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PubMed2005

A matrix isolation study of the photochemically induced reactions of nitrogen dioxide with 1,2-dibromoethene and 1,2-dichloroethene using Fourier transform infrared spectroscopy.

Photolyses of matrices of either BrCHCHBr/NO2/Ar or ClCHCHCl/NO2/Ar using quartz-filtered radiation (lambda>240 nm) led to the appearance of infrared bands attributable to carbonyl, carbon monoxide, and ketene species; no bands belonging to a precursor complex NO2cdots, three dots, centeredXCHCHX (where X=Br or Cl) were observed upon matrix deposition. The possible reaction pathway is discussed.

Bromine↗
PubMed1996

Evidence for load-dependent and load-independent determinants of cardiac natriuretic peptide production.

BACKGROUND: In hypertension with cardiac hypertrophy, the specific contributions to increased production of the cardiac natriuretic peptides (NP) atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) by load and the hypertrophic process are not known. In the present work we determine ANF and BNP synthesis and secretion in the aortic-banded rat treated with dosage schedules of the ACE inhibitor ramipril that result in the prevention or regression of both hypertension and hypertrophy (high dosage) or in the prevention or regression of hypertrophy alone with persistent hypertension (low dosage). Myosin heavy chain (MHC) isoform switch was studied as an indicator of ventricular cardiocyte hypertrophy as well as the levels of collagen III mRNA as a measure of changes in extracellular matrix. METHODS AND RESULTS: Ramipril was administered for 6 weeks just after suprarenal aortic banding, or rats were banded for 6 weeks, after which ramipril was administered during the following 6 weeks. Banding caused an increase in blood pressure, left ventricular weight-to-body weight ratio, plasma and ventricular NP, ventricular NP mRNA, collagen III, and beta-MHC mRNA. Ramipril at 1 mg/kg normalized all these parameters while ramipril at 10 micrograms/kg normalized left ventricular weight-to-body weight ratio but not blood pressure. Plasma and ventricular NP content and mRNA levels were partially normalized by ramipril (10 micrograms/kg). Ramipril (10 micrograms/kg) prevented increased collagen III mRNA levels but did not affect beta-MHC mRNA levels. CONCLUSIONS: (1) NP production and secretion in aortic-banded rats are independently related to increased blood pressure and hypertrophy. (2) A load-dependent component is more important than a load-independent component in regulating left ventricular NP production. (3) ANF production is more sensitive than BNP production to the load-independent component. (4) Low-dose ramipril treatment reverses hypertrophy and the increased collagen III expression but does not reverse the increased beta-MHC isoform expression, suggesting that these are independently regulated processes. (5) Aortic banding and ACE inhibition do not affect atrial NP production and content.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed2001

Integration of matrix-assisted laser desorption ionization-time of flight mass spectrometry and molecular cloning for the identification and functional characterization of mobile ortho-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2.

Protein mass spectrometry and molecular cloning techniques were used to identify and characterize mobile o-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2 and Pseudomonas huttiensis strain D1. Proteins induced in strains JB2 and D1 by growth on 2-chlorobenzoate (2-CBa) were extracted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two bands gave significant matches to OhbB and OhbA, which have been reported to be the alpha and beta subunits, respectively, of an ortho-1,2-halobenzoate dioxygenase of P. aeruginosa strain 142 (T. V. Tsoi, E. G. Plotnikova, J. R. Cole, W. F. Guerin, M. Bagdasarian, and J. M. Tiedje, Appl. Environ. Microbiol. 65:2151-2162, 1999). PCR and Southern hybridization experiments confirmed that ohbAB were present in strain JB2 and were transferred from strain JB2 to strain D1. While the sequences of ohbA from strains JB2, D1, and 142 were identical, the sequences of ohbB from strains JB2 and D1 were identical to each other but differed slightly from that of strain 142. PCR analyses and Southern hybridization analyses indicated that ohbAB were conserved in strains JB2 and D1 and in strain 142 but that the regions adjoining these genes were divergent. Expression of ohbAB in Escherichia coli resulted in conversion of o-chlorobenzoates to the corresponding (chloro)catechols with the following apparent affinity: 2-CBa approximately 2,5-dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate. The activity of OhbAB(JB2) appeared to differ from that reported for OhbAB(142) primarily in that a chlorine in the para position posed a greater impediment to catalysis with the former. Hybridization analysis of spontaneous 2-CBa(-) mutants of strains JB2 and D1 verified that ohbAB were lost along with the genes, suggesting that all of the genes may be contained in the same mobile element. Strains JB2 and 142 originated from California and Russia, respectively. Thus, ohbAB and/or the mobile element on which they are carried may have a global distribution.

