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Results for “Pathway analysis”

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PubMed1979

Effect of sulphated glycopeptides on kinetics of 3-O-methyl glucose and 2-deoxyglucose transport by epithelial cells isolated from rabbit small intestine.

Suspensions of cells isolated from rabbit small intestine were prepared which showed a considerable rate of respiration after 4 h of their isolation. Rabbit intestinal cells accumulated 3-O-methyl-glucose by an active transport mechanism which was Na+-dependent whereas 2-deoxyglucose was transported by a saturable pathway. Analysis of the initial transport data indicated that the uptake of both sugars by cells followed Michaelis-Menten-type kinetics. These cells also showed a considerable degree of 2-deoxyglucose phosphorylation and the transport of the sugar into the cells appeared to be a rate-limiting factor for its phosphorylation. Sulphated glycopeptide had no effect on the respiration rate of these cells but it increased the transport of 3-O-methyl-glucose and 2-deoxyglucose into cells by many fold compared with untreated cells. Kinetic analysis of the data indicated that the increase in the transport was due to increased V while the Km remained unchanged. Presence of sulphated glycopeptide had no effect on the kinetics of phosphorylation of 2-deoxyglucose. These effects of sulphated glycopeptide on the transport activity of the cells have been discussed as a result of an increase in the number of sugar sites in the cell membrane due to unmasking or activation of preexisting sites owing to the interaction between the cell membrane components and highly polyanionic sulphated glycopeptides.

Animals↗

PubMed1991

A multivariate study of simultaneous escape from suppression by dexamethasone of urinary free cortisol, plasma cortisol, adrenocorticotropic hormone and beta-endorphin in melancholic patients.

To investigate the relationships between pre- and postdexamethasone hypothalamic-pituitary-adrenal (HPA) axis functioning in depression, we measured the levels of baseline and postdexamethasone urinary free cortisol (UFC), plasma cortisol, adrenocorticotropic hormone (ACTH) and beta-endorphin. We found that dexamethasone significantly suppressed all hormone levels. All 4 postdexamethasone hormones--but not their baseline levels--were significantly higher in melancholic subjects than in minor and simple major depressives. We have accumulated evidence that the melancholic and minor depression groups form discrete classes in postdexamethasone HPA axis hormone levels; this supports the biological heterogeneity hypothesis of melancholia. We found that a combination of the postdexamethasone UFC and beta-endorphin values yielded the most significant diagnostic tool for melancholia. Our results suggest that the measurements of both hormones may constitute the most accurate index reflecting the HPA axis escape from suppression by dexamethasone in melancholia. By means of pathway analysis, we determined the causal relationships between age, dexamethasone circulating levels, diagnostic depression classification and the various baseline and postdexamethasone hormone values.

Adrenocorticotropic Hormone↗

PubMed1978

Factors influencing nickel dermatitis. II.

The nickel concentrations in urine and other data of a very hypersensitive female patient have been followed during two periods exceeding 30 days each. Only a limited degree of correlation was found between the course of the nickel concentration in urine and the clinical activity of the dermatitis. In order to better evaluate the measure of the correlation and the influence of some other factors upon the activity of the dermatitis, a pathway analysis scheme has been constructed. Consideration of this scheme reveals the need for more extensive data regarding the nickel ion climate in the body.

Dermatitis, Contact↗

PubMed2026

Natural leaf shape variation reveals diverse transcriptional targets of GmJAG1 during soybean leaf development.

