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P Perez

Publications and source records attributed to P Perez.

At least 37 records · Page 2Linked to original sources
PubMed2000

Schizosaccharomyces pombe rho2p GTPase regulates cell wall alpha-glucan biosynthesis through the protein kinase pck2p.

Schizosaccharomyces pombe rho1(+) and rho2(+) genes are involved in the control of cell morphogenesis, cell integrity, and polarization of the actin cytoskeleton. Although both GTPases interact with each of the two S. pombe protein kinase C homologues, Pck1p and Pck2p, their functions are distinct from each other. It is known that Rho1p regulates (1,3)beta-D-glucan synthesis both directly and through Pck2p. In this paper, we have investigated Rho2p signaling and show that pck2 delta and rho2 delta strains display similar defects with regard to cell wall integrity, indicating that they might be in the same signaling pathway. We also show that Rho2 GTPase regulates the synthesis of alpha-D-glucan, the other main structural polymer of the S. pombe cell wall, primarily through Pck2p. Although overexpression of rho2(+) in wild-type or pck1 delta cells is lethal and causes morphological alterations, actin depolarization, and an increase in alpha-D-glucan biosynthesis, all of these effects are suppressed in a pck2 delta strain. In addition, genetic interactions suggest that Rho2p and Pck2p are important for the regulation of Mok1p, the major (1-3)alpha-D-glucan synthase. Thus, a rho2 delta mutation, like pck2 delta, is synthetically lethal with mok1-664, and the mutant partially fails to localize Mok1p to the growing areas. Moreover, overexpression of mok1(+) in rho2 delta cells causes a lethal phenotype that is completely different from that of mok1(+) overexpression in wild-type cells, and the increase in alpha-glucan is considerably lower. Taken together, all of these results indicate the presence of a signaling pathway regulating alpha-glucan biosynthesis in which the Rho2p GTPase activates Pck2p, and this kinase in turn controls Mok1p.

Cell Wall↗
PubMed2000

TrkA immunoglobulin-like ligand binding domains inhibit spontaneous activation of the receptor.

The extracellular region of the nerve growth factor (NGF) receptor, TrkA, contains two immunoglobulin (Ig)-like domains that are required for specific ligand binding. We have investigated the possible role of these two Ig-like domains in receptor dimerization and activation by using different mutants of the TrkA extracellular region. Deletions of each Ig-like domain, of both, and of the entire extracellular region were made. To probe the structural constraints on ligand-independent receptor dimerization, chimeric receptors were generated by swapping the Ig-like domains of the TrkA receptor for the third or fourth Ig-like domain of c-Kit. We also introduced single-amino-acid changes in conserved residues within the Ig-like domains of TrkA. Most of these TrkA variants did not bind NGF, and their expression in PC12nnr5 cells, which lack endogenous TrkA, promoted ligand-independent neurite outgrowth. Some TrkA mutant receptors induced malignant transformation of Rat-1 cells, as assessed by measuring proliferation in the absence of serum, anchorage-independent growth, and tumorigenesis in nude mice. These mutants exhibited constitutive phosphorylation and spontaneous dimerization consistent with their biological activities. Our data suggest that spontaneous dimerization of TrkA occurs when the structure of the Ig-like domains is altered, implying that the intact domains inhibit receptor dimerization in the absence of NGF.

Amino Acid Substitution↗
PubMed2000

SB-253514 and analogues; novel inhibitors of lipoprotein-associated phospholipase A2 produced by Pseudomonas fluorescens DSM 11579. I. Fermentation of producing strain, isolation and biological activity.

Two new classes of inhibitors of LpPLA2 have been identified in fermentations of Pseudomonas fluorescens. The two structurally isomeric series differ in the geometry of closure of the bicyclic carbamate and comprise a range of compounds varying only in length of their lipophilic sidechain. The most abundant species were extracted from the cells and purified by silica and C18 chromatography. Members of the more stable class were shown to be potent and selective competitive inhibitors of LpPLA2.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗
PubMed1999

Increased expression of p50-NF-kappaB and constitutive activation of NF-kappaB transcription factors during mouse skin carcinogenesis.

