Mutational study of the bacterial hemoglobin distal heme pocket

Ligand binding experiments on three mutants in the distal heme pocket of Vitreoscilla hemoglobin (GlnE7His, ProE8Ala, and GlnE7His,ProE8Ala) were used to probe the role of GlnE7 and ProE8 in the pocket's unusual structure. The oxygen dissociation constants for the wild type, E8Ala mutant, and E7His mutant proteins were 4.5, 4.7, and 1.7microM, respectively; the K(d) for the double mutant was not determinable by our technique. Visible-Soret spectra of the carbonyl and cyanyl forms and FT-IR of the carbonyl form of the E8 mutant were similar to those of the wild type; the opposite was true for the GlnE7His and GlnE7His,ProE8Ala mutants, which also differed from wild type in the visible-Soret spectra of their oxidized forms. Models of the effects of the mutations on distal pocket structure were consistent with the experimental findings, particularly the larger effects of the GlnE7His change.     

Antioxidant and anti-stress compounds improve regrowth of cryopreserved <Emphasis Type="Italic">Rubus</Emphasis> shoot tips

Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ∼25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols.     

A meta-analysis of the traits affecting dispersal ability in butterflies: can wingspan be used as a proxy?

1. Dispersal ability of a species is a key ecological characteristic, affecting a range of processes from adaptation, community dynamics and genetic structure, to distribution and range size. It is determined by both intrinsic species traits and extrinsic landscape-related properties. 2. Using butterflies as a model system, the following questions were addressed: (i) given similar extrinsic factors, which intrinsic species trait(s) explain dispersal ability? (ii) can one of these traits be used as a proxy for dispersal ability? (iii) the effect of interactions between the traits, and phylogenetic relatedness, on dispersal ability. 3. Four data sets, using different measures of dispersal, were compiled from published literature. The first data set uses mean dispersal distances from capture-mark-recapture studies, and the other three use mobility indices. Data for six traits that can potentially affect dispersal ability were collected: wingspan, larval host plant specificity, adult habitat specificity, mate location strategy, voltinism and flight period duration. Each data set was subjected to both unifactorial, and multifactorial, phylogenetically controlled analyses. 4. Among the factors considered, wingspan was the most important determinant of dispersal ability, although the predictive powers of regression models were low. Voltinism and flight period duration also affect dispersal ability, especially in case of temperate species. Interactions between the factors did not affect dispersal ability, and phylogenetic relatedness was significant in one data set. 5. While using wingspan as the only proxy for dispersal ability maybe problematic, it is usually the only easily accessible species-specific trait for a large number of species. It can thus be a satisfactory proxy when carefully interpreted, especially for analyses involving many species from all across the world.     

Recent trends in remote homology detection: an Indian Medley

The development of remote homology detection methods is a challenging area in Bioinformatics. Sequence analysis-based approaches that address this problem have employed the use of profiles, templates and Hidden Markov Models (HMMs). These methods often face limitations due to poor sequence similarities and non-uniform sequence dispersion in protein sequence space. Search procedures are often asymmetrical due to over or under-representation of some protein families and outliers often remain undetected. Intermediate sequences that share high similarities with more than one protein can help overcome such problems. Methods such as MulPSSM and Cascade PSI-BLAST that employ intermediate sequences achieve better coverage of members in searches. Others employ peptide modules or conserved patterns of motifs or residues and are effective in overcoming dependencies on high sequence similarity to establish homology by using conserved patterns in searches. We review some of these recent methods developed in India in the recent past.     

Targeted deletion of fatty acid transport protein-4 results in early embryonic lethality

Fatty acid transport protein-4 (FATP4) is the major FATP in the small intestine. We previously demonstrated, using in vitro antisense experiments, that FATP4 is required for fatty acid uptake into intestinal epithelial cells. To further examine the physiological role of FATP4, mice carrying a targeted deletion of FATP4 were generated. Deletion of one allele of FATP4 resulted in 48% reduction of FATP4 protein levels and a 40% reduction of fatty acid uptake by isolated enterocytes. However, loss of one FATP4 allele did not cause any detectable effects on fat absorption on either a normal or a high fat diet. Deletion of both FATP4 alleles resulted in embryonic lethality as crosses between heterozygous FATP4 parents resulted in no homozygous offspring; furthermore, no homozygous embryos were detected as early as day 9.5 of gestation. Early embryonic lethality has been observed with deletion of other genes involved in lipid absorption in the small intestine, namely microsomal triglyceride transfer protein and apolipoprotein B, and has been attributed to a requirement for fat absorption early in embryonic development across the visceral endoderm. In mice, the extraembryonic endoderm supplies nutrients to the embryo prior to development of a chorioallantoic placenta. In wild-type mice we found that FATP4 protein is highly expressed by the epithelial cells of the visceral endoderm and localized to the brush-border membrane of extraembryonic endodermal cells. This localization is consistent with a role for FATP4 in fat absorption in early embryogenesis and suggests a novel requirement for FATP4 function during development.     

Cu-mediated N-arylation of 1,2,3-triazin-4-ones: synthesis of fused triazinone derivatives as potential inhibitors of chorismate mutase

A rapid and direct access to N-aryl substituted fused triazinone derivatives has been accomplished via N-arylation of 1,2,3-triazin-4-one ring involving a Cu-mediated coupling between triazinone derivatives and aryl boronic acids. A combination of Cu(OAc)(2)-Et(3)N in 1,2-dichloroethane was found to be effective and various fused triazinone derivatives have been prepared by using this methodology. Molecular structure of a representative compound was confirmed by single crystal X-ray diffraction study. The scope and limitations of this reaction is discussed. Some of the compounds synthesized were tested for chorismate mutase inhibitory properties in vitro. The in vitro dose response study of an active compound is presented.     

Continuous biosorption of cadmium by Moringa olefera in a packed column

The biosorption of Cd(II) by Moringa oleifera using a batch system and a continuous up flow mode in a fixed bed column was studied. Batch adsorption experiments were performed as a function of pH, biosorbent dose, contact time, volume of the solution, and initial metal concentration. The adsorption isotherms obtained fitted well into the Freundlich and Langmuir isotherms. The dynamic removal of cadmium by powdered seed of the Moringa oleifera was studied in a packed column. The effect of bed height (4 and 8 cm) and flow rate (2 and 5mL/min) on biosorption process was investigated and the experimental breakthrough curves were obtained. Results showed that by increasing the bed height and decreasing the flow rate, the breakthrough and exhaustion times increased. The break-through time was considered as a measure of the column performance. The maximum break-through time of 320 min was achieved at the operating condition of 2 mL/min influent flow rate and bed height of 8 cm.