Correlation with larval body size of mRNA levels of growth hormone, growth hormone receptor I and insulin-like growth factor I in larval torafugu Takifugu rubripes.

The full-length of insulin-like growth factor (IGF) complementary (c)DNAs encoded by igf-I and igf-II from torafugu pufferfish Takifugu rubripes were cloned in the present study. The deduced amino acid sequences of the two genes showed c. 80% identity each with those of Igf-I and Igf-II from other teleosts, respectively. Two growth hormone (GH) receptors, ghr1 and ghr2, were also cloned in silico using the T. rubripes Fugu genome database. The transcripts of T. rubripes igf-I were detected in slow muscle, heart, skin, gill, liver and intestine but not in fast muscle, spleen and testis of adult fish, whereas those of igf-II were found in all tissues examined. Subsequently, the accumulated messenger (m)RNA levels of igf-I and igf-II were investigated in an F(2) population derived from a male of an apparent fast-growing T. rubripes strain and a wild female T. rubripes together with those of other growth-related genes encoding Gh, Ghr1 and Ghr2, and with those of prolactin (Prl) and leptin (Lep) previously reported. The accumulated mRNA levels of igf-I, gh and ghr1 were significantly correlated to growth rate at larval stages in the population, but not for those of igf-II, prl, ghr2 and lep. Although it is unclear whether or not this phenotype is directly related to the heredity of the fast-growing strain, the findings suggest that the expression of igf-I, gh and ghr1 is involved in the regulation of growth rate at larval stages in T. rubripes.

Incidence of and risk factors for incisional hernia after closure of temporary ileostomy for colorectal malignancy.

PURPOSE: Incisional hernia is a major complication after stoma closure and can cause uncomfortable symptoms. In this study, we evaluated the risk factors for hernia formation with the aim of reducing the incidence of incisional hernia. METHODS: A total of 134 oncology patients underwent closure of a temporary loop ileostomy between May 2004 and December 2013. The incidence of incisional hernia was determined by routine follow-up computed tomography scanning every 6 months. The relationships between patients' characteristics, including age, sex, obesity, diabetes mellitus, surgical site infection, chronic obstructive pulmonary disease, hypertension, hypoalbuminemia, smoking, and presence of a midline hernia and the occurrence of incisional hernia were retrospectively evaluated. RESULTS: The median follow-up time was 47 months (range 8-130). Hernias occurred in 23.9% of patients (32/134). The median time to detection of hernias was 8 months (range 2-39). The Chi-squared test revealed significant differences in obesity (P = 0.0003), hypertension (P = 0.0057), and incisional hernia history (P = 0.0000) between patients with and without incisional hernia. Multivariable analysis and univariate analysis revealed that hypertension and the presence of midline incisional hernia were risk factors for incisional hernia. CONCLUSIONS: Hypertension and the presence of a midline incisional hernia were the major risk factors for incisional hernia after loop ileostomy closure. These risk factors can be addressed before planning surgery.

DFF45/ICAD restores cisplatin-induced nuclear fragmentation but not DNA cleavage in DFF45-deficient neuroblastoma cells.

We have previously defined a homozygously deleted region at chromosome 1p36.2-p36.3 in human neuroblastoma cell lines, NB-1 and NB-C201, and identified six genes including DFF45/ICAD within this region. In this study, we found that NB-C201 cells are much more resistant to various genotoxic stresses such as cisplatin (CDDP) than CHP134 and SH-SY5Y cells that do not have the homozygous deletion. To examine a role(s) of DFF45 in the regulation of apoptosis in response to CDDP, we have established stably DFF45-expressing NB-C201 cell clones (DFF45-1 and DFF45-3) and a control cell clone (NB-C201-C) using a retrovirus-mediated gene transfer. In contrast to NB-C201-C cells, DFF45-3 cells displayed apoptotic nuclear fragmentation in response to CDDP. Although CDDP-induced proteolytic cleavage of procaspase-3 and DFF45 in DFF45-3 cells, we could not detect a typical apoptotic DNA fragmentation. Additionally, deletion analysis revealed that C-terminal region of DFF45 is required for inducing nuclear fragmentation. Unexpectedly, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays demonstrated that DFF45 has undetectable effect on CDDP sensitivity of NB-C201 cells. Taken together, our present results suggest that DFF45/DFF40 system may be sufficient for CDDP-induced nuclear fragmentation but not DNA cleavage.

