Iron supplementation promotes in vitro shoot induction and multiplication of <Emphasis Type="Italic">Baptisia australis</Emphasis>

C₄ plants can efficiently accumulate CO₂ in leaves and thus reduce wasteful oxygen fixation by the RuBisCO enzyme. Three C₄ enzymes, namely carbonic anhydrase (CA), phosphoenol pyruvate (PEPC) and pyruvate orthophosphate dikinase (PPDK), were over expressed in Oryza sativa L. ssp. indica var. Khitish under the control of green tissue specific promoters PD_54o, PEPC and PPDK, respectively. Integration of these genes was confirmed by Southern hybridization. The relative expression of PEPC, CA and PPDK were, respectively, 6.75, 6.57 and 3.6-fold higher in transgenic plants compared to wild type plants (control). Photosynthetic efficiency of the transgenic plants increased significantly along with a 12?% increase in grain yield compared to wild type plants. Compared to control plants, transgenic plants also showed phenotypic changes such as increased leaf blade size, root biomass, and plant height and anatomical changes such as greater leaf vein number, bundle sheath cells, and bulliform cells. Our findings indicate that the combined over expression of these three enzymes is an efficient strategy for incorporating beneficial physiological and anatomical features that will enable subsequent yield enhancement in C₃ rice plants.     

Morphological and molecular divergence of Indian hill trout,Barilius bendelisis (Hamilton, 1822) stocks from different Rivers in Indo‐Burma biodiversity hotspot: Does river altitude and Dam play a role?

The present study explains the intraspecific variation in Indian Hill trout (Barilius bendelisis) on the basis of image based truss network system and D‐loop region of mtDNA. A total of 210 samples were collected from three different rivers (Teesta, Kameng and Myntudu River) of North East India in Indo‐Burma Biodiversity Hotspot. By using the software applications (tpsDig version 2.1 and PAST), a total of 25 morphometric measurements were generated from 10 landmarks. The Analysis of Variance (ANOVA), Factor Analysis (FA) and Discriminate Function Analysis (DFA) showed, out of the total variations, factor 1 explained 46.74% while factor 2 and factor 3 explained 27.14% and 11.92%, respectively. Using these variables 83.33% of the cross‐validated specimens were classified into distinct groups. Analysis of Molecular Variance (AMOVA) and pairwise Fst value for D‐loop region of mtDNA also showed high to medium level of genetic variation among the stocks and within the stocks. We conclude that the observed discrete stocks might be the result of changing environmental conditions in different rivers of the hotspot as the rivers are present at different altitudinal labels. It is also believed that the variation might be due to the construction of barrages across the river which hinder the mixing among the stocks.     

Treatment of low strength complex wastewater using an anaerobic baffled reactor (ABR)

Treatment of a low strength complex wastewater of chemical oxygen demand (COD) around 500mg/L was studied in a 10L capacity laboratory scale anaerobic baffled reactor (ABR). It was operated at hydraulic retention times (HRTs) of 20, 15, 10, 8 and 6h. Corresponding organic loading rates (OLRs) were 0.6, 0.8, 1.2, 1.5 and 2kg COD/m(3)d. At every HRT (or OLR), pseudo steady state (PSS) was achieved. Even at maximum OLR of 2kg COD/m(3)d, COD and biochemical oxygen demand (BOD) removals exceeded 88%. Removal of particulate fraction of organics was found to be greater than soluble fraction. Compartment-wise studies of various parameters revealed that if the OLR was larger, the number of initial compartments played significant role in the removal of organics. The values of volatile fatty acids (VFA) demonstrated that hydrolysis and acidogenesis were the main biochemical activities in the initial few compartments. Based on the tracer studies, dead space in the ABR was found to range from 23% to 34%. The flow pattern in the ABR was classified as intermediate between plug flow and perfectly mixed flows. Observations from scanning electron micrographs (SEM) also suggested that distinct phase separation takes place in an ABR. Study of organic and hydraulic shock loads revealed that ABR was capable of sustaining the type of shock loads generally experienced at a sewage treatment plant (STP).     

Allosteric Communication between the Pyridoxal 5′-Phosphate (PLP) and Heme Sites in the H2S Generator Human Cystathionine β-Synthase

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5′-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ∼2- to ∼500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H₂S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H₂S-generating reactions catalyzed by CBS.     

