Archives of Medical Research - Articles in Press

Amino Acids Potentiate Insulin Signaling in CHO-K1 at High Glucose Conditions - Uncorrected Proof

2012-05-18

Background and Aims: Amino acids reportedly increase the glucose uptake under high glucose conditions. However, there are controversies in the role of amino acids in diabetes mellitus. The present study explores the insulin signaling pathway involved in glucose uptake mediated by amino acids in CHO-K1 cells.Methods: CHO-K1 cells were exposed to normal (7 mM) and high glucose (17 and 27 mM) in the presence and absence of amino acid mixtures (AAM) in varying concentration (5 and 20 mM) followed by the assay insulin receptor tyrosine kinase (IRTK) and phosphatidylinositol 3 kinase (PI3K) by autoradiography, protein kinase B (Akt) and glucose transporter (GLUT4) by Western blot and glycogen synthase (GS) by HPLC.Results: The addition of 5 and 20 mM AAM significantly increased IRTK and PI3K activity (ANOVA p = 0.025, p = 0.003, respectively) with increasing glucose concentration. Addition of 5 mM AAM in the presence of normal glucose significantly increased the levels of phosphorylated Akt Ser473 (p = 0.02) with no significant change at high glucose. At 20 mM AAM there was a significant decrease in Akt phosphorylation (p = 0.035) that was increased by high glucose concentration. GLUT4 protein levels were increased with AAM (5 mM) along with increase in glycogen synthase activity at all glucose concentrations (p <0.05).Conclusions: Amino acids as a mixture is beneficial in augmenting insulin signaling pathway via IRTK/PI3K/GLUT4 pathway along with activation of GS in CHO-K1 cells, thereby ensuring increased intracellular glucose availability.

Identification of Two Novel Mutations in the SLC4A1 Gene in Two Unrelated Chinese Families with Distal Renal Tubular Acidosis - Uncorrected Proof

2012-05-18

Background and Aims: Distal renal tubular acidosis (dRTA) is characterized by a reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Mutations in the SLC4A1 gene have been found to cause either autosomal dominant (AD) or autosomal recessive (AR) dRTA.Methods: Four affected individuals and nine healthy family members from two unrelated Chinese families with dRTA were clinically studied. The SLC4A1 gene was screened and analyzed, and the mutations were confirmed using molecular genetic techniques.Results: In family1, the affected individuals had novel compound heterozygous SLC4A1 G494S/G701D mutations inherited from their clinically normal heterozygous father and mother, respectively. In family 2, the affected individuals exhibited a novel 3-bp duplication (c.2715_2717dupCGA) in exon 20 of SLC4A1 that led to the D905dup mutation. The age of presentation was younger, hypokalemia was more severe, and growth retardation was more severe in recessive patients in family 1 than patients with AD dRTA in family 2.Conclusions: This is the first report of dRTA patients with compound heterozygous conditions in mainland China. Two novel SLC4A1 mutations (G494S and D905dup) were identified. Our results confirm the importance of the C-terminal residues of the SLC4A1 gene product in normal acidification processes and indicate that mutations in this region are likely to result in AD dRTA. Our study extends the mutation spectrum of dRTA and is helpful in early molecular diagnoses of dRTA.

Reply: Upregulation of Target Genes by MiRNAs Is a Previously Found Phenomenon - Uncorrected Proof

Jie Dong, Yu-Pei Zhao, Li Zhou, Tai-Ping Zhang, Ge Chen — 2012-05-17

Thank you very much for your kindness for our manuscript entitled “Bcl-2 upregulation induced by miR-21 via a direct interaction is associated with apoptosis and chemoresistance in MIA PaCa-2 pancreatic cancer cells” . I have carefully read the comments offered by the readers. I hereby address their comments as below.

