Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments

Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a 'coiled-coil'-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent K(D) of approx. 0.5 muM and a stoichiometry of approx. 5:1 [actin/C(1-498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.     

Separation and Purification of Antioxidant Peptides from Enzymatically Prepared Scorpion (Buthus martensii Karsch) Protein Hydrolysates

International Journal of Peptide Research and Therapeutics - The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein...     

Biosynthesis of NAAG by an enzyme-mediated process in rat central nervous system neurons and glia

The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.     

Quantifying the response of free-ranging mammalian herbivores to the interplay between plant defense and nutrient concentrations

While trying to achieve their nutritional requirements, foraging herbivores face the costs of plant defenses, such as toxins. Teasing apart the costs and benefits of various chemical constituents in plants is difficult because their chemical defenses and nutrient concentrations often co-vary. We used an approach derived from predator–prey studies to quantitatively compare the foraging response of a free-ranging mammalian herbivore, the swamp wallaby (Wallabia bicolor), through three feeding trials with artificial diets that differed in their concentrations of (1) the terpene 1,8-cineole, (2) primary constituents (including nitrogen and fiber), and (3) both the terpene and the primary constituents. Applying the giving-up density (GUD) framework, we demonstrated that the foraging cost of food patches increases with higher dietary cineole concentration and decreases with higher dietary nutrient concentration. The effect of combined differences in nutrients and cineole concentrations on GUD was interactive, and high nutrient food required more cineole to achieve the same patch value as low nutrient food. Our results indicate that swamp wallabies equate low nutrient, poorly defended food with high nutrient, highly defended food, providing two contrasting diets with similar cost–benefit outcomes. This behavior suggests that equal concentrations of chemical defenses provide nutrient-poor plants with relatively greater protection as nutrient-rich plants. Nutrient-rich plants may therefore face the exacerbated problem of being preferred by herbivores and therefore need to produce more defense compounds to achieve the same level of defense as nutrient-poor plants. Our findings help explain the difference in anti-herbivore strategy of nutrient-poor and rich plants, i.e., tolerance versus defense.     

Association of ‘Candidatus Phytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province,Saudi Arabia

Typical symptoms of phytoplasma such as whitening of the leaves, shortening of the stolons on Bermuda grass, variegated leaves, yellows, stunting, little leaves and yellows on Giant reed, Cooba and sand olive shrub were observed in Qassim province, Saudi Arabia, during the autumn season of 2015. When tested for phytoplasma by universal primers P1/P7 followed by R16mF2/R16mR2, products of approximately 1400?bp (as expected) were amplified from 16 plants with symptoms but not from symptomless plants. Based on sequencing, phylogenetic analysis and virtual restriction fragment length polymorphism patterns of the 16S rDNA F2nR2 fragments of seven Qassim phytoplasma isolates, bermuda grass isolates 170, 175 and 177, giant reed isolate 180, sand olive isolates 181 and 182 and cooba isolate 185, the associated phytoplasma was identified as a member of ‘Candidatus Phytoplasma cynodontis’ which belong to the 16SrXIV-A subgroup. The 16S rDNA gene sequences of seven Qassim phytoplasma isolates exhibited over 99.2% identity with members of ‘Ca. Phytoplasma cynodontis’ group of phytoplasmas. This is the first report of characterization of ‘Ca. phytoplasma cynodonties’ (16SrXIV) associated with Cynodon dactylon in Saudi Arabia and its new hosts, Dodonaea angustifolia, Arundo donax and Acacia salicia.     

Human arterial smooth muscle cells in culture: Inverse relationship between proliferation and expression of contractile proteins

Summary Human arterial smooth muscle cells (hASMC) from explants of the inner media of uterine arteries were studied in secondary culture. We had previously found that these cells depend on exogenous platelet-derived growth factor (PDGF) for proliferation in vitro. Deprivation of the serum mitogen(s) by culture in plasma-derived serum or bovine serum albumin (BSA) caused a true growth arrest that was reversible upon reexposure to the mitogen(s). When added to serum-containing medium, heparin caused a reversible growth arrest which could be competed for by increasing concentrations of serum. In the current study we used a set of smooth muscle-specific actin and myosin, antibodies to study the expression of contractile proteins in stress fibers under indirect immunofluorescence on hASMC in culture. Even in sparse culture, grwoth-arrested hASMC expressed stress fibers containing these actin and myosin epitopes. This was true irrespective of whether growth arrest was achieved by culture in media containing only BSA or a combination of heparin and whole blood serum. hASMC proliferating in whole blood serum in sparse culture did not express such strees fibers, as judged by immunofluorescent staining. This was true also for cells that were restimulated to proliferate in serum after a growth arrest. Utilizing a monoclonal antibody against a nuclear antigen expressed in proliferating human cells, we were able to demonstrate an inverse relationship between the expression of this antigen and the SMC-specific contractile proteins, respectively. Under these culture conditions, the reversible transition between defifferentiated and differentiated hASMC was almost complete and terminated about 1 wk after the change in culture condition. We conclude that hASMC in vitro respond, to exogenous PDGF by proliferation and dedifferetiation as a single population of cells. We also conclude that this modulation is reversible, because the cells become uniformly quiescent and differentiated when the mitogenic stimulus is blocked or removed. This study was supported by grants from the Swedish Medical Research Council (Project no. 4531 and 6816), the Swedish Association against Heart and Chest Diseases, the King Gustaf V and Queen Victoria Foundation, the National Institutes of Health, Bethesda, MD (grant HL 29873) and the Swedish National Board for Laboratory Animals.     

