Corticosteroid biosynthesis in vitro by testes of the grouper (Epinephelus coioides) after 17alpha-methyltestosterone-induced sex inversion

This study reports the unique compartmentalization of cortisol and 11-deoxycortisol biosynthesis in vitro from [(3)H]17alpha-hydroxyprogesterone (17P) in testicular tissues of groupers after sex inversion induced by 17alpha-methyltestosterone (MT). Before MT implantation, the ovarian tissues produced only nonpolar metabolites. Following sex inversion some 6 months later, synthesis of these nonpolar metabolites was not detectable. Instead, cortisol and 11-deoxycortisol, with yields of about 3% and 14%, respectively, were synthesized together with two other polar metabolites. The corticosteroids and polar metabolites were distinctly nondetectable in ovarian tissues of the control fish throughout the experiment. While the significance of this testicular synthesis of corticosteroids is presently unclear, it could be related to the increased energy demands arising from the reorganization of gonadal tissues during sex inversion.     

Identification of a substrate for Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus by a novel method for substrate identification

Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases.     

Defects in nuclear and cytoskeletal morphology and mitochondrial localization in spermatozoa of mice lacking nectin-2, a component of cell-cell adherens junctions

Nectin-2 is a cell adhesion molecule encoded by a member of the poliovirus receptor gene family. This family consists of human, monkey, rat, and murine genes that are members of the immunoglobulin gene superfamily. Nectin-2 is a component of cell-cell adherens junctions and interacts with l-afadin, an F-actin-binding protein. Disruption of both alleles of the murine nectin-2 gene resulted in morphologically aberrant spermatozoa with defects in nuclear and cytoskeletal morphology and mitochondrial localization. Homozygous null males are sterile, while homozygous null females, as well as heterozygous males and females, are fertile. The production by nectin-2(-/-) mice of normal numbers of spermatozoa containing wild-type levels of DNA suggests that Nectin-2 functions at a late stage of germ cell development. Consistent with such a role, Nectin-2 is expressed in the testes only during the later stages of spermatogenesis. The structural defects observed in spermatozoa of nectin-2(-/-) mice suggest a role for this protein in organization and reorganization of the cytoskeleton during spermiogenesis.     

Absence of trabecular meshwork-inducible stretch response (TISR)/oculomedin gene and proximal promoter mutation in primary open angle glaucoma patients

We investigated the coding exon and promoter sequence in the trabecular meshwork-inducible stretch response (TISR)/oculomedin gene for mutations in Chinese primary open angle glaucoma (POAG) subjects. The entire TISR/oculomedin coding sequence, together with 138 bp of promoter sequence 5' to the start codon and 170 bp of the 3' untranslated region in 110 Chinese POAG patients and 108 unrelated control subjects without glaucoma, aged 50 years or above, were screened for alterations by DNA sequencing. One heterozygous sequence alteration, K28E, was identified in one control subject, and two homozygous sequence alterations, K28K and 135+36delC, were universally found in every sample. As a result, no common TISR/ oculomedin coding sequence nor any proximal promoter mutation that causes POAG was found. The effect of TISR/ oculomedin in glaucoma has yet to be established.     

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters

The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.     

Hybrid vector designs to control the delivery, fate and expression of transgenes

One of the greatest challenges to gene therapy is the targetting of gene delivery selectively to the sites of disease and regulation of transgene expression without adverse effects. Ultimately, the successful realization of these goals is dependent upon improvements in vector design. Over the years, viral vector design has progressed from various types of replication-defective viral mutants to replication-conditioned viruses and, more recently, to 'gutted' and hybrid vectors, which have, respectively, eliminated expression of non-relevant or toxic viral genes and incorporated desired elements of different viruses so as to increase the efficacy of gene delivery in vivo. This review will focus on the different viral and cellular elements which have been incorporated into virus vectors to: improve transduction efficiencies; alter the entry specificity of virions; control the fate of transgenes in the host cells; and regulate transgene expression.     

