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PubMed1987

Increased expression of a high molecular weight matrix component in rat hepatocellular carcinoma.

The urea extract of the glycoproteins from the extracellular matrix of rat liver has been compared with that of Morris hepatoma 7777. A high molecular weight glycoprotein present in Morris hepatoma 7777 was not found in the extract of liver matrix. Under reducing conditions in SDS-gel electrophoresis this component gave two glycoprotein bands with Mr 53 k and 56 k. The indirect immunofluorescence staining with a monospecific antiserum directed against the component showed its abundant presence in Morris hepatoma 7777 as well as in the less malignant Morris hepatoma 9121 in form of extracellular network structures. The antigen also densely filled some cumuli of cells. In contrast the liver tissue showed only very weak staining of the extracellular areas. The overall distribution of the component could be correlated with the distribution of several hydrolases in the tumor matrix, notably beta-D-glucuronidase.

Animals↗
PubMed1990

Relationship of one form of human histamine-releasing factor to connective tissue activating peptide-III.

We have previously reported purification of three forms of histamine-releasing factors (HRFs) from mixtures of streptokinase-streptodornase stimulated human mononuclear cells and platelets with apparent molecular masses of 10-12, 15-17, and 40-41 kD (1989. J. Clin. Invest. 83:1204-1210). We have also prepared mouse MAbs against the 10-12-kD HRF (1989. J. Allergy Clin. Immunol. 83:281). Affinity-purified 10-12-kD HRF appears as a broad band upon polyacrylamide gel electrophoresis in the presence of SDS. We determined the NH2-terminal amino acid sequence of the top and bottom halves of this broad band. Sequence analysis revealed striking homology between this HRF and connective tissue activating peptide-III (CTAP-III), a platelet-derived 8-10-kD protein known to cause mitogenesis and extracellular matrix formation in fibroblast cultures. 19 of 21 NH2-terminal residues in the top half of the HRF band were identical to the NH2-terminal sequence of CTAP-III. 20 of 21 NH2-terminal residues in the bottom half were identical to the NH2-terminal sequence of neutrophil-activating peptide-2, which is derived from CTAP-III by proteolytic cleavage between residues 15 and 16. Purified CTAP-III also released histamine from basophils. Rabbit antiserum raised against either native or recombinant CTAP-III recognized affinity-purified HRF in immunodot blot assays, and MAb against HRF recognized CTAP-III in both dot blot and microtiter plate based immunoassays. These data demonstrate the first structural, functional, and immunologic relationship between one form of human HRF and a previously described cell product.

Amino Acid Sequence↗
PubMed1998

High-Resolution Infrared Study of the nu14, nu17, and nu18 Bands of 11B2H6 and 10B11BH6.

Using high-resolution Fourier transform spectra, a thorough analysis of the nu14 c-type, nu17 a-type, and nu18 a-type bands of both 11B2H6 and 10B11BH6 has been carried in the 10.3-, 6.2-, and 8.5-µm spectral regions, respectively. From this analysis a large set of precise ground state combination differences with J values up to 36 (31) and Ka values extending to 18 (18) was derived for 11B2H6(10B11BH6). These data were fitted using a Watson-type Hamiltonian leading to accurate ground state rotational constants. An rs value for the B-B distance has been determined to be 1.7645(10) Å. The determination of upper state Hamiltonian constants proved to be much more difficult since the corresponding rotational levels of each of the bands are strongly perturbed by nearby dark states. To account for these strong localized resonances, it was necessary to introduce the relevant interacting terms in the Hamiltonian matrix. As a result it was possible to calculate the upper state energy levels quite satisfactorily. From these fits, estimates of the band centers and a few of the rotational constants of the resonating dark states were obtained. Copyright 1998 Academic Press.

Journal Article↗
PubMed1992

Homocarnosinosis patients and great apes have a serum protein that cross-reacts with human serum carnosinase.

A specific polyclonal antiserum to human serum carnosinase was raised in rabbits and was used to prepare an agarose-protein A-antibody matrix. An antigen capture procedure showed that sera from homocarnosinosis patients, which lack carnosinase activity, contain an immunoreactive protein (M(r) 75,000) indistinguishable from the carnosinase band from normal serum. Other higher primates have active serum carnosinase and a similar immunoreactive M(r) 75,000 protein. The immunoaffinity matrix was used in a facile procedure to isolate pure carnosinase from human plasma with a yield of 69%. The antiserum inhibited human serum carnosinase strongly, but the maximum inhibition attained averaged only 71%. The antiserum inhibited human and chimpanzee serum carnosinases more effectively than gorilla or other higher primate serum carnosinases.