Parameterization and classification of the protein universe via geometric techniques

We present a scheme for the classification of 3487 non-redundant protein structures into 1207 non-hierarchical clusters by using recurring structural patterns of three to six amino acids as keys of classification. This results in several signature patterns, which seem to decide membership of a protein in a functional category. The patterns provide clues to the key residues involved in functional sites as well as in protein-protein interaction. The discovered patterns include a "glutamate double bridge" of superoxide dismutase, the functional interface of the serine protease and inhibitor, interface of homo/hetero dimers, and functional sites of several enzyme families. We use geometric invariants to decide superimposability of structural patterns. This allows the parameterization of patterns and discovery of recurring patterns via clustering. The geometric invariant-based approach eliminates the computationally explosive step of pair-wise comparison of structures. The results provide a vast resource for the biologists for experimental validation of the proposed functional sites, and for the design of synthetic enzymes, inhibitors and drugs.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

Restoration of ultrastructural and biochemical changes in alloxan-induced diabetic rat sciatic nerve on treatment with Na3VO4 and Trigonella—a promising antidiabetic agent

Vanadium has been reported to have broad pharmacological activity both in vitro and in vivo. Vanadium compound, sodium orthovanadate, Na₃VO₄, is well known for its hypoglycaemic effects. However, Na₃VO₄ exerts these effects at relatively high doses (0.6 mg/ml) and exhibit several toxic effects. In the present study lower doses of Na₃VO₄ (0.2 mg/ml) are combined with Trigonella foenum graecum seed powder (TSP), another hypoglycaemic agent, to reduce its toxicity without compromising its antidiabetic potential. The efficacy of the lower doses of Na₃VO₄ has been investigated in restoring the altered glucose metabolism and histological structure in the sciatic nerves in 21 and 60 days alloxan diabetic rats. A portion of the glucose was found to be channelled from the normal glycolytic route to polyol pathway, evident by the reduced hexokinase activity and increased polyol pathway enzymes aldose reductase and sorbitol dehydrogenase activity causing accumulation of sorbitol and fructose in diabetic conditions. Ultrastructural observation of the sciatic nerve showed extensive demylination and axonal loss after eight weeks of diabetes induction. Blood glucose levels increased in diabetic rats were normalized with the lower dose of vanadium and Trigonella treatment. The treatment of the diabetic rats with vanadium and Trigonella prevented the activation of the polyol pathway and sugar accumulations. The sciatic nerves were also protected against the structural abnormalities found in diabetes with Trigonella foenum graecum as well as Na₃VO₄. Results suggest that lower doses of Na₃VO₄ may be used in combination with TSP as an efficient antidiabetic agent to effectively control the long-term complications of diabetes in tissues like peripheral nerve.     

A cybernetic model to predict the effect of freely available nitrogen substrate on rifamycin B production in complex media

It is well-known that secondary metabolite production is repressed by excess nitrogen substrate available in the fermentation media. Although the nitrogen catabolite repression has been known, quantitative process models have not been reported to represent this phenomenon in complex medium. In this paper, we present a cybernetic model for rifamycin B production via Amycolatopsis mediterranei S699 in complex medium, which is typically used in industry. Nitrogen substrate is assumed to be present in two forms in the medium; available nitrogen (S _ANS) such as free amino acids and unavailable nitrogen (S _UNS) such as peptides and proteins. The model assumes that an inducible enzyme catalyzes the conversion of S _UNS to S _ANS. Although S _ANS is required for growth and product formation, high concentrations were found to inhibit rifamycin production. To experimentally validate the model, five different organic nitrogen sources were used that differ in the ratio of S _ANS/S _UNS. The model successfully predicts higher rifamycin B productivity for nitrogen sources that contain lower initial S _ANS. The higher productivity is attributed to the sustained availability of S _ANS at low concentration via conversion of S _UNS to S _ANS, thereby minimizing the effects of nitrogen catabolite repression on rifamycin production. The model can have applications in model-based optimization of substrate feeding recipe and in monitoring and control of fed batch processes.     

Insight into trichomonas vaginalis genome evolution through metabolic pathways comparison

Trichomonas vaginalis causes the trichomoniasis, in women and urethritis and prostate cancer in men. Its genome draft published by TIGR in 2007 presents many unusual genomic and biochemical features like, exceptionally large genome size, the presence of hydrogenosome, gene duplication, lateral gene transfer mechanism and the presence of miRNA. To understand some of genomic features we have performed a comparative analysis of metabolic pathways of the T. vaginalis with other 22 significant common organisms. Enzymes from the biochemical pathways of T. vaginalis and other selected organisms were retrieved from the KEGG metabolic pathway database. The metabolic pathways of T. vaginalis common in other selected organisms were identified. Total 101 enzymes present in different metabolic pathways of T. vaginalis were found to be orthologous by using BLASTP program against the selected organisms. Except two enzymes all identified orthologous enzymes were also identified as paralogous enzymes. Seventy-five of identified enzymes were also identified as essential for the survival of T. vaginalis, while 26 as non-essential. The identified essential enzymes also represent as good candidate for novel drug targets. Interestingly, some of the identified orthologous and paralogous enzymes were found playing significant role in the key metabolic activities while others were found playing active role in the process of pathogenesis. The N-acetylneuraminate lyase was analyzed as the candidate of lateral genes transfer. These findings clearly suggest the active participation of lateral gene transfer and gene duplication during evolution of T. vaginalis from the enteric to the pathogenic urogenital environment.     

Isolation and cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant

Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708. ³⁵S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli. This suggested that A. lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system. A genomic library of A. lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E. coli. Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected. Selected recombinant cosmids hybridized with a proU gene probe from E. coli. Complementation of E. coli proU mutant with the A. lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.