Blocking the short isoform of augmenter of liver regeneration inhibits proliferation of human multiple myeloma U266 cells via the MAPK/STAT3/cell cycle signaling pathway

Multiple myeloma (MM) is the second most common haematological malignancy and remains an incurable disease, with most patients relapsing and requiring further treatment. Augmenter of liver regeneration (ALR) is a vital protein affecting fundamental processes such as energy transduction, cell survival and regeneration. Silencing ALR inhibits cell proliferation and triggers apoptosis in human MM U266 cells. However, little is known about the role of 15-kDa-ALR on MM. In the present study, the role of 15-kDa-ALR in human MM cells was investigated. Blocking extracellular 15-kDa-ALR with an anti-ALR monoclonal antibody (McAb) decreased the proliferation and viability of U266 cells. However, the results of flow cytometry revealed no changes in apoptosis, and the expression levels of Bax, Bcl-2, caspase-3 and cleaved caspase-3 were not affected. However, combined treatment with anti-ALR McAb and epirubicin increased the apoptosis of U266 cells. RNA sequencing results indicated that the ERK1/2, JNK-MAPK and STAT3 signaling pathways, as well as the cell cycle, were associated with the mechanism of action of the anti-ALR McAb, and PCR, western blotting and cell cycle analysis confirmed these results. The present findings suggested that blocking extracellular 15-kDa-ALR in U266 cells with an anti-ALR McAb decreased cell proliferation via the MAPK, STAT3 and cell cycle signaling pathways without increasing apoptosis. Thus, 15-kDa-ALR may be a new therapeutic target for myeloma.     

糖尿病周围神经病变与内皮型一氧化氮含酶基因、终末糖化产物受体基因、对氧磷酶基因及醛糖还原酶基因多态性的关联研究

目的　明确终末糖化产物受体基因 (RAGE)的Gly82Ser多态、对氧磷酶基因 (PON1)的Gln191Arg多态及Leu5 4Met多态、内皮型一氧化氮合酶基因 (eNOS)的Glu2 98Asp多态以及醛糖还原酶基因 (ALR2 ) 5′端上游的二核苷酸 (CA)n串联重复序列多态与糖尿病周围神经病变的关系。方法　采用PCR RFLP及放射自显影技术对 83名健康人和 2 11例糖尿病患者的上述多态进行病例对照 关联研究。结果　①糖尿病周围神经病变与eNOS基因Glu2 98Asp多态、PON1基因Leu5 4Met多态以及RAGE基因Gly82Ser多态均不相关 (P >0 .0 5 ) ;②PON1基因Gln191Arg多态的B等位基因频率及BB基因型频率随着糖尿病周围神经病变的进展呈降低趋势(趋势分析 ,P =0 .196 ) ,等位基因频率在伴 /不伴糖尿病微血管病变组间差异有显著性 (P =0 .0 4 6 ) ,进一步分析显示这种差异主要来源于糖尿病视网膜病变 ;③ALR2基因 5′上游 (CA)n二核苷酸串联重复序列多态的Z +6等位基因频率在不伴糖尿病周围神经病变组中较伴有周围神经病变组中高 ,两者间差异有显著性 (P =0 .0 38)。④以糖尿病周围神经病变为应变量 ,各基因多态为自变量进行Logistic回归分析后显示 ,各个基因多态与糖尿病周围神经病变均无独立相关。结论　上述基因在糖尿病周围神经病变的发病中不起     

肝特异性生物活性肽研究进展

肝脏是人体内最重要的器官之一,具有强大的再生能力。肝再生的调控机制非常复杂,众多因子参与其中,但大多数因子,类似肝细胞生长因子（HGF）、表皮生长因子（EGF）都不具备肝细胞作用的特异性。近年来,本实验室从新生小牛的肝脏中分离得到了能够特异性地促进肝细胞增殖、促进70%肝切除小鼠肝再生的物质并将其命名为HPPCn。因此,结合本实验室的工作对肝刺激物（HSS）、肝再生增强因子（ALR）、HPS、HPPCn等具有肝特异性作用的生物活性肽的研究进展以及相互之间的异同加以综述。     

重组人ALR单克隆抗体的制备、鉴定和初步应用

目的 制备抗重组人肝再生增强因子(ALR)的单克隆抗体(McAb)。方法 以基因重组人ALR二聚体(drhALR)为抗原，免疫Balb／c小鼠，制备单克隆抗体。用常规法制备抗ALR单体(mrhALR)多抗，建立夹心ELISA法测定ALR，初步确定ALR在小鼠组织的分布；用荧光免疫方法对胎鼠各组织进行ALR检测。结果 筛选出3株稳定分泌抗drhALR的单抗杂交瘤细胞株，免疫球蛋白亚类分别为IgG1、IgG2a、IgG2b，特异性高，用于rhALR的免疫印迹及ELISA检测，可同时识别rhALR单体(mrhALR)和二聚体(drhALR)；夹心ELISA法检测小鼠肝、肾、脾提取液ALR含量与鼠龄无关，各脏器的ALR含量不同，分别为肝0．36μg/g，肾0．11μg/g，脾0．05μg/g；免疫荧光检测胎鼠，各器官有不同程度ALR的存在，无器官特异性。结论 用drhALR为抗原制备的抗rhALR单克隆抗体，能够用于ALR的体内外免疫学检测，为rhALR及生化提取ALR的药理学研究及质量标准奠定了基础。     

mt-Nd2 Allele of the ALR/Lt mouse confers resistance against both chemically induced and autoimmune diabetes