The JAGGED transcription factor family regulates lateral organ development across angiosperms. In soybean (Glycine max Merr.), a D9H mutation in the EAR repression motif of GmJAG1 causes a narrow-leaflet phenotype and explains over 70% of phenotypic variance in leaf shape. Because this mutation does not affect the zinc finger DNA-binding domain, both alleles bind identical targets but differ in repressor recruitment. Previous studies mapped GmJAG1 binding sites, but the functional targets controlling leaf morphology are uncharacterized. Here, we used comparative transcriptomics across four soybean genotypes with contrasting leaf shape, spanning a developmental time series from shoot apex to mature leaf, and identified 1567 putative candidate target genes. GmJAG1 expression was confined to the shoot apex, yet 99.1% of candidate targets maintained differential expression throughout development. We found that neither Kip-Related Protein (KRP) cell cycle inhibitors nor Cyclin-Dependent Kinases (CDKs) showed differential expression despite binding evidence in Arabidopsis. However, D-type cyclins were upregulated in narrow-leaf genotypes suggesting that soybean GmJAG1 acts through cyclin-mediated rather than KRP-mediated cell cycle regulation described in Arabidopsis- a divergence in regulatory logic between the two species. Pathway analysis revealed enrichment of auxin (1.8-fold, P = 0.02) and salicylic acid (fourfold, P = 0.016) genes among JAG1D9H targets. Filtering by differential expression, binding data, phenotype correlation, and co-expression network membership identified 79 high-confidence targets, including orthologs of NPH3 (phototropin-mediated leaf flattening), MIK2 (cell wall integrity sensing), RD22 (ABA-responsive stress signaling), and SCL23 (GRAS transcription factor in bundle sheath development). These candidates provide targets for functional validation and breeding in legumes.

Glycine max↗

PubMed1995

Electron tunneling in proteins: coupling through a beta strand.

Electron coupling through a beta strand has been investigated by measurement of the intramolecular electron-transfer (ET) rates in ruthenium-modified derivatives of the beta barrel blue copper protein Pseudomonas aeruginosa azurin. Surface histidines, introduced on the methionine-121 beta strand by mutagenesis, were modified with a Ru(2,2'-bipyridine)2(imidazole)2+ complex. The Cu+ to Ru3+ rate constants yielded a distance-decay constant of 1.1 per angstrom, a value close to the distance-decay constant of 1.0 per angstrom predicted for electron tunneling through an idealized beta strand. Activationless ET rate constants in combination with a tunneling-pathway analysis of the structures of azurin and cytochrome c confirm that there is a generally efficient network for coupling the internal (native) redox center to the surface of both proteins.

Azurin↗

PubMed1996

Demethylation of dimethylsulfoniopropionate to 3-S-methylmercaptopropionate by marine sulfate-reducing bacteria.

The initial step in the anaerobic degradation of the algal osmolyte dimethylsulfoniopropionate (DMSP) in anoxic marine sediments involves either a cleavage to dimethylsulfide and acrylate or a demethylation to 3-S-methylmercaptopropionate. Thus far, only one anaerobic bacterial strain has been shown to carry out the demethylation, namely, Desulfobacterium sp. strain PM4. The aims of the present work were to study how common this property is among certain groups of anaerobic bacteria and to obtain information on the affinities for DMSP of DMSP-demethylating strains. Screening of several pure cultures of sulfate-reducing and acetogenic bacteria showed that Desulfobacterium vacuolatum DSM 3385 and Desulfobacterium niacini DSM 2059 are also able to demethylate DMSP; a very slow demethylation of DMSP was observed with a salt-tolerant strain of Eubacterium limosum. From a 10(5) dilution of intertidal sediment a new marine DMSP-demethylating sulfate-reducing bacterium (strain WN) was isolated. Strain WN was a short, gram-negative, nonmotile rod that grew on betaine, sarcosine, palmitate, H2 plus CO2, and several alcohols, organic acids, and amino acids. Extracts of betaine-grown cells had hydrogenase, formate dehydrogenase, and CO dehydrogenase activities but no alpha-ketoglutarate oxidoreductase activity, indicating the presence of the acetyl coenzyme A-CO dehydrogenase pathway. Analysis of the 16S rRNA gene sequence of strain WN revealed a close relationship with Desulfobacter hydrogenophilus, Desulfobacter latus, and Desulfobacula toluolica. Strain PM4 was shown to group with Desulfobacterium niacini. The K(m) of strain WN for DMSP, as derived from substrate progress curves in cell suspensions, was approximately 10 microM. A similar value was found for D. niacini PM4.

