scholarly journals Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties

2018 ◽  
Vol 38 (2) ◽  
Author(s):  
Shohei Yokoo ◽  
Seiya Inoue ◽  
Nana Suzuki ◽  
Naho Amakawa ◽  
Hidenori Matsui ◽  
...  
Keyword(s):  
Biochemical Properties ◽  
Stress Conditions ◽  
The Other ◽  
Protein Accumulation ◽  
Transcriptional Level ◽  
Multiple Sequence ◽  
Primary And Secondary Metabolites ◽  
Transit Peptides ◽  
Alignment Analysis ◽  
Structural Mechanisms

Isochorismate synthase (ICS) converts chorismate into isochorismate, a precursor of primary and secondary metabolites including salicylic acid (SA). SA plays important roles in responses to stress conditions in plants. Many studies have suggested that the function of plant ICSs is regulated at the transcriptional level. In Arabidopsis thaliana, the expression of AtICS1 is induced by stress conditions in parallel with SA synthesis, and AtICS1 is required for SA synthesis. In contrast, the expression of NtICS is not induced when SA synthesis is activated in tobacco, and it is unlikely to be involved in SA synthesis. Studies on the biochemical properties of plant ICSs are limited, compared with those on transcriptional regulation. We analyzed the biochemical properties of four plant ICSs: AtICS1, NtICS, NbICS from Nicotiana benthamiana, and OsICS from rice. Multiple sequence alignment analysis revealed that their primary structures were well conserved, and predicted key residues for ICS activity were almost completely conserved. However, AtICS1 showed much higher activity than the other ICSs when expressed in Escherichia coli and N. benthamiana leaves. Moreover, the levels of AtICS1 protein expression in N. benthamiana leaves were higher than the other ICSs. Construction and analysis of chimeras between AtICS1 and OsICS revealed that the putative chloroplast transit peptides (TPs) significantly affected the levels of protein accumulation in N. benthamiana leaves. Chimeric and point-mutation analyses revealed that Thr531, Ser537, and Ile550 of AtICS1 are essential for its high activity. These distinct biochemical properties of plant ICSs may suggest different roles in their respective plant species.

scholarly journals Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8300
Author(s):  
Xiangzhu Wang ◽  
Chanchan Chen ◽  
Ting Shen ◽  
Jiangying Zhang
Keyword(s):  
Kinetic Parameters ◽  
Streptococcus Mutans ◽  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Structure Characterization ◽  
Multiple Sequence ◽  
Chromatography Method ◽  
Native Page ◽  
Aggregation State ◽  
Glutamate Racemase

Background Glutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome. Methods Structure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS–PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis–Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures. Results The glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (Km, d-Glu→l-Glu = 0.3631 ± 0.3205 mM, Vmax, d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1, kcat, d-Glu→l-Glu = 0.0306 ± 0.0065 s−1, kcat/Km, d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1, with d-glutamate as substrate; Km, l-Glu→d-Glu = 0.8077 ± 0.5081 mM, Vmax, l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1, kcat, l-Glu→d-Glu = 0.0378 ± 0.0056 s−1, kcat/Km, l-Glu→d-Glu = 0.0468 ± 0.0176 s−1 mM−1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0. Conclusion The findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.

Protein Accumulation in Early Chick Embryos Grown under Different Conditions of Explantation

Development ◽  
1959 ◽  
Vol 7 (1) ◽  
pp. 66-72
Author(s):  
L. Gwen Britt ◽  
Heinz Herrmann
Keyword(s):  
Chick Embryo ◽  
Slow Rate ◽  
The Other ◽  
Protein Accumulation ◽  
Chick Embryos ◽  
Developmental Processes ◽  
Embryonic Tissues ◽  
Somite Formation ◽  
Explanted Chick Embryo

The recent development of techniques originally devised by Waddington (1932) for the maintenance of the explanted chick embryo (Spratt, 1947; New, 1955; Wolff & Simon, 1955) has opened the possibility of determining quantitatively some parameters of the developmental processes occurring in embryonic tissues under these conditions. As a result of such measurements, protein accumulation in explanted embryos was found to be much smaller than in embryos developing in the egg. On the other hand, the progress of somite formation was found to take place at similar rates in embryos developing as explants or in situ (Herrmann & Schultz, 1958). The slow rate of protein accumulation in the explanted embryos made it seem desirable to investigate whether under some other conditions of explantation protein accumulation would approach more closely the rate of protein formation observed in the naturally developing embryo.

