but hyperphagia continued post-migration in NDT birds. The study suggests that constantly suboptimal temperature despite increasing daylength, NDT, appeared to affect feeding and body weight of migratory buntings as evident from continued hyperphagia and body weight gain, even after concluding migrating activity.     

One-step purification of chitinase from Serratia marcescens NK1, a soil isolate

AIMS: A simple single step technique of gel filtration was developed for the purification of chitinase from Serratia marcescens NK1. METHODS AND RESULTS: Chitinase from Ser. marcescens NK1 was purified to homogeneity by gel filtration chromatography with 9.2% recovery. The enzyme had a pH optimum of 6.2 and a temperature optimum of 47 degrees C. It was stable in a wide pH range of 3.0 to 10.0, retaining 60% activity at pH 3.0 and 65% activity at pH 10.5. It retained 70% activity at 28 degrees C after 72 h and nearly 50% activity at 50 degrees C up to 24 h. CONCLUSION: The chitinase from Ser. marcescens NK1 can be efficiently purified in a single step by gel filtration chromatography. The chitinase of Ser. marcescens NK1, a soil isolate, is highly stable and as active as that of other reported isolates of Ser. marcescens. SIGNIFICANCE AND IMPACT OF THE STUDY: This purification scheme is advantageous because of its simplicity and can therefore be applied for the purification of other enzymes. The yield is sufficient for initial characterization studies of the enzyme, and an improved resolution can be obtained if the chromatography is done under fast flow systems.     

Genetic Diversity in Lens Species Revealed by EST and Genomic Simple Sequence Repeat Analysis

Low productivity of pilosae type lentils grown in South Asia is attributed to narrow genetic base of the released cultivars which results in susceptibility to biotic and abiotic stresses. For enhancement of productivity and production, broadening of genetic base is essentially required. The genetic base of released cultivars can be broadened by using diverse types including bold seeded and early maturing lentils from Mediterranean region and related wild species. Genetic diversity in eighty six accessions of three species of genus Lens was assessed based on twelve genomic and thirty one EST-SSR markers. The evaluated set of genotypes included diverse lentil varieties and advanced breeding lines from Indian programme, two early maturing ICARDA lines and five related wild subspecies/species endemic to the Mediterranean region. Genomic SSRs exhibited higher polymorphism in comparison to EST SSRs. GLLC 598 produced 5 alleles with highest gene diversity value of 0.80. Among the studied subspecies/species 43 SSRs detected maximum number of alleles in L. orientalis. Based on Nei’s genetic distance cultivated lentil L. culinaris subsp. culinaris was found to be close to its wild progenitor L. culinaris subsp. orientalis. The Prichard’s structure of 86 genotypes distinguished different subspecies/species. Higher variability was recorded among individuals within population than among populations.     

Antifungal lactic acid bacteria with potential to prolong shelf-life of fresh vegetables

AIM: The aim of this study was to isolate and identify antifungal lactic acid bacteria from fresh vegetables, and evaluate their potential in preventing fungal spoilage of vegetables. METHODS AND RESULTS: Lactic acid bacteria from fresh vegetables were enriched in MRS (de Man Rogosa Sharpe) broth and isolated by plating on MRS agar. All the isolates (359) were screened for activity against Aspergillus flavus of which 10% showed antifungal activity. Potent antifungal isolates were identified by phenotypic characters and confirmed by partial 16S rRNA gene sequencing. These were screened against additional spoilage fungi viz. Fusarium graminearum, Rhizopus stolonifer, Sclerotium oryzae, Rhizoctonia solani, Botrytis cinerea and Sclerotinia minor by overlay method. Most of the isolates inhibited wide range of spoilage fungi. When fresh vegetables were inoculated with either cell suspension (10(4) cells ml(-1)) or cell-free supernatant of Lact. plantarum, followed by application of vegetable spoilage fungi (A. flavus and F. graminearum, R. stolonifer, B. cinerea each with 10(4) conidia ml(-1)) the vegetable spoilage was significantly delayed than control. CONCLUSIONS: Fresh vegetables constitute a good source of lactic acid bacteria with ability to inhibit wide range of spoilage fungi. Such bacteria can be applied to enhance shelf-life of vegetables. In the present study, we report for the first time the antifungal activity of Weissella paramessenteroides and Lact. paracollinoides isolated from fresh vegetables, against wide range of food spoilage fungi. SIGNIFICANCE AND IMPACT OF THE STUDY: Fresh vegetables can be used as a source of antifungal lactic acid bacteria. Their exploitation as biopreservative will help in prolonging shelf-life of fresh vegetables.     

Defective molecular timer in the absence of nucleotides leads to inefficient caspase activation

In the intrinsic death pathway, cytochrome C (CC) released from mitochondria to the cytosol triggers Apaf-1 apoptosome formation and subsequent caspase activation. This process can be recapitulated using recombinant Apaf-1 and CC in the presence of nucleotides ATP or dATP [(d)ATP] or using fresh cytosol and CC without the need of exogenous nucleotides. Surprisingly, we found that stored cytosols failed to support CC-initiated caspase activation. Storage of cytosols at different temperatures led to the loss of all (deoxy)nucleotides including (d)ATP. Addition of (d)ATP to such stored cytosols partially restored CC-initiated caspase activation. Nevertheless, CC could not induce complete caspase-9/3 activation in stored cytosols, even with the addition of (d)ATP, despite robust Apaf-1 oligomerization. The Apaf-1 apoptosome, which functions as a proteolytic-based molecular timer appeared to be defective as auto-processing of recruited procaspase-9 was inhibited. Far Western analysis revealed that procaspase-9 directly interacted with Apaf-1 and this interaction was reduced in the presence of physiological levels of ATP. Co-incubation of recombinant Apaf-1 and procaspase-9 prior to CC and ATP addition inhibited CC-induced caspase activity. These findings suggest that in the absence of nucleotide such as ATP, direct association of procaspase-9 with Apaf-1 leads to defective molecular timer, and thus, inhibits apoptosome-mediated caspase activation. Altogether, our results provide novel insight on nucleotide regulation of apoptosome.     

RhoA as a mediator of clinically relevant androgen action in prostate cancer cells

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.     

Biophysical and mechanistic insights into novel allosteric inhibitor of spleen tyrosine kinase

Extracellular stimulation of the B cell receptor or mast cell FcεRI receptor activates a cascade of protein kinases, ultimately leading to antigenic or inflammation immune responses, respectively. Syk is a soluble kinase responsible for transmission of the receptor activation signal from the membrane to cytosolic targets. Control of Syk function is, therefore, critical to the human antigenic and inflammation immune response, and an inhibitor of Syk could provide therapy for autoimmune or inflammation diseases. We report here a novel allosteric Syk inhibitor, X1, that is noncompetitive against ATP (K(i) 4 ± 1 μM) and substrate peptide (K(i) 5 ± 1 μM), and competitive against activation of Syk by its upstream regulatory kinase LynB (K(i) 4 ± 1 μM). The inhibition mechanism was interrogated using a combination of structural, biophysical, and kinetic methods, which suggest the compound inhibits Syk by reinforcing the natural regulatory interactions between the SH2 and kinase domains. This novel mode of inhibition provides a new opportunity to improve the selectivity profile of Syk inhibitors for the development of safer drug candidates.