Study of the Interaction between Bacteriophage T4 asiA and Escherichia coli ς70, Using the Yeast Two-Hybrid System: Neutralization of asiA Toxicity to E. coli Cells by Coexpression of a Truncated ς70 Fragment

The interaction of T4 phage-encoded anti-sigma factor, asiA, and Escherichia coli sigma(70) was studied by using the yeast two-hybrid system. Truncation of sigma(70) to identify the minimum region involved in the interaction showed that the fragment containing amino acid residues proximal to the C terminus (residues 547 to 603) was sufficient for complexing to asiA. Studies also indicated that some of the truncated C-terminal fragments (residues 493 to 613) had higher affinity for asiA as judged by the increased beta-galactosidase activity. It is proposed that the observed higher affinity may be due to the unmasking of the binding region of asiA on the sigma protein. Advantage was taken of the increased affinity of truncated sigma(70) fragments to asiA in designing a coexpression system wherein the toxicity of asiA expression in E. coli could be neutralized and the complex of truncated sigma(70) and asiA could be expressed in large quantities and purified.     

Putative Calmodulin-Binding Domains in Aflatoxin Biosynthesis–Regulatory Proteins

The inhibition of aflatoxin production by trifluoperazine, an anticalmodulin (CaM) agent and the relevance of Ca²⁺/CaM-dependent phosphorylation and dephosphorylation during aflatoxin biosynthesis was previously reported. To identify proteins that may be regulated by CaM, an in silico analysis for putative CaM-binding domains (CaMBDs) in the aflatoxin-related proteins of Aspergillus parasiticus was performed using the CaM target database. Interestingly, the key regulators of aflatoxin biosynthesis such as AflR and AflJ contained predicted CaMBDs at their C-termini. Furthermore, potential phosphorylation sites for CaM-kinase II were present within these CaMBDs. In addition to other aflatoxin biosynthesis enzymes—such as Vbs, DmtA and OmtA, and the VeA protein (known to regulate the expression of AflJ and AflR)—also showed the presence of putative CaMBDs. Although the present report reaffirms earlier observations on CaM-mediated regulation of aflatoxin biosynthesis, it also opens new avenues for identifying the specific targets of CaM and elucidating the exact mechanism of initiation and regulation of aflatoxin biosynthesis.     

Synthesis and antibacterial evaluation of isoxazolinyl oxazolidinones: Search for potent antibacterial

A series of (5S) N-(3-{3-fluoro-4-[4-(3-aryl-4,5-dihydro-isoxazole-5-carbonyl)-piperazin-1-yl]-phenyl}-2-oxo-oxazolidin-5-ylmethyl)-acetamide(6a–o) were synthesized and their in vitro antibacterial activity against various resistant Gram-positive and Gram-negative bacteria were evaluated. Most of the synthesized compounds showed 2 to 10 fold lower MIC values compared to linezolid against Staphylococcus aureus ATCC 25923, ATCC 70069, ATCC 29213, Bacillus cereus MTCC 430, Enterococcus faecalis MTCC439, Klebsiella pneumoniae ATCC 27736, and Streptococcus pyogens.     

Effect of probiotic fermented milk and chlorophyllin on gene expressions and genotoxicity during AFB₁-induced hepatocellular carcinoma

The aim of this study was to investigate the chemopreventive effect of probiotic fermented milk and chlorophyllin on aflatoxin B? (AFB?) induced hepatocellular carcinoma. In vivo trials were conducted on 200 Wistar rats allocated to eight groups. Rats in the positive control group were given intraperitoneal injection of aflatoxin B? at 450 μg/kg body weight twice a week for 6 weeks. The rats were sacrificed and dissected at 25th week of the experiment, and comet assay was carried out in hepatic cells to assess the genotoxicity or DNA damage. The tumour incidence was decreased by approximately one-third than AFB? control group. The expression of c-myc bax, bcl-2, cyclin D1, p53 and rasp-21 genes was also studied. A significant (P<0.05) reduction in DNA damage was observed in probiotic fermented milk with chlorophyllin group as compared to aflatoxin B? control group. The c-myc, bcl-2, cyclin D1 and rasp-21 level was found to be highest in AFB? control group as compared to the treatment group. The results advocate the enhanced protective potential of probiotic fermented milk and chlorophyllin against AFB?-induced molecular alterations in hepatic cells during carcinogenesis.     

The Kub5-Hera/RPRD1B interactome: a novel role in preserving genetic stability by regulating DNA mismatch repair

Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by concomitantly minimizing persistent R-loops and promoting repair of DNA double strand breaks (DSBs). We used tandem affinity purification-mass spectrometry, co-immunoprecipitation and gel-filtration chromatography to define higher-order protein complexes containing K-H scaffolding protein to gain insight into its cellular functions. We confirmed known protein partners (Ku70, RNA Pol II, p15RS) and discovered several novel associated proteins that function in RNA metabolism (Topoisomerase 1 and RNA helicases), DNA repair/replication processes (PARP1, MSH2, Ku, DNA-PKcs, MCM proteins, PCNA and DNA Pol δ) and in protein metabolic processes, including translation. Notably, this approach directed us to investigate an unpredicted involvement of K-H in DNA mismatch repair (MMR) where K-H depletion led to concomitant MMR deficiency and compromised global microsatellite stability. Mechanistically, MMR deficiency in K-H-depleted cells was a consequence of reduced stability of the core MMR proteins (MLH1 and PMS2) caused by elevated basal caspase-dependent proteolysis. Pan-caspase inhibitor treatment restored MMR protein loss. These findings represent a novel mechanism to acquire MMR deficiency/microsatellite alterations. A significant proportion of colon, endometrial and ovarian cancers exhibit k-h expression/copy number loss and may have severe mutator phenotypes with enhanced malignancies that are currently overlooked based on sporadic MSI+ screening.     