Perinatal Iodine Deficiency and Hypothyroidism Increase Cell Apoptosis and Alter Doublecortin and Reelin Protein Expressions in Rat Cerebellum - Uncorrected Proof

2012-05-17

Background and Aims: Adequate thyroid hormone is critical for cerebellar development. Developmental hypothyroidism induced by iodine deficiency during the perinatal period results in permanent impairments of cerebellar development with an unclear mechanism. In the present study we investigated effects of perinatal iodine deficiency and hypothyroidism on cerebellar cell apoptosis, doublecortin (Dcx) and reelin. Apoptosis is an essential part of neural development. Dcx and reelin are two important molecules involved in neuronal migration, structure, and development in cerebellum.Methods: Two developmental rat models were created by administering dam rats with either iodine-deficient diet or propylthiouracil (PTU, 5 ppm or 15 ppm)-added drinking water from gestational day (GD) 6 until postnatal day (PND) 28. TUNEL assay and protein levels of Dcx and reelin in cerebella were assessed on PND14, 21 and 28.Results: Apoptotic cells were increased in the iodine-deficient and PTU-treated rats. Dcx protein levels in the cerebella of iodine-deficient and PTU-treated rats were significantly downregulated on PND14. Interestingly, iodine deficiency and PTU treatment upregulated the levels of Dcx protein on PND21 and 28. Reelin expressions in iodine-deficient and PTU-treated rats were significantly decreased on PND14 and 21. On PND28, reelin expressions of three treated groups were still lower than control group, although without significant difference.Conclusions: These findings may implicate alterations in cell apoptosis and levels of Dcx and reelin in the impairments of cerebellar development induced by developmental iodine deficiency and hypothyroidism.

Serum Total Bilirubin Concentration Is Inversely Correlated with Framingham Risk Score in Koreans - Uncorrected Proof

2012-05-16

Background and Aims: Compelling evidence suggests that bilirubin, via its antioxidant potential, has anti-atherogenic properties, and that serum bilirubin concentrations within the reference range for the general population may provide some protection against coronary artery disease (CAD). This study examined the association between serum total bilirubin concentration and Framingham risk score (FRS) in the Korean population.Methods: This cross-sectional study was performed on 19,792 Koreans. In addition to FRS, data on body mass index, fasting blood glucose, liver enzymes, lipid profile, uric acid, gamma glutamyltransferase, high-sensitive C-reactive protein and total bilirubin were used.Results: Negative correlations were established between log-transformed total bilirubin concentration and FRS (females; r = −0.067, p <0.001, males; r = −0.128, p <0.001). Analyses relating total bilirubin to FRS ≥10% utilized multiple confounder adjusted logistic regression. Unadjusted odd ratios for FRS ≥10% were 0.325 (95% CI: 0.160–0.659, p = 0.002) and 0.342 (95% CI: 0.281–0.417, p <0.001) for log-transformed total bilirubin in females and males, respectively. These inverse relationships remained significant after adjustments for multiple confounders in both genders.Conclusions: Increased total bilirubin concentrations are associated with the decrease in FRS. Serum total bilirubin may be helpful to decrease the future risk of CAD.

Serious Error Regarding the Article ‘Bcl-2 Upregulation Induced by miR-21 Via a Direct Interaction Is Associated with Apoptosis and Chemoresistance in MIA PaCa-2 Pancreatic Cancer Cells’ - Uncorrected Proof

Tian-Hong Pan, Xiao-Yang Wu — 2012-05-07

We read the study by Dong et al. entitled “Bcl-2 upregulation induced by miR-21 via a direct interaction is associated with apoptosis and chemoresistance in MIA PaCa-2 pancreatic cancer cells” with great interest. We were very surprised that entirely incorrect and seemingly impossible results were reported.

