Effects of dietary digestible amino acids and a novel exogenous sfericase protease on growth performance of broilers.

Two experiments (Exp.) were conducted to evaluate the effects of a novel exogenous sfericase protease on growth performance and ileal digestibility of broiler chickens until day 35 of age. In Exp. 1, 1350 one-day-old male chicks (Cobb 500) were allocated in 54 floor pens and fed one of the three dietary treatments, with 18 replicates of 25 birds each in a completely randomized design. Diets consisted of positive control [PC; commercially relevant ME and balanced amino acids (AA)]; negative control (NC; with reduction of 6% dig. Lys and proportional reductions for adjacent AA compared to the PC), and NC supplemented with sfericase protease [30,000 New Feed Protease units (NFP)/kg]. On day 35, ileal digesta was collected to determine apparent ileal digestibility of dry matter and nitrogen (N). In Exp. 2, 1620 one-day-old male chicks (Cobb 500) were allocated in 54 floor pens having three treatments and 18 replicates of 30 birds each in a completely randomized design from day 1-35. Broilers were fed a control basal diet (Control); Control supplemented with sfericase at 30,000 NFP/kg and at 60,000 NFP/kg. In Exp. 1, from day 1-35, body weight gain (BW gain) and feed conversion ratio (FCR) of broilers improved 3.4 and 2.5% when diets were supplemented with sfericase, respectively, whereas the digestibility of N increased by 2.7% compared to the NC. In Exp. 2, diets with usual protein and AA levels and supplemented with 30,000 NFP/kg had 2.3 and 1.75% improved BW gain and FCR from day 1-35, respectively. When diets were supplemented with 60,000 NFP/kg, BW gain and FCR were enhanced by 3.9 and 3.2%, respectively compared to the Control. In conclusion, these results demonstrate that the novel sfericase protease could be successfully used in corn-soy diets with protein and AA reductions or in feed formulations with usual digestible AA levels to enhance growth performance of broilers.

Benefits of Vitamin D Supplementation on Pregnancy of Rats with Pregestational Diabetes and Their Offspring.

Studies on vitamin D supplementation have been performed in experimental and clinical investigations considering gestational diabetes and/or vitamin D deficiency in pregnancy. However, the results are controversial and few present the effects and mechanisms of this micronutrient on pregestational diabetes. The objective of this study was to evaluate the effect of vitamin D on the pregnancy of rats with pre-existing diabetes and their fetuses. Pregestational diabetes was induced in Sprague-Dawley rats at birth. The adult diabetic and nondiabetic rats were orally administered with vitamin D (cholecalciferol) throughout the pregnancy. The diabetes status was monitored during pregnancy by an oral glucose tolerance test (OGTT). At the end of the pregnancy, pancreas and blood samples were collected for morphological analyses and lipid peroxidation measurements, respectively. The influence of vitamin D treatment on reproductive outcomes, fetal growth, and development were compared to those of untreated diabetic and nondiabetic pregnant rats. P < 0.05 was considered a significant statistical limit. The diabetic rats given vitamin D had a greater number of insulin-positive cells, contributing to reduced blood glucose levels and thiobarbituric acid reactive substance concentrations (TBARS-an indicator of membrane lipid peroxidation), and increased reduced thiol group levels, contributing to suitable intrauterine conditions for better fetal development, which was confirmed by higher fetal viability rates. Thus, this study shows the effects and mechanisms of vitamin D supplementation on pre-existing diabetes in pregnant rats, confirming its beneficial effects on maternal redox status and glycemic control, and the decline of adverse maternal-fetal repercussions.

Genetic Variants of VEGFA and FLT4 Are Determinants of Survival in Renal Cell Carcinoma Patients Treated with Sorafenib.

