The impact of lactating Hu sheep's dietary protein levels on lactation performance, progeny growth and rumen development.

This study aimed to investigate whether lactating Hu sheep's dietary protein levels could generate dynamic effects on the performance of their offspring. Twelve ewes with similar parity were fed iso-energy diets which contained different protein levels (P1: 9.82%, P2: 10.99%) (n = 6), and the corresponding offspring were divided into SP1 and SP2 (n = 12). At 60 days, half of the lambs were harvested for further study: the carcass weight (p = 0.043) and dressing percentage (p = 0.004) in the SP2 group were significantly higher than SP1. The acetic acid (p = 0.007), propionic acid (p = 0.003), butyric acid (p < 0.001) and volatile fatty acids (p < 0.001) in rumen fluid of SP2 were significantly lower than SP1. The expression of MCT2 (p = 0.024), ACSS1 (p = 0.039) and NHE3 (p = 0.006) in the rumen of SP2 was lower than SP1, while the HMGCS1 (p = 0.026), HMGCR (p = 0.024) and Na+/K+-ATPase (p = 0.020) was higher than SP1. The three dominant phyla in the rumen are Bacteroidetes, Proteobacteria and Firmicutes. The membrane transport, amino acid metabolism and carbohydrate metabolism of SP1 were relatively enhanced, the replication and repair function of SP2 was relatively enhanced. To sum up, the increase of dietary protein level significantly increased the carcass weight and dressing percentage of offspring and had significant effects on rumen volatile fatty acids, acetic acid activation and cholesterol synthesis related genes. HIGHLIGHTSIn the early feeding period, the difference in ADG of lambs was mainly caused by the sucking effect.The increase in dietary protein level of ewes significantly increased the carcass weight and dressing percentage of offspring.The dietary protein level of ewes significantly affected the volatile fatty acids (VFAs) and genes related to acetic acid activation and cholesterol synthesis in the rumen of their offspring.The membrane transport, amino acid metabolism and carbohydrate metabolism of the offspring of ewes fed with a low protein diet were relatively enhanced.The replication and repair function of the offspring of ewes fed with a high protein diet was relatively strengthened.

Predicting hepatocellular carcinoma development for cirrhosis patients via methylation detection of heparocarcinogenesis-related genes.

Background: Most hepatocellular carcinoma (HCC) patients have undergone a progression from chronic hepatitis, then liver cirrhosis (LC), and finally to carcinoma. The objective of this study was to elucidate risk factors to predict HCC development for cirrhosis patients. Methods: Multiple methylated specific PCR (MSP) was applied to determine methylation status of heparocarcinogenesis-related genes in 396 tissue and plasma specimens and multivariate cox model was used to analyze the relationship between risk variables and HCC development among cirrhosis patients, followed up in a median period of 30 months. Results: Among 105 LC cases, HCC incidence rate at 30 months was 30.48% (32/105), which were statistically associated with patients' age and aberrant methylation of p16, SFRP, and LINE1 (p<0.05). Receiver operating characteristic (ROC) curve showed the overall predictive accuracy reached the highest (90.7%) if the four risk variables were concurrent to predict HCC development. Moreover, along with the growth of age from 0-40, 40-55, to 55-70 years or the increased number of aberrantly-methylated gene from 0-1 to 2-3, the HCC incidence rate of cirrhosis patients rised from 10.00%, 12.28% to 82.14% and 17.44% to 89.47%, separately. Thus, based on combined analysis with diverse age and number of aberrantly-methylated gene, 105 cases were divided into five groups and computed their respective HCC incidecne rate to categorize them into different risk groups. Of note, A significant lifting of HCC incidence rate in the high-risk group (40-55 years coupled with 2-3 aberrantly-methylated genes, 55-70 years coupled with 0-1 aberrantly-methylated gene, 55-70 years coupled with 2-3 aberrantly-methylated genes; n=33) was observed compared with the low-risk group (0-40 years coupled with 0-1 aberrantly-methylated gene, 40-55 years coupled with 0-1 aberrantly-methylated gene; (n=72) (p<0.01). Conclusions: Ultimately, high-risk cirrhosis patients with 55-over years or 2-3 aberrantly-methylated genes should be paid more attention to be regularly screened with HCC development.

