Sequence analysis of the 5′-flanking region of the gene encoding human N-acetylglucosaminyltransferase III

We have isolated and characterized the immediate (1651 bp) 5′-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5′-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2-binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.     

Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

Allantoinase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of (S)-allantoin into allantoate, the final step in the ureide pathway. Despite limited sequence similarity, biochemical studies of the enzyme suggested that allantoinase belongs to the amidohydrolase family. In this study, the crystal structure of allantoinase from Escherichia coli was determined at 2.1 Å resolution. The enzyme consists of a homotetramer in which each monomer contains two domains: a pseudo-triosephosphate-isomerase barrel and a β-sheet. Analogous to other enzymes in the amidohydrolase family, allantoinase retains a binuclear metal center in the active site, embedded within the barrel fold. Structural analyses demonstrated that the metal ions in the active site ligate one hydroxide and six residues that are conserved among allantoinases from other organisms. Functional analyses showed that the presence of zinc in the metal center is essential for catalysis and enantioselectivity of substrate. Both the metal center and active site residues Asn94 and Ser317 play crucial roles in dictating enzyme activity. These structural and functional features are distinctively different from those of the metal-independent allantoinase, which was very recently identified.     

A Modest Protective Effect of Thyrotropin against Bone Loss Is Associated with Plasma Triiodothyronine Levels

Background

The independent skeletal effect of thyrotropin (thyroid stimulating hormone, TSH) has been suggested in animal studies. However, clinical data on the association between bone loss and variations in TSH levels is inconsistent. This study aimed to investigate the relationship between TSH levels and bone mineral density (BMD).

Methods

We conducted a cross-sectional study with 37,431 subjects (33,052 cases with euthyroidism and 4,379 cases with subclinical thyroid dysfunction) aged over 35 years. We performed thyroid function tests and measured BMD at the lumbar spine, femur neck, and total hip.

Results

Levels of TSH and T3 were positively correlated in women (r = 0.076, P = 0.001) and uncorrelated in men. In both men and women, TSH levels correlated positively and T3 levels correlated negatively with BMD at all skeletal sites in age and body mass index adjusted analyses. BMD increased steadily with TSH levels from the subclinical hyperthyroid to subclinical hypothyroid range in subjects with T3 levels in the highest tertile (119.5–200.0 ng/dL), but was no longer significant in subjects with lower plasma T3 levels.

Conclusions

The variations in TSH levels within the euthyroid and subclinical range were positively correlated with BMD in healthy men and women. The negative effect of T3 on BMD appears to be compensated for by increased TSH in subjects with plasma T3 levels in the upper normal range.     

Use of selected lactic acid bacteria in the eradication of Helicobacter pylori infection

Helicobacter pylori is among the major pathogenic bacteria that cause chronic gastritis and peptic ulcer disease and is related to the development of gastric cancer. Several chemicals, including antibiotics, have been used to eradicate H. pylori; however, they do not always curb the infection. Ten representative type strains of lactic acid bacteria (LAB) were screened for antagonism toward H. pylori via inhibition of urease activity. Strains inhibiting the binding of H. pylori to human gastric cell line cells and suppressing H. pylori-induced interleukin-8 (IL-8) production were also screened. Of these, Pediococcus pentosaseus (SL4), which inhibited the adhesion of H. pylori to MKN-45 gastric cancer cells, Bifidobacterium longum (BG7), with urease inhibiting activity, and Lactococcus lactis (SL3), and Enterococcus faecalis (SL5), which suppressed H. pylori-induced IL-8 production within MKN-45 and AGS cells, were selected. In mouse model, these LAB stains in combination significantly suppressed IL-8 levels in serum. Gastric pH also recovered to normal values after the administration of these LAB. These stains effectively suppressed H. pylori viability, although not to the extent of antibiotic treatment. When used as probiotics, LAB may help decrease the occurrence of gastritis and reduce the risk of H. pylori infection without, inducing side effects.     

