Effect of surfactants and inducers on increased uricase production under submerged fermentations by Bacillus cereus

Uricase is a clinical enzyme used for the oxidation of uric acid crystals in gout disease. The present study aimed to increase the suitable surfactant-mediated uricase production on induction by different concentrations of inducers. The efficiency of Bacillus cereus to produce extracellular uricase enzyme was studied in uric acid-containing agar plates. Among the studied inducers, uric acid is the potential inducer for uricase production under submerged fermentations (SMF), which induced 19.41?U/ml uricase in medium containing 2.0?g/L of uric acid, however further increase in the uric acid concentration decreased uricase production, which could be because of substrate inhibition. The physical parameters including agitation speed (rpm) and time duration (h) of uricase production were optimized and found to produce optimum uricase at 150?rpm in 26?h of SMF. Among the studied surfactants, nonionic surfactant, polyvinyl alcohol has shown a remarkable increase in the uricase production of 31.58?U/ml, which is a 61% increase under optimized conditions in SMF. The stability of produced uricase was found at pH 7.5 and temperature 30°C. Also the effects of various metal ions (1?mM) on the uricase activity were studied and observed to be inhibitory in nature in the descending order K⁺?>?Ca²⁺?>?Zn²⁺?>?Fe³⁺?>?Ni²⁺?>?Mg²⁺?>?Mn²⁺?>?Cu²⁺.     

Xanthoferrin,the α‐hydroxycarboxylate‐type siderophore of Xanthomonas campestris pv. campestris,is required for optimum virulence and growth inside cabbage

Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin‐type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α‐hydroxycarboxylate‐type siderophore (named xanthoferrin), which is required for growth under low‐iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low‐iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore–iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe³⁺) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin–Fe³⁺ complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low‐iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe³⁺.     

Lipid classification, structures and tools

The study of lipids has developed into a research field of increasing importance as their multiple biological roles in cell biology, physiology and pathology are becoming better understood. The Lipid Metabolites and Pathways Strategy (LIPID MAPS) consortium is actively involved in an integrated approach for the detection, quantitation and pathway reconstruction of lipids and related genes and proteins at a systems-biology level. A key component of this approach is a bioinformatics infrastructure involving a clearly defined classification of lipids, a state-of-the-art database system for molecular species and experimental data and a suite of user-friendly tools to assist lipidomics researchers. Herein, we discuss a number of recent developments by the LIPID MAPS bioinformatics core in pursuit of these objectives.     

A LIN28-dependent structural change in pre-let-7g directly inhibits dicer processing

Several recent studies have provided evidence that LIN28, a cytoplasmic RNA-binding protein, inhibits the biogenesis of members of the let-7 microRNA family at the Dicer step in both mammals and Caenorhabditis elegans. However, the precise mechanism of inhibition is still poorly understood. Here we report on an in vitro study, which combined RNase footprinting, gel shift binding assays, and processing assays, to investigate the molecular basis and function of the interaction between the native let-7g precursor (pre-let-7g) and LIN28. We have mapped the structure of pre-let-7g and identified some regions of the terminal loop of pre-let-7g that physically interact with LIN28. We have also identified a conformational change upon LIN28 binding that results in the unwinding of an otherwise double-stranded region at the Dicer processing site of pre-let-7g. Furthermore, we showed that a mutant pre-let-7g that displays an open upper stem inhibited pre-let-7g Dicer processing to the same extent as LIN28. The data support a mechanism by which LIN28 can directly inhibit let-7g biogenesis at the Dicer processing step.     

Coordination polymers and monomers based on new aminocarboxylate ligands: A cadmium(II) polymer containing dimeric aqua-bridged cadmium complex governed by polymeric chain

The synthesis and characterization of novel coordination polymers [Co(HCCB)(H₂O)₂]_n (1), [Zn(HCCB)(H₂O)₂]_n (2), {[Cd(HCCB)₂]·0.5[Cd(μ-H₂O)(H₂O)₄]₂}_n (3) and [Cu(HCCB)(H₂O)₂]_n (4) based on 3-(carboxymethylamino)-4-chlorobenzoic acid (H₃CCB) and mononuclear complexes [Cu(HBCCB)(H₂O)]·H₂O (5), [Co(HBCCB)(H₂O)]·H₂O (6), [Zn(HBCCB)(H₂O)] (7) and [Cd(HBCCB)(H₂O)] (8) containing 3-bis(carboxymethylamino)-4-chlorobenzoic acid (H₃BCCB) have been described. The compounds under investigation have been characterized by elemental analyses, spectral studies and structures of 1-3 and 5 determined crystallographically. Structural data of 1 and 2 revealed that the deprotonated HCCB²⁻ bridges metal centers leading to a double stranded 1D chain. On the other hand, the HCCB²⁻ coordinated thorough carboxylate oxygen and amino nitrogen in 3 to afford a 1D chain whose charge neutrality is maintained by inclusion of aqua-bridged dimer [{Cd(μ-H₂O)(H₂O)₄}₂]⁴⁺. Strong Cu?Cl interaction (2.754 Å) in 5 imposes a coordination geometry that is half-way between the square planar and square pyramidal. The H₃CCB, H₃BCCB and 1-3 and 5 are fluorescent at rt. Thermal studies (TG and DSC) on 1-3 suggested higher stability of 2 relative to 1 and 3 [ΔH_f (kcal/mol), ΔS_f = 152.17, 0.60, 1; 195.56: 0.86, 2; 69.33:0.36, 3].     

