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A pentanucleotide TTTCA repeat insertion into a polymorphic TTTTA repeat element in SAMD12 causes benign adult familial myoclonic epilepsy. Although the precise determination of the entire SAMD12 repeat sequence is important for molecular diagnosis and research, obtaining this sequence remains challenging when using conventional genomic/genetic methods, and even short-read and long-read next-generation sequencing technologies have been insufficient. Incomplete information regarding expanded repeat sequences may hamper our understanding of the pathogenic roles played by varying numbers of repeat units, genotype-phenotype correlations, and mutational mechanisms. Here, we report a new approach for the precise determination of the entire expanded repeat sequence and present a workflow designed to improve the diagnostic rates in various repeat expansion diseases. We examined 34 clinically diagnosed benign adult familial myoclonic epilepsy patients, from 29 families using repeat-primed PCR, Southern blot, and long-read sequencing with Cas9-mediated enrichment. Two cases with questionable results from repeat-primed PCR and/or Southern blot were confirmed as pathogenic using long-read sequencing with Cas9-mediated enrichment, resulting in the identification of pathogenic SAMD12 repeat expansions in 76% of examined families (22/29). Importantly, long-read sequencing with Cas9-mediated enrichment was able to provide detailed information regarding the sizes, configurations, and compositions of the expanded repeats. The inserted TTTCA repeat size and the proportion of TTTCA sequences among the overall repeat sequences were highly variable, and a novel repeat configuration was identified. A genotype-phenotype correlation study suggested that the insertion of even short (TTTCA)14 repeats contributed to the development of benign adult familial myoclonic epilepsy. However, the sizes of the overall TTTTA and TTTCA repeat units are also likely to be involved in the pathology of benign adult familial myoclonic epilepsy. Seven unsolved SAMD12-negative cases were investigated using whole-genome long-read sequencing, and infrequent, disease-associated, repeat expansions were identified in two cases. The strategic workflow resolved two questionable SAMD12-positive cases and two previously SAMD12-negative cases, increasing the diagnostic yield from 69% (20/29 families) to 83% (24/29 families). This study indicates the significant utility of long-read sequencing technologies to explore the pathogenic contributions made by various repeat units in complex repeat expansions and to improve the overall diagnostic rate., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Vitamin D plays a protective role in ulcerative colitis (UC) patients through unclear mechanisms. Cathelicidin is an antimicrobial peptide induced by 1,25(OH)D2. Our goal was to evaluate the link between cathelicidin and vitamin D-associated clinical outcomes in UC patients, explore vitamin D induction of cathelicidin in human colon cells, and evaluate the effects of intrarectal human cathelicidin on a murine model of colitis., Methods: Serum and colonic cathelicidin levels were measured in UC patients and correlated with clinical and histologic outcomes. Human colon cells were treated with 1,25(OH)2D and production of cathelicidin and cytokines were quantified. Antimicrobial activity against Escherichia coli from cell culture supernatants was measured. Mice were treated with intrarectal cathelicidin, and its effects on DSS colitis and intestinal microbiota were evaluated., Results: In UC patients, serum 25(OH)D positively correlated with serum and colonic cathelicidin. Higher serum cathelicidin is associated with decreased risk of histologic inflammation and clinical relapse but not independent of 25(OH)D or baseline inflammation. The 1,25(OH)2D treatment of colon cells induced cathelicidin and IL-10, repressed TNF-α, and suppressed Escherichia coli growth. This antimicrobial effect was attenuated with siRNA-cathelicidin transfection. Intrarectal cathelicidin reduced the severity of DSS colitis but did not mitigate the impact of colitis on microbial composition., Conclusions: Cathelicidin plays a protective role in 25(OH)D-associated UC histologic outcomes and murine colitis. Cathelicidin is induced by vitamin D in human colonic epithelial cells and promotes antimicrobial activity against E. coli. Our study provides insights into the vitamin D-cathelicidin pathway as a potential therapeutic target., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Recent studies have suggested that the enteric nervous system can modulate gut immunity. Ecto-nucleoside triphosphate diphosphohydrolases [E-NTPDases] regulate purinergic signalling by sequential phosphohydrolysis of pro-inflammatory extracellular adenosine 5'-triphosphate [ATP]. Herein, we test the hypothesis that E-NTPDases modulate gut inflammation via neuro-immune crosstalk., Design: We determined expression patterns of NTPDase2 and NTPDase3 in murine and human colon. Experimental colitis was induced by dextran sodium sulphate [DSS] in genetically engineered mice deficient in NTPDase2 or NTPDase3. We compared plasma adenosine diphosphatase [ADPase] activity from Crohn's patients and healthy controls, and linked the enzyme activity to Crohn's disease activity., Results: NTPDase2 and -3 were chiefly expressed in cells of the enteric nervous system in both murine and human colon. When compared with wild type, DSS-induced colitis was exacerbated in Entpd2, and to a lesser extent, Entpd3 null mice as measured by disease activity score and histology, and marked anaemia was seen in both. Colonic macrophages isolated from Entpd2 null mice displayed a pro-inflammatory phenotype compared with wild type. In human plasma, Crohn's patients had decreases in ADPase activity when compared with healthy controls. The drop in ADPase activity was likely associated with changes in NTPDase2 and -3, as suggested by inhibitor studies, and were correlated with Crohn's disease activity., Conclusions: NTPDase2 and -3 are ecto-enzymes expressed in the enteric nervous system. Both enzymes confer protection against gut inflammation in experimental colitis and exhibit alterations in Crohn's disease. These observations suggest that purinergic signalling modulated by E-NTPDases governs neuro-immune interactions that are relevant in Crohn's disease., (Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com)

