Expression of bovine trypsin in Lactococcus lactis

Bovine trypsin is widely used in the production of bioactive peptides from milk proteins. The objective of this study was to express bovine trypsin in Lactococcus lactis to produce peptides by fermentation. The bovine trypsin with bias codon of L. lactis was synthesized, cloned into a secretory expressive vector pSEC-E7, and then introduced to L. lactis strain NZ9000 by electroporation. The expression of trypsin induced by nisin was identified by Western blot. Western blot analysis revealed a band in the protoplast fraction corresponding to SP_Usp45 (signal peptide of usp45)-trypsin. Biological activity of expressed trypsin was confirmed by the single substrate overlay assay. These results suggest that the expression of biologically active bovine trypsin has been detected in the protoplast fraction of L. lactis strain NZ9000 (pSEC:trypsin).     

牛胎盘提取物促进淋巴细胞增殖活性的研究

探讨牛胎盘中提高机体免疫力活性成分的分离纯化及其活性验证。分别采用离子交换层析色谱、凝胶层析色谱和反相层析色谱对活性成分进行分离纯化：采用体外检测脾脏淋巴细胞增殖活性，筛选活性成分；采用反相层析图谱和毛细管电泳验证活性成分达到一定纯度。     

Differential allele-specific accumulation of bovine kappa-casein mRNA throughout lactation

A differential allele-specific accumulation of kappa-casein mRNA that is not linked to the kappa-casein protein variants is described in Holstein cows. Actually, cows genotyped kappa-casein AB were a mixed population. For the first group of kappa-casein AB cows, allele A-specific kappa-casein mRNA contents within mammary epithelial cells were lower than the allele B-specific ones (cows LH), suggesting that the allele A-specific kappa-casein gene was expressed with lower efficiency in mRNA. For the other group of kappa-casein AB cows, allele A- and B-specific kappa-casein mRNA accumulated to a similar level within mammary epithelial cells (cows HH). The objective of this study was to determine whether the accumulation of allele-specific kappa-casein mRNA remained constant throughout lactation for the two groups of cows. Quantitative RT-PCR was used to monitor Holstein cows kappa-casein AB genotyped HH and LH throughout lactation for the proportion of allele B-specific mRNA accumulation relative to the total kappa-casein encoded mRNA within mammary epithelial cells: RNA was extracted from milk somatic cells known to contain a small proportion of mammary epithelial cells. Mean values of allele B-specific mRNA content were 50.6+/-0.5 and 54.0+/-0.9%, for cows HH and cows LH, respectively, and did not vary during lactation (P> 0.10). This suggests that the phenotypic expression of the genetic mutation that causes the differential allele-specific accumulation of kappa-casein mRNA was not affected by physiological and environmental factors, which tend to vary considerably throughout lactation.     

Occurrence of selenoprotein enzyme activities and mRNA in bovine mammary tissue

To elucidate the possible role of selenoproteins for milk formation and mammary gland physiology, the activities of selenoprotein enzymes and the expression of selenoprotein genes were studied in the bovine mammary gland. Messenger RNA was demonstrated for selenoprotein P, thioredoxin reductase 1, and for glutathione peroxidase (GPx) 1, 3, and 4. Significant differences in mRNA expression between the cows were seen for GPx 1 and GPx 3. The enzyme activity of glutathione peroxidase varied approximately 16-fold among cows, and the activity of thioredoxin reductase and the concentration of soluble Se varied approximately 6-fold among cows. There were positive correlations between glutathione peroxidase activity, thioredoxin reductase activity, and soluble Se, the correlation between glutathione peroxidase activity and soluble Se being the strongest. Furthermore, selenoprotein P expression correlated with GPx 1 mRNA expression and with soluble Se. There was also a correlation between glutathione peroxidase activity and the mRNA expression of GPx 1. The general conclusion from the data was that the activity of glutathione peroxidase and thioredoxin reductase and the mRNA expression of selenoprotein P and GPx 1 and 3 were influenced by Se status, but the expression of GPx 4 and thioredoxin reductase 1 were not. These results indicate that the Se status in mammary tissue is an important regulator of selenoprotein activity and expression, but that other factors are also in operation.     

Rumen-protected B vitamin complex supplementation during the transition period and early lactation alters endometrium mRNA expression on day 14 of gestation in lactating dairy cows

