Some studies on characterization of three phase partitioned chitosan

Three phase partitioning (TPP) is generally carried out by adding ammonium sulfate and t-butanol to a solution of a macromolecule. Chitosan could be obtained as an interfacial precipitate with 88% yield by subjecting 0.2% (w/v) chitosan solution to TPP with 45% (w/v) ammonium sulfate, with an equal volume of t-butanol at 40 °C. TPP resulted in structural changes which could be seen in its UV spectra, FT-IR spectra and solubility characteristics. TPP-treated chitosan also showed decreased susceptibility towards hydrolysis by chitinase. Thus, TPP can be used as a useful way of altering the properties of chitosan.     

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.     

Germline Allele-Specific Expression of DAPK1 in Chronic Lymphocytic Leukemia

We previously reported a rare germline variant (c.1-6531) that resulted in allele–specific expression (ASE) of death-associated protein kinase 1 (DAPK1) and predisposition to chronic lymphocytic leukemia (CLL). We investigated a cohort of CLL patients lacking this mutation for the presence of ASE of DAPK1. We developed a novel strategy that combines single-nucleotide primer extension (SNuPE) with MALDI-TOF mass spectrometry, and detected germline DAPK1 ASE in 17 out of 120 (14.2%) CLL patients associated with a trend towards younger age at diagnosis. ASE was absent in 63 healthy controls. Germline cells of CLL patients with ASE showed increased levels of DNA methylation in the promoter region, however, neither genetic nor further epigenetic aberrations could be identified in the DAPK1 5′ upstream regulatory region, within distinct exons or in the 3′-UTR. We identified B-lymphoid malignancy related cell line models harboring allelic imbalance and found that allele-specific methylation in DAPK1 is associated with ASE. Our data indicate that ASE at the DAPK1 gene locus is a recurrent event, mediated by epigenetic mechanisms and potentially predisposing to CLL.     

Molecular characterization of <Emphasis Type="Italic">Vigna radiata</Emphasis> (L.) Wilczek genotypes based on nuclear ribosomal DNA and RAPD polymorphism

Mungbean germplasm characterization, evaluation and improvement are fundamentally based on morpho-agronomic traits. The lack of break-through in mungbean production has been due to non-availability of genetic variability for high yield potential. Forty-four genotypes of mungbean [Vigna radiata (L.)Wilczek] were subjected to random amplified polymorphic DNA (RAPD) analysis to assess the genetic diversity and relationships among the genotypes. Multilocus genotyping by twelve RAPD primers generated 166 markers and detected an average of intraspecific variation amounting to 82% polymorphism in banding patterns. Dendrogram obtained from cluster analysis delineated all the 44 genotypes into six clusters. Higher values of Nei’s gene diversity (h) and Shannon information index (i) and genetic distance analysis validate existence of wide genetic diversity among mungbean genotypes tested. Besides internal transcribed spacer (ITS) length variations, single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELS) were detected at number of sites in nuclear rDNA region and the sequences of representatives of each sub-cluster and all distinct genotypes have been submitted to NCBI database and assigned Gen accession numbers HQ 148136-148147. Multiple sequence alignment revealed further lineages of distinct genotypes with main RAPD clusters. The measures of relative genetic distances among the genotypes of mungbean did not completely correlate the geographical places of their development. The homogeneous phenotypic markers proved insufficient in exhibiting genetic divergence among mungbean genotypes studied. RMG-62, RMG-976, and NDM-56 have been identified as potential source of parents for crop improvement. RAPD primers, OPA-9 and OPA-2 as polymorphic genetic markers and number of pods/plant and number of seeds/plant as dependable phenotypic markers have been identified for improving yield potentials. This genetic diversity will be of significance in developing intraspecific crosses in mungbean crop improvement programme.     

Human galanin expresses amphipathic properties that modulate its vasoreactivity in vivo

The purpose of this study was to determine whether human galanin, a pleiotropic 30-amino acid neuropeptide, expresses amphipathic properties in vitro and, if so, whether these properties modulate its vasoactive effects in the intact peripheral microcirculation. We found that human galanin aggregates in an aqueous solution and forms micelles with a critical micellar concentration (CMC) of 0.4 microM. In addition, the peptide interacted with model membrane as indicated by long and significant increase of the surface pressure of the biomimetic monolayer membrane in vitro. Interactions of human galanin with sterically stabilized phospholipid micelles (SMM) were not associated with a significant change in peptide conformation. Using intravital microscopy, we found that suffusion of human galanin alone elicited significant concentration-dependent vasoconstriction in the intact hamster cheek pouch. This response was amplified when human galanin in SSM was suffused onto the cheek pouch. The effects of human galanin alone and in SSM were mediated by galanin receptors because galantide, a galanin receptor antagonist, abrogated galanin-induced vasoconstriction. Collectively, these data show that human galanin expresses amphipathic properties in the presence of phospholipids which in turn amplifies its vasoactive effects in the intact peripheral microcirculation.