Animals↗
PubMed1994

Identification of lactoferrin-binding proteins from Treponema pallidum subspecies pallidum and Treponema denticola.

Lactoferrin-binding or -associated proteins were identified in Treponema pallidum subspecies pallidum and Treponema denticola by affinity column chromatography using human lactoferrin and detergent-solubilized, radiolabelled spirochaetes. Two discrete polypeptides of T. pallidum with masses of 45 and 40 kDa and a broad band from 29-34 kDa exhibited association with human apo- and partially ferrated lactoferrin. T. denticola produced two proteins that associated with a lactoferrin affinity matrix (50 and 35 kDa). T. pallidum and T. denticola did not associate with soluble, human transferrin in parallel experiments. Soluble human lactoferrin competed with all lactoferrin-associated proteins from T. pallidum and T. denticola in competitive-binding assays. However, the T. denticola proteins dissociated from a lactoferrin-affinity matrix in the presence of differing concentrations of unlabelled, soluble lactoferrin competitor. Treatment with phospholipase D altered migration of the diffuse 29-34 kDa band of T. pallidum suggesting that the polypeptide was lipid-modified. Each of the lactoferrin-binding proteins from T. pallidum and T. denticola reacted with pooled rabbit syphilitic antisera. The lactoferrin-binding proteins of T. pallidum reacted with human sera from patients at all stages of syphilis. In addition, a monoclonal antibody generated against the 45 kDa polypeptide of T. pallidum crossreacted with the 29-34 kDa protein.

Antibodies, Bacterial↗
PubMed2006

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

Bacterial Adhesion↗
PubMed2006

Ultrastructure of the reticular basement membrane in asthmatic adults, children and infants.

Reticular basement membrane (RBM) thickening in asthma is considered to be the result of subepithelial fibrosis. Thus, the RBM in asthma should contain an excess of fibrils identified as interstitial collagen and the ratio of fibril to matrix should be increased above normal levels. Electron micrographs of the RBM were compared with those of interstitial collagen deeper in the bronchial wall using endobronchial biopsy specimens from adult asthmatics (aged 18-41 yrs (n = 10)), children with difficult asthma (aged 6-16 yrs (n = 10)), wheezy infants with reversible airflow limitation (aged 0.3-2 yrs (n = 10)) and age-matched nonasthmatic controls: 10 adults, nine children and nine symptomatic infants with normal lung function. Fibrils in the RBM were significantly thinner (median (range) width 39 (30-52) nm versus 59 (48-73) nm), and fewer fibrils were banded than in the interstitial collagen (ratio of banded to non-banded fibrils 0.08 (0-0.17) versus 0.22 (0-1.3)). The ratio of fibrils to matrix in the thickened RBM of asthmatics did not differ from that of their respective controls (1.34 (0.63-2.49) versus 1.18 (0.31-2.6)). The ratio of fibril to matrix in the thickened reticular basement membrane of asthmatics is normal, and, contrary to what is expected in fibrosis, the fibrils do not resemble those of interstitial collagen.

Adolescent↗
PubMed2001

Regression of left ventricular hypertrophy after surgical therapy for aortic stenosis is associated with changes in extracellular matrix gene expression.

BACKGROUND: Regression of left ventricular hypertrophy (LVH) after surgical correction for aortic stenosis is not fully understood on the molecular level. The aim of this study was to examine whether there is an association between LVH regression and extracellular matrix (ECM) gene expression. METHODS AND RESULTS: A standard model of controlled LVH induction by supracoronary banding (A=baseline) was applied in 44 growing sheep (age, 6 to 8 months). Surgical correction to release the pressure gradient was performed 8.3+/-1 months later (B). The animals were killed after another 10.1+/-2 months (C). At all time points, hemodynamic evaluations and quantitative analysis of mRNA and protein expression for matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) was performed. Left ventricular mass index was 82+/-21 (A) versus 150+/-33 (B), P<0.01, and 78+/-18 g/m(2) (C), P<0.01. Left ventricular function and cardiac index remained stable. Myocardial fiber diameter index was 9.1+/-1.2 (A) versus 12.3+/-1.4 (B), P<0.01, and 8.4+/-1.3 micrometer/m(2) (C), P<0.01. In parallel to the development of LVH at B, gene expression was increased significantly for MMP-1, MMP-2, MMP-3, and MMP-9 and for TIMP-1 and TIMP-2 and decreased significantly for TIMP-3. After surgical correction (C), there was a complete regression of gene expression to baseline measures. CONCLUSIONS: Controlled induction of compensated LVH leads to significant increase in ECM gene expression. The regression of LVH after surgical therapy is associated with complete regression of ECM gene expression. However, no cause-and-effect relation could be demonstrated.

