Electron microscopic mapping of monoclonal antibodies on the tail region of Dictyostelium myosin

The binding sites of five monoclonal antibodies against myosin of Dictyostelium discoideum have been mapped. These antibodies bind to the tail region of the myosin molecule. By rotary shadowing, images of myosin-antibody complexes were obtained in which the mean distance of the midpoint of an antibody molecule from the myosin heads was localized with a precision better than 2 nm (90% confidence limit). Other quantitative data extracted from electron micrographs provided information on the stoichiometry of antibody-myosin interaction. Certain antibodies interacted with myosin molecules only at a ratio of 1:1. Other antibodies formed complexes of two molecules bound to homologous sites on a double-stranded myosin tail. Affinities were estimated and the abilities of different antibodies to cross-connect two myosin molecules were evaluated.     

Definitive Evidence for the existence of tight junctions in invertebrates

Extensive and unequivocal tight junctions are here reported between the lateral borders of the cellular layer that circumscribes the arachnid (spider) central nervous system. This account details the features of these structures, which form a beltlike reticulum that is more complex than the simple linear tight junctions hitherto found in invertebrate tissues and which bear many of the characteristics of vertebrate zonulae occludentes. We also provide evidence that these junctions form the basis of a permeability barrier to exogenous compounds. In thin sections, the tight junctions are identifiable as punctate points of membrane apposition; they are seen to exclude the stain and appear as election- lucent moniliform strands along the lines of membrane fusion in en face views of uranyl-calcium-treated tissues. In freeze-fracture replicas, the regions of close membrane apposition exhibit P-face (PF) ridges and complementary E-face (EF) furrows that are coincident across face transitions, although slightly offset with respect to one another. The free inward diffusion of both ionic and colloidal lanthanum is inhibited by these punctate tight junctions so that they appear to form the basis of a circumferential blood-brain barrier. These results support the contention that tight junctions exist in the tissues of the invertebrata in spite of earlier suggestions that (a) they are unique to vertebrates and (b) septate junctions are the equivalent invertebrate occluding structure. The component tight junctional 8- to 10-nm-particulate PF ridges are intimately intercalated with, but clearly distinct from, inverted gap junctions possessing the 13-nm EF particles typical of arthropods. Hence, no confusion can occur as to which particles belong to each of the two junctional types, as commonly happens with vertebrate tissues, especially in the analysis of developing junctions. Indeed, their coexistance in this way supports the idea, over which there has been some controversy, that the intramembrane particles making up these two junctional types must be quite distinct entities rather than products of a common precursor.     

In vivo investigations of glucose transport in Saccharomyces cerevisiae

In the present study, the glucose transport into the yeast Saccharomyces cerevisiae has been investigated. The approach suggested is based on a rapid sampling technique for studying the dynamic response of the yeast to rapid changes in extracellular glucose concentrations. For this purpose a concentrated glucose solution has been injected into a continuous culture at steady state growth conditions resulting in a shift of the extracellular glucose level. Samples have been taken every 5 s for determination of extracellular glucose and intracellular glucose-6-phosphate concentrations. Attempts to fit the experimental observations with simulations from existing models failed. The mechanism then proposed is based on a facilitated diffusion of glucose superimposed by an inhibition of glucose-6-phosphate. The use of the so-called in vivo approach suggested in this article appears to be proper, because the investigations can be performed at defined physiological states of the microbial cultures. Furthermore, the experimental observations are not being corrupted by the preparation of the samples for the transport studies as it happens during radioactive measurements. (c) 1996 John Wiley & Sons, Inc.     

Cryptococcal Cell Morphology Affects Host Cell Interactions and Pathogenicity

Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 µm. Cell enlargement was observed in vivo, producing cells up to 100 µm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3 a Δ pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.     

Factors governing macrozoobenthic assemblages in perennial springs in north-western Switzerland

Springs are important freshwater habitats that provide refuge for many rare species. In this study, the fauna and abiotic parameters of 20 perennial springs in north-western Switzerland were investigated. Correlation of abiotic and macrozoobenthos data showed that physicochemical parameters had little impact on macrozoobenthic composition, whereas specific substrate parameters strongly influenced the composition of the macrofauna. Surprisingly, nonmetric multidimensional scaling did not reveal a grouping of springs with similar substrate composition or macrozoobenthic assemblages. However, discharge was identified as the factor significantly determining substrate and the composition of macroinvertebrate assemblages. This justifies the hypothesis that, variation in discharge is the disturbance factor governing the macrofaunal composition temporally and spatially within and between patches. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

ZFN,TALEN and CRISPR-Cas9 mediated homology directed gene insertion in Arabidopsis: A disconnect between somatic and germinal cells

