scholarly journals Structural features of the exocellular polysaccharides of Mycobacterium tuberculosis

1994 ◽  
Vol 297 (2) ◽  
pp. 351-357 ◽  
Author(s):  
A Lemassu ◽  
M Daffé
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mimicry ◽  
Degradation Products ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Structural Features ◽  
Two Dimensional ◽  
Phagocytic Cells ◽  
Molecular Masses ◽  
Branched Structure

The cell envelope which surrounds pathogenic mycobacteria is postulated to be a defence barrier against phagocytic cells and its outermost constituents have a tendency to accumulate in the culture medium. The present work demonstrates that the exocellular material of Mycobacterium tuberculosis contains large amounts of polysaccharides with only traces, if any at all, of lipids. Three types of polysaccharides were purified by anion-exchange and gel-filtration chromatography; all were found to be neutral compounds devoid of acyl substituents. They consisted of D-glucan, D-arabino-D-mannan and D-mannan, which were eluted from gel-filtration columns in positions corresponding to molecular masses of 123, 13 and 4 kDa respectively. Their predominant structural features were determined by the characterization of the per-O-methyl derivatives of enzymic, acetolysis and Smith-degradation products and by 1H- and 13C-n.m.r. spectroscopy of the purified polysaccharides, using mono- and two-dimensional homonuclear chemical-shift correlated spectroscopy and two-dimensional heteronuclear (1H/13C) spectroscopy. The glucan which represented up to 90% of the polysaccharides was composed of repeating units of five or six-->4-alpha-D-Glcp-1--> residues and a -->4-alpha-D-Glcp substituted at position 6 with an alpha-D-Glcp, indicating a glycogen-like highly branched structure not related to the so-called polysaccharide-II previously identified in tuberculin. The arabinomannan consisted of a mannan segment composed of a -->6-alpha-D-Man-1--> core substituted at some positions 2 with an alpha-D-Manp. The arabinan termini of the arabinomannan were found to be extensively capped with mannosyl residues. The possibility that these polysaccharides contribute to the persistence of the tubercle bacillus in the macrophage by molecular mimicry is discussed.

scholarly journals Purification of a Crenarchaeal ATP Synthase in the Light of the Unique Bioenergetics ofIgnicoccusSpecies

2019 ◽  
Vol 201 (7) ◽  
Author(s):  
Lydia J. Kreuter ◽  
Andrea Weinfurtner ◽  
Alexander Ziegler ◽  
Julia Weigl ◽  
Jan Hoffmann ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Cell Envelope ◽  
Gel Filtration ◽  
Cellular Membrane ◽  
Content Type ◽  
The Pacific ◽  
Native Electrophoresis ◽  
Molecular Masses ◽  
The Pacific Ocean

ABSTRACTIn this study, the ATP synthase ofIgnicoccus hospitaliswas purified, characterized, and structurally compared to the respective enzymes of the otherIgnicoccusspecies, to shed light on energy conservation in this unique group of archaea. The crenarchaeal genusIgnicoccuscomprises three described species, i.e.,I. hospitalisandIgnicoccus islandicusfrom hot marine sediments near Iceland andIgnicoccus pacificusfrom a hydrothermal vent system in the Pacific Ocean. This genus is unique among all archaea due to the unusual cell envelope, consisting of two membranes that enclose a large intermembrane compartment (IMC).I. hospitalisis the best studied member of this genus, mainly because it is the only known host for the potentially parasitic archaeonNanoarchaeum equitans.I. hospitalisgrows chemolithoautotrophically, and its sole energy-yielding reaction is the reduction of elemental sulfur with molecular hydrogen, forming large amounts of hydrogen sulfide. This reaction generates an electrochemical gradient, which is used by the ATP synthase, located in the outer cellular membrane, to generate ATP inside the IMC. The genome ofI. hospitalisencodes nine subunits of an A-type ATP synthase, which we could identify in the purified complex. Although the maximalin vitroactivity of theI. hospitalisenzyme was measured around pH 6, the optimal stability of the A1AOcomplex seemed to be at pH 9. Interestingly, the soluble A1subcomplexes of the differentIgnicoccusspecies exhibited significant differences in their apparent molecular masses in native electrophoresis, although their behaviors in gel filtration and chromatography-mass spectrometry were very similar.IMPORTANCETheCrenarchaeotarepresent one of the major phyla within theArchaeadomain. This study describes the successful purification of a crenarchaeal ATP synthase. To date, all information about A-type ATP synthases is from euryarchaeal enzymes. The fact that it has not been possible to purify this enzyme complex from a member of theCrenarchaeotauntil now points to significant differences in stability, possibly caused by structural alterations. Furthermore, the study subjectI. hospitalishas a particular importance among crenarchaeotes, since it is the only known host ofN. equitans. The energy metabolism in this system is still poorly understood, and our results can help elucidate the unique relationship between these two microbes.