Immunomagnetic separation of Cryptosporidium parvum oocysts using MACS MicroBeads and high gradient separation columns

We evaluated the MACS immunomagnetic separation (IMS) system for concentrating Cryptosporidium parvum. Oocysts were first labeled with fluorescein isothiocyanate (FITC) or rabbit anti-C. parvum antibodies, then linked to MicroBeads coated with anti-FITC or anti-rabbit IgG, and separated through a high gradient separation column. Results indicated that over 95% of oocysts were recovered and their fluorescence and infectivity were retained. The presence of MicroBeads showed no effect on genomic DNA extraction and subsequent polymerase chain reaction (PCR)-based analyses, as sensitivity of PCR (10 oocysts) and the band pattern of randomly amplified polymorphic DNA (RAPD) were identical to those using DNAs extracted from normally purified oocysts. IMS-PCR consistently detected as few as 10 oocysts from 100 ml of apple juice or homogenized milk and IMS-IFA could detect 100 oocysts from 1 g of deer manure, demonstrating the efficiency of IMS in recovering oocysts from environmental and food samples. Our results suggest that the MACS IMS system could be used for multiple applications in Cryptosporidium research.     

Enzyme-Linked Immunofiltration Assay To Estimate Attachment of Thiobacilli to Pyrite

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10⁴ bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 10⁵ bacteria mg⁻¹ of pyrite at a particle size of 56 to 65 μm. The geometric coverage ratio of pyrite by T. ferrooxidans ranged from 0.25 to 2.25%. This suggests an attachment of T. ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces.     

Architecture of the sugar binding sites in carbohydrate binding proteins-a computer modeling study

Different sugars, Gal, GalNAc and Man were docked at the monosaccharide binding sites of Erythrina corallodenron (EcorL), peanut lectin (PNA), Lathyrus ochrus (LOLI), and pea lectin (PSL). To study the lectin-carbohydrate interactions, in the complexes, the hydroxymethyl group in Man and Gal favors, gg and gt conformations respectively, and is the dominant recognition determination. The monosaccharide binding site in lectins that are specific to Gal/GalNAc is wider due to the additional amino acid residues in loop D as compared to that in lectins specific to Man/Glc, and affects the hydrogen bonds of the sugar involving residues from loop D, but not its orientation in the binding site. The invariant amino acid residues Asp from loop A, and Asn and an aromatic residue (Phe or Tyr) in loop C provides the basic architecture to recognize the common features in C4 epimers. The invariant Gly in loop B together with one or two residues in the variable region of loop D/A holds the sugar tightly at both ends. Loss of any one of these hydrogen bonds leads to weak interaction. While the subtle variations in the sequence and conformation of peptide fragment that resulted due to the size and location of gaps present in amino acid sequence in the neighborhood of the sugar binding site of loop D/A seems to discriminate the binding of sugars which differ at C4 atom (galacto and gluco configurations). The variations at loop B are important in discriminating Gal and GalNAc binding. The present study thus provides a structural basis for the observed specificities of legume lectins which uses the same four invariant residues for binding. These studies also bring out the information that is important for the design/engineering of proteins with the desired carbohydrate specificity.     

High rate of DNA loss in the Drosophila melanogaster and Drosophila virilis species groups

We recently proposed that patterns of evolution of non-LTR retrotransposable elements can be used to study patterns of spontaneous mutation. Transposition of non-LTR retrotransposable elements commonly results in creation of 5' truncated, "dead-on-arrival" copies. These inactive copies are effectively pseudogenes and, according to the neutral theory, their molecular evolution ought to reflect rates and patterns of spontaneous mutation. Maximum parsimony can be used to separate the evolution of active lineages of a non-LTR element from the fate of the "dead-on-arrival" insertions and to directly assess the relative frequencies of different types of spontaneous mutations. We applied this approach using a non-LTR element, Helena, in the Drosophila virilis group and have demonstrated a surprisingly high incidence of large deletions and the virtual absence of insertions. Based on these results, we suggested that Drosophila in general may exhibit a high rate of spontaneous large deletions and have hypothesized that such a high rate of DNA loss may help to explain the puzzling dearth of bona fide pseudogenes in Drosophila. We also speculated that variation in the rate of spontaneous deletion may contribute to the divergence of genome size in different taxa by affecting the amount of superfluous "junk" DNA such as, for example, pseudogenes or long introns. In this paper, we extend our analysis to the D. melanogaster subgroup, which last shared a common ancestor with the D. virilis group approximately 40 MYA. In a different region of the same transposable element, Helena, we demonstrate that inactive copies accumulate deletions in species of the D. melanogaster subgroup at a rate very similar to that of the D. virilis group. These results strongly suggest that the high rate of DNA loss is a general feature of Drosophila and not a peculiar property of a particular stretch of DNA in a particular species group.