Identification of bacteria from clinical samples using Bartonella alpha-Proteobacteria growth medium

In an effort to overcome historical problems associated with the isolation of Bartonella species from animal and human blood samples, our laboratory developed a novel, chemically modified, insect-based, liquid culture medium (Bartonella alpha-Proteobacteria growth medium, BAPGM). In this study, we describe the isolation of non-Bartonella bacteria from aseptically obtained human blood and tissue samples that were inoculated into BAPGM pre-enrichment culture medium, and were obtained during attempts to define each individuals Bartonella infection status. After incubation for at least 7 days in liquid BAPGM, pre-enriched inoculums were sub-cultured onto a BAPGM/blood agar plate. Bacterial DNA was extracted from pooled plated colonies and amplified using conventional PCR targeting the 16S rRNA gene. Subsequently, amplicons were cloned, sequenced and compared to GenBank database sequences using the BLAST program. Regardless of the patient's Bartonella status, seventeen samples generated only one 16S rDNA sequence, representing the following genera: Arthrobacter, Bacillus, Bartonella, Dermabacter, Methylobacterium, Propionibacterium, Pseudomonas, Staphylococcus and bacteria listed as "non-cultured" in the GenBank database. Alkalibacterium, Arthrobacter, Erwinia, Kineococcus, Methylobacterium, Propionibacterium, Sphingomonas, and Staphylococcus were isolated from nine Bartonella-infected individuals. Co-isolation of Acinetobacter, Sphingomonas, Staphylococcus spp. and bacteria listed as "non-cultured" in the GenBank database was achieved for four samples in which Bartonella spp. were not detected. Despite the phylogenetic limitations of using partial 16S rRNA gene sequencing for species and strain identification, the investigational methodology described in this study may provide a complementary approach for the isolation and identification of bacteria from patient samples.     

Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited

Mitosis requires precise coordination of multiple global reorganizations of the nucleus and cytoplasm. Cyclin-dependent kinase 1 (Cdk1) is the primary upstream kinase that directs mitotic progression by phosphorylation of a large number of substrate proteins. Cdk1 activation reaches the peak level due to positive feedback mechanisms. By inhibiting Cdk chemically, we showed that, in prometaphase, when Cdk1 substrates approach the peak of their phosphorylation, cells become capable of proper M-to-G1 transition. We interfered with the molecular components of the Cdk1-activating feedback system through use of chemical inhibitors of Wee1 and Myt1 kinases and Cdc25 phosphatases. Inhibition of Wee1 and Myt1 at the end of the S phase led to rapid Cdk1 activation and morphologically normal mitotic entry, even in the absence of G2. Dampening Cdc25 phosphatases simultaneously with Wee1 and Myt1 inhibition prevented Cdk1/cyclin B kinase activation and full substrate phosphorylation and induced a mitotic "collapse," a terminal state characterized by the dephosphorylation of mitotic substrates without cyclin B proteolysis. This was blocked by the PP1/PP2A phosphatase inhibitor, okadaic acid. These findings suggest that the positive feedback in Cdk activation serves to overcome the activity of Cdk-opposing phosphatases and thus sustains forward progression in mitosis.     

Jack bean α-mannosidase (Jbα-man): tolerance to alkali, chelating and reducing agents and energetics of catalysis and inhibition

Investigations of the catalytic and structural transitions of jack bean α-mannosidase (Jbα-man) are described in the present paper. The enzyme was maximally stable at pH 5.0; however, when incubated in the pH range of 11.0-12.0, showed 1.3 times higher activity and also stability for longer time. The free amino group at or near the active site was probably involved in the stability and activation mechanism. The active site is constituted by the association of two unidentical subunits connected by disulfide linkages. The metalloenzyme has Zn²⁺ ions tightly bound and chelation reduces the thermal stability of the protein. Energetics of catalysis and thermodynamics of inhibition of the enzyme were also carried out.     

