scholarly journals Formation of Protoanemonin from 2-Chloro-cis,cis-Muconate by the Combined Action of Muconate Cycloisomerase and Muconolactone Isomerase

2002 ◽  
Vol 184 (19) ◽  
pp. 5402-5409 ◽  
Author(s):  
Anke Skiba ◽  
Volker Hecht ◽  
Dietmar Helmut Pieper
Keyword(s):  
Ralstonia Eutropha ◽  
Minor Importance ◽  
Enzyme Preparations ◽  
Cell Extracts ◽  
Combined Action ◽  
Rate Limiting ◽  
Muconate Cycloisomerase ◽  
Predominant Product ◽  
Chloromuconate Cycloisomerase

ABSTRACT Muconate cycloisomerases are known to catalyze the reversible conversion of 2-chloro-cis,cis-muconate by 1,4- and 3,6-cycloisomerization into (4S)-(+)-2-chloro- and (4R/5S)-(+)-5-chloromuconolactone. 2-Chloromuconolactone is transformed by muconolactone isomerase with concomitant dechlorination and decarboxylation into the antibiotic protoanemonin. The low k cat for this compound compared to that for 5-chloromuconolactone suggests that protoanemonin formation is of minor importance. However, since 2-chloromuconolactone is the initially predominant product of 2-chloromuconate cycloisomerization, significant amounts of protoanemonin were formed in reaction mixtures containing large amounts of muconolactone isomerase and small amounts of muconate cycloisomerase. Such enzyme ratios resemble those observed in cell extracts of benzoate-grown cells of Ralstonia eutropha JMP134. In contrast, cis-dienelactone was the predominant product formed by enzyme preparations, in which muconolactone isomerase was in vitro rate limiting. In reaction mixtures containing chloromuconate cycloisomerase and muconolactone isomerase, only minute amounts of protoanemonin were detected, indicating that only small amounts of 2-chloromuconolactone were formed by cycloisomerization and that chloromuconate cycloisomerase actually preferentially catalyzes a 3,6-cycloisomerization.

scholarly journals Role oftfdCIDIEIFIandtfdDIICIIEIIFIIGene Modules in Catabolism of 3-Chlorobenzoate by Ralstonia eutropha JMP134(pJP4)

2000 ◽  
Vol 66 (4) ◽  
pp. 1602-1608 ◽  
Author(s):  
D. Pérez-Pantoja ◽  
L. Guzmán ◽  
M. Manzano ◽  
D. H. Pieper ◽  
B. González
Keyword(s):  
Ralstonia Eutropha ◽  
Regulatory Gene ◽  
Plasmid Vector ◽  
Liquid Cultures ◽  
Cell Extracts ◽  
Dienelactone Hydrolase ◽  
Gene Modules ◽  
Maleylacetate Reductase ◽  
Chloromuconate Cycloisomerase

ABSTRACT The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4,tfdCIDIEIFI (module I) andtfdDIICIIEIIFII (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putidaKT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containingtfdR-tfdCIDIEIFI grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containingtfdR-tfdDIICIIEIIFII . In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.

A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III

1994 ◽  
Vol 14 (1) ◽  
pp. 822-830
Author(s):  
G Rameau ◽  
K Puglia ◽  
A Crowe ◽  
I Sethy ◽  
I Willis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Structural Motif ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Polymerase Iii ◽  
Rate Limiting

In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.

scholarly journals A mutation in the second largest subunit of TFIIIC increases a rate-limiting step in transcription by RNA polymerase III.