Base Sequence↗

PubMed1986

Isolation of a mutation resulting in constitutive synthesis of L-fucose catabolic enzymes.

A ribitol-positive transductant of Escherichia coli K-12, JM2112, was used to facilitate the isolation and identification of mutations affecting the L-fucose catabolic pathway. Analysis of L-fucose-negative mutants of JM2112 enabled us to confirm that L-fucose-1-phosphate is the apparent inducer of the fucose catabolic enzymes. Plating of an L-fuculokinase-negative mutant of JM2112 on D-arabinose yielded an isolate containing a second fucose mutation which resulted in the constitutive synthesis of L-fucose permease, isomerase, and kinase. This constitutive mutation differs from the constitutive mutation described by Chen et al. (J. Bacteriol. 159:725-729, 1984) in that it is tightly linked to the fucose genes and appears to be located in the gene believed to code for the positive activator of the L-fucose genes.

Aldose-Ketose Isomerases↗

PubMed1990

Complementation of Escherichia coli sigma 54 (NtrA)-dependent formate hydrogenlyase activity by a cloned Thiobacillus ferrooxidans ntrA gene.

The ntrA gene of Thiobacillus ferrooxidans was cloned by complementation of an Escherichia coli ntrA mutant that was unable to produce gas via the sigma 54 (NtrA)-dependent formate hydrogenlyase pathway. Analysis of the DNA sequence showed that the T. ferrooxidans ntrA gene coded for a protein of 475 amino acids (calculated Mr, 52,972). The T. ferrooxidans NtrA protein had 49, 44, 33, and 18% amino acid similarity with the NtrA proteins of Klebsiella pneumoniae, Azotobacter vinelandii, Rhizobium meliloti, and Rhodobacter capsulatus, respectively. The ability of the T. ferrooxidans NtrA protein to direct transcription from sigma 54-dependent promoters was demonstrated in E. coli by using fdhF-lacZ and nifH-lacZ fusions. An open reading frame coding for a protein of 241 amino acids (calculated Mr, 27,023) was situated 12 base pairs upstream of the T. ferrooxidans ntrA gene. Comparison of this protein with the product of the open reading frame ORF1, located upstream of the R. meliloti ntrA gene, showed that the two proteins had 55% amino acid similarity. The cloned T. ferrooxidans ntrA gene was expressed in E. coli from a promoter located within the ORF1 coding region.

Acidithiobacillus thiooxidans↗

PubMed1992

MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins.

Shigella flexneri causes bacillary dysentery by invading epithelial cells of the colonic mucosa. The invasion process requires the synthesis and secretion of the virulence plasmid-encoded Ipa proteins. Using TnphoA mutagenesis, we have identified two virulence plasmid genes, mxiJ and mxiM, that encode proteins exported by the general export pathway. Analysis of the MxiJ and MxiM deduced amino acid sequences suggested that mxiJ and mxiM might encode lipoproteins, which was confirmed by [3H]palmitate labeling of MxiJ:PhoA and MxiM:PhoA fusion proteins. A mxiJ mutant was unable to invade HeLa cells, to induce the formation of plaques on confluent monolayers of HeLa cells, and to provoke keratoconjunctivitis in guinea pigs. In addition, secretion of seven polypeptides, including IpaA, IpaB, and IpaC, was abolished in the mxiJ mutant. Sequence comparisons indicated that MxiJ and MxiH, which is encoded by a gene located upstream from mxiJ, are homologous to the Yersinia enterocolitica YscJ and YscF proteins, respectively.

Adhesins, Bacterial↗

PubMed2000

Characterization of tpp1(+) as encoding a main trehalose-6P phosphatase in the fission yeast Schizosaccharomyces pombe.

