A role for glutamate transporters in the regulation of insulin secretion

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.     

Characterisation and subcellular localisation of the GroEL-like and DnaK-like proteins isolated from Fusobacterium nucleatum ATCC 10953

Fusobacterium nucleatum is associated with periodontitis in humans, and is a central member of the dental biofilm. Heat shock proteins (HSPs) of many different bacteria have been considered to play important roles during inflammations and infections. We have identified and characterised the HSP60 and HSP70, the Escherichia coli GroEL and DnaK homologues, respectively, in F. nucleatum ATCC 10953. The N-terminal 22 amino acid residues of HSP60 exhibited up to 63.6% identity with members of the HSP60 heat shock protein family of some selected bacterial species, while the N-terminal of 25 residues of HSP70 revealed up to 80% identity with members of the HSP70 family. The subcellular localisation of HSP60 and HSP70 was analysed by immunoblotting of bacterial cell fractions and immunoelectron microscopy of whole cells. HSP60 and HSP70 were localised in the cytosol, associated with membranes and extracellular fractions. These results are consistent with localisation for HSPs found in other micro-organisms, which further lead to the suggestion of a potential role in the pathogenesis of infectious diseases.     

The phylogeny of orthoretroviral long terminal repeats (LTRs)

LTRs are sequence elements in retroviruses and retrotransposons which are difficult to align due to their variability. One way of handling such cases is to use Hidden Markov Models (HMMs). In this work HMMs of LTRs were constructed for three groups of orthoretroviruses: the betaretroviruslike human MMTV-like (HML) endogenous retroviruses, the lentiviruses, including HIV, and gammaretroviruslike human endogenous retroviruses (HERVs). The HMM-generated LTR alignments and the phylogenetic trees constructed from them were compared with trees based on alignments of the pol gene at the nucleic acid level. The majority of branches in the LTR and pol based trees had the same order for the three retroviral genera, showing that HMM methods are successful in aligning and constructing phylogenies of LTRs. The HML LTR tree deviated somewhat from the pol tree for the groups HML3, HML7 and HML6. Among the gammaretroviruslike proviruses, the exogenous Mouse Leukemia Virus (MLV) was highly related to HERV-T in the pol based tree, but not in the LTR based tree. Aside from these differences, the similarity between the trees indicates that LTRs and pol coevolved in a largely monophyletic way.     

Molecular Evidence that Only Two Opsin Subfamilies,the Blue Light- (SWS2) and Green Light-Sensitive (RH2), Drive Color Vision in Atlantic Cod (Gadus morhua)

Teleosts show a great variety in visual opsin complement, due to both gene duplication and gene loss. The repertoire ranges from one subfamily of visual opsins (scotopic vision) including rod opsin only retinas seen in many deep-sea species to multiple subfamilies of visual opsins in some pelagic species. We have investigated the opsin repertoire of Atlantic cod (Gadus morhua) using information in the recently sequenced cod genome and found that despite cod not being a deep sea species it lacks visual subfamilies sensitive towards the most extreme parts of the light spectra representing UV and red light. Furthermore, we find that Atlantic cod has duplicated paralogs of both blue-sensitive SWS2 and green-sensitive RH2 subfamilies, with members belonging to each subfamily linked in tandem within the genome (two SWS2-, and three RH2A genes, respectively). The presence of multiple cone opsin genes indicates that there have been duplication events in the cod ancestor SWS2 and RH2 opsins producing paralogs that have been retained in Atlantic. Our results are supported by expressional analysis of cone opsins, which further revealed an ontogenetic change in the array of cone opsins expressed. These findings suggest life stage specific programs for opsin regulation which could be linked to habitat changes and available light as the larvae is transformed into an early juvenile. Altogether we provide the first molecular evidence for color vision driven by only two families of cone opsins due to gene loss in a teleost.     

Finding Markers That Make a Difference: DNA Pooling and SNP-Arrays Identify Population Informative Markers for Genetic Stock Identification

Genetic stock identification (GSI) using molecular markers is an important tool for management of migratory species. Here, we tested a cost-effective alternative to individual genotyping, known as allelotyping, for identification of highly informative SNPs for accurate genetic stock identification. We estimated allele frequencies of 2880 SNPs from DNA pools of 23 Atlantic salmon populations using Illumina SNP-chip. We evaluated the performance of four common strategies (global F _ST, pairwise F _ST, Delta and outlier approach) for selection of the most informative set of SNPs and tested their effectiveness for GSI compared to random sets of SNP and microsatellite markers. For the majority of cases, SNPs selected using the outlier approach performed best followed by pairwise F _ST and Delta methods. Overall, the selection procedure reduced the number of SNPs required for accurate GSI by up to 53% compared with randomly chosen SNPs. However, GSI accuracy was more affected by populations in the ascertainment group rather than the ranking method itself. We demonstrated for the first time the compatibility of different large-scale SNP datasets by compiling the largest population genetic dataset for Atlantic salmon to date. Finally, we showed an excellent performance of our top SNPs on an independent set of populations covering the main European distribution range of Atlantic salmon. Taken together, we demonstrate how combination of DNA pooling and SNP arrays can be applied for conservation and management of salmonids as well as other species.     