Aims/hypothesis ALR/Lt, a mouse strain with strong resistance to type 1 diabetes, is closely related to autoimmune type 1 diabetes-prone NOD/Lt mice. ALR pancreatic beta cells are resistant to the beta cell toxin alloxan, combinations of cytotoxic cytokines, and diabetogenic NOD T-cell lines. Reciprocal F1 hybrids between either ALR and NOD or ALR and NON/Lt, showed that alloxan resistance was transmitted to F1 progeny only when ALR was the maternal parent. Here we show that the mitochondrial genome (mtDNA) of ALR mice contributes resistance to diabetes.Methods When F1 progeny from reciprocal outcrosses between ALR and NOD were backcrossed to NOD, a four-fold lower frequency of spontaneous type 1 diabetes development occurred when ALR contributed the mtDNA. Because of the apparent interaction between nuclear and mtDNA, the mitochondrial genomes were sequenced.Results An ALR-specific sequence variation in the mt-Nd2 gene producing a leucine to methionine substitution at amino acid residue 276 in the NADH dehydrogenase 2 was discovered. An isoleucine to valine mutation in the mt-Co3 gene encoding COX3 distinguished ALR and NOD from NON and ALS. All four strains were distinguished by variation in a mt-encoded arginyl tRNA polyadenine tract. Shared alleles of mt-Co3 and mt-Tr comparing NOD and ALR allowed for exclusion of these two genes as candidates, implicating the mt-Nd2 variation as a potential ALR-derived type 1 diabetes protective gene.Conclusions/interpretation The unusual resistance of ALR mice to both ROS-mediated and autoimmune type 1 diabete stresses reflects an interaction between the nuclear and mt genomes. The latter contribution is most likely via a single nucleotide polymorphism in mt-Nd2.     

Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase

BACKGROUND: Augmenter of Liver Regeneration is an important secondary hepatic growth factor. Augmenter of liver regeneration protein has been shown to control mitochondrial gene expression and the lytic activity of liver-resident Natural Killer cells through the levels of interferon-gamma, but the precise enzymatic function of this protein is unknown. AIMS: To define the enzymatic activity of augmenter of liver regeneration protein. The carboxy terminus of augmenter of liver regeneration protein contains a special CXXC motif characteristic for redox proteins and with faint homologies to the redox-active site of sulfhydryl oxidases. Tests were, therefore, carried out to establish whether isolated augmenter of liver regeneration protein can also function in the formation of sulfur bridges. METHODS: Purified augmenter of liver regeneration proteins from rat and human were tested in enzyme assays for the ability to introduce disulfide bonds into protein substrates. The isolated proteins were tested for the formation of dimers and the presence of bound FAD was investigated spectroscopically. The function of the conserved CXXC motif was investigated by in vitro mutagenesis experiments and subsequent enzyme assays. RESULTS: In this study, we demonstrate that rat and human augmenter of liver regeneration protein are flavin-linked sulfhydryl oxidases that catalyze the formation of disulfide bonds in reduced protein substrates. A flavin moiety is firmly but not covalently attached to the protein. In human cell cultures augmenter of liver regeneration protein is expressed in a long and short form that both exist as covalently linked dimers. The active site of the enzyme is associated with a conserved CXXC motif in the carboxy-terminal domain, that is present in the homologous proteins from yeast to humans and also in the human Q6 growth regulator protein. In vitro mutagenesis of one cysteine residue in the CXXC motif results in loss of enzymatic function and the mutated protein no longer binds FAD. CONCLUSIONS: For the first time, these data assign an enzymatic activity to the important hepatic growth factor augmenter of liver regeneration protein. The finding that augmenter of liver regeneration protein acts as a FAD-linked sulfhydryl oxidase is essential to identify the molecular targets inside liver cells and to elucidate the precise role of mammalian augmenter of liver regeneration protein in hepatic cell growth, liver disease and regeneration.     

Spectrum of MLL2 (ALR) mutations in 110 cases of Kabuki syndrome

Kabuki syndrome is a rare, multiple malformation disorder characterized by a distinctive facial appearance, cardiac anomalies, skeletal abnormalities, and mild to moderate intellectual disability. Simplex cases make up the vast majority of the reported cases with Kabuki syndrome, but parent-to-child transmission in more than a half-dozen instances indicates that it is an autosomal dominant disorder. We recently reported that Kabuki syndrome is caused by mutations in MLL2, a gene that encodes a Trithorax-group histone methyltransferase, a protein important in the epigenetic control of active chromatin states. Here, we report on the screening of 110 families with Kabuki syndrome. MLL2 mutations were found in 81/110 (74%) of families. In simplex cases for which DNA was available from both parents, 25 mutations were confirmed to be de novo, while a transmitted MLL2 mutation was found in two of three familial cases. The majority of variants found to cause Kabuki syndrome were novel nonsense or frameshift mutations that are predicted to result in haploinsufficiency. The clinical characteristics of MLL2 mutation-positive cases did not differ significantly from MLL2 mutation-negative cases with the exception that renal anomalies were more common in MLL2 mutation-positive cases. These results are important for understanding the phenotypic consequences of MLL2 mutations for individuals and their families as well as for providing a basis for the identification of additional genes for Kabuki syndrome.