To elucidate the possible role of NF-kappaB in mouse skin carcinogenesis we studied the expression of p50 (NF-kappaB1), p52 (NF-kappaB2), p65 (RelA) and IkappaB-alpha inhibitor as well as kappaB-binding activity in adult SENCAR mouse skin, skin papillomas, and squamous cell carcinomas (SCC) generated by a two-stage carcinogenesis protocol. We found that in normal epidermis all of the above proteins were mostly expressed in the cytoplasm of basal cells. Western blot analysis revealed a dramatic increase of p50 and p52 expression in mouse skin tumors starting from the middle stage of promotion. We also found that the level of IkappaB-alpha protein in many late papillomas and SCC was lower than in normal epidermis. Results of EMSA showed an increase in kappaB-binding activity in mouse skin tumors and suggested that p50 is the major component of constitutive kappaB-binding complexes in normal epidermis and in tumors. It has been shown that nuclear IkappaB protein Bcl-3 is able to increase p50/p50 homodimer binding to the different kappaB sites in mouse thymocytes. Our finding on Bcl-3 overexpression in late papillomas and SCC could explain the selective increase of p50-related kappaB-binding in mouse skin tumors. Thus, our results strongly suggest the important role of p50 in skin carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1999

Interactions of surface-confined DNA with electroreduced mitomycin C comparison with acid-activated mitomycin C.

The anticancer activity of the antineoplastic drug mitomycin C (MC) was investigated using transfer stripping cyclic voltammetry (TSCV) with single-stranded DNA-modified hanging mercury drop electrode (HMDE). Reductive activation of MC is necessary for drug covalent binding to DNA, and we have found that some potential-controlled interactions of MC with DNA occur at the electrode, i.e. MC can be activated by electroreduction. Acid and electroreductive MC activations were compared and different adducts were subsequently generated, suggesting that the drug can bind to DNA in more than one way. Under conditions of acid activated MC, a monofunctional adduct between C-1 of MC and N-7 of guanine was formed on the electrode surface, reduced at - 0.44 V (vs. SCE). However, when the DNA-modified electrode was immersed in a MC solution and potentials corresponding to the quinone moiety reduction (- 0.3 V or more negative vs. SCE) were applied, an intrastrand bifunctional adduct between C-1 and C-10 of MC and two N-7 of a pair of adjacent guanines in ssDNA were formed at the electrode, reduced at - 0.49 V, i.e. 50 mV more negative than the monoadduct. The results presented in this paper show for the first time electrochemical detection of DNA-MC adducts at the hanging mercury drop electrode.

Animals↗
PubMed1999

Changes in glutathione status and the antioxidant system in blood and in cancer cells associate with tumour growth in vivo.

The relationship among cancer growth, the glutathione redox cycle and the antioxidant system was studied in blood and in tumour cells. During cancer growth, the glutathione redox status (GSH/GSSG) decreases in blood of Ehrlich ascites tumour-bearing mice. This effect is mainly due to an increase in GSSG levels. Two reasons may explain the increase in blood GSSG: (a) the increase in peroxide production by the tumour that, in addition to changes affecting the glutathione-related and the antioxidant enzyme activities, can lead to GSH oxidation within the red blood cells; and (b) an increase of GSSG release from different tissues into the blood. GSH and peroxide levels are higher in the tumour cells when they proliferate actively, however GSSG levels remain constant during tumour growth in mice. These changes associate with low levels of lipid peroxidation in plasma, blood and the tumour cells. The GSH/GSSG ratio in blood also decreases in patients bearing breast or colon cancers and, as it occurs in tumour-bearing mice, this change associates with higher GSSG levels, especially in advanced stages of cancer progression. Our results indicate that determination of glutathione status and oxidative stress-related parameters in blood may help to orientate cancer therapy in humans.

