Anthocyanin enhances adipocytokine secretion and adipocyte-specific gene expression in isolated rat adipocytes

Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte-specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. In this study, we demonstrated that anthocyanins (cyanidin or cyanidin 3-glucoside) have the potency of a unique pharmacological function in isolated rat adipocytes. Treated adipocytes with anthocyanins enhanced adipocytokine (adiponectin and leptin) secretion and up-regulated the adipocyte specific gene expression without activation of PPARgamma in isolated rat adipocytes. The gene expression of adiponectin was also up-regulated in white adipose tissue in mice fed an anthocyanin supplemented diet. As one of the possible mechanisms, AMP-activated protein kinase activation would be associated with these changes, nevertheless, the AMP:ATP ratio was significantly decreased by administration of the anthocyanins. These data suggest that anthocyanins have a potency of unique therapeutic advantage and also have important implications for preventing obesity and diabetes.     

Adipokine gene expression in dog adipose tissues and dog white adipocytes differentiated in primary culture.

Obesity and its associated disorders are increasing in companion animals, particularly in dogs. We have investigated whether genes encoding key adipokines, some of which are implicated in the pathologies linked to obesity, are expressed in canine adipose tissues. Using RT-PCR, mRNAs encoding the following adipokines were detected in dog white adipose tissue: adiponectin, leptin, angiotensinogen, plasminogen activator inhibitor-1, IL-6, haptoglobin, metallothionein-1 and 2, and nerve growth factor. The adipokine mRNAs were present in all fat depots examined. Fractionation of adipose tissue by collagenase digestion showed that each gene was expressed in mature adipocytes. The mRNA for TNFalpha was not evident in adipose tissue, but was detected in isolated adipocytes. Fibroblastic preadipocytes from gonadal white fat were differentiated into adipocytes in primary culture and adipokine expression examined before and after differentiation (days 0 and 11, respectively). Each adipokine gene expressed in dog white adipocytes was also expressed in the differentiated cells. These results demonstrate that dog white adipose tissue expresses major adipokine genes, expression being in the adipocytes. Investigation of adipokine production and function will provide insight into the mechanisms involved in obesity-related pathologies in dogs and serve as a model for the related human diseases.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Regulation of metallothionein gene expression and secretion in rat adipocytes differentiated from preadipocytes in primary culture.

The gene encoding metallothionein, a low mol. wt. metal binding and stress response protein, is expressed in white adipose tissue. In the present study, metallothionein (MT-1) gene expression and factors regulating metallothionein production have been examined in adipocytes induced to differentiate from fibroblastic preadipocytes in primary cell culture. On the induction of differentiation, the metallothionein-1 gene was strongly expressed in the cells and metallothionein released into the medium. A peak in metallothionein-1 mRNA level and metallothionein secretion occurred at 2 and 10 days post-differentiation, respectively, with a decrease in protein release after this time. The metallothionein-1 gene was expressed in the adipocytes prior to the adipsin and lipoprotein lipase genes, suggesting that it is an early marker of adipocyte differentiation. The addition of the glucocorticoid, dexamethasone, led to a substantial increase in metallothionein-1 mRNA in the cells and metallothionein secretion. Insulin and leptin also stimulated metallothionein production, although the effect was small. Neither noradrenaline nor the beta3-adrenoceptor agonist, BRL 37 344, altered metallothionein release but forskolin and bromo-cAMP were stimulatory, markedly increasing both metallothionein-1 level and metallothionein secretion. It is suggested that metallothionein is a novel secretory product of the differentiated white adipocyte and that its production is regulated particularly by glucocorticoids and through a cAMP-dependent pathway.     

结核分枝杆菌espK基因G-四链体核酸序列多态性与表达调控功能研究

DNA的G-四链体(G-quadruplex,G4)是由富含串联重复的鸟嘌呤(guanine,G)的核酸序列折叠形成的四链体螺旋结构,目前认为其与基因表达调控和基因组稳定性有关。已有研究表明,结核分枝杆菌(Mycobacterium tuberculosis)的espK(Rv3879c)是构成ESX-1分泌系统的一个重要元件,其蛋白序列具有串联重复的GTPITP氨基酸序列多态性。本研究经核酸序列比对分析,确定该氨基酸序列多态性区域对应的模板链上存在G4序列,且该G4序列仅存在于结核分枝杆菌复合群。通过比对结核分枝杆菌临床分离株espK基因的核酸序列,发现espK基因的高频率G1573C突变位于G4序列。为研究该G4结构及基因表达调控功能,首先利用圆二色谱检测其核酸片段在钾离子存在条件下的光谱学特征,证实其可在体外形成具有顺式平行结构特征的G4,同义点突变G4会使其结构稳定性下降。采用重叠聚合酶链反应(overlapping polymerase chain reaction,overlapping PCR)构建含有G4突变的espK表达质粒,获得重组表达菌株。通过实时定量PCR测定espK重组表达菌株中基因转录水平变化,发现同义点突变G4后,其基因转录水平比野生型espK重组菌株提升 1.5 倍(P＜0.05)。此外,临床分离株中espK出现的高频率G1573C突变会破坏G4结构,但蛋白免疫印迹检测结果显示espK G1573C突变导致EspK蛋白表达水平上升。以上结果提示,espK的G4结构具有表达调控功能,该G4区域的序列多态性可能通过影响EspK表达水平来调节ESX-1分泌系统的活性。     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Organization of Xenopus histone gene variants within clusters and their transcriptional expression

