The transcribed-ultraconserved regions in prostate and gastric cancer: DNA hypermethylation and microRNA-associated regulation.

The transcribed-ultraconserved regions (T-UCRs) are a novel class of non-coding RNAs, which are absolutely conserved (100%) between the orthologous regions of the human, rat and mouse genomes. Previous studies have described that several T-UCRs show differential expressions in cancers and might be involved in cancer development. We investigated the transcriptional levels of representative 26 T-UCRs and determined the regions that were differently expressed in prostate cancer (PCa) and gastric cancer (GC). A quantitative reverse transcription-polymerase chain reaction analysis revealed the downregulation of Uc.158+A expression by a DNA methylation-associated mechanism, which was restored by 5-Aza-dC (5-aza-2'-deoxycytidine) treatment. Bisulfite genomic sequencing using cell lines and tissue samples demonstrated cancer-specific CpG hypermethylation in both GC and PCa. However, Uc.416+A was only overexpressed in GC and we identified an miR-153 binding site in the possible regulatory region of Uc.416+A using online databases. Along with a forced expression or knockdown of miR-153 in MKN-74 GC cells, the transcriptional levels of Uc.416+A were significantly disturbed. A luciferase reporter gene assay supported the direct regulation of Uc.416+A expression by miR-153. Furthermore, Uc.416+A was associated with cell growth through the regulation of IGFBP6 (insulin-like growth factor-binding protein 6) in GC. These findings suggest an oncogenic role of Uc.416+A in GC, which suggests that our approach would provide new insights into functional studies of T-UCRs in cancer biology.

A case of polycystic ovary syndrome conceived by intracytoplasmic sperm injection following laparoscopic ovarian drilling.

Polycystic ovary syndrome (PCOS) is a disease in which an ovulation disorder is the main cause of infertility. Clomifene citrate (CC) is the treatment of first choice for ovulation induction in PCOS. If ovulation cannot be induced by CC, then either laparoscopic ovarian drilling (LOD) or gonadotropin therapy is selected as a subsequent treatment. Assisted reproductive technology (ART) is indicated for women with PCOS, similar to other infertility patients, when pregnancy is not achieved by intrauterine insemination (IUI). In this study, we experienced a case of PCOS in which pregnancy was achieved by ART following LOD. The case pertains to a 26-year-old patient. She consulted our hospital with a chief complaint of primary infertility. IUI with administration of CC plus recombinant follicle-stimulating hormone (rFSH) was carried out; however, pregnancy was not achieved. Subsequently, ART was carried out. In the first attempt, the development of several follicles was observed under the gonadotropin releasing hormone (GnRH) agonist long protocol. However, a fertilized oocyte was not obtained. In the second attempt, an ovum could not be collected after CC-rFSH ovarian stimulation. In the third attempt, a good quality embryo could not be obtained under the GnRH antagonist protocol, and therefore pregnancy could not be achieved. We performed LOD using a harmonic scalpel for the purpose of preventing severe OHSS and improving the quality of embryos. Following the operation, ovarian stimulation was performed under the CC-rFSH-antagonist protocol. Eighteen follicles were aspirated, six oocytes were picked-up, and five oocytes were normally fertilized. As a result, four embryos from day 2 culture were cryopreserved. Cryopreserved-thawed embryo transfer was thereafter performed, and a single pregnancy was achieved. LOD is a clinically effective treatment for PCOS requiring ART.

Effusion and solid lymphomas have distinctive gene and protein expression profiles in an animal model of primary effusion lymphoma.