Animals↗
PubMed1999

[Cost-benefit analysis of screening strategies by human immunodeficiency virus in French blood donors].

BACKGROUND: The residual risk of human immunodeficiency virus (HIV) infection from screened blood transfusion was estimated to be 1.7/10(6) between 1993 and 1995 in France. To orient blood safety policies, we have evaluated what would be, from the perspective of blood banks, the best screening strategy in terms of gain in effectiveness and added costs. METHODS: A cost-effectiveness analysis compared 20 HIV-testing protocols using (1) available data for performances of the current screening tests; and, (2) national insurance estimates for the cost of tests. Results were expressed as the number of false negative donations that would be avoided and the cost by avoided false negative donation. RESULTS: For 3 million donated blood units a year and a prevalence of 24 per million, there would be 72 infectious donated blood units, 70.56 of which would be detected by the current screening strategy. The number of additional donated blood infections avoided in all other strategies would be low (between 0.25 and 1.28) with a very high cost (280 million French francs per added false negative avoided or more). CONCLUSION: A change in screening strategies for blood donations in France is not currently justified. If such a change was to be done, adding p24 antigen detection to the current screening strategy would be one of the worst solutions.

AIDS Serodiagnosis↗
PubMed1999

Structure of two maize phytase genes and their spatio-temporal expression during seedling development.

Up to 80% of Zea mays L. grain phosphorus is stored in the form of phytin in the embryo. Our objective is to determine the control of phytin mobilization during germination and seedling growth. A maize phytase cDNA, phy S11, has been previously characterized (Maugenest et al., Biochem J 322: 511-517, 1997). In the present work, phy S11 was used to screen a maize genomic library and two distinct genes, PHYT I and PHYT II, were isolated and sequenced. The transcribed sequences of these two genes presented a strong homology whereas the untranscribed upstream and downstream sequences appeared very different. Northern blot analysis and in situ hybridization showed a high accumulation of phytase mRNA at the early steps of germination in the coleorhiza, radicle cortex and coleoptile parenchyma. Phytase expression was also detected at a lower extent in the scutellum. In adult plants, northern blot analyses revealed low but significant levels of phytase mRNA in the roots. In situ hybridizations on root cross-sections localized phytase mRNA in rhizodermis, endodermis and pericycle layers. Immunolocalization analysis showed phytase accumulation at the same sites as its mRNA. A RT-PCR approach was used in an attempt to discriminate between the transcripts from each gene in the different situations. These experiments indicate that both genes are expressed during germination, whereas only PHYT I is expressed in adult roots. This suggests that signals responsible for phytase gene expression in roots are different from those responsible for gene expression during germination.

6-Phytase↗
PubMed1999

The 'Folic Acid Campaign': has the message got through? A questionnaire study.

There is now clear evidence that folic acid reduces the risk of neural tube defects. In February 1996 the Health Education Authority launched a publicity campaign to inform women of the benefits of periconceptual folic acid. We have surveyed 1000 women to assess the compliance of pregnant women with the Department of Health's recommendations about taking folic acid. Of the women, 761 (76%) said they knew about the benefits of folic acid but only 433 (43%) of them took it before pregnancy. Of the 567 women who did not take folic acid before conception, 227 had not planned their pregnancy and 239 did not know about the benefits. Of the 644 women who planned their pregnancy and knew about the benefits of folic acid before conception 211 still did not take folic acid pre-pregnancy. These findings have important implications for public policy and health professionals if the incidence of neural tube defect is to be reduced further.

Journal Article↗
PubMed1999

Schizosaccharomyces pombe protein kinase C homologues, pck1p and pck2p, are targets of rho1p and rho2p and differentially regulate cell integrity.

