PubMed Health⌕ Search

Biomedical subjects

S Wasi

Publications and source records attributed to S Wasi.

At least 37 records · Page 2Linked to original sources
PubMed1989

An enzyme immunoassay for the determination of low levels of IgA in human serum.

An enzyme immunoassay (EIA) has been developed which facilitates the detection of low levels of immunoglobulin A in human serum. IgA is captured by an anti-IgA antibody linked to micron-sized polyacrylamide beads and subsequently detected by an anti-IgA horseradish peroxidase conjugate. The standard curve is linear in the region between 25-1000 ng/ml IgA. The assay is particularly suited to measure IgA antibodies in sera from IgA-deficient individuals and IgA contaminants in blood products.

Animals↗
PubMed1989

A rapid procedure for the purification of IgA1 and IgA2 subclasses from normal human serum using protein G and jackfruit lectin (jacalin) affinity chromatography.

Immunoglobulin A (IgA) from pooled normal human sera was purified using antibody and protein G affinity chromatography and gel filtration high-pressure liquid chromatography (HPLC). This high purity product was separated into IgA1 and IgA2 subclasses utilizing the agarose-bound lectin 'jacalin'. Evaluation of product homogeneity by immunological testing confirmed greater than 95% purity. The total IgA1 and IgA2 recovered from sera was approximately 26% of the initial antibody present.

Chromatography, Affinity↗
PubMed1988

Characterization of porcine osteonectin extracted from foetal calvariae.

Osteonectin, extracted from foetal porcine calvariae with 0.5 M-EDTA, was purified to homogeneity by using gel filtration and polyanion anion-exchange fast protein liquid chromatography under dissociative conditions without the need of reducing agents. The purified protein migrated with an Mr of 40,300 on SDS/polyacrylamide gels and was similar to bovine osteonectin in both amino acid composition and in its ability to bind to hydroxyapatite in the presence of 4 M-guanidinium hydrochloride (GdmCl). However, unlike the bovine protein, porcine osteonectin did not bind selectively to hydroxyapatite when EDTA tissue extracts were used. In addition, purified porcine osteonectin did not show any apparent affinity for either native or denatured type I collagen, but did bind to serum albumin. Primary sequence analysis revealed an N-terminal alanine residue, with approximately one-half of the subsequent 35 residues identified as small hydrophobic amino acids and one-quarter as acidic amino acids. The only significant difference between the N-terminal sequences of the bovine and porcine proteins was the deletion of the tripeptide Val-Ala-Glu in porcine osteonectin. In contrast with bovine osteonectin, far-u.v.c.d. of porcine osteonectin revealed considerable secondary structure, of which 27% was alpha-helix and 39% was beta-sheet. Cleavage of the molecule with CNBr under non-reducing conditions generated five fragments, of which two major fragments (Mr 27,900 and 12,400) stained blue with Stains All, a reagent that stains sialic-acid-rich proteins/phosphate-containing proteins and/or Ca2+-binding proteins blue while staining other proteins pink. The 12,400-Mr fragment bound 45Ca2+ selectively, indicating a Ca2+-binding site in this part of the molecule. The 27,900-Mr fragment did not bind Ca2+, and since biosynthetic studies with 32PO4(3-) did not show phosphorylation of porcine osteonectin, this fragment is likely to be highly acidic. The incomplete cleavage of the molecule with CNBr and the ability of the molecule to regain its secondary structure after exposure to 7 M-urea are features consistent with the molecule having a compact structure that is stabilized by numerous disulphide bridges. The chemical and binding properties of porcine osteonectin are closely similar to the recently described 'culture shock', SPARC and BM-40 proteins, indicating that these are homologous proteins.

Amino Acid Sequence↗
PubMed1987

Characterization of a monoclonal antibody recognizing small collagenous proteins in fetal bone.

