Polymorphic sites away from the Bw4 epitope that affect interaction of Bw4+ HLA-B with KIR3DL1

KIR3DL1 is a polymorphic, inhibitory NK cell receptor specific for the Bw4 epitope carried by subsets of HLA-A and HLA-B allotypes. The Bw4 epitope of HLA-B*5101 and HLA-B*1513 is determined by the NIALR sequence motif at positions 77, 80, 81, 82, and 83 in the alpha(1) helix. Mutation of these positions to the residues present in the alternative and nonfunctional Bw6 motif showed that the functional activity of the Bw4 epitopes of B*5101 and B*1513 is retained after substitution at positions 77, 80, and 81, but lost after substitution of position 83. Mutation of leucine to arginine at position 82 led to loss of function for B*5101 but not for B*1513. Further mutagenesis, in which B*1513 residues were replaced by their B*5101 counterparts, showed that polymorphisms in all three extracellular domains contribute to this functional difference. Prominent were positions 67 in the alpha(1) domain, 116 in the alpha(2) domain, and 194 in the alpha(3) domain. Lesser contributions were made by additional positions in the alpha(2) domain. These positions are not part of the Bw4 epitope and include residues shaping the B and F pockets that determine the sequence and conformation of the peptides bound by HLA class I molecules. This analysis shows how polymorphism at sites throughout the HLA class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1. This influence is likely mediated by changes in the peptides bound, which alter the conformation of the Bw4 epitope.     

Placental Alpha Hemoglobin Stabilizing Protein (AHSP) and recurrent miscarriage

AHSP inhibits cellular production of the reactive oxygen species. Reduced AHSP indicates reduced protection against oxidative stressors. Our objective was to investigate AHSP levels in recurrent miscarriage (RM). Trophoblast was collected from women of 10 weeks gestation: voluntary abortion controls (VA, n = 10); spontaneous first miscarriage with subsequent normal pregnancy (SMSN, n = 15) or with subsequent miscarriage (SMSM, n = 5); RM previously investigated (RMPS, n = 5) or not previously investigated (RM, n = 5). AHSP mRNA and protein were determined using real-time quantitative polymerase chain reaction (PCR) and Western blot, respectively. One-way ANOVA was performed to assess statistical significance (p < 0.05). ahsp mRNA levels were maximally reduced in RM and RMPS (8.0 × 10⁻⁶ ± 1.3 and 8.1 × 10⁻⁶ ± 0.7, respectively) compared with SMSN and VA (16.1 × 10⁻⁶ ± 2.3 and 26.1 × 10⁻⁶ ± 2.7, respectively). SMSM showed levels significantly reduced as well (9.0 × 10⁻⁶ ± 2.3). In RM, a reduced defense from oxidative stressors is evident at first miscarriage, identifying women at high risk for subsequent eventful pregnancy. Reduced AHSP may identify women at risk of experiencing further miscarriages. Monica Emanuelli and Monia Cecati contributed equally to this paper.     

A model with space-varying regression coefficients for clustering multivariate spatial count data

Multivariate spatial count data are often segmented by unobserved space-varying factors that vary across space. In this setting, regression models that assume space-constant covariate effects could be too restrictive. Motivated by the analysis of cause-specific mortality data, we propose to estimate space-varying effects by exploiting a multivariate hidden Markov field. It models the data by a battery of Poisson regressions with spatially correlated regression coefficients, which are driven by an unobserved spatial multinomial process. It parsimoniously describes multivariate count data by means of a finite number of latent classes. Parameter estimation is carried out by composite likelihood methods, that we specifically develop for the proposed model. In a case study of cause-specific mortality data in Italy, the model was capable to capture the spatial variation of gender differences and age effects.     

Matrix metalloproteinase 3 and 9 as genetic biomarkers for the occurrence of cardiovascular complications in coronary artery disease: a prospective cohort study

Molecular Biology Reports - Matrix metalloproteinases (MMPs) are widely expressed in atherosclerosis lesions. The disequilibrium of MMPs driving to an overexpression or a lack of its level can be...     

