High-affinity L-arabinose transport operon. Nucleotide sequence and analysis of gene products

The nucleotide sequence of the "high-affinity" L-arabinose transport operon has been determined 3' from the regulatory region and found to contain three open reading frames designated araF, araG and araH. The first gene 3' to the regulatory region, araF, encodes the 23-residue signal peptide and the 306-residue mature form of the L-arabinose binding protein (33,200 Mr). The binding protein, which has been described elsewhere, is hydrophilic, soluble and found in the periplasm of Escherichia coli. This gene is followed by an intragenic space of 72 nucleotides, which contains a region of dyad symmetry 23 nucleotides long capable of forming an 11-member stem-loop. The second gene, designated araG, contains an open reading frame capable of encoding an equally hydrophilic protein containing 504 residues (55,000 Mr). Following a 14-nucleotide spacer, which does not appear to have any secondary structure, the third open reading frame, herein designated araH, is capable of encoding a hydrophobic protein containing 329 residues (34,000 Mr) that can only be envisioned as having an integral membrane location. 3' to araH there is a T-rich region containing a 24-nucleotide area of dyad symmetry centered 55 nucleotides from the termination codon. Analysis of the derived primary sequences of the araG and araH products indicates the nature and potential features of these components. The araG protein was found to possess internal homology between its amino and carboxyl-terminal halves, suggesting a common origin. The araG gene product has been shown to be homologous to the rbsA gene product, the hisP product, the ptsB product and the malK product, all of which presumably play similar roles in their respective transport systems. Putative ATP binding sites are observed within the regions of homology. The araH gene product has been shown to be homologous to the rbsC gene product, which is the first observed homology between two purported membrane proteins.     

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.     

Mechanisms in bradykinin stimulated arachidonate release and synthesis of prostaglandin and platelet activating factor

Regulatory mechanisms in bradykinin (BK) activated release of arachidonate (ARA) and synthesis of prostaglandin (PG) and platelet activating factor (PAF) were studied in bovine pulmonary artery endothelial cells (BPAEC). A role for GTP binding protein (G-protein) in the binding of BK to the cells was determined. Guanosine 5-O- (thiotriphosphate), (GTPtauS), lowered the binding affinity for BK and increased the Kd for the binding from 0.45 to 1.99 nM. The Bmax remained unaltered at 2.25 x 10(-11) mole. Exposure of the cells to aluminium fluoride also reduced the affinity for BK. Bradykinin-induced release of ARA proved pertussis toxin (PTX) sensitive, with a maximum sensitivity at 10 ug/ml PTX. GTPtauS at 100 muM increased the release of arachidonate. The effect of GTPtauS and BK was additive at suboptimal doses of BK up to 0.5 nM but never exceeded the levels of maximal BK stimulation at 50 nM. PTX also inhibited the release of ARA induced by the calcium ionophore, A23187. Phorbol 12-myristate 13-acetate or more commonly known as tetradecanoyl phorbol acetate (TPA) itself had little effect on release by the intact cells. However, at 100 nM it augmented the BK activated release. This was downregulated by overnight exposure to TPA and correlated with down-regulation of protein kinase C (PKC) activity. The down-regulation only affected the augmentation of ARA release by TPA but not the original BK activated release. TPA displayed a similar, but more potent amplification of PAF synthesis in response to both BK or the calcium ionophore A23187. These results taken together point to the participation of G-protein in the binding of BK to BPAEC and its activation of ARA release. Possibly two types of G-protein are involved, one associated with the receptor, the other activated by Ca(2+) and perhaps associated with phospholipase A(2) (PLA(2)). Our results further suggest that a separate route of activation, probably also PLA(2) related, takes place through a PKC catalysed phosphorylation.     

Prostaglandin synthesis by cells comprising the calf pulmonary artery

Prostaglandin (PG) production was evaluated in the three cell types (endothelial, smooth muscle, and fibroblast) comprising the bovine pulmonary artery. Prostacyclin (PGI2) was the predominant prostaglandin (PG) produced by endothelial, smooth muscle, and fibroblast cells as they exist in culture or in freshly excised tissue fragments. In addition to PGI2, measurable amounts of PGE2, PGF2a, and thromboxane A2 (TXA2) were also produced by these cells. Endothelial cells were the most active producers of PGs. However, the type of PG produced was characteristic of the particular cell type, while the level of production was dependent on external factors. Prostaglandin production by cultured cells, both under basal conditions and in response to stimulatory agents, was quite similar to that of the respective freshly excised tissue fragments containing a given cell type. These cells in culture could be stimulated to produce PGI2 by both angiotensin and bradykinin at very low (physiological) concentrations, a further indication of the retention of the physiological responsiveness of these cells in culture. Endothelial cells and fibroblasts were activated by bradykinin at concentrations as low as 10(-12) M but did not respond to angiotensin. Smooth muscle cells in primary and first passage cultures were activated by both bradykinin and angiotensin at 10(-12) M concentrations. Serial subcultivations of smooth muscle cells resulted in a progressive loss in their responsiveness to bradykinin stimulation. The state of cell growth proved to be an important determinant of PG production. Actively growing cells in culture synthesized less PG when compared to cells which had entered into a "quiescent" nongrowth state.     