Fibrin-I Degradation Products with a Molecular Weight Higher than That of Fibrinogen

1973 ◽  
Vol 29 (01) ◽  
pp. 122-129 ◽  
Author(s):  
René von Hugo ◽  
Henner Graeff
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Degradation Products ◽  
Gel Filtration ◽  
Agarose Gel ◽  
Two Dimensional ◽  
Plasma Clot ◽  
Two Dimensional Gel Electrophoresis ◽  
Parent Molecule

SummaryThe observation of intravascular lysis of fibrin deposits and of fibrinogen derivatives with a molecular weight higher than the parent molecule in human cases of disseminated intravascular coagulation (DIC) initiated the following in vitro study. Following streptokinase induced plasma clot solubilization fibrinogen derivatives were investigated after ß-alanine precipitation of the plasma samples by polyacrylamide (PAA) gel electrophoresis, intra gel immunoprecipitation, two dimensional gel electrophoresis and by agarose gel filtration. Three fibrin-i degradation products were observed and characterized according to their relative electrophoretic mobility in 5% PAA gel: 0.23, 0.35, 0.46 (fibrinogen: 0.43) x 10-5 cm2/V x sec. They could also be demonstrated after electrophoresis in the presence of 5 M urea. Agarose gel filtration yielded one peak at 180 ml of effluent volume. The 0.23 derivative was eluted in the peak fractions, whilst the 0.35 and 0.46 derivatives were eluted together at approximately 201 ml of the effluent volume (fibrinogen: 225 ml). This indicates, that the three fibrin-i degradation products described are molecular entities with molecular weights higher than fibrinogen and, that the 0.46 derivative has an increased charge/molecular size ratio in comparison with fibrinogen. Corresponding data were obtained by two dimensional gel electrophoresis in gels of different pore size.

scholarly journals Sulfolipid Deficiency Does Not Affect the Virulence of Mycobacterium tuberculosis H37Rv in Mice and Guinea Pigs

2003 ◽  
Vol 71 (8) ◽  
pp. 4684-4690 ◽  
Author(s):  
Cécile Rousseau ◽  
Oliver C. Turner ◽  
Erik Rush ◽  
Yann Bordat ◽  
Tatiana D. Sirakova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Guinea Pigs ◽  
Cell Envelope ◽  
Biological Effects ◽  
Phagocytic Cells ◽  
Definitive Evidence ◽  
Mycobacterium Tuberculosis H37rv ◽  
Complex Lipids ◽  
Methyl Branched Fatty Acids ◽  
First Time

ABSTRACT Lipids that are found only in the cell envelope of pathogenic mycobacteria, such as those containing multiple methyl-branched fatty acids, have long been thought to play a role in pathogenesis. Among these complex lipids, sulfolipids have been the most extensively studied over the last 50 years. The numerous biological effects exhibited by purified sulfolipids on phagocytic cells led to the idea that these molecules are probably important virulence factors facilitating the intracellular survival of Mycobacterium tuberculosis. However, definitive evidence to support this concept has been lacking. The recent construction of an isogenic sulfolipid-deficient mutant of M. tuberculosis H37Rv (Sirakova et al., J. Biol. Chem. 276:16833-16839, 2001) has for the first time provided the opportunity to directly assess the contribution of these complex lipids to pathogenesis. In the present study, we show that against all expectations, sulfolipid deficiency does not significantly affect the replication, persistence, and pathogenicity of M. tuberculosis H37Rv in mice and guinea pigs or in cultured macrophages.

scholarly journals Targeting MmpL3 for anti-tuberculosis drug development

2020 ◽  
Vol 48 (4) ◽  
pp. 1463-1472
Author(s):  
Jani R. Bolla
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Membrane Protein ◽  
Drug Development ◽  
Crystal Structures ◽  
Therapeutic Target ◽  
Cell Envelope ◽  
Structural Features ◽  
Lipid Transport ◽  
Attractive Therapeutic Target ◽  
Made In

The unique architecture of the mycobacterial cell envelope plays an important role in Mycobacterium tuberculosis (Mtb) pathogenesis. A critical protein in cell envelope biogenesis in mycobacteria, required for transport of precursors, trehalose monomycolates (TMMs), is the Mycobacterial membrane protein large 3 (MmpL3). Due to its central role in TMM transport, MmpL3 has been an attractive therapeutic target and a key target for several preclinical agents. In 2019, the first crystal structures of the MmpL3 transporter and its complexes with lipids and inhibitors were reported. These structures revealed several unique structural features of MmpL3 and provided invaluable information on the mechanism of TMM transport. This review aims to highlight the recent advances made in the function of MmpL3 and summarises structural findings. The overall goal is to provide a mechanistic perspective of MmpL3-mediated lipid transport and inhibition, and to highlight the prospects for potential antituberculosis therapies.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

1989 ◽  
Vol 61 (03) ◽  
pp. 409-414 ◽  
Author(s):  
M Rånby ◽  
G Nguyen ◽  
P Y Scarabin ◽  
M Samama
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Degradation Products ◽  
Gel Filtration ◽  
Free Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Turnover of Human Extrinsic (Tissue-Type) Plasminogen Activator in Rabbits