Polysomal organization in growing and resting cultures and its relation to protein synthesis in Tetrahymena

1983 ◽  
Vol 59 (1) ◽  
pp. 121-131
Author(s):  
P. Isberner ◽  
G. Cleffmann
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Growth Conditions ◽  
The Other ◽  
Protein Accumulation ◽  
Resting Cells ◽  
Amino Acid Incorporation ◽  
Total Rna ◽  
Acid Incorporation ◽  
Other Hand

Cytosol from Tetrahymena cells growing at different rates was isolated and separated by centrifugation into polysomal and non-polysomal fractions. The RNAs of either fraction were separated chromatographically into poly(A)+ RNA and poly(A)-RNA. It was found that in resting cultures the total RNA per cell is only about half of that of rapidly growing cultures. All fractions of RNA were reduced proportionally. Thus, the percentage of polysomally bound total RNA (70% of cytosol RNA) and polysomally bound poly(A)+ RNA (72% of cytosol poly(A)+ RNA) is the same in growing and resting cultures. Differences, however, were found in the polysomal structure. Polysomes from resting cultures contained significantly fewer ribosomes. The amounts of RNA bound to polysomes were related to the rate of protein synthesis under different growth conditions. The decrease in cellular RNA corresponded well with the reduction in amino acid incorporation in resting cells. The rate of protein accumulation in resting cells, on the other hand, was considerably less, suggesting that polypeptides in resting cultures are less stable.

scholarly journals Bacterial tRNA 2′-O-methylation is dynamically regulated under stress conditions and modulates innate immune response

2020 ◽  
Vol 48 (22) ◽  
pp. 12833-12844
Author(s):  
Adeline Galvanin ◽  
Lea-Marie Vogt ◽  
Antonia Grober ◽  
Isabel Freund ◽  
Lilia Ayadi ◽  
...  
Keyword(s):  
Gene Expression Regulation ◽  
Current Knowledge ◽  
Protein Translation ◽  
Stress Conditions ◽  
Transcriptional Level ◽  
Trna Modification ◽  
Rna Modifications ◽  
Swarming Motility ◽  
E Coli ◽  
Response To Stress

Abstract RNA modifications are a well-recognized way of gene expression regulation at the post-transcriptional level. Despite the importance of this level of regulation, current knowledge on modulation of tRNA modification status in response to stress conditions is far from being complete. While it is widely accepted that tRNA modifications are rather dynamic, such variations are mostly assessed in terms of total tRNA, with only a few instances where changes could be traced to single isoacceptor species. Using Escherichia coli as a model system, we explored stress-induced modulation of 2′-O-methylations in tRNAs by RiboMethSeq. This analysis and orthogonal analytical measurements by LC-MS show substantial, but not uniform, increase of the Gm18 level in selected tRNAs under mild bacteriostatic antibiotic stress, while other Nm modifications remain relatively constant. The absence of Gm18 modification in tRNAs leads to moderate alterations in E. coli mRNA transcriptome, but does not affect polysomal association of mRNAs. Interestingly, the subset of motility/chemiotaxis genes is significantly overexpressed in ΔTrmH mutant, this corroborates with increased swarming motility of the mutant strain. The stress-induced increase of tRNA Gm18 level, in turn, reduced immunostimulation properties of bacterial tRNAs, which is concordant with the previous observation that Gm18 is a suppressor of Toll-like receptor 7 (TLR7)-mediated interferon release. This documents an effect of stress induced modulation of tRNA modification that acts outside protein translation.

scholarly journals Study on the treatments and countermeasures for liquefiable foundation

2019 ◽  
Vol 272 ◽  
pp. 01012
Author(s):  
Binhua Xu ◽  
Ning He ◽  
Denghua Li
Keyword(s):  
Early Stage ◽  
Soil Liquefaction ◽  
Stress Conditions ◽  
The Other ◽  
Advantages And Disadvantages ◽  
Ground Liquefaction

