Structural biology of the aldo-keto reductase family of enzymes: catalysis and cofactor binding

The aldo-keto reductases (AKR) comprise a large family of oxidoreductases with importance to both health and industrial applications. The redox chemistry of the AKRs is dependent on NAD(P)H as a cofactor. Despite a wealth of structural and biochemical data relating to the interaction of AKRs with specific inhibitors, much less is known regarding the interactions with cofactor or substrate. In particular, while many X-ray structures are available for AKR/inhibitor complexes, they are only a few examples where apo- and holo- forms can be directly compared. Thus, while the role of the cofactor in the redox chemistry is generally understood, the details of the structural dynamics associated with cofactor binding are less clear. Likewise, the structural details of both cofactor and substrate specificity are limited. In this review, we focus on details of the structural biology and molecular dynamics associated with catalysis, cofactor, and substrate binding as elucidated for those AKRs for which apo- and holo- structures are available. Understanding such dynamics may identify a new direction in the design of specific inhibitors.     

The effect of cooling rate on the survival of cryopreserved bull,ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines

Spermatozoa from three species, bovine, ovine, and porcine, were frozen using standard techniques in two controlled-rate cooling machines, a commercial instrument and a custom-built device. Ice crystallisation was induced mechanically by touching the straws with a pre-cooled rod. The sperm samples were stored 24h, and then thawed rapidly and evaluated for motility, viability, and acrosomal integrity in the membrane-intact population. The custom-built controlled-rate cooling machine proved significantly better at all cooling rates for all species. This was particularly evident for the ram and the boar spermatozoa. In general, -30 or -50 degrees C/min were better than -1 degrees C/min, with a slight advantage being evident for -30 degrees C/min. However, this became very apparent for boar spermatozoa. It is clear that the higher cooling rates are necessary for successful freezing of spermatozoa from these species, and that careful control of the cooling rate is essential for maximal recovery of viable and functional cells. This is best achieved when the cooling profile is controlled from within a dummy sample.     

Catheterization of pulmonary artery in rats with an ultraminiature catheter pressure transducer

Utilizing new materials and miniaturization techniques, an ultraminiature catheter pressure transducer for catheterization of the pulmonary artery (PA) has been developed and applied in intact, spontaneously breathing, anesthetized rats. The catheter arrangement consists of three components: 1) an SPR-671 ultraminiature pressure transducer (measuring catheter), 2) a plastic introducer (sheath) that is slipped over the measuring catheter, and 3) an external wire mounted on the outside of the introducer for bending its tip. The measuring catheter is first inserted through the right jugular vein into the right ventricle. The introducer is then slipped over it. The tip of the introducer is bent so that there is an angle of approximately 90 degrees or less to the shaft. The measuring catheter is advanced across the pulmonary valve into the PA. Measurements of pulmonary arterial pressure were made in five male Long Evans (364 +/- 7 g body wt) and five female Sprague-Dawley (244 +/- 7 g body wt) rats under control conditions. The effects of infusion of norepinephrine (0.1 mg.kg(-1).h(-1) iv for 20-min duration) were tested in Long Evans rats. Pulmonary arterial systolic pressure measurements were 34.0 +/- 0.8 and 29.5 +/- 0.4 mmHg, and diastolic pressure values were 23.6 +/- 0.8 and 18.1 +/- 0.6 mmHg in male Long Evans and female Sprague-Dawley rats, respectively. Norepinephrine induced an increase in pulmonary arterial systolic (40.8 +/- 0.1 mmHg) and diastolic (28.6 +/- 0.4 mmHg) pressures and an elevation in pulmonary vascular resistance from a control value of 0.093 +/- 0.003 to 0.103 +/- 0.004 mmHg.kg.min.ml(-1).     