Physiological concentration of Mg 2+ , Cu 2+ , and Zn 2+ accelerated AGE formation only in glucose-mediated conditions, which was effectively inhibited by chelating ligands. Only quercetin (10) inhibited MGO-mediated AGE formation as well as glucose- and ribose-mediated AGE formation among 10 polyphenols (1-10) tested. We performed an additional structure-activity relationship (SAR) study on flavanols (10, 11, 12, 13, and 14). Morin (12) and kaempherol (14) showed inhibitory activity against MGO-mediated AGE formation, whereas rutin (11) and fisetin (13) did not. These observations indicate that 3,5,7,4'-tetrahydroxy and 4-keto groups of 10 are important to yield newly revised mono-MGO adducts (16 and 17) and di-MGO adduct (18) having cyclic hemiacetals, while 3'-hydroxy group is not essential. We propose here a comprehensive inhibitory mechanism of 10 against AGE formation including chelation effect, trapping of MGO, and trapping of reactive oxygen species (ROS), which leads to oxidative degradation of 18 to 3,4-dihydroxybenzoic acid (15) and other fragments.

Objective: Antibodies against 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) have recently been associated with immune-mediated necrotizing myopathy, especially in patients with statin exposure. As the data are very limited concerning phenotypes and treatment in paediatric patients, we aimed to identify the paediatric patients positive for anti-HMGCR antibodies and clarify their features and therapeutic strategies., Methods: We screened 62 paediatric patients who were clinically and/or pathologically suspected to have inflammatory myopathy for anti-HMGCR antibodies. We further re-assessed the clinical and histological findings and the treatment of the patients positive for anti-HMGCR antibodies., Results: We identified nine paediatric patients with anti-HMGCR antibodies (15%). This was more frequent than anti-signal recognition particle antibodies (four patients, 6%) in our cohort. The onset age ranged from infancy to 13 years. Five patients were initially diagnosed with muscular dystrophy, including congenital muscular dystrophy. Most patients responded to high-dose corticosteroid therapy first but often needed adjuvant immunosuppressants to become stably controlled., Conclusion: Paediatric necrotizing myopathy associated with anti-HMGCR antibodies may not be very rare. Phenotypes are similar to those of adult patients, but a chronic slowly progressive course may be more frequent. Some patients share the clinicopathological features of muscular dystrophy indicating that recognizing inflammatory aetiology would be challenging without autoantibody information. On the other hand, most patients responded to treatment, especially those who were diagnosed early. Our results suggest the importance of early autoantibody testing in paediatric patients who have manifestations apparently compatible with muscular dystrophy in addition to those who have typical features of inflammatory myopathy., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Rheumatology.)

Background: Extracellular vesicles (EVs) are membrane-enclosed particles released by cells as a means of intercellular communication. They are potential novel biomarkers, as they are readily isolated from body fluids, and their composition reflects disease pathways. Whether these particles are released from sites of intestinal inflammation in inflammatory bowel disease (IBD) has not previously been determined., Methods: EVs were isolated by ultracentrifugation of colonic luminal fluid aspirates and characterized according to surface proteins, and constituent mRNA and proteins. The effects of EVs on colonic epithelial cells and macrophages in culture were assessed at the transcriptional, translational, and functional levels., Results: Intestinal luminal aspirates contained abundant EVs, at a mean concentration of 4.3 × 10 particles/mL and with a mean diameter of 146 nm. EVs from patients with IBD with a high endoscopic score (≥1) contained significantly higher mRNA and protein levels of interleukin 6 (IL-6), IL-8, IL-10, and tumor necrosis factor α than EVs from healthy controls. EVs were absorbed by cultured colonic epithelial cells, leading to an increased translation of IL-8 protein by recipient cells when treated with EVs from patients with IBD. EVs and EV-treated epithelial cells induced migration of a significantly greater number of macrophages than epithelial cells alone., Conclusions: EVs shed from sites of intestinal inflammation in patients with IBD have a distinct mRNA and protein profile from those of healthy individuals. These EVs have proinflammatory effects on the colonic epithelium, in vitro. Their stability in luminal samples and their mRNA and protein content identify them as a potential fecal biomarker that reflects mucosal inflammatory pathways.

Because the mitotic kinesin Eg5 is essential for the formation of bipolar spindles during eukaryotic cell division, it has been considered as a potential target for cancer treatment. A number of specific and potent inhibitors of Eg5 are known. S-trityl-L-cysteine is one of the inhibitors of Eg5 whose molecular mechanism of inhibition was well studied. The trityl group of S-trityl-L-cysteine was shown to be a key moiety required for potent inhibition. In this study, we synthesized a novel photochromic S-trityl-L-cysteine analogue, 4-(N-(2-(N-acetylcysteine-S-yl) acetyl) amino)-4'- (N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (ACTAB), composed of a trityl group, azobenzene and N-acetyl-L-cysteine, which exhibits cis-trans photoisomerization in order to photocontrol the function of Eg5. ACTAB exhibited cis-trans photoisomerization upon alternating irradiation at two different wavelengths in the visible range, 400 and 480 nm. ACTAB induced reversible changes in the inhibitory activity of ATPase and motor activities correlating with the cis-trans photoisomerization. Compared with cis-ACTAB, trans-ACTAB reduced ATPase activity and microtubule gliding velocity more significantly. These results suggest that ACTAB could be used as photochromic inhibitor of Eg5 to achieve photocontrol of living cells.

