Analysis of the distribution and phosphorylation state of ZO-1 in MDCK and nonepithelial cells

We have examined the distribution and extent of phosphorylation of the tight junction-associated protein ZO-1 in the epithelial MDCK cell line, and in three cell types that do not form tight junctions: S180 (sarcoma) cells, S180 cells transfected with E-cadherin (S180L), and primary cultures of astrocytes. In shortterm calcium chelation experiments on MDCK cells, removal of extracellular calcium caused cells to pull apart. However, ZO-1 remained concentrated at the plasma membrane and no change in ZO-1 phosphorylation was observed. Maintenance of MDCK cells in low calcium medium, conditions where no tight junctions are found, resulted in altered ZO-1 distribution and lower total phosphorylation of the protein. In S180 cells, ZO-1 was diffusely distributed along the entire cell surface, with concentration of the antigen in motile regions of the cell. Cell-cell contact was not a prerequisite for ZO-1 localization at the plasma membrane in this cell type, and the phosphate content of ZO-1 was found to be lower in S180 cells relative to MDCK cells. Expression of Ecadherin in S180L cells did not alter either the distribution or phosphorylation of ZO-1. In contrast to S180 cells, ZO-1 in primary cultures of astrocytes was concentrated at sites of cell-cell contact, and the phosphorylation state was the same as that in control MDCK cells. Comparison of one-dimensional proteolytic digests of ³²P-labeled ZO-1 revealed the phosphorylation of two peptides in control MDCK cells that was absent in both MDCK cells grown in low calcium and in S180 cells.We would like to thank Cheryl Richards for her help with the cell culture and immunohistochemistry; David Begg, Gary Firestone, Vik Maraj, Manijeh Pasdar and Colin Rasmussen for helpful discussions; Jaclyn Peebles and Greg Morrison for help with graphics and photography; and Grace Martin and Bob Campenot for rat tail collagen. We are grateful to all the members of our laboratories for their friendship, advice and support. This work was supported by an Establishment Award to B.R.S. from the Alberta Heritage Foundation for Medical Research and grants to B.R.S. from the Kidney Foundation of Canada and the Medical Research Council of Canada. A.H. is funded by a Studentship from the AHFMR. K.L.S. was supported by a grant from the National Institutes of Health (DK-42799) to Gary L. Firestone. B.R.S. is a Medical Research Council of Canada and AHFMR Scholar.     

Use of synthetic lethal mutants to clone and characterize a novel CTP synthetase gene in Saccharomyces cerevisiae

In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5-triphosphate (UTP) to cytidine 5-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.     

Genetic interaction between the Ras-CAMP pathway and the Dis2s1/Glc7 protein phosphatase in Saccharomyces cerevisiae

The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1 ^ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.     

Genetics and biochemistry of phenol degradation byPseudomonas sp. CF600

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid locateddmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-knownmeta-cleavage pathway for catechol, and include the recently discoveredmeta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of thesemeta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to themeta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations betweenmeta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.     

Microbial transformation of ethylpyridines

Pyridine and its derivatives have been found as pollutants in the environment. Although alkylpyridines constitute the largest class of pyridines contaminating the environment, little information is available concerning the fate and transformation of these compounds. In this investigation ethylpyridines have been used as model compounds for investigating the biodegradability of alkylpyridines. A mixed culture of ethylpyridine-degrading microorganisms was obtained from a soil that had been exposed to a variety of pyridine derivatives for several decades. The enrichment culture was able to degrade 2-, 3-, and 4-ethylpyridine (100 mg/L) at 28° C and pH 7 within two weeks under aerobic conditions. The degradation rate was greatest for 2-ethylpyridine and least for 3-ethylpyridine. Transformation of ethylpyridines was dependent on substrate concentration, pH, and incubation temperature. Studies on the metabolic pathway of 4-ethylpyridine revealed two products; these chemicals were identified by MS and NMR analyses as 4-ethyl-2(1H)-pyridone and 4-ethyl-2-piperidone. 6-Ethyl-2(1H)-pyridone was determined to be a product of 2-ethylpyridine degradation. These results indicate that the transformation mechanism of ethylpyridines involves hydroxylation and reduction of the aromatic ring before ring cleavage.     