This paper summarizes the current treatments and countermeasures for liquefiable foundations, and divides the existing anti-liquefaction countermeasures into two categories. One of the ideas is proceeding from the properties of liquefiable foundation soils, by the means of improvement for the soil’s qualities to enhance the capacity of soil’s anti-liquefaction in the early stage. The other idea is considering from the stress conditions of liquefiable foundation soils, and to reduce the liquefaction-induced disasters by changing the stress conditions of the soil. The advantages and disadvantages of various anti-liquefaction measures were analysed by verifying the effectiveness of field applications of anti-liquefaction measures against ground liquefaction hazards, and the applicable conditions of various anti-liquefaction measures were classified. This paper provides experience for resisting soil liquefaction disasters.

scholarly journals Profiling of primary metabolites in grapes of interspecific grapevine varieties: sugars and organic acids

2011 ◽  
Vol 29 (No. 4) ◽  
pp. 361-372 ◽  
Author(s):  
P. Pavloušek ◽  
M. Kumšta
Keyword(s):  
Cluster Analysis ◽  
Organic Acids ◽  
Vitis Vinifera ◽  
Hplc Method ◽  
The Other ◽  
Vitis Vinifera L ◽  
Primary Metabolites ◽  
Primary And Secondary Metabolites ◽  
The Individual

The quality of grapes is determined above all by the contents of the primary and secondary metabolites. The primary metabolites involve sugars and organic acids, and just these compounds are dealt with in this study. Its objective was to analyse and critically evaluate the primary metabolites in new interspecific varieties and, based on a comparison with European varieties of grapevine (Vitis vinifera L.), to find out the similarities and also possible differences between them. The study evaluates and compares 4 conventional varieties of Vitis vinifera with 11 new interspecific cultivars. The contents and compositions of the individual sugars and acids were estimated by means of the HPLC method. Most of these varieties belong to the group with either medium or low content of malic acid, i.e. with a medium to high β ratio. This corroborates the similarity of interspecific varieties to those of V. vinifera. The cluster analysis identified the existence of two interesting groups of varieties: the first one involved the varieties Riesling, Nativa, Marlen, and Kofranka while the other group consisted of varieties Blaufränkisch, Blauer Portugieser, and Laurot. This observation also indicates similarity between Vitis vinifera L. varieties and interspecific cultivars and demonstrates that the contents of the primary metabolites (i.e. sugars and organic acids) are also comparable.

scholarly journals Regulation of gbpC Expression in Streptococcus mutans

2007 ◽  
Vol 189 (18) ◽  
pp. 6521-6531 ◽  
Author(s):  
Indranil Biswas ◽  
Laura Drake ◽  
Saswati Biswas
Keyword(s):  
Streptococcus Mutans ◽  
Dna Sequences ◽  
Promoter Region ◽  
Response Regulator ◽  
Surface Protein ◽  
Maximum Level ◽  
Stress Conditions ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Transcriptional Level

ABSTRACT Streptococcus mutans, the principal causative agent of dental caries, produces four glucan-binding proteins (Gbp) that play major roles in bacterial adherence and pathogenesis. One of these proteins, GbpC, is an important cell surface protein involved in biofilm formation. GbpC is also important for cariogenesis, bacteremia, and infective endocarditis. In this study, we examined the regulation of gbpC expression in S. mutans strain UA159. We found that gbpC expression attains the maximum level at mid-exponential growth phase, and the half-life of the transcript is less than 2 min. Expression from PgbpC was measured using a PgbpC-gusA transcriptional fusion reporter and was analyzed under various stress conditions, including thermal, osmotic, and acid stresses. Expression of gbpC is induced under conditions of thermal stress but is repressed during growth at low pH, whereas osmotic stress had no effect on expression from PgbpC. The results from the expression analyses were further confirmed using semiquantitative reverse transcription-PCR analysis. Our results also reveal that CovR, a global response regulator in many Streptococcus spp., represses gbpC expression at the transcriptional level. We demonstrated that purified CovR protein binds directly to the promoter region of PgbpC to repress gbpC expression. Using a DNase I protection assay, we showed that CovR binds to DNA sequences surrounding PgbpC from bases −68 to 28 (where base 1 is the start of transcription). In summary, our results indicate that various stress conditions modulate the expression of gbpC and that CovR negatively regulates the expression of the gbpC gene by directly binding to the promoter region.

scholarly journals Arabidopsis Nuclear-Encoded Plastid Transit Peptides Contain Multiple Sequence Subgroups with Distinctive Chloroplast-Targeting Sequence Motifs

