In the course of surveying for the carbapenem-hydrolyzing metallo-␤-lactamase gene bla IMP in pathogenic bacteria by the PCR method, we detected a gene encoding a variant metallo-␤-lactamase, designated IMP-3, which differed from IMP-1 by having low hydrolyzing activity for penicillins and carbapenems. PCR product direct sequencing of a 2.2-kb segment revealed that the gene bla IMP-3 was located on a cassette inserted within a class I integron in the pMS390 plasmid. The 741-bp nucleotide sequence of bla IMP-3 was identical to that of bla IMP-1 , except for seven base substitutions. Among these were two, at nucleotide positions 314 and 640, which caused amino acid alterations. Hybrid bla genes were constructed from bla IMP-3 and bla IMP-1 by recombinant DNA techniques, and ␤-lactamases encoded by these genes were compared with those of the parents IMP-3 and IMP-1 under the same experimental conditions. The kinetic parameters indicated that the inefficient hydrolysis of benzylpenicillin, ampicillin, imipenem, and ceftazidime by IMP-3 was due to the substitution of glycine for serine at amino acid residue 196 in the mature enzyme. This alteration corresponded to the presence of guanine instead of an adenine at nucleotide position 640 of the bla IMP-3 gene. This indicated that extension of the substrate profile in the metallo-␤-lactamase IMP-1 compared to IMP-3 is the result of a one-step single-base mutation, suggesting that the gene bla IMP-3 is an ancestor of bla IMP-1 .␤-Lactamases are enzymes that hydrolyze ␤-lactam antibiotics, conferring resistance to a variety of these antibiotics for most pathogenic bacteria. These enzymes have been classified phylogenetically based on their functional and molecular characteristics (5).Molecular class B metallo-␤-lactamases belonging to functional group 3a subclass B1 are characteristic in their broad substrate spectrum, which extends to most ␤-lactam antibiotics, except for monobactams, and have activities as penicillinases, cephalosporinases, and carbapenemases (5,15,23). They have been reported in Bacillus cereus, alkalophilic Bacillus sp., Bacteroides fragilis, Pseudomonas aeruginosa, Serratia marcescens, and Klebsiella pneumoniae (23). Among this group of metallo-␤-lactamases, ESP from P. aeruginosa GN17203, IMP-1 from S. marcescens TN9106, and DK4 from K. pneumoniae are all plasmid mediated and were found to be the same enzyme because the nucleotide sequences of their genes are identical (14, 22; GenBank accession number D29636).Genes bla ESP and bla IMP , respectively encoding ESP and IMP-1 ␤-lactamase, were identified in the cassettes inserted in the integrons on plasmids (24). Both cassettes had the same nucleotide sequence, but they were found to be inserted into different integrons, class 1 integron 0 (In0) for the bla ESP cassette (2, 14) and the class 3 integron for the bla IMP cassette (1). This fact suggested that the bla IMP (bla ESP ) cassette has been disseminated among different integrons. Since In0 is reported to be widespread among clinical isolates of ...

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The package insert of the antithrombotic agent warfarin warns users of its interaction with azole antifungals. However, information on the frequency or degree of these interactions is limited. In particular, the time to onset of azole-mediated prothrombin time prolongation, expressed as the international normalized ratio ( The antithrombotic agent, warfarin, shows large inter-and intra-individual differences in its pharmacokinetic and pharmacodynamic properties.1) Prothrombin time, expressed as the international normalized ratio (INR), is commonly used to adjust the individual maintenance dose of warfarin.1) Many drug-drug interactions also contribute to individual differences in warfarin dose. Sufficient understanding of these is therefore necessary for the safe and effective administration of warfarin.2) Details of interactions between warfarin and other drugs, such as the timing of onset or the degree of interaction, have yet to be elucidated. Therefore, it is difficult to decide whether or when INR should be measured.The warfarin package insert in Japan and some other reports suggest an increased risk of bleeding following coadministration of warfarin and azoles. [3][4][5][6] We recently encountered a patient at the Kyoto University Hospital, in whom the INR abruptly increased to >5 when a combination of warfarin and fluconazole (FLCZ) was administered. The inhibition of hepatic drug-metabolizing enzymes is described as the mechanism of this interaction between warfarin and azoles such as FLCZ, voriconazole (VRCZ), and itraconazole (ITCZ).3) However, we were unable to find specific information on the frequency of these interactions, their degree, or the time of onset of INR prolongation for each azole. To the best of our knowledge, there are no previous reports comparing the changes of INR following coadministration of azoles and warfarin in patients. Therefore, we retrospectively compared changes in the anticoagulant effects of warfarin in Japanese patients concomitantly administered with FLCZ, VRCZ, or ITCZ. PATIENTS AND METHODS Study PopulationPatients who were administered warfarin in combination with FLCZ, VRCZ, or ITCZ from April 2010 to March 2013 were analyzed. Patients were excluded under the following circumstances: (1) No INR data; (2) Altered dosage of other agents that were considered highly probable or probable to interact with warfarin 2) (e.g., miconazole oral gel, levofloxacin, and loxoprofen); (3) Increasing warfarin dosage after azole coadministration; (4) Observation of abnormal liver function. Liver function was considered abnormal if the aspartate aminotransferase or alanine aminotransferase level was≥the upper limit of normal×3, in accordance with the Common Terminology Criteria for Adverse Events v4.0.Study Protocol Patient age, sex, warfarin and azole dosing information, the reasons for administration of warfarin and azoles, and INR values were obtained from medical charts retrospectively. INR values were obtained within 2 weeks before commencing coadministration of warfarin and azol...

Antiserum directed to the amphibian bombesin was raised in a guinea pig using the conjugate of N"-glycyl-[1-Glutamine]-bombesin with bovine serum albumin. The major immunologic determinant of this antiserum GP-3303 was shown to reside in the 6-7 sequence of bombesin. Bombesin-like immunoreactivities in gastrointestinal tissue extracts of the pig, dog, monkey, rat and guinea pig were found to be extremely low when measured by the present radioimmunoassay system using antiserum GP-3303. The porcine gastrin releasing peptide recently isolated has the same decapeptide sequence as bombesin (5-14) in the C-terminal region except for the amino acid in position 7 of the bombesin molecule. The very low concentrations of bombesin-like immunoreactivity in the mammalian tissue extracts examined in the present study seem to reflect, at least in part, this amino acid substitution in the position corresponding to position 7 of bombesin, since the amino acid is involved in the major immunologic determinant of antiserum GP-3303. The present result also suggests that the bombesin-like immunoreactivity detected in mammalian tissues may be due to a new group of peptides in which the porcine gastrin releasing peptide is included.KEY WORDS bombesin-like immunoreactivity / porcine gastrin releasing peptide / radioimmunoassay Several of the polypeptides isolated from the amphibian skin have been found to have their counterpart in the mammalian tissues (4). Bombesin was isolated from the skin of the frog Bombina bombina (1) and immunohistochemical (7, 9) and radioimmunological (2, 3, 8) studies have revealed the presence of bombesinlike immunoreactivity in mammalian tissue and blood. McDonald er al. (6) very recently characterized a gastrin releasing peptide from porcine non-antral gastric tissue. The C-terminal region of the peptide showed striking homology with bombesin. This may be interpreted as a