Tissue distribution and early developmental expression patterns of aldolase A, B, and C in grass carp Ctenopharyngodon idellus.

Aldolase is a key enzyme involved in glycolysis, gluconeogenesis, and the pentose phosphate pathway. To establish the expression patterns of all three aldolase isozyme genes in different tissues and during early embryogenesis in lower vertebrates, as well as to explore the functional differences between these three isozymes, the grass carp was selected as a model owing to its relatively high glucose-metabolizing capability. Based on the cDNA sequences of the aldolase A, B, and C genes, the expression patterns of these three isozymes were analyzed in different tissues and during early embryogenesis using quantitative real-time polymerase chain reaction (qRT-PCR). Sequence analysis of cDNAs indicated that aldolase A, B, and C (GenBank accession numbers: KM192250, KM192251, and KM192252) consist of 364, 364, and 363 amino acids, respectively. The qRT-PCR results showed that the expression levels of aldolase A, B, and C were highest in the muscle, liver, and brain, respectively. Aldolase A and C exhibited similar expression patterns during embryogenesis, with high levels observed in unfertilized and fertilized eggs and at the blastocyst stage, followed by a decline and then increase after organogenesis. In contrast, aldolase B transcript was not detected during the unfertilized egg stage, and appeared only from gastrulation; the expression increased markedly during the feeding period (72 h after hatching), at which point the level was higher than those of aldolase A and C. These data suggest that the glucose content of grass carp starter feed should be adjusted according to the metabolic activity of aldolase B.

Dynamic relationship between SIPA1 gene and protein expression and the development of gastric cancer.

Association of signal-induced proliferation-associated 1 (SIPA) gene and protein expression with gastric cancer development was examined. SIPA1 mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and western blot, respectively, in 40 gastric tumor and tumor-adjacent normal tissues. SIPA1, VEGF-A, and FVIII levels in 60 gastric tumor and 40 tumor-adjacent normal tissues were examined by immunohistochemical staining. Correlations between SIPA1, VEGF-A, and microvessel density (MVD) were analyzed. SIPA1 mRNA levels were significantly lower in tumor tissues than in tumor-adjacent normal tissues (P < 0.05). Similarly, protein levels were significantly lower in tumor tissues (0.3043 ± 0.1062) than in tumor-adjacent normal tissues (0.5423 ± 0.0682, P < 0.05). Positive staining rates of SIPA1 (48.3%) and VEGF-A (36.7%) were lower and higher, respectively, in tumor tissues than in tumor-adjacent normal tissues (65.0 and 2.5%, P < 0.05). Positive protein staining rates in tumor tissues correlated with the degree of differentiation, lymph node metastases, and clinical grading (P < 0.05) and not with sex, age, or tumor size (P > 0.05). Significantly higher MVD (57.4 ± 9.3) was observed in tumor tissues displaying positive SIPA1 staining than in tumor-adjacent normal tissues (41.2 ± 5.7, P < 0.05). SIPA1 and VEGF-A expression in tumor tissues were negatively correlated (r = -0.736, P < 0.05). SIPA1 and its protein may play important roles in gastric cancer invasion, metastasis, and biological behavior. Low SIPA1 levels in gastric cancer may accelerate tumor development and progression by promoting VEGF-A expression to increase vascular density.

Association between expression of DNA mismatch repair genes and clinical features and prognosis of patients with radical resection of colon cancer.

The aim of this study was to investigate the clinical significance of the expression of DNA mismatch repair (MMR) genes in patients subjected to radical surgical removal of colon cancer, as well as their correlation with disease prognosis. Ninety stage II and III colon cancer patients who received laparoscopic radical resection of colon cancer at our hospital were recruited in this study. The expression of hMLH1, hMSH2, hMSH6, and hPMS2 in the resected tumor tissues was examined by SP immunohistochemistry, in order to analyze the relationship between defective DNA MMR (dMMR) and the clinico-pathological features and prognosis of colon cancer. Patients were followed up over a period of 5-35 months, and the Kaplan-Meier survival curve was plotted. dMMR was confirmed in 27 subjects (30.0%), among whom recurrence with metastasis and death was reported in 5 (18.5%) and 2 (7.4%) patients, respectively. The remaining 63 subjects displayed proficient DNA MMR (pMMR); among these, 19 (30.2%) and 7 (11.1%) recurrences with metastasis and death were reported, respectively. dMMR showed no significant correlation with gender, age, or therapeutic modality (P > 0.05), but was significantly correlated with the degree of differentiation, tumor location, number of resected lymph nodes, presence of ileus, and TNM stage (P < 0.05). The prognosis of patients with dMMR was better than that of patients with pMMR. dMMR serves as a biomarker for the prognosis of stage II/III colon cancers.

Aldehyde dehydrogenase 2 protects human umbilical vein endothelial cells against oxidative damage and increases endothelial nitric oxide production to reverse nitroglycerin tolerance.

