Supplementation with beta-1,3-glucan improves productivity, immunity and antioxidative status in transition Holstein cows.

Dairy cows undergo dramatic physiological changes during the transition from late pregnancy to early lactation, which make them vulnerable to metabolic stress and immune dysfunction. The objective of this study was to evaluate the effects of a commercial beta-1,3-glucan product (Aleta™, containing 50% beta-1,3-glucan) on productivity, immunity and antioxidative status in transition cows. Fifty-four multiparous Holstein cows received a control diet or a diet supplemented with 5 or 10 g of beta-1,3-glucan per cow per day from 21 days before expected calving to 21 days after parturition. Blood samples were collected at day -21, 1, and 21 relative to calving. Colostrum and milk were collected at day 1 and 21 after calving, respectively. Data showed that supplementation with beta-1,3-glucan had no effect on milk composition, but increased milk production. Beta-1,3-glucan treatment also improved the milk quality, as shown by reduced milk somatic cell count and increased immunoglobulin levels in colostrum. Notably, beta-1,3-glucan markedly reduced serum levels of pro-inflammatory cytokines and C-reactive protein, while elevated serum immunoglobulin levels, indicating its immunity enhancement in transition cows. Moreover, beta-1,3-glucan addition reduced the serum malondialdehyde level and enhanced the activities of serum superoxide dismutase and catalase, which enhanced the antioxidative capacity in transition cows. In summary, supplementation with beta-1,3-glucan improves productivity, immunity and antioxidative status in transition dairy cows.

Effects of beta-1,3-glucan supplementation on concentrations of serum metabolites in transition Holstein cows.

Objectives of this study were to evaluate the alleviating effects of a commercial beta-1,3-glucan product (Aleta, containing 50% beta-1,3-glucan, Kemin Industries) on metabolic stress in transition Holstein cows as reflected by circulating metabolites and enzymes. Fifty-four multiparous Holstein cows were randomly allocated to three groups with 18 cows each. Cows in each group received a commercial basal diet or the basal diet supplemented with Aleta calculated to supply 5 or 10 g of Aleta per cow per day. Blood samples were collected at day -21, 1, and 21 relative to calving for determination of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDLC), very low density lipoprotein (VLDL), glucose, insulin, ß-hydroxybutyric acid (BHBA), aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyl transpeptidase (GGT), and non-esterified fatty acid (NEFA). Supplementation with Aleta markedly elevated serum concentrations of TG, TC, HDLC, LDL-C and VLDL, implying its positive effect on lipid metabolism in transition dairy cows. Aleta treatment significantly decreased the serum concentrations of NEFA and BHBA, but markedly elevated the serum concentrations of glucose and insulin. Also, Aleta treatment significantly elevated the dry matter intake and milk production in postpartum cows, indicating the alleviating effect of Aleta on negative energy balance in transition cows. Moreover, Aleta treatment significantly reduced the serum activities of AST, ALT and GGT, indicating its hepatoprotective effect on transition cows. These results suggest that Aleta supplementation may help to improve fat metabolism disorder initiated by negative energy balance in transition dairy cows.

[Database Establishing and Data Mining of Pulmonary Diseases Based on Clinical Works by Modern Famous Veteran Doctors of Chinese Medicine].

OBJECTIVE: To explore syndrome and treatment laws for treating diseases of the pulmonary system by establishing database based on clinical works by modern famous veteran doctors of Chinese medicine (CM). METHODS: Clinical experience and literature of medical records in clinical works by modern famous veteran doctors of CM were taken as data source. Database was established by fields and program design. On these bases, data mining methods such as frequency analysis, cluster analysis, factor analysis, and correlation laws were performed in syndrome and treatment laws for treating diseases of the pulmonary system. RESULTS: Established were database capable of literature searching, information statistics, data mining of modern famous veteran doctors of CM. A total of 34,414 data were input, including medical records and notes 28,045 items (81.49%) and clinical experience 6,369 items (18.51%). In medical records and notes, there were 14,048 items (50.09%) in male and 9,466 items (33.75%) in female, and the ratio of male to female was 1.48:1. There were 4,531 items (16.16%) with no marked gender in medical records or notes. Data mining such as correlation analysis, cluster analysis, factor analysis, correlation laws in more fields could be realized. CONCLUSIONS: Medical records and notes were dominated in data collected in this paper. The prevalence of pulmonary diseases was obviously higher in males than in females. The trend of concentrated manifestations in related fields for pulmonary diseases could be surfed by this database. Diagnosis and treatment laws for treating diseases of the pulmonary system could be found by various adaptive data mining targeting different fields. Multi-variables of symptoms, syndromes, prescriptions, and herbal drugs could be data mined in large samples of clinical literatures.