Cloning of the mouse gene for D-dopachrome tautomerase.

D-Dopachrome tautomerase converts 2-carboxy-2,3-dihydroindole-5, 6-quinone (D-dopachrome) into 5,6-dihydroxyindole. The amino acid sequence of this protein is 27% identical with that of macrophage migration inhibitory factor, which is known as a cytokine, pituitary hormone, and glucocorticoid-induced immunomodulator. In this study, we isolated and sequenced a 3490 bp-long genomic DNA of mouse D-dopachrome tautomerase that consists of three exons and two introns. By two procedures, 5' rapid amplification of cDNA ends and cap site labeling, we determined the transcription initiation site, which is located 46 bp upstream of the translation initiation site. The possible polyadenylation sequence (AATAAA) is located 180 bp downstream of the termination codon. Computer-assisted analysis of the nucleotide sequence revealed a number of regulatory motifs, including multiple sites for Sp1, C/EBP, NF-Y, and USF. Although the precise pathophysiological functions of D-dopachrome tautomerase remain to be elucidated, the present results will contribute not only to elucidation of the mechanism of gene expression, but also to understanding of the molecular function of this protein.

Structure and expression of the mouse S10 gene.

We previously reported the cloning of a human S10 cDNA which encodes a small GTP-binding protein belonging to the Rab subfamily. Here we describe a mouse S10 cDNA and its genomic structure. Mouse S10 is 92.3% homologous at the nucleotide level and 98.3% identical at the amino acid level compared to human S10. The mouse S10 gene is comprised of two exons and a single intron. Northern blotting of tissue RNAs indicates that the S10 gene is predominantly expressed in brain.

Estrogenic activity of 37 components of commercial sunscreen lotions evaluated by in vitro assays.

Thirty-seven chemical components of commercial sunscreen lotions were evaluated for estrogen agonistic and/or antagonistic activity using two in vitro assays, (1) an ELISA-based estrogen receptor competitive binding assay (ER-ELISA) and (2) a modified yeast two-hybrid estrogen assay, with and without addition of a rat liver preparation, S9 mix. Eleven compounds, most of which were benzophenone derivatives and parabens, showed binding affinity to ER by ER-ELISA without S9 mix. Although the activities of almost all of the compounds were attenuated by addition of S9 mix, 4-octylphenylsalicylate and 2,2'-dihydroxy-4,4'-dimethoxybenzophenone acquired estrogenic activity, suggesting metabolic activation of these compounds. Two benzophenones showed agonistic activity in the yeast two-hybrid assay without S9 mix. The activity of one of these was reduced by S9 treatment and a further two benzophenones was activated. Eight parabens were active in this assay without S9 exposure, but their activities were eliminated by S9 treatment. Benzophenones with para-phenolic hydroxyl groups and parabens with branched and/or longer linear chains were generally more potent in both bioassays. In addition, weak antagonistic activity of 4-t-butylphenyl-salicylate, 2-ethylhexyl 4-dimethylaminobenzoate and (+/-)-alpha-tocopherolacetate was observed with S9 treatment. In vivo testing of the compounds reported here to have estrogen agonistic and antagonistic activities is required to confirm their effects at an organismal level.

Molecular cloning of a cDNA encoding a novel small GTP-binding protein.

A cDNA encoding a small GTP-binding protein, S10, was cloned from Jurkat cells. The deduced amino acid sequence of S10 had the structural features characteristic to this family of proteins with highest homology to rab subfamily. Northern blot analysis revealed that this gene is expressed only in lymphoid cell lines and a histiocytic leukemia, U937. Hence, it should have a specialized function in cells derived from the hematopoietic stem cell.

Regulatory sequences required for hst-1 expression in embryonal carcinoma cells.

The hst-1 gene, which is implicated in mammalian embryonic development and morphological transformation of NIH3T3 cells, is expressed in undifferentiated F9 cells, but not in differentiated F9 and other well-differentiated cells, such as PYS-2, NIH3T3 and HeLa cells. An octamer element present in the 3' untranslated region acts as an enhancer. Although Oct3 is down-regulated when F9 cells are differentiated, transient expression of Oct3 did not enhance the hst-1 promoter activity in HeLa, NIH3T3 or PYS-2 cells. Thus, the role of Oct3 on hst-1 expression remains elusive, and an additional transcription factor which interacts may regulate hst-1 transcription in association with Oct1, Oct3 or both.

