Immunocytochemical localization of POMC-derived peptides (adrenocorticotropic hormone, α-melanocyte-stimulating hormone and β-endorphin) in the pituitary,brain and olfactory epithelium of the frog,Rana esculenta,during development

Developmental stages of Rana esculenta, starting with the posterior limb-bud stage (stage 26) up to a few days after metamorphosis, were examined immunohistochemically to localize cells and fibers producing some POMC-derived peptides, namely, -MSH, ACTH and -END. Anti ACTH and anti -MSH revealed a positive reaction in the pars intermedia during all stages of development included in this study, whereas no immunoreactivity in this pituitary zone was ever evidenced with anti -END. In the pars distalis strongly positive cells were seen with anti ACTH and anti -END, while anti -MSH yielded weakly positive cells. Interestingly, these peptides were colocalized in the same cells. Immunoreactivity for -MSH was no longer present in the pars distalis during metamorphic climax and postmetamorphosis. In the brain of premetamorphic tadpoles, belonging to stages 26 to 30, a few neurons in the posterior telencephalon showed a positive reaction only with anti -MSH,but from stage 31 (prometamorphosis) onwards, ACTH and -endorphin-like peptide producing cells, together with -MSH-immunoreactive cells, were seen in this region and in the anterior preoptic area and infundibulum. This situation persisted in the subsequent stages of development. Anti -MSH also revealed weakly positive cells in the olfactory epithelium in premetamorphic tadpoles; strong immunoreactivity with anti -MSH was seen in olfactory epithelium cells in animals during prometamorphosis, metamorphic climax and postmetamorphosis. The possible significance of these findings is briefly discussed.     

Germanium tetra(tertiary butoxide): Synthesis, structure and its utility as a precursor for the preparation of europium doped germanium oxide nanoparticles

Germanium tetra(tertiary butoxide), [Ge(O^tBu)₄], has been prepared by the reaction of GeCl₄ with KOBu^t in benzene. It is a monomeric crystalline solid having a distorted tetrahedral configuration, defined by the coordination of four OBu^t groups around germanium atom. The TG analysis showed that the compound is thermally stable and volatilizes at around 130 °C. Europium doped and un-doped germanium oxide nanoparticles were prepared based on the urea hydrolysis of Ge(O^tBu)₄/Eu(OOCCH₃)₃ in ethylene glycol medium at 150 °C followed by heating the resulting product at 750 °C. The nanoparticles were characterized by XRD, TEM and PL measurements. The europium doped nanoparticles, which were nearly monodispersed (∼30 nm), showed luminescence and the Eu³⁺ ions were occupying the surface of the GeO₂ nanoparticles.     

Nephrin regulates lamellipodia formation by assembling a protein complex that includes Ship2, filamin and lamellipodin

Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5' inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.     

Study of the experimental conditions for the lipase production by a newly isolated strain of Pseudomonas aeruginosa for the enantioselective hydrolysis of (±)-methyl trans-3(4-methoxyphenyl) glycidate

A lipase producing bacterium has been isolated from the soil to enantiospecifically hydrolyze the (±)-methyl trans-3(4-methoxyphenyl) glycidate (MPGM), an intermediate in the synthesis of cardiovascular drug, diltiazem. This hydrolysis provided the desired (−)-MPGM in 44% yield with 99% enantiomeric excess. The organism was identified and confirmed as Pseudomonas aeruginosa by 16S rRNA sequencing. The various physiochemical parameters have been optimized for the maximum production of lipase in shake flask. Beef extract was found to be the best nitrogen source for lipase production. The optimized cultivation conditions were 30°C with an initial medium pH 8 in shake flask. Both inoculum age and inoculum concentration have positive effect on the lipase production and (±)-MPGM (3 mM) was found to be the optimal inducer.An erratum to this article can be found at     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

Molecular dynamics simulation study of DNA triplex formed by mixed sequences in solution

The unrestrained molecular dynamics simulation of the triple helical DNA with mix sequences d(GACTGGTGAC).d(CTGACCACTG)*d (GACTGGTGAC), using the particle mesh Ewald sum, is presented here. The Ewald summation method effectively eliminates the usualcut-of of the long range interactions and allowed us to evaluate the full effect of the electrostatic forces. The AMBER5.0 force field has been used during the simulation in solvent. The MD results support a dynamically stable model of DNA triplex over the entire length of the trajectory. The duplex structure assumes the conformation, which is very close to B-DNA. In mixed sequences the purine bases occurs in both strand of DNA duplex. The bases of third strand do not favor the Hoogsteen or/and reverse Hoogsteen type of Hydrogen bonding but they form hydrogen bonds with the bases of both the strand of DNA duplex. The orientation of the third strand is parallel to one of the strand of duplex and all nucleotides (C, A, G & T) show isomorphic behavior with respect to the DNA duplex. The conformation of all the three strands is almost same except few exceptions. Due to interaction of third strand the conformational change in the duplex structure and a finite amount of displacement in the W-C base pairs have been observed. The conformational variation of the back bone torsion angles and helicoidal parameters, groove widths have been discussed. The sequence dependent effects on local conformation, helicoidal and morphological structure, width of the grooves of DNA helix may have important implication for understanding the functional energetics and specificity of interactions of DNA and its triplexes with proteins, pharmaceutical agents and other ligands.     

Cellular localization, oligomerization, and membrane association of the hereditary spastic paraplegia 3A (SPG3A) protein atlastin

Hereditary spastic paraplegias comprise a group of clinically heterogeneous syndromes characterized by lower extremity spasticity and weakness, with distal axonal degeneration in the long ascending and descending tracts of the spinal cord. The early onset hereditary spastic paraplegia SPG3A is caused by mutations in the atlastin/human guanylate-binding protein-3 gene (renamed here atlastin-1), which codes for a 64-kDa member of the dynamin/Mx/guanylate-binding protein superfamily of large GTPases. The atlastin-1 protein is localized predominantly in brain, where it is enriched in pyramidal neurons in the cerebral cortex and hippocampus. In cultured cortical neurons, atlastin-1 co-localized most prominently with markers of the Golgi apparatus, and immunogold electron microscopy revealed a predominant localization of atlastin-1 to the cis-Golgi. Yeast two-hybrid analyses and co-immunoprecipitation studies demonstrated that atlastin-1 can self-associate, and gel-exclusion chromatography and chemical cross-linking studies indicated that atlastin-1 exists as an oligomer in vivo, most likely a tetramer. Membrane fractionation and protease protection assays revealed that atlastin-1 is an integral membrane protein with two predicted transmembrane domains; both the N-terminal GTP-binding and C-terminal domains are exposed to the cytoplasm. Together, these findings indicate that the SPG3A protein atlastin-1 is a multimeric integral membrane GTPase that may be involved in Golgi membrane dynamics or vesicle trafficking.