Expression of proteasome subunits low molecular mass polypeptide (LMP) 2 and LMP7 in the endometrium and placenta of rhesus monkey (Macaca mulatta) during early pregnancy.

Previous studies have demonstrated that the ubiquitin-proteasome pathway plays an important role in embryo implantation. Low molecular mass polypeptide (LMP) 2 and LMP7 are the two subunits of 20S proteasome, which are critical for proteasome activity. To further elucidate the roles of LMP2 and LMP7 in embryo implantation during early pregnancy, we cloned partial sequences of the LMP2 and LMP7 genes and studied the spatiotemporal expression of LMP2 and LMP7 in rhesus monkey (Macaca mulatta) uteri on Days 12, 18, and 26 of pregnancy. The results showed that the 349-base pair (bp) LMP2 fragment and the 340-bp LMP7 fragment were 97% and 99% identical, respectively, to those of human homologues. From the statistical results of reverse transcription-polymerase chain reaction, in situ hybridization, and immunohistochemistry, we found that the expression levels of LMP2 and LMP7 significantly increased with the elongation of pregnancy. The LMP2 and LMP7 mRNAs were mainly expressed in the luminal and glandular epithelium on Day 12 of pregnancy. On Days 18 and 26 of pregnancy, strong signals of LMP2 and LMP7 mRNAs were detected in the placental villi, trophoblastic column, and arterial endothelial cells close to the implantation site, and moderate expressions were found in the trophoblastic shell and glandular epithelium. The LMP2 and LMP7 mRNAs were extensively distributed in the stroma on Day 26 of pregnancy. The expression patterns of LMP2 and LMP7 proteins were similar to those of their transcripts, but weak immunostaining of LMP2 and LMP7 proteins was detected in stroma at all stages of pregnancy. These results suggest that LMP2 and LMP7 may be involved in some key processes of trophoblastic invasion, angiogenesis, degradation of the extracellular matrix, immune tolerance, and glandular secretion.

Involvement of ERK1/2 pathway in TGF-beta1-induced VEGF secretion in normal human cytotrophoblast cells.

Transforming growth factor-beta1 (TGF-beta1) plays a pivotal role in the angiogenesis during the development of placenta, but the intracellular signaling mechanism by which TGF-beta1 stimulates this process remains poorly understood. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to TGF-beta1 stimulated the secretion of the VEGF gene encoding vascular endothelial growth factor, which is a key factor in angiogenesis. Meanwhile, treatment of normal human cytotrophoblast cells with TGF-beta1-induced expression of HIF-1a, the regulated subunit of hypoxia-inducible factor 1, a known transactivator of the VEGF gene. Our data indicated that TGF-beta1 induced extracellular signal- regulated kinase (ERK) 1/2 phosphorylation in normal human cytotrophoblast cells. Moreover, treating cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited TGF-beta1 stimulation of VEGF secretion and HIF-1a protein expression. These data indicated that in normal human cytotrophoblast cells, TGF-beta1 induced HIF-1a-mediated VEGF secretion, and TGF-beta1-stimulated-ERK1/2 activation may be involved in this process.

Expression of Smad2 and Smad4, transforming growth factor-beta signal transducers in rat endometrium during the estrous cycle, pre-, and peri-implantation.

SMADs are intracellular signaling molecules that transmit signals elicited by members of transforming growth factor-beta (TGF-beta) superfamily. To decipher the mechanism of TGF-beta signaling during the estrous cycle and implantation, we performed in situ hybridization to investigate the expression patterns of mRNAs for Smad2 and Smad4 in rat endometrium during the estrous cycle and on Days 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, and 6.5 of pregnancy. Intense epithelial expression of Smad2 mRNA at diestrus and proestrus was reduced at estrus and metaestrus, while Smad4 maintained its constitutive expression during the estrous cycle. During pre-implantation, both Smads were accumulated in the luminal epithelium and the glandular epithelium. Contrary to the dramatic Smad4 expression, Smad2 was highly down-regulated on Day 2.5 and was increased on Day 3.5. During peri-implantation, both Smads were expressed in the luminal epithelium, subepithelial stroma, and the primary decidual zone. Smad4 was down-modulated on Day 5.5. These results suggest that (a) both Smads are involved in the tissue remodeling of cycling and pregnant rat uteri; (b) TGF-beta signaling functions mainly in the epithelium during pre-implantation and Smad2 is involved in the endometrial switch from the neutral phase to the receptive phase; (c) TGF-beta signaling is down-regulated at the time when trophoblast invasion begins and both Smads are involved in the formation of the primary decidual zone.