Identification of rabaptin-5, rabex-5, and GM130 as putative effectors of rab33b, a regulator of retrograde traffic between the Golgi apparatus and ER.

The role of rab33b, a Golgi-specific rab protein, was investigated. Microinjection of rab33b mutants stabilised in the GTP-specific state resulted in a marked inhibition of anterograde transport within the Golgi and in the recycling of glycosyltransferases from the Golgi to the ER, respectively. A GST-rab33b fusion protein stabilised in its GTP form was found to interact by Western blotting or mass spectroscopy with Golgi protein GM130 and rabaptin-5 and rabex-5, two rab effector molecules thought to function exclusively in the endocytic pathway. A similar binding was seen to rab1 but not to rab6, both Golgi rabs. In contrast, rab5 was as expected, shown to bind rabaptin-5 and rabex-5 as well as the endosomal effector protein EEA1 but not GM130. No binding of EEA1 was seen to any of the Golgi rabs.

Potentiation of antitumor effect of NKT cell ligand, alpha-galactosylceramide by combination with IL-12 on lung metastasis of malignant melanoma cells.

The combined therapeutic effect of natural killer T (NKT) cell ligand alpha-galactosylceramide (alpha-GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of alpha-GalCer or IL-12, the combined treatment of tumor-bearing mice with alpha-GalCer plus IL-12 caused a super-induction of serum IFN-gamma levels, though alpha-GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-gamma production, the natural killing activity against YAC-1 cells and syngeneic B16-BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by alpha-GalCer plus IL-12 were enriched in both NK1.1+ TCRalphabeta+ NKT cells and NK1.1+ TCRalphabeta- NK cells. The preventing effect of alpha-GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of alpha-GalCer was totally abolished in NKT-deficient mice. However, IL-12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that alpha-GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands alpha-GalCer and IL-12.

Interleukin-4-dependent induction of preproenkephalin in antigen-specific T helper-type 2 (Th2) cells.

Naive Th cells obtained from OVA(323-339)-specific DO11.10 TCR-Tg mice did not express preproenkephalin (PPE) mRNA. However, culture of naive Th cells with OVA(323-339) peptide (OVA-pep) plus IL-2 under Th2-inducing conditions for 7 days resulted in an induction of PPE mRNA. The PPE mRNA was also induced by culturing with OVA-pep plus IL-2 (neutral condition). However, PPE mRNA induction under neutral conditions was totally abrogated by addition of anti-IL-4 mAb. The existence of methionine-enkephalin was also demonstrated in peptidase-digested peptides derived from Th2 cell lysate. These results demonstrate that IL-4 is a critical factor for the induction of PPE mRNA in freshly expanded antigen-specific Th2 cells.

Pronounced inhibition by a natural anthocyanin, purple corn color, of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-associated colorectal carcinogenesis in male F344 rats pretreated with 1,2-dimethylhydrazine.

The potential of purple corn color (PCC), a natural anthocyanin, to modify colorectal carcinogenesis was investigated in male F344/DuCrj rats, initially treated with 1,2-dimethylhydrazine (DMH), receiving 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the diet. After DMH initiation, PCC was given at a dietary level of 5.0% in combination with 0.02% PhIP until week 36. No PCC-treatment-related changes in clinical signs, body weight and food consumption were found. Incidences and multiplicities of colorectal adenomas and carcinomas in rats initiated with DMH were clearly increased by PhIP. In contrast, lesion development was suppressed by PCC administration. Furthermore, in the non-DMH initiation groups, induction of aberrant crypt foci by PhIP tended to be decreased by the PCC supplementation. The results thus demonstrate that while PhIP clearly exerts promoting effects on DMH-induced colorectal carcinogenesis, these can be reduced by 5.0% PCC in the diet, under the present experimental conditions.

Preliminary study of fortnightly irinotecan hydrochloride plus cisplatin therapy in patients with advanced gastric and colorectal cancer.

