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Biomedical subjects

D Engelberg

Publications and source records attributed to D Engelberg.

At least 19 recordsLinked to original sources
PubMed2006

Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

Active Transport, Cell Nucleus↗
PubMed2001

Isolation of hyperactive mutants of the MAPK p38/Hog1 that are independent of MAPK kinase activation.

Mitogen-activated protein kinases (MAPKs) play pivotal roles in growth, development, differentiation, and apoptosis. The exact role of a given MAPK in these processes is not fully understood. This question could be addressed using active forms of these enzymes that are independent of external stimulation and upstream regulation. Yet, such molecules are not available. MAPK activation requires dual phosphorylation, on neighboring Tyr and Thr residues, catalyzed by MAPK kinases (MAPKKs). It is not known how to force MAPK activation independent of MAPKK phosphorylation. Here we describe a series of nine hyperactive (catalytically and biologically), MAPKK-independent variants of the MAPK Hog1. Each of the active molecules contains just a single point mutation. Six mutations are in the conserved L16 domain of the protein. The active Hog1 mutants were obtained through a novel genetic screen that could be applied for isolation of active MAPKs of other families. Equivalent mutations, introduced to the human p38alpha, rendered the enzyme active even when produced in Escherichia coli, showing that the mutations increased the intrinsic catalytic activity of p38. It implies that the activating mutations could be directly used for production of active forms of MAPKs from yeasts to humans and could open the way to revealing their biological functions.

Amino Acid Sequence↗
PubMed2001

Gcn2 mediates Gcn4 activation in response to glucose stimulation or UV radiation not via GCN4 translation.

In mammalian cells transcription factors of the AP-1 family are activated by either stress signals such as UV radiation, or mitogenic signals such as growth factors. Here we show that a similar situation exists in the yeast Saccharomyces cerevisiae. The AP-1 transcriptional activator Gcn4, known to be activated by stress signals such as UV radiation and amino acids starvation, is also induced by growth stimulation such as glucose. We show that glucose-dependent Gcn4 activation is mediated through the Ras/cAMP pathway. This pathway is also responsible for UV-dependent Gcn4 activation but is not involved in Gcn4 activation by amino acid starvation. Thus, the unusual phenomenon of activation of mitogenic pathways and AP-1 factors by contradictory stimuli through Ras is conserved from yeast to mammals. We also show that activation of Gcn4 by glucose and UV requires Gcn2 activity. However, in contrast to its role in amino acid starvation, Gcn2 does not increase eIF2alpha phosphorylation or translation of GCN4 mRNA in response to glucose or UV. These findings suggest a novel mechanism of action for Gcn2. The finding that Gcn4 is activated in response to glucose via the Ras/cAMP pathway suggests that this cascade coordinates glucose metabolism with amino acids and purine biosynthesis and thereby ensures availability of both energy and essential building blocks for continuation of the cell cycle.

Alcohol Oxidoreductases↗
PubMed2001

Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.

Recently we reported an unusual multicellular organization in yeast that we termed stalk-like structures. These structures are tall (0.5 to 3 cm long) and narrow (1 to 3 mm in diameter). They are formed in response to UV radiation of cultures spread on high agar concentrations. Here we present an anatomical analysis of the stalks. Microscopic inspection of cross sections taken from stalks revealed that stalks are composed of an inner core in which cells are dense and vital and a layer of cells (four to six rows) that surrounds the core. This outer layer is physically separated from the core and contains many dead cells. The outer layer may form a protective shell for the core cells. Through electron microscopy analysis we observed three types of cells within the stalk population: (i) cells containing many unusual vesicles, which might be undergoing some kind of cell death; (ii) cells containing spores (usually one or two spores only); and (iii) familiar rounded cells. We suggest that stalk cells are not only spatially organized but may undergo processes that induce a certain degree of cell specialization. We also show that high agar concentration alone, although not sufficient to induce stalk formation, induces dramatic changes in a colony's morphology. Most striking among the agar effects is the induction of growth into the agar, forming peg-like structures. Colonies grown on 4% agar or higher are reminiscent of stalks in some aspects. The agar concentration effects are mediated in part by the Ras pathway and are related to the invasive-growth phenomenon.

