Urinary beta-trace protein gene expression analysis in type 2 diabetes mellitus patients / Análise da expressão do gene da proteína beta-traço na urina de pacientes com diabetes mellitus tipo 2

ABSTRACT Objective: To evaluate the gene expression of beta-trace protein in urine of diabetic patients, with no reduction in glomerular filtration rate, which was defined as below 60mL/min/1.73m2. Methods: Type 2 diabetes mellitus patients were recruited, and a group of non-diabetic individuals served as control. Beta-trace protein gene expression was analyzed by quantitative PCR. Blood samples were collected to establish glucose levels and baseline kidney function. Accuracy was analyzed using ROC curves. Results: Ninety type 2 diabetes mellitus patients and 20 non-diabetic individuals were recruited. The area under the curve was 0.601, sensitivity of 20% and specificity of 89.47%. Among diabetic participants, 18% showed an expression above the cutoff point. Conclusion: These results of accuracy of beta-trace protein gene expression in urine of diabetic patients are promising, although they did not achieve a higher area under the curve level.

RESUMO Objetivo: Avaliar a expressão do gene da proteína beta-traço na urina de pacientes diabéticos, sem redução na taxa de filtração glomerular, definida como abaixo de 60mL/min/1,73m2. Métodos: Foram recrutados pacientes com diabetes mellitus tipo 2, e um grupo de indivíduos não diabéticos serviu como controle. A expressão do gene da proteína beta-traço foi analisada por PCR quantitativa. Amostras de sangue foram coletadas para estabelecer níveis de glicemia e função renal inicial. A acurácia foi analisada utilizando curvas ROC. Resultados: Foram recrutados 90 pacientes com diabetes mellitus tipo 2 e 20 não diabéticos. A área sob a curva foi de 0,601, com sensibilidade de 20% e especificidade de 89,47%. Entre os diabéticos, 18% apresentaram expressão acima do ponto de corte. Conclusão: Estes resultados de acurácia da expressão do gene da proteína beta-traço na urina de pacientes diabéticos são promissores, apesar de não terem atingido um nível alto na área sob a curva.

Involvement of Src tyrosine kinase and protein kinase C in the expression of macrophage migration inhibitory factor induced by H2O2 in HL-1 mouse cardiac muscle cells

Macrophage migration inhibitory factor (MIF), a pleiotropic cytokine, plays an important role in the pathogenesis of atrial fibrillation; however, the upstream regulation of MIF in atrial myocytes remains unclear. In the present study, we investigated whether and how MIF is regulated in response to the renin-angiotensin system and oxidative stress in atrium myocytes (HL-1 cells). MIF protein and mRNA levels in HL-1 cells were assayed using immunofluorescence, real-time PCR, and Western blot. The result indicated that MIF was expressed in the cytoplasm of HL-1 cells. Hydrogen peroxide (H2O2), but not angiotensin II, stimulated MIF expression in HL-1 cells. H2O2-induced MIF protein and gene levels increased in a dose-dependent manner and were completely abolished in the presence of catalase. H2O2-induced MIF production was completely inhibited by tyrosine kinase inhibitors genistein and PP1, as well as by protein kinase C (PKC) inhibitor GF109203X, suggesting that redox-sensitive MIF production is mediated through tyrosine kinase and PKC-dependent mechanisms in HL-1 cells. These results suggest that MIF is upregulated by HL-1 cells in response to redox stress, probably by the activation of Src and PKC.

Variable number tandem repeats in the promoter region of prostacyclin synthase gene in choline deficient rats

Weanling Sprague-Dawley rats were fed on a choline-deficient diet with hydrogenated vegetable oil and corn oil as lipids develop acute renal failure. Pathogenesis of the latter is controversial and an ischemic mechanism has been proposed. Arachidonic acid derivatives are involved in the regulation of vascular tonus. Vasospasm could be due to an increase in tromboxane A2-mediated vasoconstriction or to a decrease in prostacyclin-induced vasodilatation. Enzymes involved in the synthesis of both compounds are tromboxane A2- and prostacyclin-synthase respectively. The aim of this study was to identify the variable number tandem repeats (VNTR) in the promoter region of prostacyclin synthase gene and verify if there exists a relationship between the occurrence of VNTR in those choline-deficient rats which die because of acute renal failure and those which do not. We verified the presence of the VNTR in the prostacyclin synthase rat gene, but we did not find any difference in the molecular weight of the alleles between experimental and control rats. Renal reparation of the acute kidney injury due to choline deficiency in some rats is not related with differences in VNTR in the promoter region of the prostacyclin synthase gene.