Medical nitroglycerin (glyceryl trinitrate, GTN) use is limited principally by tolerance typified by a decrease in nitric oxide (NO) produced by biotransformation. Such tolerance may lead to endothelial dysfunction by inducing oxidative stress. In vivo studies have demonstrated that aldehyde dehydrogenase 2 (ALDH2) plays important roles in GTN biotransformation and tolerance. Thus, modification of ALDH2 expression represents a potentially effective strategy to prevent and reverse GTN tolerance and endothelial dysfunction. In this study, a eukaryotic expression vector containing the ALDH2 gene was introduced into human umbilical vein endothelial cells (HUVECs) by liposome-mediated transfection. An indirect immunofluorescence assay showed that ALDH2 expression increased 24 h after transfection. Moreover, real-time polymerase chain reaction and western blotting revealed significantly higher ALDH2 mRNA and protein expression in the gene-transfected group than in the two control groups. GTN tolerance was induced by treating HUVECs with 10 mM GTN for 16 h + 10 min, which significantly decreased NO levels in control cells, but not in those transfected with ALDH2. Overexpression of ALDH2 increased cell survival against GTN-induced cytotoxicity and conferred protection from oxidative damage resulting from nitrate tolerance, accompanied by decreased production of intracellular reactive oxygen species and reduced expression of heme oxygenase 1. Furthermore, ALDH2 overexpression promoted Akt phosphorylation under GTN tolerance conditions. ALDH2 gene transfection can reverse and prevent tolerance to GTN through its bioactivation and protect against oxidative damage, preventing the development of endothelial dysfunction.

Association between interleukin-10 gene polymorphisms and susceptibility to diabetic nephropathy in a Chinese population.

In this study, we investigated the association between the interleukin (IL)-10 -592C/A, -819C/T, and -1082G/A genetic variations and susceptibility to diabetic nephropathy in a Chinese population. The IL-10 -592C/A, -819C/T, and -1082G/A polymorphisms were genotyped in diabetic nephropathy patient and control samples by polymerase chain reaction-restriction fragment length polymorphism. The results were then statistically analyzed using SPSS 17.0. The results of the χ(2) test revealed a significant difference in the frequencies of the GG, GA, and AA genotypes of IL-10 -1082G/A between patients with diabetic nephropathy and control subjects (χ(2) = 10.03, P = 0.007). Unconditional logistic regression analysis revealed that the AA genotype of IL-10 -1082G/A significantly increased the susceptibility to diabetic nephropathy [adjusted odds ratio (OR) = 2.52, 95% confidence interval (CI) = 1.31-4.82] compared to the wild-type genotype. Moreover, the A allele of this polymorphism was associated with an increased risk of diabetic nephropathy compared to the G allele (adjusted OR = 1.51, 95%CI = 1.15-1.99). However, the IL-10 -819T/C and -592A/C genetic polymorphisms did not increase the risk of diabetic nephropathy. In conclusion, the IL-10 -1082G/A polymorphism was found to be correlated with the development of diabetic nephropathy.

Upregulation of salivary α2 macroglobulin in patients with type 2 diabetes mellitus.

We investigated the expression of salivary α2-macroglobulin (α2-MG) in patients with type 2 diabetes mellitus (T2DM) to investigate its value for predicting damage to the salivary glands. A total of 116 patients with T2DM and 60 patients with impaired fasting glucose (IFG) were included in this study. Sixty health volunteers were enrolled as a control group. Unstimulated saliva was collected at 8 a.m. prior to breakfast. Expression of α2-MG was determined using an enzyme-linked immunosorbent assay. The correlation between salivary α2-MG, serum α2-MG, and concentration of fasting glucose was analyzed using Pearson correlation analysis. No significant difference was observed in the expression of serum α2-MG in the T2DM group, IFG group, and control group (P > 0.05). Compared with the control group and IFG group, a statistical difference was observed in the salivary α2-MG in the T2DM group (P < 0.01). No statistical difference was observed in the salivary α2-MG in the IFG group compared with the control group (P > 0.05). In the patients with T2DM, a close correlation was identified in the expression of serum α2-MG and salivary α2-MG (r = 0.52, P < 0.01). A poor correlation was identified between salivary α2-MG and blood sugar level (r = -0.12, P = 0.199). The expression of salivary α2-MG showed a remarkable increase in T2DM patients, which may be associated with functional disorders of the salivary gland.

Transcriptome analysis of the grass carp (Ctenopharyngodon idella) using 454 pyrosequencing methodology for gene and marker discovery.

Total RNA isolated from the brain, muscle, liver, gonad, and intestinal tissues of grass carp was pooled to construct cDNA libraries. Using 454 pyrosequencing, a total of 738,604 high-quality reads were generated from the normalized cDNAs of the pooled individuals. Clustering and assembly of these reads produced a set of 37,086 all-unigene sequences after BLAST. Of these, 24,010 (64.74%) were annotated in the National Center for Biotechnology Information database, and 3715 simple sequence repeats and 2008 single nucleotide polymorphisms were identified in this EST dataset as potential molecular markers. This study provides new data for functional genomic and biological research on grass carp. The markers identified in this study will enrich the currently used molecular markers and facilitate marker-assisted selection in grass carp-breeding programs. These results also demonstrate that transcriptomic analysis based on 454 sequencing is a powerful tool for gene discovery and molecular marker development in non-model species.