2008 ◽  
Vol 20 (6) ◽  
pp. 1603-1622 ◽  
Author(s):  
Dong Wook Lee ◽  
Jong Kyoung Kim ◽  
Sumin Lee ◽  
Seungjin Choi ◽  
Sanguk Kim ◽  
...  
Keyword(s):  
Sequence Motifs ◽  
Multiple Sequence ◽  
Chloroplast Targeting ◽  
Transit Peptides

scholarly journals Identification of a Hydrophobin Gene That is Developmentally Regulated in the Ectomycorrhizal Fungus Tricholoma terreum

2002 ◽  
Vol 68 (3) ◽  
pp. 1408-1413 ◽  
Author(s):  
Angela Mankel ◽  
Katrin Krause ◽  
Erika Kothe
Keyword(s):  
Transcriptional Control ◽  
Forest Ecology ◽  
Schizophyllum Commune ◽  
Aerial Mycelium ◽  
Ectomycorrhizal Fungus ◽  
Protein Accumulation ◽  
Transcriptional Level ◽  
Developmentally Regulated ◽  
Accumulation Pattern ◽  
Tricholoma Terreum

ABSTRACT The symbiosis between ectomycorrhizal fungi and trees is an essential part of forest ecology and depends entirely on the communication between the two partners for establishing and maintaining the relationship. The identification and characterization of differentially expressed genes is a step to identifying such signals and to understanding the regulation of this process. We determined the role of hydrophobins produced by Tricholoma terreum in mycorrhiza formation and hyphal development. A hydrophobin was purified from culture supernatant, and the corresponding gene was identified. The gene is expressed in aerial mycelium and in mycorrhiza. By using a heterologous antiserum directed against a hydrophobin found in the aerial mycelium of Schizophyllum commune, we detected a hydrophobin in the symbiosis between T. terreum and its native pine host Pinus sylvestris. The hydrophobin was found in aerial mycelium of the hyphal mantle and also in the Hartig net hyphae, which form the interface between both partners. Interestingly, this was not the case in the interaction of T. terreum with a host of low compatibility, the spruce Picea abies. The differential expression with respect to host was verified at the transcriptional level by competitive PCR. The differential protein accumulation pattern with respect to host compatibility seen by immunofluorescence staining can thus be attributed at least in part to transcriptional control of the hyd1 gene.

scholarly journals A potential protective mechanism of soya isoflavones against 7,12-dimethylbenz[a]anthracene tumour initiation

2003 ◽  
Vol 90 (2) ◽  
pp. 457-465 ◽  
Author(s):  
Ho Yee Chan ◽  
Lai K. Leung
Keyword(s):  
Enzyme Activities ◽  
Animal Studies ◽  
Breast Cancer Incidence ◽  
Epidemiological Studies ◽  
Significant Inhibition ◽  
The Other ◽  
Protective Mechanism ◽  
Transcriptional Level ◽  
Two Phase ◽  
Dna Mutation

Epidemiological studies indicate that Asian women have a lower breast cancer incidence compared with their counterparts in the West, and the difference has been related to soya consumption. Animal studies have suggested that soya may prevent dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in the breast. In the present study a cell culture model was developed to address the effect of soya isoflavones on the DMBA-induced DNA damage. DMBA is metabolized into a DNA-attacking moiety by two phase I cytochrome P450 (CYP) enzymes CYP1A1 and CYP1B1. DNA mutation caused by this genotoxic agent is a crucial step in cancer initiation. Substances that interfere with the CYP1 enzyme activities can affect the initiation. In the present study, genistein was found to be an effective inhibitor of recombinant human CYP1A1 and CYP1B1 with Ki of 15·35 and 0·68 μmol/l. The other soya isoflavone daidzein, on the other hand, did not demonstrate any significant inhibition of the enzyme activities. At the transcriptional level, DMBA induced the CYP1 enzyme expressions by stimulating the xenobiotic response element (XRE)-dependent transactivation pathway. When genistein (25 μmol/l) was co-administered with DMBA, the XRE-Luc activity the CYP1 mRNA abundances were significantly suppressed. The present study illustrated that the soya isoflavone genistein, but not daidzein, protected against DMBA genotoxicity.

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