Mutation of the N-terminal proline 9 of BLMA from Streptomyces verticillus abolishes the binding affinity for bleomycin

AbstractA gene, blmA, from bleomycin (Bm)-producing Streptomyces verticillus, encodes a Bm-binding protein, designated BLMA. The expression of BLMA conferred resistance to Bm in the Escherichia coli host, whereas a mutant protein, designated Pro-9/Leu, with the N-terminal proline 9 residue in BLMA replaced by leucine, did not. We created a fusion protein between the maltose-binding protein (MBP) and a mutant protein Pro-9/Leu/Leu with Met-94 in Pro-9/Leu replaced by leucine. Pro-9/Leu/Leu from the fusion protein, obtained by digestion with CNBr digestion, did not inhibit DNA-cleaving and antibacterial activities of Bm. Native-polyacrylamide gel electrophoresis (PAGE) and gel filtration column chromatographic analysis showed that the molecular size of Pro-9/Leu/Leu is roughly half of that of BLMA, suggesting that the mutant protein cannot form dimeric structure. Furthermore, Far-UV circular dichroism (CD) spectrum of Pro-9/Leu/Leu was quite different from that of BLMA and similar to the spectra obtained from unordered proteins [Venyaminov, S.Y. and Vassilenko, K.S. (1994) Anal. Biochem. 222, 176–184], suggesting that the secondary structure of Pro-9/Leu/Leu is disrupted. These results indicate that the mutation abolishes not only dimer formation but also the secondary structure of BLMA, which results in the loss of its function as a Bm-resistance determinant

FACTOR ANALYSIS OF SPRINT PHASES ON THE SPEED CURVE OF THE 100M DASH

Previous studies have indicated that the speed curve of the 100m dash consists of some distinct phases which can be used to analyze an athlete's performance. The purpose of this study was to introduce a method using a portable computer as a device for the measurement of sprint time and the illustration of the speed curve, and to clarify a simple model of sprint phases on the factor structure. Based on the data of 133 participants, principal factor solution was given to the correlation matrix, and varimax rotation was applied to simplify the factorial structure of sprint phases. Finally, two factors were extracted and interpreted. It is suggested that this method is useful for measurement and evaluation in the 100m dash, and that a simple model of sprint phases may be explained by these two factors. These findings are important in predicting the ability of 100m sprinters and in considering coaching methods in terms of technique, training, strategy, etc

Cloning and Characterization of a Streptomyces Single Module Type Non-ribosomal Peptide Synthetase Catalyzing a Blue Pigment Synthesis

In the present study, we cloned a gene, designated bpsA, which encodes a single module type non-ribosomal peptide synthetase (NRPS) from a d-cycloserine (DCS)-producing Streptomyces lavendulae ATCC11924. A putative oxidation domain is significantly integrated into the adenylation domain of the NRPS, and the condensation domain is absent from the module. When S. lividans was transformed with a plasmid carrying bpsA, the transformed cells produced a blue pigment, suggesting that bpsA is responsible for the blue pigment synthesis. However, to produce the blue pigment in Escherichia coli, the existence of the 4′-phosphopantetheinyl transferase (PPTase) gene from Streptomyces was necessary, in addition to bpsA. The chemical structure of the pigment was determined as 5,5′-diamino-4,4′-dihydroxy-3,3′-diazadiphenoquinone-(2,2′), called indigoidine. The bpsA gene product, designated BPSA, was overproduced in an E. coli host-vector system and purified to homogeneity, demonstrating that the recombinant enzyme prefers l-Gln as a substrate. The in vitro experiment using l-Gln also showed that the blue pigment was formed by the purified BPSA only when the enzyme was phosphopantetheinylated by adding a Streptomyces PPTase purified from E. coli cells. Each site-directed mutagenesis experiment of Lys598, Tyr601, Ser603, and Tyr608, which are seen in the oxidation domain of BPSA, suggests that these residues are essential for the binding of FMN to the protein and the synthesis of the blue pigment

Autonomic Dysreflexia during a Bowel Program in Patients with Cervical Spinal Cord Injury.

The purpose of the present study was to investigate the relationship between bowel maneuvers and autonomic dysreflexia (AD) in patients with cervical spinal cord injuries (CSCI). Fifteen consecutive, clinically stable patients with CSCI participated. We evaluated changes in blood pressure (BP), pulse rate (PR) and classic symptoms of AD before, during and after a bowel program involving the manual removal of stool in lateral recumbency. The insertion of rectal medication induced a significant increase in systolic BP, which persisted during additional digital rectal stimulation. Furthermore, the manual removal of stool induced AD, with maximal increases of systolic BP (169.1(+-)19.5 mmHg, mean(+-)SD). However, the insertion of a finger into the anus after the end of stool flow did not cause a further increase in systolic BP. Systolic BP recovered to pre-program values within 5 min after defecation. Our study demonstrated that the combined effects of rectal and/or anal sphincter distension and uninhibited rectal contraction in response to the manual removal of stool might induce AD. We recommend avoiding, if at all possible, the manual removal of stool in order to prevent AD in patients with CSCI

Negative regulation of hepatitis B virus replication by forkhead box protein A in human hepatoma cells

AbstractHepatitis B virus (HBV) replication is controlled by liver-enriched transcriptional factors, including forkhead box protein A (FOXA) members. Here, we found that FOXA members are directly and indirectly involved in HBV replication in human hepatic cells. HBV replication was elevated in HuH-7 treated with individual FOXA members-specific siRNA. Reciprocally, the downregulation of HBV replication was observed in FOXA-induced HuH-7. However, the mechanism of downregulation is different among FOXA members at the level of HBV RNA transcription, such as precore/pg RNA and 2.1kb RNA. In addition, FOXA1 and FOXA2 suppressed nuclear hormone receptors, such as HNF4α, that are related to HBV replication

Behavior of Latent Vector of Trivariate Wishart Matrix

This paper is concerned with the probability density function of the latent vector corresponding to the largest latent root of Wishart matrix. The latent vector may be expressed by the polar coordinates. Sugiyama (1966) give the exact expression of the probability density function of the polar coordinates. The function contained the alternating series, thus the function may not be converged on the domain of definition, numerically. In this paper we derived an improved expression of the function to be the positive series, for which we provide graphs of a population latent vector and latent roots.【査読有

A novel reductive amino cyclization method and its application for the syntheses of pyrrolidine and piperidine nucleus

金沢大学大学院自然科学研究科生理活性物質科学金沢大学薬学

Three Japanese patients with congenital pituitary hormone deficiency and ophthalmological anomalies

The clinical phenotype of congenital pituitary hormone deficiency is variable and can be associated with a number of structural abnormalities of the central nervous system. We report three Japanese patients with congenital pituitary hormone deficiency and ophthalmological anomalies. Two of the patients initially showed strabismus and unilateral optic nerve hypoplasia. Thereafter, growth failure became evident, leading to the diagnosis of pituitary hormone deficiency. The other patient had severe congenital hypopituitarism with respiratory distress and hypoglycemia from the first day of life. In addition, he had prolonged jaundice and impaired liver function with bilateral optic nerve hypoplasia. Neuroimaging of the pituitary region in all three patients demonstrated a small anterior pituitary lobe and no pituitary stalk. Our findings indicate that clinical variability of congenital hypopituitarism must be considered. In a patient with ophthalmological symptoms, endocrine evaluation and neuroimaging of the CNS including the pituitary region should be considered