ABSTRACT
We evaluated the effects of antimicrobial drugs on four strains of Pseudomonas aeruginosa that are resistant to eight widely used antipseudomonal drugs (piperacillin, piperacillin-tazobactam, imipenem, meropenem, ceftazidime, aztreonam, amikacin, ciprofloxacin) and colistin. In the killing test, colistin (2 microg/ml) was the most effective, followed by aztreonam (48 microg/ml), piperacillin-tazobactam (192-4 microg/ml), piperacillin (192 microg/ml), and a three drug combination of azetreonam (16 microg/ml), ceftazidime (16 microg/ml), and amikacin (4 microg/ml). Six hours after drug addition, colistin (2 microg/ml), aztreonam (48 microg/ml), piperacillin-tazobactam (192-4 microg/ml), piperacillin (192 microg/ml), and the above three drug combination had bacteriostatic effects on all four strains. Colistin, three time breakpoint of aztreonam, piperacillin, or piperacillin-tazobactam, and the three drug combination of aztreonam, ceftazidime, and amikacin were effective in vitro.
Subject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial/drug effects , Pseudomonas aeruginosa/drug effects , Cross Infection/microbiology , DNA, Bacterial/genetics , Drug Combinations , Humans , Microbial Sensitivity Tests , Pseudomonas Infections/microbiology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Zona pellucida-3 is an essential molecule for the binding of sperm to the egg envelope and for induction of the acrosome reaction in mice. Its homologous molecules, ZPCs, have been widely identified in the eggs of many vertebrates, except for urodeles. In this study, to investigate the participation of ZPC in newt fertilization, we cloned the cDNA of newt ZPC from the ovary of Cynops pyrrhogaster by reverse transcription polymerase chain reaction (RT-PCR). The cDNA was constructed from 1,397 nucleotides and included one open reading frame corresponding to a sequence of 439 amino acids. The deduced amino acid sequence had identities at 52, 47 and 45% with Xenopus gp41, mouse ZP3 and medaka L-SF, respectively. It included four potential N-linked glycosylation sites and 12 highly conserved cysteine residues of mammalian ZP3/ZPC molecules. This result suggests that CynopsZPC (cpZPC) has molecular features similar to those of mammalian ZP3/ZPCs. Messenger RNA for cpZPC was detected in the ovary and faintly in the testis. Two bands corresponding to 84 kDa and 70 kDa in the egg envelopes were detected by immunoblotting with an antiserum raised against a 9 amino acid peptide in the C-terminus domain of cpZPC. The molecular size of 84 kDa fits with the size of a putative sperm-binding protein reported by Nakai et al. (1999), suggesting that cpZPC may contribute to sperm binding to the egg envelope in C. pyrrhogaster. The results of immunohistochemistry suggest that cpZPC was localized in the inner surface of the egg envelope. Similar localization is seen only in fish, suggesting that cpZPC is a unique molecule which may allow us to investigate the functional evolution of the egg envelope in vertebrates.
Subject(s)
Egg Proteins/metabolism , Membrane Glycoproteins/metabolism , Ovary/physiology , Receptors, Cell Surface , Salamandridae/metabolism , Zona Pellucida/physiology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA Primers/chemistry , DNA, Complementary , Egg Proteins/genetics , Female , Immunoblotting , Immunoenzyme Techniques , Male , Membrane Glycoproteins/genetics , Molecular Sequence Data , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Salamandridae/genetics , Salamandridae/growth & development , Sequence Homology, Amino Acid , Zona Pellucida GlycoproteinsABSTRACT
l-Amino acid oxidase (LAO) was purified from mouse milk. LAO reacted with l-amino acids in an apparent order of Phe > Met, Tyr > Cys, Leu > His other 11 amino acids tested and produced H(2)O(2) in a dose- and time-dependent manner. LAO in milk had a molecular mass of about 113 kDa and was converted to a 60-kDa protein by SDS-PAGE. LAO consisted of two subunits. The N- and C-terminal amino acid sequence determination followed by cDNA cloning showed that the 60-kDa protein consisted of 497 amino acids. LAO mRNA spanned about 2.0 kb, and its expression was found only in the mammary epithelial cells. Glucocorticoid was essential for LAO gene expression. Thus, the LAO gene is expressed acutely upon the onset of milk synthesis. LAO mRNA increased 1 day before parturition, peaked during early to mid-lactation, and decreased at the end of lactation. This is the first demonstration showing that LAO is present in milk. Mastitis is caused by an intramammary bacterial infection. As mouse milk produced H(2)O(2) using endogenous free amino acids, we suggest that LAO, together with free amino acids, is responsible for killing bacteria in the mammary gland.
