Functional control of the Candida albicans cell wall by catalytic protein kinase A subunit Tpk1.

The cyclic AMP protein kinase A pathway governs numerous biological features of the fungal pathogen Candida albicans. The catalytic protein kinase A subunits, Tpk1 (orf19.4892) and Tpk2 (orf19.2277), have divergent roles, and most studies indicate a more pronounced role for Tpk2. Here we dissect two Tpk1-responsive properties: adherence and cell wall integrity. Homozygous tpk1/tpk1 mutants are hyperadherent, and a Tpk1 defect enables biofilm formation in the absence of Bcr1, a transcriptional regulator of biofilm adhesins. A quantitative gene expression-based assay reveals that tpk1/tpk1 and bcr1/bcr1 genotypes show mixed epistasis, as expected if Tpk1 and Bcr1 act mainly in distinct pathways. Overexpression of individual Tpk1-repressed genes indicates that cell surface proteins Als1, Als2, Als4, Csh1 and Csp37 contribute to Tpk1-regulated adherence. Tpk1 is also required for cell wall integrity, but has no role in the gene expression response to cell wall inhibition by caspofungin. Interestingly, increased expression of the adhesin gene ALS2 confers a cell wall defect, as manifested in hypersensitivity to the cell wall inhibitor caspofungin and a shallow cell wall structure. Our findings indicate that Tpk1 governs C. albicans cell wall properties through repression of select cell surface protein genes.

Analysis of PRA1 and its relationship to Candida albicans- macrophage interactions.

Phagocytosis of Candida albicans by either primary bone marrow-derived mouse macrophages or RAW 264.7 cells upregulated transcription of PRA1, which encodes a cell wall/membrane-associated antigen previously described as a fibrinogen binding protein. However, a pra1 null mutant was still able to bind fibrinogen, showing that Pra1p is not uniquely required for fibrinogen binding. As well, Pra1 tagged with green fluorescent protein did not colocalize with AlexaFluor 546-labeled human fibrinogen, and while PRA1 expression was inhibited when Candida was grown in fetal bovine serum-containing medium, Candida binding to fibrinogen was activated by these conditions. Therefore, it appears that Pra1p can play at most a minor role in fibrinogen binding to C. albicans. PRA1 gene expression is induced in vitro by alkaline pH, and therefore its activation in phagosomes suggested that phagosome maturation was suppressed by the presence of Candida cells. LysoTracker red-labeled organelles failed to fuse with phagosomes containing live Candida, while phagosomes containing dead Candida underwent a normal phagosome-to-phagolysosome maturation. Immunofluorescence staining with the early/recycling endosomal marker transferrin receptor (CD71) suggested that live Candida may escape macrophage destruction through the inhibition of phagolysosomal maturation.

Calnexin-dependent regulation of tunicamycin-induced apoptosis in breast carcinoma MCF-7 cells.

The endoplasmic reticulum (ER) has evolved specific mechanisms to ensure protein folding as well as the maintenance of its own homeostasis. When these functions are not achieved, specific ER stress signals are triggered to activate either adaptive or apoptotic responses. Here, we demonstrate that MCF-7 cells are resistant to tunicamycin-induced apoptosis. We show that the expression level of the ER chaperone calnexin can directly influence tunicamycin sensitivity in this cell line. Interestingly, the expression of a calnexin lacking the chaperone domain (DeltaE) partially restores their sensitivity to tunicamycin-induced apoptosis. Indeed, we show that DeltaE acts as a scaffold molecule to allow the cleavage of Bap31 and thus generate the proapoptotic p20 fragment. Utilizing the ability of MCF-7 cells to resist tunicamycin-induced apoptosis, we have characterized a molecular mechanism by which calnexin regulates ER-stress-mediated apoptosis in a manner independent of its chaperone functions but dependent of its binding to Bap31.

The MAP kinase phosphatase-1 MKP-1/DUSP1 is a regulator of human liver response to transplantation.

