Improved determination of urinary oxalate with alkylamine glass bound barley oxalate oxidase

The measurement of oxalate in urine was improved by employing barley oxalate oxidase immobilized on alkylamine glass beads affixed in a glass beaker. The minimum detection limit was 3.6 mg l(-1) urine. The recovery of added oxalate was 88.9+/-9.2%. Within- and between-assay coefficients of variation (CV) were <4.0 and <9.4%, respectively. The urinary oxalate values were obtained by a commercial kit method and the present method showed a good correlation (0.999). The method is free from tedious handling of glass beads and Cl- interference.     

Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells

In chromaffin cells the number of large dense-core vesicles (LDCVs) which can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, we overexpressed Munc13-1 in chromaffin cells and stimulated secretion by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, were increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, was increased by the same factor. The response to a second flash, however, was greatly reduced, indicating a depletion of release-competent vesicles. Since there was no apparent change in the number of docked vesicles, we conclude that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles.     

KC production in the cornea in response to Pseudomonas aeruginosa challenge

Pseudomonas aeruginosa can cause ulcerative bacterial keratitis. A feature of keratitis is the rapid infiltration of the avascular corneal stroma by neutrophils. KC is a potent neutrophil chemokine. The present study used a mouse model of ocular infection to assess the relationship between KC and inflammation in the cornea in response to challenge with a strain of P. aeruginosa causing keratitis. Low levels of KC mRNA and protein were detected by in situ hybridization and ELISA, respectively, in unchallenged corneas. Dramatically increased numbers of KC mRNA+ cells were present in P. aeruginosa strain 6294-challenged corneas. Expression of KC mRNA was found to be up-regulated in the corneal epithelium in response to wounding alone. The KC mRNA+ cells were located in the epithelium and corresponding to infiltrating neutrophils cells in the stroma. Quantification of KC protein at different time points showed peak levels at 8 h of bacterial challenge. These results suggest that KC may be involved with the regulation of leucocyte infiltration early during bacterial keratitis.     

Genetic risk assessment in hookah smokers

The genotoxic effect of hookah smoke was investigated on somatic chromosomes of 35 occupationally nonexposed male hookah smokers. These were compared with an equal number of nonsmokers matched with respect to age, sex, drug intake, if any, and socio-economic status. The mitotic index (MI), chromosomal aberrations (CA), sister chromatid exchanges (SCE) and satellite associations (SA) were analysed. All the parameters showed a significant increase (p < 0.01) in the smokers compared with control individuals, viz MI, 3.88-5.41; CA, 0.94-2.22; SCE, 3.59-5.66; and SA, 5.2-8.65. A distinct time and dose effect relationship was observed. Hookah smoke is thus, both clastogenic and genotoxic for human beings.     

Discrete analysis of plasma oxalate with alkylamine glass bound sorghum oxalate oxidase and horseradish peroxidase

We have reported a simple method of determination of plasma oxalate using a Cl(-) and NO(3)(-) insensitive oxalate oxidase purified from grain sorghum leaf and commercially available peroxidase from horseradish [Pundir et al., Ind. J. Biochem. Biophys., 35 (1998) 120-122]. The present report describes the immobilization of both the enzymes onto alkylamine glass, their kinetic properties and application for discrete analysis of plasma oxalate. In the analytic method, H(2)O(2) generated from plasma oxalate by immobilized oxalate oxidase is measured colorimetrically at 520 nm by oxidative coupling with 4-aminophenazone, and phenol catalyzed by immobilized peroxidase. The minimum detection limit of the method is 2.5 micromol/l. Analytic recovery of added oxalate in plasma was 89. 5+/-4.1% (mean+/-S.D.). The within and between day CV for plasma oxalate measurement were <9.37 and <11.0%, respectively. The normal range of plasma oxalate as measured by the present method was 3.6 to 5.7 micromol/l. The method is not only free from interference by plasma Cl(-) and NO(3)(-) but also provides the reuse of glass beads and thus reduces the cost of analysis for routine.     

Rapid in vitro propagation of Matteuccia struthiopteris (L.) Todaro – an edible fern

A rapid and efficient in vitro plant regeneration method was developed for Matteuccia struthiopteris (L.) Todaro (Ostrich fern). Side shoots, originating in meristems of sectioned rhizomes, were used as explant material. A very high rate of meristem multiplication was achieved by culturing the explants in half-strength MS liquid medium supplemented with 2.0 mg/l N-(4-Pyridyl)-N′-phenylurea (4-PU) and 0.5 mg/l thidiazuron (TDZ). Multiplication of the shoot primordia was faster in suspension culture than on solid medium. Rhizogenesis and growth of regenerants were best achieved on hormone-free one-quarter-strength MS solid medium amended with 0.4% agar and 1.0% activated charcoal. Regenerated plantlets continued to grow after transfer to soil in a phytotron. Received: 19 March 1998 / Revision received: 17 July 1998 / Accepted: 3 August 1998     

Proteomic analysis of oil body membrane proteins accompanying the onset of desiccation phase during sunflower seed development

A noteworthy metabolic signature accompanying oil body (OB) biogenesis during oilseed development is associated with the modulation of the oil body membranes proteins. Present work focuses on 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based analysis of the temporal changes in the OB membrane proteins analyzed by LC-MS/MS accompanying the onset of desiccation (20–30 d after anthesis; DAA) in the developing seeds of sunflower (Helianthus annuus L.). Protein spots unique to 20–30 DAA stages were picked up from 2-D gels for identification and the identified proteins were categorized into 7 functional classes. These include proteins involved in energy metabolism, reactive oxygen scavenging, proteolysis and protein turnover, signaling, oleosin and oil body biogenesis-associated proteins, desiccation and cytoskeleton. At 30 DAA stage, exclusive expressions of enzymes belonging to energy metabolism, desiccation and cytoskeleton were evident which indicated an increase in the metabolic and enzymatic activity in the cells at this stage of seed development (seed filling). Increased expression of cruciferina-like protein and dehydrin at 30 DAA stage marks the onset of desiccation. The data has been analyzed and discussed to highlight desiccation stage-associated metabolic events during oilseed development.     

Structure and Genetic Diversity of Natural Populations of Morus alba in the Trans-Himalayan Ladakh Region

Sequence-related amplified polymorphism markers were used to assess the genetic structure in three natural populations of Morus alba from trans-Himalaya. Multilocation sampling was conducted across 14 collection sites. The overall genetic diversity estimates were high: percentage polymorphic loci 89.66%, Nei’s gene diversity 0.2286, and Shannon’s information index 0.2175. At a regional level, partitioning of variability assessed using analysis of molecular variance (AMOVA), revealed 80% variation within and 20% among collection sites. Pattern appeared in STRUCTURE, BARRIER, and AMOVA, clearly demonstrating gene flow between the Indus and Suru populations and a geographic barrier between the Indus-Suru and Nubra populations, which effectively hinders gene flow. The results showed significant genetic differentiation, population structure, high to restricted gene flow, and high genetic diversity. The assumption that samples collected from the three valleys represent three different populations does not hold true. The fragmentation present in trans-Himalaya was more natural and less anthropogenic.