Differential regulation of thyrotropin-releasing hormone receptor mRNA levels by thyroid hormone in vivo and in vitro (GH3 cells).

We studied the effect of thyroid status on thyrotropin-releasing hormone receptor (TRH-R) mRNA levels both in vivo and in vitro (GH3 cells) using a cloned rat TRH-R cDNA by RT-PCR. Experimental hypothyroid rats were produced by total thyroidectomy and were then killed 7 days after the operation. TRH receptor binding in the anterior pituitary and serum TSH level were elevated approximately 2-fold and 8-fold, respectively, in 7 day thyroidectomized rats. TRH-R mRNA levels in hypothyroid rats were also increased significantly compared with those of normal rats. In GH3 cells, however, no significant change of TRH-R mRNA level was observed between cultures treated with triiodothyronine (T3, 10(-9) and 10(-7) M) and the untreated group. The present data indicate that 1) the in vivo effects of thyroid status on TRH-R mRNA levels differ from the in vitro one, and that 2) the down regulation of TRH-R binding by thyroid hormone in GH3 cells may be mediated by translational or post-translational mechanisms.     

Twinning across the Developing World

Background

Until now, little was known about the variation in incidence of twin births across developing countries, because national representative data was lacking. This study provides the first comprehensive overview of national twinning rates across the developing world on the basis of reliable survey data.

Methods

Data on incidence of twinning was extracted from birth histories of women aged 15–49 interviewed in 150 Demographic and Health Surveys, held between 1987 and 2010 in 75 low and middle income countries. During the interview, information on all live births experienced by the women was recorded, including whether it was a singleton or multiple birth. Information was available for 2.47 million births experienced by 1.38 million women in a period of ten years before the interview. Twinning incidence was measured as the number of twin births per thousand births. Data for China were computed on the basis of published figures from the 1990 census. Both natural and age-standardized twinning rates are presented.

Results/Conclusions

The very low natural twinning rates of 6–9 per thousand births previously observed in some East Asian countries turn out to be the dominant pattern in the whole South and South-East Asian region. Very high twinning rates of above 18 per thousand are not restricted to Nigeria (until now seen as the world''s twinning champion) but found in most Central-African countries. Twinning rates in Latin America turn out to be as low as those in Asia. Changes over time are small and not in a specific direction.

Significance

We provide the most complete and comparable overview of twinning rates across the developing world currently possible.     

The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress

Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear localization regions in the N-terminal region of RECQL4 and a nucleolar localization signal at amino acids 376-386. RECQL4 localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and gamma irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 accumulated in nucleoli. Using a T7 phage display screen, we determined that RECQL4 interacts with poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and signaling pathways. The RECQL4 nucleolar localization was inhibited by pretreatment with a PARP-1 inhibitor. The C-terminal portion of RECQL4 was found to be an in vitro substrate for PARP-1. These results demonstrate changes in the intracellular localization of RECQL4 in response to oxidative stress and identify an interaction between RECQL4 and PARP-1.     

A unique enzyme of acetic acid bacteria, PQQ-dependent alcohol dehydrogenase, is also present in Frateuria aurantia

A membrane-bound, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) was purified from Frateuria aurantia LMG 1558^T. Although F. aurantia belongs to a group of γ-Proteobacteria, the characteristics of its PQQ-ADH were similar to the enzyme characteristics of the typical high-acetic acid-resistant bacterium Gluconacetobacter europaeus from the group of α-Proteobacteria. The PQQ-dependent ADH was solubilized from the membranes and purified after anionic, cationic, and affinity chromatography with specific activity of 117 U/mg. The purified enzyme was estimated to be composed of two subunits of ca. 72 and 45 kDa, as judged by SDS-polyacrylamide gel electrophoresis. The purified enzyme had maximum activity at pH 4.5 and showed the highest substrate specificity to ethanol, isoamyl alcohol, 1-butanol, and 1-propanol. The deduced sequences of cloned genes adhA and adhB encoding subunits I and II of PQQ-ADH showed 80 % amino acid (AA) identity to AdhA and 68 % AA identity to AdhB of Ga. europaeus V3 (LMG 18494). Because of the high similarity between genes encoding subunits I and II of PQQ-ADH and its homologous genes found in a distantly related taxonomic group of acetic acid bacteria, the results suggest the possibility of horizontal gene transfer between these two groups of genera.     

Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test

Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed.     

The Purification of a GroEL-Like Stress Protein from Aerobically Adapted Campylobacter jejuni

From plate cultures of Campylobacter jejuni grown in room air a particulate protein of 62 kDa was isolated by ion-exchange chromatography. The protein had a square shape from the side view but when viewed from the top it had a star-shaped structure. The molecular size of the whole particle determined by gel filtration was 850 kDa which suggested the presence of 14 subunits of 62 kDa in each particle. The N-terminal 37 amino residues showed more than 80% homology with the sequence of these heat shock protein (HSP) 60 homologs of Chlamydia trachomatis, Helicobacter pylori, and Escherichia coli (GroEL). This protein is immunologically cross-reactive with the antiserum for the 60-kDa HSP of Yersinia enterocolitica. Production of the 62-kDa protein increased under heat stress and growth in an aerobic atmospheric environment. From these observations we concluded that the 62-kDa protein is a Campylobacter stress protein (Cj62) which belongs to the HSP 60 family.     

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline (o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containing NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and alpha-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.     

Gain-of-function screen identifies a role of the Sec61alpha translocon in Drosophila postmitotic neurotoxicity

To elucidate the intrinsic mechanisms of neurotoxicity induction, including those underlying neural cell death and neurodegeneration, we developed a gain-of-function screen for gene products causing neural cell loss. To identify novel genes with a cell-death-related function in neurons, we screened 4,964 Drosophila GS lines, in which one or two genes from much of the Drosophila genome can be overexpressed. Approximately 0.68% of the GS lines produced phenotypes involving a loss of postmitotic neurons. Of these, we identified and characterized the endd2 gene, which encodes the Drosophila ortholog of Sec61alpha (DSec61alpha), an endoplasmic reticulum protein with protein translocation activity. Ectopic expression of DSec61alpha caused neural cell death accompanied by the accumulation of ubiquitinated proteins, which was mediated by DSec61alpha's translocon activity. This supported our previous observation that the DSec61alpha translocon contributes to expanded polyglutamine-mediated neuronal toxicity, which is also associated with ubiquitinated protein accumulation. These data suggest that the translocon may be a novel component of neural cell death and degeneration pathways. Our approach can be used to identify potential neurotoxic factors within the whole genome, which will increase our understanding of the molecular mechanisms of various types of cell death, including those associated with human neurodegenerative diseases.     

Crystalline 2-Ketogluconate Reductase from Acetobacter ascendens,the Second Instance of Crystalline Enzyme in Genus Acetobacter

Crystalline 2-ketogluconate reductase in genus Acetobacter was prepared from cell free extract of Acetobacter ascendens. Crystalline enzyme was purified 13,000-fold with a yield of 15%. Affinity chromatography on blue-dextran Sepharose 4B column successfully purified the enzyme. The enzyme was composed of three identical subunits with a molecular weight of 40,000. Substrate specificity of 2-ketogluconate reductase from two genera of acetic acid bacteria was compared using highly purified enzyme preparations, and it was confirmed that gluconate oxidation activity of the enzyme was intrinsically weak or absent in genus Acetobacter and intense in Gluconobacter. This fact must be a useful criterion for classification of acetic acid bacteria.