Modulation of tyrosine phosphorylation of p36 and other substrates by the S-100 protein

The inhibitory effects of Ca2+-binding proteins on tyrosine phosphorylation of p36 protein isolated from bovine intestinal epithelium by immunoprecipitated p130fps were investigated. S-100 protein dose dependently inhibited the p36 phosphorylation, and calmodulin weakly depressed the phosphorylation, whereas parvalbumin and troponin C had no significant effects. The S-100 preparation purified from bovine brain did not contain phosphatase activity or ATPase activity. The concentration of ATP did not affect the S-100-mediated inhibition of phosphorylation but the substrate protein, p36, reversed the inhibition. S-100 similarly inhibited the tyrosine phosphorylation of p36 by p60src but did not affect the p36 phosphorylation by protein kinase C. S-100 inhibited the tyrosine kinase activity of p130fps using the other substrates tested as well. These results suggest that S-100 interacts with the substrate binding site of retroviral tyrosine-specific protein kinases and may play a regulatory role in the tyrosine phosphorylation.     

G-estimation of structural nested restricted mean time lost models to estimate effects of time-varying treatments on a failure time outcome

G-estimation of structural nested models (SNMs) plays an important role in estimating the effects of time-varying treatments with appropriate adjustment for time-dependent confounding. As SNMs for a failure time outcome, structural nested accelerated failure time models (SNAFTMs) and structural nested cumulative failure time models have been developed. The latter models are included in the class of structural nested mean models (SNMMs) and are not involved in artificial censoring, which induces several difficulties in g-estimation of SNAFTMs. Recently, restricted mean time lost (RMTL), which corresponds to the area under a distribution function up to a restriction time, is attracting attention in clinical trial communities as an appropriate summary measure of a failure time outcome. In this study, we propose another SNMM for a failure time outcome, which is called structural nested RMTL model (SNRMTLM) and describe randomized and observational g-estimation procedures that use different assumptions for the treatment mechanism in a randomized trial setting. We also provide methods to estimate marginal RMTLs under static treatment regimes using estimated SNRMTLMs. A simulation study evaluates finite-sample performances of the proposed methods compared with the conventional intention-to-treat and per-protocol analyses. We illustrate the proposed methods using data from a randomized controlled trial for cardiovascular disease with treatment changes. G-estimation of SNRMTLMs is a useful tool to estimate the effects of time-varying treatments on a failure time outcome.     

Spectrophotometric determination of pyridoxal and pyridoxal 5′-phosphate with 3-methyl-2-benzothiazolone hydrazone hydrochloride, and their selective assay

A spectrophotometric method with 3-methyl-2-benzothiazolone hydrazone hydrochloride was developed for the determination of pyridoxal and pyridoxal 5'-phosphate, and for the selective determination of each in the presence of the other. Pyridoxal and pyridoxal 5'-phosphate react with the reagent to yield the azine derivatives, which give characteristic absorption spectra. The highest extinction values are obtained when pyridoxal and pyridoxal 5'-phosphate are incubated at pH values of about 3.4 and 8.0 respectively; their maxima are at 430nm. (in 2.74x10(4)) and 380nm. (in 2.24x10(4)) respectively. The azine of pyridoxal is only slightly soluble under the neutral and alkaline conditions, whereas that of pyridoxal 5'-phosphate is substantially insoluble in the acid pH range. This difference in solubility of the azines made possible the selective determination of pyridoxal and pyridoxal 5'-phosphate. alpha-Oxoglutarate and pyruvate are among the substances shown not to interfere with the assay of pyridoxal; their derivatives absorb appreciably only at wavelengths below 420nm. For the assay of pyridoxal 5'-phosphate in the presence of these compounds measurement at 390nm. is necessary.     

Competence-dependent endogenous DNA rearrangement and uptake of extracellular DNA give a natural variant of Streptococcus mutans without biofilm formation

The production of water-insoluble glucan (WIG) enables Streptococcus mutans to survive and persist in the oral niche. WIG is produced from sucrose by glucosyltransferase encoded tandemly by the highly homologous gtfB and gtfC genes. Conversely, a single hybrid gene from the endogenous recombination of gtfB and gtfC is easily generated using RecA, resulting in S. mutans UA159 WIG- (rate of ～1.0×10(-3)). The pneumococcus recA gene is regulated as a late competence gene. comX gene mutations did not lead to the appearance of WIG- cells. The biofilm collected from the flow cell had more WIG- cells than among the planktonic cells. Among the planktonic cells, WIG- cells appeared after 16 h and increased ～10-fold after 32 h of cultivation, suggesting an increase in planktonic WIG- cells after longer culture. The strain may be derived from the biofilm environment. In coculture with donor WIG+ and recipient WIG- cells, the recipient cells reverted to WIG+ and acquired an intact gtfBC region from the environment, indicating that the uptake of extracellular DNA resulted in the phenotypic change. Here we demonstrate that endogenous DNA rearrangement and uptake of extracellular DNA generate WIG- cells and that both are induced by the same signal transducer, the com system. Our findings may help in understanding how S. mutans can adapt to the oral environment and may explain the evolution of S. mutans.     