Role of MDR1 and MRP1 in trophoblast cells,elucidated using retroviral gene transfer

Natural differences in expression and retroviral transduction techniques were used to test the hypothesis that MDR1 P-glycoprotein (P-gp) and MRP1 (multidrug resistance-related protein) contribute to xenobiotic handling by placental trophoblast. RT-PCR and Western blotting in placenta, primary cytotrophoblast cell cultures, and BeWo, JAr, and JEG choriocarcinoma cell lines showed that MRP1 was ubiquitously expressed, whereas MDR1 was absent or minimally expressed in BeWo and JEG cell lines. In syncytiotrophoblast, P-gp was localized predominantly to the microvillous, maternal facing plasma membrane, and MRP1 to the basal, fetal facing plasma membrane. Functional studies showed that cyclosporin A-sensitive accumulation of [³H]vinblastine by cells containing both transport proteins was significantly different from those expressing predominantly MRP1. Retroviral gene transfer of MDR1 to BeWo cells confirmed that this difference was due to the relative expression of MDR1. Therefore, both P-gp and MRP1 contribute to xenobiotic handling by the trophoblast. Localization of P-gp to the microvillous membrane suggests an essential role in preventing xenobiotic accumulation by the syncytiotrophoblast and, therefore, in protecting the fetus. placenta; multidrug resistance; xenobiotic     

Kakadumycins,novel antibiotics from Streptomyces sp NRRL 30566, an endophyte of Grevillea pteridifolia

An endophytic streptomycete (NRRL 30566) is described and partially characterized from a fern-leaved grevillea (Grevillea pteridifolia) tree growing in the Northern Territory of Australia. This endophytic streptomycete produces, in culture, novel antibiotics - the kakadumycins. Methods are outlined for the production and chemical characterization of kakadumycin A and related compounds. This antibiotic is structurally related to a quinoxaline antibiotic, echinomycin. Each contains, by virtue of their amino acid compositions, alanine, serine and an unknown amino acid. Other biological, spectral and chromatographic differences between these two compounds occur and are given. Kakadumycin A has wide spectrum antibiotic activity, especially against Gram-positive bacteria, and it generally displays better bioactivity than echinomycin. For instance, against Bacillus anthracis strains, kakadumycin A has minimum inhibitory concentrations of 0.2-0.3 microg x ml(-1) in contrast to echinomycin at 1.0-1.2 microg x ml(-1). Both echinomycin and kakadumycin A have impressive activity against the malarial parasite Plasmodium falciparum with LD(50)s in the range of 7-10 ng x ml(-1). In macromolecular synthesis assays both kakadumycin A and echinomycin have similar effects on the inhibition of RNA synthesis. It appears that the endophytic Streptomyces sp. offer some promise for the discovery of novel antibiotics with pharmacological potential.     

Endogenously produced endothelial lipase enhances binding and cellular processing of plasma lipoproteins via heparan sulfate proteoglycan-mediated pathway

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family, which includes lipoprotein lipase (LpL) and hepatic lipase (HL). Enzymatic activity of EL has been studied before. Here we characterized the ability of EL to bridge lipoproteins to the cell surface. Expression of EL in wild-type Chinese hamster ovary (CHO)-K1 but not in heparan sulfate proteoglycan (HSPG)-deficient CHO-677 cells resulted in 3-4.4-fold increases of 125I-low density lipoprotein (LDL) and 125I-high density lipoprotein 3 binding (HDL3). Inhibition of proteoglycan sulfation by sodium chlorate or incubation of cells with labeled lipoproteins in the presence of heparin (100 microg/ml) abolished bridging effects of EL. An enzymatically inactive EL, EL-S149A, was equally effective in facilitating lipoprotein bridging as native EL. Processing of LDL and HDL differed notably after initial binding via EL to the cell surface. More than 90% of the surface-bound 125I-LDL was destined for internalization and degradation, whereas about 70% of the surface-bound 125I-HDL3 was released back into the medium. These differences were significantly attenuated after HDL clustering was promoted using antibody against apolipoprotein A-I. At equal protein concentration of added lipoproteins the ratio of HDL3 to VLDL bridging via EL was 0.092 compared with 0.174 via HL and 0.002 via LpL. In summary, EL mediates binding and uptake of plasma lipoproteins via a process that is independent of its enzymatic activity, requires cellular heparan sulfate proteoglycans, and is regulated by ligand clustering.     