Culture Media↗
PubMed2001

Enhanced ROS production in oncogenically transformed cells potentiates c-Jun N-terminal kinase and p38 mitogen-activated protein kinase activation and sensitization to genotoxic stress.

Many primary tumors as well as transformed cell lines display high sensitivity to chemotherapeutic drugs and radiation. The molecular mechanisms that underlie this sensitivity are largely unknown. Here we show that the sensitization of transformed cells to stress stimuli is due to the potentiation of the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Activation of these pathways by the antitumor drug cis-platin (CDDP) and by other stress agents is markedly enhanced and is induced by lower stress doses in NIH 3T3 cells overexpressing epidermal growth factor receptor, HER1-2 kinase, or oncogenic Ras than in nontransformed NIH 3T3 cells. Inhibition of stress kinase activity by specific inhibitors reduces CDDP-mediated cell death in transformed cells, whereas overactivation of stress kinase pathways augments cells death. Potentiation of stress kinases is a common feature of cells transformed by different oncogenes, including cells derived from human tumors, and is shown here to be independent of the activity of the particular transforming oncoprotein. We further show that the mechanism that underlies potentiation of stress kinases in transformed cells involves reactive oxygen species (ROS), whose production is elevated in these cells. JNK/p38 activation is inhibited by antioxidants and in particular by inhibitors of the mitochondrial respiratory chain and NADPH oxidase. Conversely, by artificially elevating ROS levels in nontransformed NIH 3T3 cells we were able to induce potentiation of JNK/p38 activation. Taken together, our findings suggest that ROS-dependent potentiation of stress kinase pathways accounts for the sensitization of transformed cells to stress and anticancer drugs.

3T3 Cells↗
PubMed2001

The potential impacts of climate variability and change on health impacts of extreme weather events in the United States.

Extreme weather events such as precipitation extremes and severe storms cause hundreds of deaths and injuries annually in the United States. Climate change may alter the frequency, timing, intensity, and duration of these events. Increases in heavy precipitation have occurred over the past century. Future climate scenarios show likely increases in the frequency of extreme precipitation events, including precipitation during hurricanes, raising the risk of floods. Frequencies of tornadoes and hurricanes cannot reliably be projected. Injury and death are the direct health impacts most often associated with natural disasters. Secondary effects, mediated by changes in ecologic systems and public health infrastructure, also occur. The health impacts of extreme weather events hinge on the vulnerabilities and recovery capacities of the natural environment and the local population. Relevant variables include building codes, warning systems, disaster policies, evacuation plans, and relief efforts. There are many federal, state, and local government agencies and nongovernmental organizations involved in planning for and responding to natural disasters in the United States. Future research on health impacts of extreme weather events should focus on improving climate models to project any trends in regional extreme events and as a result improve public health preparedness and mitigation. Epidemiologic studies of health effects beyond the direct impacts of disaster will provide a more accurate measure of the full health impacts and will assist in planning and resource allocation.

Climate↗
PubMed2000

The effects of changing weather on public health.

Many diseases are influenced by weather conditions or display strong seasonality, suggestive of a possible climatic contribution. Projections of future climate change have, therefore, compelled health scientists to re-examine weather/disease relationships. There are three projected physical consequences of climate change: temperature rise, sea level rise, and extremes in the hydrologic cycle. This century, the Earth has warmed by about 0.5 degrees centigrade, and the mid-range estimates of future temperature change and sea level rise are 2.0 degrees centigrade and 49 centimeters, respectively, by the year 2100. Extreme weather variability associated with climate change may especially add an important new stress to developing nations that are already vulnerable as a result of environmental degradation, resource depletion, overpopulation, or location (e.g. low-lying coastal deltas). The regional impacts of climate change will vary widely depending on existing population vulnerability. Health outcomes of climate change can be grouped into those of: (a) direct physical consequences, e.g. heat mortality or drowning; (b) physical/chemical sequelae, e.g. atmospheric transport and formation of air pollutants; (c) physical/biological consequences, e.g. response of vector- and waterborne diseases, and food production; and (d) sociodemographic impacts, e.g. climate or environmentally induced migration or population dislocation. Better understanding of the linkages between climate variability as a determinant of disease will be important, among other key factors, in constructing predictive models to guide public health prevention.