Optimization of l‐DOPA production by Brevundimonas sp. SGJ using response surface methodology

l ‐DOPA (3,4‐dihydroxyphenyl‐l ‐alanine) is an extensively used drug for the treatment of Parkinson's disease. In the present study, optimization of nutritional parameters influencing l ‐DOPA production was attempted using the response surface methodology (RSM) from Brevundimonas sp. SGJ. A Plackett–Burman design was used for screening of critical components, while further optimization was carried out using the Box–Behnken design. The optimized levels of factors predicted by the model were pH 5.02, 1.549 g l^?1 tryptone, 4.207 g l^?1 l ‐tyrosine and 0.0369 g l^?1 CuSO₄, which resulted in highest l ‐DOPA yield of 3.359 g l^?1. The optimization of medium using RSM resulted in a 8.355‐fold increase in the yield of l ‐DOPA. The anova showed a significant R² value (0.9667), model F‐value (29.068) and probability (0.001), with insignificant lack of fit. The highest tyrosinase activity observed was 2471 U mg^?1 at the 18th hour of the incubation period with dry cell weight of 0.711 g l^?1. l ‐DOPA production was confirmed by HPTLC, HPLC and GC‐MS analysis. Thus, Brevundimonas sp. SGJ has the potential to be a new source for the production of l ‐DOPA.     

Chemical evolution of iron containing enzymes: Mixed ligand complexes of iron as intermediary steps

Activities of the iron complexes of evolutionary importance like K₄[Fe(CN)₆], K₄[Fe(CN)₅(gly)], and K₄[Fe(CN)₅(trigly)] have been tested towards some redox reactions of biological significance, namely, decomposition of hydrogen peroxide, dehydrogenation of NADH and ascorbic acid both coupled with reduction of methylene blue. It has been observed that the catalytic activities of iron (II) complexes towards the redox reactions studied at pH 9.18 followed the order, K₄[Fe(CN)₆]4[Fe(CN)₅(gly)]4[Fe(CN)₅(trigly)]. Decomposition of H₂O₂ catalysed by cyanocomplexes of iron (II) has been discussed through the formation of an innersphere complex in which loosly bound ligands like, glycine and triglycine are replaced by hydroperoxide ion. A tentative mechanism for the catalysed decomposition of H₂O₂ has been discussed.Based upon the experimental observations a hypothesis on the evolution of iron containing enzymes has been envisaged as: iron(II) ion iron(II) cyanide complexes mixed ligand iron(II) cyanide and amino acid complexes iron(II) complexes of macromolecules proenzyme or early enzyme containing iron(II).     

Abiotic and microbial decomposition of pre- and post-bloom leaves of water hyacinth (Eichhornia crassipes (mart.) Solms)

Rates of abiotic and microbial decomposition in pre- and post-bloom leaves of water hyacinth are determined under laboratory conditions. Decomposition in all types of hyacinth leaves is dominated by physical leaching in an initial phase of 4 days duration, and later by microbial processes. The largest part of physical leaching takes place within the first 4 h. Thereafter, the weight loss due to physical leaching declines exponentially. The weight loss by microbial decomposition is minimal in the initial phase but increases exponentially in the later phase. Pre-bloom leaves decompose significantly faster than post-bloom leaves, and post-bloom green leaves decompose faster than post-bloom brown leaves. The rate constants of abiotic decomposition are significantly higher in post-bloom leaves as compared with pre-bloom leaves, while microbial decomposition is significantly higher in pre-bloom leaves. After 30 days, the dry mass loss by abiotic and microbial decomposition is 15% and 55%, respectively, in pre-bloom leaves, 33% and 19% in post-bloom green leaves, and 24% and 6% in post-bloom brown leaves.     

Role of transition metal ferrocyanides (II) in chemical evolution

Due to ease of formation of cyanide under prebiotic conditions, cyanide ion might have formed stable complexes with transition metal ions on the primitive earth. In the course of chemical evolution insoluble metal cyano complexes, which settled at the bottom of primeval sea could have formed peptide and metal amino acid complexes through adsorption processes of amino acids onto these metal cyano complexes.Adsorption of amino acids such as glycine, aspartic acid, and histidine on copper ferrocyanide and zinc ferrocyanide have been studied over a wide pH range of 3.6 – 8.5. Amino acids were adsorbed on the metal ferrocyanide complexes for different time periods. The progress of the adsorption was followed spectro-photometrically using ninhydrin reagent. Histidine was found to show maximum adsorption on both the adsorbents at neutral pH. Zinc ferrocyanide exhibits good sorption behaviour for all the three amino acids used in these investigations.