Efectividad de clorogenos destinadosa agua de consumo / Efectiveness of chlorogenic products destinated to drinking waters

Se propone una metodología para la evaluación de productos clorógenos destinados a la desinfección de aguas para consumo, utilizando materia orgánica para simular el consumo de cloro que tienen las aguas no tratadas. Se evaluó el comportamiento de suspensión de levadura de cerveza, peptona y extracto de levadura frente a dosis usuales (10 mg/L, como cloro total) de comprimidos de la sal disódica de la dicloro-S-triazina-triona. Como comparación se utilizó agua de río.Se determinó la concentración de cada sustancia orgánica que despues de 30 minutos dejó 0,1 mg/L de cloro libre en la solución de ensayo. Posteriormente se ensayó microbiologicamente la efectividad del clorógeno en presencia de dichas sustancias según metodología AFNOR (AU)

A 48-kDa, S-antigen-like phosphoprotein in yeast DNA-replicative complex preparations.

A 48-kDa protein from the budding yeast Saccharomyces cerevisiae is antigenically and structurally similar to S-antigen from retina. Eight anti-S-antigen monoclonal antibodies, directed against distinct epitopes, cross-reacted with a yeast 48-kDa protein. Structural similarity between the bovine and yeast proteins was further demonstrated by comparison of tryptic peptide fragments containing one of these epitopes. This 48-kDa yeast protein appears to be a component of the replicative complex of the cell. It was found associated with immunoaffinity-purified yeast DNA polymerase I-primase and with yeast DNA-replicative complex. The 48-kDa protein was phosphorylated by a protein kinase activity endogenous to the replicative complex preparation. This phosphorylation was dependent on the cell division cycle gene CDC7. In addition, authentic bovine S-antigen, when added to yeast DNA polymerase I-primase, stimulated polymerase activity. These findings suggest that the yeast S-antigen-like protein may play a role in replication, and they raise the possibility that it may be involved in traversal of the G1/S boundary of the cell cycle.

Glucan structure in a fragile mutant of Saccharomyces cerevisiae.

The phenotype of VY1160 fragile Saccharomyces cerevisiae mutant is characterized by cell lysis upon transfer to hypotonic solutions and increased permeability of cells growing in osmotically stabilized media. Two mutations, srb1 and ts1, have been identified in VY1160 cells and previous studies have shown that the increased permeability is due to the ts1 mutation which causes a shortening of mannan side-chains. Here we report that the srb1 mutation, which is the genetic determinant of cell lysis, is responsible for quantitative and structural changes of glucans. Experiments with isogenic single mutation strains, genetic studies coupled with quantitative measurements of glucan content per cell, and methylation analysis of glucans provide evidence that srb1 mutation leads to i) formation of mechanically unstable cell wall network made of insoluble glucan fibrils which are shorter and contain beta(1-6) inter-residue linkages and ii) insufficient filling of the space between the fibrils due to a shortage of the alkali-soluble glucan. Although growing exponentially in osmotically stabilized media, the srb1 cells cannot resist an osmotic shock and, hence, burst immediately.

C-myc and the yeast transcription factor PHO4 share a common CACGTG-binding motif.

The basic-helix-loop-helix (b-HLH) motif is common to a number of proteins involved in transcriptional regulation and cell-type determination. The b-HLH motif is also present in the S. cerevisiae transcription factor PHO4 which positively regulates the acid phosphatase gene PHO5. In this report we show that the b-HLH region of PHO4 is sufficient to confer specific DNA-binding to the sequence CACGTG and, by comparison of the basic regions of PHO4 with those of other recently isolated CACGTG-binding proteins, we identify a specific subset of conserved amino acids in the basic region likely to confer DNA-binding specificity. On the basis of these observations we predict successfully the effect of substituting the PHO4 basic region with that from c-myc and show that the chimaeric protein activates transcription from the CACGTG elements present in the PHO5 UAS. From these data it is clear that the myc basic region confers specific binding to the sequence CACGTG.

