scholarly journals Characterization of human pre-elafin mutants: full antipeptidase activity is essential to preserve lung tissue integrity in experimental emphysema

2007 ◽  
Vol 405 (3) ◽  
pp. 455-463 ◽  
Author(s):  
Alain Doucet ◽  
Dominique Bouchard ◽  
Marie France Janelle ◽  
Audrey Bellemare ◽  
Stéphane Gagné ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Neutrophil Elastase ◽  
Tight Binding ◽  
Biochemical Properties ◽  
Van Der Waals Interactions ◽  
Tissue Destruction ◽  
Methionine Residue ◽  
Experimental Emphysema

Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1′ methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1′ subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.

scholarly journals Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT)

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2904
Author(s):  
Margot Gautier ◽  
Cécile Thirant ◽  
Olivier Delattre ◽  
Isabelle Janoueix-Lerosey
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Biochemical Properties ◽  
Poor Overall Survival ◽  
Cell Identity ◽  
Mesenchymal Transition ◽  
Neuroblastoma Cell Lines

Neuroblastoma, a pediatric cancer of the peripheral sympathetic nervous system, is characterized by an important clinical heterogeneity, and high-risk tumors are associated with a poor overall survival. Neuroblastoma cells may present with diverse morphological and biochemical properties in vitro, and seminal observations suggested that interconversion between two phenotypes called N-type and S-type may occur. In 2017, two main studies provided novel insights into these subtypes through the characterization of the transcriptomic and epigenetic landscapes of a panel of neuroblastoma cell lines. In this review, we focus on the available data that define neuroblastoma cell identity and propose to use the term noradrenergic (NOR) and mesenchymal (MES) to refer to these identities. We also address the question of transdifferentiation between both states and suggest that the plasticity between the NOR identity and the MES identity defines a noradrenergic-to-mesenchymal transition, reminiscent of but different from the well-established epithelial-to-mesenchymal transition.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

Differential Regulation of Articular Cartilage Biomechanical and Biochemical Properties by IGF-1 and TGF-β1 During In Vitro Growth

2009 ◽  
Author(s):  
Michael E. Stender ◽  
Christian R. Flores ◽  
Kristin J. Dills ◽  
Gregory M. Williams ◽  
Kevin M. Stewart ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Growth Models ◽  
Biochemical Properties ◽  
Low Friction ◽  
Cartilage Growth ◽  
Detailed Characterization ◽  
Naturally Occurring ◽  
Tgf Β1

Articular cartilage (AC) is a load bearing material that provides a low friction wear resistant interface in synovial joints. Naturally-occurring and stimulated intrinsic repair of damaged AC is ineffective. Thus, there is a desire to engineer effective replacement tissue that could be used for AC repair. Previous studies [1] have shown that culture of immature cartilage with medium including TGF-β1 will result in a more mature tissue than culture with IGF-1. Detailed characterization of tissue mechanical properties would be helpful for development of cartilage growth models [2].

scholarly journals In vitro fusion of Acanthamoeba phagolysosomes. II Quantitative characterization of in vitro vacuole fusion by improved electron microscope and new light microscope techniques.

1978 ◽  
Vol 79 (1) ◽  
pp. 217-234 ◽  
Author(s):  
P J Oates ◽  
O Touster
Keyword(s):  
Electron Microscope ◽  
Light Microscope ◽  
Fusion Reaction ◽  
Biochemical Properties ◽  
New Techniques ◽  
Vacuole Fusion ◽  
Low Concentrations ◽  
Reproducible Manner

To investigate the properties of phagolysosome (PL) fusion in Acanthamoeba homogenates, it was necessary to develop reliable methods for measuring in vitro PL fusion. The need to distinguish PL fusion from PL adhesion was met by the development of a quantitative electron microscope assay. Initial characterization of the fusion reaction by this method was followed by the development of a more rapid light microscope assay. Results obtained by the two methods were found to be in close agreement. By use of these new techniques, the in vitro PL fusion reaction was demonstrated to occur in a quantitatively reproducible manner. Under the present conditions employed, PL breakdown was not detected at any time during the in vitro incubation, while PL fusion was observed to proceed linearly for approximately 10 min, at which time the reaction ceased. Incubation of mixtures of two distinct PL types resulted in increases in hybrid PL types that were paralleled by decreases in nonhybrid PL types. The relative changes in PL concentrations observed were quantitatively consistent with PL fusion occurring randomly with respect to PL type. PL fusion was strongly inhibited by low concentrations of KF (50% inhibition at 2.7 mM), and by approximately tenfold higher concentrations of KCl, while KCN and 2,4-dinitrophenol (2,4-DNP) had little effect. In addition to further defining the nature of the PL fusion reaction in this system, these results demonstrate that, by use of the techniques described, quantitative study of the biochemical properties of this reaction is now possible.

scholarly journals Purification and characterization of NAD+:ADP-ribosyltransferase (polymerizing) from Dictyostelium discoideum

1993 ◽  
Vol 293 (1) ◽  
pp. 275-281 ◽  
Author(s):  
B Kofler ◽  
E Wallraff ◽  
H Herzog ◽  
R Schneider ◽  
B Auer ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Time Course ◽  
Affinity Purification ◽  
Tight Binding ◽  
Purification Scheme ◽  
Adp Ribosyltransferase ◽  
Dna Ends ◽  
Lower Eukaryote