A monoclonal antibody (MBP-322) that recognizes two small collagenous, apatite-binding (SCAB) proteins associated with the mineral phase of fetal bone, has been prepared. The SCAB proteins, which are quantitatively extracted from bone with EDTA, have been shown by immunotransfer analyses to have MrS of 28K and 25K and both were selectively degraded by bacterial collagenase. Amino acid analysis of the collagenase-digested protein revealed a hypro:pro:gly ratio of approximately 0.5:1:0.7 for both proteins and indicated that one-third of the protein could have a collagen-like sequence. The SCAB proteins, unlike other collagens and collagen fragments tested bound quantitatively to hydroxylapatite in the presence of 4M guanidine hydrochloride and appear to be unique to bone. The antibody, however, was not specific for the SCAB proteins and showed comparable immunoreactivity against denatured alpha 1 chains of types I, II and III collagens and the alpha 2 chains of types I and V collagens but not type IV collagen nor native collagens I-V. The epitope was further localized to the CB6 fragment in the alpha 1(I) chain and the CB5 fragment of alpha 1(III) chain, and was present in both the TCA and TCB fragments of alpha 2(I). Despite the immunological reactivity, the properties of the SCAB proteins were not consistent with their being derived from known collagen types. Immunocytochemical staining of permeabilized bone cells with MPB-322 showed a perinuclear, punctate staining pattern in most cells with some cells showing specific nuclear staining. In non-permeabilized cells, the antibody stained various sized spherical particles, many of which were closely associated with the cell surface. Immunoblots of cell proteins revealed a number of immunoreactive proteins sensitive to collagenase digestion including two proteins with MrS similar to the SCAB proteins. The MBP-322 antibody appears useful for identifying sequence homology in various collagens, and for recognizing denatured collagen and specific collagen fragments in tissues, as well as being important for the further characterization of the SCAB proteins.

Amino Acids↗
PubMed1986

Osteonectin is a minor component of mineralized connective tissues in rat.

Osteonectin is a major glycoprotein of porcine and bovine bones and teeth that is found associated with hydroxylapatite crystal surfaces. From the ability of osteonectin to bind calcium ions, it has been proposed as a possible nucleator of hydroxylapatite crystal formation. Analysis of hydroxylapatite-bound proteins of rat bone and dentine, however, has revealed that osteonectin represents only 2.5 +/- 1.5% of the hydroxylapatite-bound protein in long bones, 0.9 +/- 0.5% in calvariae, and less than 0.1% in incisor dentine of animals of different ages. Further, in vivo pulse-chase studies carried out in young adult rats have shown osteonectin to be synthesized at low levels in these tissues. Similarly, low levels of osteonectin were synthesized by rat calvarial cells in vitro. In contrast, fibroblastic cells from periodontal ligament and gingiva synthesized significantly greater amounts of osteonectin. These studies indicate that the low quantities of osteonectin in rat mineralized tissues are a consequence of low rates of formation rather than being due to rapid turnover. The virtual absence of osteonectin in incisor dentine correlates with the lack of peritubular dentine in rat, whereas the low osteonectin content of rat bones may reflect differences in their structure and biophysical properties compared with bones of larger mammals.

Animals↗
PubMed1985

Cell adhesion to low-Mr proteins extractable from mineralized and soft connective tissues.

Guanidinium chloride (4 M) containing proteinase inhibitors was used to extract proteins from porcine calvariae and long bones. The extracted proteins were separated on polyacrylamide slab gels and transferred electrophoretically to nitrocellulose strips. Proteins with cell-adhesion properties were identified by incubating the strips with cells and staining with Amido Black. In addition to binding to fibronectin, both bone cells and fibroblast-like cells adhered to proteins of Mr approximately 30 000 and approximately 14 000-17 000. 4 M-Guanidinium chloride extracts of porcine skin and gingiva yielded cell-binding proteins with similar Mr values. These data suggest that these low-Mr proteins may have a general cell-adhesion function in both soft and mineralized connective tissues.

Animals↗
PubMed1985

Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues.

