scholarly journals A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8

1990 ◽  
Vol 271 (3) ◽  
pp. 797-801 ◽  
Author(s):  
B C Beaubien ◽  
P D Collins ◽  
P J Jose ◽  
N F Totty ◽  
J Hsuan ◽  
...  
Keyword(s):  
Peritoneal Cavity ◽  
Inflammatory Reaction ◽  
Gel Filtration ◽  
Reversed Phase ◽  
Interleukin 8 ◽  
Terminal Sequence ◽  
Rabbit Skin ◽  
Sds Page

An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

Crotalin, a vWF and GP Ib Cleaving Metalloproteinase from Venom of Crotalus atrox

2001 ◽  
Vol 86 (12) ◽  
pp. 1501-1511 ◽  
Author(s):  
Wen-Bin Wu ◽  
Hui-Chin Peng ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Adhesion ◽  
Early Stage ◽  
Gel Filtration ◽  
Platelet Glycoprotein ◽  
Antithrombotic Effect ◽  
Binding Motifs ◽  
Sds Page ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryBinding of von Willebrand factor (vWF) to a variety of extracellular matrix (ECM) components and to platelet glycoprotein (GP) Ib-IX-V complex is important in mediating platelet adhesion and aggregation in the early stage of hemostasis. We previously purified a potent antithrombotic protein, named crotalin, functionally acting as a GP Ib antagonist (1). In this study, we further characterized crotalin as a P-I metalloproteinase with a molecular mass of 25 kDa as determined by gel filtration and two-dimensional SDS-PAGE. Crotalin is a vWF binding and cleaving metalloproteinase. In addition, crotalin cleaved platelet GP Ib as judged by flow cytometry and Western blotting. The multiple effects of crotalin on vWF and platelet GP Ib antagonized ristocetin-, but not collagen and thrombin-induced platelet aggregation, suggesting that its effect is specific. We also found that crotalin auto-proteolytically degraded to ~14 and ~10 kDa fragments in the presence of SDS. Interestingly, both degradation fragments, intact and reduced crotalin were able to bind vWF, suggesting the binding of crotalin to vWF is conformation-independent. In conclusion, the results presented further explain the potent antithrombotic effect of crotalin in vivo. In addition, the multiple effects of crotalin may be used as a tool to determine the binding motifs that are responsible for the vWF-ECMs or vWF-GP Ib interaction.

scholarly journals Isolation and Characterization of Two Proteins fromMoraxella catarrhalis That Bear a Common Epitope

1998 ◽  
Vol 66 (9) ◽  
pp. 4374-4381 ◽  
Author(s):  
John C. McMichael ◽  
Michael J. Fiske ◽  
Ross A. Fredenburg ◽  
Deb N. Chakravarti ◽  
Karl R. VanDerMeid ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Bacterial Attachment ◽  
Vaccine Candidates ◽  
Isolation And Characterization ◽  
Sds Page

ABSTRACT The UspA1 and UspA2 proteins of Moraxella catarrhalisare potential vaccine candidates for preventing disease caused by this organism. We have characterized both proteins and evaluated their vaccine potential using both in vitro and in vivo assays. Both proteins were purified from the O35E isolate by Triton X-100 extraction, followed by ion-exchange and hydroxyapatite chromatography. Analysis of the sequences of internal peptides, prepared by enzymatic and chemical cleavage of the proteins, revealed that UspA1 and UspA2 exhibited distinct structural differences but shared a common sequence including an epitope recognized by the monoclonal antibody 17C7. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), purified UspA1 exhibited a molecular weight of approximately 350,000 when unheated and a molecular weight of 100,000 after being heated for 10 min at 100°C. In contrast, purified UspA2 exhibited an apparent molecular weight of 240,000 by SDS-PAGE that did not change with the length of time of heating. Their sizes as determined by gel filtration were 1,150,000 and 830,000 for UspA1 and UspA2, respectively. Preliminary results indicate the proteins have separate functions in bacterial pathogenesis. Purified UspA1 was found to bind HEp-2 cells, and sera against UspA1, but not against UspA2, blocked binding of the O35E isolate to the HEp-2 cells. UspA1 also bound fibronectin and appears to have a role in bacterial attachment. Purified UspA2, however, did not bind fibronectin but had an affinity for vitronectin. Both proteins elicited bactericidal antibodies in mice to homologous and heterologous disease isolates. Finally, mice immunized with each of the proteins, followed by pulmonary challenge with either the homologous or a heterologous isolate, cleared the bacteria more rapidly than mock-immunized mice. These results suggest that UspA1 and UspA2 serve different virulence functions and that both are promising vaccine candidates.

