Quantification of exocytosis kinetics by DIC image analysis of cortical lawns

Cortical lawns prepared from sea urchin eggs have offered a robust in vitro system for study of regulated exocytosis and membrane fusion events since their introduction by Vacquier almost 40 years ago (Vacquier in Dev Biol 43:62–74, 1975). Lawns have been imaged by phase contrast, darkfield, differential interference contrast, and electron microscopy. Quantification of exocytosis kinetics has been achieved primarily by light scattering assays. We present simple differential interference contrast image analysis procedures for quantifying the kinetics and extent of exocytosis in cortical lawns using an open vessel that allows rapid solvent equilibration and modification. These preparations maintain the architecture of the original cortices, allow for cytological and immunocytochemical analyses, and permit quantification of variation within and between lawns. When combined, these methods can shed light on factors controlling the rate of secretion in a spatially relevant cellular context. We additionally provide a subroutine for IGOR Pro® that converts raw data from line scans of cortical lawns into kinetic profiles of exocytosis. Rapid image acquisition reveals spatial variations in time of initiation of individual granule fusion events with the plasma membrane not previously reported.     

Antioxidant activity and phenolic content of the Indian wheat varieties

In this study, Indian wheat varieties grown under different agro-climatic zones were evaluated for their antioxidant potential. Different grain fractions (bran, flour, shorts) and the whole meal were tested using two free radicals (ABTS and DPPH) for their radical scavenging activities. More variation was observed in the antioxidant activities from different zones using DPPH assay. Irrespective of the method used, the whole meal and the bran of central zone varieties showed the highest and the north western plains zone varieties showed the lowest antioxidant activities. Within each growing zone, both the effect of genotype and environment was observed on the antioxidant activity. Both free and bound phenolic compounds were extracted from the bran of varieties representing different zones. Total phenolic content (TPC) varied from 2,900 to 5,650 μg Gallic acid equivalents/g of bran. Bound phenolic content was found to be more strongly correlated to the TPC than the free phenolic content. Highly significant genotypic differences were observed in the total phenolic content. This study therefore indicates that Indian wheat varieties have good antioxidant activity and high content of phenolic compounds and can be used further in breeding programmes to increase the content of phytochemicals responsible for antioxidant activity.     

Production of Laccase and Optimization of Its Production by Bacillus sp. Using Distillery Spent Wash as Inducer

A bacillus sp. isolated from the sediments of a distillery mill was used for laccase production under optimized culture conditions. The distillery effluent was used as an inducer for overproduction of laccase by employing the Taguchi approach. Screening of different medium components and their effect on laccase production was studied using an M-16 orthogonal array. The formation of laccase was considerably increased by addition of 1 mM copper sulfate (51.95 U/ml), which was further enhanced by the use of different inducers. The usefulness of the Taguchi method for optimization of culture conditions was investigated with five selected factors at four levels, and it was observed that the optimized medium resulted in a 9-fold increase in extracellular laccase production compared with the control. The optimized medium composition for laccase production was dextrose (1%), tryptone (0.1%), CuSO₄ (1 mM), and an inducer (distillery effluent 10% [v/v]) at pH 7, which altogether resulted in 107.32 U/ml extracellular laccase activity. Hence, the Taguchi approach proved to be a reliable tool in optimizing culture conditions and achieving the best possible combination for enhanced laccase production.     

The Activity of GAT107, an Allosteric Activator and Positive Modulator of α7 Nicotinic Acetylcholine Receptors (nAChR), Is Regulated by Aromatic Amino Acids That Span the Subunit Interface

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.     

Glutathione utilization by Candida albicans requires a functional glutathione degradation (DUG) pathway and OPT7, an unusual member of the oligopeptide transporter family

Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1-OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a K(m) of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence.     

Effect of aberrant promoter methylation of FHIT and RASSF1A genes on susceptibility to cervical cancer in a North Indian population

As current evidence suggests the involvement of epigenetic modification of tumour suppressor genes in human cancer, we investigated the aberrant promoter methylation of FHIT and RASSF1A genes in human papillomavirus (HPV)-mediated cervical cancer in Indian women. We analysed 60 cervical cancer tissue biopsies of different clinical stage and histological grading and 23 healthy control samples with normal cervical cytology. Methylation-specific polymerase chain reaction (MSP) was performed to analyse the methylation status of FHIT and RASSF1A genes and confirmed by sequencing. Both patients and controls were screened for HPV infection and 98% of the HPV-infected cases showed positivity for HPV type 16. Aberrant promoter methylation of the FHIT gene was found in 28.3% (17/60) of cases and of the RASSF1A gene in 35.0% (21/60) of cases; promoter methylation of both the genes was found in 13.3% (8/60) of cervical cancer cases. Methylation was significantly (p<0.01) associated with the cervical cancer cases compared with controls. None of the 23 controls was found to be methylated in either of these genes. This is the first study indicating a correlation between the promoter methylation of FHIT and RASSF1A genes and the clinical stage and histological grading of cervical carcinoma in Indian women. Future studies are underway to examine the practical implications of these findings for use as a biomarker.     