Orthotopic liver transplantation (OLT) continues to be the only remedy for end-stage liver disease. In an attempt to decrease the ever-widening gap between organ donor and recipient numbers, and ultimately make more livers amenable to transplantation, we characterized the healthy human liver's response to ischemia and reperfusion-induced injury during transplantation. This was carried out by transcriptional profiling using cDNA microarray to identify genes whose expression was modulated at the 1-h postreperfusion time point. We observed that the map kinase phosphatase-1/dual-specificity phosphatase-1 (MKP-1/DUSP1) mRNA was strongly and significantly upregulated. Validation of this observation was carried out using reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblotting and immunohistochemistry. In addition, we characterized the signaling pathways regulating MKP-1 expression using the human hepatoma cell line HepG2. Finally, by combining MKP-1 silencing with reperfusion-associated stresses, we reveal the preferential role of this protein in attenuating the activity of the JNK and p38(MAPK) pathways, and the resulting apoptosis, making MKP-1 a potential target for therapeutic intervention.

Specific inhibition by hGRB10zeta of insulin-induced glycogen synthase activation: evidence for a novel signaling pathway.

Grb10 is a member of a family of adapter proteins that binds to tyrosine-phosphorylated receptors including the insulin receptor kinase (IRK). In this study recombinant adenovirus was used to over-express hGrb10zeta, a new Grb10 isoform, in primary rat hepatocytes and the consequences for insulin signaling were evaluated. Over-expression of hGrb10zeta resulted in 50% inhibition of insulin-stimulated IRK autophosphorylation and activation. Analysis of downstream events showed that hGrb10zeta over-expression specifically inhibits insulin-stimulated glycogen synthase (GS) activity and glycogen synthesis without affecting insulin-induced IRS1/2 phosphorylation, PI3-kinase activation, insulin like growth factor binding protein-1 (IGFBP-1) mRNA expression, and ERK1/2 MAP kinase activity. The classical pathway from PI3-kinase through Akt-PKB/GSK-3 leading to GS activation by insulin was also not affected by hGrb10zeta over-expression. These results indicate that hGrb10zeta inhibits a novel and presently unidentified insulin signaling pathway leading to GS activation in liver.

One test microbial diagnostic microarray for identification of Mycoplasma mycoides subsp. mycoides and other Mycoplasma species.

The present study describes the use of microarray technology for rapid identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. A microarray containing genetic sequences of 55 different bacterial species from Acholeplasma, Mycoplasma, Spiroplasma and Ureaplasma genera was constructed. Sequences to genes of interest were collected in FASTA format from NCBI. The collected sequences were processed with OligoPicker software. Oligonucleotides were then checked for their selectivity with BLAST searches in GenBank. The microarray was tested with ATCC/NCTC strains of Mycoplasma spp. of veterinary importance in ruminants including Mycoplasma belonging to the mycoides cluster as well as Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri field strains. The results showed that but one ATCC/NCTC reference strains hybridized with their species-specific sequences showed a profile/signature different and distinct from each other. The heat-map of the hybridization results for the nine genes interrogated for Mycoplasma mycoides subsp. mycoides demonstrated that the reference strain Mycoplasma mycoides subsp mycoides PG1 was positive for all of the gene sequences spotted on the microarray. CBPP field, vaccine and reference strains were all typed to be M. mycoides subsp. mycoides, and seven of the nine strains gave positive hybridization results for all of the nine genes. Two Italian strains were negative for some of the genes. Comparison with non-Mycoplasma mycoides subsp. mycoides reference strains showed some positive signals or considerable homology to Mycoplasma mycoides subsp. mycoides genes. As expected, some correlations were observed between the strictly genetically and antigenically correlated Mycoplasma mycoides subsp. mycoides and Mycoplasma mycoides subsp. capri strains. Specifically, we observed that some Italian Mycoplasma mycoides subsp. mycoides strains were positive for two out of the three Mycoplasma mycoides subsp. capri genes, differently from what has been observed for other European or African Mycoplasma mycoides subsp. mycoides strains. This study highlighted the use of microarray technology as a simple and effective method for a single-step identification and differentiation of Mycoplasma mycoides subsp. mycoides from other mycoplasmas that may be pathogenic to ruminants, including those of the Mycoplasma mycoides cluster, genetically and antigenically strictly correlated with Mycoplasma mycoides subsp. mycoides. The opportunity to discriminate several mycoplasmas in a single analysis enhances diagnostic rapidity and may represent a useful tool to screen occasionally mycoplasmas affecting animal farming in territories where diagnostic laboratory support is limited. The heat-map of the hybridization results of the comparative genomic hybridizations DNA-designed chip clearly indicates that the microarray performs well for the identification of the tested Mycoplasma mycoides subsp. mycoides reference and field strains, discriminating them from other mycoplasmas.