1994 ◽  
Vol 14 (1) ◽  
pp. 822-830 ◽  
Author(s):  
G Rameau ◽  
K Puglia ◽  
A Crowe ◽  
I Sethy ◽  
I Willis
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Structural Motif ◽  
Cell Extracts ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Polymerase Iii ◽  
Rate Limiting

In previous studies, we have shown that the PCF1-1 mutation of Saccharomyces cerevisiae suppresses the negative effect of a tRNA gene A block promoter mutation in vivo and increases the transcription of a variety of RNA polymerase III genes in vitro. Here, we report that PCF1 encodes the second largest subunit of transcription factor IIIC (TFIIIC) and that the PCF1-1 mutation causes an amino acid substitution in a novel protein structural motif, a tetratricopeptide repeat, in this subunit. In agreement with the nature of the mutation, in vitro transcription studies with crude extracts indicate that PCF1-1 facilitates the rate-limiting step in transcription, namely, the recruitment of TFIIIB to the template. Additionally, biochemical fractionation of wild-type and mutant cell extracts shows that PCF1-1 increases the amount of the 70-kDa TFIIIB subunit detectable by Western (immunoblot) analysis in purified TFIIIB fractions and the transcription activity of a TFIIIB" fraction containing the 90-kDa subunit of this factor. We suggest that the effect of PCF1-1 on TFIIIB activity in vitro is a consequence of its increased rate of recruitment in vivo.

scholarly journals Uridine kinase activities and pyrimidine nucleoside phosphorylation in fluoropyrimidine-sensitive and -resistant cell lines of the Novikoff hepatoma

1977 ◽  
Vol 164 (2) ◽  
pp. 379-387 ◽  
Author(s):  
N Greenberg ◽  
D E Schumm ◽  
T E Webb
Keyword(s):  
Intact Cell ◽  
Nucleoside Transport ◽  
Transport Systems ◽  
Human Carcinoma ◽  
Novikoff Hepatoma ◽  
Uridine Kinase ◽  
Cell Extracts ◽  
Rate Limiting

Uridine kinase, the rate-limiting enzyme in the activation (phosphorylation) of uridine and the corresponding chemotherapeutic analogues, is present as two isoenzymes localized exclusively in the cytosol of rapidly growing neoplasms, including the S-37 sarcoma, EL-4 leukaemia, HeLa cells (a human carcinoma) and the Novikoff hepatoma. The activities of the isolated isoenzymes are markedly decreased when the concentrations of ATP, phosphate or Mg2+ that are optimum in vitro are replaced by concentrations of ATP, phosphate or Mg2+ that are optimum in vitro are replaced by concentrations approximating to those found in vivo. Further, comparisons of the Km values of isolated uridine kinases with those for cellular uptake of pyrimidine nucleosides and their rate of intracellular phosphorylation suggest that nucleoside-transport systems play a rate-limiting role in nucleoside analogue activation and consequently that it is impossible to estimate the Km of uridine kinase in the intact cell. During the development of tumour-cell resistance to 5-fluorouracil or 5-fluorouridine in vivo there was an early differential increase in the activity of a low-affinity (high-Km) uridine kinase isoenzyme, as measured in cell extracts, and a 7-fold increase in the Km values for the uptake of both uridine and 5-fluorouridine into the intact resistant cells.

In-Vitro Effects of Ricinus Communis Seed Kernel Extract On Some Antioxidant And Hydrolytic Enzymes In Nymph And Adult Zonocerus Variegatus (Grasshopper)

2019 ◽  
Vol 3 (1) ◽  
pp. 129-137
Author(s):  
Gbadebo E . Adeleke ◽  
Olaniyi T. Adedosu ◽  
Rachael O. Adeyi ◽  
John O. Fatoki
Keyword(s):  
Ricinus Communis ◽  
Hydrolytic Enzymes ◽  
Crude Enzyme ◽  
Economic Losses ◽  
Seed Kernel ◽  
Zonocerus Variegatus ◽  
Sod Activity ◽  
Enzyme Preparations ◽  
In Vitro Effects

