Identification of protein X of Escherichia coli as the recA+/tif+ gene product.

Summary Protein X, molecular weight 40,000, has been separated from the other proteins of E. coliby a two-dimensional gel electrophoretic technique which separates proteins according to isoelectric point (pI) in the firstdimension and according to molecular weight in the second. When protein X is induced in wild-type cells by mitomycin C treatmentit has a pI6.0. However, when protein X is induced in a tif-1 mutant, either by temperatureshift-up to 42° or by mitomycin C treatment at 30°, it has a pI6.2. The low level of protein X which is present inuninduced tif mutants at 30° also has a pI6.2. These results suggest thattif-1 is a mis-sense mutation in the gene coding for protein X. Since transduction andcomplementation studies indicate that tif-1 is a mutation of therecA ⁺ gene (Castellazzi, Morand, George and Buttin, 1977) it follows that protein X is the recA ⁺ gene product.A model has been formulated to account for the relationship between protein X synthesis and the recA ⁺ and lexA ⁺ genes. In this model, a repressor coded by lexA ⁺ binds to the operator of the recA ⁺ gene from whence it can normally only be removed by the combined action of an inducer and protein X, the recA ⁺ product. Thus, protein X controls its own synthesis. The tif-1 mutation leads to a temperature sensitive form of protein X which, at 42°, can spontaneously remove the repressor without the intervention of the inducer.     

Carbon and nitrogen metabolism in legume root nodules

The literature concerning the metabolism of carbon compounds during the reduction, assimilation and translocation of nitrogen in root nodules of leguminous plants is reviewed. The reduction of dinitrogen requires an energy source (ATP) and a reluctant which are both supplied by respiratory catabolism of carbohydrates produced by the host plant. Photosynthates are also required to generate the carbon skeletons for amino acid or urcide synthesis during the assimilation of ammonia produced by the bacteria within the nodule tissue. Competition for photosynthates occurs between the bacteroids, nodule tissue and the various vegetative and reproductive sinks in the host plant. The nature of carbon compounds involved in these processes, their routes of metabolism, the mechanisms of control and the partitioning of metabolises between the various sites of utilization are only poorly understood. It is apparent that dinitrogen is reduced to ammonia in the bacteroids. Both fast- and slow-growing strains of Rhizobium possess the Entner-Doudoroff pathway of glucose catabolism, and some, if not all, enzymes of the Emden-Meyerhof pathway. Some bacterial cultures also metabolize carbon through the ketogluconate pathway but only the fast-growing strains of cultured rhizobia possess the key enzyme of the pentose phosphate pathway (6-phosphogluconate dehydrogenase). The host cells are thought to contain the complete Emden-Meyerhof pathway and tricarboxylic acid cycle, which provides the carbon skeletons for assimilation of the ammonia, formed by the bacteroids, into α-amino acids. A pathway of anapleurotic carbon conservation, operative in the host cells, synthesizes oxaloacetic acid through β-carboxylation of phosphoenol pyruvate. This process could be important in the recapture and assimilation of respired CO₂ in the rhizosphere. The main route of assimilation of ammonia produced by the bacteroids would appear to be via the glutamine synthetase-glutamate synthase pathway in the host cells. However, glutamate dehydrogenase may also be involved in ammonia assimilation. These enzymes also occur in in vitro cultures of Rhizobium and in bacteroids where they presumably participate in the synthesis of amino acids for growth of the bacteria or bacteroids. Nitrogen assimilated into glutamine or glutamate is exported from the nodules in a variety of forms, which include asparagine, glutamine, aspartate, homoserine and allantoates, in proportions which depend on the legume species. Studies on regulation of the overall process have focussed on expression of bacteroid genes and on the control of enzyme activity, at the level of nitrogenase and enzymes of nitrogen assimilation in particular. However, due to the wide range of experimental techniques, environmental conditions and plant species which have been used, no clear conclusions can yet be drawn. The pathways of carbon flow in nitrogen metabolism, particularly in relation to the synthesis of ureides and the regulation of carbon metabolism, remain key areas for future research in symbiotic nitrogen fixation.     

Scaling analysis of coral reef systems: an approach to problems of scale

Dimensional analysis and scaling are related, semi-formal procedures for capturing the essential process(es) controlling the behaviour of a complex system, and for describing the functional relationships between them. The techniques involve the parameterization of natural processes, the identification of the temporal and spatial scales of variation of processes, and the evaluation of potential interactions between processes referenced to those scales using non-dimensional (scaled) parameters. Scaling approaches are increasingly being applied to a broad range of marine ecological problems, with the aims of assessing the relative importance of physical and biological parameters in controlling variation in process rates, and placing limits on the ability of one process to affect another. The value of the approach to coral reef research lies in the conceptualization of relationships between discipline-specific processes, and the evaluation of scale-dependent processes across the large range of spatial and temporal scales which pertain to coral reefs. Characteristic scales of physical, geological and biological processes exhibit different patterns of distribution along the temporal dimension. Scaling arguments based on examples from reef systems indicate that a large group of biological and biogeochemical processes are strongly influenced by hydrodynamic processe occuring at similar time scales within the range from about on hour to one year. We argue that scaling approaches to process-related problems are pre-requisite to interdisciplinary research on coral reefs.     