Patients Exposed to Mycobacterium tuberculosis Infection with a Prominent IgE Response - Uncorrected Proof

2012-05-07

Background and Aims: Even though it has been reported that chronic immune activation associated with intestinal helminthic infections results in a predominant IgE response, specific IgE antibodies that are also interleukin 4 (IL-4) dependent have been reported in tuberculosis patients; however, this fact has not been widely reported. This study was aimed at investigating the levels of circulating IgE in Warao (an indigenous population) of the Orinoco river delta, an area isolated from contact with the tubercle bacillus for millennia until the mid-1960s as compared to Creole (nonindigenous population).Methods: A total of 294 individuals were studied, 161 Warao and 136 Creole. Patient group was comprised of 86 Warao patients (WP) and 60 Creole patients (CP). Control group was comprised of 75 Warao controls (WC) and 76 Creole controls (CC). Total serum IgE and IgE and IgG4 reactivities to M. tuberculosis antigens were measured by an enzyme-linked immunosorbent assay (ELISA).Results: Levels of total serum IgE were significantly elevated in WP (13002.0 ± 11200.0 IU/mL) and WC (2763.5 ± 2596.2 IU/mL) than in CP (385.9 ± 155.1 IU/mL) and CC (356.6 ± 157.5 IU/mL) (p <0.0001). Anti-PPD and anti-H37Rv IgE were significantly higher in WP (240 ± 145 and 230 ± 155) than in CP (127 ± 152 and 97 ± 103, respectively) and also between WC (240 ± 273 and 147 ± 158) and CC (115 ± 136 and 43 ± 46, respectively) (p <0.0001). Anti-PPD and anti-H37Rv IgG4 did not show differences among groups; however, anti-H37Rv IgG4 was affected by anti-TB treatment, which could be predictive of treatment outcome.Conclusions: The findings suggest that for the Warao population there is an intrinsic propensity to produce a high IgE response, which could be incompatible with the protective response to M. tuberculosis.

Effect of Acute Extremely Low Frequency Electromagnetic Field Exposure on the Antioxidant Status and Lipid Levels in Rat Brain - Uncorrected Proof

2012-05-07

Background and Aims: It is generally accepted that electromagnetic fields (EMF) can exert biological effects; however, the mechanisms by which EMF elicits responses are still unknown. The present study was designed to assess the immediate effects of acute EMF exposure, movement restriction, and the combination of both on the antioxidant systems and lipid content in the whole brain of rat.Methods: Thirty two male Wistar rats were arranged in four groups: control, EMF exposed, movement restrained (MR), and EMF + MR for 2 h. Rats were then sacrificed and their brains analyzed for superoxide dismutase and catalase activities, reduced glutathione, nitric oxide, total cholesterol, and triacylglycerol levels, as well as plasma corticosterone concentrations.Results: Acute exposure to EMF induces reduction in catalase and superoxide dismutase activities, whereas the combination of EMF + MR also decreases both reduced glutathione and nitric oxide levels. Our results show that the acute exposure to EMF does not induce elevation of stress-hormone corticosterone but impairs the antioxidant status in rat brain.Conclusions: Plasma corticosterone concentration and antioxidant data indicate that the acute exposure to EMF appears to be a mild stressor that leads to some adaptive responses due to the activation of systems controlling the brain oxidative balance.

Adverse Cardiovascular Effects of Concomitant Use of Proton Pump Inhibitors and Clopidogrel in Patients with Coronary Artery Disease: A Systematic Review and Meta-Analysis - Uncorrected Proof

Baotao Huang, Yan Huang, Yulin Li, Hongmei Yao, Xianchao Jing, He Huang, Jing Li — 2012-05-07