Lymphoma usually forms solid tumours in patients, and high expression levels of adhesion molecules are observed in these tumours. However, Kaposi's sarcoma-associated herpesvirus (KSHV)-related primary effusion lymphoma (PEL) does not form solid tumours and adhesion molecule expression is suppressed in the cells. Inoculation of a KSHV-associated PEL cell line into the peritoneal cavity of severe combined immunodeficiency mice resulted in the formation of effusion and solid lymphomas in the peritoneal cavity. Proteomics using two-dimensional difference gel electrophoresis and DNA microarray analyses identified 14 proteins and 105 genes, respectively, whose expression differed significantly between effusion and solid lymphomas. Five genes were identified as having similar expression profiles to that of lymphocyte function-associated antigen 1, an important adhesion molecule in leukocytes. Among these, coronin 1A, an actin-binding protein, was identified as a molecule showing high expression in solid lymphoma by both DNA microarray and proteomics analyses. Western and northern blotting showed that coronin 1A was predominantly expressed in solid lymphomas. Moreover, KSHV-encoded lytic proteins, including viral interleukin-6, were highly expressed in effusion lymphoma compared with solid lymphoma. These data demonstrate that effusion and solid lymphomas possess distinctive gene and protein expression profiles in our mouse model, and suggest that differences in gene and protein expression between effusion and solid lymphomas may be associated with the formation of effusion lymphoma or invasive features of solid lymphoma. Furthermore, the results obtained using this combination of proteomics and DNA microarray analyses indicate that protein synthesis partly reflects, but does not correlate strictly with, mRNA production.

Pituitary-gonadal axis and secondary sex characters in the spontaneously masculinized pintail, Anas acuta (Anatidae, Aves), with special regard to the gonadotrophs.

This paper describes some aspects of the pituitary gland, gonads, and secondary sex characters of unusual pintails, Anas acuta, found in the wild. They were demonstrated to be females with partially masculinized plumage; i.e., their plumage showed various degrees of intersex, but the genital organs, syrinx, and electrophoretic pattern of sex-specific DNA were of the female type. Their left ovary underwent a marked involution and was associated with the mesonephros (the Wolffian body), as was the degenerated right ovary. Neither testicular tissue nor ovotestis was found in the gonad of either side. The oviduct was anatomically normal and comparable to that of the control adult. The plasma concentration of estradiol-17beta (E2) was shown to be 5.7+/-0.5 (mean+/-SE)pg/ml in the masculinized birds, 7.0+/-0.7 pg/ml in control males, and 22.5+/-6.1 pg/ml in control females, whereas plasma testosterone (T) was below the detection level in all of the samples. As to the pituitary gland, hypertrophy and/or deformity of the pars distalis was evident in the majority of the masculinized birds. Among others, hyperactive gonadotrophs, mainly luteinizing hormone (LH)- and LH/follicle-stimulating hormone (FSH)-immunoreactive cells, were prominent in the entire gland; and typical signet ring cells (castration cells) or giant gonadotrophs were frequently observed. These changes in the gonadotrophs may have been caused by a feedback response to the physiologically ovariectomized condition in the masculinized birds. Causal factor(s) of the ovarian degeneration remain to be further investigated.

Different expression of hepatic and renal cytochrome P450s between the streptozotocin-induced diabetic mouse and rat.

1. Since limited information is available about alterations of cytochrome P450 levels in diabetic animals other than rat, expression of P450s in the liver and kidney of the streptozotocin (STZ)-induced diabetic mouse was investigated. 2. The mRNA levels of CYP2B10, 3A11, 4A10 and 4A14 in the liver were increased in the STZ-induced diabetic mouse of both sexes. The CYP2B9 mRNA level was increased in the liver of the male diabetic mouse. These alterations were observed even at 2 weeks after administration. Insulin treatment restored these changes. The findings were consistent with changes reported in rat. 3. The levels of hepatic CYP1A2 and 2E1 and renal 2E1 and 4A did not change in the diabetic mouse at any time-point examined. No changes were seen in CYP2A- or 2C-related proteins in the diabetic mouse. These findings were in contrast to those in rat. 4. The results indicate that mouse P450s respond to insulin-dependent diabetes mellitus differently from those of the rat, and suggest that the expression of P450s in diabetes is not generally the same across animal species.

rDrak1, a novel kinase related to apoptosis, is strongly expressed in active osteoclasts and induces apoptosis.