Maillard reaction inhibitors could be useful therapeutics for diabetes and other age-related diseases. We isolated for the first time 4-O-demethylsilvaticol (1) and (-)-mitorubrin (2) as Maillard reaction inhibitors from Paecilomyces sp. 3193B. Among the isolated inhibitors, 2 showed most potent inhibitory effect by an SDS-PAGE assay on cross-linked protein formation and by a fluorescent assay on AGE formation.

(-)-Tulipalin B and (+)-6-tuliposide B were confirmed to inhibit MurA in vitro. However, contrary to fosfomycin, these compounds showed potent inhibitory activities against MurA overexpressing Escherichia coli, especially in the presence of UDP-GlcNAc. These observations suggest that these compounds induced bacterial cell death not through a MurA malfunction, but in such a MurA-mediated indirect manner as the inhibition of other Mur enzymes.

Total elbow arthroplasty (TEA) with the GSB III prosthesis was performed in 32 patients (36 elbows) with rheumatoid arthritis between 2001 and 2009. At final follow-up, 31 patients (35 TEAs) were available for clinical and radiological evaluation. The mean follow-up period was 6.3 (2.0-10.3) years, with a minimum follow-up of 2 years. The mean Mayo elbow performance score was significantly improved from 48 points preoperatively to 83 points at final follow-up. The radiographic loosening rate was 14.3% for humeral components and 5.7% for ulnar components. There were 4 cases of intraoperative fracture and 1 case of humeral shaft fracture at 4 months after surgery. The rates for loosening and fracture were relatively low when compared with those in other studies of linked TEA. There were 2 cases of ulnar nerve palsy, but there was no deep infection or triceps disruption. The clinical results of TEA using the GSB III prosthesis in patients with rheumatoid arthritis were found to be satisfactory.

The highly reactive α-dicarbonyl compounds, glyoxal, methylglyoxal (MGO), and 3-deoxyglucosone, react with the amino groups of proteins to form advanced glycation end-products (AGEs) which have been implicated in diabetic complications, aging, and Alzheimer's disease. We found that a test sample of terpinen-4-ol (T4) containing hydroperoxides showed cleaving activity toward an α-dicarbonyl compound, but that the freshly isolated pure sample did not. Prepared terpinen-4-ol hydroperoxide (T4-H) also efficiently cleaved the C-C bond of the α-dicarbonyl compounds via Baeyer-Villiger-like rearrangement and subsequent hydrolysis of an acid anhydride moiety in the rearranged product to give carboxylic acids. Other terpene hydroperoxides, as well as T4-H, showed significant cleaving activities, and all these hydroperoxides protected RNase A from the lowering of enzyme activity induced by MGO. The cleaving mechanism via Baeyer-Villiger-like rearrangement was confirmed by time-interval NMR measurements of the reaction mixture of the symmetrical α-dicarbonyl compound, diacetyl with T4-H.

The optimal cellular responses to DNA damage are modulated by kinase and phosphatase. The ataxia telangiectasia mutated (ATM) is a Ser/Thr kinase which is the core of the DNA damage signaling apparatus. The Ser/Thr protein phosphatase type 1 (PP1) inhibitor, tautomycetin (TC) and an antibody to the phospho-(S/T)Q sites of the ATM substrate were used to identify the common substrates for PP1 and ATM in regulating the pathway for DNA damage response. Ribosomal protein S6 (RPS6) was first identified as a substrate for PP1 and ATM. The phosphorylation at Ser247 of RPS6 was then significantly decreased by PP1-mediated dephosphorylation immediately after UV irradiation. These results suggest that PP1 specifically dephosphorylated RPS6 at phospho-Ser247 in vivo. In response to DNA damage, ATM activity was finally required for the phosphorylation of RPS6 at Ser247. We propose from these results a novel mechanism for modulating the RPS6 function by PP1 and ATM which regulates cell growth and survival in response to DNA-damage stimuli.

Mutagenized cell libraries of Corynebacterium glutamicum were screened for mutants that lost the ability to grow under low oxygen concentrations. The resulting high-oxygen-requiring mutants were used to clone wild-type DNA fragments that could complement the phenotype. Sequencing and subcloning analyses identified six genes, Cgl0807, Cgl1102, Cgl0600, Cgl1427, Cgl2857, and Cgl2859, as the genes responsible for complementation. Some of these genes showed cross-complementation of the mutants in oxygen-limiting static culture, suggesting the utility of these genes for improved growth and production under oxygen limitation.

Mycophenolic acid (MPA, 1), an inhibitor of IMP-dehydrogenase (IMPDH) and a latent PPARgamma agonist, is used as an effective immunosuppressant for clinical transplantation and recently entered clinical trials in advanced multiple myeloma patients. On the other hand, suberoylanilide hydroxamic acid (SAHA), a non-specific histone deacetylase (HDAC) inhibitor, has been approved for treating cutaneous T-cell lymphoma. MPA seemed to bear a cap, a linker, and a weak metal-binding site as a latent inhibitor of HDAC. Therefore, the hydroxamic acid derivatives of mycophenolic acid having an effective metal-binding site, mycophenolic hydroxamic acid (MPHA, 2), 7-O-acetyl mycophenolic acid (7-O-Ac MPHA, 3), and 7-O-lauroyl mycophenolic hydroxamic acid (7-O-L MPHA, 4) were designed and synthesized. All these compounds inhibited histone deacetylase with IC50 values of 1, 0.9 and 0.5 microM, and cell proliferation at concentrations of 2, 1.5 and 1 microM, respectively.