We have characterized an open reading frame of 2,454 bp on chromosome I of Schizosaccharomyces pombe as the gene encoding trehalose-6P phosphatase (tpp1(+)). Disruption of tpp1(+) caused in vivo accumulation of trehalose-6P upon heat shock and prevented cell growth at 37 to 40 degrees C. Accumulation of trehalose-6P in cells bearing a chromosomal disruption of the tpp1(+) gene and containing a plasmid with tpp1(+) under the control of the thiamine-repressible promotor correlated with tpp1(+) repression. The level of tpp1(+) mRNA rose upon heat shock, osmostress, or oxidative stress and was negatively controlled by cyclic AMP-dependent protein kinase activity. Expression of tpp1(+) during oxidative or osmotic stress, but not during heat shock, was under positive control by the wis1-sty1 (equivalent to phh1 and spc1) mitogen-activated protein kinase pathway. Analysis of Tpp1 protein levels suggests that the synthesis of trehalose-6P phosphatase may also be subjected to translational or posttranslational control.

Chromosomes, Fungal↗

PubMed2002

Genome-scale metabolic model of Helicobacter pylori 26695.

A genome-scale metabolic model of Helicobacter pylori 26695 was constructed from genome sequence annotation, biochemical, and physiological data. This represents an in silico model largely derived from genomic information for an organism for which there is substantially less biochemical information available relative to previously modeled organisms such as Escherichia coli. The reconstructed metabolic network contains 388 enzymatic and transport reactions and accounts for 291 open reading frames. Within the paradigm of constraint-based modeling, extreme-pathway analysis and flux balance analysis were used to explore the metabolic capabilities of the in silico model. General network properties were analyzed and compared to similar results previously generated for Haemophilus influenzae. A minimal medium required by the model to generate required biomass constituents was calculated, indicating the requirement of eight amino acids, six of which correspond to essential human amino acids. In addition a list of potential substrates capable of fulfilling the bulk carbon requirements of H. pylori were identified. A deletion study was performed wherein reactions and associated genes in central metabolism were deleted and their effects were simulated under a variety of substrate availability conditions, yielding a number of reactions that are deemed essential. Deletion results were compared to recently published in vitro essentiality determinations for 17 genes. The in silico model accurately predicted 10 of 17 deletion cases, with partial support for additional cases. Collectively, the results presented herein suggest an effective strategy of combining in silico modeling with experimental technologies to enhance biological discovery for less characterized organisms and their genomes.

Amino Acids↗

PubMed2005

Novel roles of ohrR-ohr in Xanthomonas sensing, metabolism, and physiological adaptive response to lipid hydroperoxide.

Lipid hydroperoxides are highly toxic to biological systems. Here, the Xanthomonas campestris pv. phaseoli sensing and protective systems against linoleic hydroperoxide (LOOH) were investigated by examining the phenotypes, biochemical and regulatory characteristics of various Xanthomonas mutants in known peroxide resistance pathways. Analysis of LOOH resistance levels indicates that both alkyl hydroperoxide reductase (AhpC) and organic hydroperoxide resistance enzyme (Ohr) have important and nonredundant roles in the process. Nonetheless, inactivation of ohr leads to a marked reduction in LOOH resistance levels. The regulatory characteristics of an ohr mutant add further support to its primary role in LOOH protection. Northern analysis shows that LOOH had differential effects on induction of ahpC and ohr expression with the latter being more sensitive to the inducer. Analysis of the ahpC and ohr promoters confirmed that the LOOH-dependent induction of these promoters is mediated by the transcription regulators OxyR and OhrR, respectively. Using the in vivo promoter assays and the in vitro gel mobility shift assay, we show that LOOH directly oxidized OhrR at the sensing residue Cys-22 leading to its inactivation. In addition, physiological analysis shows that pretreatment of X. campestris pv. phaseoli with a sublethal dose of LOOH induced high levels of resistance to subsequent exposure to lethal concentrations of LOOH. This novel LOOH-induced adaptive response requires a functional ohrR-ohr operon. These data illustrate an important novel physiological role for the ohrR-ohr system in sensing and inactivating lipid hydroperoxides.

Adaptation, Physiological↗

PubMed2006

Differential signaling networks induced by mild and lethal hemorrhagic fever virus infections.

