Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast.

STE20 encodes a protein kinase related to mammalian p65Pak which functions in several signal transduction pathways in yeast, including those involved in pseudohyphal and invasive growth, as well as mating. In addition, Ste20 plays an essential role in cells lacking Cla4, a kinase with significant homology to Ste20. It is not clear how the activity of Ste20 is regulated in response to these different signals in vivo, but it has been demonstrated recently that binding of the small GTP binding protein Cdc42 is able to activate Ste20 in vitro. Here we show that Ste20 functionally interacts with Cdc42 in a GTP-dependent manner in vivo: Ste20 mutants that can no longer bind Cdc42 were unable to restore growth of ste20 cla4 mutant cells. They were also defective for pseudohyphal growth and agar invasion, and displayed reduced mating efficiency when mated with themselves. Surprisingly, however, the kinase activity of such Ste20 mutants was normal when assayed in vitro. Furthermore, these alleles were able to fully activate the MAP kinase pathway triggered by mating pheromones in vivo, suggesting that binding of Cdc42 and Ste20 was not required to activate Ste20. Wild-type Ste20 protein was visualized as a crescent at emerging buds during vegetative growth and at shmoo tips in cells arrested with alpha-factor. In contrast, a Ste20 mutant protein unable to bind Cdc42 was found diffusely throughout the cytoplasm, suggesting that Cdc42 is required to localize Ste20 properly in vivo.     

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.     

A negative image of the quiescent centre in regenerating root apices of Zea mays

Usually the presence of the quiescent centre in roots is demonstrated by the absence of labelled nuclei following treatment of the root with appropriate radioactive markers. By modification of the pulselabelling technique, a negative image of the quiescent center, showing more intense labelling from [³H]thymidine than the surrounding area, was obtained in regenerating root apices of Zea mays L.     

Genetic Dissection of the Biochemical Activities of Recbcd Enzyme

RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.     

Organic Flocculation: an Efficient Second-Step Concentration Method for the Detection of Viruses in Tap Water

A method is described for second-step concentration of viruses from water. This method, combined with an adsorption-elution method, yields a mean recovery of about 75%     

Ca2+-dependent cross-linking processes in human platelets

When platelet cytoplasmic Ca²⁺ is increased by the ionophore A23187 in the presence of the protease inhibitor leupeptin, there is the coincident appearance of a cross-linked polymer and the partial disappearance of monomeric protein and glycoprotein units. In the absence of leupeptin only 30% of the polymer was formed. The disappearance of monomeric protein bands, as detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis, is prevented by histamine, which as a pseudodonor amine is a known inhibitor of transglutaminase-catalyzed cross-linking. [¹⁴C]Histamine, at a tracer concentration, is incorporated into the polymer as well as into myosin, glycoproteins IIB and III, actin and tropomyosin. The lose of monomeric protein bands is mostly due to their conversion into polymers. Control measurements show that leupeptin effectively inhibited platelet Ca²⁺-dependent proteases. The cross-linking processes bringing about the observed increase in polymer formation are thus the result of a Ca²⁺-dependent platelet transglutaminase activity. The latter is located in the platelet cytosol and has been identified as platelet factor XIII on the basis of its specific cross-linking of fibrin. Platelet factor XIII, upon activation, may function physiologically to couple membrane proteins to cytoplasmic structural proteins. Thus, a new concept is proposed for the stabilization of platelet membranes and platelets as they form the hemostatic plug.     

Male phenotypes in a female framework: Evidence for degeneration in sperm produced by male snails from asexual lineages

How changes in selective regimes affect trait evolution is an important open biological question. We take advantage of naturally occurring and repeated transitions from sexual to asexual reproduction in a New Zealand freshwater snail species, Potamopyrgus antipodarum, to address how evolution in an asexual context—including the potential for relaxed selection on male‐specific traits—influences sperm morphology. The occasional production of male offspring by the otherwise all‐female asexual P. antipodarum lineages affords a unique and powerful opportunity to assess the fate of sperm traits in a context where males are exceedingly rare. These comparisons revealed that the sperm produced by ‘asexual’ males are markedly distinct from sexual counterparts. We also found that the asexual male sperm harboured markedly higher phenotypic variation and was much more likely to be morphologically abnormal. Together, these data suggest that transitions to asexual reproduction might be irreversible, at least in part because male function is likely to be compromised. These results are also consistent with a scenario where relaxed selection and/or mutation accumulation in the absence of sex translates into rapid trait degeneration.     

