Endocytosis of synaptotagmin 1 is mediated by a novel,tryptophan-containing motif

The rate at which a membrane protein is internalized from the plasma membrane can be regulated by revealing a latent internalization signal in response to an appropriate stimulus. Internalization of the synaptic vesicle membrane protein, synaptotagmin 1, is controlled by two distinct regions of its intracytoplasmic C2B domain, an internalization signal present in the 29 carboxyterminal (CT) amino acids and a separate regulatory region. We have now characterized the internalization motif by mutagenesis and found that it involves an essential tryptophan in the last beta strand of the C2B domain, a region that is distinct from the AP2-binding site previously described. Internalization through the tryptophan-based motif is sensitive to eps15 and dynamin mutants and is therefore likely to be clathrin mediated. A tryptophan-to-phenylalanine mutation had no effect on internalization of the CT domain alone, but completely inhibited endocytosis of the folded C2B domain. This result suggests that recognition of sorting motifs can be influenced by their structural context. We conclude that endocytosis of synaptotagmin 1 requires a novel type of internalization signal that is subject to regulation by the rest of the C2B domain.     

Plasma matrix metalloproteinases, low density lipoprotein oxidisability and soluble adhesion molecules after a glucose load in Type 2 diabetes

Background

Type 2 diabetes mellitus (DM-2) is one of the most prevalent chronic diseases of the aged and contributes to a significant amount of cardiovascular disease morbidity and mortality. Exercise training may be beneficial in attenuating the cardiovascular maladaptations associated with DM-2. The purpose of this study was to examine the effects of exercise training on left ventricular (LV) and vascular function in a sample of postmenopausal women with DM-2.

Methods

Twenty-eight postmenopausal women with DM-2 (age: 59 ± 7 yrs) were assigned to either an exercise training (ET) (n = 17) or control group (CT) (n = 7). Cardiorespiratory fitness ( ), LV filling dynamics and arterial compliance were assessed at baseline in all participants. The ET group performed a supervised aerobic and resistance training intervention three days per week for a period of 10 weeks, while the CT group continued normal activities of daily living.

Results

Body mass index, , age and duration of diabetes were similar between the ET and CT groups at baseline. (21.3 ± 3.3 to 24.5 ± 4.2 ml·kg^-1·min^-1, p < 0.05) and large artery compliance (1.0 ± 0.4 to 1.2 ± 0.4 mL·mmHg^-1, p < 0.05), increased significantly in the ET group following training despite no change in LV filling dynamics, blood pressure, lipid profile or insulin sensitivity. All variables remained unchanged in the CT group.

Conclusions

Exercise training improves large artery compliance and cardiorespiratory fitness in postmenopausal women with DM-2, without any appreciable changes in LV filling dynamics or conventional risk factors for cardiovascular disease.     

Frequency of angiotensin II type 1 receptor gene polymorphism in Turkish acute stroke patients

This study was performed in acute stroke patients in the Turkish population to determine the frequency of the A1166C polymorphism in the AT1 gene and to examine the role of this polymorphism in acute stroke development. In this study, 257 genomic DNA samples were analysed (from 206 acute stroke patients and 51 healthy individuals). Genomic DNA was prepared from peripheral blood using the salt‐extraction method. The presence of the A1166C polymorphism in the AT1 gene was determined using the polymerase chain reaction (PCR)‐restriction fragment length polymorphism (RFLP) method. PCR products were separated by 2% agarose gel electrophoresis and visualized by a charge‐coupled device (CCD) camera. In this study, the allele frequency at the A1166C position was 92% A and 8% C for control and 97% A and 3% C for patients. This difference in allele frequency between the control group and the patient group was not statistically significant. However, genotype and allele frequencies showed a significant difference (P < 0.001) in the control and the patient groups. The results of this study show no relationship between the A1166C polymorphism in the AT1 gene and acute stroke in the Turkish population.     

A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release

The function of synaptotagmin as a Ca²⁺ sensor in neurotransmitter release involves Ca²⁺-dependent phospholipid binding to its two C₂ domains, but this activity alone does not explain why Ca²⁺ binding to the C₂B domain is more critical for release than Ca²⁺ binding to the C₂A domain. Synaptotagmin also binds to SNARE complexes, which are central components of the membrane fusion machinery, and displaces complexins from the SNAREs. However, it is unclear how phospholipid binding to synaptotagmin is coupled to SNARE binding and complexin displacement. Using supported lipid bilayers deposited within microfluidic channels, we now show that Ca²⁺ induces simultaneous binding of synaptotagmin to phospholipid membranes and SNARE complexes, resulting in an intimate quaternary complex that we name SSCAP complex. Mutagenesis experiments show that Ca²⁺ binding to the C₂B domain is critical for SSCAP complex formation and displacement of complexin, providing a clear rationale for the preponderant role of the C₂B domain in release. This and other correlations between the effects of mutations on SSCAP complex formation and their functional effects in vivo suggest a key role for this complex in release. We propose a model whereby the highly positive electrostatic potential at the tip of the SSCAP complex helps to induce membrane fusion during release.     

