The SLO-1 BK channel of Caenorhabditis elegans is critical for muscle function and is involved in dystrophin-dependent muscle dystrophy

The Caenorhabditis elegans SLO-1 channel belongs to the family of calcium-activated large conductance BK potassium channels. SLO-1 has been shown to be involved in neurotransmitter release and ethanol response. Here, we report that SLO-1 also has a critical role in muscles. Inactivation of the slo-1 gene in muscles leads to phenotypes similar to those caused by mutations of the dystrophin homologue dys-1. Notably, slo-1 mutations result in a progressive muscle degeneration when put into a sensitized genetic background. slo-1 localization was observed by gfp reporter gene in both the M-line and the dense bodies (Z line) of the C.elegans body-wall muscles. Using the inside-out configuration of the patch clamp technique on body-wall muscle cells of acutely dissected wild-type worms, we characterized a Ca2+-activated K+ channel that was identified unambiguously as SLO-1. Since neither the abundance nor the conductance of SLO-1 was changed significantly in dys-1 mutants compared to wild-type animals, it is likely that the inactivation of dys-1 causes a misregulation of SLO-1. All in all, these results indicate that SLO-1 function in C.elegans muscles is related to the dystrophin homologue DYS-1.     

Proteomic profiling of Tectona grandis L. leaf

Tectona grandis L. (teak) is one of the premier hardwood timbers in the world, ranking at present in the top five tropical hardwood species in terms of worldwide plantation area. Characterization of the proteins present in teak leaves will provide a basis for the development of new tools aimed at assisting tree selection, the monitoring of plant propagation, and the certification of clonal and phenotypic identities. In this paper, we describe the extraction, separation, and identification of leaf proteins from T. grandis using a TCA/acetone protocol, 2DE, and MALDI-TOF. After TCA/acetone protein extraction of leaves, 998 well-resolved spots were detected in Coomassie-stained gels within the 10-114 kDa relative molecular mass (Mr) range at a pH ranging from 3 to 11. A total of 120 spots were digested and subjected to MS. Of these, 100 nonredundant protein species were successfully identified. Functional classification of the identified proteins revealed that proteins involved in photosynthesis, protein translation, and energy production were the most abundant. This work is the first high-throughput attempt to study the T. grandis leaf proteome and represents a stepping stone for further differential expression proteomic studies related to growth, development, biomass production, and culture-associated physiological responses.     

Correlations between the modelled potato crop yield and the general atmospheric circulation

Biology-related indicators do not usually depend on just one meteorological element but on a combination of several weather indicators. One way to establish such integral indicators is to classify the general atmospheric circulation into a small number of circulation types. The aim of present study is to analyse connections between general atmospheric circulation and potato crop yield in Estonia. Meteorologically possible yield (MPY), calculated by the model POMOD, is used to characterise potato crop yield. Data of three meteorological stations and the biological parameters of two potato sorts were applied to the model, and 73 different classifications of atmospheric circulation from catalogue 1.2 of COST 733, domain 05 are used to qualify circulation conditions. Correlation analysis showed that there is at least one circulation type in each of the classifications with at least one statistically significant (99%) correlation with potato crop yield, whether in Kuressaare, Tallinn or Tartu. However, no classifications with circulation types correlating with MPY in all three stations at the same time were revealed. Circulation types inducing a decrease in the potato crop yield are more clearly represented. Clear differences occurred between the observed geographical locations as well as between the seasons: derived from the number of significant circulation types, summer and Kuressaare stand out. Of potato varieties, late 'Anti' is more influenced by circulation. Analysis of MSLP maps of circulation types revealed that the seaside stations (Tallinn, Kuressaare) suffer from negative effects of anti-cyclonic conditions (drought), while Tartu suffers from the cyclonic activity (excessive water).     

Chromogranin A Promotes Peptide Hormone Sorting to Mobile Granules in Constitutively and Regulated Secreting Cells: ROLE OF CONSERVED N- AND C-TERMINAL PEPTIDES*S?

