Characterization and comparative genomic analysis of intronless Adams with testicular gene expression

ADAM (a disintegrin and metalloprotease) family members with testis-specific or -predominant gene expression are divided phylogenically into two groups: ADAMs 2, 3, 5, 27, and 32 (the first group) and ADAMs 4, 6, 20, 21, 24, 25, 26, 29, 30, and 34 (the second group). We cloned and sequenced cDNAs for previously unidentified mouse Adams that belong to the second group. We found that all the Adam genes in the second phylogenic group are transcribed by both somatic and germ cells in mouse testis, representing a unique expression pattern different from that of the first-group Adams. Genomic analyses revealed that all the second-group Adam genes lack introns interrupting protein-coding sequences and many of them are present as multicopy genes, resulting in total of 14 functional mouse genes in this phylogenic group. Comparing the mouse and human ADAM genes, we found that a number of these mouse Adam genes do not have human orthologues and, even if they exist, some orthologues are pseudogenes in human. These results suggest the differential expansion of the second-group Adam genes in the mouse genome during evolution and a relationship between these Adams and male reproduction unique to mouse.     

Calcineurin in animal behavior

The conserved Ca2+/calmodulin-dependent phosphatase calcineurin has been shown to be involved in numerous and diverse functions both at the cellular and organism level. Recent genetic and pharmacological studies in animals including C. elegans, Drosophila, Aplysia, rat and mice have also implicated calcineurin in behavior, particularly in the regulation of plasticity and modulation of behaviors. These studies have not only brought a clearer understanding of the molecular contributions to behavior, but should also give insight into roles that calcineurin may be playing in the cognitive and behavioral defects observed in some diseases.     

Vitamin C and vitamin E protect the rat testes from cadmium-induced reactive oxygen species

Cadmium is an environmental and industrial pollutant that affects the male reproductive system of humans and animals. However, the mechanism of its adverse effect on Leydig cell steroidogenesis remains unknown. The present study points to the possible involvement of oxidative stress in the suppression of steroidogenesis. Cadmium administration caused an increase in reactive oxygen species (ROS) by elevating testicular malondialdehyde (MDA) and decreasing the activities of testicular antioxidant enzymes such as glutathione peroxidase and superoxide dismutase. The mRNA of Steroid Acute Regulatory (StAR) protein was substantially reduced. The activities of testicular delta5-3beta and 17-beta-hydroxysteroid dehydrogenases (HSD) as well as serum testosterone level were also lowered, suggesting that cadmium-induced ROS inhibit testicular steroidogenesis. Supplementation with vitamin C (VC) and or vitamin E (VE) reduced testicular ROS and restored normal testicular function in Cd-exposed rats. We conclude that VC and VE prevent oxidative stress and play vital roles in co-regulating StAR gene expression and steroid production in cadmium-exposed rats.     

Two isoforms of sarco/endoplasmic reticulum calcium ATPase (SERCA) are essential in Caenorhabditis elegans

SERCA (Sarco/Endoplasmic Reticulum Calcium ATPase), a membrane bound Ca(2+)- /Mg(2+)- dependent ATPase that sequesters Ca(2+) into the SR/ER lumen, is one of the essential components for the maintenance of intracellular Ca(2+) homeostasis. Here we describe the identification and functional characterization of a C. elegans SERCA gene (ser-1). ser-1 is a single gene alternatively spliced at its carboxyl terminus to form two isoforms (SER-1A and SER-1B) and displays a high homology (70% identity, 80% similarity) with mammalian SERCAs. Green fluorescent protein (GFP) and whole-mount immunostaining analyses reveal that SER-1 expresses in neuronal cells, body-wall muscles, pharyngeal and vulval muscles, excretory cells, and vulva epithelial cells. Furthermore, SER-1::GFP expresses during embryonic stages and the expression is maintained through the adult stages. Double-stranded RNA injection (also known as RNAi) targeted to each SER-1 isoform results in severe phenotypic defects: ser-1A(RNAi) animals show embryonic lethality, whereas ser-1B(RNAi) results in L1 larval arrest phenotype. These findings suggest that both isoforms of C. elegans SERCA, like in mammals, are essential for embryonic development and post-embryonic growth and survival.     

Analysis of the flt-1 Gene Encoding a ECM Protein, Flectin, in Caenorhabditis elegans

Flectin is a new type of extracellular matrix protein and its function was suggested to provide a micro-environment of great elasticity. The C. elegans genome database revealed the presence of a flectin homologue, flt-1, which shows approximately 40% similarity (20% identity) to chick flectin. Here we propose a new gene structure for the flt-1 based on our experiments and the partial cDNA clones obtained from Y. Kohara and further suggest that the previous gene prediction is incorrect. FLT-1 is shown to be expressed in various neurons, hypodermal cells, distal tip cells and vulva epithelial cells. Immunostaining results with anti-FLT-1 antibody, further confirm the FLT-1 expression in vulva epithelial cells. The lipophilic dye, DiI, was used to identify the head neurons expressing GFP and results indicated that none of the head neurons expressing GFP are the 6 chemosensory neurons. In order to determine the function of flt-1 gene, RNA-mediated interference (RNAi) experiments were conducted.     