Background: Many plants have been identified for their insecticidal properties as alternatives to synthetic ones, which are toxic to untargeted organisms and environment. Ricinus communis (Castor) has been re-ported to exhibit insecticidal properties against insect pests. Zonocerus variegatus (Grasshopper) is a notable pest of several crops, and has been linked with great economic losses to farmers. The present study investigates the in-vitro toxicity of R. communis seed kernel extract (RCSKE) on the activities of selected antioxidant and hydrolytic enzymes in nymph and adult Zonocerus variegatus (Grasshopper), using cypermethrin (CYPER-M) and chlorpyrifos (CPF) as standard conventional pesticides. Methods: Seed kernel of Ricinus communis (Castor) was subjected to acidified aqueous extraction to obtain the extract (RCSKE). Crude enzyme preparations were obtained from nymph and adult Z. variegatus grass-hoppers. The in-vitro effects of different concentrations (15, 30, 45, 60, 75, 90 and 105μg/ml) each of RCSKE, CYPER-M and CPF on the activities of superoxide dismutase (SOD), catalase (CAT), acetylcholinesterase (AChE) and carboxylesterase (CES) in crude enzyme preparations were estimated spectrophotometrically. The level of statistical significance was 0.05. Results: The RCSKE significantly reduced the in-vitro SOD activity (p < 0.05) in nymph Z. variegatus at all the concentrations, whereas both CYPER-M and CPF significantly reduced the activity only at certain concentrations. The CAT activity in the nymph was significantly decreased by RCSKE and CPF at all the concentrations, but CYPER-M decreased it only at certain concentrations. In adult Z. variegatus, SOD activity was not significantly affected (p > 0.05), while CAT activity was significantly increased (p < 0.05) by the three agents at all the concentrations. The AChE and CES activities in the nymph were significantly reduced by RCSKE, CYPER-M and CPF at all the concentrations. The RCSKE and CPF significantly increased the CES activity, while CYPER-M caused a significant decrease in the activity in adult Z. variegatus. Conclusion: The seed kernel extract of Ricinus communis is an effective pesticidal agent and hence, it could be a source of biopesticide alternative with greater potential than cypermethrin and chlorpyrifos. In addition, the antioxidant, acetylcholinesterase and carboxylesterase enzymes in the nymphs of Z. variegatus grasshoppers are more susceptible to the effect of the extract than in the adult grasshoppers.

scholarly journals Assembly of the B subunit pentamer of Escherichia coli heat-labile enterotoxin. Kinetics and molecular basis of rate-limiting steps in vitro.

1996 ◽  
Vol 271 (43) ◽  
pp. 27188
Author(s):  
Lloyd W. Ruddock ◽  
Jeremy J.F. Coen ◽  
Caroline Cheesman ◽  
Robert B. Freedman ◽  
Timothy R. Hirst
Keyword(s):  
Escherichia Coli ◽  
Molecular Basis ◽  
B Subunit ◽  
Heat Labile ◽  
Rate Limiting

Regulation of proboscipedia in Drosophila by Homeotic Selector Genes

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 183-194
Author(s):  
Douglas B Rusch ◽  
Thomas C Kaufman
Keyword(s):  
Expression Pattern ◽  
Regulatory Element ◽  
Polycomb Group ◽  
Accumulation Pattern ◽  
Cis Acting ◽  
Polycomb Group Genes ◽  
Gap Genes ◽  
Combined Action ◽  
Selector Genes

Abstract The gene proboscipedia (pb) is a member of the Antennapedia complex in Drosophila and is required for the proper specification of the adult mouthparts. In the embryo, pb expression serves no known function despite having an accumulation pattern in the mouthpart anlagen that is conserved across several insect orders. We have identified several of the genes necessary to generate this embryonic pattern of expression. These genes can be roughly split into three categories based on their time of action during development. First, prior to the expression of pb, the gap genes are required to specify the domains where pb may be expressed. Second, the initial expression pattern of pb is controlled by the combined action of the genes Deformed (Dfd), Sex combs reduced (Scr), cap'n'collar (cnc), and teashirt (tsh). Lastly, maintenance of this expression pattern later in development is dependent on the action of a subset of the Polycomb group genes. These interactions are mediated in part through a 500-bp regulatory element in the second intron of pb. We further show that Dfd protein binds in vitro to sequences found in this fragment. This is the first clear demonstration of autonomous positive cross-regulation of one Hox gene by another in Drosophila melanogaster and the binding of Dfd to a cis-acting regulatory element indicates that this control might be direct.