The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase-dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.     

Inactivation of Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae by ozone

AIMS: To clarify the inhibitory effects of ozone on Chlamydia trachomatis and C. pneumoniae. METHODS AND RESULTS: Cell culture was performed using HeLa229 cells for C. trachomatis, and Human Line cells for C. pneumoniae. C. trachomatis strain D/UW-3/Cx and C. pneumoniae strain AR-39 were used. Ozone water was generated by an ozone water dispenser and diluted to desired concentration just before each experiment. Preinoculation minimum cidal concentration (MCC) and postinoculation MCC methods were employed. In preinoculation MCC, chlamydial strains were treated with serially diluted ozone water followed by inoculation to cells. In postinoculation method, chlamydial strains were inoculated to cells and incubated for 24 h. Then infected cells were treated with ozone water, followed by additional incubation for 48 h. Complete inactivation was obtained in preinoculation MCC method at 0.5 ppm of ozone water for 30 s, or 4 ppm for 5 s. CONCLUSION: Ozone at a concentration of 4 ppm was enough for immediate inactivation of both C. trachomatis and C. pneumoniae. SIGNIFICANCE AND IMPACT OF THE STUDY: Ozone water at 4 ppm should be applicable for prevention of C. trachomatis urogenital infections.     

Fluorescence study of the high pressure-induced denaturation of skeletal muscle actin.

Ikkai & Ooi [Ikkai, T. & Ooi, T. (1966) Biochemistry 5, 1551-1560] made a thorough study of the effect of pressure on G- and F-actins. However, all of the measurements in their study were made after the release of pressure. In the present experiment in situ observations were attempted by using epsilon ATP to obtain further detailed kinetic and thermodynamic information about the behaviour of actin under pressure. The dissociation rate constants of nucleotides from actin molecules (the decay curve of the intensity of fluorescence of epsilon ATP-G-actin or epsilon ADP-F-actin) followed first-order kinetics. The volume changes for the denaturation of G-actin and F-actin were estimated to be -72 mL x mol(-1) and -67 mL x mol(-1) in the presence of ATP, respectively. Changes in the intensity of fluorescence of F-actin whilst under pressure suggested that epsilon ADP-F-actin was initially depolymerized to epsilon ADP-G-actin; subsequently there was quick exchange of the epsilon ADP for free epsilon ATP, and then polymerization occurred again with the liberation of phosphate from epsilon ATP bound to G-actin in the presence of excess ATP. In the higher pressure range (> 250 MPa), the partial collapse of the three-dimensional structure of actin, which had been depolymerized under pressure, proceeded immediately after release of the nucleotide, so that it lost the ability to exchange bound ADP with external free ATP and so was denatured irreversibly. An experiment monitoring epsilon ATP fluorescence also demonstrated that, in the absence of Mg(2+)-ATP, the dissociation of actin-heavy meromyosin (HMM) complex into actin and HMM did not occur under high pressure.     

Probable laboratory contamination of clinical specimens with Leptospira meyeri

Leptospira meyeri were isolated from the blood or the pleural effusion cultures of four patients at a commercial clinical laboratory. All isolates were identical in their 16S rDNA sequences and NotI or SfiI restriction profiles of the chromosome DNA on pulsed-field gel electrophoresis. However, intraperitoneal inoculation of the isolate (NIID1) failed to kill hamsters and none of the patients' sera reacted with L. meyeri strains isolated, indicating leptospiral contamination occurred during laboratory investigation.     

Development of a pUC19-based recombinant plasmid to serve as a positive control in PCR for Orientia tsutsugamushi

A recombinant positive control plasmid was developed to be used in PCR for Orientia tsutsugamushi. This pUC19-based plasmid contains the O. tsutsugamushi DNA sequence, part of which is replaced with Candida albicans DNA. pUC19-Posi is a PCR control that provides several advantages over currently used positive controls for both universal and serotypical PCR.