The transcription factor 7-like 2 (TCF7L2) polymorphism may be associated with focal arteriolar narrowing in Caucasians with hypertension or without diabetes: the ARIC Study

Background

To investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (IA-2A)]. Furthermore the study aimed at determining whether mutations in KCNJ11, ABCC8, HNF1A, HNF4A or INS are common in AAB negative diabetes.

Materials and methods

In 261 newly diagnosed children with type 1 diabetes, we measured residual β-cell function, ICA, GADA, and IA-2A at 1, 6 and 12 months after diagnosis. The genes KCNJ11, ABCC8, HNF1A, HNF4A and INS were sequenced in subjects AAB negative at diagnosis. We expressed recombinant K-ATP channels in Xenopus oocytes to analyse the functional effects of an ABCC8 mutation.

Results

Twenty-four patients (9.1%) tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual β-cell function (P = 0.002), lower blood glucose (P = 0.004), received less insulin (P = 0.05) and had lower HbA_1c (P = 0.02) 12 months after diagnosis. One patient had a heterozygous mutation leading to the substitution of arginine at residue 1530 of SUR1 (ABCC8) by cysteine. Functional analyses of recombinant K-ATP channels showed that R1530C markedly reduced the sensitivity of the K-ATP channel to inhibition by MgATP. Morover, the channel was highly sensitive to sulphonylureas. However, there was no effect of sulfonylurea treatment after four weeks on 1.0-1.2 mg/kg/24 h glibenclamide.

Conclusion

GAD, IA-2A, and ICA negative children with new onset type 1 diabetes have slower disease progression as assessed by residual beta-cell function and improved glycemic control 12 months after diagnosis. One out of 24 had a mutation in ABCC8, suggesting that screening of ABCC8 should be considered in patients with AAB negative type 1 diabetes.     

Divalent metal addition restores sulfide-inhibited N2O reduction in Pseudomonas aeruginosa

Hydrogen sulfide (H₂S) inhibits the last step of the denitrification process, i.e. the reduction of nitrous oxide (N₂O) to dinitrogen gas (N₂), both in natural environments (marine sediments) and industrial processes (activated sludge, methanogenic sludge, BioDeNOx process). In a previously published study, we showed that the inhibitory effect of sulfide to N₂O reduction in mixed microbial communities is reversible and can be counteracted by dosing trace amounts of copper. It remained, however, unclear if this was due to copper sulfide precipitation or a retrofitting of the copper containing N₂O-reductase (N₂OR). The present study aimed to elucidate the mechanism of the restoration of sulfide-inhibited N₂O reducing activity by metal addition to a pure Pseudomonas aeruginosa culture. This was done by using other metals (zinc, cobalt and iron) in comparison with copper. Zinc and cobalt clearly alleviated the sulfide inhibition of N₂OR to the same extent as copper and the activity restoration was extremely fast (within 15 min, Fig. 3) for zinc, cobalt and copper. This suggests that the alleviation of the inhibitory effect of sulfide is due to metal sulfide precipitation and thus not exclusively limited to Cu. This work also underlines the importance of metal speciation: supply of iron did not restore the N₂OR activity because it was precipitated by the phosphates present in the medium and thus could not precipitate the sulfide.     

A population genetics study of Anopheles darlingi (Diptera: Culicidae) from Colombia based on random amplified polymorphic DNA-polymerase chain reaction and amplified fragment lenght polymorphism markers

The genetic variation and population structure of three populations of Anopheles darlingi from Colombia were studied using random amplified polymorphic markers (RAPDs) and amplified fragment length polymorphism markers (AFLPs). Six RAPD primers produced 46 polymorphic fragments, while two AFLP primer combinations produced 197 polymorphic fragments from 71 DNA samples. Both of the evaluated genetic markers showed the presence of gene flow, suggesting that Colombian An. darlingi populations are in panmixia. Average genetic diversity, estimated from observed heterozygosity, was 0.374 (RAPD) and 0.309 (AFLP). RAPD and AFLP markers showed little evidence of geographic separation between eastern and western populations; however, the F ST values showed high gene flow between the two western populations (RAPD: F ST = 0.029; Nm: 8.5; AFLP: F ST = 0.051; Nm: 4.7). According to molecular variance analysis (AMOVA), the genetic distance between populations was significant (RAPD:phiST = 0.084; AFLP:phiST = 0.229, P < 0.001). The F ST distances and AMOVAs using AFLP loci support the differentiation of the Guyana biogeographic province population from those of the Chocó-Magdalena. In this last region, Chocó and Córdoba populations showed the highest genetic flow.     