Background and Aims: Conclusions from clinical studies and previous meta-analyses were inconsistent regarding the cardiovascular effects of concomitant use of proton pump inhibitors (PPIs) and clopidogrel. As new studies are constantly emerging, we performed this meta-analysis to further assess the cardiovascular effects of concomitant use of PPIs and clopidogrel with a focus on individual PPIs.Methods: A systematic electronic literature search was conducted in EMBASE, MEDLINE, PubMed and Chinese Biomedical Literature Database (CBM) to identify the studies reporting on the association of concomitant use of PPIs and clopidogrel with adverse cardiovascular outcomes. A hand search of reference lists was performed to identify further studies. Only studies published in English or Chinese were included in this review.Results: Twenty seven full-text articles and five abstracts with 159,998 patients were included in meta-analysis. Concomitant use of PPIs and clopidogrel is associated with an increased risk of major cardiovascular events (MACE) (HR 1.40, 95% CI 1.19–1.64; OR 1.27, 95% CI 1.13–1.42) and acute coronary syndrome (HR 1.42, 95% CI 1.14–1.77; OR 1.42, 95% CI 1.08–1.87) but not with all-cause mortality (HR 1.30, 95% CI 0.91–1.86; OR 0.92, 95% CI 0.82–1.04), cardiovascular death (HR 1.21, 95% CI 0.60–2.43) and stent thrombosis (HR 1.52, 95% CI 0.87–2.65). In the analyses of individual PPIs, none of the PPIs is associated with an increased MACE risk except for pantoprazole (HR 1.52, 95% CI 1.18–1.94).Conclusions: Concomitant use of PPIs and clopidogrel in patients with coronary artery disease is associated with an increased risk of MACE or acute coronary syndrome, but there is insufficient evidence to conclude that there is an interaction between individual PPIs and clopidogrel.

Matrix Metalloproteinase-2 and -9 in Glioblastoma: A Trio of Old Drugs—Captopril, Disulfiram and Nelfinavir—Are Inhibitors with Potential as Adjunctive Treatments in Glioblastoma - Uncorrected Proof

Richard E. Kast, Marc E. Halatsch — 2012-05-07

Given the poor prognosis of glioblastoma, we have been investigating treatments adjunctive to the current standard of resection, irradiation and temozolomide. Our focus has been on exploring already-marketed medicines that have evidence of inhibiting growth factors previously identified as active and important in glioblastoma. In this short note we describe how previous research has demonstrated that the common angiotensin-converting enzyme (ACE) inhibitor captopril used to treat hypertension and for renal protection inhibits 72-kDa matrix metalloproteinase-2 and 92-kDa matrix metalloproteinase-9, which a separate body of research shows are used by glioblastoma cells to grow and invade. We review these bodies of data and combine them to conclude that captopril may slow glioblastoma progression. Two other drugs, the aldehyde dehydrogenase inhibitor disulfiram used to treat alcoholism and the anti-HIV protease inhibitor nelfinavir also have a database supporting their incidental inhibition of matrix metalloproteinases. Given the importance of matrix metalloproteinases in helping glioblastomas grow and invade, we suggest that this trio—captopril, disulfiram, and nelfinavir—be tested for antiglioblastoma activity.

Detection of Phytoconstituents in Column Fractions of n-Hexane Extract of Goldcrest Honey Exhibiting Anti-Helicobacter pylori Activity - Uncorrected Proof

Christy E. Manyi-Loh, Anna M. Clarke, Roland N. Ndip — 2012-05-07

Background and Aims: Alternative therapy for Helicobacter pylori eradication from natural products is gaining much attention. This study sought to isolate and characterize the fraction responsible for the antibacterial activity in Goldcrest (GC) n-hexane extract.Methods: Thin-layer chromatography (TLC) of the extract was carried out on Silica gel plates to determine the presence of chemical compounds, which were separated and partially purified by column chromatography. The obtained fractions GCCL, GCF2, GCF3 and GCF4 were tested for anti-H. pylori activity using the broth microdilution method. Volatile compounds in the active fractions were identified by gas chromatography–mass spectrometry (GC-MS) analysis. MINITAB was used for statistical analysis at 95% confidence interval.Results: The best antibacterial activity was exhibited by GCF3 (5 mg/mL), which was composed of many compounds with known antimicrobial and antioxidant properties. A total of 16 volatile compounds were identified from fractions GCF2, GCF3 and GCF4 into the following families; alcohol, ketone, aliphatic acid, benzene compound, hydrocarbon, furan and pyran derivatives.Conclusions: The demonstration of antibacterial activity by the column fractions of GC n-hexane extract may provide new lead molecules that could serve as selective agents for H. pylori chemotherapy and control.

