Hepatocyte nuclear factor-1α (HNF-1α) regulates the expression of genes encoding proteins involved in glucose metabolism and insulin secretion. Mutations in the HNF-1α gene cause maturity-onset diabetes of the young Type 3. However, the mechanism leading to this disease has not been completely ascertained. Previously, we found a novel mutation in the regulatory element of the human HNF-1α gene in two Chinese diabetes pedigrees. The nucleotide at position -128 T was substituted by G (nt-128 T→G). In this study, we analysed the functional defect of nt-128 T→G in HNF-1α transcription activity.
Luciferase reporter gene assays were carried out to examine the functional characteristics of this mutant. Electrophoretic mobility shift assays and chromatin immunoprecipitation were performed to confirm the binding of nuclear proteins to oligonucleotides.
The variant construct (nt-128 T→G) had a 1.65-fold increase in promoter activity compared with that of the wild-type construct in HepG2 cells and a 1.33-fold increase in MIN6 cells, respectively. The variant resided at a FOXA/ HNF-3 binding site identified by a series of competitive electrophoretic mobility shift assays and antibody supershift analyses. The assays showed a differential binding affinity in the wild-type and the nt-128 T→G mutant fragments by FOXA/ HNF-3. Chromatin immunoprecipitation indicated that FOXA/ HNF-3 bound to this region in vivo. One nucleotide substitution in the FOXA/ HNF-3 site in the human HNF-1α regulatory element caused an increase of HNF-1α transcriptional activity.
Our data suggested that this substitution in the promoter region affects DNA–protein interaction and HNF-1α gene transcription. The mutant may contribute to the development of diabetes in these two nt-128 T→G pedigrees of Chinese.
Asymmetric dimethylarginine (ADMA) is synthesized by protein arginine methyltransferases during methylation of protein arginine residues and released into blood upon proteolysis. Higher concentrations of ADMA in blood have been observed in patients with metabolic diseases and certain cancers. However, the role of ADMA in colon cancer has not been well investigated. ADMA serum levels in human patients diagnosed with colon cancer were found to be higher than those present in healthy subjects. ADMA treatment of LoVo cells, a human colon adenocarcinoma cell line, attenuated serum starvation-induced apoptosis and suppressed the activation of the Fas (APO-1/CD95)/JNK (SAPK) (c-Jun N terminal protein kinase/stress-activated protein kinase)pathway. ADMA also suppressed the activation of JNK triggered by death receptor ligand anti-Fas mAb and exogenous C2-ceramide. Moreover, we demonstrated that ADMA pretreatment protected LoVo cells from doxorubicin hydrochloride-induced cell death and activation of the Fas/JNK pathway. In summary, our results suggest that the elevated ADMA in colon cancer patients may contribute to the blocking of apoptosis of cancer cells in response to stress and chemotherapy.
ADMA; colon cancer; apoptosis; Fas; JNK; chemotherapy
Karyopherin alpha 2 (KPNA2), a member of the karyopherin family, has a central role in nucleocytoplasmic transport and is overexpressed in many cancers. Our previous study identified KPNA2 as significantly upregulated in epithelial ovarian carcinoma (EOC), correlating with poor survival of patients. However, the precise mechanism of this effect remains unclear. The aim of the present study was to examine the role of KPNA2 in the proliferation and tumorigenicity of EOC cells, and its clinical significance in tumor progression. Real-time quantitative RT-PCR analysis revealed high expression levels of KPNA2 in 162 out of 191 (84.8%) fresh EOC tissues, which was significantly correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation, histological type, recurrence, and prognosis of EOC patients. Our results showed that upregulation of KPNA2 expression significantly increased the proliferation and tumorigenicity of EOC cells (EFO-21 and SK-OV3) in vitro and in vivo, by promoting cell growth rate, foci formation, soft agar colony formation, and tumor formation in nude mice. By contrast, knockdown of KPNA2 effectively suppressed the proliferation and tumorigenicity of these EOC cells in vitro and in vivo. Our results also indicated that the molecular mechanisms of the effect of KPNA2 in EOC included promotion of G1/S cell cycle transition through upregulation of c-Myc, enhanced transcriptional activity of c-Myc, activation of Akt activity, suppression of FOXO3a activity, downregulation of cyclin-dependent kinase (CDK) inhibitor p21Cip1 and p27Kip1, and upregulation of CDK regulator cyclin D1. Our results show that KPNA2 has an important role in promoting proliferation and tumorigenicity of EOC, and may represent a novel prognostic biomarker and therapeutic target for this disease.