Lotus (Nelumbo nuficera) flower essential oil increased melanogenesis in normal human melanocytes

In this study, the essential oil from lotus flower extract, including petals and stamens, was assessed with regard to its effects on melanogenesis in human melanocytes. The lotus flower essential oil was shown to stimulate melanin synthesis and tyrosinase activity in a dose-dependent manner. The lotus flower essential oil induced the expression of tyrosinase, microphthalmia-associated transcription factor M (MITF-M), and tyrosinase-related proten-2 (TRP-2) proteins, but not tyrosinase mRNA. Moreover, it increased the phosphorylation of ERK and cAMP response element binding protein (CREB). In order to verify the effective components of the lotus flower oil, its lipid composition was assessed. It was found to be comprised of palmitic acid methyl ester (22.66%), linoleic acid methyl ester (11.16%), palmitoleic acid methyl ester (7.55%) and linolenic acid methyl ester (5.16%). Among these components, palmitic acid methyl ester clearly induced melanogenesis as the result of increased tyrosinase expression, thereby indicating that it may play a role in the regulation of melanin content. Thus, our results indicate that lotus flower oil may prove useful in the development of gray hair prevention agents or tanning reagents.

Anti-tumor immunity elicited by adenovirus encoding AdhTrp2 or AdmTrp2 without vitiligo / 华中科技大学学报（医学）（英德文版）

To compare the difference in tumor immunity and autoimmunity elicited by adenovirus (Ad) encoding human or murine tyrosinase-related protein 2 (AdhTRP2 or AdmTRP2), and to find the most effective way to induce immunity by AdhTRP2 or AdmTRP2, C57BL/6 mice were immunized with AdhTRP2 or AdmTRP2 intramuscularly at different doses of 10(5), 10(6), 10(7) and 10(8) separately (10 mice for each dose). Two weeks after the immunization, in vivo CTL assay and intracellular staining (ICS) of IFN-gamma were carried out to analyze the dose-effect relationship. Tumor growth and vitiligo (as an sign of autoimmunity) were observed until 3 months after challenge with 10(5) B16F10 tumor cells. The results showed that Ad encoding AdmTrp2 induced weak tumor immune response. Similar immunization with AdhTrp-2 elicited stronger protective immunity. CTL activity and IFN-gamma-produced CD8+T cells were directly proportional to dose of AdhTrp2 or AdmTrp2. Moreover, AdhTrp2 group showed tumor rejection in 100% of challenged mice till the end of 3rd month while 60% of mice immunized with AdmTrp2 were protected against tumor. In the whole process of this experiment, no vitiligo was observed in mice immunized either with AdhTrp2 or AdmTrp2. It is concluded that anti-melanoma responses induced by genetic vaccination expressing xenoantigens breaks immune tolerance effectively and is able to elicit strong antigen-specific cytotoxic T cell response without vitiligo.

Stimulation of melanogenesis by glycyrrhizin in B16 melanoma cells

Glycyrrhizin (GR), triterpenoid saponin composed of one glycyrrhetinic acid (GA) and two glucuronic acids, is a main constituent of the hydrophilic fraction of licorice (Glycyrrhiza glabra) extracts and is known to have a wide range of pharmacological actions. In this study, we investigated the mechanism of GR effect on melanogenesis in B16 murine melanoma cells. The cellular levels of tyrosinase mRNA, protein, enzyme activities and melanin contents were increased by GR in a dose dependent manner. Expression of tyrosinase-related protein-2 (TRP-2) mRNA was also increased by GR, however, no significant change was observed on TRP-1. No cytotoxicity was observed at the effective concentration range of GR. GA showed no effect on melanogenesis at the equivalent nontoxic concentrations, indicating that glycoside structure is important in the stimulatory effect of GR on melanogenesis. These results indicate that GR-induced stimulation of melanogenesis is likely to occur through the transcriptional activation.

Assignments of the tyrosinase related protein-1 and -2 genes to human chromosome bands 9p23 and 13q32.1 by in situ hybridization

To determine the precise chromosomal localization of tyrosine related protein-1 and -2 (TRP-1 and TRP-2) genes by fluorescence in situ hybridization, we used DNAs isolated from human bacterial artificial chromosome clones. They contain genomic sequences with approximately 120 kb inserts for TRP-1 and TRP-2. The TRP-1 and TRP-2 genes were assigned to human chromosome bands 9p23 and 13q32.1, respectively. These results confirmed the previously mapped location for the TRP-1 gene and more precisely located the TRP-2 gene, which had previously been mapped to chromosome 13q31-q32.