Lysine intake affects gene expression of anabolic hormones in Atlantic salmon, Salmo salar

Nutritional factors influence regulation of the growth hormone (GH) and the insulin-like growth factor (IGF) system in fish, but so far there are no published studies describing how single indispensable amino acids influence these systems. Therefore, the present study aimed to test whether lysine (Lys) intake at low (LL=2.85 g/16 gN), medium (ML=4.91 g/16 gN) and high levels (HL=9.19 g/16 gN) affected the expression of genes related to the GH-IGF system (i.e. GH receptor, GH-R, IGF-I, IGF-II, IGF binding protein 1, IGFBP-1, IGF-I receptor IGF-IR) in Atlantic salmon during seawater growth phase. Salmon fed the HL diet significantly up-regulated hepatic IGF-I mRNA level by a factor of 2.2 as compared to those with medium Lys intake. In addition a significant up-regulation of 2.7-fold in muscle IGF-II mRNA was present. Low Lys intake decreased the nitrogen deposition and muscle protein accretion in fish and significantly down-regulated hepatic IGFBP-1 as well as muscle GH-R and IGF-II, as compared to those fed the ML diet. mRNA of IGF-IR on the other hand was not affected by Lys intake. High Lys intake resulted in a 7-fold up-regulation of muscle IGF-II mRNA level as compared to low Lys intake, and thus might be an important local anabolic regulator in fast muscle tissue. The single indispensable amino acid Lys indeed affected signalling through the genes of IGF-I, IGFBP-1 in hepatic tissue and GH-R, IGF-II in fast muscle in Atlantic salmon. Concomitantly the higher Lys intake increased nitrogen deposition to a certain level.     

Temporal dissociation of frequency-dependent acceleration of relaxation and protein phosphorylation by CaMKII

Frequency-dependent acceleration of relaxation (FDAR) is an important intrinsic mechanism that allows for diastolic filling of the ventricle at higher heart rates, yet its molecular mechanism is still not understood. Previous studies showed that FDAR is dependent on functional sarcoplasmic reticulum (SR) and can be abolished by phosphatase or by Ca/CaM kinase (CaMKII) inhibition. Additionally, CaMKII activity/autophosphorylation has been shown to be frequency-dependent. Thus, we tested the hypothesis that CaMKII phosphorylation of SR Ca(2+)-handling proteins (Phospholamban (PLB), Ca(2+) release channel (RyR)) mediates FDAR. Here we show that FDAR occurs abruptly in fluo-4 loaded isolated rat ventricular myocytes when frequency is raised from 0.1 to 2 Hz. The effect is essentially complete within four beats (2 s) with the tau of [Ca(2+)](i) decline decreasing by 42+/-3%. While there is a detectable increase in PLB Thr-17 and RyR Ser-2814 phosphorylation, the increase is quantitatively small (PLB<5%, RyR approximately 8%) and the time-course is clearly delayed with regard to FDAR. The low substrate phosphorylation indicates that pacing of myocytes only mildly activates CaMKII and consistent with this CaMKIIdelta autophosphorylation did not increase with pacing alone. However, in the presence of phosphatase 1 inhibition pacing triggered a net-increase in autophosphorylated CaMKII and also greatly enhanced PLB and RyR phosphorylation. We conclude that FDAR does not rely on phosphorylation of PLB or RyR. Even though CaMKII does become activated when myocytes are paced, phosphatases immediately antagonize CaMKII action, limit substrate phosphorylation and also prevent sustained CaMKII autophosphorylation (thereby suppressing global CaMKII effects).     

deltaPKC participates in the endoplasmic reticulum stress-induced response in cultured cardiac myocytes and ischemic heart

The cellular response to excessive endoplasmic reticulum (ER) stress includes the activation of signaling pathways, which lead to apoptotic cell death. Here we show that treatment of cultured cardiac myocytes with tunicamycin, an agent that induces ER stress, causes the rapid translocation of deltaPKC to the ER. We further demonstrate that inhibition of deltaPKC using the deltaPKC-specific antagonist peptide, deltaV1-1, reduces tunicamycin-induced apoptotic cell death, and inhibits expression of specific ER stress response markers such as CHOP, GRP78 and phosphorylation of JNK. The physiological importance of deltaPKC in this event is further supported by our findings that the ER stress response is also induced in hearts subjected to ischemia and reperfusion injury and that this response also involves deltaPKC translocation to the ER. We found that the levels of the ER chaperone, GRP78, the spliced XBP-1 and the phosphorylation of JNK are all increased following ischemia and reperfusion and that deltaPKC inhibition by deltaV1-1 blocks these events. Therefore, ischemia-reperfusion injury induces ER stress in the myocardium in a mechanism that requires deltaPKC activity. Taken together, our data show for the first time that deltaPKC activation plays a critical role in the ER stress-mediated response and the resultant cell death.     

Delivery of the aggregate inhibitor peptide QBP1 into the mouse brain using PTDs and its therapeutic effect on polyglutamine disease mice

The polyglutamine (polyQ) diseases are neurodegenerative diseases caused by proteins with an abnormally expanded polyQ stretch, which triggers abnormal aggregation of these proteins in the brain. We previously showed that the polyQ-binding peptide QBP1 inhibits polyQ aggregation, and further that administration of QBP1 fused with a protein transduction domain (PTD) suppresses polyQ-induced neurodegeneration in Drosophila. As the next step towards developing a therapy using QBP1, we investigated the delivery of PTD-QBP1 to the mouse brain upon its administration. Here we successfully detected delivery of PTD-QBP1 into mouse brain cells upon its single intracerebroventricular injection. In addition, long-term administration of PTD-QBP1 to polyQ disease mice improved their weight loss phenotype, suggesting a possible therapeutic effect. Our study indicates the potential of PTD-mediated delivery of QBP1 as a therapeutic strategy for the currently untreatable polyQ diseases.     