1981 ◽  
Vol 46 (03) ◽  
pp. 658-661 ◽  
Author(s):  
C Korninger ◽  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Intravenous Injection ◽  
Active Site ◽  
Half Life ◽  
Degradation Products ◽  
Gel Filtration ◽  
Tissue Type ◽  
Organ Distribution ◽  
Small Rise

SummaryThe turnover of highly purified human extrinsic plasminogen activator (EPA) (one- and two-chain form) was studied in rabbits. Following intravenous injection, EPA-activity declined rapidly. The disappearance rate of EPA from the plasma could adequately be described by a single exponential term with a t ½ of approximately 2 min for both the one-chain and two-chain forms of EPA.The clearance and organ distribution of EPA was studied by using 125I-labeled preparations. Following intravenous injection of 125I-1abeled EPA the radioactivity disappeared rapidly from the plasma also with a t ½ of approximately 2 min down to a level of 15 to 20 percent, followed by a small rise of blood radioactivity. Gel filtration of serial samples revealed that the secondary increase of the radioactivity was due to the reappearance of radioactive breakdown products in the blood. Measurement of the organ distribution of 125I at different time intervals revealed that EPA was rapidly accumulated in the liver, followed by a release of degradation products in the blood.Experimental hepatectomy markedly prolonged the half-life of EPA in the blood. Blocking the active site histidine of EPA had no effect on the half-life of EPA in blood nor on the gel filtration patterns of 125I in serial plasma samples.It is concluded that human EPA is rapidly removed from the blood of rabbits by clearance and degradation in the liver. Recognition by the liver does not require a functional active site in the enzyme. Neutralization in plasma by protease inhibitors does not represent a significant pathway of EPA inactivation in vivo.

Characterization of dissolved material during the initial phase of softwood kraft pulping

TAPPI Journal ◽  
2013 ◽  
Vol 12 (1) ◽  
pp. 37-43 ◽  
Author(s):  
HANNU PAKKANEN ◽  
TEEMU PALOHEIMO ◽  
RAIMO ALÉN
Keyword(s):  
Mass Transfer ◽  
Molecular Mass ◽  
Initial Phase ◽  
Degradation Products ◽  
Kraft Pulping ◽  
Reaction Products ◽  
Cooking Temperature ◽  
Molecular Masses ◽  
Aliphatic Carboxylic Acids

The influence of various cooking parameters, such as effective alkali, cooking temperature, and cooking time on the formation of high molecular mass lignin-derived and low molecular mass carbohydrates-derived (aliphatic carboxylic acids) degradation products, mainly during the initial phase of softwood kraft pulping was studied. In addition, the mass transfer of all of these degradation products was clarified based on their concentrations in the cooking liquor inside and outside of the chips. The results indicated that the degradation of the major hemicellulose component, galactoglucomannan, typically was dependent on temperature, and the maximum degradation amount was about 60%. In addition, about 60 min at 284°F (140°C) was needed for leveling off the concentrations of the characteristic reaction products (3,4-dideoxy-pentonic and glucoisosaccharinic acids) between these cooking liquors. Compared with low molecular mass aliphatic acids, the mass transfer of soluble lignin fragments with much higher molecular masses was clearly slower.

Exploration of Ion Channels in Mycobacterium tuberculosis: Implication on Drug Discovery and Potent Drug Targets Against Tuberculosis

2020 ◽  
Vol 14 (1) ◽  
pp. 14-29
Author(s):  
Manish Dwivedi
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Cell Envelope ◽  
Intracellular Pathogen ◽  
Host Immune System ◽  
Transporter Proteins ◽  
Channel Proteins ◽  
Medical Importance ◽  
Potent Drug ◽  
Emergence Of Resistance

Scientific interest in mycobacteria has been sparked by the medical importance of Mycobacterium tuberculosis (Mtb) that is known to cause severe diseases in mammals, i.e. tuberculosis and by properties that distinguish them from other microorganisms which are notoriously difficult to treat. The treatment of their infections is difficult because mycobacteria fortify themselves with a thick impermeable cell envelope. Channel and transporter proteins are among the crucial adaptations of Mycobacterium that facilitate their strength to combat against host immune system and anti-tuberculosis drugs. In previous studies, it was investigated that some of the channel proteins contribute to the overall antibiotic resistance in Mtb. Moreover, in some of the cases, membrane proteins were found responsible for virulence of these pathogens. Given the ability of M. tuberculosis to survive as an intracellular pathogen and its inclination to develop resistance to the prevailing anti-tuberculosis drugs, its treatment requires new approaches and optimization of anti-TB drugs and investigation of new targets are needed for their potential in clinical usage. Therefore, it is imperative to investigate the survival of Mtb. in stressed conditions with different behavior of particular channel/ transporter proteins. Comprehensive understanding of channel proteins and their mechanism will provide us direction to find out preventive measures against the emergence of resistance and reduce the duration of the treatment, eventually leading to plausible eradication of tuberculosis.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

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