Molecular markers of sorafenib efficacy in patients with metastatic renal cell carcinoma (mRCC) are not available. The purpose of this study was to discover genetic markers of survival in patients with mRCC treated with sorafenib. Germline variants from 56 genes were genotyped in 295 patients with mRCC. Variant-overall survival (OS) associations were tested in multivariate regression models. Mechanistic studies were conducted to validate clinical associations. VEGFA rs1885657, ITGAV rs3816375, and WWOX rs8047917 (sorafenib arm), and FLT4 rs307826 and VEGFA rs3024987 (sorafenib and placebo arms combined) were associated with shorter OS. FLT4 rs307826 increased VEGFR-3 phosphorylation, membrane trafficking, and receptor activation. VEGFA rs1885657 and rs58159269 increased transcriptional activity of the constructs containing these variants in endothelial and RCC cell lines, and VEGFA rs58159269 increased endothelial cell proliferation and tube formation. FLT4 rs307826 and VEGFA rs58159269 led to reduced sorafenib cytotoxicity. Genetic variation in VEGFA and FLT4 could affect survival in sorafenib-treated patients with mRCC. These markers should be examined in additional malignancies treated with sorafenib and in other angiogenesis inhibitors used in mRCC. SIGNIFICANCE: Clinical and mechanistic data identify germline genetic variants in VEGFA and FLT4 as markers of survival in patients with metastatic renal cell carcinoma.

Identification of selected therapeutic agents as inhibitors of carboxylesterase 1: potential sources of metabolic drug interactions.

A series of studies were designed and carried out in order to explore the potential for the major human hepatic hydrolase, carboxylesterase 1 (hCES1), to serve as a target of metabolic inhibition by a variety of medications. The risk of adverse drug-drug interaction(s) is present when metabolic inhibitors are combined with known or suspected substrates of a given enzyme. In the present report the abundantly expressed hepatic enzyme, hCES1, was examined as a potential target of metabolic inhibition by a number of routinely prescribed medications. hCES1 has been seldom assessed in this regard despite its role in the metabolism and detoxification of many compounds. The psychostimulant methylphenidate (MPH) was chosen as an hCES1 selective substrate. In vitro studies were performed using previously developed cell lines which overexpress hCES1 with both p-nitrophenyl acetate and d-MPH serving as known substrates. Aripiprazole, perphenazine, thioridazine, and fluoxetine were determined to be the potent hCES1 inhibitors. A complementary animal study followed in vitro screening studies to further evaluate the inhibitory effect of aripiprazole on CES1 activity in FVB mice. The results suggest that the concurrent administration of racemic (i.e. dl-) MPH with aripiprazole significantly increased the plasma concentrations of both total MPH as well as the less active l-isomer. The ratio of d-MPH and l-MPH plasma concentrations was significantly decreased in the mice treated with aripiprazole compared to the control animals, indicating an overall decrease of CES1 catalytic activity in aripiprazole treated animals. Additionally, a quantitative structure-activity relationship based analysis identified a number of structural similarities of CES1 inhibitors. In conclusion, drug-drug interactions with MPH are likely mediated via CES1 inhibition as a result of concomitant drug therapies. CES1 inhibition represents an overlooked and little studied source of variability in MPH disposition, tolerability, and response.

Discovery of geranylgeranyltransferase-I inhibitors with novel scaffolds by the means of quantitative structure-activity relationship modeling, virtual screening, and experimental validation.

Geranylgeranylation is critical to the function of several proteins including Rho, Rap1, Rac, Cdc42, and G-protein gamma subunits. Geranylgeranyltransferase type I (GGTase-I) inhibitors (GGTIs) have therapeutic potential to treat inflammation, multiple sclerosis, atherosclerosis, and many other diseases. Following our standard workflow, we have developed and rigorously validated quantitative structure-activity relationship (QSAR) models for 48 GGTIs using variable selection k nearest neighbor (kNN), automated lazy learning (ALL), and partial least squares (PLS) methods. The QSAR models were employed for virtual screening of 9.5 million commercially available chemicals, yielding 47 diverse computational hits. Seven of these compounds with novel scaffolds and high predicted GGTase-I inhibitory activities were tested in vitro, and all were found to be bona fide and selective micromolar inhibitors. Notably, these novel hits could not be identified using traditional similarity search. These data demonstrate that rigorously developed QSAR models can serve as reliable virtual screening tools, leading to the discovery of structurally novel bioactive compounds.