Genomic analysis of a classically derived L-lysine-producing mutant, Corynebacterium glutamicum B-6, identified a nonsense mutation in the mqo gene, which encodes malate:quinone oxidoreductase (MQO). The effect of mqo disruption on L-lysine production was investigated in a defined L-lysine producer, C. glutamicum AHP-3, showing approximately 18% increased production. To explore the underlying mechanisms of the increase, the mqo-disrupted strain was analyzed from the viewpoints of redox balance, activities of membrane-bound dehydrogenases, and transcriptome. The intracellular [NADH]/[NAD] ratio in the strain remained unchanged. Also, there were no significant differences in the activities of the membrane-bound dehydrogenases examined. However, transcriptome analysis showed that some TCA cycle genes, such as acn, sucC, and sucD, were down-regulated in the strain. These results suggest that the loss of MQO activity down-regulates the flux of the TCA cycle to maintain the redox balance and results in redirection of oxaloacetate into L-lysine biosynthesis.

Based on the progress in genomics, we have developed a novel approach that employs genomic information to generate an efficient amino acid producer. A comparative genomic analysis of an industrial L-lysine producer with its natural ancestor identified a variety of mutations in genes associated with L-lysine biosynthesis. Among these mutations, we identified two mutations in the relevant terminal pathways as key mutations for L-lysine production, and three mutations in central metabolism that resulted in increased titers. These five mutations when assembled in the wild-type genome led to a significant increase in both the rate of production and final L-lysine titer. Further investigations incorporated with transcriptome analysis suggested that other as yet unidentified mutations are necessary to support the L-lysine titers observed by the original production strain. Here we describe the essence of our approach for strain reconstruction, and also discuss mechanisms of L-lysine hyperproduction unraveled by combining genomics with classical strain improvement.

Toward the elucidation of advanced mechanisms of L-lysine production by Corynebacterium glutamicum, a highly developed industrial strain B-6 was analyzed from the viewpoint of gene expression. Northern blot analysis showed that the lysC gene encoding aspartokinase, the key enzyme of L-lysine biosynthesis, was up-regulated by several folds in strain B-6, while no repression mechanism exists in L-lysine biosynthesis of this bacterium. To analyze the underlying mechanisms of the up-regulation, we compared the transcriptome between strain B-6 and its parental wild-type, finding that not only lysC but also many other amino acid-biosynthetic genes were up-regulated in the producer. These results suggest that a certain global regulatory mechanism is involved in the industrial levels of L-lysine production.

Toward more efficient L-lysine production, we have been challenging genome-based strain breeding by the approach of assembling only relevant mutations in a single wild-type background. Following the creation of a new L-lysine producer Corynebacterium glutamicum AHP-3 that carried three useful mutations (lysC311, hom59, and pyc458) on the relevant downstream pathways, we shifted our target to the pentose phosphate pathway. Comparative genomic analysis for the pathway between a classically derived L-lysine producer and its parental wild-type identified several mutations. Among these mutations, a Ser-361-->Phe mutation in the 6-phosphogluconate dehydrogenase gene (gnd) was defined as a useful mutation for L-lysine production. Introduction of the gnd mutation into strain AHP-3 by allelic replacement led to approximately 15% increased L-lysine production. Enzymatic analysis revealed that the mutant enzyme was less sensitive than the wild-type enzyme to allosteric inhibition by intracellular metabolites, such as fructose 1,6-bisphosphate, D-glyceraldehyde 3-phosphate, phosphoribosyl pyrophosphate, ATP, and NADPH, which were known to inhibit this enzyme. Isotope-based metabolic flux analysis demonstrated that the gnd mutation resulted in 8% increased carbon flux through the pentose phosphate pathway during L-lysine production. These results indicate that the gnd mutation is responsible for diminished allosteric regulation and contributes to redirection of more carbon to the pentose phosphate pathway that was identified as the primary source for NADPH essential for L-lysine biosynthesis, thereby leading to improved product formation.

It has been shown that calcineurin (CN), a serine/threonine protein phosphatase type 2B (PP2B), plays an important role in the development and diseases of cardiac muscles. However, reports on CN activity in dilated cardiomyopathy (DCM) are inconsistent, since there are few good disease models and the measurement of the amount of CN is difficult. Previously, we developed a novel line of DCM hamster, J2N-k, and its healthy control counterpart, J2N-n, by crossbreeding cardiomyopathy (CM) hamsters, Bio 14.6, and Golden hamsters followed by consecutive sib mating. In this study, we identified the DCM-causative gene in J2N-k by analysis of F2 of these two lines, and then we analyzed the change in CN gene expression in the course of the disease, and the change in CN activity using a newly developed method. We show that: (i) the DCM gene of J2N-k hamster is the delta- sarcoglycan (SG) gene, (ii) CN expression and potential CN activities (CN activity fully activated with Ca(2+) and calmodulin) in the hearts of J2N-k and J2N-n hamsters are the same levels, (iii) transcription levels of natriuretic peptides, which are augmented by activation of Ca(2+)/calmodulin-dependent enzyme including CN, are significantly increased in the DCM stage in J2N-k hamster. J2N-k and J2N-n hamsters will be a useful tool for studying the pathogenesis, therapy, and prevention of human DCM. Although the total amount and potential activity of CN did not change in the cell extracts, targets of CN in vivo were activated in cardiomyocytes of DCM, suggesting that CN activity in the cells is activated by the raising of Ca(2+) concentration in cardiomyocytes of DCM, which is caused by the defect in the delta-SG gene. Our results reveal the complexity of CN regulation in the heart and indicate the need for additional experimentation.