The M10 Locus of Murine Gammaherpesvirus 68 Contributes to both the Lytic and the Latent Phases of Infection

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus and Kaposi''s sarcoma-associated herpesvirus (KSHV) and provides a small-animal model to study the pathogenesis of gammaherpesvirus (γHV) infections. According to the colinear organization of the γHV genomes, the M10 locus is situated at a position equivalent to the K12 locus of KSHV, which codes for proteins of the kaposin family. The M10 locus of MHV-68 has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) with unknown function. In addition, the M10 locus contains a lytic origin of replication (oriLyt). To elucidate the function of the M10 locus during lytic and latent infections, we investigated, both in vitro and in vivo, the following four recombinant viruses which were generated using MHV-68 cloned as a bacterial artificial chromosome: (i) a mutant virus with a deletion which affects both the coding region for M10a-c and the oriLyt; (ii) a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type; (iii) a virus with an ectopic insertion of the oriLyt, which restores the function of the oriLyt but not the M10a-c coding region; and (iv) a mutant virus with a deletion in the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced. In contrast, both the revertant virus and the virus with the ectopic oriLyt insertion grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype. We conclude that the oriLyt in the M10 locus plays an important role during infection of mice with MHV-68.Diseases caused by gammaherpesviruses continue to be a challenge for human health. The prototypic gamma-1 herpesvirus Epstein-Barr virus (EBV) is associated with lymphomas and nasopharyngeal carcinoma (22). Human herpesvirus 8 (also called Kaposi''s sarcoma-associated herpesvirus [KSHV]), a gamma-2 herpesvirus, is associated with lymphoproliferative disorders and Kaposi''s sarcoma (24). In vivo studies of gammaherpesvirus pathogenesis have been limited to clinical investigation of the infection because of the restricted host range of these viruses. The murine gammaherpesvirus 68 (MHV-68) is also a member of the gammaherpesvirus subfamily and is closely related to KSHV and EBV. Since there exist no good animal models for KSHV and EBV, MHV-68 serves as a small-animal model to investigate gammaherpesvirus pathogenesis (6, 9, 10, 13, 21, 25, 26, 30). MHV-68 is a natural pathogen of wild rodents (7) and is capable of infecting laboratory mice. The nucleotide sequence of MHV-68 is similar to that of EBV and even more closely related to that of KSHV (29). MHV-68 contains genes which are homologous to cellular genes or to genes of other gammaherpesviruses. In addition, it contains virus-specific genes. Many of the latency- and transformation-associated proteins of the gammaherpesviruses, for example, EBNA and LMP of EBV, appear to be encoded by virus-specific genes, yet it has been suggested that pathogenesis-associated genes of gammaherpesviruses may be contained in similarly positioned genome regions (29). The virus-specific genes of MHV-68 were originally designated M1 to M14 (29). The M10 locus has been predicted to code for three overlapping open reading frames (M10a, M10b, and M10c [M10a-c]) (29). While several MHV-68-specific genes have been shown to code for proteins with important functions, the function of M10 is still unknown. A more recent report even considered M10a-c rather unlikely to code for proteins (21). Importantly, the M10 locus also contains a lytic origin of replication (oriLyt) (3, 8). According to the colinear organization of the gammaherpesvirus genomes, the M10 locus is situated at a position equivalent to that of the K12 locus of KSHV. K12 encodes proteins of the kaposin family. Kaposin proteins are involved in cellular transformation and in stabilization of cytokine mRNAs (16-18,20). Of note, the K12 locus also contains an oriLyt (5).Here, we investigated the function of the M10 locus during lytic and latent infections by studying mutant viruses with deletions in the M10 loci, either affecting both the coding region for M10a-c and the oriLyt or the oriLyt only. While the mutants were slightly attenuated with regard to lytic replication in cell culture, they showed severe growth defects in vivo. Both lytic replication and latency amplification were strongly reduced in mice infected with the mutant viruses. In contrast, a revertant virus in which both the M10a-c coding region and the oriLyt were reverted to those of the wild type and a virus with an ectopic insertion of the oriLyt which restores the function of the oriLyt but not the M10a-c coding region grew very similarly to the parental wild-type virus both in vitro and in vivo. Thus, we provide genetic evidence that mutation of the oriLyt, and not of putative protein coding sequences within the M10a-c region, is responsible for the observed phenotype.     

Recent trends in microbial flavour Compounds: A review on Chemistry,synthesis mechanism and their application in food

Aroma and flavour represent the key components of food that improves the organoleptic characteristics of food and enhances the acceptability of food to consumers. Commercial manufacturing of aromatic and flavouring compounds is from the industry's microbial source, but since time immemorial, its concept has been behind human practices. The interest in microbial flavour compounds has developed in the past several decades because of its sustainable way to supply natural additives for the food processing sector. There are also numerous health benefits from microbial bioprocess products, ranging from antibiotics to fermented functional foods. This review discusses recent developments and advancements in many microbial aromatic and flavouring compounds, their biosynthesis and production by diverse types of microorganisms, their use in the food industry, and a brief overview of their health benefits for customers.     

Innate immune defense of the sponge Suberites domuncula against bacteria involves a MyD88-dependent signaling pathway. Induction of a perforin-like molecule

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates gram-negative bacteria; it is inactive against gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.