Proteomic analysis of serum marker proteins in recipient mice with liver cirrhosis after bone marrow cell transplantation

We previously found that transplantation with bone marrow cells (BMCs) improves liver function and liver fibrosis in cirrhotic mice. In the presence of liver damage induced by carbon tetrachloride (CCl4), transplanted BMC migrated into the peri-portal region and trans-differentiated into hepatocytes that produce albumin. Thus under these conditions, BMC transplantation induces liver regeneration. Detecting serum marker proteins is important to monitor the recovery of liver function of cirrhotic mice after BMC transplantation. We therefore initially resolved proteins extracted from serum samples at 48 h after BMC transplantation by 2-DE and compared spot intensity between control and BMC groups of mice. Six protein spots increased in the BMC group compared with the control group. MS revealed that these spots comprised apolipoprotein A1 (apoA1), apolipoprotein C3 (apoC3), vitamin D-binding protein, alpha-1-antitrypsin and proteasome subunit alpha type 1. We subsequently confirmed the levels of apoA1 in serum and liver samples by immunoblotting. ApoA1 increased at early stage (48 h and 1 wk) after BMC transplantation in this mouse model of liver cirrhosis. The early elevation of apoA1 might be useful to predict liver regeneration in cirrhotic mice after BMC transplantation.     

异种动物血清对体外培养人肿瘤细胞生长的影响

目的观察不同种属来源和浓度的动物血清对体外培养的肿瘤细胞（A549、MCF-7、BGC-823）生长的影响。探讨血清药理实验中血清供体动物的选择及血清添加量的问题。方法设置5种血清（牛、人、兔、大鼠、小鼠）及血清量（10%、20%、40%、60%、80%）的培养体系,用MTT法检测细胞增殖情况。结果不同种属来源的动物血清对肿瘤细胞生长的影响作用各不相同。从总的趋势来看,牛血清更适宜人肿瘤细胞生长,小鼠血清适应性最差。且随着加入量的增加,大多数血清会对肿瘤细胞的生长产生负效应。结论在肿瘤血清药理学试验中,为排除血清本身带给试验的干扰,应测定正常动物血清对肿瘤细胞增殖的影响,且血清添加量以小于20%为宜。     

Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca²⁺ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.     

Improved metabolic control by depletion of Liver X Receptors in mice

Liver X Receptors (LXRs) coordinate the regulation of lipid and carbohydrate metabolism and insulin signaling. LXR-ligands lower plasma glucose in hyperglycemic rodents and have consequently been proposed as anti-diabetic agents. We investigated the metabolic effects induced by high carbohydrate diet in LXRalpha(-/-)beta(-/-) mice. Irrespective of diets, LXRalpha(-/-)beta(-/-) mice had reduced fatty acid, insulin, and C-peptide plasma levels than wild-type controls, suggesting a lower insulin production. High carbohydrate diet decreased the plasma glucose levels and the homeostasis model assessment (HOMA)-index in LXRalpha(-/-)beta(-/-) mice and increased hepatic triglyceride content and mRNA levels of lipogenic genes in wild-type and LXRalpha(-/-)beta(-/-) mice, proportionally. In wild-type mice high carbohydrate diet was associated with induced expression of LXR (1.5-fold), despite unchanged SREBP-1c expression. LXRalpha(-/-)beta(-/-) mice responded to this diet by induction of SREBP-1c. Our study suggests that in LXRalpha(-/-)beta(-/-) mice, glucose utilization seems to be privileged possibly due to reduced circulating free fatty acid levels.     

Serum-induced expression of metallothionein isoforms in K-562 cells

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.     

Measurement of salicylic acid in human serum using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method using stable isotope dilution and gas chromatography-mass spectrometry has been developed to quantify salicylic acid (SA) at concentrations naturally occurring in biological fluids, such as in the serum of subjects not taking aspirin. After extraction of liquid-liquid with diethyl ether and ethyl acetate and preparation of the tert-butyldimethylsilyl derivative, SA content was detected using deuterated SA as internal standard. The mean recovery of SA from serum was 85 +/- 6%. Intra- and interday precision and % relative error were <15% in all cases.With a detection limit of 0.6 ng and a quantification limit of 2 ng, the method is therefore also adequate for population studies because of the small amount of blood necessary to perform the analyses.     

Elevated serum silicon levels in women with silicone gel breast implants

The metatolic fate of silicone gel leaked from an intact or ruptured prosthesis is unknown. In this study, serum was blindly assayed by inductively coupled plasma atomic emission spectroscopy (ICP-AES) for elemental silicon in 72 women with silicone gel breast implants and 55 control women (mean age 48 yr, both groups). Blood was drawn and processed using silicon-free materials. The mean silicon level in controls was 0.13±0.07 mg/L (range 0.06–0.35 mg/L), whereas in implant patients, the mean was significantly higher at 0.28±0.22 mg/L (range 0.06–0.87 mg/L) (P<0.01, Student'st-test with correction for unequal variances). Using the mean of the control group +2 SD as a cutoff for normal range (0.27 mg/L), 25/72 (34.7%) implant patients exceeded this value, compared with 2/55 (3.6%) controls. There was no significant correlation between past rupture of one or both implants, current rupture at the time of the blood draw, or the number of years with implants and silicon levels. The results suggest that serum silicon levels are elevated in many women with silicone gel breast implants. The chemical species involved and kinetics of this elevation remain to be determined.     

A new approach to touch down method using betaine as co-solvent for increased specificity and intensity of GC rich gene amplification

Tissue specific genes that contain high GC segments are difficult to amplify by standard PCR. We report an improved method for successful amplification of gene segment that has >70% GC base pairs. This new method of touch down PCR differed by having an initial annealing temperature (Ta) 1.5°C below the primers melting temperature that descended 0.2°C per cycle for 20 cycles and continued thereafter at fixed Ta for next 15 cycles. Different co-solvents were tested with this method to improve the result and betaine proved better than the other co-solvents. This new method is economical, fast and specific in amplifying GC rich region of other genes also.