3'-UTR polymorphism in the human CYP2A6 gene affects mRNA stability and enzyme expression

Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in human and participates in the metabolism of drugs and precarcinogens. The CYP2A6 gene is highly polymorphic and more than 22 different alleles have been described. We here focused on the polymorphism in the 3'-UTR region, in particular the common CYP2A6*1B allele, carrying an unequal crossover element from the pseudogene CYP2A7. Analysis of CYP2A6 expression in a human liver bank (n=46) revealed that the protein level and catalytic activity using coumarin as a substrate were all higher, following a linear gene-dose relationship, in livers carrying one or two copies of CYP2A6*1B, as compared to other CYP2A6 allelic variants. Different variants of the CYP2A6 3'-UTR were cloned into a modified pGL3 plasmid downstream of the luciferase reporter gene. The plasmids, having the proximal promoter of CYP2A6 gene, were transfected into HeLa cells or injected into the tail veins of male CD1 mice. In both systems, the 3'-UTR CYP2A6*1B constructs caused higher reporter gene activity and the CYP2A7 3'-UTR construct lower activity, compared to the CYP2A6*1 3'-UTR constructs. Two SNPs differentiating the 3'-UTR between CYP2A7 and CYP2A6*1B were found to be of importance for the expression in both systems. Analysis of reporter enzyme degradation in HeLa cells showed that luciferase-3'-UTR-CYP2A6*1A had a half-life of approximately 4.9h as compared to 6.3h for luciferase-3'-UTR-CYP2A6*1B. In conclusion, we identified polymorphic motifs in the CYP2A6 3'-UTR of importance for CYP2A6 mRNA stabilization and enzyme expression. Such polymorphism has been described to influence the in vivo rate of nicotine elimination and possibly the cigarette consumption and risk of smoking induced lung cancer.     

Large-Scale Production of Nanographite by Tube-Shear Exfoliation in Water

The number of applications based on graphene, few-layer graphene, and nanographite is rapidly increasing. A large-scale process for production of these materials is critically needed to achieve cost-effective commercial products. Here, we present a novel process to mechanically exfoliate industrial quantities of nanographite from graphite in an aqueous environment with low energy consumption and at controlled shear conditions. This process, based on hydrodynamic tube shearing, produced nanometer-thick and micrometer-wide flakes of nanographite with a production rate exceeding 500 gh^-1 with an energy consumption about 10 Whg^-1. In addition, to facilitate large-area coating, we show that the nanographite can be mixed with nanofibrillated cellulose in the process to form highly conductive, robust and environmentally friendly composites. This composite has a sheet resistance below 1.75 Ω/sq and an electrical resistivity of 1.39×10^-4 Ωm and may find use in several applications, from supercapacitors and batteries to printed electronics and solar cells. A batch of 100 liter was processed in less than 4 hours. The design of the process allow scaling to even larger volumes and the low energy consumption indicates a low-cost process.     

Scl1-dependent internalization of group A Streptococcus via direct interactions with the alpha2beta(1) integrin enhances pathogen survival and re-emergence

The molecular pathogenesis of infections caused by group A Streptococcus (GAS) is not fully understood. We recently reported that a recombinant protein derived from the collagen-like surface protein, Scl1, bound to the human collagen receptor, integrin α₂β₁. Here, we investigate whether the same Scl1 variant expressed by GAS cells interacts with the integrin α₂β₁ and affects the biological outcome of host–pathogen interactions. We demonstrate that GAS adherence and internalization involve direct interactions between surface expressed Scl1 and the α₂β₁ integrin, because (i) both adherence and internalization of the scl1- inactivated mutant were significantly decreased, and were restored by in-trans complementation of Scl1 expression, (ii) GAS internalization was reduced by pre-treatment of HEp-2 cells with anti-α₂ integrin-subunit antibody and type I collagen, (iii) recombinant α₂-I domain bound the wild-type GAS cells and (iv) internalization of wild-type cells was significantly increased in C2C12 cells expressing the α₂β₁ integrin as the only collagen-binding integrin. Next, we determined that internalized GAS re-emerges from epithelial cells into the extracellular environment. Taken together, our data describe a new molecular mechanism used by GAS involving the direct interaction between Scl1 and integrins, which increases the overall capability of the pathogen to survive and re-emerge.     

Short-Term Synaptic Plasticity Orchestrates the Response of Pyramidal Cells and Interneurons to Population Bursts

The synaptic drive from neuronal populations varies considerably over short time scales. Such changes in the pre-synaptic rate trigger many temporal processes absent under steady-state conditions. This paper examines the differential impact of pyramidal cell population bursts on post-synaptic pyramidal cells receiving depressing synapses, and on a class of interneuron that receives facilitating synapses. In experiment a significant shift of the order of one hundred milliseconds is seen between the response of these two cell classes to the same population burst. It is demonstrated here that such a temporal differentiation of the response can be explained by the synaptic and membrane properties without recourse to elaborate cortical wiring schemes. Experimental data is first used to construct models of the two types of dynamic synaptic response. A population-based approach is then followed to examine analytically the temporal synaptic filtering effects of the population burst for the two post-synaptic targets. The peak-to-peak delays seen in experiment can be captured by the model for experimentally realistic parameter ranges. It is further shown that the temporal separation of the response is communicated in the outgoing action potentials of the two post-synaptic cells: pyramidal cells fire at the beginning of the burst and the class of interneuron receiving facilitating synapses fires at the end of the burst. The functional role of such delays in the temporal organisation of activity in the cortical microcircuit is discussed.     

