scholarly journals Quaternary structure and biochemical properties of mycobacterial RNase E/G

2007 ◽  
Vol 403 (1) ◽  
pp. 207-215 ◽  
Author(s):  
Mirijam-Elisabeth Zeller ◽  
Agnes Csanadi ◽  
Andras Miczak ◽  
Thierry Rose ◽  
Thierry Bizebard ◽  
...  
Keyword(s):  
Quaternary Structure ◽  
Catalytic Domain ◽  
Biochemical Properties ◽  
Cleavage Sites ◽  
Rrna Processing ◽  
Rnase E ◽  
Human Pathogens ◽  
E Coli ◽  
Gram Positive Bacteria

The RNase E/G family of endoribonucleases plays the central role in numerous post-transcriptional mechanisms in Escherichia coli and, presumably, in other bacteria, including human pathogens. To learn more about specific properties of RNase E/G homologues from pathogenic Gram-positive bacteria, a polypeptide comprising the catalytic domain of Mycobacterium tuberculosis RNase E/G (MycRne) was purified and characterized in vitro. In the present study, we show that affinity-purified MycRne has a propensity to form dimers and tetramers in solution and possesses an endoribonucleolytic activity, which is dependent on the 5′-phosphorylation status of RNA. Our data also indicate that the cleavage specificities of the M. tuberculosis RNase E/G homologue and its E. coli counterpart are only moderately overlapping, and reveal a number of sequence determinants within MycRne cleavage sites that differentially affect the efficiency of cleavage. Finally, we demonstrate that, similar to E. coli RNase E, MycRne is able to cleave in an intercistronic region of the putative 9S precursor of 5S rRNA, thus suggesting a common function for RNase E/G homologues in rRNA processing.

scholarly journals Decreased Expression of Stable RNA Can Alleviate the Lethality Associated with RNase E Deficiency in Escherichia coli

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
P. Himabindu ◽  
K. Anupama
Keyword(s):  
Escherichia Coli ◽  
Mrna Degradation ◽  
Gram Negative Bacteria ◽  
Rrna Processing ◽  
Rnase E ◽  
Rnase R ◽  
Content Type ◽  
E Coli ◽  
Rrna Species ◽  
Rna Levels

ABSTRACT The endoribonuclease RNase E participates in mRNA degradation, rRNA processing, and tRNA maturation in Escherichia coli, but the precise reasons for its essentiality are unclear and much debated. The enzyme is most active on RNA substrates with a 5′-terminal monophosphate, which is sensed by a domain in the enzyme that includes residue R169; E. coli also possesses a 5′-pyrophosphohydrolase, RppH, that catalyzes conversion of 5′-terminal triphosphate to 5′-terminal monophosphate on RNAs. Although the C-terminal half (CTH), beyond residue approximately 500, of RNase E is dispensable for viability, deletion of the CTH is lethal when combined with an R169Q mutation or with deletion of rppH. In this work, we show that both these lethalities can be rescued in derivatives in which four or five of the seven rrn operons in the genome have been deleted. We hypothesize that the reduced stable RNA levels under these conditions minimize the need of RNase E to process them, thereby allowing for its diversion for mRNA degradation. In support of this hypothesis, we have found that other conditions that are known to reduce stable RNA levels also suppress one or both lethalities: (i) alterations in relA and spoT, which are expected to lead to increased basal ppGpp levels; (ii) stringent rpoB mutations, which mimic high intracellular ppGpp levels; and (iii) overexpression of DksA. Lethality suppression by these perturbations was RNase R dependent. Our work therefore suggests that its actions on the various substrates (mRNA, rRNA, and tRNA) jointly contribute to the essentiality of RNase E in E. coli. IMPORTANCE The endoribonuclease RNase E is essential for viability in many Gram-negative bacteria, including Escherichia coli. Different explanations have been offered for its essentiality, including its roles in global mRNA degradation or in the processing of several tRNA and rRNA species. Our work suggests that, rather than its role in the processing of any one particular substrate, its distributed functions on all the different substrates (mRNA, rRNA, and tRNA) are responsible for the essentiality of RNase E in E. coli.