Transcriptome profiling of a TGF-beta-induced epithelial-to-mesenchymal transition reveals extracellular clusterin as a target for therapeutic antibodies.

Transforming growth factor (TGF)-beta plays a dual role in tumorigenesis, switching from acting as a growth inhibitory tumor suppressor early in the process, to a tumor promoter in late-stage disease. Since TGF-beta's prometastatic role may be linked to its ability to induce tumor cell epithelial-to-mesenchymal transition (EMT), we explored TGF-beta's EMT-promoting pathways by analysing the transcriptome changes occurring in BRI-JM01 mammary tumor epithelial cells undergoing a TGF-beta-induced EMT. We found the clusterin gene to be the most highly upregulated throughout most of the TGF-beta time course, and showed that this results in an increase of the secreted form of clusterin. By monitoring several hallmark features of EMT, we demonstrated that antibodies targeting secreted clusterin inhibit the TGF-beta-induced EMT of BRI-JM01 cells, as well as the invasive phenotype of several other breast and prostate tumor cell lines (4T1, NMuMG, MDA-MB231LM2 and PC3), without affecting the proliferation of these cells. These results indicate that secreted clusterin is a functionally important EMT mediator that lies downstream within TGF-beta's EMT-promoting transcriptional cascade, but not within its growth-inhibitory pathways. To further investigate the role played by secreted clusterin in tumor metastasis, we assessed the effect of several anti-clusterin monoclonal antibodies in vivo using a 4T1 syngeneic mouse breast cancer model and found that these antibodies significantly reduce lung metastasis. Taken together, our results reveal a role for secreted clusterin as an important extracellular promoter of EMT, and suggest that antibodies targeting clusterin may inhibit tumor metastasis without reducing the beneficial growth inhibitory effects of TGF-beta.

Characterization of three rice basic/leucine zipper factors, including two inhibitors of EmBP-1 DNA binding activity.

The promoter of the wheat Em gene contains elements with a CACGTG core sequence (G-boxes), which are recognized by EmBP-1, a wheat basic/leucine zipper (bZIP) protein. G-boxes are required for Em expression in response to the phytohormone abscisic acid and for transactivation by the Viviparous-1 protein (VP1) using transient expression systems. In order to identify other factors that are part of the transcriptional complex that associates with G-boxes, we have screened a rice (Oryza sativa) cDNA library with biotinylated EmBP-1. We have isolated osZIP-1a, a homolog of EmBP-1 and other plant G-box-binding factors. We show that EmBP-1 and osZIP-1a will preferentially heterodimerize in vitro. Overexpression of osZIP-1a in rice protoplasts can enhance expression from the Em promoter only in the presence of abscisic acid. Two other clones have been identified by screening with EmBP-1: osZIP-2a and osZIP-2b. These osZIP-2 factors represent a novel class of bZIP proteins with an unusual DNA-binding domain that does not recognize G-boxes. The osZIP-2 factors can heterodimerize with EmBP-1 and prevent it from binding to the Em promoter. Interestingly, osZIP-1a does not heterodimerize with the osZIP-2 factors and its DNA binding activity is unaffected by their presence. Thus, osZIP-2 factors may be involved in sequestering a particular group of G-box-binding factors into inactive heterodimers.

