A potential oncogenic role of the commonly observed E2F5 overexpression in hepatocellular carcinoma

AIM: To explore the expression pattern of E2F5 in primary hepatocellular carcinomas (HCCs) and elucidate the roles of E2F5 in hepatocarcinogenesis. METHODS: E2F5 expression was analyzed in 120 primary HCCs and 29 normal liver tissues by immunohistochemistry analysis. E2F5-small interfering RNA was transfected into HepG2, an E2F5-overexpressed HCC cell line. After E2F5 knockdown, cell growth capacity and migrating potential were examined. RESULTS: E2F5 was significantly overexpressed in primary HCCs compared...     

FcεRIβ编码区E237G基因多态性与哮喘关系的研究

目的探讨FcεRIβ编码区E237G基因多态性与哮喘、特应性的相关性及与血清总IgE的关系。方法应用限制性片断多态性———聚合酶链技术(RELP-PCR)检测2004年10月至2005年10月山东潍坊市人民医院收治的60例哮喘患者及50名健康对照人群的E237G基因型,结合皮肤过敏原试验及血清总IgE水平分析。结果(1)哮喘组和对照组相比在E237G位点基因型频率(分别为25·00%和22·00%)及G等位基因频率(分别为13·33%和12·00%)差异无显著性意义(P均>0·05)。(2)具有特应性的哮喘患者和对照组相比在E237G位点的基因型频率(分别为34·40%和22·00%)及G等位基因频率(分别为18·97%和22·00%)差异无显著性意义(P均>0·05)。(3)通过单因素方差分析年龄、性别、E237G基因型对血清总IgE的影响差异无显著性意义(P均>0·05)。结论哮喘、特应性与E237G基因突变可能无相关性;哮喘患者E237G不同基因型对血清总IgE可能无影响。     

Functional characterization of factor V-Ile359Thr: a novel mutation associated with thrombosis

A missense mutation, FV-Ile359Thr (FV Liverpool), associated with thrombosis has recently been described. This mutation creates an additional potential N-linked glycosylation site (Asn-X-Ser/Thr) in factor V (FV) at Asn357 that could interfere with secretion and/or protein interactions. To investigate the molecular pathology of FV-Ile359Thr, the mutation was created by site-directed mutagenesis and expressed together with other mutations that could help explain the phenotype (FV-Arg306Gln/Ile359Thr/Arg679Gln, FV-Ile359Thr/Arg506Gln/Arg679Gln, and FV-Asn357Gln/Ile359Thr). The FV-Ile359Thr was secreted normally and had full procoagulant activity. Western blot analysis showed that FV-Ile359Thr migrated more slowly, while the FV-Asn357Gln/Ile359Thr was indistinguishable from FV-wild type (FV-WT), indicating that FV-Ile359Thr was expressed with an additional carbohydrate chain. Activated protein C (APC)-mediated inactivation in an FVa degradation assay showed that the Ile359Thr mutation significantly reduced the cleavage at Arg306 both in the presence and absence of protein S, whereas the cleavage at Arg506 was unaffected. When tested in an FVIIIa degradation assay, the FV-Ile359Thr variant exhibited equally low APC cofactor activity as FV Leiden (FVArg506Gln). In conclusion, the Ile359Thr mutation appears to affect anticoagulation by 2 mechanisms, impeding the APC-mediated down-regulation of the FVa molecule and additionally being a poor APC cofactor for the down-regulation of FVIIIa. These findings explain the association of the FV-Ile359Thr mutation with thrombosis.     

Functional characterization of melanocortin-3 receptor variants identify a loss-of-function mutation involving an amino acid critical for G protein-coupled receptor activation