TUMOR-ASSOCIATED FIBRINOLYSIS IN OVARIAN CARCINOMA - HPLC AND N-TERMINAL AMINO ACID ANALYSIS REVEAL THE PATHWAY OF DEGRADATION OF CROSSLINKED FIBRIN

1987 ◽  
Author(s):  
O Wilhelm ◽  
A Henschen ◽  
R Hafter ◽  
H Graeff
Keyword(s):  
Ovarian Cancer ◽  
Ovarian Carcinoma ◽  
Ascitic Fluid ◽  
High Performance ◽  
Degradation Products ◽  
Gel Filtration ◽  
Advanced Ovarian Cancer ◽  
Reversed Phase ◽  
Fibrin Degradation Products ◽  
Sds Page

Crosslinked fibrin has been demonstrated by immunohistochemi-cal tests to occur around tumor plugs, on the surface and in the stroma of the tumor in ovarian cancer. High levels of D-Dimer (200-800μg/ml), the characteristic terminal degradation product of crosslinked fibrin, are found in ascitic fluid of patients with advanced ovarian cancer. These findings suggest that fibrin polymerisation and degradation are related to and even may influence tumor growth. The kind of proteases which are responsible for degradation of crosslinked fibrin is, however, unknown.lt was the aim of this study to evaluate whether plasmin and/or other proteases are involved in tumor-associated fibrinolysis. Therefore the total high-molecular-weight fibrin degradation products in ascitic fluid were purified by protamine sulfate precipitation, gel filtration, immunoadsorption and compared with the components of plasmin-degraded crosslinked fibrin, i.e. DD,DY,YX,DXD and DXY, by direct SDS-PAGE in the absence of mercaptoethanol and after excision of the bands, mercaptolysis and re-electrophoresis. Pronounced similarity between the two sets of fragments was observed. For further information the fragments from the two sources were mercaptolysed and their polypeptide chain components separated by reversed-phase high-performance liquid chromatography, the components being identified by N-terminal sequence analysis and SDS-PAGE. Highly similar patterns were obtained and components corresponding to γ-γ ,γ-γ1, β, β2 and α1 could be recognized. The findings provide strong evidence for plasmin being the primary protease involved in ovarian carcinoma-related fibrinolysis, (supported by Deutsche Forschungsgemeinschaft.SFB 207, A2).

scholarly journals l(+)-Mandelate dehydrogenase from Rhodotorula graminis: purification, partial characterization and identification as a flavocytochrome b

1993 ◽  
Vol 293 (2) ◽  
pp. 455-460 ◽  
Author(s):  
M Yasin ◽  
C A Fewson
Keyword(s):  
Feedback Inhibition ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Maximum Activity ◽  
British Library ◽  
Terminal Sequence ◽  
Sds Page ◽  
Competitive Inhibitors ◽  
Lactate Dehydrogenases ◽  
Flavocytochrome B

L(+)-Mandelate dehydrogenase was purified to homogeneity from the yeast Rhodotorula graminis KGX 39 by a combination of (NH4)2SO4 fractionation, ion-exchange and hydrophobic-interaction chromatography and gel filtration. The amino-acid composition and the N-terminal sequence of the enzyme were determined. Comprehensive details of the sequence determinations have been deposited as Supplementary Publication SUP 50172 (4 pages) at the British Library Document Supply Centre, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1993) 289, 9. The enzyme is a tetramer as judged by comparison of its subunit M(r) value of 59,100 and native M(r) of 239,900, estimated by SDS/PAGE and gel filtration respectively. There is one molecule of haem and approx. one molecule of non-covalently bound FMN per subunit. 2,6-Dichloroindophenol, cytochrome c and ferricyanide can all serve as electron acceptors. L(+)-Mandelate dehydrogenase is stereospecific for its substrate. D(-)-Mandelate and L(+)-hexahydromandelate are competitive inhibitors. The enzyme has maximum activity at pH 7.9 and it has a pI value of 4.4. HgCl2 and 4-chloromercuribenzoate are potent inhibitors, but there is no evidence that the enzyme is subject to feedback inhibition by potential metabolic effectors. The evidence suggests that L(+)-mandelate dehydrogenase from R. graminis is a flavocytochrome b which is very similar to, and probably (at least so far as the haem domain is concerned) homologous with, certain well-characterized yeast L(+)-lactate dehydrogenases, and that the chief difference between them is their mutually exclusive substrate specificities.

scholarly journals Elevation of interleukin-6 in response to a chronic inflammatory stimulus in mice: inhibition by indomethacin