The vanadate (Vi)-mediated photocleavage reaction was used to study the interaction between the regulatory segment and the catalytic site of smooth muscle myosin light chain kinase (MLCK). When MLCK was irradiated with long-wave UV (366 nm) in the presence of ADP and Vi, kinase activity was substantially decreased, and the MLCK polypeptide of 130 kDa was cleaved into several smaller fragments with apparent molecular masses of 100, 70, 60, 32, and 28 kDa. Inhibition of kinase activity and photocleavage were both competitively antagonized by the addition of ATP. Inconsistency between the observed maximum levels of UV-induced inhibition of MLCK-mediated phosphorylation (80%) and photocleavage (15-20%) suggested that the photocleavage reaction proceeds as a two-step process. Monoclonal antibodies recognizing the C-terminus of MLCK labeled the 60- and 28-kDa fragments, indicating that MLCK was cleaved at two sites, at 28 and 60 kDa from the C-terminus, within what are believed to be the autoinhibitory region and the catalytic site, respectively. Moreover, Ca2+-calmodulin (Ca2+-CaM) protected against cleavage at the site at 28 kDa from the C-terminus. Analysis of the amino acid composition of the fragment revealed that the cleavage site at 28 kDa from C-terminus occurred at Lys 799 +/- 3 amino acid residues, which is in a region where the CaM-binding and pseudosubstrate regions overlap. These results suggest that the three-dimensional structure of MLCK brings the regulatory segment into direct contact with the ATP-binding site. Moreover, the binding of Ca2+-CaM displaces the regulatory segment away from the catalytic site.

The in-vitro activity of T-5575, 2-carboxypenam, a new parenteral antibiotic and its stability to beta-lactamases were compared with those of ceftazidime and imipenem. The activity of T-5575 was equal or superior to that of ceftazidime or imipenem against Gram-negative bacteria that produced penicillinase with the exception of the enzyme OXA-1. Against strains that produced Cephalosporinase and zinc-dependent beta-lactamase, the activity of T-5575 was superior to that of ceftazidime or imipenem. T-5575 was a poor substrate and had low affinity for beta-lactamases produced by Citrobacter freundii, Enterobacter cloacae and Pseudomonas aeruginosa. The activity of T-5575 was less influenced by the derepressed production of chromosomal enzymes than that of ceftazidime. Overall, T-5575 had excellent activity against Gram-negative pathogens that produced various types of beta-lactamases.

From October 1988 to January 1992, nine isolates of Pseudomonas aeruginosa carrying transferable plasmids encoding imipenem-hydrolyzing beta-lactamase (pI = c. 9.5) were recovered from nine different patients in a neurosurgical ward of a hospital in Japan. The beta-lactamase activities of the sonicated extracts from the transconjugants were inhibited by EDTA and this was partially reversible by the addition of zinc cation. The substrate specificity and pI of the beta-lactamase were similar to those of the metallo beta-lactamases from P. aeruginosa and Serratia marcescens TN9106. All strains were resistant to imipenem, carbenicillin and antipseudomonal cephems including ceftazidime, cefsulodin, cefpirome, while four and five strains were susceptible to piperacillin and aztreonam, respectively. Both low level imipenem resistance and high level cephem resistance were co-transferred with the production of metallo beta-lactamase, while resistance to piperacillin, aztreonam, and high level imipenem-resistance were not selected. Production of chromosomal cephalosporinase in piperacillin resistant strains was derepressed, and production of outer membrane protein of D2 was diminished in highly imipenem resistant strains. Six strains were isolated in 1991, and the amounts of antipseudomonal agents, especially imipenem, used in the neurosurgical ward increased markedly in this year. Only three of the nine isolates had the same serotype, pyocin type and phage type. Our results suggest that the repeated isolation of imipenem and cephem-resistant P. aeruginosa producing metallo beta-lactamase was related to the high usage of antipseudomonal beta-lactam antibiotics such as imipenem, and was exacerbated by the dissemination of a plasmid.

We devised an accurate procedure with which to measure the affinities of beta-lactam antibiotics for penicillin-binding protein (PBP) 2' in methicillin-resistant Staphylococcus aureus (MRSA). In the present study, we used two isogenic strains of MRSA, on heterogeneous and the other homogeneous, derived from the methicillin-susceptible strain FDA209P, harbouring the mecA gene. In these MRSA strains, PBP2' was saturated by [14C]benzylpenicillin (PCG) at a concentration of 300 mg/L. In addition, the saturation of PBP2' by [14C]PCG required an incubation period of 30 min. According to these results, the precise affinities of beta-lactam antibiotics for PBP2' were determined by the 'accurate competition assay', using a high concentration of [14C]PCG and extending the reaction time. This procedure yielded lower IC50 values of beta-lactams than the 'usual competition assay'. However, each beta-lactam had almost the same affinity for PBP2' in heterogeneous and homogeneous strains. These results suggest there is a factor(s) other than PBP2' responsible for controlling resistance levels and the heterogeneity or homogeneity of MRSA strains.