Trichostatin A Ameliorates Myocardial Ischemia/Reperfusion Injury Through Inhibition of Endoplasmic Reticulum Stress-induced Apoptosis - Uncorrected Proof

Ling Yu, Mengmeng Lu, Ping Wang, Xia Chen — 2012-05-07

Background and Aims: Trichostatin A (TSA) is a potent histone deacetylase inhibitor and widely used as a promising anticancer agent. Recently, a novel insight for TSA has been shown to protect the heart from ischemia/reperfusion (I/R) injury in mice, but the underlying mechanism remains unclear. The purpose of this study is to investigate whether TSA can influence endoplasmic reticulum stress (ERS) and whether its cardioprotective effect is mediated by inhibiting myocardial ERS-induced apoptosis in rats.Methods: Male Wistar rats were used and pretreated with saline or TSA (0.05, 0.1 and 0.2 mg·kg−1) once daily i.p. for 5 days. I/R model was established by occlusion/release of the left anterior descending coronary artery.Results: TSA significantly reduced myocardial infarct size and plasma activities of lactate dehydrogenase and creatine kinase in a dose-dependent manner in rats. Accompanied by the reduced injury, TSA also markedly reduced I/R-induced myocardial apoptosis (30 min/24 h) by the TUNEL assay. In addition, increased expression of glucose-regulated protein 78 (an ERS marker) by Western blot showed the effects of TSA on ERS. Induction of C/EBP homologous protein (CHOP), a critical mediator for ERS-induced apoptosis, was attenuated by TSA after reperfusion for 6 h and 24 h.Conclusions: Our findings showed that inhibition of histone deacetylase ameliorated I/R-induced myocardial injury in vivo and for the first time provided the evidence that suppression of CHOP expression and attenuation of the CHOP-induced apoptosis may contribute to the cardioprotection of TSA against myocardial I/R injury.

Association Between MTHFR C677T Polymorphism and Diabetic Nephropathy or Diabetes Mellitus Risk: Need for Clarification of Data in a Recent Meta-analysis - Uncorrected Proof

Wei-wei Chang, Yue-long Jin, Liu Zhang, Yan Chen, Ying-shui Yao — 2012-04-05

We read with great interest the recent article, “Is the C677T Polymorphism in Methylenetetrahydrofolate Reductase Gene a Risk Factor for Diabetic Nephropathy or Diabetes Mellitus in a Chinese Population?” published online in Archives of Medical Research . The meta-analysis by Cui et al. included 12 case–control studies on the association between C667T polymorphism in the MTHFR gene and diabetes mellitus (DM) or diabetic nephropathy (DN), which were comprised of 808 DN cases, 1134 DM cases and 826 healthy controls. The results suggested that the C677T polymorphism in the MTHFR gene may be a risk factor for DN, but not for DM, in a Chinese population. It is an interesting study. Nevertheless, there are some comments we would like to raise related to this article.

Reply: Association Between MTHFR C677T Polymorphism and Diabetic Nephropathy or Diabetes Mellitus Risk: Need for Clarification of Data in a Recent Meta-analysis - Uncorrected Proof

Wen-Peng Cui, Bing Du, Li-Ning Miao — 2012-04-04

We are grateful to the author who shared the Letter about his comments and suggestions regarding several issues of our meta-analysis to the editor . After reviewing the author's comments and carefully checking the original studies, we are pleased to reply and to clarify his questions, one by one, related to our paper published in Archives of Medical Research .

Association Between PPAR-γ and RXR-α Gene Polymorphism and Metabolic Syndrome Risk: A Case-Control Study of a Chinese Han Population - Uncorrected Proof