Animals↗
PubMed1992

Foetal antigen 2 (FA2) in relation to wound healing and fibroblast proliferation.

During wound healing of human skin the deposition of foetal antigen 2 (FA2) and basement membrane (BM) components collagen type IV and collagen VII was followed. FA2 appeared on day 8 in the cytoplasm of proliferating fibroblasts and around newly formed blood vessels. As granulation tissue was formed, FA2 was seen diffusely in the loose matrix and in proliferating fibroblasts. Re-establishment of FA2 as a broad diffuse band along the BM at the dermo-epidermal junction was seen on day 22. In contrast, type IV and VII collagen were found along the BM and in the basal cells of the newly formed epithelium and a continuous linear BM distribution of these two components were re-established by days 12 and 14, respectively. The molecular weight (Mr) analysis of FA2, isolated from human skin fibroblast culture supernatants using SDS-PAGE, revealed a Mr of 27 kDa, corresponding to that of FA2 isolated from amniotic fluid. The presence of FA2 in proliferating fibroblasts and diffusely in the newly formed matrix of granulation tissue, as well as its late appearance at the BM after this had become established, suggests that FA2 takes part in connective tissue metabolism and perhaps tissue morphogenesis.

Amniotic Fluid↗
PubMed1993

Fluoride-induced developmental changes in enamel and dentine of European roe deer (Capreolus capreolus L.) as a result of environmental pollution.

Using macroscopic, microradiographic and scanning electron-microscopic methods, the effects of increased fluoride exposure on enamel and dentine formation were studied in fluorosed mandibular premolars and molars of roe deer from the heavily industrialized Ruhr area, Germany. Macroscopically, fluorosed teeth were characterized by opaque and stained enamel and in more severe cases also by enamel surface lesions, reduction or loss of enamel ridges on their occlusal surfaces and increased wear. Microradiographically, fluorosed enamel exhibited different degrees of subsurface hypomineralization, in part apparently indicating a fluoride effect during enamel maturation. In some specimens, a pronounced but varying enhancement of the pattern of Retzius lines was observed throughout the enamel, denoting strongly intermittent fluoride exposure during enamel matrix secretion. This variation in exposure was also reflected histologically in dentine, by bands of interglobular dentine and marked accentuation of incremental lines. Microradiography of sections through enamel surface hypoplastic lesions showed the enamel forming the bottom and partly also the walls of the lesions to be highly mineralized. Scanning electron microscopy showed that the outer enamel along the more pronounced hypoplastic lesions consisted of stacked, thin layers of 'aprismatic' enamel, indicating that the ameloblasts in these areas had lost the distal (rod-forming) regions of their Tomes' processes. These observations demonstrate that the origin of enamel hypoplasias in deer clearly differs from that in rodents, where fluoride induces the formation of subameloblastic cysts. The differences in the degree of fluorotic alteration between the teeth of a single tooth row could be related to the developmental sequence of the dentition in roe deer. The roe deer is thus considered to be a very sensitive and useful bioindicator of environmental pollution by fluorides.

Animals↗
PubMed1995

Proteinases and restenosis in the human coronary artery: extracellular matrix production exceeds the expression of proteolytic activity.

To understand the balance of proteinase antiproteinase activity and the production of extracellular matrix (ECM) at the site of arterial injury, we analyzed the composition of ECM and proteinase activity in normal internal mammary arteries, tissue samples obtained from atherosclerotic coronary lesions and restenotic lesions obtained during directional coronary atherectomy. Histologically and biochemically, collagen and proteoglycans increased, and elastin decreased in samples from restenotic lesions when compared to samples taken from patients undergoing their first revascularization (de novo). In contrast, cellularity was increased in samples obtained from de novo patients as compared to samples obtained from restenotic lesions. Intrinsic activity of matrix metalloproteinases (MMPs) was measured by using zymography and scanning all the lytic bands in zymographic gel. In these gels, identical amounts of total protein were loaded in each lane. MMP activity was determined as % of the total (latent and active) MMPs after trypsin activation (100%) in the normal artery. Intrinsic MMP activity was reduced to 6% +/- 1% in atherosclerotic lesions and 1% +/- 1% in restenotic lesions, when compared to activity found in normal (10% +/- 3%) arteries. Based on solubilization of fluorescein-conjugated elastin by the extracts, the MMP-mediated elastinolytic activity was 0.2 +/- 0.1, 8.8 +/- 1.5, and 24.0 +/- 3 nmol/min/mg in restenotic, native atherosclerotic and normal tissue, respectively. The results suggested that, in arterial tissue from patients with angiographic restenosis, there is an increased production of ECM collagen and a decrease in MMP activity compared to both normal artery and atherosclerotic samples from de novo patients undergoing an initial revascularization procedure of a significant coronary artery lesion.