Greater metabolic demands in high-producing dairy cows are believed to be a cause of sub-fertility in these animals. Previously, supplementation with vitamin B complex molecules has shown benefits in improving milk production, health, and reproductive efficiency of dairy cows. The primary aim of this project was to determine the effects of rumen-protected vitamin B complex supplementation of 100 g of Transition VB (Jefo, St. Hyacinthe, QC, Canada) and 4 g of Lactation VB (VB; Jefo), during the transition and early lactation periods, respectively, compared with a control diet containing no supplementation on d 14 endometrial outcomes of pregnancy. In the vitamin B supplemented cows, we expect to see a change in the mark-up of endometrial genes important for embryo survival before implantation. Multiparous Holstein cows were enrolled into the study 3 wk before parturition and were randomly assigned to either the VB or control treatment. Twice-a-week blood samples, weekly milk samples, and daily feed intake were collected. Cows were enrolled onto a double-ovsynch protocol at 33 ± 3 d postpartum and inseminated by timed artificial insemination. Milk production and components, concentrations of BHB, haptoglobin, and progesterone in serum, and ovarian dynamics were also measured, but no treatment effect was observed. The uterus was flushed on d 14 after artificial insemination (around 72 DIM) for conceptus collection, and endometrial samples were collected at the same time. Overall, 42 cows were flushed and 13 embryos were collected. Analysis of mRNA expression of genes related to embryo development, immune system, adhesion, and regulation of vitamin B molecules showed that OXTR, MUC5B, MUC1, IL1B, SPP, TRD, FZD8, and FOLR1 genes were significantly upregulated in the VB group. Vitamin B supplementation had no effect on the size of the embryo and ovulatory follicle or corpus luteum diameter at embryo collection. In conclusion, the benefits of strategic dietary VB supplementation during the transition and early lactation might be directly linked to endometrial functions required for embryo survival during the peri-implantation period.     

淫羊藿苷影响大鼠卵巢功能与RLF mRNA表达的实验研究

目的:研究淫羊藿苷(ICA)对大鼠卵巢功能与松弛素样因子(relaxin-like factor,RLF)mRNA表达的影响,探讨其对大鼠卵巢功能的调节作用机制。方法:大鼠按ICA低、中、高剂量隔日给药,连续30d;雌二醇按雌二醇放射免疫试剂盒步骤测定;RT-PCR法测定RLF mRNA的表达量。结果:ICA能够增加大鼠卵巢重量,促进卵泡发育,提高雌激素分泌量,但不扰乱动情周期;ICA中剂量组和高剂量组均使RLF mRNA表达增加(P<0.05)。结论:ICA具有促进雌性生殖的作用,可通过上调RLF mRNA表达来实现。     

Functional maturation during bovine granulopoiesis

Granulocytic precursor cells undergo morphologic changes in the nucleus and the cytoplasm during the process of granulopoiesis, which takes place in the bone marrow. These changes are associated with the development of stage-specific proteins necessary for the highly specialized roles of polymorphonuclear leukocytes in phagocytosis, bacterial killing, and in mediating the inflammatory process. The objective of the current study was to sequence the various events that occur upon functional development of granulocytic bone marrow cells in the bovine species. Cells were obtained from the bone marrow of clinically healthy cows and separated into different stages of maturation using density gradient centrifugation. Three cellular fractions were obtained that were enriched for either early immature, late immature or mature granulocytic cells. Functions and receptor expressions assessed in the three maturation stages were:Fc-IgG2 receptor and CD11b expression, phagocytosis of Escherichia coli, respiratory burst activity, and cellular myeloperoxidase activity. Immature cells expressed already Fc-IgG2 receptor and CD11b on their cytoplasma membrane. Phagocytic ability was acquired in the myelocytic stage, but only the more mature forms were readily capable of phagocytosis. Promyelocytes, myelocytes and metamyelocytes showed no respiratory burst activity. Only band and segmented cells produced reactive oxygen species. Myeloperoxidase was present at all stages of maturity. Thus, each of the maturation stages was characterized by a selective expression of one or more functions and receptors. Therefore, sequential biochemical maturation is postulated during bovine granulopoiesis.     

Effects of bovine somatotropin on beta-casein mRNA levels in mammary tissue of lactating cows

Bovine somatotropin (bST) increases milk production in lactating cows through its effect on nutrient partition and maintenance of mammary cell function. A positive relationship between bST treatment and abundance of β-casein mRNA in mammary tissues from lactating cows was hypothesized. In mammary tissue isolated from 14 midlactation Holstein cows, β-casein mRNA was 35.4% higher among 7 cows receiving continuous bST infusions at 29 mg/d for 63 d compared with tissue from 7 untreated control cows. To investigate whether increased β-casein mRNA resulted from a direct effect of bST on the mammary gland, explants of mammary tissue from other lactating cows that had not received bST were incubated with bST and prolactin in 2 experiments. Mammary explant cultures taken from 2 lactating cows that had not been milked for 48 h were supplemented with either prolactin or bST. Both prolactin and bST stimulated higher levels of β-casein mRNA in the mammary explants compared with their non-supplemented counterparts. Explant cultures from 4 additional lactating cows were prepared from rear quarter mammary tissue subjected to milking intervals of 6 h for right rear quarters or 20 h for left rear quarters. Both bST- and prolactin-mediated increases in β-casein mRNA were dependent on milking intervals. That is, levels of β-casein mRNA were increased by bST or prolactin supplementation in explants isolated from the mammary quarters biopsied 20 h after milking but not for those biopsied at 6 h after milking. Results are consistent with a potential role for bST in up-regulating or sparing β-casein mRNA levels in lactating bovine mammary tissue in a manner similar to prolactin.     