Schizosaccharomyces pombe rho1(+) is required for maintenance of cell integrity and polarization of the actin cytoskeleton. However, no other effector besides the (1,3)beta-D-glucan synthase enzyme has been identified in S. pombe. We have further investigated if rho1(+ )signalling could be also mediated by the two protein kinase C homologues, pck1p and pck2p. We show in this study that both kinases interact with rho1p and rho2p only when bound to GTP, as most GTPase effectors do. Interestingly, the interaction was mapped in a different part of the proteins than in Saccharomyces cerevisiae Pkc1p. Thus, active rho1p binds to the amino-terminal region of the pcks where two HR1 motifs are located, and binding to the GTPase dramatically stabilizes the kinases. Detailed biochemical analysis suggests that pck2p is more important in the regulation of the enzyme (1-3)beta-D-glucan synthase. Thus, overexpression of pck2(+), but not pck1(+), caused a general increase in cell wall biosynthesis, mainly in beta-glucan, and (1-3)beta-D-glucan synthase activity was considerably augmented. When this activity was separated into soluble and membrane fractions and reconstituted, the increase caused by pck2(+) overexpression was exclusively detected in the membrane component. We also show that both protein kinase C homologues are required for the maintenance of cell integrity. pck1delta and pck2delta strains present a number of defects related to the cell wall, indicating that this structure might be co-ordinately regulated by both kinases. In addition, pck2p, but not pck1p, seems to be involved in keeping cell polarity. Genetic evidence indicates that both pck1(+) and pck2(+) interact with cps1(+) and gls2(+), two genes similar to S. cerevisiae FKS1 and FKS2 that encode membrane subunits of the (1-3)beta-D-glucan synthase. pck1(+ )also showed a genetic interaction with ras1(+) and ral1(+) suggesting the existence of a functional link between both signalling pathways.

Amino Acid Sequence↗
PubMed1999

Comparison of short-term estrogenicity tests for identification of hormone-disrupting chemicals.

The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 17beta++-estradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17beta-Estradiol, 17alpha-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds--tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p'-DDT, p,p'-DDE, endosulfan, chlomequat chloride, and ethanol--varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods.

DDT↗
PubMed1999

The human melanocyte as a particular target for UVA radiation and an endpoint for photoprotection assessment.

The induction of DNA breaks by UVA (320-400 nm) in the nucleus of normal human melanocytes in culture was investigated using single cell gel electrophoresis, also called the comet assay. Endogenous pigment and/or melanin-related molecules were found to enhance DNA breakage: comets were more intense in melanocytes than in fibroblasts, in cells with high melanin content or after stimulation of melanogenesis by supplying tyrosine in the culture medium. After UVA doses where strong comets were observed, neither cytotoxicity nor stimulation of tyrosinase activity were detected. However, the accumulation of p53 protein suggested that cells reacted to genotoxic stress under these experimental conditions. The same approach was used to compare two sunscreens with identical sun protection factors but different UVA protection factors. The results presented in this paper suggest that human melanocytes may be used as a target cell to evidence broadspectrum photoprotection. Moreover, these data appear to be helpful in getting a better understanding of the role of sunlight in the initiating steps of melanocyte transformation.

Cell Line↗
PubMed1998

Functional expression of TrkA receptors in hippocampal neurons.

Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types.

Adaptor Proteins, Signal Transducing↗
PubMed1998

[Evaluation of aa tracking system for labile blood products in the Midi-Pyrenes region].