Increases of calcium and magnesium and decreases of phosphorus and iron with aging in human uterine tubes.

To elucidate compositional changes of the uterine tube by aging, the authors studied age-related changes of elements in human uterine tubes by inductively coupled plasma-atomic emission spectrometry. The uterine tubes were resected postmortem or surgically removed from patients with uterine myoma. It was found that the contents of calcium and magnesium increased progressively with aging in uterine tubes, whereas the contents of phosphorus and iron decreased gradually with aging. The sulfur content of uterine tubes remained constant and independent of aging. Regarding relationships between elements, significant relationships were found between calcium and magnesium contents, between phosphorus and iron contents, between phosphorus and sulfur contents, and between phosphorus and sodium contents in human uterine tubes.

Age-dependent changes of elements in human trachea.

To elucidate compositional changes of human trachea by aging, element contents in tracheae were determined by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. The sulfur content of the tracheae decreased gradually with aging. In regard to calcium and phosphorus, both the contents increased to about threefold amounts in their seventies compared with those in their sixties, and decreased thereafter. The contents of calcium and phosphorus were the highest in their seventies. Therefore, it is likely that surplus calcium released from bones is deposited temporally in the trachea, and the deposits are released from the trachea at older age. Based on our results of human cartilages, there are two types in regard to calcium accumulation: The first type is that calcium accumulation occurs progressively with aging; the second one is that calcium accumulation becomes the highest in the seventies or eighties, and decreases thereafter. Therefore, the trachea belongs to the second type. Furthermore, the magnesium content remained constant through the age range.

Age-dependent decreases of phosphorus and magnesium in human Achilles' tendons.

To elucidate changes of human tendons with aging, the authors studied age-related changes of elements in human Achilles' tendons by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. It was found that the content of calcium increased progressively with aging in the Achilles' tendons, whereas the contents of phosphorus and magnesium decreased gradually with aging. The previous investigations demonstrated that the content of calcium and phosphorus increased progressively with aging in most, but not all, human tissues, except for the bones. In ligaments, such as the anterior cruciate ligament and the ligament of the head of the femur, which are histologically similar to the Achilles' tendon, it was previously found that both the contents of calcium and phosphorus increased with aging in the ligaments. It should be noted that the content of phosphorus in the Achilles' tendons decreased during the aging process. In addition, it was found that there was a very high direct correlation between phosphorus and magnesium contents in the tendons, but not between calcium and phosphorus contents.

Postnatal development of NADPH-diaphorase activity in the rat: the role of nitric oxide in the ontogeny of arginine vasopression and oxytocin.

To clarify the role of nitric oxide (NO) in the ontogeny of arginine vasopressin (AVP) and oxytocin (OXT) neurons in the hypothalamo-neurohypophysial system (HNS), we observed the coexpression pattern of NADPH-diaphorase (NADPH-d) activity and AVP- or OXT-immunoreactivity (IR) in the rat hypothalamus and posterior pituitary during the postnatal period. The enzymatic activity of NADPH-d was observed in the supraoptic nucleus (SON), paraventricular nucleus (PVN), median eminence (ME) and posterior pituitary throughout the postnatal development. AVP-containing neurons were clearly observed from postnatal day 1 in both the SON and PVN, while OXT-containing neurons were recognized from postnatal day 14. The coexistence of NADPH-d and AVP or OXT was detected in the SON from postnatal day 14. At postnatal day 21, the coexpression pattern was approximately the same as that of the SON and PVN in adult rats. Our findings indicated that the expression of NADPH-d and OXT was observed from almost the same postnatal period in both the SON and PVN. In addition, the pattern of increased numbers of NADPH-d positive fibers was similar to that of OXT-immunoreactive fibers in both the inner layer of the ME and the posterior pituitary. A good correlation was thus obtained between OXT expression and NADPH-d activity in the HNS during postnatal development. The present study suggests that NO is more closely involved in the expression and regulation of secretion of OXT than AVP.

cDNA sequence, mRNA expression, and chromosomal localization of human carbonic anhydrase-related protein, CA-RP XI.

