Lack of GPR40/FFAR1 does not induce diabetes even under insulin resistance condition.

AIMS: G protein-coupled receptor/free fatty acid receptor 1 (GPR40/FFAR1 ) regulates free fatty acid-induced insulin secretion. This study has been performed to clarify whether or not loss of GPR40/FFAR1 function exacerbates diabetes, that is, whether GPR40 has an essential physiological role in the development of diabetes or not. METHODS: We generated GPR40/FFAR1 knockout (KO) mice and analysed their phenotypes in vitro and in vivo under the condition of dietary or genetically induced insulin resistance. RESULTS: GPR40/FFAR1 KO mice kept on a high-fat diet became obese, developed glucose intolerance to a similar degree as GPR40/FFAR1 wild-type (WT) mice. In addition, the phenotype of KO mice harbouring diabetogenic KK background genes showed glucose intolerance at a level similar to level for control KK mice. In both mouse models with insulin resistance, insulin secretion after oral glucose load and homeostasis model assessment-insulin resistance (HOMA-IR) did not change between GPR40/FFAR1 KO and WT mice. Although glucose-induced insulin secretion under high palmitate concentration was significantly lower in KO than in WT islets, pancreatic insulin content and insulin secretion stimulated with glucose alone were not different between KO and WT mice. CONCLUSIONS: GPR40/FFAR1 has a major role in regulating fatty-acid-mediated insulin secretion, but the lack of GPR40/FFAR1 does not exacerbate glucose intolerance and insulin resistance induced by high-fat diet or diabetogenic KK gene. Our findings indicate that loss of GPR40/FFAR1 function does not play an important role in inducing or exacerbating diabetes.

Three-dimensional SAFT imaging for anisotropic materials using photoacoustic microscopy.

A pulsed laser illuminates a target zone that causes rapid thermoelastic expansion, generating broadband high-frequency ultrasonic wave (photoacoustic wave, PA wave). We developed a PA microscopy (PAM) with a confocal area of laser and ultrasonic wave for applications in nondestructive testing (NDT). The synthetic aperture focusing technique (SAFT) is applied in the PAM for the three-dimensional (3D) imaging of interior flaws. Here, we report proof-of-concept experiments for the NDT of a subsurface flaw in a thin laminar material. Graphical abstract (a) shows a specimen of carbon-fiber-reinforced plastic (CFRP) with an artificial delamination. Here, it should be noted that the group velocity varies directionally due to the strong anisotropy of the CFRP specimen (see Graphical abstract (b)). By considering the group velocity distribution in the SAFT, the shape and location of the subsurface delamination were accurately estimated as shown in Graphical abstract (c). Coating the surface of the CFRP specimen with a light-absorbent material improved the amplitude of the PA wave. This finding showed that the signal-to-noise ratio of the waves scattered from the flaws can be improved.

Cloning of human centromeres by transformation-associated recombination in yeast and generation of functional human artificial chromosomes.

Human centromeres remain poorly characterized regions of the human genome despite their importance for the maintenance of chromosomes. In part this is due to the difficulty of cloning of highly repetitive DNA fragments and distinguishing chromosome-specific clones in a genomic library. In this work we report the highly selective isolation of human centromeric DNA using transformation-associated recombination (TAR) cloning. A TAR vector with alphoid DNA monomers as targeting sequences was used to isolate large centromeric regions of human chromosomes 2, 5, 8, 11, 15, 19, 21 and 22 from human cells as well as monochromosomal hybrid cells. The alphoid DNA array was also isolated from the 12 Mb human mini-chromosome DeltaYq74 that contained the minimum amount of alphoid DNA required for proper chromosome segregation. Preliminary results of the structural analyses of different centromeres are reported in this paper. The ability of the cloned human centromeric regions to support human artificial chromosome (HAC) formation was assessed by transfection into human HT1080 cells. Centromeric clones from DeltaYq74 did not support the formation of HACs, indicating that the requirements for the existence of a functional centromere on an endogenous chromosome and those for forming a de novo centromere may be distinct. A construct with an alphoid DNA array from chromosome 22 with no detectable CENP-B motifs formed mitotically stable HACs in the absence of drug selection without detectable acquisition of host DNAs. In summary, our results demonstrated that TAR cloning is a useful tool for investigating human centromere organization and the structural requirements for formation of HAC vectors that might have a potential for therapeutic applications.

Aberrant expression of beta- and gamma-catenin is an independent prognostic marker in oral squamous cell carcinoma.

Alteration in expression of E-cadherin and catenins is associated with loss of differentiation, acquisition of an invasive phenotype and poor clinical outcome in many types of cancer. To identify molecular prognostic markers, membrane expression levels of E-cadherin, and alpha-, beta- and gamma-catenin in biopsy samples (n=135) of oral squamous cell carcinoma (OSCC) were evaluated immunohistochemically in relation to preoperative tumour-related features, clinical course and prognostic value, and were found to be significantly correlated with an endophytic growth pattern and pathologically proved lymph-node metastasis. Alteration of expression of E-cadherin, and alpha-, beta- and gamma-catenin was also significantly correlated with poor disease-specific 5-year survival (P=0.0096, 0.0434, 0.0005 and 0.0005, respectively). Multivariate Cox proportional hazard regression analysis showed that alteration of beta- and gamma-catenin expression was a significantly independent prognostic parameter for survival (P=0.0112 and 0.0088, respectively), as was the case with endophytic growth pattern and advanced N-category. These results indicate that patients with OSCC and absent or reduced membrane expression of beta- and gamma-catenin should be considered a high-risk group for regional lymph-node metastasis and poor prognosis.