Chromogranin A (CgA) has been proposed to play a major role in the formation of dense-core secretory granules (DCGs) in neuroendocrine cells. Here, we took advantage of unique features of the frog CgA (fCgA) to assess the role of this granin and its potential functional determinants in hormone sorting during DCG biogenesis. Expression of fCgA in the constitutively secreting COS-7 cells induced the formation of mobile vesicular structures, which contained cotransfected peptide hormones. The fCgA and the hormones coexpressed in the newly formed vesicles could be released in a regulated manner. The N- and C-terminal regions of fCgA, which exhibit remarkable sequence conservation with their mammalian counterparts were found to be essential for the formation of the mobile DCG-like structures in COS-7 cells. Expression of fCgA in the corticotrope AtT20 cells increased pro-opiomelanocortin levels in DCGs, whereas the expression of N- and C-terminal deletion mutants provoked retention of the hormone in the Golgi area. Furthermore, fCgA, but not its truncated forms, promoted pro-opiomelanocortin sorting to the regulated secretory pathway. These data demonstrate that CgA has the intrinsic capacity to induce the formation of mobile secretory granules and to promote the sorting and release of peptide hormones. The conserved terminal peptides are instrumental for these activities of CgA.Eukaryotic cells share the capacity to rapidly secrete proteins through the constitutive secretory pathway. The fundamental feature of neuroendocrine and endocrine cells is the occurrence of dense-core secretory granules (DCGs),³ which are key cytoplasmic organelles responsible for secretion of hormones, neuropeptides, and neurotransmitters through the regulated secretory pathway (RSP). Storage at high concentrations of these secretory products is required for their finely tuned release in response to extracellular stimulation (1, 2). DCG biogenesis starts with the budding of immature secretory granules (ISGs) from the trans-Golgi network (TGN) through interactions between lipid rafts and protein components, in a similar manner to constitutive vesicle budding (2, 3). The ISG budding is followed by a multistep maturation process to form the mature secretory granules, including removal of the constitutive secretory proteins and lysosomal enzymes inadvertently packaged into ISGs (4).Despite increasing knowledge of the various steps of DCG formation, the nature of the sorting signals for entry of proteins into the DCGs and the molecular machinery required to generate secretory granules are not fully elucidated (5, 6). Several recent studies highlighted the role of members of the granin family, which may represent the driving force for granulogenesis in the TGN (2), although this notion has been a matter of debate (7). Granins are soluble acidic proteins widely distributed in endocrine and neuroendocrine cells, which are characterized by the ability to aggregate at acidic pH and a high Ca²⁺ environment (8, 9). These conditions are found in the lumen of the TGN allowing granins to aggregate in this compartment and to be segregated from constitutively secreted proteins (10, 11). The granin aggregates are believed to associate directly or indirectly with lipid rafts at the TGN to induce budding and formation of the ISGs. A prominent role of chromogranin A (CgA) in the regulation of DCG formation in endocrine and neuroendocrine cells has been proposed. Thus, depletion of CgA in PC12 cells led to a dramatic decrease in the number of DCGs (12), and exogenously expressed CgA in these depleted PC12 cells, as in DCG-deficient endocrine A35C and 6T3 cells, restored DCG biogenesis (12, 13). Besides, expression of granins in non-endocrine, constitutively secreting cells such as CV-1, NIH3T3, or COS-7 cells provoked the formation of DCG-like structures that release their content in response to Ca²⁺ influx (12, 14, 15). Further investigations performed in CgA null mice and transgenic mice expressing antisense RNA against CgA also revealed a reduction in the number of DCGs in chromaffin cells that was associated with an impairment of catecholamine storage, thus demonstrating the crucial role of CgA in normal DCG biogenesis (16, 17). In CgA knockout mice, the introduction of the gene expressing human CgA restored the regulated secretory phenotype (16). A different CgA null mice strain exhibited no discernable effect on DCG formation, but elevated catecholamine secretion (18), proving that CgA deficiency is associated with hormone storage impairment in neuroendocrine cells in vivo, a finding that was confirmed in vitro (19). The CgA^-/- mice strain generated by Hendy et al. (18) exhibited a compensatory overexpression of other granins, pointing to a possible overlap in granin function in secretory granule biogenesis.We reported previously that the frog CgA (fCgA) gene is coordinately regulated with the pro-opiomelanocortin (POMC) gene in the pituitary pars intermedia during the neuroendocrine reflex of skin color change, which allows amphibia to adapt to their environment through the release of POMC-derived melanotropic peptides (20, 21). Sequence comparison of fCgA with its mammalian orthologs revealed a high conservation of the N- and C-terminal domains, and far less conservation of the central part of the protein (Fig. 1A), suggesting that these domains may play a role in DCG formation and hormone release in various species (9, 20, 21). To assess the role of fCgA and its conserved N- and C-terminal regions in hormone sorting, storage, and secretion, we engineered different constructs that produce the native unmodified (no tag added) protein and truncated forms lacking the conserved N- and C-terminal domains, and we developed an antibody that specifically recognizes the central region of fCgA. Using the constitutively secreting COS-7 cells, which are devoid of DCGs, we could demonstrate for the first time that CgA is essential for targeting peptide hormones to newly formed mobile DCG-like structures. In the CgA-expressing AtT20 cells, which exhibit an only moderate capacity to sort secretory proteins to the regulated pathway (22), the granin plays a pivotal role in the sorting and release of POMC. The conserved terminal peptides of CgA are instrumental for these activities.Open in a separate windowFIGURE 1.Specificity of the antibody directed against frog CgA. A, scheme depicting the structure of fCgA and showing the high conservation of the terminal regions and the percentages of amino acid identity between frog and human CgA sequences. The highly conserved peptide WE14 and dibasic cleavage sites are also indicated. B, Western blot showing that the antibody developed against fCgA recognized the protein and several processing intermediates in frog but not rat pituitary extracts, whereas an antibody, directed against the WE14 conserved peptide, detected CgA and its processing products in both rat and frog pituitary extracts. C, immunofluorescence analysis of frog pituitary and adrenal glands, and rat adrenal gland using the antibodies against fCgA and WE14. cx, cortex; DL, distal lobe; IL, intermediate lobe; and m, medulla. Scale bars equal 10 μm.     