scholarly journals Nerve Growth Factor (NGF) modulates in vitro induced myofibroblasts by highlighting a differential protein signature

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Graziana Esposito ◽  
Bijorn Omar Balzamino ◽  
Egidio Stigliano ◽  
Filippo Biamonte ◽  
Andrea Urbani ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Cell Signalling ◽  
Specific Protein ◽  
Conditioned Media ◽  
Cell Extracts ◽  
Dose Dependent Decrease ◽  
Significant Expression ◽  
Biomolecular Analysis ◽  
Protein Signature

AbstractWe previously described the profibrogenic effect of NGF on conjunctival Fibroblasts (FBs) and its ability to trigger apoptosis in TGFβ1-induced myofibroblasts (myoFBs). Herein, cell apoptosis/signalling, cytokines’ signature in conditioned media and inflammatory as well as angiogenic pathway were investigated. Experimental myoFBs were exposed to NGF (0.1–100 ng/mL), at defined time-point for confocal and biomolecular analysis. Cells were analysed for apoptotic and cell signalling activation in cell extracts and for some inflammatory and proinflammatory/angiogenic factors’ activations. NGF triggered cJun overexpression and phospho-p65-NFkB nuclear translocation. A decreased Bcl2:Bax ratio and a significant expression of smad7 were confirmed in early AnnexinV-positive myoFBs. A specific protein signature characterised the conditioned media: a dose dependent decrease occurred for IL8, IL6 while a selective increase was observed for VEGF and cyr61 (protein/mRNA). TIMP1 levels were unaffected. Herein, NGF modulation of smad7, the specific IL8 and IL6 as well as VEGF and cyr61 modulation deserve more attention as opening to alternative approaches to counteract fibrosis.

scholarly journals PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells

2001 ◽  
Vol 21 (11) ◽  
pp. 3738-3749 ◽  
Author(s):  
Ulf Andersson ◽  
Richard C. Scarpulla
Keyword(s):  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Peroxisome Proliferator ◽  
Nuclear Respiratory Factor ◽  
Nuclear Respiratory Factor 1 ◽  
Cell Extracts

ABSTRACT The thermogenic peroxisome proliferator-activated receptor γ (PPAR-γ) coactivator 1 (PGC-1) has previously been shown to activate mitochondrial biogenesis in part through a direct interaction with nuclear respiratory factor 1 (NRF-1). In order to identify related coactivators that act through NRF-1, we searched the databases for sequences with similarities to PGC-1. Here, we describe the first characterization of a 177-kDa transcriptional coactivator, designated PGC-1-related coactivator (PRC). PRC is ubiquitously expressed in murine and human tissues and cell lines; but unlike PGC-1, PRC was not dramatically up-regulated during thermogenesis in brown fat. However, its expression was down-regulated in quiescent BALB/3T3 cells and was rapidly induced by reintroduction of serum, conditions where PGC-1 was not detected. PRC activated NRF-1-dependent promoters in a manner similar to that observed for PGC-1. Moreover, NRF-1 was immunoprecipitated from cell extracts by antibodies directed against PRC, and both proteins were colocalized to the nucleoplasm by confocal laser scanning microscopy. PRC interacts in vitro with the NRF-1 DNA binding domain through two distinct recognition motifs that are separated by an unstructured proline-rich region. PRC also contains a potent transcriptional activation domain in its amino terminus adjacent to an LXXLL motif. The spatial arrangement of these functional domains coincides with those found in PGC-1, supporting the conclusion that PRC and PGC-1 are structurally and functionally related. We conclude that PRC is a functional relative of PGC-1 that operates through NRF-1 and possibly other activators in response to proliferative signals.

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