At the request of the regional authority, the system of traceability of blood products in the Midi-Pyrenees area in France was assessed using available documentation, visits and interviews of participants. We studied the time needed to obtain traceability data in the Blood Banks and the reactivity of the system defined as the time needed to carry out a simulated descending transfusional survey. This information allowed us to assess usefulness and cost of the system, its applicability and the timeliness of information, according to the criteria of the Centers for Disease Control. The system is based on a standardized nominative form for the delivery of blood products and specific feedback. For the participants, traceability makes transfusional surveys easier and improves transfusions safety. In two of the 130 hospitals, the system is linked to the "program of medicalization of information systems". In the 2nd semester of 1996, the traceability proportion was 97%. For a given date of distribution, 75% of blood products were traced in 14 days and 83% in a month. The study of reactivity showed that all donations coming from two randomly chosen blood collection sites could be traced in about 2 hours. This first evaluation of a regional system of traceability showed its feasibility and acceptability, based on evaluation criteria of a public health surveillance system. A continuous evaluation process would be necessary to assess the good functioning of the system and to detect and prevent possible deficiencies.

Blood Banks↗
PubMed1998

[Transfusion incidents related to bacterial contamination: review of the literature and hemovigilance data. For the working group Bacterial incidents of French Blood Agency, scientific advice of Bacthem and the French hemovigilance network].

The impact of transfusion reactions related to bacterial contamination (TRBC) of labile blood products (LBP) was identified in France soon after the implementation of the haemovigilance system. The aims of our communication were: 1) to confront published data on TRBC with available French data; 2) to describe measures implemented in France to prevent TRBC. Bacterial contamination of whole blood at the collection stage is found in 0.6 to 3% of blood donations. Systematic bacteriological controls detect frequent contaminations of LBP that vary according to studies and the nature of the product. The incidence of clinical TRBC was only estimated in recipients with malignant diseases. Based on French haemovigilance data, the incidence was estimated to two per 100,000 delivered units. The measures taken were the diffusion of information and recommendations and the implementation of studies. In the framework of the Bacthem study (a national case-control study on LBP-related bacterial contamination), standardised diagnostic criteria are applied to suspected TRBC notified to the Centre National d'Hémovigilance. Expected results should allow us to estimate the incidence of TRBC in France and to determine the recipients or situations at risk more precisely, in order to target additional preventive measures. Preliminary results, 11 months after the beginning of the study, show the feasibility and the relevance of completing the surveillance with specific studies within the haemovigilance network.

Bacterial Infections↗
PubMed1998

Characterization of the geranylgeranyl transferase type I from Schizosaccharomyces pombe.

The Schizosaccharomyces pombe cwg2+ gene encodes the beta-subunit of geranylgeranyl transferase I (GGTase I), which participates in the post-translational C-terminal modification of several small GTPases, allowing their targeting to the membrane. Using the two-hybrid system, we have identified the cwp1+ gene that encodes the alpha-subunit of the GGTase I. cwp1p interaction with cwg2p was mapped to amino acids 1-244 or 137-294 but was not restricted to amino acids 137-244. The genomic cwp1+ was isolated and sequenced. It has two putative open reading frames of 677 and 218 bp, separated by a 51 bp intron. The predicted amino acid sequence shows significant similarity to GGTase I alpha-subunits from different species. However, complementation of Saccharomyces cerevisiae ram2-1 mutant by overexpressing the cwp1+ gene was not possible. Expression of both cwg2+ and cwp1+ in Escherichia coli allowed 'in vitro' reconstitution of the GGTase I activity. S. pombe cells expressing the mutant enzyme containing the cwg2-1 mutation do not grow at 37 degrees C, but the growth defect can be suppressed by the addition of sorbitol. Actin immunostaining of the cwg2-1 mutant strain grown at 37 degrees C showed an abnormal distribution of actin patches. The cwg2-1 mutation was identified as a guanine to adenine substitution at nucleotide 604 of the coding region, originating the change A202T in the cwg2p. Deletion of the cwg2 gene is lethal; delta cwg2 spores can divide two or three times before losing viability. Most cells have aberrant morphology and septation defects. Overexpression of the rho1G15VC199R double-mutant allele in S. pombe caused loss of polarity but was not lethal and did not render the (1-3)beta-D-glucan synthase activity independent of GTP. Therefore, geranylgeranylation of rho1p is required for the appropriate function of this GTPase.

Actins↗