Molecular origin of the self-assembly of lanreotide into nanotubes: a mutational approach

Lanreotide, a synthetic, therapeutic octapeptide analog of somatostatin, self-assembles in water into perfectly hollow and monodisperse (24-nm wide) nanotubes. Lanreotide is a cyclic octapeptide that contains three aromatic residues. The molecular packing of the peptide in the walls of a nanotube has recently been characterized, indicating four hierarchical levels of organization. This is a fascinating example of spontaneous self-organization, very similar to the formation of the gas vesicle walls of Halobacterium halobium. However, this unique peptide self-assembly raises important questions about its molecular origin. We adopted a directed mutation approach to determine the molecular parameters driving the formation of such a remarkable peptide architecture. We have modified the conformation by opening the cycle and by changing the conformation of a Lys residue, and we have also mutated the aromatic side chains of the peptide. We show that three parameters are essential for the formation of lanreotide nanotubes: i), the specificity of two of the three aromatic side chains, ii), the spatial arrangement of the hydrophilic and hydrophobic residues, and iii), the aromatic side chain in the β-turn of the molecule. When these molecular characteristics are modified, either the peptides lose their self-assembling capability or they form less-ordered architectures, such as amyloid fibers and curved lamellae. Thus we have determined key elements of the molecular origins of lanreotide nanotube formation.     

Searching for the “sweet spot”: the foot rotation and parallel engagement of ankle ligaments in maximizing injury tolerance