Role of heme oxygenase-1 in demethylating effects on SKM-1 cells induced by decitabine.

We evaluated the influence of heme oxygenase-1 (HO-1) gene inhibition in myelodysplastic syndrome (MDS) cell line SKM-1 on enhancement of the demethylating effects of decitabine on p15, and explored the possible mechanism. DNMT1 gene expression in SKM-1 cells was silenced by being transfected by a constructed siRNA with liposomes. The proliferation inhibition rates after drug treatment were detected by cell counting kit-8 assay. The apoptotic rates were detected by Annexin V/PI assay with flow cytometry. The expressions of p16, p15, TP73, CDH1, ESR1, and PDLIM4 mRNAs were detected by real-time PCR, and those of HO-1, DNMT1, DNMT3A, DNMT3B, HDAC, and p15 proteins were measured by western blot. The degree of methylation of the p15 gene was analyzed by using methylation-specific PCR (MSP). CCK-8 assay showed that after HO-1 gene expression was inhibited; the proliferation rate of SKM-1 cells treated by decitabine (70.91 ± 0.05%) was significantly higher than that of the control group (53.67 ± 0.05%). Flow cytometry showed that the apoptotic rate of SKM- 1 cells treated by decitabine in combination with HO-1 expression inhibition (44.25 ± 0.05%) exceeded that of the cells treated by this drug alone (37.70 ± 0.05%). MSP showed that inhibiting HO-1 expression significantly increased the degree of methylation of the p15 gene. As suggested by western blot, the degree of methylation of the p15 protein was changed after decitabine treatment when the expression of the HO-1 protein was changed, being associated with the affected DNMT1 expression. Inhibited HO-1 expression attenuated the hypermethylation of CDKN2B by suppressing DNMT1, which was conducive to treatment by cooperating with decitabine. In conclusion, the findings of this study provide valuable experimental evidence for targeted MDS therapy, and a theoretical basis for further studies.

Effects of mycophenolate mofetil on the expression of monocyte chemoattractant protein-1 and fibronectin in high glucose cultured human mesangial cells.

The effects of high glucose on the expression of monocyte chemoattractant protein-1 (MCP-1) and the main component of the extracellular matrix, fibronectin (FN), were explored in human mesangial cells (HMCs), along with the intervention effects of mycophenolate mofetil (MMF) on these indicators. Cultured HMCs were divided into five groups: 1) normal control group (5 mM glucose); 2) high glucose group (30 mM glucose); 3) mannitol osmotic pressure control group (5 mM glucose + 25 mM mannitol); 4) high glucose + MMF-10 group (30 mM glucose + 10 µg/mL MMF); 5) high glucose + MMF-100 group (30 mM glucose + 100 µg/mL MMF). At 24, 48, and 72 h, reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay methods were used to detect the effects of MMF on MCP-1 mRNA and protein and FN expression in HMCs under high glucose conditions. MCP-1 mRNA and protein expressions and FN secretion significantly increased in HMCs of the high glucose group compared with the normal control group (P < 0.01), with the highest expression observed at 48 h. MMF could reduce the MCP-1 mRNA and protein and FN expression levels (P < 0.01), and the inhibition occurred in a dose- and time-dependent manner (P < 0.05). In conclusion, MMF could inhibit MCP-1 expression and the secretion of FN, indicating that it may delay the progression of glomerulosclerosis and interstitial fibrosis in diabetic nephropathy to ultimately achieve protective effects on the kidney.

Association of GSTTI and GSTM1 variants with acute myeloid leukemia risk.

We aimed to investigate the relationships between polymorphisms of the glutathione S-transferases (GSTs) GSTM1, GSTTI, and GSTP1 and the risk of developing acute myeloid leukemia (AML). A total of 206 AML cases and 231 controls were collected for our study. The genotyping of GSTs (GSTM1, GSTTI, and GSTP1) was based upon the duplex polymerase chain reaction with the confronting two-pair primer (PCR-CTPP) method. Individuals carrying null GSTTI and GSTM1 genotypes had a 1.52- and 1.78-fold increased risk of developing acute leukemia, respectively, compared to non-null genotype carriers (P < 0.05). A high risk was observed in those carrying a combination of null genotypes of GSTM1 and GSTTI with GSTP1-Val allele genotypes when compared with those carrying wild-type genotypes, with an odds ratio (95% confidence interval) of 3.62 (1.53-8.82) (P < 0.05). These findings indicate that genetic variants of GSTTI and GSTM1 significantly increase the risk of developing AML. Our study offers important insights into the molecular etiology of AML.