The family Arenaviridae includes several National Institutes of Allergy and Infections Diseases category A select agents which cause hemorrhagic fever. There are few vaccines available, and treatment is limited to ribavirin, which varies in efficacy. Development of new antiviral compounds has been hindered by a lack of understanding of the molecular basis of pathogenesis. We used two variants of Pichinde virus, one attenuated and one virulent in the guinea pig model, to delineate the host determinants which lead to either viral clearance or lethal disease. By analyzing protein level changes using pathway analysis, we have identified key intermediates which may be targets for therapeutic intervention.

Animals↗

PubMed2003

Tomato mosaic virus replication protein suppresses virus-targeted posttranscriptional gene silencing.

Posttranscriptional gene silencing (PTGS), a homology-dependent RNA degradation system, has a role in defending against virus infection in plants, but plant viruses encode a suppressor to combat PTGS. Using transgenic tobacco in which the expression of green fluorescent protein (GFP) is posttranscriptionally silenced, we investigated a tomato mosaic virus (ToMV)-encoded PTGS suppressor. Infection with wild-type ToMV (L strain) interrupted GFP silencing in tobacco, coincident with visible symptoms, whereas some attenuated strains of ToMV (L(11) and L(11)A strains) failed to suppress GFP silencing. Analyses of recombinant viruses containing the L and L(11)A strains revealed that a single base change in the replicase gene, which causes an amino acid substitution, is responsible for the symptomless and suppressor-defective phenotypes of the attenuated strains. An agroinfiltration assay indicated that the 130K replication protein acts as a PTGS suppressor. Small interfering RNAs (siRNAs) of 21 to 25 nucleotides accumulated during ToMV infection, suggesting that the major target of the ToMV-encoded suppressor is downstream from the production of siRNAs in the PTGS pathway. Analysis with GFP-tagged recombinant viruses revealed that the suppressor inhibits the establishment of the ToMV-targeted PTGS system in the inoculated leaves but does not detectably suppress the activity of the preexisting, sequence-specific PTGS machinery there. Taken together, these results indicate that it is likely that the ToMV-encoded suppressor, the 130K replication protein, blocks the utilization of silencing-associated small RNAs, so that a homology-dependent RNA degradation machinery is not newly formed.

Green Fluorescent Proteins↗

PubMed2000

Extracellular signal-regulated kinase binds to TFII-I and regulates its activation of the c-fos promoter.

We have previously shown that TFII-I enhances transcriptional activation of the c-fos promoter through interactions with upstream elements in a signal-dependent manner. Here we demonstrate that activated Ras and RhoA synergize with TFII-I for c-fos promoter activation, whereas dominant-negative Ras and RhoA inhibit these effects of TFII-I. The Mek1 inhibitor, PD98059 abrogates the enhancement of the c-fos promoter by TFII-I, indicating that TFII-I function is dependent on an active mitogen-activated protein (MAP) kinase pathway. Analysis of the TFII-I protein sequence revealed that TFII-I contains a consensus MAP kinase interaction domain (D box). Consistent with this, we have found that TFII-I forms an in vivo complex with extracellular signal-related kinase (ERK). Point mutations within the consensus MAP kinase binding motif of TFII-I inhibit its ability to bind ERK and its ability to enhance the c-fos promoter. Therefore, the D box of TFII-I is required for its activity on the c-fos promoter. Moreover, the interaction between TFII-I and ERK can be regulated. Serum stimulation enhances complex formation between TFII-I and ERK, and dominant-negative Ras abrogates this interaction. In addition, TFII-I can be phosphorylated in vitro by ERK and mutation of consensus MAP kinase substrate sites at serines 627 and 633 impairs the phosphorylation of TFII-I by ERK and its activity on the c-fos promoter. These results suggest that ERK regulates the activity of TFII-I by direct phosphorylation.

3T3 Cells↗

PubMed2004

TRAF family proteins link PKR with NF-kappa B activation.