An extended-spectrum beta-lactamase, the gene for which is located on plasmid pMS350 in Pseudomonas aeruginosa strains, hydrolyzes carbapenems and other extended-spectrum beta-lactam antibiotics. We cloned the pMS350 beta-lactamase gene in an Escherichia coli K-12 strain using the vector plasmid pHSG398, and subcloned it into pMS360, a plasmid with a wide host-range. This resulted in the formation of the recombinant plasmid, pMS363, containing a 4.1-kb DNA insert that includes the extended-spectrum beta-lactamase gene. Plasmid pMS363 was introduced into the P. aeruginosa PAO strain or into six species of Enterobacteriaceae, and the specific activities of the beta-lactamase and MICs of various beta-lactam antibiotics were estimated. The cloned gene was capable of expression in these strains and caused resistance to carbapenem, penem and other beta-lactam antibiotics, with the exception of aztreonam.

The in-vitro synergic activities of BRL42715, a new beta-lactamase inhibitor, clavulanic acid, sulbactam, and tazobactam combined with ampicillin, piperacillin, cephalothin, or cefoperazone were tested against various bacteria producing known types of beta-lactamase. BRL42715 showed the best synergistic activity among the inhibitors tested against strains producing penicillinases of type I, II, III, V, and that from Klebsiella pneumoniae, cephalosporinases, and oxyiminocephalosporinases (except that from Klebsiella oxytoca). Clavulanic acid combined with the beta-lactams tested showed the best synergic activity of the inhibitors against strains producing type IV penicillinase and oxyiminocephalosporinase from K. oxytoca. The 50% inhibitory doses of BRL42715 were superior to those of clavulanic acid against various types of beta-lactamases except for type IV penicillinase and the oxyiminocephalosporinase from K. oxytoca. The inhibitory activity of BRL42715 against cephalosporinases from various bacteria was 10(4) to 10(6)-fold greater than that of clavulanic acid. The synergic effects of BRL42715 and clavulanic acid on the activity of piperacillin were compared against six clinical isolates of bacteria resistant to piperacillin. The synergic activity of BRL42715 was greater than that of clavulanic acid in all six isolates.

The role of inhibin in gonadal function and reproduction has been confirmed by the measurement of plasma inhibin levels, but there has been no clinical data available on activin because of the lack of a good assay method. We measured plasma free activin levels during the normal menstrual cycle using a newly developed competitive protein binding assay with follistatin as the binding protein. Plasma inhibin levels were measured simultaneously using an alpha-subunit N-terminal fragment RIA with recombinant inhibin as the reference standard. Four normal women, aged 23-29 years, were investigated by obtaining plasma at 3-day intervals. Plasma inhibin levels showed some variation during the follicular phase, but a parallel rise in inhibin and progesterone was observed during the luteal phase. These findings confirmed those of previous studies. In contrast, plasma free activin levels did not show any substantial changes during the menstrual cycle. This study suggests that activin has no endocrine role in modulating the pituitary-gonadal axis during the normal menstrual cycle, while changes of inhibin reflect cyclic gonadal function and indicate an endocrine role for this protein in modulating gonadal activity.

The stability of the new carbapenem antibiotics, panipenem, meropenem and LJC 10,627, against porcine and human renal dehydropeptidase-I (DHP-I) was compared with that of imipenem. The order of stability to hydrolysis by renal DHP-I was: LJC 10,627 greater than meropenem greater than panipenem greater than imipenem. After incubation of the drugs with porcine or human enzyme at 30 degrees C for 4 h, the percentages of residual activity were as follows: LJC 10,627, 73.7% and 95.6%, respectively; meropenem, 0.2% and 28.7%, respectively; panipenem, 0% and 4.3%, respectively; and imipenem, 0% and 0.1%, respectively. These results demonstrate that LJC 10,627 has extremely high stability against renal DHP-I.

The activity of RP 59500 against Gram-positive cocci was determined by an agar dilution method and compared with that of erythromycin, cefotaxime and ampicillin. Of the 344 clinical isolates tested, none was resistant to RP 59500; this compound was active both against methicillin-resistant Staphylococcus aureus and against macrolide-resistant Gram-positive cocci. The bactericidal activity of RP 59500 was confirmed by killing curve determinations.

The effect of cyanide, arsenate, Mg++ or EDTA on the uptake of norfloxacin in Escherichia coli was measured. Uptake of norfloxacin was suppressed by either 0.1 mM MgSO4 or 0.1 mM EDTA, while the presence of 0.1 mM MgSO4 increased the minimum suppressive concentration of EDTA from 0.1 to 0.2 mM. Increased uptake in the presence of 10 mM cyanide was observed, but the addition of 10 mM arsenate had no significant effect. Concentration of norfloxacin in bacterial cells was observed even when uptake was suppressed by the addition of 10 mM EDTA. Uptake in mini-cells was comparable to that in whole cells. These results suggest that the uptake of norfloxacin in E. coli, in addition to influx by simple diffusion and energy-dependent efflux, is influenced by binding of norfloxacin to the cell surface as a result of chelating activity to Mg++, together with an unknown concentration step resulting from binding to cell components other than the chromosome.