Design and synthesis of novel inhibitors of human kynurenine aminotransferase-I

Herein we report 6-ethoxy-6-oxo-5-(2-phenylhydrazono) hexanoic acid and 3-(2-carboxyethyl)-1H-indole-2-carboxylic acid derivatives as synthetically accessible leads for human kynurenine aminotransferase-I (KAT-I) inhibitors. In total, 12 compounds were synthesized and their biological activities were determined using the HPLC-UV based KAT-I inhibition assay. Of the 12 compounds synthesized, 10 were found to inhibit human KAT-I and the most active compound was found to be 5-(2-(4-chlorophenyl) hydrazono)-6-ethoxy-6-oxohexanoic acid (9a) with an IC(50) of 19.8 μM.     

Functional assessment of Nramp-like metal transporters and manganese in Caenorhabditis elegans

Nramp1 (natural resistance-associated macrophage protein-1) is a functionally conserved iron-manganese transporter in macrophages. Manganese (Mn), a superoxide scavenger, is required in trace amounts and functions as a cofactor for most antioxidants. Three Nramp homologs, smf-1, smf-2, and smf-3, have been identified thus far in the nematode Caenorhabditis elegans. A GFP promoter assay revealed largely intestinal expression of the smf genes from early embryonic through adult stages. In addition, smf deletion mutants showed increased sensitivity to excess Mn and mild sensitivity to EDTA. Interestingly, these smf deletion mutants demonstrated hypersensitivity to the pathogen Staphylococcus aureus, an effect that was rescued by Mn feeding or knockdown of the Golgi calcium/manganese ATPase, pmr-1, indicating that Mn uptake is essential for the innate immune system. This reversal of pathogen sensitivity by Mn feeding suggests a protective and therapeutic role of Mn in pathogen evasion systems. We propose that the C. elegans intestinal lumen may mimic the mammalian macrophage phagosome and thus could be a simple model for studying Mn-mediated innate immunity.     

Functional and phenotypic relevance of differentially expressed proteins in calcineurin mutants of Caenorhabditis elegans

Calcineurin is a heterodimeric serine/threonine protein phosphatase, important for many cellular processes such as T-cell regulation, cardiac hypertrophy and kidney development. We previously reported the characterization of Caenorhabditis elegans calcineurin mutants as providing a simple but excellent genetic model system for studying in vivo functions of calcineurin. Calcineurin loss-of-function mutants, cnb-1(lf), and gain-of-function mutants, tax-6(gf), show certain opposite phenotypes as well as some similar phenotypes. In order to explain the phenotypic similarity observed in both loss-of-function and gain-of-function mutants, we examined the proteins that followed similar trends in both mutants relative to wild-type worms by using 2-DE. Interestingly, VHA-13, HSP-6 and phosphoenolpyruvate carboxykinase are down-regulated in both mutants. A total of 96 differentially regulated proteins were identified by MALDI-TOF/MS. Among these, 42 proteins are up-regulated and 54 proteins are down-regulated in calcineurin mutants. Furthermore, knock-down of about 30% of the genes, which are down-regulated in calcineurin mutants, showed some of the phenotypes of calcineurin-null mutants. This analysis suggests the functional relevance of these proteins to calcineurin activity in C. elegans.     

<Emphasis Type="Italic">C. elegans</Emphasis> behavior of preference choice on bacterial food

Caenorhabditis elegans is a free living soil nematode and thus in its natural habitat, C. elegans encounters many different species of soil bacteria. Although some soil bacteria may be excellent sources of nutrition for the worm, others may be pathogenic. Thus, we undertook a study to understand how C. elegans can identify their preferred food using a simple behavioral assay. We found that there are various species of soil bacteria that C. elegans prefers in comparison to the standard laboratory E. coli strain OP50. In particular, two bacterial strains, Bacillus mycoides and Bacillus soli, were preferred strains. Interestingly, the sole feeding of these bacteria to wild type animals results in extended lifespan through the activation of the autophagic process. Further studies will be required to understand the precise mechanism controlling the behavior of identification and selection of food in C. elegans.     

Enhanced 2,3-butanediol production in recombinant Klebsiella pneumoniae via overexpression of synthesis-related genes

2,3-Butanediol (2,3-BD) is a major metabolite produced by Klebsiella pneumoniae KCTC2242, which is a important chemical with wide applications. Three genes important for 2,3-BD biosynthesis acetolactate decarboxylase (budA), acetolactate synthase (budB), and alcohol dehydrogenase (budC) were identified in K. pneumoniae genomic DNA. With the goal of enhancing 2,3-BD production, these genes were cloned into pUC18K expression vectors containing the lacZ promoter and the kanamycin resistance gene to generate plasmids pSB1-7. The plasmids were then introduced into K. pneumoniae using electroporation. All strains were incubated in flask experiments and 2,3-BD production was increased by 60% in recombinant bacteria harboring pSB04 (budA and budB genes), compared with the parental strain K. pneumoniae KCTC2242. The maximum 2,3-BD production level achieved through fedbatch fermentation with K. pneumoniae SGJSB04 was 101.53 g/l over 40 h with a productivity of 2.54 g/l.h. These results suggest that overexpression of 2,3-BD synthesisrelated genes can enhance 2,3-BD production in K. pneumoniae by fermentation.