Reinvestigation of the role of snapin in neurotransmitter release

Snapin, a 15-kDa protein, has been identified recently as a binding partner of SNAP-25. Moreover, snapin is regulated by phosphorylation and enhances synaptotagmin binding to SNAREs. Furthermore, snapin and C-terminal snapin fragments have been effective in changing the release properties of neurons and chromaffin cells. Here we have reinvestigated the role of snapin using both biochemical and electrophysiological approaches. Snapin is ubiquitously expressed at low levels with no detectable enrichment in the brain or in synaptic vesicles. Using non-equilibrium and equilibrium assays including pulldown experiments, co-immunoprecipitations, and CD and fluorescence anisotropy spectroscopy, we were unable to detect any specific interaction between snapin and SNAP-25. Similarly, overexpression of a C-terminal snapin fragment in hippocampal neurons failed to influence any of the analyzed parameters of neurotransmitter release. Initial biochemical characterization of recombinant snapin revealed that the protein is a stable dimer with a predominantly alpha-helical secondary structure. We conclude that the postulated role of snapin as a SNARE regulator in neurotransmitter release needs reconsideration, leaving the true function of this evolutionarily conserved protein to be discovered.     

Conformational changes of murine polyomavirus capsid proteins induced by sialic acid binding

Murine polyomavirus (Py) infection initiates by the recognition of cell membrane molecules containing terminal sialic acid (SA) residues through specific binding pockets formed at the major capsid protein VP1 surface. VP1 Pockets 1, 2, and 3 bind terminal SA, Gal, and second branched SA, respectively. The consequence of recognition on viral cell entry remains elusive. In this work, we show that preincubation of Py with soluble compounds within Pocket 1 (N-acetyl or N-glycolyl neuraminic acids) increases Py cell binding and infectivity in murine 3T6 fibroblasts. In contrast, Gal does not significantly alter Py binding nor infectivity, whereas sialyllactose, in Pockets 1 and 2, decreases cell binding and infectivity. Binding experiments with Py virus-like particles confirmed the direct involvement of VP1 in this effect. To determine whether such results could reflect VP1 conformational changes induced by SA binding, protease digestion assays were performed after pretreatment of Py or virus-like particles with soluble receptor fragments. Binding of SA with the VP1 Pocket 1, but not of compounds interacting with Pocket 2, was associated with a transition of this protein from a protease-sensitive to a protease-resistant state. This effect was transmitted to the minor capsid proteins VP2 and VP3 in virus particles. Attachment of Py to cell monolayers similarly led to a VP1 trypsin-resistant pattern. Taken together, these data present evidence that initial binding of Py to terminal SA induces conformational changes in the viral capsid, which may influence subsequent virus cell entry steps.     

A new type of NADH dehydrogenase specific for nitrate respiration in the extreme thermophile Thermus thermophilus

A four-gene operon (nrcDEFN) was identified within a conjugative element that allows Thermus thermophilus to use nitrate as an electron acceptor. Three of them encode homologues to components of bacterial respiratory chains: NrcD to ferredoxins; NrcF to iron-sulfur-containing subunits of succinate-quinone oxidoreductase (SQR); and NrcN to type-II NADH dehydrogenases (NDHs). The fourth gene, nrcE, encodes a membrane protein with no homologues in the protein data bank. Nitrate reduction with NADH was catalyzed by membrane fractions of the wild type strain, but was severely impaired in nrc::kat insertion mutants. A fusion to a thermophilic reporter gene was used for the first time in Thermus spp. to show that expression of nrc required the presence of nitrate and anoxic conditions. Therefore, a role for the nrc products as a new type of membrane NDH specific for nitrate respiration was deduced. Consistent with this, nrc::kat mutants grew more slowly than the wild type strain under anaerobic conditions, but not in the presence of oxygen. The oligomeric structure of this Nrc-NDH was deduced from the analysis of insertion mutants and a two-hybrid bacterial system. Attachment to the membrane of NrcD, NrcF, and NrcN was dependent on NrcE, whose cytoplasmic C terminus interacts with the three proteins. Interactions were also detected between NrcN and NrcF. Inactivation of nrcF produced solubilization of NrcN, but not of NrcD. These data lead us to conclude that the Nrc proteins form a distinct third type of bacterial respiratory NDH.     

