Association of ebola virus matrix protein VP40 with microtubules

Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.     

Human hepatic triglyceride lipase: cDNA cloning, amino acid sequence and expression in a cultured cell line

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.     

Protein kinase D1 is essential for contraction-induced glucose uptake but is not involved in fatty acid uptake into cardiomyocytes

Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.     

Ecology of Microbial Saprophytes and Pathogens in Tissue Culture and Field-Grown Plants: Reasons for Contamination Problems In Vitro

This review compares published surveys of microbial populations in plant tissue and cell cultures with the microbial saprophytes and pathogens found on field grown plants and microbial populations in the laboratory environment. From this comparison and the measured reduction in contamination after improvements in working practices in the laboratory, conclusions can be drawn about the importance of the explant and the laboratory as sources of contamination.

Mechanisms of pathogenicity in vitro are described to explain why bacteria, fungi, and yeasts that are not pathogenic to plants in the field become pathogens in plant tissue cultures. Conversely, plant metabolism and its effect on the tissue culture environment are described to explain why prokaryotes, viruses, and viroids that cause disease in the field can stay latent in vitro.

Detection methods for latent contaminants in plant tissue cultures are summarized, and the strategies and methods for prevention or treatment of contamination are discussed.     

Low and annually variable migratory connectivity in a long‐distance migrant: Whinchats Saxicola rubetra may show a bet‐hedging strategy

The spatial scale of non‐breeding areas used by long‐distance migrant animals can vary from specific, relatively small non‐breeding areas for each independent breeding population (high connectivity) to a distribution over a large non‐breeding area with mixing of breeding populations (low connectivity). Measuring variation in the degree of connectivity and how it arises is crucial to predict how migratory animals can respond to global habitat and climate change because low connectivity is likely to be an adaptation to environmental uncertainty. Here, we assess whether use of non‐breeding areas in a long‐distance migrant may be stochastic by measuring the degree of connectivity, and whether it is annually variable. Twenty‐nine wintering Whinchats tagged with geolocators over 2 years within 40 km² in central Nigeria were found to be breeding over 2.55 million km² (26% of the land area of Europe), without an asymptote being approached in the relationship between area and sample size. Ranges differed in size between years by 1.51 million km² and only 15% of the total breeding range across both years overlapped (8% overlap between years when only first‐year birds were considered), well above the range size difference and below the proportion of overlap that would be predicted from two equivalent groups breeding at random locations within the observed range. Mean distance between breeding locations (i.e. migratory spread) differed significantly between years (604 ± 18 km in 2013 and 869 ± 33 km in 2014). The results showed very low and variable connectivity that was reasonably robust to the errors and assumptions inherent in the use of geolocators, but with the caveat of having only ranges of 2 years to compare, and the sensitivity of range to the breeding locations of a small number of individuals. However, if representative, the results suggest the scope for between‐year variation (cohort effects) to determine migrant distribution on a large scale. Furthermore, for species with similarly low connectivity, we would predict breeding population trends to reflect average conditions across large non‐breeding areas: thus, as large areas of Africa become subject to habitat loss, migrant populations throughout Europe will decline.     

Quantification of ventricular β2‐adrenoceptor density and ligand binding affinity in wild sockeye salmon Oncorhynchus nerka smolts using a novel modification to the tritiated ligand technique

A new, image‐based, tritiated ligand technique for measuring cardiac β₂‐adrenoceptor (β₂‐AR) binding characteristics was developed and validated with adult rainbow trout Oncorhynchus mykiss hearts so that the tissue limitation of traditional receptor binding techniques could be overcome and measurements could be made in hearts nearly 14‐times smaller than previously used. The myocardial cell‐surface (functional) β₂‐AR density of O. nerka smolts sampled at the headwaters of the Chilko River was 54·2 fmol mg protein^?1 and about half of that previously found in return migrating adults of the same population, but still more than twice that of adult hatchery O. mykiss (21·1 fmol mg protein^?1). This technique now opens the possibility of investigating cardiac receptor density in a much wider range of fish species and life stages.