Probiotic Potential of Autochthonous Bacteria Isolated from the Gastrointestinal Tract of Four Freshwater Teleosts

In this study, a total of 121 bacterial strains were isolated from the gastrointestinal tract of four teleostean species, namely striped snakehead (Channa striatus), striped dwarf catfish (Mystus vittatus), orangefin labeo (Labeo calbasu) and mrigal carp (Cirrhinus mrigala), among which 8 isolates showed promising antibacterial activity against four potential fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas sobria and Pseudomonas fluorescens and were non-hemolytic. The isolates were further screened in response to fish bile tolerance and extracellular digestive enzyme activity. Two bacterial strains MVF1 and MVH7 showed highest tolerance and extracellular enzymes activities, and selected for further studies. Antagonistic activity of these two isolates was further confirmed by in vitro growth inhibition assay against four selected fish pathogens in liquid medium. Finally, these two bacterial strains MVF1 and MVH7 were selected as potential probiotic candidates and thus identification by partial 16S rRNA gene sequence analysis. The bacterial isolates MVF1 and MVH7 were identified as two strains of Bacillus sp.     

The effect of transforming growth factor beta on human neuroendocrine tumor BON cell proliferation and differentiation is mediated through somatostatin signaling

The dual effect of the ubiquitous inflammatory cytokine transforming growth factor beta1 (TGF beta) on cellular proliferation and tumor metastasis is intriguing but complex. In epithelial cell- and neural cell-derived tumors, TGF beta serves as a growth inhibitor at the beginning of tumor development but later becomes a growth accelerator for transformed tumors. The somatostatin (SST) signaling pathway is a well-established antiproliferation signal, and in this report, we explore the interplay between the SST and TGF beta signaling pathways in the human neuroendocrine tumor cell line BON. We defined the SST signaling pathway as a determinant for neuroendocrine tumor BON cells in responding to TGF beta as a growth inhibitor. We also determined that TGF beta induces the production of SST and potentially activates the negative growth autocrine loop of SST, which leads to the downstream induction of multiple growth inhibitory effectors: protein tyrosine phosphatases (i.e., SHPTP1 and SHPTP2), p21(Waf1/Cip1), and p27(Kip1). Concurrently, TGF beta down-regulates the growth accelerator c-Myc protein and, collectively, they establish a firm antiproliferation effect on BON cells. Additionally, any disruption in the activation of either the TGF beta or SST signaling pathway in BON leads to "reversible" neuroendocrine-mesenchymal transition, which is characterized by the loss of neuroendocrine markers (i.e., chromogranin A and PGP 9.5), as well as the altered expression of mesenchymal proteins (i.e., elevated vimentin and Twist and decreased E-cadherin), which has previously been associated with elevated metastatic potential. In summary, TGF beta-dependent growth inhibition and differentiation is mediated by the SST signaling pathway. Therefore, any disruption of this TGF beta-SST connection allows BON cells to respond to TGF beta as a growth accelerator instead of a growth suppressor. This model can potentially apply to other cell types that exhibit a similar interaction of these pathways.     

In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up

The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.     

Influence of IGF-I on buffalo (Bubalus bubalis) spermatozoa motility, membrane integrity, lipid peroxidation and fructose uptake in vitro

The objective of the present experiment was to examine the influence of mean physiological concentration of insulin-like growth factor-I (IGF-I) on frozen-thawed Surti buffalo (Bubalus bubalis) spermatozoa functional parameters, i.e., motility, plasmalemma integrity, acrosomal integrity, functional membrane integrity, lipid peroxidation and fructose uptake in vitro. Frozen-thawed semen samples (n=6) were washed in tris buffer and divided into two equal parts (control and IGF-I groups). Only in the IGF-I group, IGF-I (rhIGF-I analogue) was added to a final concentration of 100 ng/ml. The samples were incubated at 37 degrees C for 2h and the assessments were made at 0, 30, 60, 90 and 120 min of incubation. The mean concentration of the buffalo seminal plasma (n=17) IGF-I was 116.83+/-28.34 ng/ml (range 41.4-198.95). IGF-I had significant effect on the total motility (P<0.01), progressive forward motility (P<0.01), functional membrane integrity (P<0.05) and lipid peroxidation levels (P<0.05) during the 120-min study period as assessed by area under curve. Treatment with IGF-I increased (P<0.01) the total spermatozoa motility at 30, 60 and 90 min as compared to the control. The progressive forward motility was significantly (P<0.01) higher at 60 and 90 min of incubation. The addition of IGF-I resulted in significant (P<0.01) increase in straight-line velocity (VSL, microm/s) as compared to the control at 60 and 90 min of incubation. The linearity (%) was significantly (P<0.01) higher in IGF-I treated semen as compared to control at 60 min of incubation. Plasmalemma integrity in IGF-I group was significantly (P<0.05) higher than control at 30 and 60 min of incubation. The functional membrane integrity differed significantly (P<0.01) between groups (control and IGF-I) at 60 and 90 min of incubation. The percentage of acrosomal intact spermatozoa decreased continuously over a period of time in both the groups. As compared to 0 min of incubation, the significant (P<0.05) loss of acrosome was observed at 60 and 90 min of incubation in control (63.87+/-3.17 vs. 58.52+/-2.54) and IGF-I (61.60+/-2.26 vs. 56.11+/-2.12) groups, respectively. Lipid peroxidation levels were significantly lower in IGF-I group at 90 min (P<0.05) and 120 min (P<0.01) of incubation than the control group. Fructose utilization was significantly higher in IGF-I group as compared to control at 30 min (P<0.05) and 60 min (P<0.01) of incubation. The present study suggests that addition of IGF-I improve spermatozoa functional parameters by reducing lipid peroxidation levels.     

