Increased Staphylococcus-killing activity of an antimicrobial peptide,lactoferricin B,with minocycline and monoacylglycerol

This study aimed to find antibiotics or other compounds that could increase the antimicrobial activity of an antimicrobial peptide, lactoferricin B (LFcin B), against Staphylococcus aureus, including antibiotic-resistant strains. Among conventional antibiotics, minocycline increased the bactericidal activity of LFcin B against S. aureus, but methicillin, ceftizoxime, and sulfamethoxazole-trimethoprim did not have such an effect. The combination of minocycline and LFcin B had synergistic effects against three antibiotic-resistant strains of S. aureus, according to result of checkerboard analysis. Screening of 33 compounds, including acids and salts, alcohols, amino acids, proteins and peptides, sugar, and lipids, showed that medium-chain monoacylglycerols increased the bactericidal activity of LFcin B against three S. aureus strains. The short-term killing test in water and the killing curve test in growing cultures showed that a combination of LFcin B and monolaurin (a monoacylglycerol with a 12-carbon acyl chain) killed S. aureus more rapidly than either agent alone. These findings may be helpful in the application of antimicrobial peptides in medical or other situations.     

Construction of BAC libraries derived from the ascidian Ciona intestinalis

Large insert genomic bacterial artificial chromosome (BAC) libraries were constructed from a basal chordate, the ascidian Ciona intestinalis. Insert analyses of randomly selected clones indicated that in the first library the mean insert size was 135 kb and predicted a 15-fold coverage of the Ciona genome, and in the second library the mean insert size was 165 kb and predicted a 5-fold coverage of the genome. These first large insert genomic libraries of the ascidian should increase the speed of genomic analyses of basal chordates.     

Diethylstilbestrol attenuates antioxidant activities in testis from male mice

It has been reported that acute exposure to diethylstilbestrol (DES) induces apoptosis in the testis, and antioxidants play a role in preventing DES-induced tissue damage. In this study, the effect of chronic exposure to DES on the antioxidants was examined in the testis and liver. Eight-week old male ICR mice were treated subcutaneously with various doses of DES for 20 days. Morphologically apparent apoptotic changes, 4-hydroxy-2-nonenal-positive cells and TUNEL-positive DNA-fragmentation, were demonstrated in the testis, but were minimal in the liver. Activities of antioxidants such as glutathione (GSH) peroxidase and GSH S -transferase decreased in both the liver and testis. The activity of Mn-superoxide dismutase (SOD) decreased in the liver but increased in the testis. The activity of Cu, Zn-SOD decreased in the liver but was unchanged in the testis. On Western and Northern blots, gamma-glutamylcysteine synthetase ( γ-GCS), a rate limiting enzyme of GSH synthesis, was increased in the liver dependent on the dose of DES. However, the expression of γ-GCS was reduced in the testis. Since quinones, metabolites of DES, generate reactive oxygen species, which damage DNA, antioxidants are important to prevent the damage. The data suggest that antioxidant activities are impaired by DES, and the levels of GSH are related to DES-induced apoptosis in the testis.     

Molecular cloning of DAX1 and SHP cDNAs and their expression patterns in the Nile tilapia,Oreochromis niloticus

Piscine DAX1 and SHP cDNAs with an open reading frame encoding 296 and 258 amino acid residues, respectively, as well as SHP partial gene fragment, were cloned from Nile tilapia. Phylogenetic analyses of DAX1s, SHPs, and homologous EST fragments indicate that DAX1 and SHP are conserved in gene structure and are present throughout vertebrates. A single band of approximately 1.4kb for DAX1 and of approximately 1.2kb for SHP was detected in the Northern blot analysis. Tissue distribution analysis by RT-PCR showed that fish DAX1 and SHP mRNAs are widely expressed in adult tissues, with the most abundant expression in gonads and liver, respectively. DAX1 and SHP were also detected in gonads of both sexes at 5-90 days after hatching (dah). However, the expression of DAX1 is weak at 5 and 10dah and then significantly up-regulated between 10 and 15dah, whereas the expression of SHP is moderate and consistent during the ontogeny.     

Identification of a new member of the GLWamide peptide family: physiological activity and cellular localization in cnidarian polyps

KPNAYKGKLPIGLWamide, a novel member of the GLWamide peptide family, was isolated from Hydra magnipapillata. The purification was monitored with a bioassay: contraction of the retractor muscle of a sea anemone, Anthopleura fuscoviridis. The new peptide, termed Hym-370, is longer than the other GLWamides previously isolated from H. magnipapillata and another sea anemone, A. elegantissima. The amino acid sequence of Hym-370 is six residues longer at its N-terminal than a putative sequence previously deduced from the cDNA encoding the precursor protein. The new longer isoform, like the shorter GLWamides, evoked concentration-dependent muscle contractions in both H. magnipapillata and A. fuscoviridis. In contrast, Hym-248, one of the shorter GLWamide peptides, specifically induced contraction of the endodermal muscles in H. magnipapillata. This is the first case in which a member of the hydra GLWamide family (Hym-GLWamides) has exhibited an activity not shared by the others. Polyclonal antibodies were raised to the common C-terminal tripeptide GLWamide and were used in immunohistochemistry to localize the GLWamides in the tissue of two species of hydra, H. magnipapillata and H. oligactis, and one species of sea anemone, A. fuscoviridis. In each case, nerve cells were specifically labeled. These results suggest that the GLWamides are ubiquitous among cnidarians and are involved in regulating the excitability of specific muscles.     

Possible role of nitric oxide in radiation-induced salivary gland dysfunction

In this study, we developed a murine model of xerostomia to elucidate the mechanism of radiation-induced salivary gland dysfunction and determined the levels of nitric oxide (NO) in the salivary glands to assess its involvement in the salivary dysfunction induced by radiation. In addition, an inhibitor of NO synthesis was administered to the model in vivo, and its effect on saliva secretion was investigated. Salivary gland irradiation at a dose of 15 Gy caused a significant decrease in secretion compared to unirradiated salivary glands. There were no marked differences between the irradiated mice and unirradiated mice in water or food consumption or in body weight changes. The NO levels in the cultured salivary gland epithelial cells were increased by treatment with a combination of interferon gamma (Ifng), interleukin 1-beta (Il1b), and tumor necrosis factor alpha (Tnfa). Irradiation increased the NO level in the salivary gland tissue. The presence of N(G)-monomethyl-l-arginine acetate (l-NMMA), an inhibitor of NO synthesis, caused a decrease in the NO level in cultured salivary gland tissues after irradiation. Administration of l-NMMA to irradiated mice improved saliva secretion. These results suggest that excessive production of NO induced by radiation is involved in the formation of radiation-induced xerostomia. The finding that administration of an inhibitor of NO synthesis ameliorated the dysfunction of irradiated salivary glands indicates that NO plays a role as a mediator of the dry mouth symptoms that occur after irradiation.