Non-verbal communication of the residents living in homes for the older people in Slovenia

Aging of the population is a growing problem in all developed societies. The older people need more health and social services, and their life quality in there is getting more and more important. The study aimed at determining the characteristics of non-verbal communication of the older people living in old people's homes (OPH). The sample consisted of 267 residents of the OPH, aged 65-96 years, and 267 caregivers from randomly selected twenty-seven OPH. Three types of non-verbal communication were observed and analysed using univariate and multivariate statistical methods. In face expressions and head movements about 75% older people looked at the eyes of their caregivers, and about 60% were looking around, while laughing or pressing the lips together was rarely noticed. The differences between genders were not statistically significant while statistically significant differences among different age groups was observed in dropping the eyes (p = 0.004) and smiling (0.008). In hand gestures and trunk movements, majority of older people most often moved forwards and clenched fingers, while most rarely they stroked and caressed their caregivers. The differences between genders were statistically significant in leaning on the table (p = 0.001), and changing the position on the chair (0.013). Statistically significant differences among age groups were registered in leaning forwards (p = 0.006) and pointing to the others (p = 0.036). In different modes of speaking and paralinguistic signs almost 75% older people spoke normally, about 70% kept silent, while they rarely quarrelled. The differences between genders were not statistically significant while statistically significant differences among age groups was observed in persuasive speaking (p = 0.007). The present study showed that older people in OPH in Slovenia communicated significantly less frequently with hand gestures and trunk movements than with face expressions and head movements or different modes of speaking and paralinguistic signs. The caregivers should be aware of this and pay a lot of attention to these two groups of non-verbal expressions. Their importance should be constantly emphasized during the educational process of all kinds of health-care professionals as well.     

Nonverbal transitive inference: Effects of task and awareness on human performance

We studied human nonverbal transitive inference in two paradigms: with choice stimuli orderable along a physical dimension and with non-orderable choice stimuli. We taught 96 participants to discriminate four overlapping pairs of choice stimuli: A+ B−, B+ C−, C+ D−, and D+ E−. Half of the participants were provided with post-choice visual feedback stimuli which were orderable by size; the other half were not provided with orderable feedback stimuli. In later testing, we presented novel choice pairs: BD, AC, AD, AE, BE, and CE. We found that transitive responding depended on task awareness for all participants. Additionally, participants given ordered feedback showed clearer task awareness and stronger transitive responding than did participants not given ordered feedback. Associative models ( [Wynne, 1995] and [Siemann and Delius, 1998]) failed to predict the increase in transitive responding with increasing awareness. These results suggest that ordered and non-ordered transitive inference tasks support different patterns of performance.     

Nucleoside diphosphate kinase B knock-out mice have impaired activation of the K+ channel KCa3.1, resulting in defective T cell activation

Nucleoside diphosphate kinases (NDPKs) are encoded by the Nme (non-metastatic cell) gene family. Although they comprise a family of 10 genes, NDPK-A and -B are ubiquitously expressed and account for most of the NDPK activity. We previously showed that NDPK-B activates the K(+) channel KCa3.1 via histidine phosphorylation of the C terminus of KCa3.1, which is required for T cell receptor-stimulated Ca(2+) flux and proliferation of activated naive human CD4 T cells. We now report the phenotype of NDPK-B(-/-) mice. NDPK-B(-/-) mice are phenotypically normal at birth with a normal life span. Although T and B cell development is normal in NDPK-B(-/-) mice, KCa3.1 channel activity and cytokine production are markedly defective in T helper 1 (Th1) and Th2 cells, whereas Th17 function is normal. These findings phenocopy studies in the same cells isolated from KCa3.1(-/-) mice and thereby support genetically that NDPK-B functions upstream of KCa3.1. NDPK-A and -B have been linked to an astonishing array of disparate cellular and biochemical functions, few of which have been confirmed in vivo in physiological relevant systems. NDPK-B(-/-) mice will be an essential tool with which to definitively address the biological functions of NDPK-B. Our finding that NDPK-B is required for activation of Th1 and Th2 CD4 T cells, together with the normal overall phenotype of NDPK-B(-/-) mice, suggests that specific pharmacological inhibitors of NDPK-B may provide new opportunities to treat Th1- and Th2-mediated autoimmune diseases.