Communicable Diseases↗
PubMed2000

GCN4-based expression system (pGES): translationally regulated yeast expression vectors.

The expression of foreign proteins in Saccharomyces cerevisiae is a powerful tool for basic research and the biotechnological industry. In spite of the potential of S. cerevisiae, only a few useful expression vectors have been developed for this yeast. These vectors are based on an increasing transcription rate in combination with an increase in gene dosage. Most vectors are maintained as plasmids, which forces growth of cultures on poor selective media. Expression of the yeast Gcn4 protein is regulated at the translational level and increases strongly under amino acid starvation. Because under these conditions protein synthesis in general ceases, it is conceivable that regulatory elements that control Gcn4 expression could support selective expression of foreign genes. We cloned DNA fragments residing upstream from the GCN4 coding sequence (including the 5' UTR) and ligated them to a cDNA that encodes the human serum albumin (HSA) gene. These GCN4 regulatory elements induced efficient HSA expression at the translational level under amino acid starvation. The GCN4/HSA cassette promoted efficient, inducible expression on either a multicopy or integrative plasmid. The integrated cassette induced a high level of HSA in dense cultures grown on rich media. Thus, the GCN4-based expression system (pGES) provides high protein quantities. pGES is the first expression vector to be induced at the translational level.

DNA-Binding Proteins↗
PubMed1999

UV-responsive genes of arabidopsis revealed by similarity to the Gcn4-mediated UV response in yeast.

A UV response that involves the Ras proteins and AP-1 transcription factors has recently been described in mammals and yeast. To test whether an equivalent response exists in plants, we monitored the expression of Arabidopsis histidinol dehydrogenase gene (HDH), a homologue of the yeast HIS4 gene, which is strongly induced by UV light and is a target of the transcriptional activator Gcn4. We show that HDH mRNA levels increase specifically in response to UV-B light. Only small increases were detected upon exposure to other wavelengths. To isolate plant genes involved in this UV response, a gcn4 mutant was transfected with an Arabidopsis thaliana cDNA library. A new type of nucleotide diphosphate kinase (NDPK Ia) with a significant homology to the human tumor suppressor protein Nm23 rescued the gcn4 phenotype. NDPK Ia specifically binds to the HIS4 promoter in vitro and induces HIS4 transcription in yeast. In Arabidopsis, the NDPK Ia protein is located in the nucleus and cytosol. Expression studies in seedlings revealed that the level of NDPK Ia mRNA, like that of HDH, increases in response to UV-B light. It appears that NDPK Ia and HDH are components of a novel UV-responsive pathway in A. thaliana.

Alcohol Oxidoreductases↗
PubMed1999

Follow-up and evaluation of skin cancer screening in British Columbia.

BACKGROUND: Skin cancer screening is thought to be a useful public health tool for the early detection of skin cancers. However, few studies have reported on follow-up and outcome of subjects who have a positive screen. OBJECTIVE: The aims of this study were to evaluate attendance at skin cancer screening clinics in British Columbia for the period 1994 and 1995 and to assess follow-up outcome among participants who were identified to have a potentially serious skin lesion that warranted further medical review. METHODS: A self-administered questionnaire was sent to participants screening positive for skin cancer and to their attending physicians. RESULTS: Five hundred twenty people were screened. Of these, 105 were referred for evaluation of a potential malignancy or precursor lesion. One melanoma, 3 basal cell carcinomas, 4 atypical nevi, and 1 actinic keratosis were histologically confirmed in 76 referred participants for whom follow-up information was available. The positive predictive values ranged from 17% to 89% depending on the screening diagnosis. Several false-positive results and one false-negative result were observed. Reasons for not seeking recommended follow-up were addressed. CONCLUSIONS: Our yield and positive predictive values for different screening diagnoses were virtually identical to those previously reported in larger US studies. Improved communication between screening physicians and screening participants may improve follow-up rates in those people who would benefit from further medical care.