Normoxic induction of the hypoxic-inducible factor-1 alpha by interleukin-1 beta involves the extracellular signal-regulated kinase 1/2 pathway in normal human cytotrophoblast cells.

During early pregnancy, an environment of relative low oxygen tension is essential for normal embryonic and placental vasculature. In low-oxygen conditions, the hypoxic-inducible factor-1 (HIF-1), composed of alpha and beta subunits, controls the expression of a number of genes such as vascular endothelial growth factor (VEGF), a key angiogenic factor. The recent studies in some tumor cells have found that the labile component, HIF-1 alpha, is not only activated by hypoxia but also by peptides such as interleukin-1 (IL-1) in normoxia. In this article, we demonstrated that exposure of normal human cytotrophoblast cells to IL-1 beta stimulated the expression of HIF-1 alpha protein. Meanwhile, IL-1 beta also induced the secretion of VEGF in normal human cytotrophoblast cells. Our data indicated that IL-1 beta induced extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. Moreover, treatment of cells with PD98059, an inhibitor of ERK1/2 signaling, inhibited the stimulation of HIF-1 alpha protein expression and VEGF secretion by IL-1 beta. These data indicate that, in normal human cytotrophoblast cells, IL-1 beta induces HIF- 1 alpha-mediated VEGF secretion and that IL-1 beta-stimulated ERK1/2 activation may be involved in this process.

Effect of ubiquitin-proteasome pathway on mouse blastocyst implantation and expression of matrix metalloproteinases-2 and -9.

Previous studies have documented that ubiquitin-related proteins are present in human, baboon, rhesus monkey, cow, sheep, and mouse pregnant uteri, indicating that the ubiquitin-proteasome pathway (UPP) may be involved in the extensive uterine remodeling during mammalian early pregnancy, but there is still no direct evidence. A mouse intrauterine injection model was employed to study the direct effect of the UPP on mouse embryo implantation and its possible mechanisms. On Day 3 of pregnancy in each mouse, one of the uterine horns in each mouse was injected with different concentrations of lactacystin, a specific proteasome inhibitor, or anti-ubiquitin antibody, and the other side was used as a control. On days 5, 6, and 7, the number of implanted embryos was counted and the expression and gelatinolytic activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 were studied. Results presented here illustrate that injection of lactacystin and anti-ubiquitin antibody significantly inhibited mouse embryo implantation. Further investigations by reverse transcription-polymerase chain reaction and gelatin zymography showed that MMP-2 and MMP-9 mRNA expression, as well as the gelatinolytic activity of MMP-9 in the lactacystin-treated uterine horn, significantly decreased, whereas the activity of MMP-2 was not significantly affected. The results obtained from this study, together with previous reports, suggest that the UPP is involved in mouse embryo implantation, and UPP's effect on embryo implantation is achieved at least in part by regulating MMP-2 and MMP-9 mRNA expression and the gelatinolytic activity of MMP-9.

Expression and functional analysis of liver receptor homologue 1 as a potential steroidogenic factor in rat ovary.

Liver receptor homologue 1 (LRH-1) is a member of the nuclear receptor superfamily originally found in liver cells. LRH-1 participates in regulation of cholesterol metabolism and bile acid synthesis. Recent studies have shown that LRH-1 is even more highly expressed in the ovary, and LRH-1 has been implicated as a key transcriptional regulator of cytochrome P450 aromatase (P450arom) in vitro. In the present study, we investigated the spatiotemporal expression patterns of LRH-1 using in situ hybridization and immunohistochemistry in ovaries from rats with a 4-day estrous cycle, from pregnant rats, from immature rats treated with eCG to stimulate follicular development, and from eCG-treated rats that were subsequently given hCG to stimulate ovulation and luteinization. To establish a potential connection between the expression of LRH-1 and that of the steroidogenic genes in vivo, we directly compared the localization patterns of LRH-1 and P450arom transcripts in consecutive ovarian sections from these animals. LRH-1 mRNA and protein were primarily localized to granulosa cells and luteinized follicles or newly formed corpora lutea (CLs) of immature and adult rats, and the levels of expression increased during eCG-hCG-induced follicular development and ovulation. In the functional CLs of pregnant rats, a biphasic change in LRH-1 mRNA content occurred throughout the gestation process, whereas LRH-1 protein was persistently detected during the entire pregnancy. In the consecutive ovarian sections, expression of LRH-1 was approximately colocalized with that of P450arom in both tertiary and Graafian follicles and the functional CLs of pregnant rats. LRH-1 mRNA and protein expression preceded those of P450arom during early follicular development. Stage-specific expression of LRH-1 in rat granulosa and luteal cells suggests a role for LRH-1 in the regulation of ovarian function. The overlapping but distinct expression patterns of LRH-1 and P450arom circumstantially support the recent finding that LRH-1 serves as a critical upstream regulator of P450arom gene expression in ovarian cells, but LRH-1 also may be a multifunctional steroidogenic factor in ovarian physiology.