Although melanocortin-4 receptor mutations are the cause of the most common monogenic form of obesity, the involvement of the melanocortin-3 receptor (MC3R) in the pathogenesis of obesity is unknown. Earlier studies failed to identify any mutations in obese patients except for the identification of two variants (K6T and I81V) that likely represent polymorphisms. However, a potential mutation (I183N) was recently reported from patients having high-fat contents. We report here the functional characterization of these variants. We show that K6T and I81V have ligand binding and signaling properties similar to wild-type (wt) MC3R, indicating that they are indeed polymorphisms. However, the other variant, I183N, completely lacks signaling in response to agonist stimulation, although it binds ligand with normal affinity and with only slightly decreased capacity. Coexpression of the wt and I183N MC3Rs showed that I183N does not exert dominant-negative activity on wt MC3R. These results provide supporting evidence for the hypothesis proposed in the original case report that MC3R mutation might be a genetic factor that confers susceptibility to obesity, likely due to haploinsufficiency. Further mutations at I183 revealed a discrete requirement for I183 in agonist-induced MC3R activation. The corresponding residue is also important for agonist-induced human melanocortin-4 receptor and lutropin receptor activation. In summary, we identify a residue that is critical for activation of G protein-coupled receptors.     

Reversion of the Jun-induced oncogenic phenotype by enhanced synthesis of sialosyllactosylceramide (GM3 ganglioside)

In the mouse fibroblast cell line C3H 10T1/2 and the chicken fibroblast cell line DF1, the ganglioside GM3 is the major glycosphingolipid component of the plasma membrane. Expression of the viral oncoprotein Jun (v-Jun) induces transformed cell clones with greatly reduced levels of GM3 and GM3 synthase (lactosylceramide α2,3-sialyltransferase) mRNA in both 10T1/2 and DF1 cell cultures. Compared with nontransformed controls, v-Jun transfectants show enhanced ability of anchorage-independent growth, and their growth rates as adherent cells are increased. When the mouse GM3 synthase gene is transfected with the pcDNA vector into v-Jun-transformed 10T1/2 cells, the levels of GM3 synthase and corresponding mRNA are restored to those of control cells. Reexpression of GM3 correlates with a reduced ability of the cells to form colonies in nutrient agar. Similarly, when the newly cloned chicken GM3 synthase gene is transfected into v-Jun-transformed DF1 with the pcDNA vector, the GM3 synthase level is restored to that of control cells, and the ability of the cells to form agar colonies is reduced. The levels of GM3 in the cell also affect membrane microdomains. The complex of GM3 with tetraspanin CD9 and integrin α5β1 inhibits motility and invasiveness. The amounts of this complex are greatly reduced in transformed cells. Expression of GM3 and consequent reversion of the transformed phenotype results in increased levels of that microdomain complex.     

Interleukin 6-174 G/C promoter gene polymorphism in centenarians: no evidence of association with human longevity or interaction with apolipoprotein E alleles

The C allele at position -174 in the promoter of the interleukin 6 (IL-6) gene has been associated with reduced gene expression and reduced plasma levels of IL-6. Given that IL-6 tracks with functional disability and age-related diseases, there may be attrition or reduction in the frequency of the homozygous subjects, who produce higher IL-6 serum levels, in older survivors in a population. In fact, a marked reduction of the IL-6*G/*G genotype was recently demonstrated in male though not female Italian centenarians compared with younger age groups. First aim of the present study was to investigate whether there was evidence of an association among IL-6 -174 G/C promoter polymorphism and extreme longevity in a population of 81 centenarians compared with a control group of 122 middle-aged healthy subjects (mean age: 51+/-18 SD; range: 19-73 years), from Apulia (Southern Italy). Secondly, we also tested possible interaction of apolipoprotein E (APOE) alleles with the IL-6 -174 G/C promoter polymorphism in view of our recent findings for reduced APOE epsilon4 allele in centenarians. No differences have been found in the IL-6 -174 G/C promoter allele and genotype frequencies between centenarians and controls nor was there any observed interaction with APOE alleles that are also reputed to be linked to longevity. Regional genetic differences in conjunction with differing environmental factors may explain in part previous results suggesting a role of this polymorphism in longevity.     

Genetic and biochemical characterization of the E32del polymorphism in human mesotrypsinogen.

