Advances in rice breeding,genetics and genomics

Molecular Breeding -     

Catfishes of the genus Sperata (Pisces:Bagridae) in India

DNA barcode data of the South Asian bagrid catfish genus Sperata indicate the presence of at least five species in the Indian subcontinent. Those results, which are supported by morphological data, show a marked increase in species diversity from the recent taxonomic and fishery literature, although each of the five species had been previously named. Two species are restricted to rivers of peninsular India south of the Godavari: Sperata aorides from the Cauvery river basin and S. seenghala from the Krishna river basin. Most literature records of S. seenghala from the Ganges-Brahmaputra-Meghna river basins likely refer to S. lamarrii, a species which appears to also be present in the Indus river basin. Some genetic data reported as S. seenghala from the Ganges-Brahmaputra-Meghna river basins refer to S. aorella. S. aor is widespread in the Ganges-Brahmaputra-Surma river basins in India and Bangladesh, extending southwards to the Godavari river.     

Microscopic insight to specificity of metal ion cofactor in DNA cleavage by restriction endonuclease EcoRV

Restriction endonucleases protect bacterial cells against bacteriophage infection by cleaving the incoming foreign DNA into fragments. In presence of Mg²⁺ ions, EcoRV is able to cleave the DNA but not in presence of Ca²⁺, although the protein binds to DNA in presence of both metal ions. We make an attempt to understand this difference using conformational thermodynamics. We calculate the changes in conformational free energy and entropy of conformational degrees of freedom, like DNA base pair steps and dihedral angles of protein residues in Mg²⁺(A)-EcoRV-DNA complex compared to Ca²⁺(S)-EcoRV-DNA complex using all-atom molecular dynamics (MD) trajectories of the complexes. We find that despite conformational stability and order in both complexes, the individual degrees of freedom behave differently in the presence of two different metal ions. The base pairs in cleavage region are highly disordered in Ca²⁺(S)-EcoRV-DNA compared to Mg²⁺(A)-EcoRV-DNA. One of the acidic residues ASP90, coordinating to the metal ion in the vicinity of the cleavage site, is conformationally destabilized and disordered, while basic residue LYS92 gets conformational stability and order in Ca²⁺(S) bound complex than in Mg²⁺(A) bound complex. The enhanced fluctuations hinder placement of the metal ion in the vicinity of the scissile phosphate of DNA. Similar loss of conformational stability and order in the cleavage region is observed by the replacement of the metal ion. Considering the placement of the metal ion near scissile phosphate as requirement for cleavage action, our results suggest that the changes in conformational stability and order of the base pair steps and the protein residues lead to cofactor sensitivity of the enzyme. Our method based on fluctuations of microscopic conformational variables can be applied to understand enzyme activities in other protein-DNA systems.     

Antibody mediated suppression of secondary IgM response in nude mice against vesicular stomatitis virus

Nude mice immunized with either of the two serotypes of vesicular stomatitis virus (VSV), VSV Indiana (VSV-Ind) or VSV-New Jersey (VSV-NJ), showed an early T cell independent immunoglobulin (Ig) M antibody response comparable with normal euthymic mice. Unlike euthymic mice, however, nude mice reinjected with the homologous serotype were unable to mount a second measurable serum neutralizing (SN) antibody response; a second injection with the heterologous serotype induced a normal primary type of SN antibody response. The serotype-specific refractoriness to a second challenge recovered at about 10 wk after primary infection. When antibody responses were assayed by enzyme-linked immunoabsorbent assay (ELISA), suppressive effects by previous immunization could be observed even after challenge with the heterologous serotypes; this finding probably reflects the known existence of common nonneutralizing determinants shared between the two serotypes. A weak 2-mercaptoethanol (2-ME)-resistant anti-VSV IgG SN antibody response was noticed during the primary response in nude mice and was also found in ELISA; after second infections, this 2-ME-resistant response did not develop. Serum transfer studies in nude and +/+ mice confirmed that the serotype-specific transitory refractoriness of a second response in nude mice was mediated through the anti-VSV-specific IgM antibodies.     

Temperature-Stress-Induced Impairment of Chlorophyll Biosynthetic Reactions in Cucumber and Wheat

Chlorophyll (Chl) biosynthesis in chill (7°C)- and heat (42°C)-stressed cucumber (Cucumis sativus L. cv poinsette) seedlings was affected by 90 and 60%, respectively. Inhibition of Chl biosynthesis was partly due to impairment of 5-aminolevulinic acid biosynthesis both in chill- (78%) and heat-stress (70%) conditions. Protochlorophyllide (Pchlide) synthesis in chill- and heat-stressed seedlings was inhibited by 90 and 70%, respectively. Severe inhibition of Pchlide biosynthesis in chill-stressed seedlings was caused by inactivations of all of the enzymes involved in protoporphyrin IX (Proto IX) synthesis, Mg-chelatase, and Mg-protoporphyrin IX monoester cyclase. In heat-stressed seedlings, although 5-aminolevulinic acid dehydratase and porphobilinogen deaminase were partially inhibited, one of the porphyrinogen-oxidizing enzymes, uroporphyrinogen decarboxylase, was stimulated and coproporphyrinogen oxidase and protoporphyrinogen oxidase were not substantially affected, which demonstrated that protoporphyrin IX synthesis was relatively more resistant to heat stress. Pchlide oxidoreductase, which is responsible for phototransformation of Pchlide to chlorophyllide, increased in heat-stress conditions by 46% over that of the control seedlings, whereas it was not affected in chill-stressed seedlings. In wheat (Triticum aestivum L. cv HD2329) seedlings porphobilinogen deaminase, Pchlide synthesis, and Pchlide oxidoreductase were affected in a manner similar to that of cucumber, suggesting that temperature stress has a broadly similar effect on Chl biosynthetic enzymes in both cucumber and wheat.     

