Effect of restricted suckling on the superovulatory response and reproductive performance in postpartum Japanese black cows.

The reproductive performance of postpartum cows is affected by factors such as suckling and nutrition. We investigated the effect of a restricted suckling period on the superovulatory response and the fertility after flushing in postpartum Japanese Black cows. Forty-seven postpartum cows were used in this study. At 7 days postpartum, the cows were divided into 2 groups: (1) continuous access to calves from birth to weaning at 3 months postpartum (ad libitum suckling group; n=20); and (2) twice daily suckling to the calves penned adjacent to them (restricted suckling group; n=27). All cows were initiated a superstimulatory treatment with a controlled internal drug releasing device and follicle stimulating hormone at 40 days postpartum. Embryos were nonsurgically collected at 7 or 8 days after estrus. After uterine flushing, the cows were again used for reproduction. There were no significant differences between the ad libitum and restricted suckling groups in terms of the numbers of transferable (6.7 ± 5.4 versus 7.9 ± 7.0) and freezable embryos (5.5 ± 4.9 versus 6.2 ± 7.0). In contrast, the interval to the first estrus after flushing in the restricted suckling group was lower (P<0.05) than that in the ad libitum suckling group (8.9 ± 5.7 days versus 27.9 ± 24.2 days). These results suggest that restricted suckling in postpartum Japanese Black cows does not affect the superovulatory response and embryo quality; however, it improves their fertility after flushing.

Candesartan-induced gene expression in five organs of stroke-prone spontaneously hypertensive rats.

To test the functional consequences of blocking the local renin-angiotensin system (RAS), we investigated the effects of an angiotensin II type 1 receptor blocker (ARB), candesartan, on the systemic gene expression profile of five important organs (brain, heart, kidney, liver and adipose tissues) in the stroke-prone spontaneously hypertensive rat (SHRSP), an established model of essential hypertension and cardiovascular disorders, and its normotensive control, the Wistar Kyoto (WKY) rat. Rats were treated with candesartan (5 mg/kg/d) for 4 weeks from 12 to 16 weeks of age. DNA microarray technology was used to identify changes in gene expression. Four weeks of treatment with candesartan significantly lowered systolic blood pressure in male rats of both the SHRSP and the WKY strains (p<0.0005). Candesartan differentially modulated the gene expression profile in an organ-specific manner in male SHRSP; of the five organs tested, gene expression was most prominently altered in the hearts of SHRSP. In contrast, candesartan treatment exerted minimal or no significant effects on the gene expression profile of the corresponding organs of male WKY rats. The inter-strain differences in gene expression changes induced by candesartan were considered to be associated with both blood pressure-dependent and independent mechanisms. These results help to delineate the mechanisms that underlie the organ or tissue protection conferred by ARB at the levels of cellular biology and genomics in the context of the local RAS. Further studies are warranted to investigate not only individual genes of interest but also genetic "networks" that involve differential organ- or tissue-specific gene expression induced by the blockade of RAS in essential hypertension. Tokyo, Japan

Dynamic changes of the renin-angiotensin and associated systems in the rat after pharmacological and dietary interventions in vivo.

To address the multiplicity of the renin-angiotensin system (RAS) with particular interest in its local, synergistic regulation, we investigate dynamic changes of the RAS and associated systems in response to external stimuli in the rat. We tested influences of the RAS blockade (candesartan and enalapril), diuretics (hydrochlorothiazide), high lipid diet, and salt loading on tissue mRNA level of 12 principal genes. Under the hemodynamic conditions appropriately predetermined, we quantitatively evaluated mRNA level changes with and without each intervention in five organs-the brain, heart, kidney, liver, and adipose tissues-of male rats (n = 5 each). A total of 250 tissues were examined by real-time PCR. Significant changes in mRNA level (P < 0.05) were found in a drug-, diet- and tissue-specific manner. For instance, 29% of genes (14 out of 48 tissues showing detectable mRNA levels) were differentially regulated by candesartan and enalapril, although both drugs reduced blood pressure to similar extents. When the overall interactions among 12 genes were compared between interventions, the RAS and associated systems appeared to change in the opposite direction between candesartan and high lipid diet in the adipose tissue and between candesartan and salt loading in the heart. Enalapril, however, induced unique patterns of perturbation in the local RAS under the corresponding conditions. Thus, this study provides a fundamental picture of gene expression profile in vivo in the RAS and associated systems. In particular, our data highlight differential regulation between candesartan and enalapril, which may reflect the individual pharmacological properties regarding clinical implications.

Systemic evaluation of gene expression changes in major target organs induced by atorvastatin.

