In contrast to the fact that alpha 1-antitrypsin (alpha 1-AT) deficiency is one of the most common hereditary disorders of Caucasians, deficient variants among Orientals have been recognized to be extremely rare. Only 12 cases of alpha 1-AT deficiency have been reported in Japan, including five cases in which the genetic defects have already been elucidated: Mnichinan (delta Phe52[TTC] and Gly148[GGG]-->Arg148[AGG]), two unrelated cases of Siiyama (Ser53[TCC]-->Phe53[TTC]), a heterozygote of Mmalton (delta Phe52[TTC]), and one additional case of 14q- syndrome (sporadic deletion of the neighboring region of the alpha 1-AT gene locus). alpha 1-AT Siiyama is a deficient variant originally identified in a 38-yr-old patient with pulmonary emphysema in Japan. The amino acid substitution in this variant occurs in a highly conserved residue of the serpin (serine protease inhibitor) backbone (Seyama K, et al. 1991. J. Biol. Chem. 266:12627-12632). We attempted to determine whether alpha 1-AT deficiency in Japan was caused by independent genetic defects or whether it shared some common mutations in the alpha 1-AT gene. We examined five of seven available families for which the genetic defects causing alpha 1-AT deficiency have not yet been explored. When the allele-specific polymerase chain reaction (PCR) was performed with a pair of oligonucleotide primers having the mutated base sequence of the alpha 1-AT Siiyama allele at the 3' end, all eight cases of alpha 1-AT deficiency among five unrelated families turned out to be homozygous carriers of the alpha 1-AT Siiyama mutation.(ABSTRACT TRUNCATED AT 250 WORDS)

Two cell lines producing monoclonal antibodies, Abp4 (IgM) and Abp7 (IgG1) against ampicillin were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. Abp4 showed broad cross-reaction to human serum albumin (HSA) conjugates of several beta-lactams, 6-aminopenicillanic acid and 7-aminocephalosporanic acid. Abp7 reacted only with ampicillin and cephalexin, which have the same acyl side chain. In ELISA inhibition tests, Abp4 inhibited binding to ampicillin-HSA by benzylpenicilloyl-epsilon-amino-n-caproic acid, and strongly inhibited the binding by penicillamine. Abp7 strongly inhibited the reaction by aminobenzylpenicilloyl-epsilon-amino-n-caproic acid, but benzylpenicilloyl-epsilon-amino-n-caproic acid was less effected. These data suggest that Abp4 recognizes the thiazolidine ring and Abp7 recognizes the acyl side chain. Therefore, the thiazolidine ring-epitope acts in broad cross-reaction among beta-lactams, and the acyl side chain acts only in the cross-reactivity between penam and cephem, which have a similar acyl side chain.

-Acute and chronic inflammatory diseases are associated with the induction of inducible nitric oxide synthase (iNOS) and inducible heme oxygenase (HO-1). These inducible enzymes are upregulated in macrophages subjected to inflammatory stimuli and oxidative stress. β 2 -Adrenoceptor (AR) agonists, which function as bronchial dilators, are widely used for the treatment of asthma and chronic obstructive pulmonary disease (COPD). We examined whether salbutamol, a classical β 2 -AR agonist, inhibits the induction of proinflammatory cytokines and stress inducible proteins. Rat macrophages obtained from the abdominal cavity were incubated with lipopolysaccharide (LPS) with or without salbutamol. Induction by LPS of tumor necrosis factor (TNF)-α and interleukin (IL)-6 was significantly inhibited (P < 0.05) by salbutamol treatment. Induction by LPS of iNOS mRNA and protein was also significantly inhibited (P < 0.05) by salbutamol. LPS-mediated increases in HO-1 mRNA and protein were not appreciably affected by salbutamol. One of the anti-inflammatory mechanisms of salbutamol was thus found to be inhibition of induction by LPS of extracellular stimulus-responsive kinase (ERK) 1/2 in macrophages. These findings suggest that salbutamol has the potential for use as an anti-inflammatory agent due to its suppression of LPS-induced TNF-α, and IL-6 and iNOS via ERK pathway without affecting HO-1 expression.Key words: Salbutamol, TNF-α, IL-6, iNOS, LPS Correspondence: Satoru Tanaka (E-mail: satoru_tanaka@pharm.kissei.co.jp) Original ArticleThe Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.35, No.3, 327-334, 2010 Vol. 35 No. 3 327 enzymes play roles in the pathogenesis of and may therefore be useful as targets in the treatment of asthma and COPD. β 2 -adrenoceptor (AR) agonists such as salbutamol are mainstay bronchodilators in the treatment of asthma and COPD. Malfait et al. (1999) demonstrated that salbutamol exhibited therapeutic effects against rheumatoid arthritis model rats with collagen-induced arthritis. In a clinical study, inhalation of a long-acting β 2 -AR agonist by patients with mild asthma yielded a novel antineutrophilic effect (Jeffery et al., 2002).Based on these findings we examined whether β 2 -AR agonists inhibit proinflammatory cytokine and stressinducible protein production by in vitro cultured abdominal macrophages, in an attempt to determine novel targets in the treatment of inflammatory diseases. MATERIALS AND METHODS MaterialsSalbutamol (hemisulfate, minimum purity 98%) and PD98059, an extracellular stimulus-responsive kinase (ERK) 1/2 inhibitor, were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). LPS from Escherichia coli and ICI118551, a selective β 2 -adrenoceptor antagonist, were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). The rat IL-6 Immunoassay KIT and rat TNF-α ELISA KIT were purchased from BioSource International Inc. (Camarillo, CA, USA). Primer sets, including those for iNOS oligo (RA008296), HO-1 oligo (RA008256), and β-actin oligo (RA006...