Molecular species of cardiolipin in relation to other mitochondrial phospholipids. Is there an acyl specificity of the interaction between cardiolipin and the ADP/ATP carrier?

Molecular species in the three major mitochondrial lipids cardiolipin, phosphatidylcholine and phosphatidylethanolamine were analysed in bovine heart and Saccharomyces cerevisiae. In both organisms cardiolipin contains mainly diacylglycerol moieties with two unsaturated chains and a significant higher proportion of C18-C18 species than phosphatidylcholine and phosphatidylethanolamine. To study whether the specific acyl composition of cardiolipin has a functional significance in lipid-protein interaction, experiments were made with the isolated ADP/ATP carrier of bovine heart mitochondria since this dimeric protein is known to be tightly associated with six molecules of cardiolipin [Beyer, K. and Klingenberg, M. (1985) Biochemistry 24, 3821-3826]. This association seems to be very strong as protein-bound cardiolipin does not exchange with soluble cardiolipin on a time scale of hours. Analysis of the species composition suggests that one carriers dimer is associated with four molecules of tetralinoleoyl cardiolipin and two molecules of trilinoleoyl-monolinolenoyl cardiolipin. Catalytic hydrogenation of the acyl chains of carrier-bound cardiolipin does not result in release of cardiolipin as judged by 31P-NMR spectroscopy. The ADP/ATP carrier was reconstituted with saturated phosphatidylcholines and spin-labelled cardiolipin whose double bonds were subsequently saturated by catalytic hydrogenation. ESR spectroscopy shows that saturation of spin-labelled cardiolipin has no significant impact on its association with the ADP/ATP carrier. However, precipitation of the detergent-solubilized ADP/ATP carrier can only be induced by addition of unsaturated but not by saturated cardiolipin. It is concluded that the specific acyl composition of cardiolipin is not a prerequisite of its high affinity for the ADP/ATP carrier, at least when the protein is reconstituted in a saturated phosphatidylcholine environment.

Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38. The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3188-3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.

A rapid, efficient method for purifying DNA-binding proteins. Denaturation-renaturation chromatography of human and yeast mitochondrial extracts.

We describe a novel method for the purification of DNA-binding proteins. Isolated mitochondria were lysed in boiling sodium dodecyl sulfate-containing buffer, the extracts were chromatographed on hydroxylapatite in the presence of sodium dodecyl sulfate, and DNA-binding activities were identified after adding a large excess of nonionic detergent (Triton X-100) and assaying fractions by a gel retardation procedure. Fractions containing DNA-binding activity were bulk renatured and chromatographed on phosphocellulose in the presence of Triton X-100. When applied to human mitochondria, the technique resulted in the purification to homogeneity of fully functional mitochondrial transcription factor 1 (mtTF1), the major activator of mammalian mitochondrial transcription. Moreover, the yield of mtTF1 purified by this method was at least 25 times higher than that obtained by conventional nondenaturing chromatographies. When yeast mitochondria were subjected to the same protein isolation scheme, a 19-kilodalton putative yeast homologue of mtTF1 was purified to homogeneity. These results suggest that the denaturation-renaturation approach may be a valuable general method for the identification and high yield purification of DNA-binding proteins.

Calcium-dependent secretory vesicle-binding and lipid-binding proteins of Saccharomyces cerevisiae.

Yeast (Saccharomyces cerevisiae) cytosol was examined for the presence of calcium-dependent membrane- or lipid-binding proteins that might play fundamental roles in membrane-associated phenomena in stimulated cells. A complex group of proteins was isolated from late log phase cultures of yeast strain YP3 on the basis of calcium-dependent association with yeast secretory vesicles isolated from the temperature-sensitive sec6-4 secretory mutant. The masses of the major proteins in this group were 32, 35, 47, 51, 55, 60 and 120 kDa. A similar group of proteins was isolated by calcium-dependent association with bovine brain lipids enriched in the predominant acidic phospholipids of the yeast secretory vesicles. The 47 kDa protein was highly purified when commercial yeast cake was used as the source of yeast cytosol. The 32 kDa and 60 kDa proteins were demonstrated to reassociate with lipids at calcium concentrations of 100 microM or higher, while no association was promoted by 2 mM-magnesium. The 47 kDa protein could be removed from lipids by reducing the calcium concentration to between 1 and 32 microM. The sequences of peptides isolated from digests of several of these proteins indicate that they are novel proteins but are insufficient to judge the possible homology of these proteins with mammalian membrane-binding proteins. The sequence data may be adequate to permit isolation and modification of the corresponding genes in order to assess the possible function of this class of proteins in stimulated cells.