PURPOSE: Irinotecan hydrochloride shows a strong activity against gastric cancer and colorectal cancer, while combined therapy with irinotecan and cisplatin is useful for gastric cancer. However, myelosuppression and diarrhea are still dose-limiting factors. To reduce such toxicities to enable therapy to be performed on an outpatient basis, we tested the effect of divided administration of cisplatin. METHODS: Irinotecan (60 mg/m2) plus cisplatin (30 mg/m2) were administered on days 1 and 15 every 4 weeks to 13 patients with advanced gastric cancer and 13 with advanced colorectal cancer. Treatment was continued if a leukocyte count > or = 3000/mm3, a platelet count > or = 100,000/mm3, and grade 0 diarrhea were confirmed. Doses were reduced if grade 3-4 hematological toxicity and grade 2 or higher nonhematological toxicity occurred. RESULTS: The major toxicity was leukopenia (neutropenia), but grade 3-4 nonhematological toxicity was not observed. The response rate was 41.7% for gastric cancer (5/12 evaluable patients) and 36.7% for colorectal cancer (4/11 evaluable patients). The median survival time was 313 days (range 29-920 days) for gastric cancer patients and 490 days (range 83-1184 + days) for colorectal cancer patients. CONCLUSION: Fortnightly administration of irinotecan and cisplatin (with a divided cisplatin dose) seems to be a useful regimen for gastrointestinal cancer. It reduces toxicity while maintaining a good antitumor effect.

The critical role of Th1-dominant immunity in tumor immunology.

To investigate the precise role of antigen-specific Th1 and Th2 cells in tumor immunity, we developed a novel adoptive tumor-immunotherapy model using OVA-specific Th1 and Th2 cells and an OVA gene-transfected tumor. This therapeutic model demonstrated that both antigen-specific Th1 and Th2 cells had strong antitumor activity in vivo with distinct mechanisms. However, immunological memory suitable for the generation of tumor-specific cytotoxic T lymphocytes was induced only when tumor-bearing mice received Th1 cell therapy, but not Th2 cell therapy. Thus it was strongly suggested that Th1-dominant immunity is critically important for the induction of antitumor cellular immunity in vivo. We also proposed that several immunomodulating protocols using interleukin (IL)-12, IL-12 gene, the natural killer T cell ligand alpha-galactosylceramide, or Th1 cytokine-conditioned dendritic cells might be useful strategies for the induction of Th1-dominant immunity essential for the development of tumor-specific immunotherapy.

Comparison of the type-2 insulin-like growth factor receptor in normal osteoblasts and osteosarcoma-derived osteoblast-like cells.

Insulin-like growth factor-II is known to stimulate the proliferation and differentiation of osteoblasts in part through activation of the type-2 insulin-like growth factor receptor. The present study examined the type-2 insulin-like growth factor receptors of three normal osteoblast-like cells and three osteosarcoma-derived osteoblast-like cells (OGA, SU, and IMAI) from humans. [125I]insulin-like growth factor-II was used for the binding studies. All of the cell types had high affinity binding sites for insulin-like growth factor-II (dissociation constants [Kd] < or = 1 nM). The concentration of these sites was 10 to 24-fold higher in normal osteoblasts than in the osteosarcoma cells studied. Unlabeled insulin-like growth factor-II inhibited the binding of [125I]insulin-like growth factor-II to the cells in a dose-dependent manner; however, unlabeled insulin-like growth factor-I and insulin were less effective. Covalent crosslinking of insulin-like growth factor-II binding sites gave molecular mass estimates of M(r) 250,000 in human osteoblast cells, 250,000 and 130,000 in OGA cells, 240,000 in SU cells, and 250,000 and 130,000 in IMAI cells. Unlabeled insulin-like growth factor-II inhibited all affinity labeling. In Northern blot analysis, the type-2 insulin-like growth factor receptor mRNA of normal osteoblasts was seen in greater abundance than it was in osteosarcoma cells. These results indicate that the numbers of type-2 insulin-like growth factor receptors differ between normal and transformed osteoblasts and that the differential expression of the receptor may be due to the differentiation of osteoblasts.

Influence of carbonate on sintering of apatites.

Sintering of carbonate apatite, prepared at 100 degrees C and pH 9.0 for 3 days, was studied by thermal analysis, x-ray diffraction, and infrared spectroscopy. The sintering temperature, at which the linear thermal shrinkage of isostatically compacted specimens increased sharply, decreased in proportion to the amount of carbonate initially present in the apatite. For example, specimens with over 8 wt% carbonate could be sintered at a temperature (650 degrees C) which was nearly 400 degrees C lower than that needed for sintering a specimen with no carbonate. Amounts of carbonate lost at the end of sintering, estimated chemically and by infra-red spectroscopy, were approximately equal to sample weight losses estimated thermogravimetrically.