Matrix metalloproteinase-28 transcript and protein are expressed in rhesus monkey placenta during early pregnancy.

The role of matrix metalloproteinases (MMP), especially newly described MMP, in trophoblast invasion during human embryo implantation is poorly understood. In this report, using a model of early pregnancy in the rhesus monkey, we have examined the expression and localization of the most recently identified MMP, MMP-28/epilysin, transcript and protein in macaque uterine samples on days 12, 18 and 26 of pregnancy. MMP-28 mRNA expression was shown by in-situ hybridization after day 12 of pregnancy, and both the syncytial and the cytotrophoblastic cell layers of placental villi, the cytotrophoblast cells of the trophoblastic column, and the extravillous trophoblast cells of trophoblastic shell were primary producers of MMP-28 transcript. Expression of MMP-28 mRNA was undetectable in the endovascular trophoblast cells, decidual cells, luminal and glandular epithelium, arterioles, and myometrium. RT-PCR analysis amplified a fragment of 258 nucleotides from rhesus monkey uterine samples containing implantation sites on days 18 and 26. The cDNA fragment, following sequencing, was confirmed to be part of the haemopexin-like domain of MMP-28. It has 95% identity with the corresponding region of human MMP-28 gene. Immunohistochemical analysis further demonstrated that the localization of MMP-28 protein was similar to that of its mRNA. The restricted distribution pattern of this novel MMP in the villous and extravillous trophoblasts during rhesus monkey early pregnancy suggests a potential role in trophoblast invasion associated with embryo implantation.

Expression and localization of RAP250 mRNA in rat ovary: possible implications in follicular development and ovulation.

The expression levels of nuclear receptor coregulators in specific tissue compartments and cells are thought to influence the expression of hormone-responsive genes involved in metabolism, development, and reproduction. RAP250 is a novel nuclear receptor coactivator highly expressed in brain and reproductive organs. To investigate the possible involvement of RAP250 in tissue-specific regulation of ovarian function, untreated immature, pregnant mare's serum gonadotropin luteinizing hormone (PMSG-LH)-primed, cycling, and pregnant rat models were used to study the localization and expression of RAP250 mRNA in rat ovary by in situ hybridization (ISH) and reverse transcriptase polymerase chain reaction (RT-PCR). The results showed that RAP250 mRNA was primarily localized to granulosa cells of healthy follicles in immature, cycling, and pregnant rats and increased during PMSG-induced follicular development. In the preovulatory and ovulatory follicles from the LH-primed rats of 48-h post-PMSG administration, the signals for RAP250 mRNA increased further and remained high until early luteal formation. Only a subset of corpora lutea during diestrus 1, diestrus 2, and initiation of pregnancy was weakly positive, and atretic follicles were largely negative. The RT-PCR results confirmed the presence of RAP250 mRNA in the rat ovary and strengthen the data from ISH. These findings suggest that RAP250 may play potential roles in follicular development and ovulation.

Expression of gelatinases and their tissue inhibitors in rat corpus luteum during pregnancy and postpartum.

Extensive tissue remodeling occurs in the corpus luteum (CL) during both formation and luteolysis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play pivotal roles in these processes. In the present study, to evaluate the potential roles of matrix degrading proteases in luteal development and regression, we examined gelatinases and TIMP-1, -2, -3 mRNA expressions, as well as gelatinase activity in rat CL during pregnancy and postpartum using Northern blot, in situ hybridization, and gelatin zymography, respectively. The results showed that MMP-2 mRNA was only expressed at the early stages of pregnancy; TIMP-2 mRNA was highly expressed at the early and late pregnancy and day 1 postpartum, but could not be detected during the mid-phase of pregnancy; TIMP-3 mRNA expression was abundant during early pregnancy and peaked at day 7, but was absent from other time points examined. MMP-9 and TIMP-1 mRNAs in rat CL were below detectable level in the current study. Furthermore, the active MMP-2 was only present during the early stages of pregnancy, and no MMP-9 activity was observed in the zymogram. Taken together, our results suggest that MMP-2 and TIMP-3 may have functional roles in rat luteal formation, while TIMP-2 may be implicated in both formation and regression of the pregnant CL.