[The Evaluation Value of Methylation Status of CpG Island of SFRP1 and LINE1 Gene Promoter Area in the Prognosis of Hepatocellular Carcinoma.]

OBJECTIVES: To investigate the relationship between aberrant promoter CpG islands methylation status of secreted frizzled related protein 1 (SFRP1) and long intersper sed nuclear element 1 (LINE1) gene and clinicopathologic parameters to determine their prognosis value for hepatocellular carcinoma (HCC). METHODS: 105 cases of HCC and 50 cases of normal people plasma were collected,and then the promoter hypermethylation status of SFRP1 and hypormethylation status of LINE1 were examined by methylation specific PCR (MSP); The relationship between SFRP1/LINE1 methylation status and patients' clinicopathologic factors was analyzed;The association between SFRP1/LINE1 methylation status and disease-free survival and overall survival was analyzed by Kaplan-Meier curves,the log-rank test,and multivariate Cox regression. RESULTS: SFRP1 gene promoter CpG islands hypermethylation and LINE1 gene promoter CpG islands hypomethylation were found in 59.05% (62/105) and 66.67% (70/105) of 105 cancerous plasma cases,repectively,SFRP1 hypermethylation status and LINE1 hypomethylation status in plasma of HCC account for 43.81%(62/105) and no positive methylation cases were detected in normal cases;The hypermethylation status of SFRP1 and hypomethylation status of LINE1 gene were related with HBsAg and α-fetoprotein (AFP) level;There was statistically significant difference between CpG islands hypermethylation of two genes and disease-free survival rate and overall survival rate;The group patients with SFRP1 hypermethylation positive and LINE1 hypomethylation positive demonstrated the worst prognosis while the group with SFRP1 hypermethylation negative and LINE1 hypomethylation negative had the best prognosis. CONCLUSIONS: The promoter methylation of SFRP1 and LINE1 is correlated with the occurrence and development of HCC.SFRP1 and LINE1 might be potential and reliable biomarkers for predicting prognosis in HCC patients.

Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1.

The regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity in the nucleus is related to factor inhibiting HIF-1 (FIH-1). FIH-1 hydrolyzes asparagine at the C-terminal of HIF-1α, preventing the interaction between HIF-1α and its associated cofactors, and leading to suppressed activation of HIF-1. FIH-1 is a cytosolic protein and its entry to the nucleus has to be coordinated with HIF-1α. The present study was undertaken to examine the correlation between HIF-1α and FIH-1 in their nuclear entry. Human umbilical vein endothelial cells were treated with dimethyloxalylglycine at a final concentration of 100 µM for 4 h, resulting in an accumulation of HIF-1α and an increase of FIH-1 in the nucleus as determined by Western blot analysis. Pretreatment of the cells with copper (Cu) chelator tetraethylenepentamine at 50 µM in cultures for 24 h reduced both HIF-1α protein levels and the HIF-1α entry to the nucleus, along with decreased FIH-1 protein levels in the nucleus but no changes in the total FIH-1 protein levels in the cells. These effects were prevented by simultaneous addition of 50 µM CuSO4 with tetraethylenepentamine. Gene-silencing of HIF-1α significantly inhibited FIH-1 entry to the nucleus, but did not affect the total protein levels of FIH-1 in the cells. This work demonstrates that the nuclear entry of FIH-1 depends on HIF-1α. Cu deficiency caused a decrease of HIF-1α, leading to suppression of FIH-1 entry to the nucleus.

Copper promotion of angiogenesis in isolated rat aortic ring: role of vascular endothelial growth factor.

