Skh1, the MEK component of the mkh1 signaling pathway in Schizosaccharomyces pombe

Skip to Next Section We previously reported the identification of Mkh1, a MEK kinase in Schizosaccharomyces pombe that is required for cell wall integrity, and we presented genetic evidence that Pmk1/Spm1, a MAP kinase, functions downstream from Mkh1 in the same pathway. Here, we report the identification of Skh1, a MEK (MAP kinase kinase) in S. pombe. The sequence of Skh1 is nearly identical to that of the recently reported Pek1 sequence. We present biochemical and genetic evidence that Skh1 is the MEK component of the Mkh1-Spm1 MAP kinase cascade. Our yeast two-hybrid results indicate that Mkh1, Skh1, and Spm1 physically interact to form a ternary complex. Deletion of mkh1, skh1 or spm1 results in identical phenotypes, including sensitivity to (beta)-glucanase treatment, growth inhibition on media containing KCl, and filamentous growth on medium containing caffeine. Double mutant strains exhibit phenotypes that are identical to the single mutant strains. Furthermore, expression of an activated HA-Skh1(DD)protein suppressed these defects in mkh1(delta) cells, and overexpression of Spm1 suppressed these defects in skh1(delta) cells. We also show that HA-Spm1 is hyper-phosphorylated on tyrosine residues in cells co-expressing the activated HA-Skh1(DD) protein. Furthermore, we found the phosphorylated/activated form of GFP-HA-Spm1 at detectable levels in wild-type cells, but not at appreciable levels in mkh1(delta) or skh1(delta) cells expressing this fusion protein. Together, our results indicate that Mkh1, Skh1 and Spm1 constitute a MAPK cascade in fission yeast

CAP1 expression is developmentally regulated in Xenopus

We have cloned and characterized a Xenopus member of the cyclase associated protein (CAP) gene family. xCAP1 is expressed as a maternal transcript, but is up-regulated prior to gastrulation and subsequently localizes to head mesenchyme, lens, otic vesicle, and trunk mesoderm including the pronephros. At different stages, the gone also appears to differentiate surface from deep (sensorial) ectoderm. As in Drosophila, Xenopus CAP1 is expressed in the developing eye, specifically in the differentiating lens. However, in distinction to Drosophila, Xenopus CAP1 does not express in periodically arrayed neural bands. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved

Alternative methods in tracking sources of microbial contamination in waters

A key factor in the management and remediation of impaired ground- and surface water is the ability to distinguish the sources of faecal contamination. Several approaches have been adopted as microbial source tracking methods (MST), which are generally classified as culturing, phenotypic, genetic, and chemical MST. None of the techniques used thus far can be considered a standard; important factors, such as the statistical correlation between the source and the faecal indicator and the understanding of the environmental fate of the faecal pollutants, still need attention. The most promising MST methods available today are based on the genetic fingerprinting of faecal micro-organisms. However, research is very active also in the investigation of pharmaceuticals and personal care products discharged in the environment together with faecal waste. An updated overview of MST methods to distinguish human from animal sources of faecal pollution is presented here, focusing particularly on the potentialities of new chemical tracers

Benzo(a)pyrene exposure causes adaptive changes in p53 and CYP1A gene expression in Brown bullhead (Ameiurus nebulosus)

The Brown bullhead (Ameiurus nebulosus) is able to survive and reproduce in high levels of environmentally contaminated areas of the Great Lakes. The purpose of this study was to establish whether there are adaptive genetic/molecular changes occurring in these fish that allow for their survival. Expression of a cell cycle regulator, p53 and the toxin metabolizing protein, CYP1A were measured in liver tissue from bullhead caught from either clean or contaminated areas of Lake Erie and surrounding areas. Wild caught fish and F1 raised offspring (whose parents originated from clean and contaminated sites) were used to measure endogenous gene expression levels. Results revealed that endogenous expression of p53 was on average 6.6× higher in contaminated fish than in fish caught from clean sites. Interestingly, when fed benzo(a)pyrene (BaP)-treated food, p53 expression increased 0.2× in clean fish and decreased 2.6× in contaminated fish. Endogenous CYP1A expression was not detectable in clean fish and low in contaminated fish. Upon exposure to BaP-treated food, CYP1A expression increased in both clean and contaminated fish, although at a higher rate in clean fish. Furthermore, when fish were cleared and then re-exposed to BaP, CYP1A expression increased from basal levels at a higher rate in clean versus contaminated fish. CYP1A and p53 expression in F1 offspring was similar to wild caught fish at the endogenous level and when fed BaP treated food. Results suggest that fish in contaminated regions may be implementing an adaptive response to severe environmental stress by maintaining high expression of p53 and low expression of CYP1A; thus lending increased protection to cells and decreasing the potential amount of carcinogens produced by contaminant metabolism

Human Scythe Contains a Functional Nuclear Localization Sequence and Remains in the Nucleus during Staurosporine-Induced Apoptosis

Human Scythe (also known as BAT3) has been implicated in the control of apoptosis and regulating heat shock protein (HSP) 70 activity. We have attempted to further characterize the role of human Scythe in HeLa cells by studying the cellular localization and functional domains of a hemagglutinin (HA) epitope-tagged Scythe protein. Several HA-Scythe deletion mutant proteins were expressed in HeLa cells and their localization was detected using indirect immunofluorescence. Our data demonstrate that full-length human Scythe is a nuclear protein that contains an active C-terminal nuclear localization sequence (NLS). Site-directed mutagenesis of the NLS leads to complete nuclear exclusion of full-length Scythe. Furthermore, induction of apoptosis by staurosporine does not cause redistribution or cleavage of Scythe, suggesting that Scythe remains localized in the nucleus during apoptosis. These results provide evidence that Scythe is a nuclear protein that probably does not interact with elements of the apoptotic machinery in the cytosol

Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization

Many extracellular signals elicit changes in the actin cytoskeleton, which are mediated through an array of signaling proteins and pathways. One family of proteins that plays a role in regulating actin remodeling in response to cellular signals are the cyclase-associated proteins (CAPs). CAPs are highly conserved monomeric actin binding proteins present in a wide range of organisms including yeast, fly, plants, and mammals. The original CAP was isolated as a component of the Saccharomyces cerevisiae adenylyl cyclase complex that serves as an effector of Ras during nutritional signaling. CAPs are multifunctional molecules that contain domains involved in actin binding, adenylyl cyclase association in yeast, SH3 binding, and oligomerization. Genetic studies in yeast have implicated CAPs in vesicle trafficking and endocytosis. CAPs play a developmental role in multicellular organisms, and studies of Drosophila have illuminated the importance of the actin cytoskeleton during eye development and in establishing oocyte polarity. This review will highlight the critical structural and functional domains of CAPs, describe recent studies that have implied important roles for these proteins in linking cell signaling with actin polymerization, and highlight their roles in vesicle trafficking and development.—Hubberstey, A. V., Mottillo, E. P. Cyclase-associated proteins: CAPacity for linking signal transduction and actin polymerization

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-μm plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu− strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-μm plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a ..., Un système de recombinaisons de plasmides a été développé, qui s\u27appuie sur les échanges entre plasmides pour assurer la viabilité cellulaire des levures. Deux types de plasmides, l\u27un porteur de l\u27allèle LEU2 inséré à l\u27intérieur des séquences du gène de l\u27actine des levures, l\u27autre porteur d\u27un ADN plasmidique de 2 μm, et un gène intact de l\u27actine ont été produits. Employés seuls, ni l\u27un ni l\u27autre n\u27a pu produire de transformant chez la souche haploïde Leu− AH22, mais une fois cotransformés, un certain nombre de colonies ont été obtenues. Des analyses de buvardage Southern ont révélé que les transformations sont survenues, suite à des événements de recombinaison à l\u27intérieur des séquences homologues de l\u27actine qui ont transféré le gène LEU2 au gène de l\u27actine sur le plastide de 2 μm. Les plastides recombinants ont pu être recouvrés et l\u27analyse des séquences d\u27un site de recombinaison a révélé que les événements reliés aux échanges avaient lieu véritablement au niveau des nucléotides. Les plastid..

Sensitive genetic biomarkers for determining apoptosis in the brown bullhead (Ameiurus nebulosus)

Biomarkers are necessary for monitoring environmentally induced alterations at the molecular level in order to assess the impact of xenobiotic compounds on organism health. Apoptosis is a highly regulated cellular process that controls programmed cell death and is involved in tumor formation. Apoptosis thus may provide the basis for developing biomarkers for use in the field of ecotoxicology to monitor non-lethal levels of xenobiotic induced cellular stress and toxicity. This study shows that a brown bullhead (Ameiurus nebulosus) fibroblast cell line (BB-2) responds to known apoptotic inducers (staurosporine, cycloheximide, and tumor necrosis factor α (TNF-α)), as characterized by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick end-labelling (TUNEL). Furthermore, we characterized the apoptotic process using a series of newly identified bullhead genetic markers. Exposure to protein kinase C inhibitors altered the transcription of TF-cell apoptosis-related protein (TFAR)-15 and p23 with no effect on p53, inhibitor of apoptosis protein (IAP), or PNAS-2. Inhibition of protein synthesis caused a consistent reduction in the transcription of p53 and PNAS-2. This study demonstrates that our novel transcriptional markers are sensitive biomarkers for the study of the effects of xenobiotic chemicals on apoptosis in the brown bullhead

Cancer therapy utilizing an adenoviral vector expressing only E1A

The human adenovirus type 5 (Ad5) early region 1A (E1A) proteins have been shown to have potent antitumor effects, due to their ability to reprogram oncogenic signalling pathways in tumor cells. The success of E1A antitumor therapy in animal models has led to its use in phase I and phase II clinical trials, where liposome-based delivery vehicles are being used to deliver a plasmid encoding E1A. To increase the efficiency of E1A delivery to tumors, we have developed an Ad vector deleted of all viral protein coding sequences (termed helper-dependent Ad vectors, hdAds) with the exception of E1A, designated hdAd-E1A. In culture, this vector mediated high-level expression of E1A gene products. A549 cells, a human lung adenocarcinoma cell line, infected with hdAd-E1A showed a reduced proliferative capacity in adherent culture, and the ability to form colonies in soft agarose was completely abolished. In contrast, A549 infected with an hdAd expressing β-gal were able to form colonies of a similar size and frequency as uninfected cells. Under serum-depleted conditions, expression of E1A within A549 led to the induction of apoptosis. Finally, A549 cells treated with hdAd-E1A showed approximately 10-fold greater sensitivity to the chemotherapeutic drug cisplatin. Taken together, these data indicate that the use of hdAd provides a simple and effective method to deliver E1A to cancer cells, and results in reduction in the tumorigenic potential of the cells, as well as increasing the cells sensitivity to anticancer drugs