cis‐Regulatory analysis for later phase of anterior neuroectoderm‐specific foxQ2 expression in sea urchin embryos

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis‐regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis‐regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half‐life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4–9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension‐class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.     

Aberrant Active cis-Regulatory Elements Associated with Downregulation of RET Finger Protein Overcome Chemoresistance in Glioblastoma

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Trend and Risk Factors of Diverticulosis in Japan: Age,Gender, and Lifestyle/Metabolic-Related Factors May Cooperatively Affect on the Colorectal Diverticula Formation

Background

Despite the marked increase of diverticulosis, its risk factors have not been adequately elucidated. We therefore aim to identify significantly associated factors with diverticulosis. We also aim to investigate the present state of diverticulosis in Japan.

Methods

We reviewed the medical records from 1990 to 2010 that included the data of consecutive 62,503 asymptomatic colonoscopy examinees from the general population in Japan. Most recent 3,327 examinees were analyzed with 16 background factors.

Results

Among the 62,503 subjects (47,325 men and 15,178 women; 52.1 ± 9.2 years old), diverticulosis was detected in 11,771 subjects (18.8%; 10,023 men and 1,748 women). The incidences of diverticulosis in 1990-2000 and 2001-2010 were respectively 13.0% (3,771 of 29,071) and 23.9% (8,000 of 33,432): the latter was much higher than the former in all age groups and for both genders. Considering the anatomical locations of colorectal diverticula, left-sided ones have markedly increased with age but not significantly changed with times. Univariate analyses of the 3,327 subjects showed significant association of diverticulosis with four basic factors (age, sex, body mass index, blood pressure), three life style-related factor (smoking, drinking, severe weight increase in adulthood), and two blood test values (triglyceride, HbA1c). The multiple logistic analysis calculating standardized coefficients (β) and odds ratio (OR) demonstrated that age (β = 0.217-0.674, OR = 1.24-1.96), male gender (β = 0.185, OR = 1.20), smoking (β = 0.142-0.200, OR = 1.15-1.22), severe weight increase in adulthood (β = 0.153, OR = 1.17), HbA1c (β = 0.136, OR = 1.15), drinking (β = 0.109, OR = 1.11), and serum triglyceride (β = 0.098, OR = 1.10) showed significantly positive association with diverticulosis whereas body mass index and blood pressure did not.

Conclusions

The large-scale data of asymptomatic colonoscopy examinees from the general population from 1990 to 2010 indicated that the prevalence of diverticulosis is still increasing in Japan. Age, male gender, smoking, severe weight increase in adulthood, serum HbA1c, drinking, and serum triglyceride showed significant positive association with diverticulosis.     

Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation

Protein phosphatase 2A (PP2A) is a conserved essential enzyme that is implicated as a tumor suppressor based on its central role in phosphorylation-dependent signaling pathways. Protein phosphatase methyl esterase (PME-1) catalyzes specifically the demethylation of the C-terminal Leu309 residue of PP2A catalytic subunit (PP2Ac). It has been shown that PME-1 affects the activity of PP2A by demethylating PP2Ac, but also by directly binding to the phosphatase active site, suggesting loss of PME-1 in cells would enhance PP2A activity. However, here we show that PME-1 knockout mouse embryonic fibroblasts (MEFs) exhibit lower PP2A activity than wild type MEFs. Loss of PME-1 enhanced poly-ubiquitination of PP2Ac and shortened the half-life of PP2Ac protein resulting in reduced PP2Ac levels. Chemical inhibition of PME-1 and rescue experiments with wild type and mutated PME-1 revealed methyl-esterase activity was necessary to maintain PP2Ac protein levels. Our data demonstrate that PME-1 methyl-esterase activity protects PP2Ac from ubiquitin/proteasome degradation.     

Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene

Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret^M919T, Ret^N396K and Ret^Y792F) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret^M919T/+ mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret^M919T/- mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.     

Characterization of two distinct feruloyl esterases, AoFaeB and AoFaeC, from Aspergillus oryzae

Two hypothetical proteins XP_001818628 and XP_001819091 (designated AoFaeB and AoFaeC, respectively), showing sequence identity with known type-C feruloyl esterases, have been found in the genomic sequence of Aspergillus oryzae. We cloned the putative A. oryzae feruloyl esterase-encoding genes and expressed them in Pichia pastoris. Both purified recombinant AoFaeB (rAoFaeB) and AoFaeC (rAoFaeC) had apparent relative molecular masses of 61,000 and 75,000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After N-deglycosylation, both proteins had a relative molecular mass of 55,000. The optimum pH for rAoFaeB was 6.0, although it was stable at pH values ranging from 3.0 to 9.0; rAoFaeC had an optimum pH of 6.0 and was stable in the pH range of 7.0–10.0. Thermostability of rAoFaeC was greater than that of rAoFaeB. Whereas rAoFaeC displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, methyl ferulate, and methyl sinapate, rAoFaeB displayed hydrolytic activity toward methyl caffeate, methyl p-coumarate, and methyl ferulate but not toward methyl sinapate. Substrate specificity profiling of rAoFaeB and rAoFaeC revealed type-B and type-C feruloyl esterases, respectively. Ferulic acid was efficiently released from wheat arabinoxylan when both esterases were applied with xylanase from Thermomyces lanuginosus. Both recombinant proteins also exhibited hydrolytic activity toward chlorogenic acid. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Discovery of substituted 2,4,4-triarylimidazoline derivatives as potent and selective neuropeptide Y Y5 receptor antagonists

Novel imidazoline derivatives were discovered to be potent neuropeptide Y Y5 receptor antagonists. High-throughput screening of Merck sample collections against the human Y5 receptor resulted in the identification of 2,4,4-triphenylimidazoline (1), which had an IC₅₀ of 54 nM. Subsequent optimization led to the identification of several potent derivatives.     

Comparison of the Arylamine <Emphasis Type="Italic">N-</Emphasis>Acetyltransferase from <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> and <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Arylamine N-acetyltansferase (NAT) from Mycobacterium tuberculosis (TBNAT) is a potential drug target for anti-tubercular therapy. Recombinant TBNAT is much less soluble and is produced in lower yields than the closely related NAT from Mycobacterium marinum (MMNAT). In order to explore MMNAT as a model for TBNAT in drug discovery, we compare the two mycobacterial NAT enzymes. Two site-directed mutants of MMNAT have been prepared and characterised: MMNAT71, Tyr → Phe and MMNAT209, Met → Thr, in which residues within 6 Å of the active-site cysteine have been replaced with the corresponding residue from TBNAT. Two chimeric proteins have also been produced in which the third domain of MMNAT has been replaced by the third domain of TBNAT and vice versa. The activity profile of the chimeric proteins suggests a role for the third domain in the evolutionary divergence of NAT between these closely related mycobacterial species.