Screening of Antimicrobial Activity and Cytotoxic Effects of Two Cladonia Species

2013 ◽  
Vol 68 (5-6) ◽  
pp. 191-197 ◽  
Author(s):  
Birkan Açıkgöz ◽  
İskender Karaltı ◽  
Melike Ersöz ◽  
Zeynep M. Coşkun ◽  
Gülşah Çobanoğlu ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Antimicrobial Activities ◽  
Chloroform Extract ◽  
Cytotoxic Effects ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Methanol Extracts ◽  
Gram Positive Bacteria

The present study explores the antimicrobial activity and cytotoxic effects in culture assays of two fruticose soil lichens, Cladonia rangiformis Hoffm. and Cladonia convoluta (Lamkey) Cout., to contribute to possible pharmacological uses of lichens. In vitro antimicrobial activities of methanol and chloroform extracts against two Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), two Gram-positive bacteria (Enterococcus faecalis and Staphylococcus aureus), and the yeast Candida albicans were examined using the paper disc method and through determination of minimal inhibitory concentrations (MICs). The data showed the presence of antibiotic substances in the chloroform and the methanol extracts of the lichen species. The chloroform extracts exhibited more signifi cant antimicrobial activity than the methanol extracts. However, a higher antifungal activity was noted in the methanol extract of C. rangiformis. The maximum antimicrobial activity was recorded for the chloroform extract of C. convoluta against E. coli. The cytotoxic effects of the lichen extracts on human breast cancer MCF-7 cells were evaluated by the trypan blue assay yielding IC50 values of ca. 173 and 167 μg/ml for the extracts from C. rangiformis and C. convoluta, respectively.

scholarly journals Fibroblast and neutrophil collagenases cleave at two sites in the cartilage aggrecan interglobular domain

1993 ◽  
Vol 295 (1) ◽  
pp. 273-276 ◽  
Author(s):  
A J Fosang ◽  
K Last ◽  
V Knäuper ◽  
P J Neame ◽  
G Murphy ◽  
...  
Keyword(s):  
Human Fibroblast ◽  
Catalytic Domain ◽  
Polymorphonuclear Leucocyte ◽  
Gelatinase A ◽  
Cleavage Sites ◽  
Similar Extent ◽  
Human Sequence ◽  
Fibroblast Collagenase ◽  
Gelatinases A And B

The actions of recombinant human fibroblast collagenase (MMP1), purified polymorphonuclear leucocyte collagenase (MMP8) and their N-terminal catalytic domain fragments against cartilage aggrecan and an aggrecan G1-G2 fragment have been investigated in vitro. After activation with recombinant human stromelysin and typsin, both collagenases were able to degrade human and porcine aggrecans to a similar extent. An N-terminal G1-G2 fragment (150 kDa) was used to identify specific cleavage sites occurring within the proteinase-sensitive interglobular domain between G1 and G2. Two specific sites were found; one at an Asn341-Phe342 bond and another at Asp441-Leu442 (human sequence). This specificity of the collagenases for aggrecan G1-G2 was identical with that of the truncated metalloproteinase matrilysin (MMP7), but different from those of stromelysin (MMP3) and the gelatinases (MMP2 or gelatinase A; MMP9 or gelatinase B) which cleave at the Asn-Phe site, but not the Asp-Leu site. In addition, collagenase catalytic fragments lacking C-terminal hemopexin-like domains were tested and shown to exhibit the same specificities for the G1-G2 fragment as the full-length enzymes. Thus the specificity of the collagenases for cartilage aggrecan was not influenced by the presence or absence of the C-terminal domain. Together with our previous findings, the results show that stromelysin-1, matrilysin, gelatinases A and B and fibroblast and neutrophil collagenases cleave at a common, preferred site in the aggrecan interglobular domain, and additionally that both fibroblast and neutrophil collagenases cleave at a second site in the interglobular domain that is not available to stromelysin or gelatinases.

scholarly journals Widespread Distribution in Pathogenic Bacteria of Di-Iron Proteins That Repair Oxidative and Nitrosative Damage to Iron-Sulfur Centers

2008 ◽  
Vol 190 (6) ◽  
pp. 2004-2013 ◽  
Author(s):  
Tim W. Overton ◽  
Marta C. Justino ◽  
Ying Li ◽  
Joana M. Baptista ◽  
Ana M. P. Melo ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Nitrosative Stress ◽  
Human Pathogens ◽  
Paramagnetic Resonance ◽  
Widespread Distribution ◽  
Iron Proteins ◽  
E Coli

ABSTRACT Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H2O2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H2O2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

Chemokine degradation by the Group A streptococcal serine proteinase ScpC can be reconstituted in vitro and requires two separate domains

2009 ◽  
Vol 422 (3) ◽  
pp. 533-542 ◽  
Author(s):  
Andrea Fritzer ◽  
Birgit Noiges ◽  
Daniela Schweiger ◽  
Angelika Rek ◽  
Andreas J. Kungl ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Serine Proteinase ◽  
Interferon Γ ◽  
Catalytic Triad ◽  
Terminal Region ◽  
Dependent Manner ◽  
Human Pathogens ◽  
Platelet Factor ◽  
Monocyte Chemoattractant Protein 1