Structures of neuroligin-1 and the neuroligin-1/neurexin-1 beta complex reveal specific protein-protein and protein-Ca2+ interactions

Neurexins and neuroligins provide trans-synaptic connectivity by the Ca2+-dependent interaction of their alternatively spliced extracellular domains. Neuroligins specify synapses in an activity-dependent manner, presumably by binding to neurexins. Here, we present the crystal structures of neuroligin-1 in isolation and in complex with neurexin-1 beta. Neuroligin-1 forms a constitutive dimer, and two neurexin-1 beta monomers bind to two identical surfaces on the opposite faces of the neuroligin-1 dimer to form a heterotetramer. The neuroligin-1/neurexin-1 beta complex exhibits a nanomolar affinity and includes a large binding interface that contains bound Ca2+. Alternatively spliced sites in neurexin-1 beta and in neuroligin-1 are positioned nearby the binding interface, explaining how they regulate the interaction. Structure-based mutations of neuroligin-1 at the interface disrupt binding to neurexin-1 beta, but not the folding of neuroligin-1 and confirm the validity of the binding interface of the neuroligin-1/neurexin-1 beta complex. Our results provide molecular insights for understanding the role of cell-adhesion proteins in synapse function.     

Optimizing Carbon Storage Within a Spatially Heterogeneous Upland Grassland Through Sheep Grazing Management

Livestock grazing is known to influence carbon (C) storage in vegetation and soil. Yet, for grazing management to be used to optimize C storage, large scale investigations that take into account the typically heterogeneous distribution of grazers and C across the landscape are required. In a landscape-scale grazing experiment in the Scottish uplands, we quantified C stored in swards dominated by the widespread tussock-forming grass species Molinia caerulea. The impact of three sheep stocking treatments (‘commercial’ 2.7 ewes ha^?1 y^?1, ‘low’ 0.9 ewes ha^?1 y^?1 and no livestock) on plant C stocks was determined at three spatial scales; tussock, sward and landscape, and these data were used to predict long-term changes in soil organic carbon (SOC). We found that tussocks were particularly dense C stores (that is, high C mass per unit area) and that grazing reduced their abundance and thus influenced C stocks held in M. caerulea swards across the landscape; C stocks were 3.83, 5.01 and 6.85 Mg C ha^?1 under commercial sheep grazing, low sheep grazing and no grazing, respectively. Measured vegetation C in the three grazing treatments provided annual C inputs to RothC, an organic matter turnover model, to predict changes in SOC over 100 years. RothC predicted SOC to decline under commercial sheep stocking and increase under low sheep grazing and no grazing. Our findings suggest that no sheep and low-intensity sheep grazing are better upland management practices for enhancing plant and soil C sequestration than commercial sheep grazing. This is evaluated in the context of other upland management objectives.     

Assessing the impact of within crop heterogeneity (‘patchiness’) in young Miscanthus × giganteus fields on economic feasibility and soil carbon sequestration

In Ireland, Miscanthus × giganteus has the potential to become a major feedstock for bioenergy production. However, under current climatic conditions, Ireland is situated on the margin of the geographical range where Miscanthus production is economically feasible. It is therefore important to optimize the yield and other ecosystem services such as carbon sequestration delivered by the crop. A survey of commercial Miscanthus fields showed a large number of areas with no Miscanthus crop cover. These patches can potentially lead to reduced crop yields and soil carbon sequestration and have a significant negative impact on the economic viability of the crop. The aim of this research is to assess patchiness on a field scale and to analyse the impacts on crop yield and soil carbon sequestration. Analysis of aerial photography images was carried out on six commercial Miscanthus plantations in south east Ireland. The analysis showed an average of 372.5 patches per hectare, covering an average of 13.7% of the field area. Using net present value models and a financial balance approach it was shown that patchiness has a significant impact on payback time for initial investments and might reduce gross margins by more than 50%. Total and Miscanthus‐derived soil organic carbon was measured in open patches and adjacent plots of high crop density showing significantly lower Miscanthus‐derived carbon stocks in open patches compared to high crop‐density patches (0.47Mg C ha^?1 ± 0.42 SD and 0.91Mg C ha^?1 ± 0.55 SD). Using geographic information system (GIS) it was shown that on a field scale Miscanthus‐derived carbon stocks were reduced by 7.38% ± 7.25 SD. However, total soil organic carbon stocks were not significantly different between open patches and high crop density plots indicating no impact on the overall carbon sequestration on a field scale over 3–4 years since establishment for these Miscanthus sites.     

Malondialdehyde,Antioxidant Enzymes,and Renal Tubular Functions in Children with Iron Deficiency or Iron-Deficiency Anemia

We aimed to investigate the effects of iron deficiency (ID) or iron-deficiency anemia (IDA) on oxidative stress and renal tubular functions before and after treatment of children. A total of 30 children with a diagnosis of IDA constituted the IDA group and 32 children with a diagnosis of ID constituted the ID group. Control group consisted 38 age-matched children. Serum ferritin, soluble transferrin receptor (sTfR), serum, and urinary sodium (Na), potassium (K), calcium (Ca), phosphorus (P), creatinine (Cr), uric acid (UA), urinary N-acetyl-β-d-glucosaminidase (NAG) levels, and intra-erythrocyte malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) levels were measured before and after iron therapy in the IDA and ID groups, whereas it was studied once in the control group. We have divided the study group in groups according to age (infants <2 years, children 3–9 years, and adolescents 10–15 years). Patients with IDA (infant, adolescent) and ID (infant, children, and adolescent) had a significantly high level of MDA in post-treatment period in comparison to those of healthy control. Patients with IDA (children, adolescent) and ID (infant, children) had a significantly high level of pre-treatment GSH-Px than controls. Post-treatment SOD was lower in IDA (children and adolescent) groups than control and post-treatment CAT was lower in IDA and ID (adolescent) groups than control. These findings show that ferrous sulfate used in the treatment of ID or IDA could lead to oxidative stress; however, a marked deterioration of in proximal renal tubular functions was not seen.