Morphological and phylogenetic analyses of <Emphasis Type="Italic">Uromyces appendiculatus</Emphasis> and <Emphasis Type="Italic">U. vignae</Emphasis> on legumes in Japan

Uromyces appendiculatus, inclusive of three varieties, is distinguished from U. vignae primarily by the position of urediniospore germ pores and putative host specificity. However, opinions concerning these morphological and physiological features as taxonomic characters have varied greatly, and distinction of these species has often been confused. To clarify the taxonomy of these two species, morphological features of urediniospores and teliospores of 225 rust fungus specimens on species of Phaseolus, Vigna, Apios, Lablab, and Dunbaria were examined by light microscopy and scanning electron microscopy. Forty-five specimens were subjected to molecular phylogenetic analyses. As a result, the position of germ pores in urediniospores and the teliospore-wall thickness were considered as good characters to separate three morphological groups. In molecular analyses, the specimens fell into two and three clades based on the nucleotide sequence at D1/D2 domain of LSU rDNA and ITS regions, respectively. One of the D1/D2 clades corresponded to one morphological group whereas another D1/D2 clade included two other morphological groups. In contrast, each of the three ITS clades corresponded to a separate morphological group. Neither morphological groups nor molecular clades were host limited. It is suggested that the three morphological groups that corresponded to three distinct ITS clades constitute distinct species.Contribution no. 186 from the Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Genomic organization of human lactate dehydrogenase-A gene.

A human genomic clone containing the lactate dehydrogenase-A (LDH-A) gene of approx. 12 kilobases in length was isolated and characterized. The protein-coding sequence is interrupted by six introns, and the positions of these introns are at the random coil regions or near the ends of secondary structures located on the surface of the LDH-A molecule. An additional intron is present at 24 nucleotides 5' to the translation initiation codon ATG, while the 3' untranslated sequence of 565 nucleotides is not interrupted. The genomic blot analysis of human placenta DNA indicates the presence of multiple LDH-A gene-related sequences.     

Marine algal fucoxanthin inhibits the metastatic potential of cancer cells

Metastasis is major cause of malignant cancer-associated mortality. Fucoxanthin has effect on various pharmacological activities including anti-cancer activity. However, the inhibitory effect of fucoxanthin on cancer metastasis remains unclear. Here, we show that fucoxanthin isolated from brown alga Saccharina japonica has anti-metastatic activity. To check anti-metastatic properties of fucoxanthin, in vitro models including assays for invasion, migration, actin fiber organization and cancer cell–endothelial cell interaction were used. Fucoxanthin inhibited the expression and secretion of MMP-9 which plays a critical role in tumor invasion and migration, and also suppressed invasion of highly metastatic B16-F10 melanoma cells as evidenced by transwell invasion assay. In addition, fucoxanthin diminished the expressions of the cell surface glycoprotein CD44 and CXC chemokine receptor-4 (CXCR4) which play roles in migration, invasion and cancer–endothelial cell adhesion. Fucoxanthin markedly suppressed cell migration in wound healing assay and inhibited actin fiber formation. The adhesion of B16-F10 melanoma cells to the endothelial cells was significantly inhibited by fucoxanthin. Moreover, in experimental lung metastasis in vivo assay, fucoxanthin resulted in significant reduction of tumor nodules. Taken together, we demonstrate, for the first time, that fucoxanthin suppresses metastasis of highly metastatic B16-F10 melanoma cells in vitro and in vivo.     

Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories.     

Paeonia lactiflora Enhances the Adhesion of Trophoblast to the Endometrium via Induction of Leukemia Inhibitory Factor Expression

In the present study, we investigated the role of Paeonia lactiflora Pall. extract on embryo implantation in vitro and in vivo. A polysaccharides depleted-water extract of P. lactiflora (PL-PP) increased LIF expression in human endometrial Ishikawa cells at non-cytotoxic doses. PL-PP significantly increased the adhesion of the human trophectoderm-derived JAr spheroids to endometrial Ishikawa cells. PL-PP-induced LIF expression was decreased in the presence of a p38 kinase inhibitor SB203580 and an MEK/ERK inhibitor U0126. Furthermore, endometrial LIF knockdown by shRNA reduced the expression of integrins β3 and β5 and adhesion of JAr spheroids to Ishikawa cells. In vivo administration of PL-PP restored the implantation of mouse blastocysts in a mifepristone-induced implantation failure mice model. Our results demonstrate that PL-PP increases LIF expression via the p38 and MEK/ERK pathways and favors trophoblast adhesion to endometrial cells.