Involvement of oxidative stress in the impairment in biliary secretory function induced by intraperitoneal administration of aluminum to rats

We have shown that aluminum (Al) induces cholestasis associated with multiple alterations in hepatocellular transporters involved in bile secretory function, like Mrp2. This work aims to investigate whether these harmful effects are mediated by the oxidative stress caused by the metal. For this purpose, the capability of the antioxidant agent, vitamin E, to counteract these alterations was studied in male Wistar rats. Aluminum hydroxide (or saline in controls) was administered ip (27 mg/kg body weight, three times a week, for 90 d). Vitamin E (600 mg/kg body weight) was coadministered, sc. Al increased lipid peroxidation (+50%) and decreased hepatic glutation levels (-43%) and the activity of glutation peroxidase (-50%) and catalase (-88%). Vitamin E counteracted these effects total or partially. Both plasma and hepatic Al levels reached at the end of the treatment were significantly reduced by vitamin E (-40% and -44%, respectively;p< 0.05). Al increased 4 times the hepatic apoptotic index, and this effect was fully counteracted by vitamin E. Bile flow was decreased in Al-treated rats (-37%) and restored to normality by vitamin E. The antioxidant normalized the hepatic handling of the Mrp2 substrates, rose bengal, and dinitrophenyl-S-glutathione, which was causally associated with restoration of Mrp2 expression. Our data indicate that oxidative stress has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage, and the impairment in liver transport function induced by Al and that vitamin E counteracts these harmful effects not only by preventing free-radical formation but also by favoring Al disposal.     

Genetic transformation of <Emphasis Type="Italic">Agave salmiana</Emphasis> by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> and particle bombardment

Agave salmiana was transformed using two different protocols: co-cultivation with Agrobacterium tumefaciens and particle bombardment. The uidA (β-glucuronidase) gene was used as a reporter gene for both methods whereas the nptII and bar genes were used as selectable markers for A. tumefaciens and biolistic transformation respectively. Previous reports for in vitro regeneration of A. salmiana have not been published; therefore the conditions for both shoot regeneration and rooting were optimized using leaves and embryogenic calli of Agave salmiana. The transgenes were detected by Polymerase Chain Reaction (PCR) in 11 month old plants. The transgenic nature of the plants was also confirmed using GUS histochemical assays. Transformation via co-cultivation of explants with Agrobacterium harbouring the pBI121 binary vector was the most effective method of transformation, producing 32 transgenic plants and giving a transformation efficiency of 2.7%. On the other hand, the biolistic method produced transgenic calli that tested positive with the GUS assay after 14 months on selective medium while still undergoing regeneration.     

A genomewide admixture mapping panel for Hispanic/Latino populations

Admixture mapping (AM) is a promising method for the identification of genetic risk factors for complex traits and diseases showing prevalence differences among populations. Efficient application of this method requires the use of a genomewide panel of ancestry-informative markers (AIMs) to infer the population of origin of chromosomal regions in admixed individuals. Genomewide AM panels with markers showing high frequency differences between West African and European populations are already available for disease-gene discovery in African Americans. However, no such a map is yet available for Hispanic/Latino populations, which are the result of two-way admixture between Native American and European populations or of three-way admixture of Native American, European, and West African populations. Here, we report a genomewide AM panel with 2,120 AIMs showing high frequency differences between Native American and European populations. The average intermarker genetic distance is ~1.7 cM. The panel was identified by genotyping, with the Affymetrix GeneChip Human Mapping 500K array, a population sample with European ancestry, a Mesoamerican sample comprising Maya and Nahua from Mexico, and a South American sample comprising Aymara/Quechua from Bolivia and Quechua from Peru. The main criteria for marker selection were both high information content for Native American/European ancestry (measured as the standardized variance of the allele frequencies, also known as "f value") and small frequency differences between the Mesoamerican and South American samples. This genomewide AM panel will make it possible to apply AM approaches in many admixed populations throughout the Americas.     

CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type

PURPOSE: Although relatively rare, uveal melanoma is the most common ocular tumor of adults. Up to half of uveal melanoma patients die of metastatic disease. CXCR4, a chemokine receptor, is a prognostic factor in cutaneous melanoma involved in angiogenesis and metastasis formation. The aim of this study was to evaluate the expression of CXCR4 in uveal melanoma. METHODS: CXCR4 was detected by immunohistochemistry in 44 samples of uveal melanoma. Staining was categorized into three semiquantitative classes based on the rate of stained (positive) tumor cells: absence of staining, <50% of cell (+) and >50% (++). Correlations between CXCR4 expression, data on patient and tumor features were studied by contingency tables and the chi2 test. Time-to-event curves were studied using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test. Ninety-five percent confidence intervals (95% CI) of hazard ratios were also reported. RESULTS: Staining for CXCR4 protein was absent in 18 tumors (40.9%), present in <50% of cells in 19 (43.2%) and in >50% of cells in 7 (15.9%) tumors. CXCR4 expression correlated to the epithelioid-mixed cell type (P=0.030). No statistically significant relation emerged between CXCR4 expression, largest tumor diameter (LTD) and extracellular matrix patterns as evaluated through histological patterns stained with periodic acid-Schiff (PAS). Events occurred in 2 out of 18 patients (11.1%) with negative tumors (2 deaths), in 3 out of 19 patients (15.8%) with <50% of positive tumor cells (2 deaths and 1 occurrence of metastases) and in 1 out of 7 patients (14.3%) with >50% of positive tumor cells (1 occurrence of metastases). The cell type (P=0.0457) but not CXCR4 showed prognostic value at univariate analysis. CONCLUSION: This study shows that CXCR4 is commonly expressed in uveal melanoma and correlates with cell type a well-established prognostic factor.     

Quantitative effects of common genetic variations in the 3′UTR of the human LDL-receptor gene and their associations with plasma lipid levels in the Atherosclerosis Risk in Communities study

The low-density lipoprotein receptor (LDLR) plays a pivotal role in cholesterol homeostasis. However, the role of genetic variations in the 3′UTR of the LDLR in relation to plasma cholesterol has been largely understudied. Six SNPs, G44243A, G44332A, C44506G, G44695A, C44857T and A44964G, within the 5′ region of the 3′UTR fall into three common haplotypes, GGCGCA, AGCACG, and GGCGTA, occurring at frequencies of 0.45, 0.31 and 0.17, respectively, in Caucasians (n = 29) and 0.13, 0.13 and 0.38, respectively, in African Americans (n = 32), with three other haplotypes occurring at lesser frequencies. In a tissue culture based system, expression of a reporter gene carrying a 3′UTR that includes the 1 kb nucleotide sequences corresponding to the AGCACG or GGCGTA was 70 or 63%, respectively, of the same sequence with GGCGCA. Genotyping of two “haplotype tagging” SNPs, C44857T and A44964G, in the Atherosclerosis Risk in Communities (ARIC) study population showed that in Caucasians, but not in African Americans, the inferred TA haplotype had a significant LDL-cholesterol lowering effect. The adjusted LDL-cholesterol levels in the TA/TA diplotypes were lower by 6.10 mg/dl in men (P < 0.001) and by 4.63 mg/dl in women (P < 0.01) than in individuals with other diplotypes. Caucasian men homozygous for CA, in contrast, showed significantly higher LDL-cholesterol (P < 0.04), lower HDL-cholesterol (P < 0.02) and higher LDL/HDL ratios (P < 0.001). Thus our data shows that 3′UTR sequences that cause higher reporter gene expression in vitro are associated in Caucasians with plasma lipid profiles indicative of higher cardiovascular risk, suggesting that further studies of quantitative variants in the LDLR gene will be valuable. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at