正Breakthroughs in the generation of programmable sequence-specific nucleases (SSNs), such as zinc finger nucleases (ZFNs),TAL effector nucleases (TALENs) and the RNA-directed nuclease CRISPR-associated protein 9 (Cas9), have greatly increased the ease of plant genome engineering (Voytas, 2013; Malzahn et al.,2017). Programmable SSNs introduce a DNA double-strand break     

Parasites of fishes in the recently inundated ephemeral Lake Liambezi,Namibia

Lake Liambezi forms the periodic connection between the upper Zambezi, Kwando and Okavango rivers. A full parasitological assessment was conducted on 86 fish, representing 14 species in six families sampled in August 2011. Parasite diversity was low and dominated by species with complex life cycles involving intermediate hosts. Most prevalent were larval nematodes (Contracaecum sp.) infecting 12 and Trypanasoma sp. infecting nine of the 14 host species. The intra-erythrocytic parasite Babesiosoma mariae was found in the blood of Coptodon rendalli and Oreochromis andersonii with prevalence of 50% and 60%, respectively. The host-specific monogenean Annulotrema hepseti was recorded only from H. cuvieri with a prevalence of 100%. Notable absences were the copepod and branchiuran parasites that have direct lifecycles and usually occur in high prevalence and abundance in the region. Because parasites with direct life cycles can only be transported into the lake on the host fish, their absence suggests limited immigration of infected fishes into the lake. This suggests that internal recruitment dominates over immigration in the fish population dynamics in Lake Liambezi.     

Metastatic bladder cancer cells distinctively sense and respond to physical cues of collagen fibril-mimetic nanotopography

Tumor metastasis is characterized by enhanced invasiveness and migration of tumor cells through the extracellular matrix (ECM), resulting in extravasation into the blood and lymph and colonization at secondary sites. The ECM provides a physical scaffold consisting of components such as collagen fibrils, which have distinct dimensions at the nanoscale. In addition to the interaction of peptide moieties with tumor cell integrin clusters, the ECM provides a physical guide for tumor cell migration. Using nanolithography we set out to mimic the physical dimensions of collagen fibrils using lined nanotopographical silicon surfaces and to explore whether metastatic tumor cells are uniquely able to respond to these physical dimensions. Etched silicon surfaces containing nanoscale lined patterns with varying trench and ridge sizes (65–500 nm) were evaluated for their ability to distinguish between a non-metastatic (253J) and a highly metastatic (253J-BV) derivative bladder cancer cell line. Enhanced alignment was distinctively observed for the metastatic cell lines on feature sizes that mimic the dimensions of collagen fibrils (65–100 nm lines, 1:1–1:1.5 pitch). Further, these sub-100 nm lines acted as guides for migration of metastatic cancer cells. Interestingly, even at this subcellular scale, metastatic cell migration was abrogated when cells were forced to move perpendicular to these lines. Compared to flat surfaces, 65 nm lines enhanced the formation of actin stress fibers and filopodia of metastatic cells. This was accompanied by increased formation of focal contacts, visualized by immunofluorescent staining of phospho-focal adhesion kinase along the protruding lamellipodia. Simple lined nanotopography appears to be an informative platform for studying the physical cues of the ECM in a pseudo-3D format and likely mimics physical aspects of collagen fibrils. Metastatic cancer cells appear distinctively well adapted to sense these features using filopodia protrusions to enhance their alignment and migration.     

Myosin heavy chain kinase inactivated by Ca2+/calmodulin from aggregating cells of Dictyostelium discoideum.

Soluble myosin heavy chain kinases (MHC kinases) were partially purified from growth phase and aggregation-competent cells of Dictyostelium discoideum. In the aggregation-competent cells, two MHC kinases were distinguishable. One of these enzymes, called MHC kinase II, was inactivated by Ca2+ and calmodulin in a highly temperature-dependent reaction. A MHC kinase found in growth phase cells did not have these regulatory properties. Substrate specificities were analysed for MHC kinase II and for the MHC kinase from growth phase cells. Both enzymes phosphorylated threonine residues of the myosin heavy chains of D. discoideum and Physarum polycephalum. Phosphopeptide mapping of D. discoideum myosin and determination of the stoichiometry of its phosphorylation suggested the presence of two phosphorylation sites per heavy chain. Both sites were contained within a 38-kd chymotryptic fragment. The inactivation of MHC kinase II by Ca2+ plus calmodulin suggests this enzyme has a role in the regulation of myosin functions during the chemotactic response of a cell. The phosphorylated myosin had about one third the actin-activated Mg2+-ATPase activity of the non-phosphorylated myosin. Previous findings indicated that stimulation of D. discoideum cells with the chemo-attractant cAMP increases the cytoplasmic Ca2+ concentration. Under these conditions MHC kinase II might be inhibited and the dephosphorylated, more active form of myosin would accumulate.