Blotting, Southern↗
PubMed2005

Rat enamel contains RP59: a new context for a protein from osteogenic and haematopoietic precursor cells.

We have recently identified a protein, RP59, in bone marrow cells and young osteoblasts, in cells involved in bone repair and in young erythroblasts and megakaryocytes. Here, we report immunohistochemical data at the light- and electron-microscope level indicating that RP59 is also present in newly secreted tooth enamel of the rat and in ameloblasts, the formative cells. In enamel matrix, RP59 was located proximal to secretory ameloblasts only, i.e. in newly secreted material. Distal enamel and enamel in association with maturation stage ameloblasts were unlabelled. Secretory ameloblasts contained RP59 in the matrix-proximal region including Tomes' processes, post-secretory ameloblasts in the cell-matrix interface. Western blotting of proteins from tooth germs identified RP59 as a band at 90 kD, co-migrating with RP59 from bone marrow and spleen. Antisera versus a chemically synthesised RP59 peptide and versus a bacteria-synthesised protein fragment reacted in the same manner. In situ hybridisation of tooth tissue revealed RP59 RNA specifically in ameloblasts. The reverse transcription/polymerase chain reaction method identified tooth RNA coding for RP59. Sequence analysis indicated that RP59 RNA from tooth and marrow had the same sequence. An internal sequence motif was found in rat RP59 resembling a signal implicated in secretion of the chicken "engrailed" gene product. The findings indicate that RP59 is a genuine product of ameloblasts and that it is secreted in the course of enamel formation together with other matrix components.

Ameloblasts↗
PubMed1989

Autometallography for histochemical visualization of rat incisor polyanions with cuprolinic blue.

Autometallography was applied to semi-thin sections of rat incisors fixed a solution of cuprolinic blue-aldehyde. The resulting reduction of silver ions to metallic silver amplifies the copper sulfide signal of the cationic dye. Silver grains were seen over the cell bodies of ameloblasts and odontoblasts but not over their processes. This was owing to the interaction of cuprolinic blue with the DNA and RNA of these cells. In the extracellular matrix, silver grains were unevenly distributed over the predentin, dentin, and forming enamel. The distal predentin near the mineralization front and a thin band of dentin located near the dentino-enamel junction displayed unexpectedly intense accumulation of silver grains, whereas all other portions of the extracellular matrix exhibited the distribution of glycosaminoglycans expected from previous studies. The present investigation constitutes a new application of autometallography to glycosaminoglycan histochemistry.

Animals↗
PubMed1987

Comparison of the Schwann cell surface and Schwann cell extracellular matrix as promoters of neurite growth.