Adult↗
PubMed1999

The yeast ras/cyclic AMP pathway induces invasive growth by suppressing the cellular stress response.

Haploid yeast cells are capable of invading agar when grown on rich media. Cells of the Sigma1278b genetic background manifest this property, whereas other laboratory strains are incapable of invasive growth. We show that disruption of the RAS2 gene in the Sigma1278b background significantly reduces invasive growth but that expression of a constitutively active Ras2p (Ras2(Val19)p) in this strain has a minimal effect on its invasiveness. On the other hand, expression of Ras2(Val19)p in another laboratory strain, SP1, rendered it invasive. These results suggest that a hyperactive Ras2 pathway induces invasive growth and that this pathway might be overactive in the Sigma1278b genetic background. Indeed, cells of the Sigma1278b are defective in the induction of stress-responsive genes, while their Gcn4 target genes are constitutively transcribed. This pattern of gene expression was previously shown to be associated with an active Ras/cyclic AMP (cAMP) pathway. We show that suppression of stress-related genes in Sigma1278b cells is a result of their inability to activate transcription through the stress response element (STRE). Disruption of RAS2, which abolished invasiveness, induced an increase in STRE activity. Further, in the SP1 genetic background, disruption of either the MSN2/4 genes (encoding activators of STRE) or the yAP-1 gene was sufficient to restore invasive growth in ras2Delta cells. We conclude that Ras2-mediated suppression of the stress response is sufficient to induce invasiveness. Accordingly, the fact that the stress response is suppressed in Sigma1278b background explains its invasiveness. It seems that invasiveness is a phenotype related to unregulated growth and is therefore manifested by cells harboring an overactive Ras/cAMP cascade. In this respect, invasiveness in yeast is reminiscent of the property of ras-transformed fibroblasts to invade soft agar.

Cell Differentiation↗
PubMed1998

Multicellular stalk-like structures in Saccharomyces cerevisiae.

Stalk formation is a novel pattern of multicellular organization. Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures. We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers. Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures. We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces. We show that other microorganisms (Candida albicans, Schizosaccharomyces pombe, and Escherichia coli) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms. Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.

Candida albicans↗
PubMed1994

Membrane targeting of the nucleotide exchange factor Sos is sufficient for activating the Ras signaling pathway.

Activation of growth factor receptors results in tyrosine autophosphorylation and recruitment of SH2 domain-containing effectors, including Grb2. Grb2 recruitment mediates activation of the Ras nucleotide exchanger Sos by an unknown mechanism. To examine the role of membrane recruitment, we prepared Sos derivatives containing either myristoylation or farnesylation signals. This resulted in plasma membrane targeting of Sos and stimulation of the Ras signaling pathway, including ERK and AP-1 activities leading to oncogenic transformation. Sos derivatives with nonfunctional myristoylation or farnesylation sequences were inactive. Farnesylation of Sos also activated Ras signaling in yeast. In both mammalian cells and yeast, membrane-targeted Sos derivatives lacking the C-terminal region were considerably more active. Therefore, targeting of Sos to the plasma membrane in the vicinity of Ras appears to be the primary mechanism leading to activation of the Ras pathway. A secondary mechanism could involve relief of the inhibitory effect of the Sos C-terminal region.

3T3 Cells↗
PubMed1994

The UV response involving the Ras signaling pathway and AP-1 transcription factors is conserved between yeast and mammals.

UV irradiation of mammalian cells activates AP-1 through a Ras-dependent pathway, independently of DNA damage. We show that the yeast S. cerevisiae has a remarkably similar UV response involving the AP-1 factor Gcn4, which is distinct from the DNA damage response. Transcriptional activation of HIS3 and HIS4 by Gcn4 is triggered by UV irradiation in a Ras-dependent fashion. Moreover, resistance of yeast to UV irradiation is correlated with the level of Ras activity and Gcn4 function. Like mammalian cells in which activated Ras leads to increased c-Jun synthesis and phosphorylation, the effects in yeast involve increased translation of GCN4 mRNA and a posttranslational event. However, this effect on GCN4 translation is different from the response to amino acid or purine starvation. Therefore, a UV signaling pathway involving Ras and AP-1 is an ancient and universal mechanism involved in protection against damage to cellular components other than DNA.