Streptococcus pyogenes is one of the most common human pathogens and possesses diverse mechanisms to evade the human immune defence. One example of its immune evasion is the degradation of the chemokine IL (interleukin)-8 by ScpC, a serine proteinase that prevents the recruitment of neutrophils to an infection site. By applying the ANTIGENome technology and using human serum antibodies, we identified Spy0416, annotated as ScpC, as a prominent antigen that induces protective immune responses in animals. We demonstrate here for the first time that the recombinant form of Spy0416 is capable of IL-8 degradation in vitro in a concentration- and time-dependent manner. Mutations in the conserved amino acid residues of the catalytic triad of Spy0416 completely abolished in vitro activity. However, the isolated predicted proteinase domain does not exhibit IL-8-degrading activity, but is dependent on the presence of the C-terminal region of Spy0416. Binding to IL-8 is mainly mediated by the catalytic domain. However, the C-terminal region modulates substrate binding, indicating that the proteolytic activity is amenable to regulation via the non-catalytic regions. The specificity for human substrates is not restricted to IL-8, since we also detected in vitro protease activity for another CXC chemokine GRO-α (growth-related oncogene α), but not for NAP-2 (neutrophil-activating protein 2), SDF (stromal-cell-derived factor)-1α, PF-4 (platelet factor 4), I-TAC (interferon-γ-inducible T-cell α-chemoattractant), IP-10 (interferon-γ-inducible protein 10) and MCP-1 (monocyte chemoattractant protein 1). The degradation of two human CXC chemokines in vitro, the high sequence conservation, the immunogenicity of the protein in humans and the shown protection in animal studies suggest that Spy0416 is a promising vaccine candidate for the prevention of infections by S. pyogenes.

scholarly journals Antimicrobial Effect Of Honey Distributed Around Pasar Minggu Jakarta Selatan

2017 ◽  
Vol 8 (2) ◽  
pp. 101-106
Author(s):  
Ruth Elenora Kristanty ◽  
Junie Suriawati ◽  
Priyanto Dwi Nugroho
Keyword(s):  
Infectious Diseases ◽  
Food Product ◽  
Antimicrobial Effect ◽  
Inhibition Zone ◽  
Diffusion Method ◽  
Bacteria Growth ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Almost All

Honey is a highly nutritious food product and consumed by almost all the population in the world. It has a function as an antimicrobial. Staphylococcus aureus (S. aureus) is a common Gram-positive bacteria in food and Escherichia coli (E. coli) is a Gram-negative bacteria that often appears in environmental sanitation issues that both can cause infectious diseases.  Some infectious diseases can be treated with antimicrobials such as honey. The purpose of this study was to test the antimicrobial effects on honey products distributed in Pasar Minggu area. The antimicrobial effect test was performed in vitro using agar diffusion method by measuring the inhibition zone formed where the bacteria growth was inhibited by the presence of sample. The concentration of samples were 25%, 50%, 75%, and 100% (not diluted) and as aquades control. The results showed that honey tested with various dilution concentrations resulted inhibition zone and. The higher concentration of the inhibited zone zone showed antimicrobial activity against S. aureus and E. coli.

scholarly journals Escherichia coli and Staphylococcus aureus responsible for bovine mastitis sensitivity to the essential oil of Algerian Thymus fontanesii Boiss. et Reut.

2020 ◽  
Vol 7 (1) ◽  
pp. 26-32
Author(s):  
Bendella Amina nor elhouda ◽  
Ghazi Kheira ◽  
Meliani Samia
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Essential Oil ◽  
Bovine Mastitis ◽  
Diffusion Method ◽  
Bacterial Strains ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Zones Of Inhibition

AbstractThe aim of this study is to test two different methods for evaluating the in vitro antibacterial effect of Thymus fontanesii Boiss. et Reut. essential oil against standard and clinical bacterial strains responsible for bovine mastitis: the disc diffusion method or the aromatogram which allows the demonstration of the antibacterial power of essential oils on the bacterial strains tested, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and two strains isolated from bovine mastitis milk S. aureus and E. coli. The inhibition activity of the essential oil of T. fontanesii on bacterial strains by the two methods shows that the antimicrobial power of this oil is very important and is characterized by bactericidal and bacteriostatic action against gram negative and gram positive bacteria. The antimicrobial evaluation by the aromatogram showed good antibacterial activity against all the strains tested, the zones of inhibition of the bacteria were between 23,33±1,527mm and 37,5±3,535 mm. The search for minimum inhibitory concentrations MIC and bactericides CMB made it possible to quantitatively assess the antimicrobial power of this essential oil. In this work, the MIC was 0,625 µl/ml for all strains tested, and the lowest CMB was that of T. fontanesii against E. coli ATCC 25922 was 0,625 µl/ml.