Phylogeny of Capparaceae and Brassicaceae based on chloroplast sequence data

Capparaceae and Brassicaceae have long been known to be closely related families, with the monophyly of Capparaceae more recently questioned. To elucidate the relationship between Brassicaceae and Capparaceae as well as to address infrafamilial relationships within Capparaceae, we analyzed sequence variation for a large sampling, especially of Capparaceae, of these two families using two chloroplast regions, trnL-trnF and ndhF. Results of parsimony and likelihood analyses strongly support the monophyly of Brassicaceae plus Capparaceae, excluding Forchhammeria, which is clearly placed outside the Brassicaceae and Capparaceae clade and suggest the recognition of three primary clades-Capparaceae subfamily (subf.) Capparoideae, subf. Cleomoideae, and Brassicaceae. Capparaceae monophyly is strongly contradicted with Cleomoideae appearing as sister to Brassicaceae. Two traditionally recognized subfamilies of Capparaceae, Dipterygioideae and Podandrogynoideae, are embedded within Cleomoideae. Whereas habit and some fruit characteristics demarcate the three major clades, floral symmetry, stamen number, leaf type, and fruit type all show homoplasy. Clades within Capparoideae show a biogeographical pattern based on this sampling. These results are consistent with several alternative classification schemes.     

Dbeta3, an atypical nicotinic acetylcholine receptor subunit from Drosophila : molecular cloning, heterologous expression and coassembly

Insect nicotinic acetylcholine receptors (nAChRs) play a central role in mediating neuronal synaptic transmission and are the target sites for the increasingly important group of neonicotinoid insecticides. Six nicotinic acetylcholine receptor (nAChR) subunits (four alpha-type and two beta-type) have been cloned previously from the model insect species Drosophila melanogaster. Despite extensive efforts, it has not been possible to generate functional recombinant nAChRs by heterologous expression of any combination of these six subunits. It has, however, been possible to express functional hybrid receptors when Drosophila alpha subunits are co-expressed with vertebrate beta subunits. This has led to the assumption that successful heterologous expression might require an, as yet, uncloned beta-type insect subunit. Examination of the recently completed Drosophila genomic sequence data has identified a novel putative nAChR beta-type subunit. Here we report the molecular cloning, heterologous expression and characterization of this putative Drosophila nAChR subunit (Dbeta3). Phylogenetic comparisons with other ligand-gated ion channel subunit sequences support its classification as a nAChR subunit but show it to be a distantly related member of this neurotransmitter receptor subunit family. Evidence that the Dbeta3 subunit is able to coassemble with other Drosophila nAChR subunits and contribute to recombinant nAChRs has been obtained by both radioligand binding and coimmunoprecipitation studies in transfected Drosophila S2 cells.     

Rab3D: a regulator of exocytosis in non-neuronal cells

Rab3D, a small Ras-like GTPase, is a key regulator of intracellular vesicle transport during exocytosis. It has been shown that Rab3 GTPases are abundant in cells with regulated secretory pathways and are thought to confer the specificity of docking and fusion during regulated exocytosis. Unlike other Rab3 isoforms, Rab3D is enriched in a number of non-neuronal tissues and is localised to secretory granules in the cytoplasm of these cells. The structure of Rab3D exhibits all of the conserved domains from the Rab family and also contains hypervariable N- and C-terminal regions. Rab3D undergoes post-translational isoprenylation and cycles between GDP- and GTP-bound forms. Apart from the factors involved in the Rab activation cycle, few Rab3D effector proteins have been identified to date. Nevertheless, it has long been suggested that Rab3D plays a role in regulated exocytotic processes as well as apically directed transcytosis. This review summarises the recent work on the biological function, structural integrity and molecular interactions of Rab3D in non-neuronal cells.