Acute lindane poisoning with development of muscle necrosis.

A 35-year-old man ingested food contaminated with lindane, an insecticide containing almost pure gamma hexachlorocyclohexane. Grand mal seizures and severe acidemia developed rapidly. The seizures recurred for nearly 2 hours, then ceased. In addition, the patient had muscle weakness and pain, headaches, episodic hypertension, myoglobinuria, acute renal failure and anemia. Pancreatitis developed 13 days after the ingestion of lindane. A muscle biopsy on the 15th day of illness demonstrated widespread necrosis and regeneration of muscle fibres. The patient's condition improved and he was discharged 24 days after the onset of his illness. During the year following the poisoning the patient noted difficulty with recent memory, loss of libido and easy fatigability. One year after lindane ingestion the results of physical examination, including those for muscle power and bulk, were normal.

Acute poisoning by selenious acid.

A 2 yo male child ingested approximately 15 ml of a Gun Blue solution containing selenious acid, nitric acid and copper nitrate. He was immediately given milk and vomited spontaneously blood-stained food with a garlic smell. He was admitted to our Centre less than 3 hr following ingestion. An esophago-gastroscopy showed a second degree burn of both esophagus and stomach. He became comatose and had to be ventilated mechanically. Metabolic acidosis, leucocytosis, hyperglycemia and hemoconcentration were also observed. During the following day he developed a severe intestinal distension, a cardiomyopathy (CPK = 1,302, cardiac arrhythmia), and moderate hepatic, renal and pulmonary dysfunctions. Plasma selenium concentration was 285 micrograms/L and the maximum urinary concentration was 28,459 micrograms/L. After 4 days, his condition had improved considerably and he was about to be extubated when he suddenly developed acute respiratory distress. A similar episode occurred 24 hr later. His lung function progressively deteriorated; later he required the use of an extracorporeal membrane lung. Legionella dumofii was found the causative agent. He died 17 d after ingestion despite aggressive treatment. Acute selenious acid poisoning and its relation to Legionnaire's disease is discussed.

Localization of endogenous Grb10 to the mitochondria and its interaction with the mitochondrial-associated Raf-1 pool.

Grb10 belongs to a small family of adapter proteins that are known to interact with a number of receptor tyrosine kinases and signaling molecules. We have recently demonstrated that the Grb10 SH2 domain interacts with both the Raf-1 and MEK1 kinases. Overexpression of Grb10 genes with mutations in their SH2 domains promotes apoptosis in cultured cells, a phenotype that is reversed by concomitant overexpression of the wild type gene. Using immunofluorescence microscopy and subcellular fractionation we now show that most of the Grb10 molecules are peripherally associated with mitochondria. Following insulin-like growth factor I or serum treatment, small pools of Grb10 can also be found at the plasma membrane and in actin-rich membrane ruffles, whereas overexpression of Grb10 leads to its mislocalization to the cytosol. Two-hybrid analysis shows that the Grb10-binding site on Raf-1 co-localizes with its Ras-binding domain. Finally, we show that the endogenous Grb10 and Raf-1 proteins can be co-immunoprecipitated from a partially purified mitochondrial extract, an interaction that is enhanced following the activation of Raf-1 by ultraviolet radiation. Thus, we infer that Grb10 may regulate signaling between plasma membrane receptors and the apoptosis-inducing machinery on the mitochondrial outer membrane by modulating the anti-apoptotic activity of mitochondrial Raf-1.

Visits to physicians before and after exposure to urea formaldehyde foam insulation.

The average number of visits to a physician made by a sample of 351 residents of homes insulated with urea formaldehyde foam insulation in Montreal in the one year period before exposure was 5.25, and in the year following 5.62, an increase of 7 per cent (odds ratio 1.07, 95% CI = 1.00, 1.15). The increase in visits in the post insulation year was limited to subjects who had the product installed in the winter (OR = 1.48, 95% CI = 1.18,1.85), and was not seen for study subjects who insulated their homes during other seasons of the year.