DQ-2556, a new cephalosporin, showed a broad antibacterial spectrum over Gram-positive and -negative organisms. The activity of DQ-2556 against recent clinical isolates of Gram-positive cocci and Enterobacteriaceae was comparable with that of cefpirome, and superior to that of ceftazidime. DQ-2556 was almost as active as cefpirome against Pseudomonas aeruginosa, but was less active than ceftazidime. With the exception of Ps. aeruginosa, DQ-2556 was bactericidal against various organisms at either the MIC or twice the MIC. DQ-2556 bound preferentially to penicillin-binding proteins (PBPs) 2, 1 and 3 of Staphylococcus aureus, PBPs 3, 1A and 1B of Escherichia coli and PBPs 1A, 3 and 4 of Ps. aeruginosa. DQ-2556 was stable to various penicillinases and cephalosporinases, but was unstable to oxyiminocephalosporinases. The Km values of DQ-2556 for the cephalosporinases of Citrobacter freundii and Enterobacter cloacae were only two- or three-fold higher than those of ceftazidime, indicating that DQ-2556 had a relatively high affinity for these enzymes compared with other recently developed cephalosporins. The MIC of DQ-2556 for Esch. coli increased four-fold in an OmpF-deficient mutant, indicating that the OmpF porin was one of the major routes for penetration of DQ-2556 into Esch. coli cells.

The in-vitro synergistic activity of tazobactam, a new beta-lactamase inhibitor, combined with piperacillin was tested against various beta-lactamase-producing strains. The beta-lactamase inhibitory activity of tazobactam against various known types of beta-lactamase was also tested in comparison with clavulanic acid or sulbactam. Tazobactam caused a remarkable reduction of the piperacillin MICs for penicillinase- and oxyiminocephalosporinase-producing strains and also showed a moderate synergistic effect against cephalosporinase-producing strains. The bactericidal activity of piperacillin was enhanced in combination with tazobactam. Tazobactam inhibited the penicillinases, and the oxyiminocephalosporinase produced by Proteus vulgaris, at low concentration. In these cases its activity was comparable with that of clavulanic acid and stronger than that of sulbactam. Tazobactam demonstrated a better inhibitory capability than sulbactam against the cephalosporinases tested. Tazobactam was able to inactivate intracellular beta-lactamase in Prot. vulgaris and Morganella morganii, confirming its ability to penetrate the cell membrane of these species.

BO-1165 has excellent antibacterial activity against Gram-negative bacteria but it is almost inactive against Gram-positive and anaerobic bacteria. BO-1165 was more active than the four reference drugs against Escherichia coli, Salmonella spp., Shigella spp., Klebsiella pneumoniae, Serratia marcescens, indole-positive and indole-negative Proteus. Also, BO-1165 exhibited high antibacterial activity against strains of Pseudomonas aeruginosa (MIC50, 3.12 mg/l) and P. cepacia (MIC50, 1.56 mg/l), but did not against Ps. maltophilia strains. BO-1165 had good stability to plasmid-mediated and chromosome-mediated beta-lactamases. However, the compound was slightly hydrolyzed by the beta-lactamases isolated from Proteus vulgaris, Ps. cepacia, Klebsiella oxytoca and Ps. maltophilia, which were capable of hydrolyzing aztreonam.

The in-vitro and in-vivo antibacterial activities of fleroxacin were compared with those of other new quinolones including NY-198. Fleroxacin showed potent activity against most members of the Enterobacteriaceae, Neisseria gonorrhoeae and Haemophilus influenzae, and good activity against Pseudomonas aeruginosa and staphylococci. Against these bacteria, the activity of fleroxacin was roughly comparable to that of norfloxacin, enoxacin, NY-198, and ofloxacin, but slightly less than that of ciprofloxacin. Fleroxacin strongly inhibited DNA supercoiling activity of DNA gyrase purified from Escherichia coli which may explain its high antibacterial activity. The protective effects of a single oral dose of fleroxacin in mice were greater than those of norfloxacin and comparable to or greater than that of ciprofloxacin, ofloxacin and NY-198 against systemic infections with Staphylococcus aureus, Esch. coli and Ps. aeruginosa, and Serratia marcescens.

Vibrio anguillarum strains were isolated from chloramphenicol-resistant bacteria in diseased fish. Plasmid Rms418, which confers chloramphenicol resistance, was transferred from V. anguillarum GN11379 to Escherichia coli K12 by conjugation. The Rms418-encoded chloramphenicol acetyltransferase (CAT) [EC 2.3.1.99] was isolated and purified to homogeneity using affinity chromatography on immobilized p-amino-chloramphenicol or ATP. The general CAT could be adsorbed by a matrix with a chloramphenicol base ligand (Zaidenzaig, Y. & Shaw, W.V. (1976) FEBS Lett. 62,266-271), but the Rms418-encoded CAT was not bound under these conditions. The specific activity of the enzyme, when measured by the spectrophotometric assay, was 71.4 units/mg protein at 37 degrees C. The molecular weight of the enzyme treated with SDS and 2-mercaptoethanol was shown to be approximately 22,000. The molecular weight of the native enzyme, as determined by gel filtration, was approximately 69,000, and the optimal pH was 7.8. The Km values for chloramphenicol and CoASAc were 34.5 and 150 microM, respectively. Enzyme activity was inhibited by HgCl2, p-chloromercuribenzoate (p-CMB), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and ethylendiaminotetraacetic acid (EDTA). The half life at 53 degrees C was approximately 100 min.