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 194-202 ◽  
Author(s):  
E Shacter ◽  
GK Arzadon ◽  
J Williams
Keyword(s):  
Plasma Cell ◽  
Peritoneal Cavity ◽  
Interleukin 6 ◽  
Chronic Administration ◽  
Inflammatory Cells ◽  
Serum Levels ◽  
Chronic Inflammatory Response

Abstract Intraperitoneal (i.p.) injection of a mineral oil such as pristane induces a chronic inflammatory response in mice. This is characterized by a large influx of macrophages and other inflammatory cells into the peritoneal cavity for months after injection of the oil. By using the B9 cell bioassay, it was found that injection of pristane caused a marked and prolonged elevation of interleukin-6 (IL-6) levels in the peritoneal cavities of the mice. IL-6 was undetectable (less than 15 U/mL) in the peritoneal fluids of unprimed mice and during the first week after injecting pristane. From 4 to 20 weeks, the concentration of IL-6 increased to an apparent plateau with concentrations ranging from 200 to 2,000 U/mL. Increasing the dose of pristane did not substantially increase the peritoneal levels of IL-6 established at 20 weeks after pristane treatment. At later times (by day 250), the level decreased to 263 +/- 217 U/mL. However, mice that developed plasma cell tumors around day 300 showed high levels of IL-6 in the ascites fluid (650 to 2,400 U/mL). Serum levels of IL-6 were also elevated in pristane-primed mice but were substantially lower than those found in the peritoneal cavity. Chronic administration of the nonsteroidal anti- inflammatory drug indomethacin decreased the levels of IL-6 by 75% to 80%. Experiments performed in vitro showed that pristane-elicited macrophages secreted low levels of IL-6 constitutively and high levels of IL-6 in the presence of lipopolysaccharide. Both IL-6 and prostaglandin E2 production were inhibited by addition of indomethacin to macrophage cultures in vitro. Treatment of mice with pristane may provide a model system for studying the inflammatory pathways that control IL-6 levels in vivo. The relevance of these results to elucidation of the role of IL-6 in plasma cell tumorigenesis is discussed.

Favorable effects in vitro and in vivo of two clinical isolates of Pseudomonas aeruginosa on nutritionally deficient Staphylococcus aureus strains

1973 ◽  
Vol 19 (8) ◽  
pp. 973-981 ◽  
Author(s):  
T. Gadbois ◽  
J. De Repentigny ◽  
L. G. Mathieu
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
Peritoneal Cavity ◽  
Pathogenic Bacteria ◽  
Bacterial Species ◽  
Mixed Cultures ◽  
Rabbit Skin ◽  
Metabolic Activities

We have studied aspects of interbacterial ecology with nutritionally dependent Staphylococcus aureus strains; they were grown in association with Pseudomonas aeruginosa in systems of mixed cultures and infections in vitro in a semisynthetic medium and in vivo in mouse peritoneal cavity and rabbit skin. In mixed cultures and in P. aeruginosa culture filtrates, thymine and tryptophan deficiencies in staphylococci were partly overcome. This is probably because P. aeruginosa supplied the essential metabolites required to ensure growth; however, other metabolic activities could also be involved. Other experiments showed that the sensitivity of thymineless staphylococci to nucleoside inhibitions was alleviated. In mixed infections with P. aeruginosa, the S. aureus thymineless strain has shown a greater ability to survive in the peritoneal cavity of mice than when injected alone, even when one species was injected after the other with different doses of bacteria. The examination of the liquid from the peritoneal cavity of infected mice by fluorescence microscopy after fluorochroming with acridine orange or auramine O has revealed that Pseudomonas endotoxin seems to damage leucocytes and consequently reduces the phagocytosis of Staphylococcus cells.Necrosis in rabbit skin was mainly due to S. aureus when both species were injected together intradermally; the thymineless strain was less harmful than the parent strain.It seems that survival and even growth of nutritionally dependent strains of a bacterial species can be favored by the metabolic activities of another species in mixed cultures and infections, in this instance S. aureus by P. aeruginosa. This phenomenon among others could be a determinant of bacterial pathogenicity for nutritionally dependent pathogenic bacteria; thus associated organisms could determine the effective pathogenicity of nutritionally dependent bacteria by contributing essential nutrilites at the site where infection is initiated.

scholarly journals The catalytic chain of human complement subcomponent C1. Purification and N-terminal amino acid sequences of the major cyanogen bromide-cleavage fragments

1982 ◽  
Vol 201 (1) ◽  
pp. 49-59 ◽  
Author(s):  
G J Arlaud ◽  
J Gagnon ◽  
R R Porter
Keyword(s):  
High Pressure ◽  
Amino Acid ◽  
Gel Filtration ◽  
Gel Permeation Chromatography ◽  
Reversed Phase ◽  
Amino Acid Sequences ◽  
Serine Proteinases ◽  
Human Complement ◽  
Terminal Sequence ◽  
Cnbr Cleavage

