Poly(ADP-ribose) polymerase-1 (PARP-1 1 ; EC 2.4.2.30) is an abundant nuclear protein that is activated by DNA strand breakage and that catalyzes the covalent attachment of poly-(ADP-ribose) (PAR) from NAD ϩ to numerous nuclear proteins and transcription factors, including histones; DNA polymerase ␣ and ␤; p53; and PARP-1, itself being the major target, via its automodification domain (1, 2). Besides PARP-1, another six PARPs have been identified: short PARP, PARP-2, PARP-3, tankylase-1/2, and vault PARP (2, 3). However, the physiological roles of poly(ADP-ribosyl)ation of nuclear proteins and transcription factors induced by PARPs are not completely understood. The initially identified subtype of the enzyme, PARP-1, has been thought to play a central role in the process of poly(ADP-ribosyl)ation because poly(ADP-ribosyl)ation is markedly reduced in most tissues of PARP-1 null mice (4). Transient poly(ADP-ribosyl)ation by PARP-1 can be induced by a wide variety of environmental stimuli, including reactive oxygen, ionizing radiation, and genotoxic stress (1, 2). Thus, PARP-1 has been suggested to regulate DNA repair (5). On the other hand, overactivation of PARP-1 by massively damaged DNA consumes NAD ϩ and consequently ATP, resulting in necrotic cell death by energy failure (3, 6).There are many reports suggesting that PARP-1 is also involved in regulation of gene expression at the transcriptional step (2, 3). PARP-1 seems to play dual roles in transcription. Poly(ADP-ribosyl)ation of transcription factors such as YinYang 1 (7), RNA polymerase II-associated factors (8), and p53 (9) results in reversible silencing of transcription by impairing the DNA binding of these proteins. In other instances, PARP-1 was found to have only one function, stimulating the DNA binding activity of transcription factors such as Oct-1 (10) and B-Myb (11). Recent reports have also shown that PARP-1 is required for specific nuclear factor-B (NF-B)-dependent gene expression and acts as a coactivator for 14). Indeed, the NF-B-dependent transcription of some inflammatory mediators in response to endotoxin (13) or pro-inflammatory cytokines such as tumor necrosis factor-␣ (TNF-␣) and interleukin-1␤ (IL-1␤) (12-14) is almost completely abrogated in PARP-1 null mice. Thus, anti-inflammatory effects of PARP-1 inhibitors have been extensively discussed in relation to various inflammation-related diseases (15, 16). However, the exact biochemical mechanism by which PARP-1 regulates NF-B-dependent transcription is obscure. To date, some groups have reported that the enzyme activity of PARP-1 might directly influence NF-B-dependent transcription. Kameoka et al. (17) showed that poly(ADP-ribosyl)ation markedly suppresses the DNA binding activity of NF-B via direct modification in vitro. demonstrated that the DNA binding activity of NF-B p50 is NAD ϩ -dependent and reversibly regulated by the automodification of PARP-1 under cell-free conditions. In contrast, Hassa et al. (14) concluded that neither the enzyme activity nor the DNA binding

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A series of tetrahydrobenzindoles was prepared, and the affinity of these compounds for the 5-hydroxytryptamine7 (5-HT7) receptor and other receptors was evaluated. Most of the compounds showed high affinity for the 5-HT7 receptor, and 2a-[4-(tetrahydropyridoindol-2-yl)butyl]tetrahydrobenzindole derivatives (26a-j) exhibited high selectivity for this receptor. The nature of the substituent at the 9-position of the tetrahydropyridindole ring affected the affinity for the 5-HT7 receptor, and the 9-carbamoyl moiety afforded increased selectivity. Compound 26j exhibited high affinity for the 5-HT7 receptor, with at least 280-fold selectivity over the 5-HT2 receptor. In a functional model of 5-HT7 receptor activation, this compound was confirmed to have 5-HT7 receptor antagonist activity. It should be a useful tool for clarifying the biological role of the 5-HT7 receptor.