A layover in Europe: Reconstructing the invasion route of asexual lineages of a New Zealand snail to North America

Non‐native invasive species are threatening ecosystems and biodiversity worldwide. High genetic variation is thought to be a critical factor for invasion success. Accordingly, the global invasion of a few clonal lineages of the gastropod Potamopyrgus antipodarum is thus both puzzling and has the potential to help illuminate why some invasions succeed while others fail. Here, we used SNP markers and a geographically broad sampling scheme (N = 1617) including native New Zealand populations and invasive North American and European populations to provide the first widescale population genetic assessment of the relationships between and among native and invasive P. antipodarum. We used a combination of traditional and Bayesian molecular analyses to demonstrate that New Zealand populations harbour very high diversity relative to the invasive populations and are the source of the two main European genetic lineages. One of these two European lineages was in turn the source of at least one of the two main North American genetic clusters of invasive P. antipodarum, located in Lake Ontario. The other widespread North American group had a more complex origin that included the other European lineage and two New Zealand clusters. Altogether, our analyses suggest that just a small handful of clonal lineages of P. antipodarum were responsible for invasion across continents. Our findings provide critical information for prevention of additional invasions and control of existing invasive populations and are of broader relevance towards understanding the establishment and evolution of asexual populations and the forces driving biological invasion.     

Fundamental differences in promoter CpG island DNA hypermethylation between human cancer and genetically engineered mouse models of cancer

Genetic and epigenetic alterations are essential for the initiation and progression of human cancer. We previously reported that primary human medulloblastomas showed extensive cancer-specific CpG island DNA hypermethylation in critical developmental pathways. To determine whether genetically engineered mouse models (GEMMs) of medulloblastoma have comparable epigenetic changes, we assessed genome-wide DNA methylation in three mouse models of medulloblastoma. In contrast to human samples, very few loci with cancer-specific DNA hypermethylation were detected, and in almost all cases the degree of methylation was relatively modest compared with the dense hypermethylation in the human cancers. To determine if this finding was common to other GEMMs, we examined a Burkitt lymphoma and breast cancer model and did not detect promoter CpG island DNA hypermethylation, suggesting that human cancers and at least some GEMMs are fundamentally different with respect to this epigenetic modification. These findings provide an opportunity to both better understand the mechanism of aberrant DNA methylation in human cancer and construct better GEMMs to serve as preclinical platforms for therapy development.     

Spermatozoa production by triploid males in the New Zealand freshwater snail Potamopyrgus antipodarum

Asexual lineages derived from dioecious taxa are typically assumed to be all female. Even so, asexual females from a variety of animal taxa occasionally produce males. The existence of these males sets the stage for potential gene flow across asexual lineages as well as between sexual and asexual lineages. A recent study showed that asexual triploid female Potamopyrgus antipodarum, a New Zealand freshwater snail often used as a model to study sexual reproduction, occasionally produce triploid male offspring. Here, we show that these triploid male P. antipodarum (1) have testes that produce morphologically normal sperm, (2) make larger sperm cells that contain more nuclear DNA than the sperm produced by diploid sexual males, and (3) produce sperm that range in DNA content from haploid to diploid, and are often aneuploid. Analysis of meiotic chromosomes of triploid males showed that aberrant pairing during prophase I probably accounts for the high variation in DNA content among sperm. These results indicate that triploid male P. antipodarum produce sperm, but the extent to which these sperm are able to fertilize female ova remains unclear. Our results also suggest that the general assumption of sterility in triploid males should be more closely examined in other species in which such males are occasionally produced. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 227–234.