Angioplasty, Balloon↗
PubMed1995

Pigmented longitudinal bands of the nail. A clinicopathologic study.

Eighteen patients with pigmented longitudinal bands of the nail were evaluated. The following clinical data were obtained: age, gender, and race, duration of the pigmented bands, description and location of the lesion, history of any clinical changes; other nail changes; and family and past history. Nail matrix and/or nail bed biopsy were performed in all cases. The histopathologic findings of 18 patients with longitudinal melanonychia showed hyperpigmentation of the epidermis with no apparent increase in the number of melanocytes in 10 cases. One case showed melanoma in situ; another showed keratinocytic proliferation with focal atypia. Three cases showed subungual hemorrhage. The cause of solitary pigmented bands is often not readily apparent, making the clinical diagnosis challenging. Melanotic macule of the nail matrix, consisting of increased pigmentation of the epidermis with no apparent increased in the number of melanocytes, seems to be the most common cause. When the cause of longitudinal melanonychia is not clinically apparent, biopsy of the nail matrix and the nail bed should help to establish it.

Adult↗
PubMedDate not supplied

Tissue engineering of heart valves: formation of a three-dimensional tissue using porcine heart valve cells.

Tissue engineering is a promising approach to obtaining lifetime durability of heart valves. The goal of this study was to develop a heart valve-like tissue and to compare the ultrastructure with normal valves. Myofibroblasts and endothelial cells were seeded on a type I collagen scaffold. The histologic organization and extracellular matrix were compared in light and electron micrographs. Radiolabeled proteoglycans were characterized by enzymatic degradation experiments. In tissue engineered specimens, cross sectional evaluation revealed that the scaffold (300 microm) was consistently infiltrated with myofibroblasts. Both sides were covered with a multicellular layer of myofibroblasts and overlaid by endothelial cells (50 microm). A newly formed extracellular matrix containing collagen fibrils and proteoglycans was found in the interstitial space. Collagen fibrils with a 60 nm banding pattern were found in both specimens. Small sized proteoglycans (65 nm) were associated and aligned at intervals of 60 nm with collagen fibrils. Large sized proteoglycans (180 nm) were located outside the collagen bundles in amorphous compartments of the extracellular matrix. The majority of glycosaminoglycans were chondroitin/dermatan sulfate, and a minority were heparan sulfate. The morphology and topography of cells and the organization of extracellular matrix in artificial tissues strongly resembles those of native valve tissues.

Animals↗
PubMed1999

Meniscus transplantation using a cryopreserved allograft. Histological and ultrastructural study of the transplanted meniscus.

We performed allogenic meniscus transplantation in rabbits and investigated the regenerative process in the transplanted meniscus with particular respect to its ultra-structure. Under a transmission electron microscope, the chondrocytes and extracellular matrix were degenerated or destroyed in week 0. Fibroblasts were observed 4-8 weeks postoperatively. Immature chondrocytes emerged at 12 postoperative weeks. At 16 weeks, the newly formed chondrocytes and extracellular matrix were virtually normal. Although the diameter of collagen fibrils still varied, their arrangement was dense and the filaments had almost normal banding. This study demonstrated that newly formed chondrocytes may differentiate from fibroblasts 8-12 weeks after transplantation. The fibroblasts synthesized collagen fiber and glycosaminoglycan, indicating that repair of the extracellular matrix may be initiated 4-8 weeks postoperatively, becoming were active after 12 weeks when the newly formed chondrocytes emerge. The extracellular matrix is injured during rapid freezing, cryopreservation, and slow thawing, and repair is delayed due to this deterioration. The extracellular matrix thus needs to be protected from injury as far as possible in order to promote its repair, since it normalizes more slowly than chondrocytes.

Animals↗
PubMed1998

Endogenous skin fluorescence includes bands that may serve as quantitative markers of aging and photoaging.