Statins have been reported to protect against end-organ damage in essential hypertension; however, detailed mechanisms underlying organ-protective actions of statins remain unclear. Statins can exert pleiotropic effects aside from lowering cholesterol and blood pressure levels through several different pathways, which may lead to distinct patterns of changes in gene expression in vital end-organs. The aim of the present study was to systemically evaluate gene expression changes in three major end-organs (the brain, heart and kidney) induced by atorvastatin at a dose that altered neither blood pressure nor plasma total cholesterol levels. The stroke-prone spontaneously hypertensive (SHRSP) rats, an established model of hypertension and end-organ damage, was treated with atorvastatin (15 mg/kg/day) for 4 weeks from 12 to 16 weeks of age. DNA microarray technology was used to identify gene expression changes in three end-organs. In the current experimental setting, 4 weeks of atorvastatin treatment lowered plasma levels of non-esterified fatty acid significantly (P=0.0012) and triglyceride modestly (P=0.07) without altering blood pressure and plasma total cholesterol levels in male SHRSP rats. The level of expression of a number of genes was changed in an organ-specific manner after 4 weeks of drug administration to SHRSP rats. Among the end-organs studied, the most prominent alteration in gene expression was observed in the heart. The identical treatment protocol was applied to age-matched normotensive control rats, Wistar Kyoto rats, and this also caused a number of genes to be differentially expressed in an organ-specific manner. These results provide new insights into the mechanisms underlying the potential efficacy of statins in protecting against end-organ damage in essential hypertension and thus lay the foundation for future studies.

Effect of total cholesterol, glucose and blood urea nitrogen on embryo quality in post-partum superovulated suckling Japanese Black cattle.

Aim: This study was conducted to examine the effect of blood metabolites on embryo quality in post-partum suckling Japanese Black cattle. Methods: Blood samples were taken from 23 cows 30 days before, at and 30 days after parturition. Cows were synchronized 40 or 41 days after calving (day 0) and divided into three groups: control (n = 6), gonadotropin-releasing hormone ([GnRH]n = 10) and estradiol benzoate ([EB]n = 7). All groups received a controlled internal drug release (CIDR) device intravaginally together with 2 mg EB i.m. on day 0 and superovulation was induced in all groups from days 5-7 with a gradually decreasing dose of follicle-stimulating hormone (FSH). Two milligrams of EB was given on day 8 and GnRH (0.1 mg) was given on day 9 of insertion of the CIDR in the EB and GnRH groups. Cows were inseminated twice after the onset of estrus and embryos were recovered 7-8 days after artificial insemination. Results: The number of corpus luteum detected by ultrasonography in the EB group was significantly higher (P < 0.05) than that in the GnRH group. The number and rate of transferable and freezable embryos did not differ significantly among the groups. Regardless of the treatments, the total cholesterol level from parturition until 30 days after parturition was significantly higher (P < 0.01) in the good category than in the poor category of cows. Conclusions: The number of transferable embryos produced by post-partum superovulated suckling Japanese Black cattle was affected by the level of total cholesterol from parturition until 30 days after parturition. Moreover, administration of EB in CIDR-treated cows increased the numbers of corpus luteum and yielded better rates of transferable and freezable embryos. (Reprod Med Biol 2008; 7: 55-62).

Substrate selectivity of monoamine oxidase A, monoamine oxidase B, diamine oxidase, and semicarbazide-sensitive amine oxidase in COS-1 expression systems.

The substrate selectivity of monoamine oxidase A (MAO-A), monoamine oxidase B (MAO-B), diamine oxidase (DAO), and semicarbazide-sensitive amine oxidase (SSAO) was investigated in the absence of chemical inhibitors using the COS-1 cells expressed with respective amine oxidase. Serotonin (5-hydroxytryptamine), 1-methylhistamine, and histamine were preferentially oxidized by MAO-A, SSAO, and DAO, respectively, at a low substrate concentration. In contrast, benzylamine, tyramine, and beta-phenylethylamine served as substrates for all of MAO-A, MAO-B, and SSAO. Each amine oxidase showed broad substrate selectivity at a high substrate concentration. The cross-inhibition was remarkable in MAO-A and MAO-B, especially in MAO-A, but not in SSAO and DAO. A study of the substrate selectivity of amine oxidases should include consideration of the effects of substrate concentration and specific chemical inhibitors.

Metabolism of nicotine in rat lung microvascular endothelial cells.