Description of microparticles in urea-formaldehyde foam insulation products.

Thousands of families living in homes insulated with urea-formaldehyde foam (UFF) have complained of various health problems. This product is now banned from the market both in Canada and the United States. Formaldehyde gas emitted by the product has been considered to be the source of these health problems but this cause-effect relationship has not been confirmed as yet. It has been suggested that other contaminants released by the foam could be involved. Work initiated to verify this possibility has permitted the observation of microparticles of less than 1 micron in diameter in foam samples obtained from various houses. The microparticles can easily be removed from the foam by passing air through it or, simply, by immersing it in water. These particles have been studied by different microscopy techniques and their morphological characteristics are described. They may represent a potential health risk.

Promoter for a Brassica napus ribulose bisphosphate carboxylase/oxygenase small subunit gene binds multiple nuclear factors and contains a negative-strand open reading frame encoding a putative transmembrane protein.

Using a fractionated genomic bank, we have cloned and characterized a Brassica napus gene (rbcSF1) encoding the small sub-unit of ribulose 1,5-bisphosphate carboxylase. The promoter of this gene contains a 29 bp direct repeat capable of forming a single or a double hairpin loop, and three elements that are recognized by leaf nuclear proteins in vitro. The most upstream are the S-box, a small A/T-rich sequence between -516 and -512, and the F-box between -492 and -475. Finally, we have also observed binding to the G-box, a regulatory element common to numerous plant promoters. The promoter of rbcSF1 also has a 113 amino acids open reading frame (ORF113) in the non-coding strand. When used to probe a northern blot of leaf RNA, this ORF hybridizes to a 1.5 kb transcript. The protein encoded by ORF113 contains a transmembrane domain.

Blood lead levels in children and pregnant women living near a lead-reclamation plant.

OBJECTIVE: To determine the effect of lead contamination around a lead-reclamation plant on the blood lead levels of children and pregnant women living in the area. DESIGN: Prevalence study. SETTING: Residents living 150 m or less (high-exposure area), 151 to 400 m (intermediate-exposure area) or 401 to 800 m (low-exposure area) southeast from the plant. PARTICIPANTS: All children aged 10 years or less and all pregnant women living in the designated area. OUTCOME MEASURES: Correlation of venous blood lead levels with soil lead concentrations in the areas in which the subjects lived and with sociodemographic and behavioural factors. MAIN RESULTS: Of the estimated 57 pregnant women 38 (67%) participated: 20 were in the high-exposure area and 18 in the other two areas; their geometric mean blood lead levels were low (0.15 and 0.13 mumol/L respectively). Of the 625 eligible children 510 (82%) participated: 169 were in the high-exposure area, 179 in the intermediate-exposure area and 162 in the low-exposure area; their geometric mean lead levels were 0.43, 0.30 and 0.26 mumol/L respectively. Within each age group children in the high-exposure area had the highest levels. The mean levels for children aged 6 months to 5 years were 0.49, 0.35 and 0.28 mumol/L in the three areas respectively. Within each exposure group children aged 1 to 2 years had the highest levels. No potential confounding variables could explain the relation between blood lead level and soil lead concentration. CONCLUSIONS: The pregnant women's blood lead levels did not seem to be affected by exposure level, but the children's levels were primarily related to the soil lead concentration.

Polychlorinated biphenyl (PCB) and dichlorodiphenyl dichloroethylene (DDE) concentrations in the breast milk of women in Quebec.