The ability of Schwann cells to influence the direction and rate of neurite growth was investigated in a tissue culture model of the bands of Büngner of injured peripheral nerve. The arrangement of this culture system allowed testing of the growth-promoting properties of the Schwann cell surface and extracellular matrix (ECM) assembled by Schwann cells rather than soluble substances secreted into conditioned medium. Various components of peripheral nerve were examined separately as substrata for regenerating neurites: (i) Schwann cells and their ECM; (ii) Schwann cells alone; (iii) Schwann cell ECM alone; (iv) Schwann cells, fibroblasts, and their assembled ECM; (v) Schwann cells, their ECM and neurites; and (vi) purified laminin. Regenerating peripheral neurites were from explants of foetal rat dorsal root ganglia, which had been cultured for several weeks to rid them of accompanying non-neuronal cells, or from explants of foetal rat superior cervical ganglia, which contained non-neuronal cells. CNS neurites from the somatosensory cortex of embryonic rats were also studied; these neurites may be either first growing or regenerating. Neurites from all types of explants studied grew longer and were guided on a substratum of Schwann cells or Schwann cell ECM compared with a collagen substratum. The presence of fibroblasts during ECM assembly did not enhance the neurite growth-promoting activity. The design of the experiments suggested that the factors by which the Schwann cells or their ECM promoted and guided neurite outgrowth were surface-bound rather than medium-borne. Electron microscopic examination showed that neurites grew on either Schwann cell surfaces or basal lamina material. Attempts to define the chemical nature of the neurite growth-promoting effect of ECM by partial enzymatic digestion did not identify any single component as essential. Purified laminin was a more effective promoter of outgrowth of peripheral neurites than were Schwann cells or Schwann cell ECM. Cortical explants also grew on laminin, but neurites were accompanied on this substratum by a massive migration of non-neuronal cells; the neurites appeared to extend primarily on the non-neuronal cells rather than by direct attachment to the laminin substratum. This characteristic outgrowth of cortical non-neuronal cells on laminin was not consistently seen on Schwann cell ECM. In conclusion, either the Schwann cell surface or the ECM produced and assembled by Schwann cells promotes neurite outgrowth and guides that outgrowth from the several types of peripheral and CNS neurons studied in this report.

Animals↗
PubMed2005

Proteomic identification of heat shock protein 90 as a candidate target for p53 mutation reactivation by PRIMA-1 in breast cancer cells.

INTRODUCTION: A loss of p53 function resulting from mutation is prevalent in human cancers. Thus, restoration of p53 function to mutant p53 using small compounds has been extensively studied for cancer therapy. We previously reported that PRIMA-1 (for 'p53 reactivation and induction of massive apoptosis') restored the transcriptional activity of p53 target genes in breast cancer cells with a p53 mutation. By using functional proteomics approach, we sought to identify molecular targets that are involved in the restoration of normal function to mutant p53. METHODS: PRIMA-1 treated cell lysates were subjected to immunoprecipitation with DO-1 primary antibody against p53 protein, and proteins bound to p53 were separated on a denaturing gel. Bands expressed differentially between control and PRIMA-1-treated cells were then identified by matrix-assisted laser desorption ionization-time-of-flight spectrometry. Protein expression in whole cell lysates and nuclear extracts were confirmed by Western blotting. The effect of combined treatment of PRIMA-1 and adriamycin in breast cancer cells was determined with a cytotoxicity assay in vitro. RESULTS: PRIMA-1 treated cells distinctly expressed a protein band of 90 kDa that was identified as heat shock protein 90 (Hsp90) by the analysis of the 90 kDa band tryptic digest. Immunoblotting with isoform-specific antibodies against Hsp90 identified this band as the alpha isoform of Hsp90 (Hsp90alpha). Co-immunoprecipitation with anti-Hsp90alpha antibody followed by immunoblotting with DO-1 confirmed that p53 and Hsp90alpha were interacting proteins. PRIMA-1 treatment also resulted in the translocation of Hsp90alpha to the nucleus by 8 hours. Treatment of cells with PRIMA-1 alone or in combination with adriamycin, a DNA-targeted agent, resulted in increased sensitivity of tumor cells. CONCLUSION: The studies demonstrate that PRIMA-1 restores the p53-Hsp90alpha interaction, enhances the translocation of the p53-Hsp90alpha complex and reactivates p53 transcriptional activity. Our preliminary evidence also suggests that PRIMA-1 could be considered in combination therapy with DNA-targeted agents for the treatment of breast cancer, especially for tumors with aberrant p53 function.