The aim of this study was to examine whether cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas-blood barrier, have the ability to metabolize nicotine. Nicotine was biotransformed to cotinine and nicotine N'-oxide by cytochrome 450 (CYP) and flavin-containing monooxyganase (FMO), respectively, in rat LMECs. The intrinsic clearance (Vmax1/Km1) for the cotinine formation was about 20 times as high as that for the trans-nicotine N'-oxide formation in the low-Km phase, indicating that oxidation by CYP was much higher than that by FMO. On the other hand, as shown in Eadie-Hofstee plots, the formation of cis-nicotine N'-oxide was monophasic, whereas the plot for the trans-nicotine N'-oxide formation was clearly biphasic. These results suggest that nicotine N'-oxide was stereoselectively metabolized to cis and trans forms. However, in the high-Km phase there was no significant difference in N'-oxidation between the cis and trans forms. Moreover, we suggest that CYP2C11 and CYP3A2 are key players in the metabolism to cotinine of nicotine in rat LMECs using the respective enzyme inhibitors (tranylcypromine and troleandomycine). On the other hand, methimazole (5 microM) caused 73 and 45% decreases in the formation of N'-oxides of cis- and trans- enantiomers, respectively, demonstrating the presence of FMO in rat LMECs. These results suggest that rat LMEC enzymes can convert substrates of exogenous origin such as nicotine for detoxication, indicating LMECs are an important barrier for metabolic products, besides hepatic cells.

Structural inhomogeneity in mammography quality control phantoms detected by refraction-enhanced synchrotron radiation imaging.

Synchrotron radiation imaging with the refraction-enhancement mode visualized structural inhomogeneities in phantoms used for image quality control of mammography. Eight phantoms were examined, all of which were manufactured in the United States and approved by the American College of Radiology as dedicated phantoms. In addition to fiber- and mass-mimicking test objects, each phantom has 5 groups of calcification specks of various sizes. Synchrotron radiation (SR) imaging was performed at Spring-8, a synchrotron radiation facility in Japan. Images were obtained with monochromatic 20-keV x-ray beams, a radiation field of 15 mm X 26 mm at a sample plane, a CCD camera with a resolution of 6 micrometers as a detector, and a sample-to-detector distance of 10 to 11 m. Two hundred and forty specks were evaluated in total in the SR images, and the surrounding area of each speck was also included. Evaluation of the images showed that 14 crack-like structures were depicted near specks, and there were 62 specks with attached void(s) or air bubble(s). Refraction-enhanced SR imaging sensitively detected structural inhomogeneities and abnormalities in phantoms which were implicitly agreed to have a homogeneous matrix and test objects without foreign substances. A possible manufacturing-dependent quality issue was identified. The effect of inhomogeneities detected by SR imaging on visual scoring of specks could not be identified in the tested phantoms; this should be assessed on images of other phantoms in a future study.

Molecular cloning and characterization of rat semicarbazide-sensitive amine oxidase.

Semicarbazide-sensitive amine oxidase (SSAO) (EC 1.4.3.6) is widely distributed in nature and catalyzes the oxidative deamination of primary amines. Although SSAO full-length cDNA sequences have been reported for some mammalian species, only a partial 5'-terminal sequence has been confirmed in the rat. In this study we isolated full-length SSAO cDNA from rat aorta and examined its mRNA expression in various rat tissues by real-time PCR, as well as the subcellular and tissue distributions of SSAO activity. The deduced amino acid sequence showed 91% and 80% identity with mouse and human SSAO, respectively. The mRNA was expressed in many rat tissues. Those findings were supported by the broad distribution of SSAO in the body. Thus, a high level of SSAO was shown in adipocytes by both mRNA expression and enzyme activity measurement. The results suggest that SSAO may play an important role in the degradation of biologically active amines in adipocytes.

Stereoselective transport of histidine in rat lung microvascular endothelial cells.

The transport characteristics of L- and D-histidine through the blood-lung barrier were studied in cultured rat lung microvascular endothelial cells (LMECs). L-Histidine uptake was a saturable process. The addition of metabolic inhibitors [2,4-dinitrophenol (DNP) and rotenone] reduced the uptake rate of L-histidine. Ouabain, an inhibitor of Na(+)-K(+)-ATPase, also reduced uptake of L-histidine. Moreover, the initial L-histidine uptake rate was reduced by the substitution of Na(+) with choline chloride and choline bicarbonate in the incubation buffer. The system N substrate, L-glutamic acid gamma-monohydroxamate, also inhibited uptake of L-histidine. However, system N-mediated transport was not pH sensitive. These results demonstrated that L-histidine is actively taken up by a system N transport mechanism into rat LMECs, with energy supplied by Na(+). Moreover, the Na(+)-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH), had an inhibitory effect on L-histidine uptake in Na(+) removal, indicating facilitated diffusion by a Na(+)-independent system L transport into the rat LMECs. These results provide evidence for there being at least two pathways for L-histidine uptake into rat LMECs, a Na(+)-dependent system N and Na(+)-independent system L process. On the other hand, the uptake of D-histidine into rat LMECs was not reduced by the addition of DNP, rotenone, or ouabain, or by Na(+) replacement. Although the uptake of D-histidine was reduced in the presence of BCH, the addition of L-glutamic acid gamma-monohydroxamate did not significantly decrease uptake of D-histidine. These results suggest that the uptake of D-histidine by rat LMECs has different characteristics compared with its isomer, L-histidine, indicating that system N transport did not involve D-histidine uptake.