Affinity-purified antibodies have been used in combination with the peroxidase-antiperoxidase technique to study the distribution of osteonectin and collagen types I and III in porcine dental tissues. Tissue sections (2 mm thick), including unerupted (fetal) or erupted (adult) teeth, were fixed in periodate-lysine-paraformaldehyde, demineralized in 12% w/v ethylenediaminetetraacetic acid, and after embedding, 6 micron sections were prepared for immunolocalization. Strong staining for osteonectin was observed in dentine of unerupted teeth and in the associated alveolar bone. Light to moderate staining was observed in the dental pulp, stratum intermedium, stellate reticulum, and the reticular elements in the endosteal spaces. In erupted teeth, osteonectin staining in dentine was concentrated around dentinal tubules and the associated alveolar bone stained with variable intensity. Cementum was poorly stained. However, the periodontal ligament and reticular material in the endosteal spaces showed moderate to strong staining. Weaker staining was apparent in the pulp and lamina propria of the gingiva. In comparison, type I collagen showed a similar distribution to osteonectin in both fetal and adult tissues, whereas type III collagen was generally restricted to the periodontal ligament, reticular elements of the endosteal spaces, and Sharpey's fibers in bone and cementum. Both odontoblast and ameloblast layers in fetal tissues stained for osteonectin and type III collagen.

Animals↗
PubMed1985

Severe microvascular injury induced by lysosomal releasates of human polymorphonuclear leukocytes. Increase in vasopermeability, hemorrhage, and microthrombosis due to degradation of subendothelial and perivascular matrices.

The purpose of this study was to assess the nature of the lesions in the microcirculation of the dermis of rabbits induced with lysosomal releasates of human polymorphonuclear leukocytes (PMNs). No attempt was made in the studies presented in this publication to deal with the offending agent in the releasate. Four parameters of microvascular injury were quantitated: increase in vascular permeability with 125I-labeled serum albumin, hemorrhage with 59Fe-labeled erythrocytes, accumulation (aggregation) of platelets with 111In-labeled platelets. In one experiment accumulation of 51Cr-PMNs was investigated. The lysosomal releasate induced a rapid increase in vasopermeability, but both hemorrhage and exudate formation peaked 1 hour after intradermal injection. Platelet accumulation was also demonstrable in these lesions, and microthrombosis was a very prominent feature. The microvascular injury, including microthrombosis, could be elicited also in animals rendered leukopenic with nitrogen mustard. Simultaneous injection of prostaglandin E2 with the releasate enhanced the microvascular injury. The morphologic changes in the microcirculation of the rabbit's dermis were assessed in lesions 5 minutes to 5 hours old. Several changes were encountered, primarily in the wall of venules and small veins and to a lesser degree in small arteries and capillaries. Ultrastructurally very early lesions (up to 15 minutes) had gaps or spaces in the endothelium, resembling those induced by mediators such as histamine or bradykinin. Older lesions were different, quite characteristic, and represent the hallmark of these lesions. Lysis and disappearance of vascular basement membrane, of perivascular collagen, and of the internal elastic lamina were a frequent finding, best demonstrable when microthrombi did not abut on vessel walls. Cellular components of vessels (endothelium, pericytes, smooth muscle) showed fragmentation, leading to complete disappearance of cellular elements. These lesions were usually walled off by platelet aggregates and fibrin. At times microthrombi occluded an entire vessel. These changes were interpreted as hemostasis. The mild accumulation of PMNs at the site of injury did not contribute significantly to the microvascular injury. The findings indicate that the unique changes in the microcirculation, not described before, may occur quite frequently, when the microvascular injury is elicited primarily by release of lysosomal constituents by phagocytic or nonphagocytic stimuli. One can conclude that the hallmark of this type of injury is disappearance of basement membrane followed secondarily by disintegration of the vascular wall, followed in turn by hemo

Animals↗
PubMed1984

Biosynthesis of osteonectin by fetal porcine calvarial cells in vitro.

The biosynthesis of osteonectin, a major glycoprotein of bone, has been studied in vitro using bone cells from fetal porcine calvariae. The calvarial cells, which were shown to produce osteonectin by immunotransfer and immunocytochemical analysis, were pulse labeled with [35S]methionine and the radiolabeled osteonectin in the cell layers and in the chase-medium was isolated by specific immunoprecipitation. The osteonectin, representing 0.1% of the radiolabeled cell layer and 1% of the medium proteins, co-migrated with authentic bovine osteonectin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reduced and nonreduced conditions (Mr approximately 40,000), and was bound selectively to hydroxyapatite in the presence of 4 M guanidine HCl. The co-migration of the osteonectins indicates that the tissue-extracted osteonectin represents an intact protein and that a pro- form of osteonectin is unlikely. However, a preosteonectin form (Mr approximately 46,000) could be immunoprecipitated from cell-free translation of calvarial mRNA. The minimal secretion time for osteonectin synthesized in serum-containing medium was found to be approximately 40 min but maximal accumulation did not occur until 2 h postlabeling. In serum-free media, extensive degradation of osteonectin, characterized by the presence of two immunoprecipitable fragments (Mr approximately 36,000 and 31,000), was evident.