Molecular cloning and functional expression of a human thyrotropin-releasing hormone (TRH) receptor gene.

In this study, we isolated genomic DNA fragments coding for the human thyrotropin-releasing hormone (TRH) receptor. Analysis of the nucleotide sequence revealed that the human TRH receptor gene had an exon-intron structure comprising at least two exons. A polypeptide encoded by the gene consisted of 398 amino acid residues with putative seven transmembrane domains. It showed high homology as a whole amino acid sequence with the rat and mouse TRH receptors except for considerable variation in the C-terminal region. Chromosomal mapping study indicated that the human TRH receptor gene was assigned to chromosome 8. Chinese hamster ovary (CHO) cells transfected with a DNA fragment containing the coding regions of the human TRH receptor bound with [3H]TRH. This binding was inhibited by adding unlabeled TRH in a dose-dependent fashion. Scatchard analysis indicated that the transfected CHO cells expressed a single class of high affinity binding sites at a dissociation constant (Kd) of approximately 1 nM. These results demonstrated that the isolated gene encoded a specific TRH receptor with high affinity.

Cloning and expression of a complementary DNA encoding the bovine receptor for pituitary adenylate cyclase-activating polypeptide (PACAP).

A cDNA encoding a pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was cloned from a bovine brain cDNA library using a synthetic oligonucleotide probe corresponding to the partial N-terminal amino acid sequence of the PACAP receptor purified from the bovine brain. The cloned cDNA encoded a polypeptide of 513 amino acid residues with seven putative transmembrane domains. The deduced amino acid sequence exactly matched the N-terminal amino acid sequence of the purified PACAP receptor. It also shared an apparent similarity with the vasoactive intestinal peptide (VIP), secretin, growth hormone releasing hormone, calcitonin, and glucagon receptors, suggesting that the PACAP receptor is a member of the secretin receptor subfamily of the guanine nucleotide-binding regulatory protein-coupled receptor family. Northern blot analysis showed that the size of the major mRNA band which hybridized with the cDNA was about 7 kb in the bovine cerebral-cortex and hippocampus. An expression vector containing the cloned cDNA for the PACAP receptor was introduced into Chinese hamster ovary (CHO) cells. The affinity of PACAP receptors expressed on the transfected CHO cells was quite similar to that of natural PACAP receptors on the bovine brain membranes. Competitive binding experiments showed that PACAP38 displaced the binding of 125I-labeled PACAP27 to the receptors on the CHO cells more efficiently than PACAP27, while VIP was less effective. In addition, both of PACAP27 and PACAP38 elevated the levels of cAMP and inositol phosphates in the transformed CHO cells. These results indicate that the PACAP receptors encoded by the cloned cDNA are identical to the purified PACAP receptors, and that they can stimulate dual signaling cascades.

Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene.

The human gene encoding pituitary adenylate cyclase activating polypeptide (PACAP) was isolated and its nucleotide sequence was determined. By comparison with a human PACAP cDNA, the exon/intron organization of PACAP gene was determined. The last exon encoded the longer form of PACAP, PACAP38 and 3'-untranslated sequences, suggesting that the shorter form of PACAP, PACAP27 is not generated by alternative splicing mechanisms. The 5'-flanking region of the PACAP gene contains several sequence motifs homologous to CRE, TRE, and GHF-1. On the basis of DNA isolated from mouse A9 microcell hybrid clone containing a single human chromosome, the PACAP gene was assigned to human chromosome 18. Furthermore, we determined the locus of the gene to be 18p11 by the chromosomal in situ hybridization technique.

One of the endothelin gene family, endothelin 3 gene, is expressed in the placenta.

A cDNA encoding human endothelin 3 (ET-3) precursor was cloned from a cDNA library from the placenta, and its nucleotide and deduced amino acid sequences were determined. This ET-3 cDNA was found to contain 2.3 kb pairs and encode prepro-ET-3 protein consisting of 224 amino acid residues. The putative big-ET-3 seems to consist of 42 amino acid residues. Two of the intron insertion sites were determined with information from nucleotide sequences of the cloned genomic ET-3 gene. This is the first direct evidence that the ET-3 gene is transcribed and expressed in the placenta.

Specific expression of human endothelin-2 (ET-2) gene in a renal adenocarcinoma cell line. Molecular cloning of cDNA encoding the precursor of ET-2 and its characterization.

Analysis of culture medium of human renal adenocarcinoma cells ACHN by RP-HPLC suggested that the cells specifically secreted human endothelin-2 (ET-2). cDNAs encoding human ET-2 precursor were cloned from a cDNA library constructed with mRNA derived from the ACHN cells, and the nucleotide and deduced amino acid sequences were determined. The ET-2 cDNA was revealed to contain 1.3 kb and encode prepro-ET-2 protein consisting of 178 amino acid residues. The ET-like sequences found in the prepro-ET-1 and -ET-3 was conserved in this prepro-ET-2. The Northern blot analysis of mRNA suggested that the transcript of ET-2 gene was 1.4 kb. This is the first direct evidence that human ET-2 gene was expressed specifically in tumor cells.

Synthesis of human endothelin-1 precursors in Escherichia coli.

Endothelin, the most potent vasoconstrictor found in nature, is thought to be important in the regulation of blood pressure and/or local blood distribution. Human placenta cDNA fragment encoding preproendothelin-1 (preproET-1) and its carboxyl terminal mature precursor (C-matured precursor) was expressed in E. coli. These products were characterized by both enzyme immunoassay and Western blot analysis.