Changes in biochemical composition of follicular fluid during reproductive acyclicity in water buffalo (Bubalus bubalis)

This study describes the changes in biochemical composition of follicular fluid during reproductive acyclicity in buffalo. A total of 73 pairs of ovaries collected from 26 reproductively acyclic and 47 reproductively cyclic buffaloes were used in the investigation. Ovarian follicles were classified into small (5.0-6.9 mm), medium (7.0-9.9 mm) and large (≥10.0 mm) sized categories depending upon their diameter. Follicular fluid was aspirated, processed and assayed for glucose, cholesterol, total protein, acid phosphatase and alkaline phosphatase. Glucose concentration was lesser in reproductively acyclic compared to cyclic buffaloes (19.3 ± 2.59 mg/dl compared to 32.6 ± 2.60 mg/dl; P<0.05), mainly due to difference in concentration between small sized follicles (12.4 ± 2.59 mg/dl compared to 28.0 ± 3.32 mg/dl; P<0.05). Cholesterol concentration was also lesser in reproductively acyclic compared to cyclic buffaloes (32.2 ± 2.14 mg/dl compared to 35.5 ± 2.16 mg/dl; P<0.05) and this was related to the lesser concentration found in large follicles (13.8 ± 3.45 mg/dl compared to 37.2 ± 4.10mg/dl; P<0.001). Total protein and acid phosphatase levels were not affected by either the reproductive cyclicity status or the follicular size (4.9 ± 1.07 g/dl to 6.0 ± 0.28 g/dl and 1.2 ± 0.17 U/dl to 2.5 ± 1.22 U/dl, respectively). An increased alkaline phosphatase activity was, however, observed in reproductively acyclic compared to cyclic buffaloes (27.5 ± 3.08 U/dl compared to 14.0 ± 1.09 U/dl; P<0.0001). In conclusion, results of the present study indicate an alteration in the biochemical composition of follicular fluid during reproductive acyclicity in buffalo. The findings provide further support to the notion that poor nutrition is an important factor triggering reproductive acyclicity in buffalo.     

Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50 % methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0–3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0–4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k _unf of native protein to be 5.407 × 10^?5 s^?1 and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.     

Host response profile of human brain proteome in toxoplasma encephalitis co-infected with HIV

Background

Toxoplasma encephalitis is caused by the opportunistic protozoan parasite Toxoplasma gondii. Primary infection with T. gondii in immunocompetent individuals remains largely asymptomatic. In contrast, in immunocompromised individuals, reactivation of the parasite results in severe complications and mortality. Molecular changes at the protein level in the host central nervous system and proteins associated with pathogenesis of toxoplasma encephalitis are largely unexplored. We used a global quantitative proteomic strategy to identify differentially regulated proteins and affected molecular networks in the human host during T. gondii infection with HIV co-infection.

Results

We identified 3,496 proteins out of which 607 proteins were differentially expressed (≥1.5-fold) when frontal lobe of the brain from patients diagnosed with toxoplasma encephalitis was compared to control brain tissues. We validated differential expression of 3 proteins through immunohistochemistry, which was confirmed to be consistent with mass spectrometry analysis. Pathway analysis of differentially expressed proteins indicated deregulation of several pathways involved in antigen processing, immune response, neuronal growth, neurotransmitter transport and energy metabolism.

Conclusions

Global quantitative proteomic approach adopted in this study generated a comparative proteome profile of brain tissues from toxoplasma encephalitis patients co-infected with HIV. Differentially expressed proteins include previously reported and several new proteins in the context of T. gondii and HIV infection, which can be further investigated. Molecular pathways identified to be associated with the disease should enhance our understanding of pathogenesis in toxoplasma encephalitis.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-39) contains supplementary material, which is available to authorized users.