KPNA2; epithelial ovarian carcinoma; proliferation; tumorigenicity; c-Myc; FOXO3a
The c-jun N-terminal kinase (JNK) proteins are encoded by three genes (Jnk1–3), giving rise to 10 isoforms in the mammalian brain. The differential roles of JNK isoforms in neuronal cell death and development have been noticed in several pathological and physiological contexts. However, the mechanisms underlying the regulation of different JNK isoforms to fulfill their specific roles are poorly understood. Here, we report an isoform-specific regulation of JNK3 by palmitoylation, a posttranslational modification, and the involvement of JNK3 palmitoylation in axonal development and morphogenesis. Two cysteine residues at the COOH-terminus of JNK3 are required for dynamic palmitoylation, which regulates JNK3's distribution on the actin cytoskeleton. Expression of palmitoylation-deficient JNK3 increases axonal branching and the motility of axonal filopodia in cultured hippocampal neurons. The Wnt family member Wnt7a, a known modulator of axonal branching and remodelling, regulates the palmitoylation and distribution of JNK3. Palmitoylation-deficient JNK3 mimics the effect of Wnt7a application on axonal branching, whereas constitutively palmitoylated JNK3 results in reduced axonal branches and blocked Wnt7a induction. Our results demonstrate that protein palmitoylation is a novel mechanism for isoform-specific regulation of JNK3 and suggests a potential role of JNK3 palmitoylation in modulating axonal branching.
c-Jun N-terminal kinase/JNK; palmitoylation; axonal branching; isoform regulation; cytoskeleton; Wnt pathway
Electrocardiographic ventricular repolarization QT parameters are independent risk factors for cardiovascular events and sudden cardiac death in diabetic patients. The aim of the study was to investigate the association of polymorphisms of the nitric oxide synthase 1 adaptor protein (NOS1AP) gene with QT interval in Chinese subjects with or without Type 2 diabetes.
Three single nucleotide polymorphisms (SNPs) (rs10494366, rs12143842 and rs12029454) were genotyped in 1240 Type 2 diabetic patients (631 men and 609 women) and 1196 normal controls (433 men and 763 women). Individuals with overt diseases other than diabetes were excluded. Heart-rate corrected QT interval (QTc) was determined by standard 12-lead ECG and Bazett formula. Sex-pooled analysis and sex-specific analysis for genotype–phenotype association were both conducted.
In the diabetic group, the rs12143842 T allele was associated with a 3.87-ms (P = 0.014, empirical P = 0.039) increase in QTc duration for each additional allele copy, while rs10494366 and rs12029454 exhibited no significant association with QTc. We found no evidence of association for the three SNPs in subjects with normal glucose regulation. No significant SNP-gender and -diabetes affection interaction was observed.
The genetic variant rs12143842 in NOS1AP is associated with QT interval duration in a Chinese population with Type 2 diabetes. Future studies in different populations are needed to validate this finding and to evaluate the impact of NOS1AP variants on cardiovascular events and sudden cardiac death in diabetic patients.
NOS1AP; QT interval; single nucleotide polymorphism
20S-protopanaxadiol (aPPD) is a metabolite of ginseng saponins, which is reported to be pro-apoptotic in some cells but anti-apoptotic in neuronal cells by regulating Akt signaling. Owing to its cholesterol-like structure, we hypothesized that aPPD may regulate Akt signaling by interacting with lipid rafts. Here, we compared Akt signaling in glioblastoma U87MG and neuroblastoma Neuro-2a cells treated with aPPD. aPPD did not change Akt activity in the total plasma membranes of each cell type, but drastically altered the activity of raft-associated Akt. Strikingly, Akt activity was decreased in the rafts of U87MG cells but increased in N2a cells by aPPD through regulating raft-associated dephosphorylation. The bidirectional regulation of raft-associated Akt signaling by aPPD enhanced the chemotoxicity of Paclitaxel or Vinblastine in U87MG cells but attenuated the excitotoxicity of N-methyl--aspartate in N2a cells. Our results demonstrated that the activity of raft-associated but not total membrane Akt determines its cellular functions. Lipid rafts differ in different types of cells, which allows for the possibility of cell-type-specific targeting for which aPPD might prove to be a useful agent.
20S-protopanaxadiol; Akt; apoptosis; lipid rafts
The neuro-steroids 3β-androstene-17α-diol (17α-AED), 3β-androstene-17β-diol (17β-AED), 3β-androstene-7α,-17β-triol (7α-AET) and 3β-androstene-7β,-17β-triol (7β-AET) are metabolites of dehydroepiandrosterone and are produced in neuro-ectodermal tissue. Both epimers of androstenediols (17α-AED and 17β-AED) and androstenetriols (7α-AET and 7β-AET) have markedly different biological functions of their chemical analogue. We investigated the cytotoxic activity of these neuro-steroids on human T98G and U251MG glioblastoma and U937 lymphoma cells. Proliferation studies showed that 17α-AED is the most potent inhibitor, with an IC50 ∼15 μM. For T98G glioma, 90% inhibition was achieved with 25 μM of 17α-AED. Other neuro-steroids tested only marginally suppressed cell proliferation. Reduced cell adherence and viability could be detected after 18 h of 17α-AED exposure. Treatment with 17α-AED induced a significant level of apoptosis in U937 lymphoma cells, but not in the glioma cells. Cytopathology of 17α-AED-treated T98G cells revealed the presence of multiple cytoplasmic vacuoles. Acridine orange staining demonstrated the formation of acidic vesicular organelles in 17α-AED-treated T98G and U251MG, which was inhibited by bafilomycin A1. These findings indicate that 17α-AED bears the most potent cytotoxic activity of the neuro-steroids tested, and the effectiveness may depend on the number of hydroxyls and their position on the androstene molecule. These cytotoxic effects may utilize a non-apoptotic pathway in malignant glioma cells.
