Cholecystokinin-8 (CCK-8) has no effect on heart rate in rats lacking CCK-A receptors.

Heart rate responses to i.v. administration of cholecystokinin-8 (CCK-8) were investigated in Otsuka Long-Evans Tokushima Fatty (OLETF) rats lacking CCK-A receptors and control Long-Evans Tokushima Otsuka (LETO) rats. The heart rate decreased after i.v. administration of 3 nmol.kg(-)(1) of CCK-8 in LETO rats, but not in OLETF rats. Bradycardia in the LETO rats disappeared after treatment with MK-329, but not after treatment with L-365,260. The expression of CCK-A receptor precursor mRNA was found exclusively in the atrium in LETO rats. These results suggest that CCK-8 decreases heart rate via CCK-A receptors located in the atrium of the rats.     

Hepatic iron accumulation is not directly associated with induction of DNA strand breaks in the liver cells of Long-Evans Cinnamon (LEC) rats.

Effects of accumulation of copper and iron on induction of DNA strand breaks were investigated in Long-Evans Cinnamon (LEC) rats that spontaneously develop fulminant hepatitis. Copper and iron accumulated in the liver of LEC rats in an age-dependent manner from 4 to 15 weeks. Low-iron diet prevented the accumulation of iron in the liver, but did not prevent accumulation of copper. The amounts of DNA strand breaks that were estimated by comet assay in the liver cells of rats fed standard diet increased with age from 4 to 15 weeks. No significant differences were observed in the proportions of LEC rat liver cells without tail and the average lengths of tail momentum in the comet images between LEC rats that had been fed standard MF diet and low-iron diet. These results support the idea that accumulation of iron is not directly associated with the induction of DNA damage in the liver cells of LEC rats.     

Pollen UDP-glucose pyrophosphorylase showing polymorphism well-correlated to the S genotype of Pyrus pyrifolia

A polymorphic protein well-correlated to the diploid S genotypes of the pollen parent was detected by two-dimensional gel electrophoresis in Pyrus pyrifolia (Japanese pear). Its molecular weight was about 50 kDa, and it was expressed primarily in pollen. Partial amino acid sequences of the polymorphic protein from 'Nijisseiki' (S2S4), a cultivar of P. pyrifolia, were determined. Based on these sequences, two cDNA sequences associated with the S2 and S4 genotypes were identified by PCR-based methods. Both encode a protein of 458 amino acids whose sequence has high similarity to eukaryotic UDP-glucose pyrophosphorylases (UGPases) (EC 2.7.7.9), so they were named UGPases PA and PC. As there are only three amino acid substitutions between UGPases PA and PC, it is unlikely that they are pollen factors that recognize self and non-self S-RNases. Although this UGPase had more than 75% sequence identity to the known plant UGPases, its C-terminal sequences differed markedly. This unique C-terminal region of UGPases PA and PC may act in their subcellular localization in the pollen or interact with some other factor(s).     

Molecular cloning and expression of a major surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis in Escherichia coli

A major immunodominant surface protein (the 75-kDa protein) of Porphyromonas (Bacteroides) gingivalis 381 has been purified and its amino-terminal amino acid sequence has been determined. Using oligonucleotide probes corresponding to the sequence, we identified a recombinant plasmid clone carrying a single 4.2-kb BamHI fragment from pUC19 libraries of P. gingivalis. The BamHI fragment transferred to the bacteriophage T7 RNA polymerase/promoter expression vector system produced a slightly larger (77-kDa) protein, a precursor form, immunoreactive to the antibody against the 75-kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 75-kDa protein. Genomic Southern analysis revealed a single copy of the 75-kDa protein gene per genome among all P. gingivalis strains tested, and that no homologous genes are present in other black-pigmented Bacteroides species. These observations suggest that the 75-kDa protein gene may be useful as a specific DNA probe to classify or to detect this organism.     

