Newly identified interferon tau-responsive Hes family BHLH transcription factor 4 and cytidine/uridine monophosphate kinase 2 genes in peripheral blood granulocytes during early pregnancy in cows.

In cattle, interferon-stimulated genes (ISGs) such as ISG15, MX1, MX2, and OAS1 are known as classic ISGs that are highly involved in the implantation process. Various molecules play a crucial role in the mechanisms underlying ISG effects. Although microarray analyses have highlighted the expression of various molecules during the implantation period, these molecules remain incompletely characterized. In the present study, various specifically expressed genes were selected and their characteristics were examined. The microarray data from peripheral blood leukocytes derived from artificially inseminated cows and granulocytes obtained from embryo-transferred cows, respectively, were used to identify new ISG candidates. Seven common genes, including ISG15 and OAS1, were confirmed, but only 4 of the 5 genes were amplified by reverse transcription quantitative polymerase chain reaction. In addition, 3 expressed sequence tags (ESTs) exhibited significantly greater expression in granulocytes from pregnant cows than that observed in bred nonpregnant cows, and the expression in granulocytes increased after interferon-tau stimulation. Sequence alignment revealed similar sequences within 2 ESTs on the Hairy and enhancer of split (Hes) family basic helix-loop-helix transcription factor 4 (HES4) gene. An additional EST was identified as cytidine/uridine monophosphate kinase 2 (CMPK2). In silico analysis facilitated the identification of transcription factor-binding sequences, including an interferon-stimulated response element and interferon regulatory factor-binding sites, within the promoter region of HES4 and CMPK2. These genes may function as new ISGs in the context of implantation and may participate in the coordination of the feto-maternal interface in cows.

Evaluation of interferon-stimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals.

Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status.

Cell toxicity, hemolytic action and clastogenic activity of asbestos and its substitutes.

The cell toxicity, hemolytic and clastogenic activity were examined in various kinds of asbestos and some asbestos substitutes with reference to the their mineralogical and physicochemical characteristics. There were thirty-five fibrous and non-fibrous samples including UICC chrysotile, size-selected samples of UICC chrysotile, chrysotile altered by heating and grinding, Yamabe (Japan) chrysotile with long and short fibers, Coalinga (U.S. A.) chrysotile with short fibers, UICC crocidolite, amosite, and 19 non-asbestos samples such as, glass fibers, calcium silicates, sepiolites and some clay minerals. The cell toxicity and the hemolytic and clastogenic activity of asbestos were the strongest for chrysotile among all of the asbestos samples tested, and their strengths varied with fiber length and with the conditions of grinding and heating. These cellular effects of Yamabe chrysotile with long fibers and size-selected UICC chrysotile with long fibers were stronger than those of chrysotile of the same origin but with short fibers. These effects were weaker in chrysotile altered by heating and grinding. Among the asbestos substitutes, the cell toxicity, hemolytic and clastogenic activities of thin glass fibers were more marked than those of thick glass fibers. The four types of sepiolite were strongly hemolytic, but their cell toxicity and clastogenicity varied according to their grade of crystallinity and/or fiber size. These effects of calcium silicates and some clay minerals were generally low but varied with mineral species. In general, the cell toxicity, hemolytic and clastogenic activities of the asbestos substitutes tested here were mild compared with those of asbestos.

Expression of ADAMTS1 mRNA in bovine endometrium and placenta during gestation.

A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is a secreted protease. Through the regulation of extracellular matrix remodeling or developmental processes or both, ADAMTS1 is involved in several biological functions, including ovulation and embryo receptivity. However, the expression and possible role of ADAMTS1 in bovine endometrium is unknown. In this study, we analyzed ADAMTS1 mRNA expression in bovine endometrium during the estrous cycle, peri-implantation period, and at different stages of gestation by using quantitative real-time RT-PCR (qPCR) and in situ hybridization. The qPCR results indicated that the expression of ADAMTS1 mRNA was not affected by the day of the estrous cycle and was similar to cyclic levels on day 35 of gestation; however, the expression was more abundant in cotyledonary tissues of the placenta during late gestation. The in situ hybridization study showed that ADAMTS1 mRNA was detected mainly in uterine luminal epithelia and stromal cells during the estrous cycle and peri-implantation period. A disintegrin and metalloproteinase with thrombospondin motifs 1 mRNA was also expressed in the peri-implantation conceptus as well as in trophoblast cells, which include binucleate cells, and increased during late gestation. Furthermore, treatment of stromal cell with progesterone (300 nM) stimulated the expression of ADAMTS1 mRNA. This study indicates that ADAMTS1 participates in bovine endometrial remodeling, which is required for implantation and placental development in coordination with ovarian steroids.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation.