Hepatitis C virus RNA and hepatitis C virus antibody in the serum of patients with abnormal liver function.

In order to elucidate the relation between hepatitis C virus (HCV) RNA and antibody to HCV (anti-HCV) in serum, we examined samples of serum collected from 228 HBsAg-negative patients, with abnormal alanine aminotransferase (ALT) values, for HCV-RNA by nested polymerase chain reaction (PCR) assay and for anti-HCV using C100 protein as the antigen. HCV-RNA was detected in 99 (92.5%) of 107 anti-HCV-IgG-positive samples, regardless of ELISA optical density cut-off value (ELISA ratio), and in 34 (28.1%) of 121 anti-HCV-IgG-negative samples in which the frequency of the presence of HCV-RNA became higher in proportion to the ELISA ratio. Among 42 discordant cases (34 anti-HCV-IgG-negative, RNA-positive cases and eight anti-HCV-IgG-positive, RNA-negative cases), 10 were positive for anti-HCV-IgM (8/34 and 2/8, respectively) irrespective of clinical status. These findings suggest that in patients with abnormal ALT values, even if they are anti-HCV-IgG negative, HCV infection cannot be excluded. Furthermore, PCR assay for detecting HCV-RNA may be more suitable for identifying patients with infectious virus than is detection of anti-HCV-IgG. Detection of anti-HCV-IgM may also be useful.

Features of IgA nephropathy in preschool children.

AIM: The aim of this study is to clarify the age-related characteristics of pediatric IgA nephropathy. PATIENTS AND METHODS: Five cases in preschool children less than 6 years old were analyzed and compared to 38 cases in older children from 6 to 15 years old. RESULTS: The group of younger children had higher incidences of gross hematuria, hypertension, proteinuria, and hypoproteinemia. Renal biopsy specimens in this group showed more intracapillary lesions including mesangial cell proliferation and endocapillary proliferation ofglomeruli, but less segmental lesions, global sclerosis, and interstitial changes. CONCLUSION: IgA nephritis in preschool children demonstrated more symptoms of acute onset and less chronic renal injury.

A thirteen-week oral toxicity study of annatto extract (norbixin), a natural food color extracted from the seed coat of annatto (Bixa orellana L.), in Sprague-Dawley rats.

A subchronic oral toxicity study of annatto extract (norbixin), a natural food color, was conducted. Groups of 10 male and 10 female Sprague-Dawley rats were fed annatto extract at dietary levels of 0, 0.1, 0.3 and 0.9% for 13 weeks. There were no treatment-related adverse effects on body weight, food and water consumption, ophthalmology and hematology data. Blood biochemical analysis revealed changes in rats of both sexes confined to the 0.9% and 0.3% groups, including increased alkaline phosphatase, phospholipid, total protein, albumin and albumin/globulin ratio. Marked elevation in absolute and relative liver weights was also found in both sexes of the 0.9% and 0.3% groups, but not the 0.1% group. Hepatocyte hypertrophy was evident and an additional electron microscopic examination demonstrated this to be linked to abundant mitochondria after exposure to a dietary level of 0.9% annatto extract for 2 weeks. Thus, the No-Observed-Adverse-Effect-Level (NOAEL) was judged to be a dietary level of 0.1% (69 mg/kg body weight/day for males, 76 mg/kg body weight/day for females) of annatto extract (norbixin) under the present experimental conditions.

Effect of fudosteine, a new cysteine derivative, on mucociliary transport.

We examined the effect of fudosteine ((-)-(R)-2-amino-3-(3-hydroxypropylthio)propionic acid) on the mucociliary transport (MCT) rate in quails. The MCT rate was estimated by ash transport velocity on the tracheal mucosa of quails. Fudosteine (500 mg kg(-1), p.o.) did not affect the normal MCT rate. However, topical application of fudosteine to the tracheal mucosa dose-dependently protected the impairment of the MCT rate caused by exposure to cigarette smoke. The results suggest that fudosteine may participate in the defence mechanism in the respiratory tract against irritant gases.