This is the first report of a novel serine/threonine kinase, rabbit death-associated protein (DAP) kinase-related apoptosis-inducing protein kinase 1 (rDRAK1), involved in osteoclast apoptosis. We searched for osteoclast-specific genes from a cDNA library of highly enriched rabbit osteoclasts cultured on ivory. One of the cloned genes has a high homology with human DRAK1 (hDRAK1), which belongs to the DAP kinase subfamily of serine/threonine kinases. By screening a rabbit osteoclast cDNA library and 5'-RACE (rapid amplification of cDNA ends), we obtained a full length of this cDNA, termed rDRAK1. The sequencing data indicated that rDRAK1 has 88.0, 44.6, 38.7, and 42.3% identity with hDRAK1, DAP kinase, DRP-1, and ZIP (zipper-interacting protein) kinase, respectively. To clarify the role of DRAK1 in osteoclasts, we examined the effect of three osteoclast survival factors (interleukin-1, macrophage colony-stimulating factor, and osteoclast differentiation-inducing factor) on rDRAK1 mRNA expression and the effect of rDRAK1 overexpression on osteoclast apoptosis. The results suggested that these three survival factors were proved to inhibit rDRAK1 expression in rabbit osteoclasts. After transfection of a rDRAK1 expression vector into cultured osteoclasts, overexpressed rDRAK1 was localized exclusively to the nuclei and induced apoptosis. Hence, rDRAK1 may play an important role in the core apoptosis program in osteoclast.

Prognostic factors of chronic rhinosinusitis under long-term low-dose macrolide therapy.

We studied the immunological and histopathological factors that affect the prognosis of chronic rhinosinusitis under long-term low-dose macrolide therapy. Sixteen patients with chronic rhinosinusitis were given 200 mg clarithromycin or 150 mg roxithromycin orally once a day without other concurrent treatments for 2-3 months. Measurement of the serum IgE level, blood cell count and differential leukocyte count of the peripheral blood, cytological assessment of the nasal smear and computed tomographic (CT) scans of the paranasal sinuses were performed before treatment. The opacity of the sinuses was estimated and scored by the CT images. After treatment, anterior ethmoidal mucosa samples were collected, an infiltrated inflammatory cells, interferon (IFN)-gamma-positive cells and interleukin (IL)-4-positive cells were examined histologically and immunohistochemically. The severity of nasal symptoms was scored before and after treatment, and the improvement rate of the score (symptomatic improvement rate) was calculated. Patients with normal levels of serum IgE (

A novel female-specific member of the CYP3A gene subfamily in the mouse liver.

Expression of a female-specific CYP3A in the adult mouse liver was observed on immunoblotting analysis. To characterize this cytochrome P450, we determined the primary structure of its cDNA and examined its expression profile. This cytochrome P450 consisted of 504 amino acids and showed 92, 68, 88, and 69% amino acid sequence identity with mouse CYP3A11, 3A13, 3A16, and 3A25, respectively, and was designated as CYP3A41, a new mouse CYP3A gene. In the female liver, levels of CYP3A41 mRNA expression were comparable to those of CYP3A11, the major CYP3A enzyme in the adult mouse liver. Expression of CYP3A41 mRNA was detected immediately after birth in the livers of animals of both sexes, but increased with age in females, whereas it was gradually reduced in males, resulting in predominantly female-specific expression in livers. Lesser amounts of CYP3A41 mRNA were detected in the kidneys of female mice, with traces in the stomach, ovary, and heart of female mice and in the testis of male mice. Gonadectomy and sex hormone treatment indicated that estradiol and testosterone were able to induce and suppress the expression of CYP3A41 mRNA in the liver, respectively. Among the classical CYP3A inducers, dexamethasone, rifampicin, and 3-methylcholanthrene did not affect the level of CYP3A41 mRNA in the liver of either sex. On the other hand, pregnenolone 16alpha-carbonitrile and phenobarbital suppressed CYP3A41 level to half that of untreated female mice. These observations indicated that CYP3A41 is a female-specific CYP3A and one of the major CYP3A forms in the female mouse liver.

Generic tendency of metal salt cytotoxicity for six cell lines.