Animals↗
PubMed1984

An osteonectinlike protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts.

Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine-HCl (GuHCl) and 4 M GuHCl - 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS-PAGE with a relative mass approximately 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented approximately 0.5% of the total labelled proteins secreted. It comigrated on SDS-PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.

Animals↗
PubMed1983

Plasminogen activator and thromboplastin activity from sheep alveolar macrophages.

Alveolar lavage cells from normal sheep were found to be composed of over 95% macrophages. When the cells were cultured, fibrinolytic and thromboplastin-like activities could be detected within 2-4 hours of incubation. As the number of cultured cells was increased the two activities in the conditioned medium increased proportionately. The cells were separated into two distinct subpopulations by means of a sedimentation velocity cell fractionation technique. The macrophage subpopulations were examined for differences in size, morphology, esterase staining and ability to release plasminogen activator and procoagulant activity respectively. These activities were confined to the large cell subpopulation. The fibrinolytic activity was shown to be plasminogen-dependent and could be inhibited by DFP. On the basis of this the fibrinolytic activity has been designated as plasminogen activator. The procoagulant activity was shown to be thromboplastin in nature because it was Factor VII dependent, inactivated by phospholipase C and not inhibited by DFP. The procoagulant activity has been designated as macrophage thromboplastin. The two activities could be distinguished on the basis of DFP inhibition.

Animals↗
PubMed1982

The production of plasminogen activator by afferent but not efferent lymph cells emigrating from chronic granulomatous lesions in sheep.

Subcutaneous granulomas were induced in sheep by injecting Freund's adjuvant. At varying times thereafter afferent and efferent lymphatics draining the granulomas were cannulated. Lymph was collected for periods up to several days or weeks from either normal skin or from inflammatory lesions 15 to 120 days after the initiation of the lesions. The fibrinolytic activity of cells and cell culture medium in which cells were grown was assayed by using 125I-fibrin plates. No detectable enzyme activity was found from normal afferent, normal efferent, or stimulated efferent lymph cells. Afferent granuloma cells produced a plasminogen-dependent, DFP inhibitable protease activity that was dependent on the cell concentration and the incubation time. Results of cell separation techniques such as sedimentation velocity, adherence on fibrin plates, and Sephadex G-10 fractionation showed that the large, macrophage-like cell population in afferent lymph was secreting the plasminogen activator. The lymph cells collected from lesions 1 to 2-mo-old were more active than an equivalent number of cells collected from earlier or later lesions. When tuberculin was injected directly into lesions the lymph cells that appeared in the subsequent 1 to 2 days produced more enzyme per cell. The relationship between plasminogen activator and other possible mediators that appear in either the cells or plasma of afferent lymph is discussed.

Animals↗
PubMed1978

An aminopeptidase of human neutrophil leucocytes--its possible role in enhanced vascular permeability.

An aminopeptidase of human leukocyte lysosomes was partially purified by chromatography on SP-Sephadex, Sephadex G-200 and QAE-Sephadex. By QAE-Sephadex and isolelectric focusing it showed microheterogeneity, focusing at pH 3.7 and 4.1. By gel filtration its molecular weight was estimated to be approx. 200,000. The enzyme had leucine amino peptidase activity and pharmacological assays indicated that it converted lysyl- or methionyl-lysyl-bradykinin to bradykinin. Conversion of lysyl-bradykinin to bradykinin could be confirmed also by chromatography on CM-cellulose. When the neutrophil-derived enzyme acted on methionyl-lysyl-bradykinin it increased its effect of enhancing vascular permeability, when injected intradermally into guinea pits. Thus the enzyme may play a role in neutrophil leukocyte-mediated vascular phenomena of the inflammatory reaction.

Aminopeptidases↗