Characterization of an alkaline active-thiol forming extracellular serine keratinase by the newly isolated Bacillus pumilus

Aims: The aim of the study was to optimize microbial degradation of keratinous waste and to characterize the alkaline active keratinase showing its biotechnological importance. Method and Results: An extracellular keratinase enzyme was purified from the culture medium of a bacterial isolate and the conditions were optimized. The molecular weight of DEAE‐Sepharose‐purified keratinase was determined by SDS‐PAGE. Instrumental analyses were investigated to study the mechanism of bovine hair hydrolysis. Isolate was identified as Bacillus pumilus based on phenotypic characteristics and 16S rDNA sequence. The optimized condition for its growth was pH 8 and 35°C. The molecular weight of the keratinase was estimated as 65 kDa. Activity inhibition by phenyl methyl sulphonyl fluoride confirmed keratinase as serine protease type. Instrumental analysis revealed the sulphitolysis and proteolysis involved mechanism in bovine hair hydrolysis. Conclusion: This study indicates that the isolated keratinase is an alkaline active serine protease with a high degree of activity towards bovine hair. Significance and Impact of the Study: This study examines a serine protease with high keratinolytic activity and degradation mechanism for bovine hair. The keratinolytic activity of the isolated strain and its reaction mechanism on bovine hair could show biotechnological potential in the leather industry.     

Poly-β-Hydroxybutyrate Metabolism Is Affected by Changes in Respiratory Enzymatic Activities Due to Cold Stress in Two Psychrotrophic Strains of Rhizobium

Cold stress resulted in a decrease in the poly-β-hydroxybutyrate (PHB) content of non-cold-acclimated Rhizobium DDSS69 cultures. Analysis of the specific activity of β-ketothiolase and β-hydroxybutyrate dehydrogenase revealed that decrease in PHB levels was a result of the inhibition of synthesis of PHB rather than an increase in its breakdown. Rhizobium ATR1, a cold-acclimated strain, revealed the presence of a stable PHB metabolism that did not show any significant differences either in PHB levels or in the activity of enzymes of the PHB metabolism under cold stress, suggesting that PHB is not involved in cold tolerance. Analysis of specific activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of the pentose phosphate pathway showed the upward regulation of alternate pathways of carbohydrate metabolism under cold stress to rapidly generate energy to overcome the stress. There is diversity in the switching mechanisms of carbon metabolism among cold-acclimated and non-cold-acclimated Rhizobium isolates. Upward regulation of malate dehydrogenase in both isolates suggests that it is a critical input for cold tolerance. Received: 26 June 2000 / Accepted: 31 July 2000     

Biochemical changes in Glomus fasciculatum colonized roots of Lycopersicon esculentum in presence of Meloidogyne incognita

Glasshouse experiments were conducted to elicit biochemical substantiation for the observed difference in resistance to nematode infection in roots colonized by mycorrhiza, and susceptibility of the fresh flush of roots of the same plant that escaped mycorrhizal colonization. Tomato roots were assayed for their biochemical profiles with respect to total proteins, total phenols, indole acetic acid, activities of polyphenol oxidase, phenylalanine ammonia lyase and indole acetic acid oxidase. The roots of the same plant (one set) received Glomus fasciculatum and G. fasciculatum plus juveniles of Meloidogyne incognita separately; and half the roots of second set of plants received G. fasciculatum while the other half of roots did not receive any treatment. Roots colonized by G. fasciculatum recorded maximum contents of proteins and phenols followed by that of the roots that received G. fasciculatum plus M. incognita. However, IAA content was lowest in the roots that received mycorrhiza or mycorrhiza plus juveniles of root-knot nematode and correspondingly. Roots that received juveniles of root-knot nematode recorded maximum IAA content and per cent increase over healthy check and mycorrhiza-inoculated roots. The comparative assay on the activities of PPO, PAL and IAA oxidase enzymes in treated and healthy roots of tomato, indicated that PAL and IAA oxidase activities were maximum in G. fasciculatum colonized roots followed by the roots that received mycorrhiza plus juveniles of root-knot nematode, while the activity of PPO was minimum in these roots. The roots that received juveniles of root-knot nematode recorded minimum PAL and IAA oxidase activities and maximum PPO activity. Since the roots of same plant that received mycorrhiza and that did not receive mycorrhiza; and the plant that received nematode alone and mycorrhiza plus nematode recorded differential biochemical contents of proteins, total phenols and IAA, and differential activities of enzymes under study, it was evident that the biochemical defense response to mycorrhizal colonization against root-knot nematodes was localized and not systemic. This explained for the response of plant that differed in root galling due to nematode infection in presence of mycorrhizal colonization. The new or fresh roots which missed mycorrhizal colonization, got infected by nematodes and developed root galls.     