A total of 99 clinical isolates of metallo-ß-lactamase-negative Pseudomonas aeruginosa collected in Japan between 1998 and 2001 were studied for their susceptibilities to carbapenem agents and corresponding oprD gene mutations. The OprD sequence of each strain was grouped into two major classes, based on the pattern of alterations. Eighty strains (80.8%) were so-called 'full length type', whose OprD proteins were fully encoded. The remaining 19 strains (19.2%) were so-called 'defective type', which possessed deletions or major alterations that might cause conformational changes in the OprD porin protein. The changes in 'defective type' strains led to 15-, 17-and 23-fold increases in the geometric mean MIC for imipenem, meropenem and biapenem compared with 'full length type' strains, respectively. 'Full length type' strains were further classified into six carbapenem susceptible types with the exception of four carbapenemresistant subtypes with additional amino acid substitutions at D43, G183, R154, G314, G316. However, 'defective type' strains were classified into four types as follows: 10 strains which contained a stop codon within the coding region; six strains which contained IS; one strain with a short deletion near the C-terminal domain; and two strains without a stop codon in the sequenced region. Western blot analysis using OprD antibody showed that binding abilities of OprD proteins against 'full length type' strains were normal, whereas those against 'defective type' strains were lost without exception. These results indicate that OprD structure and antimicrobial activities for carbapenem agents proved to be highly correlated in P. aeruginosa Key words carbapenem resistance, OprD, porin protein, Pseudomonas aeruginosa.Carbapenem antibiotics are recognized as one of the most potent agents with activity against Pseudomonas aeruginosa and remain important for the therapy of serious infections due to these pathogens. Recently, however, the strains with resistance to carbapenems have been increasing among clinical isolates of P. aeruginosa (1).Resistance to carbapenems in P. aeruginosa is often due to the impermeability that arises via the loss of the OprD porin, the overproduction of an active efflux pump system, and the production of carbapenem-hydrolyzing ß-lactamase.Carbapenem-hydrolyzing enzymes are divided into two types based on molecular classification: serine enzymes, which are derivatives of class A or class D enzymes, and metallo-enzymes, which belong to class B enzymes. The production of carbapenem-hydrolyzing enzymes lead to

The coding region of the alpha-amylase inhibitor (HaimIl) gene from the producing strain Streptomyces griseosporeus YM-25 was localized on an 800-base-pair DNA segment. The nucleotide sequence of a 1,191-base-pair region including the HaimIl gene was determined by the dideoxy-chain termination method. The nucleotide sequence data predicted an open reading frame of 363 base pairs starting with an ATG initiation codon and ending with a TGA translational stop codon. The amino acid sequence deduced from the nucleotide sequence indicated that the presumptive pre-HaimIl protein extends 37 amino acids to the amino terminus and 6 amino acids to the carboxyl terminus of the mature HaimIl protein. The pre-HaimIl protein is believed to be processed both during and after secretion. Two forms of the inhibitor, which have a higher molecular weight than that of the HaimIl protein isolated from S. griseosporeus, were partially purified from the culture filtrate of Streptomyces lividans containing the cloned HaimIl gene.Various proteinaceous alpha-amylase inhibitors from microorganisms have been isolated and characterized (3,7,9,20,33,34). These inhibitors are active against alpha-amylases of animal origin but inactive towards those of plants and microorganisms. Haim, a proteinaceous alpha-amylase inhibitor isolated from the culture filtrate of Streptomyces griseosporeus YM-25 (7, 21), also inhibits alpha-amylases of animal origin. Two isoinhibitors produced by S. griseosporeus, Haiml and Haimll, have similar inhibitory specificities and amino acid compositions but differ in their isoelectric points. The amino acid sequence of the Haimll protein has been determined by conventional sequencing methods (20) and found to bear considerable sequence homology with other alpha-amylase inhibitors from Streptomyces species, such as Hoe467A and A13688 (3, 9, 34). Furthermore, these proteins possess four cysteine residues at exactly identical positions and constitute two disulfide bonds, which make up the rigid and stable structure of the inhibitor molecules. Conceivably they have a similar configuration. The recent determination of the three-dimensional structure of the Hoe467A protein largely depended on the small and rigid structure of the molecules (14, 24). These characteristics of alpha-amylase inhibitors of Streptomyces origin appear to be amenable for the study of structure and function of protein molecules by protein engineering.The mechanism of alpha-amylase inhibition by HaimlI and the other inhibitors from Streptomyces spp. is attributed to specific binding of the inhibitor molecule to the active site of alpha-amylase (33). It is reasonable to believe that these alpha-amylase inhibitors will be useful for the study of specific protein-protein interactions.Previously we reported the successful cloning of the (25) is a composite plasmid consisting of pUC9 (37) and pIJ702 (13). YEME medium (10) was used for the cultivation of Streptomyces strains for protoplast transformation and HaimIl production. Streptomyces strains were cultiv...