Amino Acid Substitution↗
PubMed1996

Vitamin D3 regulation of stromelysin-1 (MMP-3) in chondrocyte cultures is mediated by protein kinase C.

Matrix metalloproteinases (MMPs) are a group of enzymes with the potential to degrade extracellular matrix proteins. One of the MMPs, stromelysin-1 (MMP-3) has been localized to extracellular matrix vesicles in growth plate chondrocyte cultures, suggesting involvement of this enzyme in remodeling of the extracellular matrix during endochondral development, a process which is regulated by the vitamin D metabolites, 1,25-(OH)2D3 and 24,25-(OH)2D3. To determine whether stromelysin-1 is regulated by vitamin D as well, confluent cultures of cells derived from growth zone (GC) and resting zone (RC) rat costochondral cartilage were treated with 1 alpha, 25-(OH)2D3 (1,25) and 24R,25-(OH)2D3 (24,25), respectively, and the effect on stromelysin-1 assessed by casein gel zymography and Western blots. Although stromelysin-1 activity was enriched in the matrix vesicle fraction, only the plasma membrane enzyme was affected by the treatment; 1, 25 and 24,25 caused a marked decrease in plasma membrane stromelysin-1 activity in their target cells. Since plasma membrane protein kinase C (PKC) activity is stimulated by 1,25 and 24,25, we hypothesized that stromelysin-1 activity was regulated by the vitamin D metabolites via PKC-dependent phosphorylation. To test this, membrane fractions (containing endogenous PKC alpha and zeta as well as stromelysin-1) were incubated in the presence of purified rat brain PKC and/or recombinant human (rh) stromelysin-1 and [gamma 32 P]-ATP and anti-stromelysin-1 immunoprecipitates were analyzed by autoradiography and Western blots. Immuno-phospho-stromelysin-1 was localized to a 52-kDa band in the plasma membrane fraction only; no phosphorylation was observed in the matrix vesicle fraction. Selective inhibitors of PKC activity demonstrated that phosphorylation was inhibited by H7 and low concentrations of H8, but not by HA1004, indicating that PKC, not PKA, was responsible. Protein phosphatase 2A1 (PP2A), a serine/threonine-specific phosphatase, selectively removed the radiolabel in a time-dependent manner, providing further support for a PKC-dependent phosphorylation mechanism. Incubation of resting zone cell plasma membranes with 24,25 but not 1, 25, resulted in phosphorylation of stromelysin-1, demonstrating that the nongenomic effect was metabolite-specific. This suggests that this may be one mechanism by which vitamin D metabolites regulate stromelysin-1 activity and that PKC-dependent phosphorylation inhibits the metalloproteinase.

24,25-Dihydroxyvitamin D 3↗
PubMed1996

In vitro characterization of enamel epithelium and pulp cells in mouse tooth germs.

Mixed cell populations consisting of enamel epithelium and pulp cells obtained from 18-day embryonic mouse tooth germs were cultured in vitro. Epithelial cells and pulp cells were also cultured individually and examined. Isolated cells were cultured in alpha-MEM supplemented with 10% fetal bovine serum for up to four weeks and examined morphologically using histological procedures including immunostaining, light and electron microscopy, and electron probe microanalysis. The pulp cells proliferated and differentiated in the absence of epithelium, but the number of epithelial cells showed a strong dependence on the pulp cells. Pulp cells showing fibroblastic morphology in the mixed culture gradually became elliptical, and eventually transformed into spherical cells surrounded by a calcified extracellular matrix. Alkaline phosphatase (ALPase) activity was expressed on the pulp cells prior to calcification of the extracellular matrix, as shown by von Kossa's and alizarin-red staining. Calcification deposition, which is closely associated with thick banded type-I collagen fibrils, was shown to be composed of calcium and phosphorous using electron probe microanalysis. Type-I collagen immunoreactivity was detected on the extracellular matrix after two weeks of culturing. The present results show that the proliferation and differentiation of pulp cells, and subsequent calcification of the extracellular matrix occur in the presence or absence of epithelial elements, but that the proliferation of epithelial cells depends on the presence of pulp cells.