Copper stimulation of angiogenesis at the organ system level is vascular endothelial growth factor (VEGF) dependent, but copper stimulation of vascular endothelial cell proliferation in cultures is VEGF independent. The present study was undertaken to use isolated rat aortic rings to understand the seemly controversial observations between in vivo and in vitro studies. The thoracic aorta was isolated from Sprague Dawley rats (8-10 weeks) and sectioned into 1.0-mm thick vascular rings for culturing. Copper sulfide at a final concentration of 5, 25, 50 or 100 µM was added to the cultures and maintained for 8 days. A copper chelator, tetraethylenepentamine (TEPA) at a final concentration of 25 µM, was added to some cultures to block the effect of copper. An anti-VEGF antibody was used to determine the role of VEGF in copper promotion of angiogenesis. The data obtained showed that copper at 5 µM in cultures stimulated the vascular formation; an effect was blocked by TEPA. Copper at concentrations above 50 µM lost the proangiogenesis effect. However, copper at 5 µM did not enhance the production of VEGF, and concentrations above 50 µM significantly increased VEGF production. On the other hand, the treatment with anti-VEGF antibody completely blocked the proangiogenesis effect of 5-µM copper. This study thus demonstrates that VEGF is essential for angiogenesis but the proangiogenesis effect of copper does not act through enhanced production of VEGF.

Abnormal surface markers expression on bone marrow CD34+ cells and correlation with disease activity in patients with systemic lupus erythematosus.

Defects of hematopoietic stem cells (HSCs) have been suggested to contribute to the development of systemic lupus erythematosus (SLE). The aim of this study was to investigate the phenotypic characteristics of bone marrow (BM) CD34(+) cells in patients with SLE and its relationship with SLE disease activity. Ten SLE patients and 10 healthy subjects were recruited and their BM CD34(+) cells were analyzed by flow cytometric analysis with CD45/SSC gating for the expression of CD90, CD95, CD117, CD123, CD164, CD166, FAS-L, and HLA-DR. The percentage of BM CD34(+) cells was significantly decreased in active SLE patients (1.48 +/- 0.41%, n = 7) compared to the healthy controls (2.31 +/- 0.75%, n = 10, p < 0.01), but no significant difference was found between the inactive patients (2.04 +/- 0.44%, n = 3) and the controls. The expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in SLE patients (48.31 +/- 10.59%, 44.9 +/- 21.5%, 30.9 +/- 19.54%, respectively, n = 10) when compared with the control subjects (24.33 +/- 11.1%, 19.5 +/- 4.4%, 10.7 +/- 5.5%, respectively, n = 10, p < 0.05). The increased CD123 expression was negatively correlated with the number of peripheral white blood cells (r = -0.700, p < 0.05, n = 10). The percentage of CD166 expression was found significantly correlated with the index of SLE disease activity (r = 0.472, p < 0.05, n = 10) and 24 h proteinuria (r = 0.558, p < 0.05, n = 10), but negatively correlated with serum C3 level (r = -0.712, p < 0.01, n = 10). Our study found that the surface marker expression of CD95, CD123, and CD166 on BM CD34(+) cells were significantly increased in patients. This supports the hypothesis that there are abnormalities of the HSC in SLE. Since CD166 and CD123 correlated with the overall lupus activity, their role as a biomarker of inflammatory disease activity also requires further study.

[Effects and mechanisms of endotoxin pretreatment on lung of rat in endotoxemia].