Using previously cloned Xenopus nucleosomal core histone genes as hybridization probes, a genomic DNA library of Xenopus laevis was screened for histone gene clusters. From over 200 histone-gene containing clones identified, 36 were selected as possibly containing H1 histone genes by hybridization to a probe derived from a sea urchin H1 histone gene. These 36 clones were further analyzed by hybrid-selected translation for the definitive presence of H1 histone genes. The genes for three different H1 histone variants were found: H1A , H1B and H1C . Mapping of the histone genes within each clone showed that at least three different gene arrangements can occur within a cluster and that the type of H1 histone variant present in a cluster may be related to the cluster type. S1-mapping experiments indicated that histone genes found in different cluster-types can be expressed in oocytes. Also, the H1 gene found in one cluster-type was expressed in at least three different cell-types: oocytes, gastrula-stage embryos, and erythroblasts.     

Distinct mechanisms regulate MHC class II gene expression in B cells and macrophages

In a previous series of studies, we had shown that the constitutive Ia expression in an immunoselected Ia-human B cell variant, RJ 2.2.5, could be restored by somatic cell hybridization with mouse B cells. These experiments allowed us to show the existence of a transacting activator factor(s) operating across species barriers and encoded by the aIr-1 locus located on mouse chromosome 16. The aim of the present study was to investigate whether the B cell constitutive Ia expression and the inducible Ia expression, as seen in macrophages treated with IFN-gamma, are controlled by similar intracellular factors. To this purpose, we constructed an interspecies somatic cell hybrid between the human Ia-RJ 2.2.5 B cells and the mouse Ia-P388 D1 macrophage cells. These murine cells transiently express Ia antigens when incubated with IFN-gamma. Our results show that RJ 2.2.5 X P388 D1 cell hybrids do not express either human or mouse class II gene products. Treatment with human recombinant IFN-gamma did not modify the MHC phenotype of either the hybrid cells or the human parental cells. On the other hand, treatment of the hybrid cells with murine recombinant IFN-gamma resulted in de novo expression of mouse Ia mRNA and corresponding cell surface antigens without, however, reinduction of the human class II-positive phenotype. Furthermore, treatment with the mouse lymphokine significantly increased the levels of human HLA class I mRNA and corresponding cell surface antigens in the hybrid cells, further reinforcing the notion of the existence of non-species-specific secondary mediators generated after receptor-ligand interaction in the IFN-gamma system. Together, these results indicate that in macrophages, the intracellular events taking place after binding of IFN-gamma with its own receptor and leading to the expression of a class II-positive phenotype do not operate via an activation of the aIr-1 locus and/or its products. Thus, at least in our experimental system, we can firmly establish a first, relevant distinction between constitutive and inducible class II gene expression. This difference, dictated by the specific differentiation program of each cell type, may be relevant for the understanding of the function of class II gene products.     

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.     

Sterols regulate development and gene expression in Arabidopsis

Sterols are important not only for structural components of eukaryotic cell membranes but also for biosynthetic precursors of steroid hormones. In plants, the diverse functions of sterol-derived brassinosteroids (BRs) in growth and development have been investigated rigorously, yet little is known about the regulatory roles of other phytosterols. Recent analysis of Arabidopsis fackel (fk) mutants and cloning of the FK gene that encodes a sterol C-14 reductase have indicated that sterols play a crucial role in plant cell division, embryogenesis, and development. Nevertheless, the molecular mechanism underlying the regulatory role of sterols in plant development has not been revealed. In this report, we demonstrate that both sterols and BR are active regulators of plant development and gene expression. Similar to BR, both typical (sitosterol and stigmasterol) and atypical (8, 14-diene sterols accumulated in fk mutants) sterols affect the expression of genes involved in cell expansion and cell division. The regulatory function of sterols in plant development is further supported by a phenocopy of the fk mutant using a sterol C-14 reductase inhibitor, fenpropimorph. Although fenpropimorph impairs cell expansion and affects gene expression in a dose-dependent manner, neither effect can be corrected by applying exogenous BR. These results provide strong evidence that sterols are essential for normal plant growth and development and that there is likely a BR-independent sterol response pathway in plants. On the basis of the expression of endogenous FK and a reporter gene FK::beta-glucuronidase, we have found that FK is up-regulated by several growth-promoting hormones including brassinolide and auxin, implicating a possible hormone crosstalk between sterol and other hormone-signaling pathways.