New monobactam compounds with fluoromethyl side chains at the 4-position were synthesized. These compounds showed strong antibacterial activity against Gram-negative bacteria including Pseudomonas aeruginosa and good stability to various beta-lactamases. The effect of replacement of the 1-carboxy-1-methylethoxyimino residue of aztreonam with various substituted groups, and of the configuration of the 3- and 4-position were examined. Substitution of a carboxycyclopropoxy group in the oxyimino moiety effected the most potent antibacterial activity. The cis congeners were not hydrolysed by any types of beta-lactamases including the oxyiminocephalosporin hydrolysing enzyme. Introduction of a fluorine atom in the methyl group at the 4-position increased the beta-lactamase stability of monobactams.

Rabbit antiserum was prepared to the penicillinase of Alcaligenes denitrificans subsp. xylosoxydans GN14062. The antibody gave a single precipitin line in double immunodiffusion assays with the penicillinases from strain GN14062 and from two other A. denitrificans subsp. xylosoxydans strains. The precipitin line given by the GN14062 enzyme gave a reaction of complete identity with the precipitin lines of the penicillinases from the other A. denitrificans subsp. xylosoxydans strains. Furthermore, activity of the penicillinases from all three A. dentrificans subsp. xylosoxydans strains was neutralized by the antiserum to the GN14062 enzyme. Thus it was proved that the penicillinases produced by the three A. denitrificans subsp. xylosoxydans strains were immunologically identical. Penicillinases types I, II, III, IV, V, SHV-1, TEM-1, TEM-2, and the chromosomal penicillinases of Klebsiella pneumoniae GN69 and A. faecalis GN14061 formed no immunoprecipitate with, nor were neutralized by, the antiserum to the GN14062 enzyme. From these results, it was concluded that the penicillinase produced by A. denitrificans subsp. xylosoxydans was immunologically distinct from the penicillinases produced by these other bacteria.

L-105 (sodium(-)-(6R, 7R)-7-[(Z)-2-(2-amino-4-thiazolyl)-2-methoxyimino- acetamido]-3-[1,2,3-thiadiazol-5-yl)thiomethyl]-8-oxo-5-thia-1-aza bicyclo [4.2.0]oct-2-ene-2-carboxylate) is a new semisynthetic cephalosporin derivative. The in-vitro antibacterial activity of L-105 against clinical isolates of 18 bacterial species was compared with those of cefmenoxime, cefoperazone and cefazolin. Its spectrum against Gram-negative bacteria was similar to that of cefmenoxime. Moreover, against Gram-positive bacteria, including Staphylococcus aureus, coagulase negative staphylococci, Streptococcus faecalis, Str. pneumoniae and Str. pyogenes, L-105 was more potent than cefmenoxime and cefoperazone. Against Gram-positive bacteria, including Staph. aureus which is comparatively resistant to most third-generation cephalosporins, it was nearly equal in potency to cefazolin. L-105 was stable to various penicillinases and cephalosporinases. However, this compound was slightly hydrolyzed by oxyiminocephalosporinases, i.e., the enzymes produced by Pseudomonas cepacia and Ps. maltophilia.

The in-vitro antibacterial activity of AMA-1080 against both Gram-positive and Gram-negative clinical isolates was studied in comparison with that of aztreonam, cefotaxime, ceftazidime and cefoperazone. AMA-1080 showed a potent antibacterial activity against Gram-negative bacteria, particularly, the strains of Enterobacteriaceae. In addition, AMA-1080 inhibited the strains of genus Pseudomonas at the low concentrations. However, AMA-1080 showed less active against Gram-positive bacteria. It was found that AMA-1080 was highly resistant to hydrolysis by both chromosomal and plasmid-mediated beta-lactamases.

A penicillin beta-lactamase was purified from a strain of Alcaligenes dentrificans subsp. xylosoxydans resistant to beta-lactam antibiotics. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis, and its molecular weight was 18,000 from sodium dodecylsulphate-acrylamide gel electrophoresis and gel filtration. Its isoelectric point was 9.8, the optimal pH was 8.5 and the optimal temperature was 35 degrees C. The enzyme hydrolyzed penicillin G and ampicillin more rapidly than cephalosporins. Relative rates, with penicillin G as 100, were: ampicillin, 102; carbenicillin, 15; cloxacillin, less than 1; piperacillin, 9; cephaloridine, 41; cefoperazone, 36; cefpiramide, 36 and cefmenoxime, 14. Clavulanic acid, sulbactam, imipenem, and cephamycins had low affinities for the enzyme. The enzyme activity was inhibited by iodine, Hg2+, clavulanic acid and sulbactam.

Imipenem, a derivative of thienamycin, a carbapenem antibiotic, has a broad spectrum of activity against aerobic (Gram-positive and Gram-negative) and anaerobic bacteria. It is quite stable to all tested beta-lactamases produced by various species of bacteria isolated from clinical specimens, whether plasmid or chromosomally mediated. One exception is its hydrolysis by the beta-lactamase produced by Pseudomonas maltophilia which is thus usually resistant to imipenem. Imipenem was found to be hydrolysed by renal dehydropeptidase-I residing on the luminal surface of the renal tubular epithelium. A dehydropeptidase-I inhibitor, cilastatin (MK-0791) was developed with specific inhibitory activity toward the renal dehydropeptidase-I and showed detectable effects in humans.