OBJECTIVES: This study documented the concentration of polychlorinated biphenyls (PCBs) and dichlorodiphenyl dichloroethylene (DDE) in the breast milk of women from Quebec, Canada, and assessed the impact of various sociodemographic and lifestyle factors on these levels. METHODS: From 1988 to 1990, milk samples were obtained from 536 Quebec women and analyzed for seven PCB congeners and p,p'-DDE. Information was obtained on subjects' physical, sociodemographic, and lifestyle characteristics. RESULTS: Mean concentrations were 0.52 mg/kg lipids (95% confidence interval [CI] = 0.50, 0.54) and 0.34 mg/kg lipids (95% CI = 0.32, 0.35) for PCBs (Aroclor 1260) and DDE, respectively. Age and history of breast-feeding showed statistically significant correlations with PCB and DDE concentrations. CONCLUSIONS: Concentrations of PCBs and DDE measured in this study are at the lower end of the concentration range recently reported for women living in industrialized countries. The modulating factors identified here should be considered when conducting studies on organochlorine exposure and disease.

Interaction of the Grb10 adapter protein with the Raf1 and MEK1 kinases.

Grb10 and its close homologues Grb7 and Grb14, belong to a family of adapter proteins characterized by a proline-rich region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen using the MEK1 kinase as a bait. We show that this unusual SH2 domain interacts, in a phosphotyrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-activated protein kinase in vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenesis of the SH2 domain demonstrated that the Arg-betaB5 and Asp-EF2 residues are necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a mutation in Ser-betaB7 affects binding only to the receptors, while a mutation in Thr-betaC5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can be competed by co-expression of the wild type protein, suggesting that these mutants act by sequestering necessary signaling components.

A conserved domain of the viviparous-1 gene product enhances the DNA binding activity of the bZIP protein EmBP-1 and other transcription factors.

The maize VP1 protein is a seed-specific regulator of gene expression that effects the expression of a subset of abscisic acid (ABA)-regulated genes that are expressed during the maturation program of the seed. In addition, VP1 has pleiotropic effects on seed development that are not related to ABA. In transient expression assays, VP1 has been shown to transactivate gene expression through at least two distinct promoter elements: the G boxes from the ABA-inducible wheat Em gene and the SphI box from the maize C1 gene. We have investigated how VP1 can transactivate gene expression through diverse promoter elements by analyzing its association in vitro with EmBP-1, a factor that binds the Em promoter. We demonstrate that VP1 can greatly enhance the DNA binding activity of EmBP-1 in a gel retardation assay. This enhancing activity has also been observed on transcription factors as diverse as Opaque-2, Max, Sp1, and NF-kappaB. Deletion of a small but highly conserved region (BR2) in VP1 eliminates the enhancement in vitro as well as the ability of VP1 to transactivate Em gene expression in a transient expression assay. A 40-amino acid fragment from VP1 sandwiched between the maltose-binding protein and LacZ can confer the enhancement function to this fusion protein in vitro. A weak and relatively nonspecific interaction between BR2 and DNA is demonstrated by UV cross-linking. The in vitro properties we observe for VP1 might explain the regulatory effects of VP1 on a diverse set of genes and why mutations in the vp1 locus have pleiotropic effects.

Argyremia in septal cauterization with silver nitrate.

OBJECTIVE: The purpose of this study was to demonstrate silver absorption in blood and hair specimens after septal cauterization with silver nitrate and to discuss the potential toxicity of silver. METHOD: A prospective study of 11 volunteers without any known occupational exposure to silver products or past history of septal cauterization with silver nitrate was undertaken. Subjects were recruited in an academic tertiary care centre from October 1996 to September 1997. The study population consisted of five patients with anterior epistaxis and six healthy volunteers without any bleeding problem. Cauterization was done with one or two silver nitrate applicators directly on the bleeding vessel or Kiesselbach's area. Blood was sampled before cauterization and at specified times after application, while hair strands were sampled only 3 months later. Measurements of silver concentration in whole blood and in hair segments were obtained by inductively coupled plasma mass spectrometry. RESULTS: Silver concentrations in whole blood increased significantly after cauterization (p = .02). The measured peak level seemed to correlate with the number of applicators used. No significant increase in silver concentration was observed in hair samples. CONCLUSIONS: Effective silver absorption occurs with only one or two silver nitrate applicators. Hair has not been as reliable as whole blood to document an acute and fragmentary exposure. The indiscriminate use of silver nitrate is a potential source of silver intoxication.