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.

Expression of endogenous retrovirus-like transcripts in bovine trophoblastic cells.

UNLABELLED: Endogenous retrovirus envelope elements are considered to participate in trophoblastic cell fusion and multinucleate cell formation in humans, mice, and sheep. However, there is limited information about their roles in the ruminant placenta. OBJECTIVES: We explore and identify the endogenous retrovirus envelope element genes expressed in bovine trophoblasts. METHODS: The NCBI UniGene database (Build #97 Bos taurus) was screened by in silico analysis. After cloning endogenous retrovirus envelope element-like transcript (ERVE), expression profiles were analyzed with quantitative RT-PCR and in situ hybrizaidation. RESULTS: Two UniGene clusters, UniGene ID: Bt.68042 and Bt.85243, were detected, and ERVE-A gene was cloned. Weak expression of this gene was first detected on Day 20 of gestation, and the intensity of its expression increased up to Day 70 of gestation. The intensity of its expression was maintained throughout gestation in the placenta, and its specific expression in trophoblastic binucleate cells was confirmed by in situ hybridization. CONCLUSIONS: bERVE-A has a similar sequence to human syncytin-1, although it lacks an intact envelope sequence, and is specifically expressed in binucleate cells. This is the first evidence that endogenous retrovirus envelope element genes are expressed in bovine binucleate cells.

Changes of the activities of superoxide dismutase after exposure to the fume of heavy metals and the significance of zinc in the tissue.

Pathological changes induced by cadmium aerosol had features common to the changes evoked by oxidants. Female rats were exposed to fumes of lead, antimony, zinc and cadmium (15-100 nmoles/m3). One hour after termination of exposure, superoxide dismutase (SOD) activity in erythrocytes of the exposed rats lowered by 15-40%. SOD activity of lung lavage fluid also lowered by 20-35% and the 2nd day after the exposure. The inverse value of SOD activity (l/SOD) in erythrocytes and of lung lavage fluid were proportional to the molar exposure level adjusted by the particle size (Dixon plot), irrespective of the difference of the exposed substance. The ratio of dry weight to wet weight of the lung was 4.3-26% lower than the control value on the later period after the exposure. With the heavy metal exposure, the uptake of the exposed metal was found to be proportional to the endogenous zinc concentration, which was correlated well with the change of SOD in the lung and in erythrocytes. Cadmium decreased the zinc concentration after the exposure.

Effects of cemented tungsten carbide dust on rat lungs following intratracheal injection of saline suspension.

In order to examine the effect of cemented tungsten carbide dust on the animal lung, saline suspensions were intratracheally administered into the lungs of rats in a single dosage. About one-fifth of the animals died during the first three days. The acute response of the lungs was hemorrhagic edema with intense alveolar congestion. The animals killed at six months all presented pulmonary lesions of patchy fibrosis in the vicinity of the deposited dusts, occasionally associated with focal traction emphysema and bronchobronchiolar ectasia. At twelve months, two-third of the animals had neither fibrosis nor dust deposition, although the remaining animals showed pulmonary lesions similar to those seen in the six-months responders. Fibrosis of the lungs seemed to consist of collapsed alveoli with condensation of the preexistent reticulin fibers, but without noticeable collagenization. It is supposed that both the early toxic and the late fibrogenic effects of the carbide dust are attributable to the cytotoxic action of cobalt present in the dust particles. It is possible that recovery of the pulmonary lesions results from removal of the dusts from the lesions.

A note on saturable and inhibitory kinetics of p-aminohippurate renal transport in rabbits.

In order to establish quantitative expressions of inhibitory kinetics in renal tubular secretion of p-aminohippurate (PAH), bolus i.v. injection and constant i.v. infusion of PAH were loaded to rabbits. Time courses of plasma concentration and urinary excretion rate were analyzed using compartment model with saturable rate process. Effects of sulfamethizole (SMZ) constant infusion and bolus injection on PAH renal excretion were also examined. Multi-compartment model for concurrent disposition of PAH and SMZ is presented. Excretory processes are composed of non-saturable filtration, saturable secretion which is subject to mutual inhibition, and reabsorption.