Mediation of protein kinase C zeta in μ-opioid receptor activation for increase of glucose uptake into cultured myoblast C2C12 cells

The present study is designed to investigate the role of atypical protein kinase C (PKC) in the signaling of μ-opioid receptors (MOR) for glucose uptake in myoblast C₂C₁₂ cells. Loperamide enhanced the uptake of radioactive deoxyglucose into C₂C₁₂ cells in a concentration-dependent manner that was abolished in cells pre-incubated with GF109203X at concentrations sufficient to block PKC. Inhibition of the atypical zeta (ζ) isoform of PKC using myristoylated PKC pseudosubstrate resulted in a concentration-dependent decrease of loperamide-stimulated glucose uptake into C₂C₁₂ cells. In addition, loperamide elicited the phosphorylation of PKC-ζ in C₂C₁₂ cells in a concentration-dependent manner that was abolished by pretreatment with naloxonazine at concentrations sufficient to block MOR. These results suggest the mediation of PKC-ζ in MOR signaling for glucose uptake in C₂C₁₂ cells. Activation of PKC-ζ by MOR stimulation is highly relevant to the search for therapeutic targets for glucose transport in insulin-sensitive tissues.     

N-terminally His-tagged hepatitis B core antigens: Construction, expression, purification and antigenicity

The core antigen of the hepatitis B virus (HBcAg) has been used widely as a diagnostic reagent for the identification of the viral infection. However, purification using the conventional sucrose density gradient ultracentrifugation is time consuming and costly. To overcome this, HBcAg particles displaying His-tag on their surface were constructed and produced in Escherichia coli. The recombinant His-tagged HBcAgs were purified using immobilized metal affinity chromatography. Transmission electron microscopy and enzyme-linked immunosorbent assay (ELISA) revealed that the displayed His-tag did not impair the formation of the core particles and the antigenicity of HBcAg.     

蛋白激酶CK2α对鼻咽癌细胞增殖和侵袭能力的影响

目的 探讨蛋白激酶CK2α对鼻咽癌细胞增殖和侵袭的作用,并探讨其可能机制.方法 通过RNA干扰技术下调鼻咽癌5-8F细胞中CK2α蛋白的表达,利用Western blot方法验证干扰效果;采用四甲基偶氮唑盐体外增殖实验、体外肿瘤侵袭实验方法检测CK2α下调后对鼻咽癌细胞增殖和侵袭的影响;应用流式细胞术检测细胞增殖周期的变化;Western blot方法分析CK2α对Akt蛋 白磷酸化的影响.结果 通过RNA干扰技术可以有效的沉默CK2α的表达.CK2α表达下调后,鼻咽癌细胞增殖和侵袭能力显著下降.细胞周期结果显示,CK2α沉默后,G0/G1期细胞比率增加,S期细胞比率降低.Western blot结果显示,CK2α沉默后下调了磷酸化Akt蛋白的表达水平.结论 蛋白激酶CK2α与鼻咽癌增殖、侵袭密切相关,CK2α可能是一个有潜力的鼻咽癌治疗靶点.     

ER and aging—Protein folding and the ER stress response

The endoplasmic reticulum (ER) is a multifunctional organelle which co-ordinates protein folding, lipid biosynthesis, calcium storage and release. Perturbations that disrupt ER homeostasis lead to the misfolding of proteins, ER stress and up-regulation of a signaling pathway called the ER stress response or the unfolded protein response (UPR). The UPR is characterized by the induction of chaperones, degradation of misfolded proteins and attenuation of protein translation. Age-related declines and activity in key molecular chaperones and folding enzymes compromise proper protein folding and the adaptive response of the UPR. This review will highlight age-related changes in the protein folding machinery and in the UPR.     

消化道恶性肿瘤中多种肿瘤标志物蛋白芯片联合检测的研究进展

目的：探讨应用蛋白芯片法联合检测多种肿瘤标志物在消化道恶性肿瘤临床应用。方法：查阅和分析近年有关多肿瘤标志物检测在消化道恶性肿瘤的诊断治疗和预后判断中应用的相关文献,并作进一步综合分析。结果：证实了多种肿瘤标志物检测在消化道恶性肿瘤的诊断治疗和预后判断中临床应用的可行性。结论：多肿瘤标志物检测在消化道恶性肿瘤的临床诊治中有广阔的应用前景,值得进一步研究。     

Human astrocytes and aortic endothelial cells actively convert the oxidized form of albumin to the reduced form: reduced albumin might participate in redox regulation of nerve and blood vessel systems

Human serum albumin (HSA) is a mixture of mercaptalbumin (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form), i.e., a protein-thiol redox couple in the extracellular fluid (ECF), and it might have antioxidant properties. Forty-two patients with orthopedic disorders participated in this study and were divided into two groups according to their age (young and older groups). By using HPLC to separate HSA into HMA and HNA, we analyzed the percentages of HMA and HNA in serum and lumbar cerebrospinal fluid (CSF). We also examined the redox activity of cultured normal human astrocytes, aortic endothelial cells, and dermal fibroblasts for HSA-thiol. The mean HMA value from the serum of the older group was significantly lower than that of the young group, whereas that from CSF was not significantly different between the two groups; CSF albumin is almost completely in the reduced form, and no age-related differences were observed. Cultured astrocytes and aortic endothelial cells showed conversion of HNA to HMA, whereas dermal fibroblasts showed no such redox activity. From the results obtained from in-vivo and in-vitro studies, HMA is considered to participate in redox regulation in the ECF, for example in the CSF that surrounds the central nervous system (CNS), and in blood serum.