Use of skin biopsies for assessing levels of organochlorines in walruses (Odobenus rosmarus)

Skin and blubber samples of ten adult male Pacific walruses (Odobenus rosmarus divergens) from Alaska were used to investigate the relationship between organochlorine (OC) levels in skin and blubber of individuals. For analyses we selected 11 components that were quantified in the blubber of all individuals: hexachlorocyclohexanes (αHCH and βHCH), the DDT (dichlorodiphenyltrichloroethane) metabolite p,p′DDE, oxychlordane, and 7 individual PCB congeners, 28, 99, 105, 118, 138, 153 and 180. The correlation between the levels in the two types of tissues was significant and the relation was isometric for all components. The regression coefficient between levels in blubber (dependent variable) and levels in skin (independent variable) was different from 1 for only four of the components. The mean levels in the two types of tissues were significantly different for 3 of the 11 chemical components (βHCH, oxychlordane, and PCB28). Although this analysis is based on only ten individuals, we propose that skin samples taken by biopsy darts can be used to monitor OC levels in walruses. In August 1993 skin biopsies were collected from 25 adult male Atlantic walruses (O. r. rosmarus) at haul-out sites in southeastern Svalbard in the Norwegian Arctic and from 28 walruses of different sex and age at haul-out sites at Franz Josef Land in the Russian Arctic. The mean levels of OCs were 2–10 times higher at Svalbard than at Franz Josef Land. The dominant OC component was PCB153 in both areas. A principal component analysis detected differences between areas in OC levels but not in patterns. Since the Franz Josef Land samples were mainly taken from females and young individuals and the Svalbard samples were taken largely from adult males, we believe the differences in tissue OC levels observed from these areas can be explained by differences in sex and age of the walrus sampled. Comparable organochlorine levels in skin samples from walruses from other areas are not available. However, compared to the corresponding OC levels found in walrus blubber in other areas, the OC levels from Svalbard and Franz Josef Land are higher. The high levels of OCs in walruses from Svalbard and Franz Josef Land may be a combined effect of high pollution level in the environment and seal-eating habits. In the present study we show that it is possible to use skin biopsies taken by a non-destructive method to assess OC levels in walruses. Accepted: 24 October 1999     

NT-proBNP independently predicts long term mortality after acute exacerbation of COPD – a prospective cohort study

Background

Cardiovascular disease is prevalent and frequently unrecognized in patients with chronic obstructive pulmonary disease (COPD). NT-proBNP is an established risk factor in patients with heart failure. NT-proBNP may also be released from the right ventricle. Thus serum NT-proBNP may be elevated during acute exacerbations of COPD (AECOPD). The prognostic value of NT-proBNP in patients hospitalized with AECOPD is sparsely studied. Our objective was to test the hypothesis that NT-proBNP independently predicts long term mortality following AECOPD.

Methods

A prospective cohort study of 99 patients with 217 admissions with AECOPD. Clinical, electrocardiographic, radiological and biochemical data were collected at index and repeat admissions and analyzed in an extended survival analysis with time-dependent covariables.

Results

Median follow-up time was 1.9 years, and 57 patients died during follow-up. NT-proBNP tertile limits were 264.4 and 909 pg/mL, and NT-proBNP in tertiles 1 through 3 was associated with mortality rates of 8.6, 35 and 62 per 100 patient-years, respectively (age-adjusted log-rank p<0.0001). After adjustment for age, gender, peripheral edema, cephalization and cTnT in a multivariable survival model, the corresponding hazard ratios for dying were 2.4 (0.95-6.0) and 3.2 (1.3-8.1) (with 95% confidence intervals in parentheses, p-value for trend 0.013).

Conclusions

NT-proBNP is a strong and independent determinant of mortality after AECOPD.     

Population cycles of autumnal moth, Epirrita autumnata, in relation to birch mast seeding

We investigated the relationship between flowering peaks of the mountain birch, Betula pubescens ssp. czerepanovii, and population levels of the autumnal moth Epirrita autumnata in Ammarnäs, Swedish Lapland, during 1968-2000, and in Budal, Central Norway, during 1972-2000. There was a significant correlation between the two moth series, both of which showed three well-defined population cycles during the study period. In both areas, the population growth index of the moth was negatively related to population size, but also to the number of years since the previous flowering peak of mountain birch. In the northern study area, Ammarnäs, there was an additional positive effect of the winter temperature index, probably due to increased mortality of moth eggs during cold winters. No significant relationships were found between the number of birch female catkins and larval density in the previous 2 years. Both in Ammarnäs and in an area without moth outbreaks in south-eastern Norway, birch flowering was positively related to temperatures during flower bud formation and to the number of years since the previous flowering peak. The results support the idea of a lower content of chemical defence compounds in birch leaves after a mast reproduction, though we cannot exclude the possibility that the negative relationship between flowering and moth population levels was influenced or caused by stress associated with defoliation during moth outbreaks.