1. The a- and b-chains of reduced and alkylated human complement subcomponent C1r were separated by high-pressure gel-permeation chromatography and isolated in good yield and in pure form. 2. CNBr cleavage of C1r b-chain yielded eight major peptides, which were purified by gel filtration and high-pressure reversed-phase chromatography. As determined from the sum of their amino acid compositions, these peptides accounted for a minimum molecular weight of 28 000, close to the value 29 100 calculated from the whole b-chain. 3. N-Terminal sequence determinations of C1r b-chain and its CNBr-cleavage peptides allowed the identification of about two-thirds of the amino acids of C1r b-chain. From our results, and on the basis of homology with other serine proteinases, an alignment of the eight CNBr-cleavage peptides from C1r b-chain is proposed. 4. The residues forming the ‘charge-relay’ system of the active site of serine proteinases (His-57, Asp-102 and Ser-195 in the chymotrypsinogen numbering) are found in the corresponding regions of C1r b-chain, and the amino acid sequence around these residues has been determined. 5. The N-terminal sequence of C1r b-chain has been extended to residue 60 and reveals that C1r b-chain lacks the ‘histidine loop’, a disulphide bond that is present in all other known serine proteinases.

scholarly journals Purification and partial characterization of a peroxidase from plant cell cultures of Cassia didymobotrya and biotransformation studies1

1998 ◽  
Vol 331 (2) ◽  
pp. 513-519 ◽  
Author(s):  
Alberto VITALI ◽  
Bruno BOTTA ◽  
Giuliano DELLE MONACHE ◽  
Sabrina ZAPPITELLI ◽  
Paola RICCIARDI ◽  
...  
Keyword(s):  
Cell Wall ◽  
Culture Medium ◽  
Gel Filtration ◽  
Cell Suspension Cultures ◽  
High Specificity ◽  
Coniferyl Alcohol ◽  
Terminal Sequence ◽  
Sds Page ◽  
Plant Peroxidases

An acidic peroxidase (EC 1.11.1.7) produced by cell suspension cultures of Cassia didymobotrya(wild senna) was purified from culture medium collected on the 29th day. The enzyme was shown to be a glycoprotein with a pI of 3.5, a molecular mass of approx. 43 kDa by SDS/PAGE and 50 kDa by gel filtration. The N-terminal sequence was very similar to those of other plant peroxidases. The peroxidase was characterized by a high specificity towards coniferyl alcohol and other natural phenolics such as guaiacol and ferulic and caffeic acids. These findings suggest that the enzyme is involved in lignification processes of the cell wall. Moreover, the enzyme was able to catalyse the oxidation of 4,3´,4´-trihydroxychalcone and 4,3´,4´-trihydroxy-3-methoxychalcone to the corresponding 3,3´-biflavanones, as mixtures of racemic and mesoforms.

Lysosomes and the "toxicity" of Rickettsiales. V. In vivo relationship of peritoneal phagocytes and egg-attenuated C. psittaci 6BC

1973 ◽  
Vol 19 (11) ◽  
pp. 1417-1423 ◽  
Author(s):  
Nonna Kordová ◽  
John C. Wilt ◽  
Linda Poffenroth
Keyword(s):  
Acid Phosphatase ◽  
Peritoneal Cavity ◽  
Inflammatory Reaction ◽  
Chronic Infection ◽  
Epitheloid Cells ◽  
Intraperitoneal Infection ◽  
Inhibitory Activities ◽  
Relationship Of

The investigations reported here follow the lines of our earlier studies and represent an attempt to identify the inhibitory activities of the lysosomes of mononuclear and polymorphonuclear phagocytes (PMN's) in the peritoneal cavity of mice after intraperitoneal infection with an egg-attenuated C. psittaci 6BC strain (avirulent for mice). Presumptive evidence indicates that lysosomes of macrophages are refractory to "nontoxic" chlamydial particles that are initially ingested, while the adsorption to, and (or) uptake of, these agents by PMN's is followed by disintegration of PMN's which causes release of lytic substances from them. Macrophages infected in vivo are nonspecifically "activated" and some of them "transform" after maintenance in vitro into large epitheloid cells containing numerous well-demarcated chlamydial inclusions in the cytoplasm. Except for the inclusions neither damage nor lysosomal acid phosphatase release was detected in these cells. The Chlamydiae-induced inflammatory reaction and the possible persistence of "nontoxic" Chlamydiae in chronic infection are discussed.

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