A full-length cDNA clone of a human carbonic anhydrase-related protein, CA-RP XI encoded by CA11, was obtained and sequenced. The cDNA sequence was 1475 bp long and predicted to encode a 328-amino acid polypeptide with a molecular mass of 36200 Da. The deduced amino acid sequence of CA-RP XI showed an overall similarity of 42-53% to the active site residues of other active CA isozymes; however, it lacked three zinc-binding histidine residues, raising questions regarding its CA catalytic activity. Northern blot analysis demonstrated strong expression of an approx. 1.5 kb transcript in the human brain, particularly in the cerebellum, cerebral cortex, and putamen. A single copy of the CA11 gene was localized to the human chromosome 19q13.2-3. These results suggest that CA-RP XI plays a general role in the human central nervous system.

The effects of oestrogen and progesterone on serotonin and its metabolite in the lateral septum, medial preoptic area and ventromedial hypothalamic nucleus of female rats.

The effects of sex steroid hormones on serotonin and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA) in the lateral septal nucleus (LS), the medial preoptic area (MPA) and the ventromedial nucleus of the hypothalamus (VMH) of female rats were investigated, using immunohistochemistry and high-performance liquid chromatography (HPLC). Female rats were divided into three groups: ovariectomized rats (OVX group); OVX-rats treated with estradiol benzoate alone (E2 group); and OVX-rats treated with E2 plus progesterone (E2 + P group). We analysed the density of serotonin-immunoreactive fibres with a computer-assisted image analysis system, and measured the tissue concentrations of serotonin and 5-HIAA. Many serotonin-immunoreactive fibres were observed in the LS, MPA and VMH in all three groups. The density of serotonin-immunoreactive fibres in the MPA and VMH was significantly lower in the E2 and E2+P groups compared to the OVX group, whereas the LS showed no detectable differences among the three groups. In the HPLC study, the concentrations of serotonin in the MPA and VMH of the E2 and E2+P groups were significantly lower than that in the OVX group. There was no significant difference in the concentration of serotonin in the LS. The concentration of 5-HIAA and the ratio of 5-HIAA/serotonin in the LS, MPA and VMH showed no significant differences among the OVX, E2 and E2+P groups. The present results suggest that E2 priming for sexual behaviour can affect the serotonergic system by decreasing serotonin content, but not the turnover rate, in the MPA and VMH of female rats.

The perinatal ontogeny of estrogen receptor-immunoreactivity in the developing male and female rat hypothalamus.

Developmental expression of the estrogen receptor (ER) in rat hypothalamus was examined using immunohistochemistry. In the medial preoptic nucleus and ventromedial nucleus ER-immunoreactivity was detected as early as E17, whereas ER protein expression in the periventricular preoptic nucleus and arcuate nucleus was delayed until E19. These results show that following a region specific onset of the ER protein expression sex differences in ER levels are already detectable during the perinatal period.

Region-specific changes of tyrosine hydroxylase-immunoreactivity by estrogen treatment in female rat hypothalamus.

The effect of 17 beta-estradiol (E2) treatment for 28 days on tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the periventricular preoptic nucleus (PPN) and medial preoptic area (MPA) of ovariectomized (OVX) rats was examined by morphometric analysis. The number of TH-IR neurons in the PPN of the E2-treated group was smaller than that of the OVX group, whereas the opposite result was found in the MPA; the number of TH-IR neurons in the MPA of the E2-treated group was larger than that of the OVX group. Numerous TH-IR neurons were found in the ventromedial portion of the MPA of the E2-treated group. In both the OVX and E2-treated groups, TH-IR neurons contained many short processes up to 40 microns in length. E2 treatment caused a significant decrement of the number of neurons containing the processes in the range of 10-40 microns length in the PPN, however it caused a significant increment of the number of neurons containing the processes in the range of 5-10 microns length in the MPA. These results suggested that immunoreactivity of TH in the PPN and MPA neuron are affected by E2 treatment and that E2 might modulate the production of TH in a region-specific pattern within the hypothalamus of the female rat.