Binding of yeast TFIIIC to tRNA gene bipartite internal promoters: analysis of physical effects on the intervening DNA.

Complexes between transcription factor TFIIIC and eukaryotic tRNA gene internal promoter A and B boxes are unusual in that the binding to the two distinct sites tolerates considerable variation in both distance and helical orientation between the sites. Electrophoretic mobility of Saccharomyces cerevisiae TFIIIC complexes with circularly permuted tRNA gene fragments and sensitivity of the complexes to a single stranded-specific reagent, potassium permanganate, indicated that no significant bend or distortion was introduced into the DNA by simultaneous binding to both internal promoters. These data support a model in which variability in the relative positions of the two binding sites is compensated by flexibility in the structure of TFIIIC.

On-line determination of glucose in biotechnological processes: comparison between FIA and an in situ enzyme electrode.

Two different analysis techniques for on-line monitoring of glucose in biotechnological processes have been tested: an in situ enzyme electrode and a flow injection analysis system (FIA). The measuring ranges, detection limits, response times and the reliabilities of each system have been compared during monitoring of batch and continuous cultures of Saccharomyces cerevisiae.

Phospholipid synthesis and lipid composition of subcellular membranes in the unicellular eukaryote Saccharomyces cerevisiae.

Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.

Cellular morphogenesis in the Saccharomyces cerevisiae cell cycle: localization of the CDC3 gene product and the timing of events at the budding site.

Budding cells of the yeast Saccharomyces cerevisiae possess a ring of 10-nm-diameter filaments, of unknown biochemical nature, that lies just inside the plasma membrane in the neck connecting the mother cell to its bud. Electron microscopic observations suggest that these filaments assemble at the budding site coincident with bud emergence and disassemble shortly before cytokinesis (Byers, B. and L. Goetsch. 1976. J. Cell Biol. 69:717-721). Mutants defective in any of four genes (CDC3, CDC10, CDC11, or CDC12) lack these filaments and display a pleiotropic phenotype that involves abnormal bud growth and an inability to complete cytokinesis. We showed previously by immunofluorescence that the CDC12 gene product is probably a constituent of the ring of 10-nm filaments (Haarer, B. and J. Pringle. 1987. Mol. Cell. Biol. 7:3678-3687). We now report the use of fusion proteins to generate polyclonal antibodies specific for the CDC3 gene product. In immunofluorescence experiments, these antibodies decorated the neck regions of wild-type and mutant cells in patterns suggesting that the CDC3 gene product is also a constituent of the ring of 10-nm filaments. We also used the CDC3-specific and CDC12-specific antibodies to investigate the timing of localization of these proteins to the budding site. The results suggest that the CDC3 protein is organized into a ring at the budding site well before bud emergence and remains so organized for some time after cytokinesis. The CDC12 product appears to behave similarly, but may arrive at the budding site closer to the time of bud emergence, and disappear from that site more quickly after cytokinesis, than does the CDC3 product. Examination of mating cells and cells responding to purified mating pheromone revealed novel arrangements of the CDC3 and CDC12 products in the regions of cell wall reorganization. Both proteins were present in normal-looking ring structures at the bases of the first zygotic buds.

Saccharomyces cerevisiae proteins involved in hybrid DNA formation in vitro.

