Depletion of hsp90beta induces multiple defects in B cell receptor signaling

Hsp90 participates in many distinct aspects of cellular functions and accomplishes these roles by interacting with multiple client proteins. To gain insight into the interactions between Hsp90 and its clients, here we have reduced the protein level of Hsp90 in avian cells by gene targeting in an attempt to elicit the otherwise undetectable (because of the vast amount of cellular Hsp90) Hsp90-interacting proteins. Hsp90beta-deficient cells can grow, albeit more slowly than wild-type cells. B cell antigen receptor signaling is multiply impaired in these mutant cells; in particular, the amount of immunoglobulin M heavy chain protein is markedly reduced. Furthermore, serum activation does not promote ERK phosphorylation in Hsp90beta-deficient cells. These multifaceted depressive effects seem to be provoked independently of each other and possibly recapitulate the proteome-wide in vivo functions of Hsp90. Reintroduction of the Hsp90beta gene efficiently restores all of the defects. Unexpectedly, however, introducing the Hsp90alpha gene is also effective in restoration; thus, these defects might be caused by a reduction in the total expression of Hsp90 rather than by loss of Hsp90beta-specific function.     

Comparison between basal and apical dendritic spines in estrogen-induced rapid spinogenesis of CA1 principal neurons in the adult hippocampus

Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Here, we demonstrated the rapid effect of 17beta-estradiol on the density and morphology of spines in the stratum oriens (s.o., basal side) and in the stratum lacunosum-moleculare (s.l.m., apical side) by imaging Lucifer Yellow-injected CA1 neurons in adult male rat hippocampal slices, because spines in s.o. and s.l.m. have been poorly understood as compared with spines in the stratum radiatum. The application of 1nM estradiol-induced a rapid increase in the density of spines of pyramidal neurons within 2h. This increase by estradiol was blocked by Erk MAP kinase inhibitor and estrogen receptor inhibitor in both regions. Effect of blockade by agonists of AMPA receptors and NMDA receptors was different between s.o. and s.l.m. In both regions, ERalpha agonist PPT induced the same enhancing effect of spinogenesis as that induced by estradiol.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

Toxic and nontoxic components of botulinum neurotoxin complex are evolved from a common ancestral zinc protein

Zinc atoms play an essential role in a number of enzymes. Botulinum neurotoxin (BoNT), the most potent toxin known in nature, is a zinc-dependent endopeptidase. Here we identify the nontoxic nonhemagglutinin (NTNHA), one of the BoNT-complex constituents, as a zinc-binding protein, along with BoNT. A protein structure classification database search indicated that BoNT and NTNHA share a similar domain architecture, comprising a zinc-dependent metalloproteinase-like, BoNT coiled-coil motif and concanavalin A-like domains. Inductively coupled plasma-mass spectrometry analysis demonstrated that every single NTNHA molecule contains a single zinc atom. This is the first demonstration of a zinc atom in this protein, as far as we know. However, the NTNHA molecule does not possess any known zinc-coordinating motif, whereas all BoNT serotypes possess the classical HEXXH motif. Homology modeling of the NTNHA structure implied that a consensus K-C-L-I-K-X(35)-D sequence common among all NTNHA serotype molecules appears to coordinate a single zinc atom. These findings lead us to propose that NTNHA and BoNT may have evolved distinct functional specializations following their branching out from a common ancestral zinc protein.     

Histone chaperone activity of Fanconi anemia proteins, FANCD2 and FANCI, is required for DNA crosslink repair

Fanconi anaemia (FA) is a rare hereditary disorder characterized by genomic instability and cancer susceptibility. A key FA protein, FANCD2, is targeted to chromatin with its partner, FANCI, and plays a critical role in DNA crosslink repair. However, the molecular function of chromatin-bound FANCD2-FANCI is still poorly understood. In the present study, we found that FANCD2 possesses nucleosome-assembly activity in vitro. The mobility of histone H3 was reduced in FANCD2-knockdown cells following treatment with an interstrand DNA crosslinker, mitomycin C. Furthermore, cells harbouring FANCD2 mutations that were defective in nucleosome assembly displayed impaired survival upon cisplatin treatment. Although FANCI by itself lacked nucleosome-assembly activity, it significantly stimulated FANCD2-mediated nucleosome assembly. These observations suggest that FANCD2-FANCI may regulate chromatin dynamics during DNA repair.     

Production of truncated protein by the crosslink formation of mRNA with 2'-OMe oligoribonucleotide containing 2-amino-6-vinylpurine

The development of convenient methods for controlling the protein expression is an important challenge in the postgenomic era. We applied the crosslink forming oligonucleotide (CFO) as a terminator of the ribosomal translation. In this study, we demonstrated that the improved reactivity of our CFO under physiological conditions enabled the sequence-specific introduction of a steric block for a ribosome on mRNAs. In vitro and in cell translation experiments revealed that the crosslinked mRNA can produce the truncated proteins in which the translation terminates at the desired position.     

The effects of postprandial glucose and insulin levels on postprandial endothelial function in subjects with normal glucose tolerance

Background

Type 2 diabetes mellitus (T2DM) patients are at increased risk of developing cardiovascular events. Unfortunately traditional risk assessment scores, including the Framingham Risk Score (FRS), have only modest accuracy in cardiovascular risk prediction in these patients.