A novel affinity-purification scheme based on the tight binding of NAD+:ADP-ribosyltransferase (polymerizing) [pADPRT; poly(ADP-ribose) polymerase; EC 2.4.2.30] to single-strand nicks in DNA, single-stranded patches and DNA ends has been developed to facilitate the purification of this enzyme from the lower eukaryote Dictyostelium discoideum. Two homogeneous forms of the enzyme, with M(r) values of 116,000 and 90,000, were prepared from D. discoideum by using poly(A) hybridized to oligo(dT)-cellulose as affinity material. The Km is 20 microM NAD+ for the 90,000-M(r) protein and 77 microM NAD+ for the 116,000-M(r) protein. The optimum conditions for the enzyme activity in vitro are 6-10 degrees C and pH 8. The time course is linear during the first 10 min of the reaction only. As in enzymes of higher eukaryotes, the activity is dependent on DNA and histone H1 and is inhibited by 3-methoxybenzamide, nicotinamide, theophylline, caffeine and thymidine.

scholarly journals Characterization of Streptococcus gordonii SecA2 as a Paralogue of SecA

2009 ◽  
Vol 191 (11) ◽  
pp. 3482-3491 ◽  
Author(s):  
Barbara A. Bensing ◽  
Paul M. Sullam
Keyword(s):  
Protein Transport ◽  
Maximal Activity ◽  
Biochemical Properties ◽  
Streptococcus Gordonii ◽  
Lower Solubility ◽  
Highly Sensitive ◽  
Sec System ◽  
High Degree

ABSTRACT The accessory Sec system of Streptococcus gordonii is essential for transport of the glycoprotein GspB to the bacterial cell surface. A key component of this dedicated transport system is SecA2. The SecA2 proteins of streptococci and staphylococci are paralogues of SecA and are presumed to have an analogous role in protein transport, but they may be specifically adapted for the transport of large, serine-rich glycoproteins. We used a combination of genetic and biochemical methods to assess whether the S. gordonii SecA2 functions similarly to SecA. Although mutational analyses demonstrated that conserved amino acids are essential for the function of SecA2, replacing such residues in one of two nucleotide binding folds had only minor effects on SecA2 function. SecA2-mediated transport is highly sensitive to azide, as is SecA-mediated transport. Comparison of the S. gordonii SecA and SecA2 proteins in vitro revealed that SecA2 can hydrolyze ATP at a rate similar to that of SecA and is comparably sensitive to azide but that the biochemical properties of these enzymes are subtly different. That is, SecA2 has a lower solubility in aqueous solutions and requires higher Mg2+ concentrations for maximal activity. In spite of the high degree of similarity between the S. gordonii paralogues, analysis of SecA-SecA2 chimeras indicates that the domains are not readily interchangeable. This suggests that specific, unique contacts between SecA2 and other components of the accessory Sec system may preclude cross-functioning with the canonical Sec system.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

The origin, evolution and diversification of multiple isoforms of light-dependent protochlorophyllide oxidoreductase (LPOR): focus on angiosperms

2020 ◽  
Vol 477 (12) ◽  
pp. 2221-2236
Author(s):  
Michal Gabruk ◽  
Beata Mysliwa-Kurdziel
Keyword(s):  
Biochemical Characterization ◽  
Duplication Event ◽  
Biochemical Properties ◽  
Protochlorophyllide Oxidoreductase ◽  
Independent Manner ◽  
Duplication Events ◽  
Multiple Species

Light-dependent protochlorophyllide oxidoreductase (LPOR) catalyzes the reduction of protochlorophyllide to chlorophyllide, which is a key reaction for angiosperm development. Dark operative light-independent protochlorophyllide oxidoreductase (DPOR) is the other enzyme able to catalyze this reaction, however, it is not present in angiosperms. LPOR, which evolved later than DPOR, requires light to trigger the reaction. The ancestors of angiosperms lost DPOR genes and duplicated the LPORs, however, the LPOR evolution in angiosperms has not been yet investigated. In the present study, we built a phylogenetic tree using 557 nucleotide sequences of LPORs from both bacteria and plants to uncover the evolution of LPOR. The tree revealed that all modern sequences of LPOR diverged from a single sequence ∼1.36 billion years ago. The LPOR gene was then duplicated at least 10 times in angiosperms, leading to the formation of two or even more LPOR isoforms in multiple species. In the case of Arabidopsis thaliana, AtPORA and AtPORB originated in one duplication event, in contrary to the isoform AtPORC, which diverged first. We performed biochemical characterization of these isoforms in vitro, revealing differences in the lipid-driven properties. The results prone us to hypothesize that duplication events of LPOR gave rise to the isoforms having different lipid-driven activity, which may predispose them for functioning in different locations in plastids. Moreover, we showed that LPOR from Synechocystis operated in the lipid-independent manner, revealing differences between bacterial and plant LPORs. Based on the presented results, we propose a novel classification of LPOR enzymes based on their biochemical properties and phylogenetic relationships.

scholarly journals The Glutathione/Glutaredoxin System Is Essential for Arsenate Reduction in Synechocystis sp. Strain PCC 6803

2009 ◽  
Vol 191 (11) ◽  
pp. 3534-3543 ◽  
Author(s):  
Luis López-Maury ◽  
Ana María Sánchez-Riego ◽  
José Carlos Reyes ◽  
Francisco J. Florencio
Keyword(s):  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Arsenate Reductase ◽  
Arsenic Resistance ◽  
Arsenate Resistance ◽  
Mutant Strains ◽  
Pcc 6803

ABSTRACT Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes in which arsC codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in Synechocystis sp. strain PCC 6803, which we have designed arsI1 and arsI2, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in Synechocystis sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the grxA gene have impaired arsenic resistance.

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