Ameloblasts↗
PubMed2004

Association of matrix metalloproteinase-9 in eosinophilic meningitis of BALB/c mice caused by Angiostrongylus cantonensis.

Induction of gelatinase in eosinophilic meningitis of BALB/c-strain mice was caused by Angiostrongylus cantonensis. Time-course studies showed that the molecular weight of 94-kDa gelatinase was detected at day 10 post-inoculation (PI), and reached a high intensity from days 15 to 25 PI. The 94-kDa gelatinase activity was clearly inhibited by EDTA and 1,10-phenanthroline, but not by leupeptin and phenylmethanesulphonyl fluoride. When immunoblots were performed using specific antiserums against the 94-kDa gelatinase B (matrix metalloproteinase-9; MMP-9) with cerebrospinal fluid (CSF), the 94-kDa immunopositive band was MMP-9. Immunohistochemistry studies demonstrated MMP-9 localisation within eosinophils and macrophages. The increased MMP-9 activity was closely associated with the rapid rise of CSF eosinophils, and the inflammatory reaction of the subarachnoid space. In contrast to changes in MMP-9, MMP-2 activity was constitutive and unaffected in this parasitic meningitis. These results show that MMP-9 was associated with eosinophilic meningitis, and that the enzyme may be a useful marker for angiostrongyliasis meningitis.

Angiostrongylus cantonensis↗
PubMed1982

Ultrastructure of the human intervertebral disc: II. Cells of the nucleus pulposus.

The cells of the intervertebral disc exist in a unique environment; not only are discs subject to large mechanical loads, they are the largest avascular structures in the body. To describe the ultrastructure and age changes in cells from human nucleus pulposus, we studied these cells in individuals ranging in age from the 26th week of fetal life to 91 years. Viable chondrocyte-like cells existed in specimens from all ages. The presence of Golgi cisternae and well-developed endoplasmic reticulum in these cells suggests that they are capable of producing and maintaining the extracellular matrix. Necrotic cells were also present in all samples, and many cells which appeared viable when examined by light microscopy proved to be necrotic when examined by electron microscopy. The percentage of necrotic cells increased with age from 2% or less in fetal specimens to over 50% in adults. In addition, with age, a distinct pericellular matrix or "nest," consisting of collagen fibrils, fine filaments, dense particles, and banded structures, formed around most cells with no apparent preference for viable or necrotic cells. Nest formation and increasing density of the cell nests may reflect accumulation of cell products.

Adolescent↗
PubMedDate not supplied

Isoelectric focusing and immunoblotting of the platelet membrane glycoprotein complex IIb. IIIA following urea solubilization.

Effective solubilization of the major platelet membrane component, the glycoprotein IIb.IIIa complex, can be achieved with 8 M urea. By avoiding nonionic detergents in the separation medium it is possible to obtain clear immunoblot patterns without interference from the isoelectric focusing matrix. Upon running on a pH 4.25-5.25 immobilized pH gradient, immunoreactive bands corresponding to the nonreduced IIb.IIIa complex stain between pH 4.5 and 5.0. The method appears of significant potential utility in evaluating glycoprotein IIb.IIIa polymorphisms under different clinical conditions.

Humans↗
PubMed2001

Regulation of angiotensin II-stimulated osteopontin expression in cardiac microvascular endothelial cells: role of p42/44 mitogen-activated protein kinase and reactive oxygen species.