Isolation of esterified fatty acids bound to serum albumin purified from human plasma and characterised by MALDI mass spectrometry.

Human serum albumin (HSA) is the most abundant protein in plasma. It is known to transport drugs as well as endogenous ligands, like free fatty acids (FFA). A mass spectrometry based method was applied to analyze the albumin bound lipid ligands. HSA was isolated from a human plasma pool by cold ethanol fractionation and ion exchange chromatography. HSA was defatted using a solvent extraction method to release the copurified lipids bound to the protein. The extracts were then analyzed by matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Using this method, phospholipids and acylglycerols were detected. The phospholipids were identified to be lyso-phosphatidylcholine (lyso-PC) with distribution of different fatty acids (palmitic, stearic, oleic, and linoleic acids). An abundant species in the HSA lipid extract was found to be a diacylglycerol, composed of two linoleic and/or oleic acid chains. The identified motifs reflect structures that are known to be present in plasma. The binding of lysophospholipids has already been described but it is the first ever-reported evidence of native diacylglycerol ligands bound to HSA. Besides the native ligands from plasma a triacylglycerol was detected that has been added during the albumin preparation steps.     

Harmony Search Algorithm for Word Sense Disambiguation

Word Sense Disambiguation (WSD) is the task of determining which sense of an ambiguous word (word with multiple meanings) is chosen in a particular use of that word, by considering its context. A sentence is considered ambiguous if it contains ambiguous word(s). Practically, any sentence that has been classified as ambiguous usually has multiple interpretations, but just one of them presents the correct interpretation. We propose an unsupervised method that exploits knowledge based approaches for word sense disambiguation using Harmony Search Algorithm (HSA) based on a Stanford dependencies generator (HSDG). The role of the dependency generator is to parse sentences to obtain their dependency relations. Whereas, the goal of using the HSA is to maximize the overall semantic similarity of the set of parsed words. HSA invokes a combination of semantic similarity and relatedness measurements, i.e., Jiang and Conrath (jcn) and an adapted Lesk algorithm, to perform the HSA fitness function. Our proposed method was experimented on benchmark datasets, which yielded results comparable to the state-of-the-art WSD methods. In order to evaluate the effectiveness of the dependency generator, we perform the same methodology without the parser, but with a window of words. The empirical results demonstrate that the proposed method is able to produce effective solutions for most instances of the datasets used.     

Cold storage effects on flesh quality of rainbow trout Oncorhynchus mykiss (Walbaum, 1792) fed diets containing different vegetable oils

The intention of this experiment was to assess the effects of different sources of dietary lipid on the fatty acid composition of the fillet and liver and the flesh quality traits of rainbow trout (Oncorhynchus mykiss) after a 70‐day feeding period. Four iso‐nitrogenous (approx. 51% crude protein) and iso‐lipidic (approx. 14% crude lipid) experimental diets were formulated. The control diet contained only fish oil (FO) as the primary lipid source. In the other three dietary treatments, fish oil was replaced by 100% (LO30/SO35/SFO35) and 70% (FO30/LO35/SO35 or FO30/SO35/SFO35) sesame oil (SO), linseed oil (LO), or sunflower oil (SFO). Triplicate groups of 40 rainbow trout (~46 g) held under similar culture conditions were hand‐fed daily to apparent satiation for 70 days. At the end of the feeding trials, no difference in growth performance among experimental groups was noted (P > 0.05). There were some differences in the proximate composition of fish fillets (P < 0.05): the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) levels were highest in fish fed the control diet (P < 0.05); and EPA and DHA levels in fish fed the FO30/LO35/SO35 diet were closest to the control diet (P < 0.05). In contrast, fish fed the diet containing 100% plant oils (LO30/SO35/SFO35) had the highest level of total n‐6 fatty acids in the fillet and liver. In a 12‐day refrigerated storage at 1°C the thiobarbituric acid (TBA), trimethylamin nitrogen (TMA‐N) and pH values gradually increased in all dietary groups (P < 0.05). The chemical indicators of spoilage, TBA, TMA‐N, and pH values were within the limit of acceptability for human consumption.     

DISTRIBUTION AND ABUNDANCE OF THE AMAZON RIVER DOLPHIN (INIA GEOFFRENSIS) AND THE TUCUXI (SOTALIA FLUVIATILIS) IN THE UPPER AMAZON RIVER

A boat survey was conducted from 5 to 26 June 1993 to estimate the abundance of the Amazon river dolphin ( Inia geoffrensis ) and the tucuxi ( Sotalia fluviatilis ) along ca . 120 km of the Amazon River bordering Colombia, Peru, and Brazil. Two survey methods were used: line transects during 5 d and strip transects during 15 d. The line transects were used to estimate the abundance of both species in the main channels of the Amazon at distances greater than 200 m from river banks and islands, and strip transects were used to estimate abundance in the remainder of the habitat. A total of 29 sightings was obtained using line transects, including 8 of Inia , 15 of Sotalia , and 6 with both species present. The total number of sightings made while using strip transects was 143, including 78 of Inia , 51 of Sotalia , and 14 with both species present. The distributions of sightings with respect to distance from the nearest bank were not significantly different between the two species. Based on the results from the two methods, we estimate that there are 346 (CV = 0.12) Inia and 409 (CV = 0.13) Sotalia in the study area. Overall, the mean group size for Inia was 2.9 individuals and for Sotalia was 3.9 individuals. Inia density (dolphin/km²) was highest in tributaries (4.8), followed by areas around islands (2.7) and along main banks (2.0); while Sotalia density was highest in lakes (8.6), followed by areas along main banks (2.8) and around islands (2.0). These are among the highest densities measured to date for any cetacean.