Catabolism of homophthalic acid by a soil bacterium

A microorganism capable of degrading homophthalic acid as a sole source of carbon was isolated from soil. The strain was tentatively identified as Pseudomonas sp. Oxygen uptake studies were carried out with possible intermediates. Assays for several different enzymes were performed. Homophthalic acid may be metabolized by this bacterium via p-hydroxyphenyl acetic acid and homogentisic acid intermediates.     

Exclusion of candidate genes for canine SRY-negative XX sex reversal

In mammals, the Y-linked SRY gene is normally responsible for testis induction, yet testis development can occur in the absence of Y-linked genes, including SRY. The canine model of SRY-negative XX sex reversal could lead to the discovery of novel genes in the mammalian sex determination pathway. The autosomal genes causing testis induction in this disorder in dogs, humans, pigs, and horses are presently unknown. In goats, a large deletion is responsible for sex reversal linked to the polled (hornless) phenotype. However, this region has been excluded as being causative of the canine disorder, as have WT1 and DMRT1 in more recent studies. The purpose of this study was to determine whether microsatellite marker alleles near or within five candidate genes (GATA4, FOG2, LHX1, SF1, SOX9) are associated with the affected phenotype in a pedigree of canine SRY-negative XX sex reversal. Primer sequences flanking nucleotide repeats were designed within genomic sequences of canine candidate gene homologues. Fluorescence-labeled polymorphic markers were used to screen a subset of the multigenerational pedigree, and marker alleles were determined by software. Our results indicate that the mutation causing canine SRY-negative XX sex reversal in this pedigree is unlikely to be located in regions containing these candidates.     

Exploiting the intracellular compartmentalization characteristics of the S. cerevisiae host cell for enhancing primary purification of lipid‐envelope virus‐like particles

This article demonstrates how the intracellular compartmentalization of the S. cerevisiae host cell can be exploited to impart selectivity during the primary purification of lipid‐envelope virus‐like particles (VLPs). The hepatitis B surface antigen (HBsAg) was used as the VLP model in this study. Expressed HBsAg remain localized on the endoplasmic reticulum and the recovery process involves treating cell homogenate with a detergent for HBsAg liberation. In our proposed strategy, a centrifugation step is introduced immediately following cell disruption but prior to the addition of detergent to allow the elimination of bulk cytosolic contaminants in the supernatant, achieving ～70% reduction of contaminating yeast proteins, lipids, and nucleic acids. Recovery and subsequent treatment of the solids fraction with detergent then releases the HBsAg into a significantly enriched product stream with a yield of ～80%. The selectivity of this approach is further enhanced by operating under moderate homogenization pressure conditions (～400 bar). Observed improvements in the recovery of active HBsAg and reduction of contaminating host lipids were attributed to the low‐shear conditions experienced by the HBsAg product and reduced cell fragmentation, which led to lower coextraction of lipids during the detergent step. As a result of the cleaner process stream, the level of product capture during the loading stage of a downstream hydrophobic interaction chromatography stage increased by two‐fold leading to a concomitant increase in the chromatography step yield. The lower level of exposure to contaminants is also expected to improve column integrity and lifespan. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Comprehensive study of interaction between biocompatible PEG‐InP/ZnS QDs and bovine serum albumin

Polyethylene glycol (PEG) surface modified biocompatible InP/ZnS quantum dots (QDs) act as a potential alternative for conventional carcinogenic cadmium‐based quantum dots for in vivo and in vitro studies. Comprehensively, we studied the interaction between a model protein bovine serum albumin (BSA) and PEGylated toxic free InP/ZnS QDs using various spectroscopic tools such as absorption, fluorescence quenching, time resolved and synchronous fluorescence spectroscopic measurements. These studies principally show that tryptophan (Trp) residues of BSA have preferable binding affinity towards PEG‐InP/ZnS QDs surface and a blue shift in Trp fluorescence emission is a signature of conformational changes in its hydrophobic microenvironment. Photoluminescence (PL) intensity of Trp is quenched by ground state complex formation (static quenching) at room temperature. However, InP/ZnS@BSA conjugates become unstable with increasing temperature and PL intensity of Trp is quenched via dynamic quenching by PEG‐InP/ZnS QDs. Experimentally determined thermodynamic parameters for these conjugates have shown spontaneity, entropy driven and exothermic nature of bio‐conjugation. The calculated binding affinity (n ? 1, Hill coefficient) suggest that the affinity of InP/ZnS QDs for a BSA protein is not dependent on whether or not other BSA proteins are already bound to the QD surface. Energy transfer efficiency (E), Trp residue to InP/ZnS QDs distances and energy transfer rate (k_T) were all obtained from FÖrster resonance energy.     

Degradation of homophthalic acid byAspergillus niger

The fungusAspergillus niger degraded homophthalic acid through the involvement ofo-hydroxyphenylacetic acid and homogentisic acid as the metabolic intermediates. Isolation of intermediates was carried out by extracting the spent medium and by using inhibitor in replacement culture techniques. Metabolites were characterized by various physicochemical methods. Oxygen uptake studies and enzyme investigations also confirmed that the degradation of homophthalic acid follows through these intermediates in the fungus.     

Metabolism of benzalphthalide by Pseudomonas sp.

A Pseudomonas sp. degraded benzalphthalide to o-phthalate and benzoate. A tentative pathway for the metabolism of benzalphthalide in this Pseudomonas sp. is proposed on the basis of isolated metabolites, oxygraphic assay and enzymatic studies.     

Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism. This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate. The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand. We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein. The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 microM. The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P. testosteroni.