The role of adenosine 3′:5′-cyclic monophosphate in the division of WI 38 cells. The cellular response to prostaglandin E1 and the effects of an cyclic adenosine 3′:5′-cyclic monophosphate analogue and prostaglandin E1 on cell division

Inhibition of growth and DNA synthesis was observed in WI 38 cells incubated with 8-methylthioadenosine 3':5'-cyclic monophosphate or prostaglandin E(1). The effect of both compounds on cell growth was reversible. On removal of these compounds from culture media the cells initiated DNA synthesis and divided. In addition, prostaglandin E(1) stimulated cyclic AMP formation in these cells to over 40 times the normal basal value. The increase in cyclic AMP concentration in WI 38 cells after addition of prostaglandin E(1) showed a marked variation. Cells that had recently been treated with trypsin and plated at a lower cell density exhibited a smaller response to addition of prostaglandin E(1) than cells that had divided and reached confluence.     

Effect of prolonged prostaglandin exposure on prostaglandin synthesis by human lung fibroblasts

Pretreatment of human lung fibroblasts with PGE₂ but not PGF_2α enhanced synthesis of prostaglandins (PGs). The effect of the pretreatment on PG synthesis was related to the concentration of PGE₂ that was added to the culture medium. Pretreatment with PGE₂ at 5 × 10⁻¹²M did not enhance PG synthesis whereas pretreatment with PGE₂ at 5 × 10⁻⁶M induced a maximal effect. Production of PGs was increased following 1 day of pretreatment with PGE₂ and was increased further following 3 days of pretreatment. The PGE₂ treated cells showed only a slight increase in the bradykinin-induced release of radioactivity from cells prelabeled with [³H]arachidonic acid but showed a dramatic increase in the bradykinin-induced synthesis of radio-labeled PGs. The conversion of free arachidonate to PGs in both intact cells and in a cell-free preparation was increased by PGE₂ pretreatment. The presence of cyclohexamide during the pretreatment did not inhibit the PGE₂-induced activation of PG synthesis. Taken together, the results indicate that pretreatment of cells with PGE₂ increased PG synthesis by augmenting the conversion of arachidonate to PGs.     

The influence of gamma radiation on arachidonic acid release and prostacyclin synthesis

Cultured pulmonary artery endothelial cells produce PGI₂ as their primary prostaglandin. Conditions which inhibit cell division have been shown to accelerate the synthesis of this compound. Exposure of endothelial cells to γ raidation results in an irreversible cessation of growth and enhanced production of PGI₂. The level of PGI₂ measured after radiation exposure exceeds that observed in cultures rendered quiescent by serum reduction. This indicates a role for γ radiation in the elevation of PGI₂ levels which is distinct from its effect on cell division. Result presented indicate that exposure to γ radiation does not, in and of itself, elevate PG levels but capacitates cells for enhanced production when presented with appropriate stimuli. Increased PGI₂ synthesis appears to be a result of an observed increase in arachidonic acid release and an activation of cyclooxygenase.     

Characterization of a variant of carcinoembryonic antigen (CEA) using monoclonal and polyclonal anti-CEA antibodies

A variant of the carcinoembryonic antigen (CEA) with lower molecular weight than a CEA reference preparation has been separated from CEA. Using a polyclonal, spleen absorbed anti-CEA antiserum, the variant crossreacts with reference CEA in immunodiffusion. The CEA-activity of the variant has been demonstrated using an enzyme-immunoassay with monoclonal CEA specific antibodies. There is sufficient immunological evidence that this variant is a distinct antigen different from the crossreactive antigens described so far. The reactivity of the polyclonal anti-CEA antiserum with the CEA variant was abolished by absorption against the immobilized variant.     

Analysing the yeast complexome—the Complex Portal rising to the challenge

The EMBL-EBI Complex Portal is a knowledgebase of macromolecular complexes providing persistent stable identifiers. Entries are linked to literature evidence and provide details of complex membership, function, structure and complex-specific Gene Ontology annotations. Data are freely available and downloadable in HUPO-PSI community standards and missing entries can be requested for curation. In collaboration with Saccharomyces Genome Database and UniProt, the yeast complexome, a compendium of all known heteromeric assemblies from the model organism Saccharomyces cerevisiae, was curated. This expansion of knowledge and scope has led to a 50% increase in curated complexes compared to the previously published dataset, CYC2008. The yeast complexome is used as a reference resource for the analysis of complexes from large-scale experiments. Our analysis showed that genes coding for proteins in complexes tend to have more genetic interactions, are co-expressed with more genes, are more multifunctional, localize more often in the nucleus, and are more often involved in nucleic acid-related metabolic processes and processes where large machineries are the predominant functional drivers. A comparison to genetic interactions showed that about 40% of expanded co-complex pairs also have genetic interactions, suggesting strong functional links between complex members.