Using spontaneously hypertensive and aortic banded rats, we have shown that expression of myocardial osteopontin, an extracellular matrix protein, coincides with the development of heart failure and is inhibited by captopril, suggesting a role for angiotensin II (ANG II). This study tested whether ANG II induces osteopontin expression in adult rat ventricular myocytes and cardiac microvascular endothelial cells (CMEC), and if so, whether induction is mediated via activation of mitogen-activated protein kinases (p42/44 MAPK) and involves reactive oxygen species (ROS). ANG II (1 microM, 16 h) increased osteopontin expression (fold increase 3.3+/-0.34, n = 12, P < 0.01) in CMEC as measured by northern analysis, but not in ARVM. ANG II stimulated osteopontin expression in CMEC in a time- (within 4 h) and concentration-dependent manner, which was prevented by the AT1 receptor antagonist, losartan. ANG II elicited robust phosphorylation of p42/44 MAPK as measured using phospho-specific antibodies, and increased superoxide production as measured by cytochrome c reduction and lucigenin chemiluminescence assays. These effects were blocked by diphenylene iodonium (DPI), an inhibitor of the flavoprotein component of NAD(P)H oxidase. PD98059, an inhibitor of p42/44 MAPK pathway, and DPI each inhibited ANG II-stimulated osteopontin expression. Northern blot analysis showed basal expression of p22phox, a critical component of NADH/NADPH oxidase system, which was increased 40-60% by exposure to ANG II. These results suggest that p42/44 MAPK is a critical component of the ROS-sensitive signaling pathways activated by ANG II in CMEC and plays a key role in the regulation of osteopontin gene expression. Published 2001 Wiley-Liss, Inc.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗
PubMed1984

Evidence for an extracellular plasmin-dependent proteolytic system in mineralizing matrices.

Plasminogen activator, which specifically catalyzes the extracellular conversion of plasminogen to plasmin, was identified in the cell-free mineralizing matrices of enamel, dentin, cementum, and bone by a fibrinolytic overlay technique. The spatial separation in developing teeth of successive stages of dentinogenesis allowed us to identify predentin as a major site of plasminogen activator activity. In addition, plasminogen, the natural substrate for the activator, was demonstrated in predentin by immunohistochemical techniques. Extraction of human dentin/predentin with neutral demineralizing buffers solubilized the activator along with inhibitory components capable of blocking the activation of plasminogen. When resolved by polyacrylamide electrophoresis under dissociative conditions, however, the activator emerged in active form as two closely spaced bands at Mr 66,000 and 62,000. In the mineralizing enamel matrix of continuously forming rodent incisors, activator activity was limited to a 3-5 mm wide segment which marks the transition between "soft" and "chalky" enamel, whereas the entire overlying enamel organ was rich in activator activity at all developmental stages. This suggests that the activator is transported to the enamel matrix only in a narrow zone which coincides with the most rapidly mineralizing site. The coincident expression of plasminogen activator activity and mineral accretion suggests that plasmin-dependent proteolysis may play a role in the extracellular regulation of matrix mineralization.

Animals↗
PubMed1997

FTIR microspectroscopic analysis of normal human cortical and trabecular bone.

Fourier transform infrared microspectroscopy (FTIRM) has been used to study the changes in mineral and matrix content and composition in replicate biopsies of nonosteoporotic human cortical and trabecular bone. Changes in osteonal bone in these same samples were reported previously. Spectral maps along and across the lamellae were obtained from iliac crest biopsies of two necropsy cases. Mineral:matrix ratios, calculated from the integrated areas of the phosphate nu1, nu3 band at 900-1200 cm-1 and the amide I band at approximately 1585-1725 cm-1, respectively, were relatively constant in both directions of analysis, i.e., along and across the lamellae. Analysis of the components of the nu1, nu3 phosphate band with a combination of second-derivative spectroscopy and curve fitting revealed the presence of 11 major underlying moieties. Of these, the ratio of the relative areas of the two underlying bands at approximately 1020 and approximately 1030 cm-1 has been shown to be a sensitive index of variation in crystal perfection in both human osteonal bone and in synthetic, poorly crystalline apatites. This ratio was calculated in both cortical and trabecular bone from human iliac crest biopsies along and across the lamellae. The ratio decreased, going from the periosteum to the medullary cavity in the cortical bone, and from the periphery towards the center of trabeculae. These observations were consistent within serial sections obtained from the same biopsy, multiple biopsies obtained from the same necropsy specimen, and biopsies obtained from the two different necropsy specimens. The results presented here along with previously reported changes in osteonal bone show a relation between bone age and "crystallinity/maturity" (a parameter dependent on crystallite size, hydroxyapatite-like stoichiometry, abundance of substituting ions such as CO32-; the more crystalline/mature, the more hydroxyapatite-like stoichiometry, the bigger the crystallite size, the less the ion substitution by ions such as CO32-) as deduced by the 1020/1030 cm-1 ratio. Invariably, younger normal bone is less mature/crystalline than older. These results provide a "baseline" for description of mineral properties, to which diseased bones may be compared.