Amitrole↗
PubMed1994

The yeast and mammalian Ras pathways control transcription of heat shock genes independently of heat shock transcription factor.

Yeast strains in which the Ras-cyclic AMP (cAMP) pathway is constitutively active are sensitive to heat shock, whereas mutants in which the activity of this pathway is low are hyperresistant to heat shock. To determine the molecular basis for these differences, we examined the transcriptional induction of heat shock genes in various yeast strains. Activation of heat shock genes was attenuated in the strains in which the Ras-cAMP pathway is constitutively active. In contrast, in a strain deficient in cAMP production, several heat shock genes were induced by removal of cAMP from the medium. These results indicate that the Ras-cAMP pathway affects the induction of heat shock genes. In all of the mutants, heat shock transcription factor expression and activity were identical to those in wild-type cells. The response to heat shock in Ha-ras-transformed rat fibroblasts was also studied. While no induction of Hsp68 was observed in Ha-ras-transformed cells, proper regulation of heat shock transcription factor was found. Therefore, in mammals, as in Saccharomyces cerevisiae, the Ras pathway controls the transcription of heat shock genes via a mechanism not involving the heat shock transcription factor.

3T3 Cells↗
PubMed1992

Interaction between the Saccharomyces cerevisiae CDC25 gene product and mammalian ras.

In order to characterize the interaction between the Saccharomyces cerevisiae Cdc25 protein and Harvey-ras (p21H-ras), we have constructed a yeast strain disrupted at the RAS1 and RAS2 loci, expressing both p21H-ras and the catalytic domain of the bovine GTPase activating protein (GAP) and containing the cdc25-2 mutation. Such a strain exhibits a temperature-sensitive phenotype. The shift to the nonpermissive temperature is accompanied by the loss of guanyl nucleotide-dependent activity of adenylylcyclase in vitro. The temperature-sensitive phenotype can be rescued by CDC25 itself, as well as by a plasmid containing a truncated SDC25 gene. In addition, wild type CDC25 significantly improves the guanyl nucleotide response observed in the background of the cdc25ts allele at the permissive temperature in a dosage-dependent manner and restores the guanyl nucleotide response at the restrictive temperature. Both CDC25 and a truncated SDC25 also restored p21H-ras-dependent guanyl nucleotide response in a strain isogenic to the one described above but containing a disrupted CDC25 locus instead of the temperature-sensitive allele. These results suggest that the S. cerevisiae Cdc25 protein interacts with p21H-ras expressed in yeast by promoting GDP-GTP exchange. It follows that the yeast system can be used for characterizing the interaction between guanyl nucleotide exchangers of Ras proteins and mammalian p21H-ras.

Adenylyl Cyclases↗
PubMed1992

dOct2, a Drosophila Oct transcription factor that functions in yeast.

Oct factors are members of the POU family of transcription factors that are shown to play important roles during development in mammals. Here we report the cDNA cloning and expression of a Drosophila Oct transcription factor. Whole mount in situ hybridization experiments revealed that the spatial expression patterns of this gene during embryonic development have not yet been observed for any other gene. In early embryogenesis, its transcripts are transiently expressed as a wide uniform band from 20% to 40% of the egg length, very similar to that of gap genes. This pattern progressively resolves into a series of narrower stripes followed by expression in 14 stripes. Subsequently, transcripts from this gene are expressed in the central nervous system and the brain. When expressed in the yeast Saccharomyces cerevisiae, this Drosophila factor functions as a strong, octamer-dependent activator of transcription. Our data strongly suggest possible functions for the Oct factor in pattern formation in Drosophila that might transcend the boundaries of genetically defined segmentation genes.

Amino Acid Sequence↗