The Chediak-Higashi protein interacts with SNARE complex and signal transduction proteins

BACKGROUND:Chediak-Higashi syndrome (CHS) is an inherited immunodeficiency disease characterized by giant lysosomes and impaired leukocyte degranulation. CHS results from mutations in the lysosomal trafficking regulator (LYST) gene, which encodes a 425-kD cytoplasmic protein of unknown function. The goal of this study was to identify proteins that interact with LYST as a first step in understanding how LYST modulates lysosomal exocytosis. MATERIALS AND METHODS: Fourteen cDNA fragments, covering the entire coding domain of LYST, were used as baits to screen five human cDNA libraries by a yeast two-hybrid method, modified to allow screening in the activation and the binding domain, three selectable markers, and more stringent confirmation procedures. Five of the interactions were confirmed by an in vitro binding assay. RESULTS: Twenty-one proteins that interact with LYST were identified in yeast two-hybrid screens. Four interactions, confirmed directly, were with proteins important in vesicular transport and signal transduction (the SNARE-complex protein HRS, 14-3-3, and casein kinase II). CONCLUSIONS:On the basis of protein interactions, LYST appears to function as an adapter protein that may juxtapose proteins that mediate intracellular membrane fusion reactions. The pathologic manifestations observed in CHS patients and in mice with the homologous mutation beige suggest that understanding the role of LYST may be relevant to the treatment of not only CHS but also of diseases such as asthma, urticaria, and lupus, as well as to the molecular dissection of the CHS-associated cancer predisposition.     

The inhibitory activity of natural products on plant p-hydroxyphenylpyruvate dioxygenase

The inhibitory activity of 34 natural products of various structural classes on hydroxyphenylpyruvate dioxygenase (HPPD), the target site for triketone herbicides, and the mode of interaction of selected natural products were investigated. Recombinant HPPD from arabidopsis is sensitive to several classes of natural compounds including, in decreasing order of sensitivity, triketones, benzoquinones, naphthoquinones and anthraquinones. The triketone natural products acted as competitive tight-binding inhibitors, whereas the benzoquinones and naphthoquinones did not appear to bind tightly to HPPD. While these natural products may not have optimal structural features required for in vivo herbicidal activity, the differences in their kinetic behavior suggest that novel classes of HPPD inhibitors may be developed based on their structural backbones.     

Vesicle formation and trafficking in endothelial cells and regulation of endothelial barrier function

Endothelial barrier function is regulated in part by the transcellular transport of albumin and other macromolecules via endothelial caveolae (i.e., this process is defined as transcytosis). Using pulmonary microvascular endothelial cells, we have identified the specific interactions between a cell surface albumin-docking protein gp60 and caveolin-1 as well as components of the signaling machinery, heterotrimeric G protein (G(i))- and Src-family tyrosine kinase. Ligation of gp60 on the apical membrane induces the release of caveolae from the apical membrane and activation of endocytosis. The formed vesicles contain the gp60-bound albumin and also albumin and other solutes present in the fluid phase. Vesicles are transported in a polarized manner to the basolateral membrane, releasing their contents by exocytosis into the subendothelial space. The signaling functions of G(i) and Src are important in the release of caveolae from the plasma membrane. The Src-induced phosphorylation of caveolin-1 is crucial in regulating interactions of caveolin-1 with other components of the signaling machinery such as G(i), and key signaling entry of caveolae into the cytoplasm and endocytosis of albumin and other solutes. This review addresses the basis of transcytosis in endothelial cells, its central role as a determinant of endothelial barrier function, and signaling mechanisms involved in regulating fission of caveolae and trafficking of the formed vesicles from the luminal to abluminal side of the endothelial barrier.     

New 2,N6-disubstituted adenosines: potent and selective A1 adenosine receptor agonists

A number of adenosine analogues substituted in the 2- and N6-positions were synthesized and evaluated for affinity, functional potency and intrinsic activity at the A1 and A2A adenosine receptors (AR). Three classes of N6-substituents were tested; norbornen-2-yl (series 1), norborn-2-yl (series 2) and 5,6-epoxynorborn-2-yl (series 3). The halogens; fluoro, bromo, and iodo were evaluated as C-2 substituents. All compounds showed relatively high affinity (nanomolar) for the A1AR and high potency for inhibiting (-)isoproterenol-stimulated cAMP accumulation in hamster smooth muscle DDT1 MF-2 cells with the 2-fluoro derivatives from each series having the highest affinity. All of the derivatives showed the same intrinsic activity as CPA. At the A2AAR, all of the derivatives showed relatively low affinity and potency (micromolar) for stimulating cAMP accumulation in rat pheochromocytoma PC-12 cells. The intrinsic activity of the derivatives compared to CGS 21680 was dependent upon the halogen substituent in the C-2 position with most showing partial agonist activity. Of particular interest is 2-iodo-N6-(2S-endo-norborn-2-yl)adenosine (5e), which is over 100-fold selective for the A1AR, is a full agonist at this receptor subtype and has no detectable agonist activity at the A2AAR.     