Metaphase-specific cell death in meiotic spermatocytes in mice

Apoptosis of male germ cells is a widespread but little-understood phenomenon in many animal species. The elucidation of its mechanisms could be useful in the understanding of male infertility. We have examined the distribution of dying cells with the terminal transferase-mediated nick-end labeling (TUNEL) method and by an electron-microscopic procedure in the testes of 10 mouse strains, viz., C57BL/10 (B10), SL/NiA (SL), C57BL/6 (B6), C3H/He (C3H), BALB/c (BALB), DBA2 (DBA), CBA/J (CBA), MRL/MpJ(-)+/+ (M+), MRL/MpJ-lpr/lpr (lpr), and wild-type NJL mice (Mus musculus musculus). In the testes of the B10, NJL, SL, B6, C3H, BALB, DBA, and CBA mice, very few TUNEL-positive cells are distributed in the seminiferous tubules, whereas in the testes of the M+ and lpr mice, many TUNEL-positive cells, which are restricted to stage XII seminiferous tubules, have been identified. The most important finding is that many metaphases of meiotic spermatocytes show a marked TUNEL-positive reaction. Some metaphases show apoptotic morphology electron-microscopically. These results suggest that the testes of MRL strains will provide a useful model for the study of the mechanism of metaphase-specific apoptosis in meiotic spermatocytes.     

Contribution of endogenous G-protein-coupled receptor kinases to Ser129 phosphorylation of α-synuclein in HEK293 cells

The majority of α-synuclein (αS) deposited in Lewy bodies, the pathological hallmark of Parkinson’s disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of αS has been demonstrated to enhance the αS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of αS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of αS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of αS; however, knockdown of GRK2 or GRK5 did not decrease αS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of αS in HEK293 cells.     

Human disease-associated extracellular matrix orthologs ECM3 and QBRICK regulate primary mesenchymal cell migration in sea urchin embryos

Sea urchin embryos have been one of model organisms to investigate cellular behaviors because of their simple cell composition and transparent body. They also give us an opportunity to investigate molecular functions of human proteins of interest that are conserved in sea urchin. Here we report that human disease-associated extracellular matrix orthologues ECM3 and QBRICK are necessary for mesenchymal cell migration during sea urchin embryogenesis. Immunofluorescence has visualized the colocalization of QBRICK and ECM3 on both apical and basal surface of ectoderm. On the basal surface, QBRICK and ECM3 constitute together a mesh-like fibrillar structure along the blastocoel wall. When the expression of ECM3 was knocked down by antisense-morpholino oligonucleotides, the ECM3-QBRICK fibrillar structure completely disappeared. When QBRICK was knocked down, the ECM3 was still present, but the basally localized fibers became fragmented. The ingression and migration of primary mesenchymal cells were not critically affected, but their migration at later stages was severely affected in both knock-down embryos. As a consequence of impaired primary mesenchymal cell migration, improper spicule formation was observed. These results indicate that ECM3 and QBRICK are components of extracellular matrix, which play important role in primary mesenchymal cell migration, and that sea urchin is a useful experimental animal model to investigate human disease-associated extracellular matrix proteins.     

High-resolution CT scoring system-based grading scale predicts the clinical outcomes in patients with idiopathic pulmonary fibrosis

Background

The 2011 idiopathic pulmonary fibrosis (IPF) guidelines are based on the diagnosis of IPF using only high-resolution computed tomography (HRCT). However, few studies have thus far reviewed the usefulness of the HRCT scoring system based on the grading scale provided in the guidelines. We retrospectively studied 98 patients with respect to assess the prognostic value of changes in HRCT findings using a new HRCT scoring system based on the grading scale published in the guidelines.

Methods

Consecutive patients with IPF who were diagnosed using HRCT alone between January 2008 and January 2012 were evaluated. HRCT examinations and pulmonary function tests were performed at six-month intervals for the first year after diagnosis. The HRCT findings were evaluated using the new HRCT scoring system (HRCT fibrosis score) over time. The findings and survival rates were analyzed using a Kaplan-Meier analysis.

Results

The HRCT fibrosis scores at six and 12 months after diagnosis were significantly increased compared to those observed at the initial diagnosis (p < 0.001). The patients with an elevated HRCT fibrosis score at six months based on a receiver operating characteristic (ROC) curves analysis had a poor prognosis (log-rank, hazard ratio [HR] 2.435, 95% CI 1.196-4.962; p = 0.0142). Furthermore, among the patients without marked changes in %FVC, those with an elevated score above the cut-off value had a poor prognosis (HR 2.192, 95% CI 1.003-4.791; p = 0.0491).

Conclusions

Our data demonstrate that the HRCT scoring system based on the grading scale is useful for predicting the clinical outcomes of IPF and identifying patients with an adverse prognosis when used in combination with spirometry.