Aging and photoaging cause distinct changes in skin cells and extracellular matrix. Changes in hairless mouse skin as a function of age and chronic UVB exposure were investigated by fluorescence excitation spectroscopy. Fluorescence excitation spectra were measured in vivo, on heat-separated epidermis and dermis, and on extracts of mouse skin to characterize the absorption spectra of the emitting chromophores. Fluorescence excitation spectra obtained in vivo on 6 wk old mouse skin had maxima at 295, 340, and 360 nm; the 295 nm band was the dominant band. Using heat separated tissue, the 295 nm band predominantly originated in the epidermis and the bands at 340 and 360 nm originated in the dermis. The 295 nm band was assigned to tryptophan fluorescence, the 340 nm band to pepsin digestable collagen cross-links fluorescence and the 360 nm band to collagenase digestable collagen cross-links fluorescence. Fluorescence excitation maxima remained unchanged in chronologically aged mice (34-38 wk old), whereas the 295 nm band decreased in intensity with age and the 340 nm band increased in intensity with age. In contrast, fluorescence excitation spectra of chronically UVB exposed mice showed a large increase in the 295 nm band compared with age-matched controls and the bands at 340 and 350 nm were no longer distinct. Two new bands appeared in the chronically exposed mice at 270 nm and at 305 nm. These reproducible changes in skin autofluorescence suggest that aging causes predictable alterations in both epidermal and dermal fluorescence, whereas chronic UV exposure induces the appearance of new fluorphores.

Animals↗
PubMed1999

Disaccharide analysis and molecular mass determination to microgram level of single sulfated glycosaminoglycan species in mixtures following agarose-gel electrophoresis.

The separation of sulfated glycosaminoglycans in mixtures by agarose-gel electrophoresis and the recovery of single polysaccharide bands has been applied to the characterization of polysaccharides extracted from tissues without previous purification of single species. Sulfated glycosaminoglycans, heparin with its two components, slow-moving and fast-moving, heparan sulfate, dermatan sulfate, and chondroitin sulfate, were separated to microgram level by conventional agarose-gel electrophoresis. After their separation, they were fixed in the agarose-gel matrix by precipitation in a cetyltrimethylammonium bromide solution, making them visible on a dark background. After recovery of gel containing the fixed bands, high temperatures (90 degrees C for 15 min) were necessary to dissolve the gel matrix, and a solution of NaCl (3 M) was used to release sulfated polysaccharides from the complex with cetyltrimethylammonium. After precipitation of glycosaminoglycans in the presence of ethanol, the recovery of slow-moving heparin, fast-moving heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate was from 1 to 10 microg, with a percentage greater than 45% and a purity above 90%. Sulfated glycosaminoglycans in mixtures recovered from gel matrix as single species were evaluated for purity and characterized for unsaturated disaccharides after treatment with bacterial lyases (heparinases for heparin and heparan sulfate samples, and chondroitinases for dermatan sulfate and chondroitin sulfate) and molecular mass. Bovine lung and heart Glycosaminoglycans were extracted and separated into single species by agarose-gel electrophoresis and recovered from gel matrix after treatment in cetyltrimethylammonium solution. Unsaturated disaccharides pattern, the sulfate to carboxyl ratio, and the molecular mass of each single polysaccharide species were determined.

Animals↗
PubMed2001

Experimental and theoretical evidence for the existence of absolute acoustic band gaps in two-dimensional solid phononic crystals.

Experimental measurements of acoustic transmission through a solid-solid two-dimensional binary-composite medium constituted of a triangular array of parallel circular steel cylinders in an epoxy matrix are reported. Attention is restricted to propagation of elastic waves perpendicular to the cylinders. Measured transmitted spectra demonstrate the existence of absolute stop bands, i.e., band gaps independent of the direction of propagation in the plane perpendicular to the cylinders. Theoretical calculations of the band structure and transmission spectra using the plane wave expansion and the finite difference time domain methods support unambiguously the absolute nature of the observed band gaps.

Journal Article↗
PubMed1990

[Identification of a protein with properties of tenascin in the matrix of embryonal cartilage tissue and in a culture of human embryonal fibroblasts].

A protein with properties of tenascine has been discovered in extracellular matrix of human embryonic cartilage tissue. Its Mr, as determined by electrophoresis in PAAG with SDS, is 205 and 195 kDa. Immunoblotting with monoclonal antibodies raised against tenascine, revealed 2 precipitation bands, which permitted to identify the discovered protein as tenascine. This is the first report of detection of tenascine in rib cartilage matrix of human embryos. A protein identified as tenascine, has been also detected in a culture of human embryonic fibroblasts.

Antibodies, Monoclonal↗