Ligament sprains, defined as tearing of bands of fibrous tissues within ligaments, account for a majority of injuries to the foot and ankle complex in field-based sports. External rotation of the foot is considered the primary injury mechanism of syndesmotic ankle sprains with concomitant flexion and inversion/eversion associated with particular patterns of ligament trauma. However, the influence of the magnitude and direction of loading vectors to the ankle on the in situ stress state of the ligaments has not been quantified in the literature. The objective of the present study was to search for the maximum injury tolerance of a human foot with an acceptable subfailure distribution of individual ligaments. We used a previously developed and comprehensively validated foot and ankle model to reproduce a range of combined foot rotation experienced during high-risk sports activities. Biomechanical computational investigation was performed on initial foot rotation from \(20{^{\circ }}\) of plantar flexion to \(15{^{\circ }}\) of dorsiflexion, and from \(15{^{\circ }}\) of inversion to \(15{^{\circ }}\) of eversion prior to external rotation. Change in initial foot rotation shifted injury initiation among different ligaments and resulted in a wide range of injury tolerances at the structural level (e.g., 36–125 Nm of rotational moment). The observed trend was in agreement with a parallel experimental study that initial plantar flexion decreased the incidence of syndesmotic injury compared to a neutral foot. A mechanism of distributing even loads across ligaments subjected to combined foot rotations was identified. This mechanism is potential to obtain the maximum load-bearing capability of a foot and ankle while minimizing the injury severity of ligaments. Such improved understanding of ligament injuries in athletes is necessary to facilitate injury management by clinicians and countermeasure development by biomechanists.     

Most of the extant mtDNA boundaries in South and Southwest Asia were likely shaped during the initial settlement of Eurasia by anatomically modern humans

Background

Recent advances in the understanding of the maternal and paternal heritage of south and southwest Asian populations have highlighted their role in the colonization of Eurasia by anatomically modern humans. Further understanding requires a deeper insight into the topology of the branches of the Indian mtDNA phylogenetic tree, which should be contextualized within the phylogeography of the neighboring regional mtDNA variation. Accordingly, we have analyzed mtDNA control and coding region variation in 796 Indian (including both tribal and caste populations from different parts of India) and 436 Iranian mtDNAs. The results were integrated and analyzed together with published data from South, Southeast Asia and West Eurasia.

Results

Four new Indian-specific haplogroup M sub-clades were defined. These, in combination with two previously described haplogroups, encompass approximately one third of the haplogroup M mtDNAs in India. Their phylogeography and spread among different linguistic phyla and social strata was investigated in detail. Furthermore, the analysis of the Iranian mtDNA pool revealed patterns of limited reciprocal gene flow between Iran and the Indian sub-continent and allowed the identification of different assemblies of shared mtDNA sub-clades.

Conclusions

Since the initial peopling of South and West Asia by anatomically modern humans, when this region may well have provided the initial settlers who colonized much of the rest of Eurasia, the gene flow in and out of India of the maternally transmitted mtDNA has been surprisingly limited. Specifically, our analysis of the mtDNA haplogroups, which are shared between Indian and Iranian populations and exhibit coalescence ages corresponding to around the early Upper Paleolithic, indicates that they are present in India largely as Indian-specific sub-lineages. In contrast, other ancient Indian-specific variants of M and R are very rare outside the sub-continent.     

Non-stochastic and stochastic linear indices of the 'molecular pseudograph's atom adjacency matrix': application to 'in silico' studies for the rational discovery of new antimalarial compounds

Malaria is one of the most deadly diseases, affecting million of people especially in developing countries. Because of the rapidly increasing threat worldwide of malaria epidemics multidrugs resistant to therapies, there is an urgent global need to discover new classes of antimalarial compounds. In an effort to overcome this problem, we have investigated the use of structure-based classification models for the 'rational' selection/identification or design/optimization of new lead antimalarials from virtual combinatorial data sets. In this sense, TOpological MOlecular COMputer Design strategy (TOMOCOMD approach) has been introduced in order to obtain two quantitative models for the discrimination of antimalarials. A collected data set containing 597 antimalarial compounds is presented as a helpful tool not only for theoretical chemist but for other researchers in this area. The validated models (including non-stochastic and stochastic indices) classify correctly more than 90% of compounds in both training and external prediction data sets. They showed high Matthews' correlation coefficients; 0.87 and 0.82 for training and 0.86 and 0.79 for test set. The TOMOCOMD-CARDD approach implemented in this work was successfully compared with two of the most useful models for antimalarials selection reported so far. Thus we expect that these two QSAR models can be used in the identification of previously un-known antimalarials compounds.