RecA-like activities that can form hybrid DNA in vitro have been identified in a wide variety of organisms. We have previously described the strand exchange protein 1 (SEP1) from the yeast Saccharomyces cerevisiae that can form hybrid DNA in vitro. Purified as an Mr 132,000 polypeptide, recent molecular and immunological studies have now shown that the native form is an Mr 175,000 polypeptide containing strand exchange activity. The gene encoding SEP1 has been cloned and sequenced. The primary sequence failed to reveal any significant sequence homology to other sequences in data base searches. In vivo SEP1 was found to be essential for normal meiosis as cells containing a homozygous insertion mutation in the SEP1 gene failed to sporulate. In order to identify additional factors that are involved in hybrid DNA formation in S cerevisiae, we used an in vitro stimulation assay to identify proteins that reconstitute strand exchange activity in reactions containing limiting amounts of SEP1. We have identified two proteins that functionally interact with SEP1. First, an Mr 34,000 single-stranded DNA binding protein stimulated the reaction by lowering the requirement for SEP1 about 3-4 fold. This protein is a fragment of the large subunit of a hetero-trimeric complex called yRP-A (yRF-A) which is thought to be the functional eukaryotic equivalent of single-stranded DNA binding proteins in prokaryotes. The gene encoding this protein (RPA1) is essential for growth. Second, an Mr 33,000 polypeptide, termed Stimulatory Factor 1 (SF1), dramatically stimulated the SEP1 catalyzed reaction by lowering the requirement for SEP1 about 300 fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Domains of yeast plasma membrane and ATPase-associated glycoprotein.

In yeast homogenates the plasma membrane H(+)-ATPase and a major surface glycoprotein of about 115 kDa are present in two membrane fractions with peak densities in sucrose gradients of 1.17 and 1.22. Immunogold electron microscopy of frozen yeast sections indicates that the ATPase is exclusively (greater than 95%) present at the surface membrane. Therefore the two ATPase-containing fractions appear to correspond to different domains of the plasma membrane. The 115 kDa glycoprotein is tightly associated with the ATPase during solubilization and purification of the enzyme. However, in a mutant lacking the glycoprotein the activity of the plasma membrane H(+)-ATPase is similar to wild type, suggesting that this association is fortuitous. The ATPase and the glycoprotein are difficult to separate by electrophoresis and therefore binding of concanavalin A to the ATPase cannot be unambiguously demonstrated in wild-type yeast. By utilizing the mutant without glycoprotein it was shown that the ATPase band of 105 kDa binds concanavalin A.

Biochemical studies of homologous and nonhomologous recombination in human cells.

Purified and partially purified protein fractions from human cells have been developed that promote homologous and nonhomologous recombination reactions in vitro. Homologous pairing of model DNA substrates is catalyzed by the homologous pairing protein HPP-1 in a magnesium-dependent, ATP-independent reaction that requires stoichiometric amounts of the protein. Addition of the human single-strand binding (SSB) holoprotein complex hRP-A reduces the requirement of HPP-1 in the reaction up to 20-fold. Although the combination of homologous pairing and SSB activities is similar to the bacterial strand-exchange process, the numbers, size, and requirements of the human reaction appear to preclude any detailed comparisons. We have used Z-DNA affinity chromatography as a major step in isolation of human recombination proteins and found that the activities appear to elute as a complex form in approximate multiples of 500 kDa. Associated with the homologous recombination complex is a potent blunt-end ligation activity that appears to mimic the nonhomologous joining functions that are frequently seen following transfection of DNA into mammalian cells. A simple scheme for the association of homologous and nonhomologous recombination functions in mammalian cells is discussed.

Effect of nystatin, amphotericin B and amphotericin B methyl ester on Saccharomyces cerevisiae with different lipid composition.

Saccharomyces cerevisiae was cultured under anaerobiosis in semi-complete medium to which either palmitoleic or oleic acid was added. Cells were grown at 20 degrees C or 30 degrees C. The levels of total lipids, total sterols, and phospholipids were higher in cells grown at 20 degrees C than at 30 degrees C. The effects of nystatin (NYS), amphotericin B (AMB), and amphotericin B methyl ester (AME) were evaluated by determining cell viability and liberation of intracellular compounds. The loss of cell viability is higher in the first 30 minutes of incubation with the drugs and is the same regardless of the type of cells obtained. Low molecular weight compounds and ions such as K+ are liberated a few minutes after incubation with the drugs whereas proteins and substances absorbing at 260 nm are liberated later. Phosphate liberation comes after K+ and before compounds of higher molecular weights.