androstene neuro-steroids; glioblastoma; lymphoma; malignant glioma; programmed cell death
A recently cloned tumour suppressor candidate, p33ING1, has been shown in vitro to collaborate with p53 to execute growth arrest and apoptosis. However, it is unclear as to how the expression of ING1 is regulated in normal and stress conditions. Using a p53-knockout mouse model, we investigated if the expression of ING1 was dependent on p53. We found that there was no difference in ING1 mRNA and protein levels between p53+/+ and p53–/– murine organs. In addition, when normal human epithelial keratinocytes (NHEK) and a keratinocyte cell line, HaCaT, which lacks wild-type p53 function, were exposed to UVB irradiation, the expression levels of ING1 were elevated in both NHEK and HaCaT cells. It is interesting, however, that UVB irradiation did not induce ING1 expression in dermal fibroblasts isolated from p53+/+ and p53–/– mice. Based on our findings, we therefore conclude that the expression of ING1 is independent of p53 status. UV induction of ING1 in keratinocytes suggests that ING1 may play a role in cellular stress response and skin carcinogenesis. © 2000 Cancer Research Campaign http://www.bjcancer.com
p33ING1; p53; in vivo expression; tumour suppressor gene; UV irradiation
The Ark and Mass serotype-specific epitopes of infectious bronchitis virus were studied by immunofluorescence and immunoprecipitation of mutant and recombinant spike glycoproteins (S protein) expressed in mouse L cells. Serotype-specific monoclonal antibodies could bind to the recombinant proteins of Ark99 and Mass41 expressed from the chimeras in which the N-terminal thirds of the S1 sequences were reciprocally exchanged. Therefore, it appears that the respective serotype-specific epitopes of both strains were localized within the C-terminal two-thirds of the S1 proteins. Deletion and insertion of a five-amino-acid fragment on the S1 proteins of Ark99 and Mass41, altered the serotype-specific epitopes. This result implies that the five-amino-acid insertion on the S1 protein of the Ark serotype was involved in determining the conformation of the protein, probably acting as a spacer. In addition, it appears that an interaction between sequences of the N-terminal third and the remaining portion of the S1 protein determines the tertiary structure of the protein as well as the conformation of the serotype-specific epitope.
Recent observational studies have reported that body fat distribution might be differentially associated with subclinical atherosclerosis. We previously reported that visceral fat area (VFA) ⩾80 cm2 is the optimal cutoff for identifying abdominal obesity in Chinese subjects. We examined whether VFA ⩾80 cm2 reflects the association between abdominal obesity and subclinical atherosclerosis, and if determination of the visceral fat quantity is useful for assessing subclinical atherosclerosis in asymptomatic individuals.
Methods and results:
Participants (N=1005, men 515, women 490, 34–66 years) free of cardiovascular disease underwent magnetic resonance imaging and carotid ultrasound assessment to quantify VFA and carotid intima–media thickness (C-IMT). Overweight/obese subjects (body mass index (BMI) ⩾25.0 kg m−2) had a higher C-IMT than lean subjects (BMI <25.0 kg m−2) (P<0.01). Subjects with VFA ⩾80 cm2 had significantly higher C-IMT than those without abdominal obesity regardless of BMI (P<0.01). By multivariate regression analysis adjusted for anthropometric measurements and cardiovascular risk factors, waist circumference but not BMI was independently correlated with C-IMT in men (P<0.001). Similar findings were observed with an accurate obesity indices adjusted model, which showed that VFA was an independent risk factor for increased C-IMT in men but not in women.
VFA ⩾80 cm2 effectively identified carotid atherosclerosis for both lean and obese individuals in middle-aged Chinese men.
atherosclerosis; intima–media thickness; visceral fat; magnetic resonance imaging
Schizophrenia is a severe mental disorder that affects 0.5–1% of the population worldwide. Current diagnostic methods are based on psychiatric interviews, which are subjective in nature. The lack of disease biomarkers to support objective laboratory tests has been a long-standing bottleneck in the clinical diagnosis and evaluation of schizophrenia. Here we report a global metabolic profiling study involving 112 schizophrenic patients and 110 healthy subjects, who were divided into a training set and a test set, designed to identify metabolite markers. A panel of serum markers consisting of glycerate, eicosenoic acid, β-hydroxybutyrate, pyruvate and cystine was identified as an effective diagnostic tool, achieving an area under the receiver operating characteristic curve (AUC) of 0.945 in the training samples (62 patients and 62 controls) and 0.895 in the test samples (50 patients and 48 controls). Furthermore, a composite panel by the addition of urine β-hydroxybutyrate to the serum panel achieved a more satisfactory accuracy, which reached an AUC of 1 in both the training set and the test set. Multiple fatty acids and ketone bodies were found significantly (P<0.01) elevated in both the serum and urine of patients, suggesting an upregulated fatty acid catabolism, presumably resulting from an insufficiency of glucose supply in the brains of schizophrenia patients.
schizophrenia; biomarker; metaobonomics; GC-TOFMS; NMR