Metabolic responses in discrete regions of rat brain following acute administration of glutamate

Glutamate is a major excitatory neurotransmitter in the mammalian brain. Nevertheless, high extracellular levels of this amino acid have been shown to be toxic to several neuronal populations, but no data are available to show how glutamate homeostasis is altered in response to local infusion of glutamate. In the present study, 1 M of glutamate was stereotactically injected into cerebral cortex, striatum, and hippocampus of adult rat brain, and the activities of key metabolic enzymes, lactate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase were evaluated by postmortem analysis in tissue homogenates. The results show that glutamate bolus, induced significant alterations in vivo glutamate and energy metabolism, as evidenced by marked alterations in these enzyme activities, whereas dizocilpine, a glutamate receptor antagonist, negated many of the effects induced by high glutamate. However, the degree of involvement of these observations in glutamate-induced neurotoxicity remains to be ascertained.     

A comparative study on the interaction of acridine and synthetic bis-acridine with G-quadruplex structure

DNA from the telomeres contains a stretch of simple tandemly repeated sequences in which clusters of G residues alternate with clusters of T/A sequences along one DNA strand. Model telomeric G-clusters form four-stranded structures in presence of Na(I), K(I) and NH(4)(I) ions. Electrophoretic and spectroscopic studies were made with the telomeric related sequences d(T6G16) or d(G4T2G4T2G4T2G4). It was noticed earlier that G-quadruplex may either be inter-molecular, or intra-molecular, or a mixture of both. CD spectral characteristics of various G-quadruplex DNA suggests that the CD maximum at 293 nm corresponds to that of an intra-molecular G-quadruplex structure or hairpin dimers. Fluorescence titration studies also show that acridine and the bis-acridine are interacting with G-quadruplex DNA and destabilize the K(I)-quadruplex structure more efficiently than the quadruplex formed by NH(4)(I) ion. Among the two drugs studied, acridine is more capable of breaking the G-quadruplex structure than bis-acridine. This result is further confirmed by the CD experiments.     

Molecular cytogenetic analysis of a familial 8p23.1 deletion associated with minimal dysmorphic features,seizures, and mild mental retardation

Summary We report a family in which three members presented with minimal phenotypic abnormalities, normal intelligence to mild mental retardation, and a cytogenetically terminal chromosome deletion at band 8p23.1 Whole chromosomal painting with a chromosome 8-specific DNA library confirmed this familial chromosome abnormality as a deletion, while fluorescence in situ hybridization with telomeric probes demonstrated the presence of telomeres at the deletion site. Coagulation studies were additionally performed to evaluate the purported location of the coagulation factor VII regulator gene at 8p23.1. A review of the clinical findings of seven cases of del(8)(p23.1) is presented.     

Production of IgM specific recombinant dengue multiepitope protein for early diagnosis of dengue infection

Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME-M) protein specific to IgM in E. coli was produced in a 5-L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME-M protein was purified under denaturing conditions using single-step nickel nitrilotriacetate (Ni-NTA) affinity chromatography. The final yield of purified rDME-M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME-M protein was checked by SDS-PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme-linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in-house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan-Bio) and rapid immunochromatography (IC) test (Pan-Bio). These results show that the in-house dipstick ELISA using rDME-M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.     