Expression of the insulin-like growth factor (IGF) system in the bovine oviduct at oestrus and during early pregnancy

Early mammalian embryo development in vitro can be enhanced by co-culture with oviductal cells and by the addition of insulin-like growth factors (IGFs). This study examined the expression patterns of the oviductal IGF system in cattle in relation to the number of days after oestrus and the presence or absence of embryos. Oviducts were collected from: (i) 66 nulliparous heifers on day 3, day 6 or day 16 after insemination and from (ii) ten non-pregnant, lactating cows on day 0 or day 1 of the oestrous cycle. Oviducts were coiled, frozen whole and sectioned for in situ hybridization. Expression patterns of mRNAs encoding IGF-I, IGF-II, type 1 IGF receptor (IGF-1R), and the IFG binding proteins (IGFBP)-1, -3 and -5 were determined from autoradiographs. Separate measurements were made for the mucosa and muscle layers of the infundibulum, ampulla and isthmus. None of the parameters measured differed between heifers with or without the presence of an embryo. mRNAs encoding IGF-I and IGF-1R were present in the mucosa and muscle of all three oviductal regions, and the highest value of IGF-I mRNA was measured in heifers on day 3. IGF-II mRNA was expressed predominantly in the muscle wall. IGFBP-1 mRNA was not detectable, whereas mRNAs encoding IGFBP-3 and -5 were expressed in both the muscle and mucosa. IGFBP-3 expression was higher in cows on day 0 and day 1 of the oestrous cycle than in heifers on day 3, day 6 and day 16 after insemination. A peak of IGFBP-5 expression was reached on day 6. Locally or systemically produced IGFs, regulated by IGFBPs, may act directly on the embryo or indirectly via modulation of oviductal secretions and muscular activity to influence the success of early embryo development.     

Mammary gland development of dairy heifers during their first gestation.

Mammary gland development was determined by analysis of udders at thirteen stages of the first gestation of nine pairs of identical twin dairy heifers. Stained sagittal sections of udders showed that gland parenchyma spread into and displaced adipose tissue so that total udder weight does not reveal extent of gland development. Changes in composition of mammary gland during gestation reflected gross and histological changes. Percentages of fat and deoxyribonucleic acid decreased while percentages of nitrogen, water, dry fat-free tissue, and ribonucleic acid increased. Development of the portion of the udder which was mammary gland appeared to be a continuous exponential process through gestation, and increases in quantitative development followed the general formula for organ growth, Y = Aekt (In Y = In A + kt), in which k is the rate constant for growth by months (t). Analysis of variance showed in addition to stage of gestation that body weight and pairs, but not age, affected the regression. Adjusting for body weight only produced rate constants for growth similar to those from adjusting for pairs and weight. Rates of development were approximately 33% per month for nitrogen, dry fat-free tissue, and internal surface area of mammary glands, and approximately 25% for parenchyma weight and deoxyribonucleic acid. Average rate of increase in total udder weight was only about .5 that for mammary glands only.     

Expression and regulation of glucose transporters in the bovine mammary gland

Glucose is the primary precursor for the synthesis of lactose, which controls milk volume by maintaining the osmolarity of milk. Glucose uptake in the mammary gland plays a key role in milk production. Glucose transport across the plasma membranes of mammalian cells is carried out by 2 distinct processes: facilitative transport, mediated by a family of facilitative glucose transporters (GLUT); and sodium-dependent transport, mediated by the Na+/glucose cotransporters (SGLT). Transport kinetic studies indicate that glucose transport across the plasma membrane of the lactating bovine mammary epithelial cell has a K(m) value of 8.29 mM for 3-O-methyl-D-glucose and can be inhibited by both cytochalasin-B and phloretin, indicating a facilitative transport process. This is consistent with the observation that in the lactating bovine mammary gland, GLUT1 is the predominant glucose transporter. However, the bovine lactating mammary gland also expresses GLUT3, GLUT4, GLUT5, GLUT8, GLUT12, and sodium-dependent SGLT1 and SGLT2 at different levels. Studies of protein expression and cellular and subcellular localizations of these transporters are needed to address their physiological functions in the mammary gland. From late pregnancy to early lactation, expression of GLUT1, GLUT8, GLUT12, SGLT1, and SGLT2 mRNA increases from at least 5-fold to several hundred-fold, suggesting that these transporters may be regulated by lactogenic hormones and have roles in milk synthesis. The GLUT1 protein is detected in lactating mammary epithelial cells. Its expression level decreases from early to late lactation stages and becomes barely detectable in the nonlactating gland. Both GLUT1 mRNA and protein levels in the lactating mammary gland are not significantly affected by exogenous bovine growth hormone, and, in addition, GLUT1 mRNA does not appear to be affected by leptin.