[Experimental pneumoconiosis induced by cemented tungsten and sequential concentrations of cobalt and tungsten in the lungs of the rat (author's transl)].

Experimental pneumoconiosis was induced by intratracheal injection of dusts of presintered cemented tungsten carbide, G2 (WC : Co=98 : 2) and TX20 (WC : Co : TiC : TaC=64 : 16 : 6 : 14) into the lungs of rats in order to document the pathological changes in lung tissues associated with environmental cobalt and tungsten. The following results were obtained. 1) Six months after the administration of G2 and TX20 dusts, marked fibrotic foci were noted and tungsten was detected in the lung tissues of all of the experimental animals. 2) Twelve months after the administration of both dusts, both the fibrotic changes and the tungsten levels were reduced in both test groups, but the reduction was more notable in the G2 group. The cases with fibrotic changes were relatively concomitant with the cases in which tungsten was detected. 3) On examination of tissue levels of cobalt derived from the dust, the cobalt levels in the lungs and bone tissues were less influenced by the dust of the G2 group at any point of investigation, whereas only the pulmonary cobalt levels at six months after the administration of the TX20 group showed a remarkable influence from the dust cobalt (p less than 0.01). 4) In the cases where tungsten was detected six months after the administration of both groups and twelve months after the administration of the TX20 group, pulmonary cobalt levels had not reached the value that should have been gained by the addition of expected values (dust cobalt levels calculated from the tungsten levels) to the control values. 5) The above results indicate that both G2 and TX20 dusts induced marked fibrotic changes in rat pulmonary tissues. However, these changes were reversible to some extent. In addition, a portion of the dust cobalt was dissolved in the body fluid and disappeared from the pulmonary fields.

Cerebro-hepato-renal syndrome of Zellweger. Clinical and autopsy findings and a review of previous cases in Japan.

An autopsy case of cerebro-hepato-renal syndrome of Zellweger, which occurred in a 14-year-old Japanese girl, is reported. The autopsy revealed widely distributed cystic changes in addition to renal blastema of both kidneys, and the liver was cirrhotic. The case was complicated by anomalies such as high forehead, strabismus, and partial defect of chorioidea. So far there have been only 10 reported cases of cerebro-hepato-renal syndrome of Zellweger in Japan.

Validation study of the in vitro micronucleus test in a Chinese hamster lung cell line (CHL/IU).

We conducted a collaborative validation study, under the auspices of the Japanese Ministry of Labour, on the in vitro micronucleus test to see if it could be used as an alternative to the in vitro chromosome aberration test for evaluation of chemical safety. We used the Chinese hamster lung cell line (CHL/IU), which is the most widely used system for the latter test in Japan, and evaluated 66 chemicals, including clastogens and polyploidy inducers. The cytochalasin B cytokinesis blocking method, which is commonly used in human lymphocyte culture, was applied to the established cell line, but did not improve the detection of chemically-induced micronuclei in continuously growing cells. The highest micronucleus frequencies were obtained at 48 or 72 h continuous treatments. In short treatments (6 h), a 42 h recovery time yielded the best responses. Concordance between the results of the micronucleus test and the chromosomal aberration test was satisfactorily high (88.7%), and we concluded that the in vitro micronucleus test could be used in place of the chromosomal aberration test as a simple and rapid method for detecting clastogens and aneugens in vitro. We also propose a protocol for the test.

A comparative study of methods of estimating renal size in normal adults.

One hundred live related voluntary kidney donors were studied prospectively. During donor nephrectomy the actual kidney bipolar length was measured and compared to the renal bipolar length estimated from abdominal sonogram, abdominal plain X-ray, intravenous pyelogram, and renal angiogram. Ultrasound was found to measure the kidney more accurately (mean difference between estimated size and actual = -3.4 mm +/- SD 6.96), than plain X-ray (mean difference from actual 13mm +/- SD 5.24), IVP (mean difference from actual 16.9 mm +/- SD 5.74), and renal angiogram (mean difference from actual 15.2 mm +/- SD 5.77).