scholarly journals Reliable method for high quality His-tagged and untagged E. coli phosphoribosyl phosphate synthase (Prs) purification

2019 ◽  
Author(s):  
Walter Beata Maria ◽  
Szulc Aneta ◽  
Glinkowska Monika
Keyword(s):  
Quaternary Structure ◽  
Physiological State ◽  
Building Blocks ◽  
High Yield ◽  
Phosphoribosyl Pyrophosphate ◽  
Protein Variant ◽  
E Coli ◽  
Nickel Affinity Chromatography

ABSTRACTPrs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks – purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation.Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state.The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.

scholarly journals Synthesis and Pharmacological Evaluation of 2-(4-(3 (Substituted Phenyl) Acryloyl) Phenoxy)-N, N Diphenylacetamides

2020 ◽  
Vol 10 (5) ◽  
pp. 274-292
Author(s):  
Rohit Kumar ◽  
Sushil Kumar ◽  
Mohammad Asif Khan
Keyword(s):  
Antibacterial Activity ◽  
Schiff Bases ◽  
Biological Activities ◽  
Diffusion Method ◽  
Gram Positive ◽  
Standard Drug ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Pharmacological Evaluation

Recently a series of Schiff bases of diphenylamine derivatives have been synthesized and evaluated in vitro for their antibacterial activity against pathogenic both Gram-positive bacteria B. subtitles and Gram-negative bacteria E. coli using ciprofloxacin as standard drug at conc. of 50 μg/ml and 100 μg/ml. Literature review revels that chalcones possesses various biological activities like antimicrobial, antiviral, anti-inflammatory, anticancer and sedative etc. Therefore the present study was designed on synthesis and pharmacological evaluation of 2-(4-(3 (Substituted Phenyl) Acryloyl) Phenoxy)-N, N Diphenylacetamides. Target compound was synthesized by reaction of chloroacetylchloride with diphenylamine to afford 2-chloro-N, N-diphenylacetamide which further by reaction with substituted Chalcones and characterized following recrystallization and evaluated for anti-microbial potential through cup-diffusion method. In results, the target compounds were tested for activity against B. Subtilis, E.Coli and C. albicans. The chalcones having the lipophilic 4-chloro group (RKCT2) showed the greatest antimicrobial activity (zone of inhibition 20 & 22 mm against. B. subtilis, E. Coli, C. Albicans respectively. It suggests further researchers to go through anti-microbial evaluations against a more varieties of bacteria and fungi. Keywords: Schiff bases of diphenylamine derivatives, antibacterial activity, Gram-positive bacteria, 2-(4-(3 (Substituted Phenyl) Acryloyl) Phenoxy)-N, N Diphenylacetamides

scholarly journals Autokinase Activity of Casein Kinase 1 δ/ε Governs the Period of Mammalian Circadian Rhythms

2019 ◽  
Vol 34 (5) ◽  
pp. 482-496 ◽  
Author(s):  
Gaili Guo ◽  
Kankan Wang ◽  
Shan-Shan Hu ◽  
Tian Tian ◽  
Peng Liu ◽  
...  
Keyword(s):  
Circadian Rhythms ◽  
Conformational Changes ◽  
Catalytic Domain ◽  
Biochemical Properties ◽  
Period Change ◽  
Casein Kinase ◽  
Full Length ◽  
Casein Kinase 1 ◽  
Autokinase Activity

Circadian rhythms exist in nearly all organisms. In mammals, transcriptional and translational feedback loops (TTFLs) are believed to underlie the mechanism of the circadian clock. Casein kinase 1δ/ε (CK1δ/ε) are key kinases that phosphorylate clock components such as PER proteins, determining the pace of the clock. Most previous studies of the biochemical properties of the key kinases CK1ε and CK1δ in vitro have focused on the properties of the catalytic domains from which the autoinhibitory C-terminus has been deleted (ΔC); those studies ignored the significance of self-inhibition by autophosphorylation. By comparing the properties of the catalytic domain of CK1δ/ε with the full-length kinase that can undergo autoinhibition, we found that recombinant full-length CK1 showed a sequential autophosphorylation process that induces conformational changes to affect the overall kinase activity. Furthermore, a direct relationship between the period change and the autokinase activity among CK1δ, CK1ε, and CK1ε-R178C was observed. These data implicate the autophosphorylation activity of CK1δ and CK1ε kinases in setting the pace of mammalian circadian rhythms and indicate that the circadian period can be modulated by tuning the autophosphorylation rates of CK1δ/ε.

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