The allelic isozymes of hexose-6-phosphate dehydrogenase isolated from Fundulus heteroclitus: physical characteristics and kinetic properties

Hexose-6-phosphate dehydrogenase (H6PDH-A2; beta-D-glucose:NAD(P)+ oxido-reductase; E.C. 1.1.1.47) of the teleost Fundulus heteroclitus (L.) shows clinal allelic variation along the east coast of North America. Three of the major allelic isozymes have been purified and compared for native molecular weight, subunit molecular weight, isoelectric point, thermal stability, and steady-state kinetic properties (pH 8.0 and 25 degrees C). Significant differences were found among the allelic isozymes for isoelectric point, thermal stability, and some kinetic parameters. The predominant allelic isozyme in northern populations (H6PDH-AcAc) was found to be more sensitive to heat denaturation than were the predominant homozygous allelic isozymes isolated from southern populations (H6PDH-AaAa and H6PDH-AbAb). The H6PDH-AcAc allelic isozyme had both a significantly greater Km for glucose-6-phosphate than did either of the southern phenotypes and a significantly greater Km for NADP+ and Ki of NAD+ than did one of the southern phenotypes (H6PDH-AaAa). While the allelic isozymes are functionally nonequivalent, it is not yet known whether these differences are reflected at higher levels of biological organization.      

Dynamic compression counteracts IL-1β induced inducible nitric oxide synthase and cyclo-oxygenase-2 expression in chondrocyte/agarose constructs

Background  

Nitric oxide and prostaglandin E₂ (PGE₂play pivotal roles in both the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. These mediators are influenced by both IL-1β and mechanical loading, and involve alterations in the inducible nitric oxide synthase (iNOS) and cyclo-oxygenase (COX)-2 enzymes. To identify the specific interactions that are activated by both types of stimuli, we examined the effects of dynamic compression on levels of expression of iNOS and COX-2 and involvement of the p38 mitogen-activated protein kinase (MAPK) pathway.     

Ligand supplementation as a method to increase soluble heterologous protein production

Ligand interactions are central to enzyme or receptor function, constituting a cornerstone in biochemistry and pharmacology. Here we discuss a ligand application that can be exploited to significantly increase the proportion of recombinant protein expressed in soluble form, by including ligands during the culture. Provided that a sufficiently soluble, cell-permeable and avid ligand is available, one can use it to stabilize nascently synthesized proteins, and in this manner promote solubility and prevent aggregation. To our knowledge, this concept has not been explored systematically and we provide here the first data on ligand supplementation in expression experiments across a whole human protein family: the short-chain dehydrogenases/reductases (SDR). We identified glycerrhitinic acid and its hemisuccinate ester, carbenoxolone (CBX), as ligands with variable affinities ranging from low nanomolar to micromolar binding constants against several SDRs. CBX was utilized as a culture additive in Escherichia coli expression systems against a total of approximately 500 constructs derived from 65 SDR targets, and significantly higher levels of soluble protein were obtained for more than four distinct targets. One of these, the glucocorticoid-activating enzyme type 1 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD1), was solubly expressed only at a very low level (<10 microg/l culture) in the absence of ligand; however, soluble expression could be enhanced to mg/l levels by inclusion of CBX or other inhibitors. Other compounds with different chemical scaffolds were used against 11 beta-HSD1 in equivalent expression experiments yielding similar results. Taken together, if suitable ligands for a given protein are available, this approach could be tested quickly and might represent an easy and effective strategy to enhance soluble protein production, suitable for structural and functional characterization studies.     

Reinstatement of Phyllanthus retroflexus Brade (Phyllanthaceae)

Phyllanthus retroflexus Brade is reinstated. It and the very similar P. subemarginatus Müll. Arg. are described, illustrated, and their relationships are discussed, based on a morphological analysis of herbarium specimens. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 78–81.     

MPID: MHC-Peptide Interaction Database for sequence-structure-function information on peptides binding to MHC molecules

SUMMARY: Binding of short antigenic peptides to Major histocompatibility complex (MHC) proteins is the first step in T-cell mediated immune response. To understand the structural principles governing MHC-specific peptide recognition and binding, we have developed the MHC-Peptide Interaction Database (MPID), containing sequence-structure-function information. MPID (version 1.2) contains curated x-ray crystallographic data on 86 MHC peptide complexes, with precomputed interaction parameters (solvent accessibility, hydrogen bonds, gap volume and gap index). A user-friendly web interface and query tools will facilitate the development of predictive algorithms for MHC-peptide binding from a structural viewpoint. AVAILABILITY: Freely accessible from http://surya.bic.nus.edu.sg/mpid.