Zinc finger nuclease‐mediated targeting of multiple transgenes to an endogenous soybean genomic locus via non‐homologous end joining

Emerging genome editing technologies hold great promise for the improvement of agricultural crops. Several related genome editing methods currently in development utilize engineered, sequence‐specific endonucleases to generate DNA double strand breaks (DSBs) at user‐specified genomic loci. These DSBs subsequently result in small insertions/deletions (indels), base substitutions or incorporation of exogenous donor sequences at the target site, depending on the application. Targeted mutagenesis in soybean (Glycine max) via non‐homologous end joining (NHEJ)‐mediated repair of such DSBs has been previously demonstrated with multiple nucleases, as has homology‐directed repair (HDR)‐mediated integration of a single transgene into target endogenous soybean loci using CRISPR/Cas9. Here we report targeted integration of multiple transgenes into a single soybean locus using a zinc finger nuclease (ZFN). First, we demonstrate targeted integration of biolistically delivered DNA via either HDR or NHEJ to the FATTY ACID DESATURASE 2‐1a (FAD2‐1a) locus of embryogenic cells in tissue culture. We then describe ZFN‐ and NHEJ‐mediated, targeted integration of two different multigene donors to the FAD2‐1a locus of immature embryos. The largest donor delivered was 16.2 kb, carried four transgenes, and was successfully transmitted to T₁ progeny of mature targeted plants obtained via somatic embryogenesis. The insertions in most plants with a targeted, 7.1 kb, NHEJ‐integrated donor were perfect or near‐perfect, demonstrating that NHEJ is a viable alternative to HDR for gene targeting in soybean. Taken together, these results show that ZFNs can be used to generate fertile transgenic soybean plants with NHEJ‐mediated targeted insertions of multigene donors at an endogenous genomic locus.     

Abundant and diverse fungal microbiota in the murine intestine

Enteric microbiota play a variety of roles in intestinal health and disease. While bacteria in the intestine have been broadly characterized, little is known about the abundance or diversity of enteric fungi. This study utilized a culture-independent method termed oligonucleotide fingerprinting of rRNA genes (OFRG) to describe the compositions of fungal and bacterial rRNA genes from small and large intestines (tissue and luminal contents) of restricted-flora and specific-pathogen-free mice. OFRG analysis identified rRNA genes from all four major fungal phyla: Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota. The largest assemblages of fungal rRNA sequences were related to the genera Acremonium, Monilinia, Fusarium, Cryptococcus/Filobasidium, Scleroderma, Catenomyces, Spizellomyces, Neocallimastix, Powellomyces, Entophlyctis, Mortierella, and Smittium and the order Mucorales. The majority of bacterial rRNA gene clones were affiliated with the taxa Bacteroidetes, Firmicutes, Acinetobacter, and Lactobacillus. Sequence-selective PCR analyses also detected several of these bacterial and fungal rRNA genes in the mouse chow. Fluorescence in situ hybridization analysis with a fungal small-subunit rRNA probe revealed morphologically diverse microorganisms resident in the mucus biofilm adjacent to the cecal and proximal colonic epithelium. Hybridizing organisms comprised about 2% of the DAPI (4',6-diamidino-2-phenylindole, dihydrochloride)-positive organisms in the mucus biofilm, but their abundance in fecal material may be much lower. These data indicate that diverse fungal taxa are present in the intestinal microbial community. Their abundance suggests that they may play significant roles in enteric microbial functions.     

Iodine-mediated one-pot intramolecular decarboxylation domino reaction for accessing functionalised 2-(1,3,4-oxadiazol-2-yl)anilines with carbonic anhydrase inhibitory action

A practical and transition metal-free one-pot domino synthesis of diversified (1,3,4-oxadiazol-2-yl)anilines has been developed employing isatins and hydrazides as the starting materials, in the presence of molecular iodine. The prominent feature of this domino process involves consecutive condensation, hydrolytic ring cleavage, and an intramolecular decarboxylation, in a one-pot process that leads to the oxidative formation of a C–O bond. Fluorescence properties of some of the representative molecules obtained in this way were studied. The synthesised 2-(1,3,4-oxadiazolo-2-yl)aniline-benzene sulphonamides (8a–o) were screened for their carbonic anhydrase (CA, EC 4.2.1.1) inhibitory activity. Most of the compounds exhibited low micromolar to nanomolar activity against human (h) isoforms hCA I, hCA II, hCA IV, and XII, with some compounds displaying selective CA inhibitory activity towards hCA II with K_Is of 6.4–17.6?nM.