The double-stranded RNA (dsRNA)-dependent protein kinase PKR activates NF-kappa B via the I kappa B kinase (IKK) complex, but little is known about additional molecules that may be involved in this pathway. Analysis of the PKR sequence enabled us to identify two putative TRAF-interacting motifs. The viability of such an interaction was further suggested by computer modeling. Here, we present evidence of the colocalization and physical interaction between PKR and TRAF family proteins in vivo, as shown by immunoprecipitation and confocal microscopy experiments. This interaction is induced upon PKR dimerization. Most importantly, we show that the binding between PKR and TRAFs is functionally relevant, as observed by the absence of NF-kappa B activity upon PKR expression in cells genetically deficient in TRAF2 and TRAF5 or after expression of TRAF dominant negative molecules. On the basis of sequence information and mutational and computer docking analyses, we favored a TRAF-PKR interaction model in which the C-terminal domain of TRAF binds to a predicted TRAF interaction motif present in the PKR kinase domain. Altogether, our data suggest that TRAF family proteins are key components located downstream of PKR that have an important role in mediating activation of NF-kappa B by the dsRNA-dependent protein kinase.

3T3 Cells↗

PubMed2005

c-Jun N-terminal kinase contributes to aberrant retinoid signaling in lung cancer cells by phosphorylating and inducing proteasomal degradation of retinoic acid receptor alpha.

Retinoic acid (RA) is the ligand for nuclear RA receptors (RARs and RXRs) and is crucial for normal epithelial cell growth and differentiation. During malignant transformation, human bronchial epithelial cells acquire a block in retinoid signaling caused in part by a transcriptional defect in RARs. Here, we show that activation of c-Jun N-terminal kinase (JNK) contributes to RAR dysfunction by phosphorylating RARalpha and inducing degradation through the ubiquitin-proteasomal pathway. Analysis of RARalpha mutants and phosphopeptide mapping revealed that RARalpha residues Thr181, Ser445, and Ser461 are phosphorylated by JNK. Mutation of these residues to alanines prevented efficient ubiquitination of RARalpha and increased the stability of the protein. We investigated the importance of RARalpha phosphorylation by JNK as a mediator of retinoid resistance in lung cancer. Mice that develop lung cancer from activation of a latent K-ras oncogene had high intratumoral JNK activity and low RARalpha levels and were resistant to treatment with an RAR ligand. JNK inhibition in a human lung cancer cell line enhanced RARalpha levels, ligand-induced activity of RXR-RAR dimers, and growth inhibition by RA. These findings point to JNK as a key mediator of aberrant retinoid signaling in lung cancer cells.

3T3 Cells↗

PubMed1991

Inheritance of phenotype in mammalian cells: genetic vs. epigenetic mechanisms.

Inherited phenotypic changes in cultured cells, as observed during differentiation and transformation, reflect alterations in gene expression and have both a genetic and epigenetic basis. The causes of specific changes are often difficult to define especially when observing phenomenological end points. Although such observations are an important step in defining the phenotypic changes that endure for multiple generations, it is necessary to analyze cells at the molecular level to characterize the pathways leading to changes in phenotype. Gene expression can be regulated at multiple levels, i.e., DNA structure, gene transcription, and/or posttranscriptional modifications. Four genetic mechanisms (DNA point mutations, deletion, rearrangement, and amplification) and two epigenetic mechanisms (DNA methylation and the preservation of DNA-protein complexes) can account for the majority of enduring changes observed in cultured cells. Genetic alterations in DNA sequence appear to be largely responsible for altered growth regulation associated with transformation, but there is also evidence to suggest that epigenetic mechanisms play a role in transformation. Differentiation of cultured cells is often associated with lack of growth, and has been ascribed in part to epigenetic mechanisms. However, differentiation and transformation are not mutually exclusive but may be regulated by parallel multistep pathways. Analysis of the causes underlying transformation has been complicated by the use of aneuploid cells, but it is clear that the tools for overcoming the ambiguities associated with phenomenological analysis are available.

Animals↗