Heavy Metal (Hg,Cr, Cd,and Pb) Contamination in Urban Areas and Wildlife Reserves: Honeybees as Bioindicators

The degree of heavy metal (Hg, Cr, Cd, and Pb) pollution in honeybees (Apis mellifera) was investigated in several sampling sites around central Italy including both polluted and wildlife areas. The honeybee readily inhabits all environmental compartments, such as soil, vegetation, air, and water, and actively forages the area around the hive. Therefore, if it functions in a polluted environment, plant products used by bees may also be contaminated, and as a result, also a part of these pollutants will accumulate in the organism. The bees, foragers in particular, are good biological indicators that quickly detect the chemical impairment of the environment by the high mortality and the presence of pollutants in their body or in beehive products. The experiment was carried out using 24 colonies of honeybees bred in hives dislocated whether within urban areas or in wide countryside areas. Metals were analyzed on the foragers during all spring and summer seasons, when the bees were active. Results showed no presence of mercury in all samples analyzed, but honeybees accumulated several amounts of lead, chromium, and cadmium. Pb reported a statistically significant difference among the stations located in urban areas and those in the natural reserves, showing the highest values in honeybees collected from hives located in Ciampino area (Rome), next to the airport. The mean value for this sampling station was 0.52 mg kg⁻¹, and July and September were characterized by the highest concentrations of Pb. Cd also showed statistically significant differences among areas, while for Cr no statistically significant differences were found.     

Potential application of two thermostable lichenases from a newly isolated Bacillus licheniformis UEB CF: Purification and characterization

Two thermostable and alkali-stable β-1,3–1,4 glucanases (EC 3.2.1.73) EG1 and EG2 from a newly isolated Bacillus licheniformis UEB CF were purified. The molecular weights of EG1 and EG2 enzymes determined by SDS–PAGE were approximately 30 kDa and 55 kDa, respectively. The N-terminal amino acid sequences of EG1 and EG2 β-glucanases were determined to be GAAPIKKGTTKLN and DINGGGATLPQK, respectively. The optimum temperature, optimum pH, k_m and V_max of EG1 were 70 °C, 5.0, 2.1 mg/ml and 21.25 μmol/min/mg, respectively. These values for EG2 were 60 °C, 7.0, 1.82 mg/ml and 18.54 μmol/min/mg, respectively.Both endoglucanases were highly active against barley β-glucan and lichenan. However, they were inactive against CMC and laminarin. The purified β-glucanases were found to be relatively stable toward non-ionic surfactants and oxidizing agents. In addition, both enzymes showed excellent stability and compatibility with a wide range of commercial solid detergents suggesting that they are a potential candidate in detergent industries formulation.     

Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is a major contributing factor to hepatic insulin resistance in type 2 diabetes. Diacylglycerol acyltransferase (Dgat), of which there are two isoforms (Dgat1 and Dgat2), catalyzes the final step in triglyceride synthesis. We evaluated the metabolic impact of pharmacological reduction of DGAT1 and -2 expression in liver and fat using antisense oligonucleotides (ASOs) in rats with diet-induced NAFLD. Dgat1 and Dgat2 ASO treatment selectively reduced DGAT1 and DGAT2 mRNA levels in liver and fat, but only Dgat2 ASO treatment significantly reduced hepatic lipids (diacylglycerol and triglyceride but not long chain acyl CoAs) and improved hepatic insulin sensitivity. Because Dgat catalyzes triglyceride synthesis from diacylglycerol, and because we have hypothesized that diacylglycerol accumulation triggers fat-induced hepatic insulin resistance through protein kinase C epsilon activation, we next sought to understand the paradoxical reduction in diacylglycerol in Dgat2 ASO-treated rats. Within 3 days of starting Dgat2 ASO therapy in high fat-fed rats, plasma fatty acids increased, whereas hepatic lysophosphatidic acid and diacylglycerol levels were similar to those of control rats. These changes were associated with reduced expression of lipogenic genes (SREBP1c, ACC1, SCD1, and mtGPAT) and increased expression of oxidative/thermogenic genes (CPT1 and UCP2). Taken together, these data suggest that knocking down Dgat2 protects against fat-induced hepatic insulin resistance by paradoxically lowering hepatic diacylglycerol content and protein kinase C epsilon activation through decreased SREBP1c-mediated lipogenesis and increased hepatic fatty acid oxidation.     

Specific reduction of hepatic glucose 6-phosphate transporter-1 ameliorates diabetes while avoiding complications of glycogen storage disease

D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.