Design and synthesis of 3-(azol-1-yl)phenylpropanes as microbicidal spermicides for prophylactic contraception

We designed a series of 25 3-(azol-1-yl)phenylpropanes which yielded 10 compounds (3, 4, 7, 8, 13, 14, 19, 21, 23, 26) that irreversibly immobilized 100% human sperm at 1% (w/v) concentration in 60 s; 12 compounds (8, 9, 15, 16, 19-21, 23-25, 27, 28) that showed potent microbicidal activity at 12.5-50 μg/mL against Trichomonas vaginalis; and 17 compounds (3-11, 13, 15, 19, 21, 23, 26, 28, 30) that exhibited potent anticandida activity with minimum inhibitory concentration (MIC) of 12.5-50 μg/mL. Almost all the compounds exhibited high level of safety towards normal vaginal flora (Lactobacillus) and human cervical (HeLa) cells in comparison to the marketed spermicide nonoxynol-9 (N-9). All the biological activities were evaluated in vitro. Two compounds (4, 8) with good safety profile exhibited multiple (spermicidal, antitrichomonas and anticandida) activities, warranting further lead optimization for furnishing a prophylactic vaginal contraceptive.     

Lactobacillus rhamnosus GG antagonizes Giardia intestinalis induced oxidative stress and intestinal disaccharidases: an experimental study

The present study describes the in vivo modulatory potential of Lactobacillus rhamnosus GG (LGG), an effective probiotic, in Giardia intestinalis-infected BALB/c mice. Experimentally, it was observed that oral administration of lactobacilli prior or simultaneous with Giardia trophozoites to mice, efficiently (p < 0.05) reduced both the severity and duration of giardiasis. More specifically, probiotics fed, Giardia-infected mice, showed a significant increase in the levels of antioxidants [reduced glutathione (GSH) and superoxide dismutase (SOD)] and intestinal disaccharidases [sucrase and lactase] and decreased levels of oxidants in the small intestine, in comparison with Giardia-infected mice. Histopathological findings also revealed almost normal cellular morphology of the small intestine in probiotic-fed Giardia-infected mice compared with fused enterocytes, villous atrophy and increased infiltration of lymphocytes in Giardia-infected mice. The results of the present study has shed new light on the anti-oxidative properties of LGG in Giardia mediated tissue injury, thereby suggesting that the effects of probiotic LGG are biologically plausible and could be used as an alternative microbial interference therapy.     

EvaGreen-based Multiplex Real-time PCR Assay for Rapid Differentiation of Wild-Type and Glycoprotein E-Deleted Bovine Herpesvirus-1 Strains

Bovine herpesvirus-1 (BoHV-1) is an important viral pathogen causing significant economic losses to the cattle industry. Glycoprotein E-deleted marker vaccines form the basis for BoHV-1 control programs widely, wherein detection and differentiation of wild-type and gE-deleted vaccine strains is of crucial importance for proper disease management. In the present study, we report an EvaGreen-based multiplex real-time polymerase chain reaction (EGRT-PCR) assay for rapid differentiation of wild-type and glycoprotein E-deleted strains of BoHV-1. The EGRT-PCR assay could simultaneously detect two viral genes (glycoprotein B and E) and an internal positive control gene (bovine growth hormone- bGH), in a single-tube reaction. The analytical sensitivity of the EGRT-PCR assay was as little as 10 copies of the BoHV-1 DNA per reaction. The modified real-time PCR assay could successfully differentiate wild-type and gE-deleted BoHV-1 strains based on gene specific melting temperatures (T_m) peaks. Our results have shown that the EGRT-PCR developed in this study might prove to be a promising tool in disease management by enabling rapid differentiation of wild-type and gE-deleted strains of BoHV-1.     

Molecularly imprinted polyaniline molecular receptor–based chemical sensor for the electrochemical determination of melamine

Molecularly imprinted polymer‐modified glassy carbon electrode (GCE)‐based electrochemical sensor is prepared using the electropolymerization of aniline in the presence of melamine (MA) as a template. In this work, the advantages of molecularly imprinted conducting polymers (MICPs) and electroanalytical methods were combined to obtain an electronic device with better performances. The sensor performance was evaluated by cyclic voltammetry (CV) and square wave voltammetry (SWV) with the linear range of 0.6‐16 × 10^?9M, quantification limit of 14.9 × 10^?10M, and detection limit of 4.47 × 10^?10M (S/N = 3). The selectivity of the sensor was tested in the presence of acetoguanamine (AGA), diaminomethylatrazine (DMT), casein, histidine, and glycine interfering molecules taken at the triple concentration with MA that demonstrated too small current response compared with that of the analyte indicating high specificity of the sensor towards the template. The sensor was successfully applied to determine MA in infant formula samples with significant recovery greater than 96% and relative standard deviation (RSD) less than 4.8%. Moreover, the good repeatability, recyclability, and stability make this sensor device promising for the real‐time monitoring of MA in different food stuffs.