2012-04-03

Background: Polymorphisms in peroxisome proliferator activated receptor-γ (PPAR-γ) and retinoid X receptor-α (RXR-α) gene may alter metabolic syndrome (MetS) risks by increasing or decreasing the human adiponectin promoter activity in cells. To test this statement, three potentially functional SNPs of PPAR-γ and four SNPs of RXR-α with minor allele frequency (MAF) ≥0.05 in the Chinese Han population were identified from NCBI dbSNPs database to evaluate their associations with MetS.Methods: TaqMan assay was performed to test the genotypes in MetS patients (n = 901) and normal controls (n = 1009). Serum adiponectin concentration was measured by ELISA kit.Results: The variant genotypes rs2920502CG and CG/CC, rs4240711GG and AG/GG, rs4842194CC and CT/CC, rs3132291CT, CC and CT/CC were associated with MetS. Furthermore, in the haplotype of PPAR-γ gene, compared with the most common haplotype GC, haplotype CC was associated with an increased risk of MetS (crude p = 0.017). In the haplotype of RXR-α gene, haplotype GCGC was associated with a significant protective effect for MetS [adjusted p = 0.002, OR (95% CI) = 0.718 (0.585–0.882)] compared with the most common haplotype GTAT. After taking smoking, alcohol consumption and physical activity as environmental adjustment factors into the analysis, the result showed A1 A2 A4 A5 A6 A7 B1 (rs3856806, rs2920502, rs180128, rs1045570, rs3132291, rs4240711, rs4842194) was the best model (cross-validation consistency 10/10, p = 0.0107).Conclusions: The present study suggested that the variant genotypes in PPAR-γ gene could increase the risk of MetS; however, genotypes in RXR-α gene could decrease the risk of MetS in a Chinese Han population.

Interaction Between Endothelial Nitric Oxide Synthase Gene Polymorphisms (−86T>C, 894G>T and Intron 4 a/b) and Cardiovascular Risk Factors in Acute Coronary Syndrome - Uncorrected Proof

2012-04-03

Background and Aims: Endothelial rupture of coronary plaque can represent the pathomorphological substratum of acute coronary syndrome (ACS). Polymorphisms in the NOS3 gene (eNOS) –786T>C, 894G>T and intron 4 a/b VNTR can be associated with a higher susceptibility for ACS. The present study is focused on the investigation of the interaction of these polymorphisms and cardiovascular risk factors in 135 patients with ACS and 115 control subjects.Methods: Case–control study where the allele and genotype frequencies of the polymorphisms –786T> C, 894G> T and intron 4 VNTR of the gene encoding eNOS were determined by PCR-RFLP associated with cardiovascular risk factors.Results: An association of the 894TT genotype and 894GT+GG (OR 1.4; 95% CI 1.0–1.8) in ACS has been observed. Subjects without dyslipidemia and intron 4 a/b genotype present a lower chance for ACS development, whereas subjects without diabetes and 894TT genotype show a higher risk for ACS (OR 1.7; 95% CI 1.2–2.3). In patients without dyslipidemia, the 894GG genotype presented a tendency to behave as a protector factor against ACS. Also, the 894GG genotype has been a protective factor for ACS in females (OR 0.5; CI 95% 0.2–0.9).Conclusions: Our results suggest that eNOS polymorphisms may be an additional risk factor in development of ACS.

Targeted Next Generation Sequencing Reveals a Novel Intragenic Deletion of the TPO Gene in a Family with Intellectual Disability - Uncorrected Proof

2012-03-02

Backgrounds and Aims: Next generation sequencing (NGS) approaches have revolutionized the identification of mutations underlying genetic disorders. This technology is particularly useful for the identification of mutations in known and new genes for conditions with extensive genetic heterogeneity. In the present study we investigated a consanguineous Pakistani family with intellectual disability (ID).Methods: Genotyping was carried out using 250k and 6k SNP microarrays in order to perform homozygosity mapping and copy number variation (CNV) analysis. Targeted NGS was performed to identify the genetic defect in this family. qPCR was performed to validate and confirm the NGS result.Results: Homozygosity mapping positioned the causative defect on chromosome 2p25.3–p25.2. Subsequent targeted NGS revealed an intragenic deletion of five exons of the gene TPO.Conclusions: NGS is a powerful method to uncover submicroscopic structural variations. This result demonstrates that an unbiased screening approach such as NGS can help to identify even unexpected disease-causing mutations.