Isolation,purification and characterization of Cardiolipin synthase from Mycobacterium phlei {PRIVATE}

It has been observed that mycobacterial species has high content of cardiolipin (CL) in their cell membranes more so pathogenicmycobacteria and in bacteria CL activates polymerases, gyrases by removing the bound ADP. Therefore, in the present studycardiolipin synthase (cls) which catalyses the formation of CL was isolated purified and characterized from the cell membrane ofMycobacterium phlei. The purified cls obtained from C-18 RP-HPLC column had a molecular weight of 58 kDa with an isoelectricpoint of 4.5. The enzyme activity (11.5+0.15 µM of CL phosphorous. ml-1 minute-1 for PG as substrate and 14+0.35µM of CLphosphorous. ml-1 minute-1 for CDP-DG as substrate) was optimal at pH 4.8 and showed K_M values of 55+0.05µM and 2.56+0.04µMfor phosphatidyl glycerol and CDP-diacylglycerol, respectively, with an absolute requirement of Mg²⁺ and Mn²⁺ ions for its activityhowever, Ca²⁺ ions inhibited the activity of the cls. The partial amino acid sequence of cls showed significant homology with pgsA3gene of M. tuberculosis and in this organism the CL biosynthesis is very high having three genes coding for PLs biosynthesistherefore, enzymes involved in CL biosynthesis may be an attractive drug target in the development of new antimycobacterialdrugs.     

Biofilm formation and acyl homoserine lactone production in the Burkholderia cepacia complex

Acyl homoserine lactone (acyl-HSL)-mediated gene regulation has been shown to influence biofilm formation in one Burkholderia cepacia cystic fibrosis isolate, but it is not known whether this relationship is a consistent feature of the several genomic species that make up the B. cepacia complex (BCC). We screened strains belonging to genomovars I to V of the BCC for biofilm formation on an abiotic surface and for acyl-HSL synthesis. We determined that organisms from each of these genomovars were capable of biofilm formation. Similarly, acyl-HSL was synthesized by organisms from each of genomovars I to V, with most isolates producing octanoyl-HSL in greatest abundance. When biofilms were grown in Luria broth, acyl-HSL synthesis and biofilm formation appeared to be associated, but these phenotypes were independent when the biofilms were grown in basal salts containing citrate. Genomovar V strains synthesized the greatest quantities of acyl-HSL, and genomovar II and III-A strains elaborated the most abundant biofilms. Quorum sensing may play a role in BCC pathogenesis, but it may not regulate biofilm formation under all growth conditions.     

Nano-hydroxyapatite-thermally denatured small intestine sub-mucosa composites for entheses applications

The objective of the present in vitro study was to estimate the adhesion strength of nanometer crystalline hydroxyapatite (HA)-small intestine sub-mucosa (SIS) composites on model implant surfaces. Techniques of thermal denaturation (60 degrees C, 20 min) of SIS were used to enhance the adhesion strength of entheses materials to underlying implants. Specifically, results indicated that the adhesion strength of thermally denatured SIS was 2-3 times higher than that for normal unheated SIS. In addition, aqua-sonicated, hydrothermally treated nano-HA dispersions enhanced the adhesion strength of SIS on implant surfaces. Importantly, results of the present study demonstrated that human skeletal muscle cell (hSkMC) numbers were not affected by thermally denaturing SIS in nano-HA composite coatings; however, they increased on aqua-sonicated nano-HA/SIS composites compared with SIS alone. Interestingly, thermally denatured SIS that contained aqua-sonicated, hydrothermally treated nano-HA decreased human osteoblasts (hOBs) numbers compared with respective unheated composites; all other composites when thermally denatured did not influence hOB numbers. Results also showed that the number of hOBs increased on nano-HA/SIS composites compared with SIS composites alone. Human mesenchymal stem cell (hMSC) numbers were not affected by the presence of nano-HA in SIS composites. For these reasons, the collective results of this in vitro study demonstrated a technique to increase the coating strength of entheses coatings on implant surfaces (using thermally denatured SIS and aqua-sonicated, hydrothermally prepared nano-HA) while, at the same time, supporting cell functions important for entheses regeneration.     

Ca2+-dependent calmodulin binding to FcRn affects immunoglobulin G transport in the transcytotic pathway

The Fcγ receptor FcRn transports immunoglobulin G (IgG) so as to avoid lysosomal degradation and to carry it bidirectionally across epithelial barriers to affect mucosal immunity. Here, we identify a calmodulin-binding site within the FcRn cytoplasmic tail that affects FcRn trafficking. Calmodulin binding to the FcRn tail is direct, calcium-dependent, reversible, and specific to residues comprising a putative short amphipathic α-helix immediately adjacent to the membrane. FcRn mutants with single residue substitutions in this motif, or FcRn mutants lacking the cytoplasmic tail completely, exhibit a shorter half-life and attenuated transcytosis. Chemical inhibitors of calmodulin phenocopy the mutant FcRn defect in transcytosis. These results suggest a novel mechanism for regulation of IgG transport by calmodulin-dependent sorting of FcRn and its cargo away from a degradative pathway and into a bidirectional transcytotic route.