New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates.

Two derivatives of pancreatic ribonuclease and endonuclease of Staphylococcus aureus, insolubilized on corn cob, have been used to reduce the percentage of nucleic acids in single cell protein (SCP) concentrates from yeasts. These derivatives are thermostable and active at 45 degrees C. At these temperatures the contamination by bacteria is negligible. The thermostability is remarkable, since the native nuclease is deactivated at above 39 degrees C. The hydrolysis of the nucleic acids in SCP is carried out first with the ribonuclease derivative followed by the endonuclease derivative. The catalytic activity of the insolubilized derivatives is similar to that of the native enzymes in the hydrolysis of RNA but not of DNA. The percentage of nucleic acids is reduced from 5-15 to 0.5%, with a loss of protein of 6%. These percentages are lower than those previously reported.

Mitochondrial phosphate transport. N-ethylmaleimide insensitivity correlates with absence of beef heart-like Cys42 from the Saccharomyces cerevisiae phosphate transport protein.

The mitochondrial phosphate transport protein (PTP) has been purified in a reconstitutively active form from Saccharomyces cerevisiae and Candida parapsilosis. ADP/ATP carriers that copurify have been identified. The PTP from S. cerevisiae migrates as a single band (35 kDa) in sodium dodecyl sulfate gels with the same mobility as the N-ethylmaleimide-alkylated beef heart PTP. It does not cross-react with anti-sera against beef heart PTP. The CNBr peptide maps of the yeast and beef proteins are very different. The rate of unidirectional phosphate uptake into reconstituted proteoliposomes is stimulated about 2.5-fold to a Vmax of 170 mumol of phosphate min-1 (mg PTP)-1 (22 degrees C) by increasing the pHi of the proteoliposomes from 6.8 (same as pHe) to 8.0. The Km for Pi of this reconstituted activity is 2.2 mM. The transport is sensitive to mersalyl (50% inhibition at 60 microM) and insensitive to N-ethylmaleimide. We have purified peptides matching the highly conserved motif Pro-X-(Asp/glu)-X-X-(Lys/Arg)-X-(Arg/lys) (X is an unspecified amino acid) of the triplicate gene structure sequence of the beef heart PTP. The N-ethylmaleimide-reactive Cys42 of the beef heart protein, located between the two basic amino acids of this motif (Lys41-Cys42-Arg43), is replaced with a Thr in the yeast protein. This substitution most likely is responsible for the lack of N-ethylmaleimide sensitivity of the yeast protein and mersalyl thus reacts with another cysteine to inhibit the transport. Finally it is concluded that Cys42 has no essential role in the catalysis of inorganic phosphate transport by the mitochondrial phosphate transport protein.

Components of the yeast spindle and spindle pole body.

Yeast spindle pole bodies (SPBs) with attached nuclear microtubles were enriched approximately 600-fold from yeast cell extracts. 14 mAbs prepared against this enriched SPB fraction define at least three components of the SPB and spindle. Immunofluorescent staining of yeast cells showed that throughout the cell cycle two of the components (110 and 90 kD) were localized exclusively to the SPB region, and the other (80 kD) was localized both to the SPB region and to particulate dots in short spindles. Immunoelectron microscopy confirmed and extended most of these findings. Thus the 110-kD component was localized to a layer in the SPB just to the nuclear side of the plane of the inner nuclear membrane. The 90-kD component was localized in a layer across the cytoplasmic face of intact SPBs, and, in SPBs where nuclear microtubules were removed by extraction with DEAE-dextran, the 90-kD component was also found in an inner nuclear layer close to where spindle microtubules emerge. In intact SPBs with attached nuclear microtubules the anit-80-kD mAb labels microtubules, particularly those close to the SPB. These results begin to provide a preliminary molecular map of the SPB and should also enable the corresponding genes to be isolated.