Changes in dormancy of Sisymbrium officinaleseeds do not depend on changes in respiratory activity

Effects of dark incubation at different temperatures were studied on dormancy and respiratory activity of seeds of Sisymbrium officinale (L.) Scop. Because germination of this species absolutely depends on the simultaneous action of light and nitrate, changes in dormancy could be studied in darkness without the interference of early germination events. Upon the start of incubation rates of O₂ uptake and CO₂ release rose. This was followed by a gradual decrease until stable levels of O₂ uptake and CO₂ release were achieved. Seeds kept for prolonged periods at 24^°C, showed neither a change in germination capacity nor in rates of O₂ uptake and CO₂ release. Respiratory quotients were 0.55–0.7. The initial rise in O₂ uptake correlated with the rate of water uptake and with breaking of primary dormancy. However, the subsequent decline in O₂ uptake was not generally linked to induction of secondary dormancy. An increased O₂ uptake was not required during breaking of secondary dormancy. It is concluded that changes in dormancy are not generally related to changes in respiratory activity. However, germination strongly depends on respiration. The increase in O₂ uptake started well before radicle protrusion. A far red irradiation only reversed this increase when it was given before germination escaped from its red light antagonising action. The contribution of different respiratory pathways was followed during prolonged incubation at 24^°C in darkness. KCN at 1.5 mM was needed to inhibit the cytochrome pathway (CP) and benzohydroxamic acid (BHAM) at 30 mM to inhibit the alternative pathway (AP). These concentrations did not exert any side effects. Electron flow was predominantly via the CP, maximally 10% was via the AP. Flow through the CP declined during the first 6 days and residual respiration remained constant. Therefore, the contribution of residual respiration became relatively more important with prolonged incubation. KCN at concentrations that almost completely inhibited flow through the CP, did not dramatically reduce germination. BHAM already inhibited germination at concentrations that do not inhibit oxygen uptake.     

Insights into the secretory pathway and vesicular transport in plant cells

Summary— The vectorial transport of membrane and macromolecules within the cytoplasm of eukaryotic cells has been the subject of intense investigation over the last decade. In this paper we review some of the recent advances made in our understanding of vesicle transport and the secretory pathway in plant cells.     

Gluconeogenesis from pyruvate in the hyperthermophilic archaeon Pyrococcus furiosus: involvement of reactions of the Embden-Meyerhof pathway

The hyperthermophilic archaeon Pyrococcus furiosus was grown on pyruvate as carbon and energy source. The enzymes involved in gluconeogenesis were investigated. The following findings indicate that glucose-6-phosphate formation from pyruvate involves phosphoenolpyruvate synthetase, enzymes of the Embden-Meyerhof pathway and fructose-1,6-bisphosphate phosphatase.Cell extracts of pyruvate-grown P.furiosus contained the following enzyme activities: phosphoenolpyruvate synthetase (0.025 U/mg, 50 °C), enolase (0.9 U/mg, 80 °C), phosphoglycerate mutase (0.13 U/mg, 55 °C), phosphoglycerate kinase (0.01 U/mg, 50 °C), glyceraldehyde-3-phosphate dehydrogenase reducing either NADP⁺ or NAD⁺ (NADP⁺: 0.019 U/mg, NAD⁺: 0.009 U/mg; 50 °C), triosephosphate isomerase (1.4 U/mg, 50 °C), fructose-1,6-bisphosphate aldolase (0.0045 U/mg, 55 °C), fructose-1,6-bisphosphate phosphatase (0.026 U/mg, 75 °C), and glucose-6-phosphate isomerase (0.22 U/mg, 50 °C). Kinetic properties (V _max values and apparent K _m values) of the enzymes indicate that they operate in the direction of sugar synthesis. The specific enzyme activities of phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase (NADP⁺-reducing) and fructose-1,6-bisphosphate phosphatase in pyruvate-grown P. furiosus were by a factor of 3, 10 and 4, respectively, higher as compared to maltose-grown cells suggesting that these enzymes are induced under conditions of gluconeogenesis. Furthermore, cell extracts contained ferredoxin: NADP⁺ oxidoreductase (0.023 U/mg, 60 °C); phosphoenolpyruvate carboxylase (0.018 U/mg, 50 °C) acts as an anaplerotic enzyme.Thus, in P. furiosus sugar formation from pyruvate involves reactions of the Embden-Meyerhof pathway, whereas sugar degradation to pyruvate proceeds via a modified non-phosphorylated Entner-Doudoroff pathway.