Methods

We sought to determine the prognostic values of different non-invasive markers of atherosclerosis, including brachial artery endothelial function, carotid artery atheroma burden, ankle-brachial index, arterial stiffness and computed tomography coronary artery calcium score (CACS) in 151 T2DM Chinese patients that were identified low-intermediate risk from the FRS recalibrated for Chinese (<20% risk in 10?years). Patients were prospectively followed-up and presence of atherosclerotic events documented for a mean duration of 61?±?16?months.

Results

A total of 17 atherosclerotic events in 16 patients (11%) occurred during the follow-up period. The mean FRS of the study population was 5.0?±?4.6% and area under curve (AUC) from receiver operating characteristic curve analysis for prediction of atherosclerotic events was 0.59?±?0.07 (P?=?0.21). Among different vascular assessments, CACS?>?40 had the best prognostic value (AUC 0.81?±?0.06, P?<?0.01) and offered significantly better accuracy in prediction compared with FRS (P?=?0.038 for AUC comparisons). Combination of FRS with CACS or other surrogate vascular markers did not further improve the prognostic values over CACS alone. Multivariate Cox regression analysis identified CACS?>?40 as an independent predictor of atherosclerotic events in T2DM patients (Hazards Ratio 27.11, 95% Confidence Interval 3.36-218.81, P?=?0.002).

Conclusions

In T2DM patients identified as low-intermediate risk by the FRS, a raised CACS?>?40 was an independent predictor for atherosclerotic events.     

Sequence heterogeneity in NS5A of hepatitis C virus genotypes 2a and 2b and clinical outcome of pegylated-interferon/ribavirin therapy

Pegylated-interferon plus ribavirin (PEG-IFN/RBV) therapy is a current standard treatment for chronic hepatitis C. We previously reported that the viral sequence heterogeneity of part of NS5A, referred to as the IFN/RBV resistance-determining region (IRRDR), and a mutation at position 70 of the core protein of hepatitis C virus genotype 1b (HCV-1b) are significantly correlated with the outcome of PEG-IFN/RBV treatment. Here, we aimed to investigate the impact of viral genetic variations within the NS5A and core regions of other genotypes, HCV-2a and HCV-2b, on PEG-IFN/RBV treatment outcome. Pretreatment sequences of NS5A and core regions were analyzed in 112 patients infected with HCV-2a or HCV-2b, who were treated with PEG-IFN/RBV for 24 weeks and followed up for another 24 weeks. The results demonstrated that HCV-2a isolates with 4 or more mutations in IRRDR (IRRDR[2a]≥4) was significantly associated with rapid virological response at week 4 (RVR) and sustained virological response (SVR). Also, another region of NS5A that corresponds to part of the IFN sensitivity-determining region (ISDR) plus its carboxy-flanking region, which we referred to as ISDR/+C[2a], was significantly associated with SVR in patients infected with HCV-2a. Multivariate analysis revealed that IRRDR[2a]≥4 was the only independent predictive factor for SVR. As for HCV-2b infection, an N-terminal half of IRRDR having two or more mutations (IRRDR[2b]/N≥2) was significantly associated with RVR, but not with SVR. No significant correlation was observed between core protein polymorphism and PEG-IFN/RBV treatment outcome in HCV-2a or HCV-2b infection. CONCLUSION: The present results suggest that sequence heterogeneity of NS5A of HCV-2a (IRRDR[2a]≥4 and ISDR/+C[2a]), and that of HCV-2b (IRRDR[2b]/N≥2) to a lesser extent, is involved in determining the viral sensitivity to PEG-IFN/RBV therapy.     

Forecasting Japan's Physician Shortage in 2035 as the First Full-Fledged Aged Society

Introduction

Japan is rapidly becoming a full-fledged aged society, and physician shortage is a significant concern. The Japanese government has increased the number of medical school enrollments since 2008, but some researchers warn that this increase could lead to physician surplus in the future. It is unknown how many physicians will be required to accommodate future healthcare needs.

Materials and Methods

We simulated changes in age/sex composition of the population, fatalities (the number of fatalities for the consecutive five years), and number of physicians from 2010 to 2035. Two indicators were defined: fatalities per physician and fatalities by physician working hour, based on the data of the working hours of physicians for each tuple of sex and age groups. We estimated the necessary number of physicians in 2035 and the number of new physicians to maintain the indicator levels in 2010.

Results

The number of physicians per 1,000 population is predicted to rise from 2·00 in 2010 to 3·14 in 2035. The number of physicians aged 60 years or older is expected to increase from 55,375 (20% of physicians) to 141,711 (36%). In 2010 and 2035, fatalities per physician were 23·1 and 24·0 for the total population, and 13·9 and 19·2 for 75 years or older, respectively. Fatalities per physician working hour are predicted to rise from 0·128 to 0·138. If working hours are limited to 48 hours per week in 2035, the number of fatalities per physician working hour is expected to be 0·196, and the number of new physicians must be increased by 53% over the current pace.

Discussion

The number of physicians per population continues to rise, but the estimated supply will not fulfill the demand for healthcare in the aging society. Strategies to increase the number of physicians and improve working conditions are urgently needed.