Systematic cytotoxicity evaluation of various metallic elements may contribute to the development of new metallic biomaterials with superior biocompatibility. It is generally reported that the cytotoxicity of a chemical differs with cell lines. However, our previous study revealed a high correlation in the cytotoxicity of 43 metal salts between two murine cell lines. If there is any generic tendency toward metal salt cytotoxicity for many kinds of cells, that information is helpful for the determination of the chemical composition of new metallic biomaterials that are expected to have lower cytotoxicity. In this study, the cytotoxicity of 12 metal salts was evaluated using four cell lines, and the results were compared, including those for two other cell lines obtained in our previous study. A metal salt concentration that reduced cell viability to 50% of that without any metal salt (IC(50)) was used as an index to compare the metal salt cytotoxicity between cell lines. The correlation was statistically proved by the IC(50)s of 12 metal salts among these cell lines (p < 0.01), suggesting the existence of a generic tendency to metal salt cytotoxicity beyond cell lines. The metal salt order of toxicity from the highest was K(2)Cr(2)O(7), AgNO(3), VCl(3), SbCl(3), CuCl(2), CoCl(2), NiCl(2), ZnCl(2), Cr(NO(3))(3), FeCl(3), TiCl(4), and Al(NO(3))(3). The sensitivity for metal salt cytotoxicity differed with cell lines; IMR-32 had the highest sensitivity among the six cell lines.

Cytotoxicity evaluation of 43 metal salts using murine fibroblasts and osteoblastic cells.

Metallic biomaterials are generally used for replacement of structural components of the human body such as bones, joints, and tooth roots. When they are implanted inside a body, metallic biomaterials may corrode and/or wear, releasing metal ions and debris which may have toxic effects on tissues and organs. Since it is important for biomaterials to have no toxicity against a living body, a systematic and quantitative evaluation of the cytotoxicity of metallic elements is required for the development of new metallic biomaterials with superior biocompatibility. In this study, the cytotoxicity of 43 metal salts were evaluated by the colony formation method using two kinds of cultured cells. The effects of the difference in valence numbers of metallic elements in the salts on cytotoxicity were examined. The cytotoxicity of the salts of metallic elements' oxo acids was also investigated. As a result, the intensity of metal salts' cytotoxicity tends to be quite similar between MC3T3-E1 and L929 (the correlation coefficient of metal salts' IC50s is 0.82). The intensity of metal salts' cytotoxicity depends on the kinds of metallic elements, their chemical states, and concentrations. The IC50 of the highest toxic salt is 1.36 x 10(-6) mol L-1, which differs four orders of magnitude from the IC50 of the lowest toxic salt. K2Cr2O7, CdCl2, VCl3, AgNO3, HgCl2, SbCl3, BeSO4, and InCl3 are high toxic salts in which IC50s are smaller then 10(-5) mol L-1 for both or either of the cell lines. HgCl, Tl(NO3)3, GaCl3, CuCl2, MnCl2, CoCl2, ZnCl2, NiCl2, SnCl2, IrCl4, TlNO3, CuCl, RhCl3, Pb(NO3)2, Cr(NO3)3 and Bi(NO3)3 are relatively high toxic salts in which IC50s are smaller than 10(-4) mol L-1 for both or either cell lines.

Isolation of cysteine protease in the crude mite extract, Dermatophagoides farinae.

In order to study the relationship between cysteine protease and Der f I, which is one of the major allergens in the mite, Dermatophagoides farinae, isolation of cysteine protease was attempted using various column chromatographies. Both the potent cysteine protease activity and the allergenic activity were detected in the same fractions by anion exchange chromatography on a DEAE-Sephacel, gel chromatographies and chelating Sepharose 6B chromatography. In the double immunodiffusion test, the finally isolated fraction and rabbit anti-Der f I sera reacted to give a single precipitation line which fused completely with the precipitation line formed by Der f I and anti-Der f I sera. Sequence analysis for the first 10 N-terminal amino acids from cysteine protease and Der f I were identical. These results strongly suggest that cysteine protease of mites may be Der f I allergen and that measuring cysteine protease activity may possibly become a beneficial method for detecting Der f I allergens.