-Nicardipine hydrochloride (Nic), a calcium channel antagonist, is used for the treatment of hypertension. In the present study, we estimated its effects on the levels and activities of hepatic cytochrome P450 isoforms in spontaneously hypertensive rats given p.o. with Nic at a dose of 0.5, 2.5, 5, or 12.5 mg/kg at 24-hr intervals for 14 days. Therapeutic effects on the development of hypertension were observed at doses of 5 and 12.5 mg/kg/day. Significant increases in the levels of mRNAs and enzyme activities of hepatic P450 isoforms, CYP1A1 and/or CYP1A2, by 14-day repetitive treatment with Nic were observed at lower therapeutic doses, whereas the increase in protein levels for CYP1A2 was observed at a higher therapeutic dose of 12.5 mg/kg/day. Likewise, the activities of hepatic CYP2B and CYP3A subfamily enzymes were increased by the 14-day-treatment of Nic only at a therapeutic dose (12.5 mg/kg/day), whereas their mRNA and protein levels were increased at lower therapeutic doses. To date, the dihydropyridine family, including Nic, has been believed to have inhibitory effects on the activity of various cytochrome P450 enzymes, especially human CYP3A4. However, the present findings demonstrate for the first time that Nic-repetitive treatments at a therapeutic dose result in significant increases in the expressions and activities of hepatic CYP1A, CYP2B, and CYP3A subfamily enzymes. Therefore, the effects of dihydropyridine family on cytochrome P450 enzymes have to be further validated to provide information on its safe and beneficial therapeutic application.

Three cell lines producing monoclonal antibodies, Az-1 (IgG1), Az-2 (IgG1) and Az-3 (IgM) against aztreonam were established. The epitopes and the cross-reactions of the antibodies with various beta-lactams, which were conjugated with human serum albumin (HSA), were examined by enzyme-linked immunosorbent assay (ELISA) and ELISA inhibition test. In ELISA, Az-1 and Az-2 reacted only with aztreonam and ceftazidime, which have the same acyl side chain. Furthermore, Az-2 showed a strong cross-reaction with carumonam. In the ELISA inhibition test, Az-1 and Az-2 were inhibited from binding to aztreonam-HSA by aztreonam, ceftazidime, aztreonam hydrolysate, aztreo-nam-ε-amino-n-caproic acid (EACA) and ceftazidime-EACA. Az-2 was also inhibited with carumonam. From the above results, it seems that Az-1 can recognize only the degraded structure of monobactam nucleus, and Az-2 can recognize the degraded nucleus moiety and the acyl side chain. On the other hand, Az-3 displayed broad cross-reaction to various beta-lactams in ELISA. Furthermore, the MAb showed no inhibitory reaction with various beta-lactams except aztreonam- and ceftazidime-EACA conjugates in the ELISA inhibition test, suggesting that Az-3 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. The above results indicate that antibodies can recognize at least three epitopes of the degraded product(s) of aztreonam nucleus, acyl side chain and NAD in aztreonam-protein conjugate.