OBJECTIVE: To observe the effects and mechanisms of endotoxin pretreatment on the rat lung in endotoxemia. METHODS: Eighty-four male Wistar rats were divided into seven groups (each group containing 12 rats): saline control and lipopolysaccharide (LPS)-treated 2 h, 4 h, 6 h groups and LPS-pretreated 2 h, 4 h, 6 h groups. LPS-pretreated rats were administrated with intraperitoneal injection of 0.25 mg/kg LPS. After 24 hours, they were injected with 0.5 mg/kg of LPS. Saline control and LPS-treated rats received an equivalent amount of saline. After 72 hours, LPS-treated and LPS-pretreated rats were intravenously injected with 10 mg/kg of LPS. An equivalent amount of saline was injected in the control rats. Blood was drawn from the carotid artery in LPS-treated and LPS-pretreated rats and sacrificed after intravenous injection of LPS 2, 4, 6 hours. Following saline injection of control rats, blood was drawn from the carotid artery after 6 hours. Arterial blood was drawn for blood gas analysis. The lungs were removed for detecting the mRNA levels of intercellular adhesion molecule-1 (ICAM-1) by reverse transcription polymerase chain reaction and the protein levels of inhibitor kappa B-alpha (I kappa B-alpha) by immunohistochemical staining. Bronchoalveolar lavage was performed in the right lung. Cell counts were evaluated with a light microscopy. The supernatant of bronchoalveolar lavage fluid (BALF) was assayed for the level of protein. The whole lung was weighed and the value was used to determine the lung-body index. The tissue was homogenized and centrifuged for the determination of myeloperoxidase enzyme (MPO) activity. RESULTS: The rats exposed to LPS alone demonstrated an increase in lung-body index, protein in BALF, and MPO activity in the lung tissue. In contrast, the rats exposed to LPS pretreatment exhibited a significant decrease in lung-body index, protein in BALF, and MPO activity. There was a significant decrease in the level of arterial bicarbonate in the LPS-treated rats in comparison with saline-treated and LPS-pretreated animals at 2 hours to 6 hours after LPS administration. The decrease of arterial bicarbonate was compensated by alveolar hyperventilation in LPS-treated animals, with a significant decrease in partial pressure of carbon dioxide. At the same time, partial pressure of oxygen decreased significantly compared with saline control animals and LPS-pretreated animals. LPS-treated rats showed a significant gradually increase in ICAM-1mRNA in the lung in comparison with the saline group. In contrast, ICAM-1mRNA levels in rats pretreated with LPS was lower than that in LPS-treated rats. In LPS-treated animals, LPS caused a decrease of I kappa B-alpha protein expression at 2 hours, returned to control level at 4 hours, and remained at 6 hours. There was no decrease of I kappa B-alpha protein expression in LPS-pretreated animals. CONCLUSION: The results in this study showed that administration of a small dose of LPS 72 hours before endotoxemia caused a attenuation effect on lung injury, which may be correlated to I kappa B-alpha expression induced by LPS pretreatment.

Vasodilative action of carbon monoxide on rat pulmonary artery in vitro.

The present study investigates the vasodilative action of carbon monoxide on rat pulmonary artery in vitro. After isolation of the pulmonary artery rings (PAR) from Wistar rats, an ACh concentration-response curve was generated; the PARs were incubated with the NOS inhibitor L-NAME (30 micromol/L, n=10) or the heme oxygenase inhibitor ZnPPIX (10 micromol/L)+L-NAME (30 micromol/L, n=10) for 30 min. After that, a second ACh concentration-response curve was elicited. Other isolated PARs were randomly divided into two groups: endothelium-intact group (n=8) and endothelium-denuded group (n=8). The effect of exogenous carbon monoxide (CO) on pulmonary arterial vessel tone was observed. The results showed that ACh induced a concentration-dependent pulmonary vasorelaxation. This relaxation disappeared after endothelium was denuded. The ACh induced relaxation was attenuated after pretreatment with 30 micromol/L L-NAME, and attenuated further after pretreatment with 10 micromol/L ZnPPIX+30 micromol/L L-NAME. Exogenous carbon monoxide relaxed pulmonary artery in both the endothelium-intact group and the endothelium-denuded group. These data suggest that ZnPPIX inhibits ACh induced endothelium-dependent pulmonary artery relaxation and that CO is an endothelium-derived relaxation factor, and exogenous CO can relax pulmonary artery.

[The roles of carbon monoxide on hypoxic pulmonary vasoconstriction].

AIM AND METHODS: To study the roles of carbon monoxide on hypoxic pulmonary vasoconstriction (HPV) by investigating the effects of exogenous carbon monoxide and heme oxygenase inhibitor ZnPPIX on hypoxic vasoconstriction reaction of isolated rat pulmonary arterial rings (PAR). RESULTS: Hypoxia caused constriction in PAR preconstricted by PE. Both ZnPPIX and carbon monoxide inhibited hypoxic pulmonary constriction significantly by increasing the cGMP level after hypoxia. CONCLUSION: ZnPPIX and exogenous carbon monoxide can inhibit HPV. The reduction of cGMP induced by the decreased of CO may be one of reasons of HPV.