Inverse electrocardiographic transformations: dependence on the number of epicardial regions and body surface data points

The inverse problem of electrocardiography, the computation of epicardial potentials from body surface potentials, is influenced by the desired resolution on the epicardium, the number of recording points on the body surface, and the method of limiting the inversion process. To examine the role of these variables in the computation of the inverse transform, Tikhonov's zero-order regularization and singular value decomposition (SVD) have been used to invert the forward transfer matrix. The inverses have been compared in a data-independent manner using the resolution and the noise amplification as endpoints. Sets of 32, 50, 192, and 384 leads were chosen as sets of body surface data, and 26, 50, 74, and 98 regions were chosen to represent the epicardium.The resolution and noise were both improved by using a greater number of electrodes on the body surface. When 60% of the singular values are retained, the results show a trade-off between noise and resolution, with typical maximal epicardial noise levels of less than 0.5% of maximum epicardial potentials for 26 epicardial regions, 2.5% for 50 epicardial regions, 7.5% for 74 epicardial regions, and 50% for 98 epicardial regions. As the number of epicardial regions is increased, the regularization technique effectively fixes the noise amplification but markedly decreases the resolution, whereas SVD results in an increase in noise and a moderate decrease in resolution. Overall the regularization technique performs slightly better than SVD in the noise-resolution relationship.There is a region at the posterior of the heart that was poorly resolved regardless of the number of regions chosen. The variance of the resolution was such as to suggest the use of variable-size epicardial regions based on the resolution.     

Phylogenetic analysis of aerobic freshwater and marine enrichment cultures efficient in hydrocarbon degradation: effect of profiling method

Aerobically grown enrichment cultures derived from hydrocarbon-contaminated seawater and freshwater sediments were generated by growth on crude oil as sole carbon source. Both cultures displayed a high rate of degradation for a wide range of hydrocarbon compounds. The bacterial species composition of these cultures was investigated by PCR of the 16S rDNA gene using multiple primer combinations. Near full-length 16S rDNA clone libraries were generated and screened by restriction analysis prior to sequence analysis. Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) was carried out using two other PCR primer sets targeting either the V3 or V6-V8 regions, and sequences derived from prominent DGGE bands were compared to sequences obtained via cloning. All data sets suggested that the seawater culture was dominated by alpha-subgroup proteobacteria, whereas the freshwater culture was dominated by members of the beta- and gamma-proteobacteria. However, the V6-V8 primer pair was deficient in the recovery of Sphingomonas-like 16S rDNA due to a 3' terminal mismatch with the reverse primer. Most 16S rDNA sequences recovered from the marine enrichment were not closely related to genera containing known oil-degrading organisms, although some were detected. All methods suggested that the freshwater enrichment was dominated by genera containing known hydrocarbon-degrading species.     

Polymorphism in intron 1 of the interferon-gamma gene influences both serum immunoglobulin E levels and the risk for chronic hepatitis B virus infection in Polynesians

Intron 1 of the interferon-gamma (IFNG) gene contains two polymorphisms. The 12 CA-repeat allele of the +875 IFNGCA microsatellite and the T allele of the +A874T single nucleotide polymorphism (SNP) have been associated with increased in vitro IFNG production and a variety of clinical phenotypes. The purpose of this study was to determine whether these polymorphisms influence total serum IgE levels [tsIgE] and the outcome of a hepatitis B virus (HBV) infection. IFNGCA and +A874T were typed in 186 asthmatics of Niuean ancestry and in Polynesian women with a chronic HBV infection (n = 60) and with natural immunity to the HBV (n = 66). The IFNGCA genotype was associated with [tsIgE] in asthmatic children (n = 51, p = 0.004) but not adults (n = 135, p = 0.87). The data were consistent with a co-dominant influence of the 12 CA-repeat allele on high [tsIgE]. The IFNGCA genotype was also associated with the risk for chronic HBV infection (χ ² = 11.6, p = 0.003) because of a dominant effect of the 12 CA-repeat allele on developing natural immunity in homozygotes (OR = 5.8, p = 0.003) and heterozygotes (OR = 2.7, p = 0.01). Similar associations were found for the T allele of the +A874T SNP. The possibility that these associations were due to linked alleles in the adjacent 783 bp of the promoter and 3′-untranslated region of the IFNG gene was excluded by direct sequencing. In summary, high-IFNG-producing alleles in intron 1 of the IFNG locus are associated with high [tsIgE] in asthmatic children from Niue and with natural immunity to the HBV in Polynesian women. These findings are consistent with a previous report of an association between +875 IFNGCA and [tsIgE] and provide preliminary evidence of a new association with the outcome of an HBV infection.