Aged↗
PubMed2004

Reduced activity of matrix metalloproteinase-9 in trypanosoma cruzi-infected mouse embryo hepatocyte cell.

In this work, we are reporting differences in the proteolytic profile of Trypanosoma cruzi-infected and non-infected primary cultures of mouse embryo hepatocyte cells. In gelatin-SDS-PAGE, ours results showed the presence of a 100kDa metalloproteinase in the supernatant and in the cells of both systems and an 85kDa extracellular metalloproteinase found only in the non-infected hepatocyte cultures. An enzymatic assay using gelatin as substrate showed a decrease of 74 and 70% in metalloproteinase activity in the culture supernatant and in the cell hepatocyte system infected with T. cruzi, respectively. Western blotting analysis using anti-matrix metalloproteinase-9 (MMP-9) antibody recognized the 100 and 85kDa protein bands, indicating that hepatocyte metalloproteinases correspond to the latent and active forms of the gelatinase MMP-9, respectively. The localization of MMP-9 was established by immunocytochemistry analysis in the cytoplasm of the non-infected and infected hepatocyte cells. In normal and infected hepatocyte cells, cysteine-proteinases migrating in gelatin-SDS-PAGE at 60kDa were detected and should correspond to lysosomal cysteine-proteinases of T. cruzi (cruzipain) and hepatocytes. In T. cruzi-infected hepatocytes an increase of approximately 50% in this enzymatic activity was observed, possibly due to parasite's cruzipain.

Journal Article↗
PubMed1993

Adherence of Actinomyces pyogenes to HeLa cells mediated by hydrophobic surface proteins.

Determination of cell-surface hydrophobicity of Actinomyces pyogenes by hydrophobic interaction chromatography on phenyl-Sepharose revealed that all 42 cultures examined were strongly hydrophobic. The hydrophobic surface proteins were solubilized by mutanolysin treatment of the bacteria and isolated by hydrophobic interaction chromatography. In SDS-PAGE, they appeared with numerous protein bands and blocked the adhesion of whole bacterial cells to the gel matrix. The A. pyogenes cultures attached to HeLa cells in varying degrees. This attachment of A. pyogenes was greatly reduced in the presence of isolated hydrophobic proteins and in the presence of specific antibodies produced against hydrophobic surface proteins. The results of the present study demonstrate that hydrophobic surface proteins promote the capacity of A. pyogenes to adhere to HeLa cells.

Actinomyces↗
PubMed2002

Identification of human placental wheat germ agglutinin-immunoreactive protein by mass spectrometry.

A preparation of polyclonal antibodies to human placental calcium-dependent, carbohydrate-binding glycoprotein, previously identified as wheat germ agglutinin-immunoreactive protein, was applied as the ligand in an immunoaffinity procedure. Following electrophoretic separation of purified material, the specific 66-kDa antigen band was excised and subjected to in-gel protein cleavage. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of the tryptic digest yielded 42 isotopically resolved peptide fragments from mass 860 to 2,690. Empirical data from MALDI-MS were analyzed by computer assistance using ProteoMetrics PROFOUND software. Protein candidates reported in the specified identification database search are discussed in relation to the biochemical characteristics of the protein analyzed and its possible antigenic relatedness to wheat germ agglutinin. We speculate that a fibulin-like member of the epidermal growth factor-repeat protein family might be selected as a positive match.

Amino Acid Sequence↗