BACKGROUND/AIMS: Mesotrypsin is a minor pancreatic digestive enzyme that degrades dietary trypsin inhibitors in the gut. In this study, we tested the hypothesis that the E32del genetic variant of mesotrypsin might represent a risk factor for the development of chronic pancreatitis, as a result of enhanced degradation of pancreatic secretory trypsin inhibitor. METHODS: We screened 97 German patients with chronic pancreatitis of alcoholic etiology and 109 healthy controls for the presence of the E32del variant and characterized the biochemical properties of E32del mesotrypsinogen. RESULTS: Higher allele frequency of the E32del variant was detected in the control population (25.7 vs. 18.0%), but the difference was not significant (p = 0.062). Recombinant E32del mesotrypsin exhibited normal catalytic activity, characteristic inhibitor resistance and inability to activate pancreatic zymogens. Degradation of trypsin inhibitors was unaffected by the E32del genotype. Interestingly, mesotrypsinogen-E32del was biochemically distinguishable from mesotrypsinogen by its faster activation with bovine enterokinase, while activation by human enterokinase, trypsin or cathepsin B was unchanged. CONCLUSION: The results classify E32del mesotrypsinogen as a frequent polymorphic variant, which is not associated with chronic alcoholic pancreatitis.     

The N370S mutation in the glucocerebrosidase gene of Portuguese type 1 Gaucher patients: Linkage to the PvuII polymorphism

Summary The mutation N370S accounts for 63% of the mutated glucocerebrosidase alleles of Portuguese type 1 Gaucher patients. It has been shown previously that this mutation is linked to the Pv1.1^– form of the PvuII polymorphism and suggested that the N370S mutation in glucocerebrosidase alleles has an Ashkenazi Jewish origin. We have found that in Portuguese type 1 Gaucher patients this mutation is also invariably associated with the Pv1.1^– haplotype, despite the fact that there is no evidence of Ashkenazi Jewish background in this population.     

Familial gastrointestinal stromal tumor with hyperpigmentation: association with a germline mutation of the c-kit gene

We describe 2 siblings with multiple gastrointestinal stromal tumors (GISTs) and cutaneous hyperpigmentation. Both had a point mutation of the c-kit gene. The patients were sisters who had exhibited cutaneous hyperpigmentation since their late teens, but the diagnosis of multiple gastrointestinal submucosal tumors was not made until they were 41 and 45 years old. Histologic examination showed that these tumors were GISTs expressing CD34 and Kit protein. Both patients died of GISTs. Single-strand conformation polymorphism analysis showed a mutation of c-kit in tumor DNA extracted from paraffin-embedded specimens. Direct sequencing analysis showed that the point mutation occurred at codon 559 of exon 11 (Val-->Ala). The same single-point mutation was detected in DNA extracted from peripheral leukocytes obtained from the younger sister and her 2 children (who had similar general hyperpigmentation) as well as in DNA from a skin biopsy specimen taken from the older sister. The germline mutation at codon 559 of the c-kit gene found in the present familial GISTs differed from that in a previously reported case of familial GISTs. We propose that GISTs caused by a germline mutation of the c-kit gene should be referred to as GIST-cutaneous hyperpigmentation disease.     

Functional characterization of a novel missense mutation identified in a Turkish patient affected by severe coagulation factor V deficiency

Summary. Factor V (FV) deficiency is a rare coagulation disorder, characterized by a bleeding phenotype varying from mild to severe. To date, 115 mutations have been described along the gene encoding for FV (F5) but only few of them have been functionally characterized. Aim of this study was the identification and the molecular characterization of genetic defects underlying severe FV deficiency in a 7‐month‐old Turkish patient. Mutation detection was performed by sequencing the whole F5 coding region, exon–intron boundaries and about 300 bp of the promoter region. Functional analysis of the identified missense mutation was conducted by transient expression of wild‐type and mutant FV recombinant molecules in COS‐1 cells. Two novel mutations: a missense (Pro132Arg) and a 1‐bp deletion (Ile1890TyrfsX19) were identified in the F5 gene. While the frameshift mutation is responsible for the introduction of a premature stop codon, likely triggering F5 mRNA to nonsense‐mediated mRNA degradation, the demonstration of the pathogenic role of the Pro132Arg mutation required an experimental validation. Expression experiments showed that the missense mutation causes a significant reduction in FV secretion and in the specific activity of the residual secreted molecule (77% and 78% decrease, respectively). This paper reports the identification of two novel mutations responsible for FV deficiency, thus widening the mutational spectrum of the F5 gene. The Pro132Arg mutation adds to the only other two functionally characterized missense defects in the FV A1 domain.