Vasopressin- and oxytocin-immunoreactive hypothalamic neurones of inbred polydipsic mice.

In the late 1950s the inbred polydipsic mice, STR/N, was discovered. The early studies indicated that the extreme polydipsia was not due to a lack of vasopressin but probably due to innate thirst of unknown origin. Because the recent investigation has revealed the presence of some functional abnormality in the brain of the STR/N mouse, we now investigated, using immunohistochemical techniques, distribution of vasopressin (AVP)- and oxytocin (OXT)-containing neurones in the hypothalamus of polydipsic strain of mouse and compared with that of the control. The pattern of distribution of AVP- and OXT-immunoreactive neurones in the paraventricular (PV), supraoptic (SO), and suprachiasmatic nuclei (SCN) of the STR/N polydipsic mouse was similar to that of the control, but the number of AVP-immunoreactive neurones was more numerous in the PVN and SON and less in the SCN in the polydipsic mouse than in the control. In addition, a discrete group of AVP- and OXT-containing neurones that was not clearly seen in the control was discovered in the STR/N. These results implicate that abnormal distribution in the brain AVP and OXT contribute to the mechanism responsible for the polydipsia shown by the strain STR/N.

[A clinical study on spontaneous passage of ureteral stone--effect of urocalun and jumping exercise to ureteral stone].

The effects of Urocalun and jumping exercise upon the passage of calculi were studied. Urocalun was administered to 47 patients with ureteral stones in a dosage of 6 capsules per day and they did jumping rope skipping 50 times twice a day. The size of the calculi was grouped according to the report of Minami et al. Namely the 47 cases were divided into the following groups according to their radiographic shadow; 1) small group (not greater than 0.5 cm in diameter), 27 cases (57.4%); 2) middle-sized group (not greater than 1.0 x 0.6 cm), 11 cases (23.4%); 3) large group (larger than 1.0 x 0.6 cm), 9 cases (19.2%). As a result, the rate of spontaneous passage was 80.9% which was considerably higher than expected. There was spontaneous passage of calculus in 25 cases of small group (85.2%), in 8 cases of middle-sized group (72.7%) and in 7 cases of large group (77.8%). Especially in the large group the rate of passage was higher than described before. On the other hand the period of calculi passage was earlier than that in Minami's report. In the large group 6 of the 7 discharged stones (85.7%) were discharged within six months. Therefore, it must be observed for six months regardless of calculus size with combination therapy if possible. The effects of this prescription on the calculi passage were better than those described on other drugs and Urocalun independently. Then the strict combination therapy of the drug and the exercise was useful for the passage of calculi.

Immunohistochemical studies on the distribution of endogenous digitalis-like substance (EDLS)-containing neurons in the rat hypothalamus, with special consideration on the possibility of their coexistence with posterior lobe hormones.

The distribution of the perikarya of endogenous digitalis-like substance (EDLS)-containing neurons in the rat hypothalamus was studied by immunohistochemistry using digoxin and digitoxin antiserum. In addition, the possible coexistence of EDLS and posterior lobe hormones was examined using an immunohistochemical double-staining technique. Digoxin-like immunoreactive neurons were demonstrated in the paraventricular and supraoptic nuclei and other hypothalamic areas. However, digitoxin-like immunoreactive neurons were not detected in the hypothalamus. A portion of the digoxin-like immunoreactive neurons showed immunoreactivity for vasopressin or oxytocin. From these results, it is suggested that these digoxin-like immunoreactive neurons correspond to "EDLS-producing neurons," and that EDLS seems to act not only as a natriuretic substance but also as a neurohormone or neurotransmitter.