Crystal structure of human cystatin D, a cysteine peptidase inhibitor with restricted inhibition profile

Cystatins are natural inhibitors of papain-like (family C1) and legumain-related (family C13) cysteine peptidases. Cystatin D is a type 2 cystatin, a secreted inhibitor found in human saliva and tear fluid. Compared with its homologues, cystatin D presents an unusual inhibition profile with a preferential inhibition cathepsin S > cathepsin H > cathepsin L and no inhibition of cathepsin B or pig legumain. To elucidate the structural reasons for this specificity, we have crystallized recombinant human Arg(26)-cystatin D and solved its structures at room temperature and at cryo conditions to 2.5- and 1.8-A resolution, respectively. Human cystatin D presents the typical cystatin fold, with a five-stranded anti-parallel beta-sheet wrapped around a five-turn alpha-helix. The structures reveal differences in the peptidase-interacting regions when compared with other cystatins, providing plausible explanations for the restricted inhibitory specificity of cystatin D for some papain-like peptidases and its lack of reactivity toward legumain-related enzymes.     

Polar bear (<Emphasis Type="Italic">Ursus maritimus</Emphasis>) maternity den distribution in Svalbard,Norway

The aim of the current study was to identify maternity denning areas and to describe aspects of denning behaviour in polar bears (Ursus maritimus) in Svalbard, Norway. Den observations made during aerial and ground surveys (n = 553), observations of recently emerged family groups made during capture and tagging of bears (n = 206), and dens recorded by satellite telemetry (n = 76 dens) were used to describe the distribution of dens in the Barents Sea region. Data were collected during March, April, and May from 1972 to 2010, and while most dens were abandoned during the first 2 weeks of April, there was substantial variation. The highest number of dens was recorded on the islands in the eastern and northern parts of Svalbard with fewer dens found further west on the island Spitsbergen. Females with cubs-of-the-year or their tracks indicated some denning in southern, eastern, and northern parts of Spitsbergen. Telemetry also revealed that females captured in Svalbard and the Barents Sea den in Franz Josef Land, Russia. The majority of dens (62%) in Svalbard were located on land within ca. 1 km of the shore with the most inland den 19 km from shore. Heavy exploitation of polar bears before 1973, when a hunting moratorium was implemented, likely led to reduced denning in some areas. Our observations of den distribution indicate that denning is now more widespread in the archipelago than 50 years ago and reflects a reestablishment of denning areas following years of protection.     

Polyisoprenoid epoxides stimulate the biosynthesis of coenzyme Q and inhibit cholesterol synthesis

In our search for compounds that up-regulate the biosynthesis of coenzyme Q (CoQ), we discovered that irradiation of CoQ with ultraviolet light results in the formation of a number of compounds that influence the synthesis of mevalonate pathway lipids by HepG2 cells. Among the compounds that potently stimulated CoQ synthesis while inhibiting cholesterol synthesis, derivatives of CoQ containing 1-4 epoxide moieties in their polyisoprenoid side chains were identified. Subsequently, chemical epoxidation of all-trans-polyprenols of different lengths revealed that the shorter farnesol and geranylgeraniol derivatives were without effect, whereas the longer derivatives of solanesol enhanced CoQ and markedly reduced cholesterol biosynthesis. In contrast, none of the modified trans-trans-poly-cis-polyprenols exerted noticeable effects. Tocotrienol epoxides were especially potent in our system; those with one epoxide moiety in the side-chain generally up-regulated CoQ biosynthesis by 200-300%, whereas those with two such moieties also decreased cholesterol synthesis by 50-90%. Prolonged treatment of HepG2 cells with tocotrienol epoxides for 26 days elevated their content of CoQ by 30%. In addition, the levels of mRNA encoding enzymes involved in CoQ biosynthesis were also elevated by the tocotrienol epoxides. The site of inhibition of cholesterol synthesis was shown to be oxidosqualene cyclase. In conclusion, epoxide derivatives of certain all-trans-polyisoprenoids cause pronounced stimulation of CoQ synthesis and, in some cases, simultaneous reduction of cholesterol biosynthesis by HepG2 cells.     

Leukotriene Production Is Increased in Abdominal Obesity

Obesity is a major risk factor for insulin resistance and type-2 diabetes. A chronic low grade inflammatory state has been described during obesity and associated with insulin resistance pathogenesis. Results from animal studies are in favor of a role of the leukotriene (LT) pathway in obesity induced-insulin resistance. However, there is a paucity of data regarding this association in human obesity. Therefore, the aim of this study was to investigate whether LT production was associated with insulin resistance and other metabolic parameters in a cohort of obese subjects. Forty-six (70% females) obese subjects (BMI≧30 kg/m²) without known diabetes and without inflammatory disease (CRP<10 mg/l) were included. Median age was 44 years (16–80) with a median BMI of 36.8 kg/m² (30–51). Insulin resistance was evaluated by HOMA-IR index and glucose tolerance test. Urinary LTE₄ (U-LTE₄) concentration was measured by enzyme immune assay. Screening for obstructive sleep apnea was performed. There was a positive association of U-LTE₄ with waist to hip ratio, systolic blood pressure and HOMA-IR in univariate analysis. Further, waist to hip ratio remained the only parameter significantly correlated with U-LTE₄, in adjusted multivariate analysis. Taken together, these results confirm the previously established notion of chronic low grade inflammation in obesity and further suggests a role for the LT pathway in obesity-associated development of insulin resistance in humans.