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli , V. cholerae Δ relA Δ spoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae . By repeated subculturing of a V. cholerae Δ relA Δ spoT mutant, a suppressor strain with (p)ppGpp⁰ phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene ( VC1224 ), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV . Using E. coli Δ relA or Δ relA Δ spoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (Δ relA Δ spoT relV676 ), which seems to be responsible for the (p)ppGpp⁰ phenotype. Analysis of a V. cholerae Δ relA Δ spoT Δ relV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.     

Predicting Gene Expression Level from Relative Codon Usage Bias: An Application to Escherichia coli Genome

We present an expression measure of a gene, devised to predictthe level of gene expression from relative codon bias (RCB).There are a number of measures currently in use that quantifycodon usage in genes. Based on the hypothesis that gene expressivityand codon composition is strongly correlated, RCB has been definedto provide an intuitively meaningful measure of an extent ofthe codon preference in a gene. We outline a simple approachto assess the strength of RCB (RCBS) in genes as a guide totheir likely expression levels and illustrate this with an analysisof Escherichia coli (E. coli) genome. Our efforts to quantitativelypredict gene expression levels in E. coli met with a high levelof success. Surprisingly, we observe a strong correlation betweenRCBS and protein length indicating natural selection in favourof the shorter genes to be expressed at higher level. The agreementof our result with high protein abundances, microarray dataand radioactive data demonstrates that the genomic expressionprofile available in our method can be applied in a meaningfulway to the study of cell physiology and also for more detailedstudies of particular genes of interest.     

Analysis of oligomeric proteins during unfolding by pH and temperature

During thermal transition and variation of pH, structural properties of 35 proteins and their complexes (bound with substrate and co-factor) were analyzed in detail. During pH alteration, these proteins were shown to have substantial differences in conformations. pH conformers were analyzed in detail. Free energy and other energy parameters were also estimated for these proteins at various pH and temperatures. Detailed structural analysis and binding interfaces of various substrates, inhibitors and cofactor of these proteins were also investigated using docking and molecular dynamic simulation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Propagation and Conservation of Picrorhiza kurrooa Royle ex Benth.: An Endangered Himalayan Medicinal Herb of High Commercial Value

Picrorhiza kurrooa Royle ex Benth., a high value medicinal herb of alpine Himalaya and a source of hepatoprotective picrosides, is listed as ‘endangered’ due to heavy collection from its natural habitat. The present report deals with successful propagation of this species using both conventional and in vitro techniques. Vegetative propagation was achieved by rooting runner cuttings with indole-3-butyric acid (IBA) or α-naphtheleneacetic acid (NAA) treatment before planting. Nearly 87% rooting success was achieved by treatment of cuttings with 50.0 μM IBA. Seeds were given a presoaking treatment with gibberellic acid (GA₃), 6-benzylaminopurine (BAP) or a combination of both to influence germination. More than 11-fold improvement in germination was recorded in seeds treated with 250.0 μM GA₃. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segment. Multiple shoots were formed following culture for 3 weeks on Murashige and Skoog (MS; 1962. Physiologia Plantarum 15: 473–497) medium containing 1.0 μM BAP. Cent percent rooting success, without basal callus formation, was observed when individual microshoots were placed in MS medium supplemented with IBA. The plantlets raised using conventional as well as tissue culture methods were hardened and successfully established in the experimental field located at 2450 m elevation. In addition, strategies have been discussed to encourage cultivation and in situ conservation of this highly valued medicinal herb so as to reduce pressure on its natural populations.     

Expression of an Acid Urease with Urethanase Activity in <Emphasis Type="Italic">E. coli</Emphasis> and Analysis of Urease Gene

Urea in alcoholic beverage is a precursor of ethyl carbamate (EC), which is carcinogenic. Enzymatic elimination of urea has attracted much research interest. Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications. In the present work, the structural genes of urease from Providencia rettgeri JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) in an active form (apourease) exhibiting both urease and urethanase (hydrolyze EC) activities. The specific activities of the purified apourease were comparatively low, which were 2.1 U/mg for urease and 0.6 U/mg for urethanase, respectively. However, apourease exhibited good resistance toward ethanol and acidic conditions. The relative activities of urease and urethanase remained over 80% in the buffers within pH 4–7. And the recoveries of both urease and urethanase activities were more than 50% in 5–25% ethanol solution. Apourease was utilized to eliminate urea in wine, and the residual urea in model wine was less than 50% after treatment with apourease for 30 h. Then 3D structure of UreC was predicted, and it was docked with urea and EC, respectively. The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease, whereas only one hydrogen bond can be formed between EC and the active center. Moreover, EC exhibited greater steric hindrance than urea when combined with the active site. Due to the low specific activities of apourease, both structural genes and accessory genes of urease were co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8 U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities.