WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.     

A new method for enzymatic preparation of isopentenyladenine-type and<Emphasis Type="Italic"> trans</Emphasis>-zeatin-type cytokinins with radioisotope-labeling

We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-(E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor–ligand assays and cell metabolism studies.     

A subfamily of RNA-binding DEAD-box proteins acts as an estrogen receptor alpha coactivator through the N-terminal activation domain (AF-1) with an RNA coactivator, SRA

One class of the nuclear receptor AF-2 coactivator complexes contains the SRC-1/TIF2 family, CBP/p300 and an RNA coactivator, SRA. We identified a subfamily of RNA-binding DEAD-box proteins (p72/p68) as a human estrogen receptor alpha (hER alpha) coactivator in the complex containing these factors. p72/p68 interacted with both the AD2 of any SRC-1/TIF2 family protein and the hER alpha A/B domain, but not with any other nuclear receptor tested. p72/p68, TIF2 (SRC-1) and SRA were co-immunoprecipitated with estrogen-bound hER alpha in MCF7 cells and in partially purified complexes associated with hER alpha from HeLa nuclear extracts. Estrogen induced co-localization of p72 with hER alpha and TIF2 in the nucleus. The presence of p72/p68 potentiated the estrogen-induced expression of the endogenous pS2 gene in MCF7 cells. In a transient expression assay, a combination of p72/p68 with SRA and one TIF2 brought an ultimate synergism to the estrogen-induced transactivation of hER alpha. These findings indicate that p72/p68 acts as an ER subtype-selective coactivator through ER alpha AF-1 by associating with the coactivator complex to bind its AF-2 through direct binding with SRA and the SRC-1/TIF2 family proteins.     

New PEG2 type polyethylene glycol derivatives for protein modification

Although proteins with 2,4-bis (o-methoxypolyethylene glycol)-6-chloro-s-triazine (PEG2-Cl) as a divalent PEG modification have some advantages compared to proteins with the linear PEG modification, PEG2Cl cannot react with amino groups at neutral pH. Therefore, we have prepared new PEG2 derivatives that have an activated ester as the functional group. We confirmed that these derivatives are useful for the divalent modification of proteins, such as bSOD and rhG-CSF. © Rapid Science Ltd. 1998     

Fear Conditioning Induced by Interpersonal Conflicts in Healthy Individuals

Psychophysiological markers have been focused to investigate the psychopathology of psychiatric disorders and personality subtypes. In order to understand neurobiological mechanisms underlying these conditions, fear-conditioning model has been widely used. However, simple aversive stimuli are too simplistic to understand mechanisms because most patients with psychiatric disorders are affected by social stressors. The objective of this study was to test the feasibility of a newly-designed conditioning experiment using a stimulus to cause interpersonal conflicts and examine associations between personality traits and response to that stimulus. Twenty-nine healthy individuals underwent the fear conditioning and extinction experiments in response to three types of stimuli: a simple aversive sound, disgusting pictures, and pictures of an actors’ face with unpleasant verbal messages that were designed to cause interpersonal conflicts. Conditioned response was quantified by the skin conductance response (SCR). Correlations between the SCR changes, and personality traits measured by the Zanarini Rating Scale for Borderline Personality Disorder (ZAN-BPD) and Revised NEO Personality Inventory were explored. The interpersonal conflict stimulus resulted in successful conditioning, which was subsequently extinguished, in a similar manner as the other two stimuli. Moreover, a greater degree of conditioned response to the interpersonal conflict stimulus correlated with a higher ZAN-BPD total score. Fear conditioning and extinction can be successfully achieved, using interpersonal conflicts as a stimulus. Given that conditioned fear caused by the interpersonal conflicts is likely associated with borderline personality traits, this paradigm could contribute to further understanding of underlying mechanisms of interpersonal fear implicated in borderline personality disorder.     

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.     

Cumulative Incidence and Predictors of Progression in Corticosteroid-Na?ve Patients with Sarcoidosis

Background

Assessment of the clinical course of sarcoidosis requires long-term observation. However, the appropriate period of follow-up for sarcoidosis remains unclear, especially in patients without indication of corticosteroid therapy at the time of diagnosis.

Objective

This study aimed to clarify the cumulative incidence and identify risk factors for disease progression in corticosteroid-naïve sarcoidosis patients.

Methods

The clinical courses of 150 Japanese patients with sarcoidosis, who were followed for more than 2 years and had no indication for corticosteroid therapy at diagnosis, were retrospectively reviewed. Disease progression was defined as worsening of pulmonary sarcoidosis, development of new organ involvement, or extrapulmonary organ damage. The cumulative incidence of progression was estimated by generating a cumulative incidence curve with the Fine and Gray method.

Results

The median follow-up duration was 7.7 years (interquartile range, 4.7–13.6 years). Thirty-two (21%) patients experienced disease progression. New organ involvement appeared in 16 patients (11%). The 6-month, and 1-, 5-, 10-, and 15-year cumulative incidence of progression was 2%, 5%, 15%, 28%, and 31%, respectively. The number of organs involved at diagnosis was an independent predictor for progression with a multifactorial adjusted hazard ratio of 1.71 (95% confidence interval, 1.11–2.62). The optimal cut-off of the number of organs involved at diagnosis to identify future progression was three.

Conclusions

In corticosteroid-naïve sarcoidosis patients, the risks of disease progression are comparable from 0–5 years and 5–10 years after diagnosis. The number of organs involved at diagnosis is a useful predictor for progression of sarcoidosis.     

Hepatitis E Virus in Cambodia: Prevalence among the General Population and Complete Genome Sequence of Genotype 4

Hepatitis E virus (HEV) is a growing public health problem in many countries. In this study, we investigated HEV seroprevalence among the general population in the Siem Reap province, Cambodia, and performed HEV genetic analysis with the aim to develop an HEV prevention strategy. This seroepidemiological cross-sectional study conducted from 2010 to 2014 included 868 participants from four different locations in Siem Reap province, Cambodia. They answered questionnaires and provided blood samples for the analysis of hepatitis virus infections. Among the participants (360 men and 508 women; age range, 7–90 years), the prevalence of anti-HEV IgG was 18.4% (95% confidence interval: 15.9–21.0); HEV RNA was detected in two participants (0.23%) and was classified as genotype 3 and 4. Full-length genome of the genotype 4 isolate, CVS-Sie10, was sequenced; it contained 7,222 nucleotides and three ORFs and demonstrated high sequence identity with the swine China isolates swGX40 (95.57%), SS19 (94.37%), and swDQ (91.94%). Multivariate logistic regression analysis revealed that men, elderly people, and house workers were risk groups significantly associated with the positivity for anti-HEV IgG. This is the first report on the detection of HEV genotype 4 in humans in Cambodia and on the complete genome sequence of HEV genotype 4 from this country. Our study demonstrates that new HEV infection cases occur frequently among the general population in Cambodia, and effective preventive measures are required.