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PubMed1995

Infrared spectroscopy of matrix-isolated polycyclic aromatic hydrocarbon cations. 3. The polyacenes anthracene, tetracene, and pentacene.

Gaseous, ionized polycyclic aromatic hydrocarbons (PAHs) are thought to be responsible for a very common family of interstellar infrared emission bands. Unfortunately, very little infrared spectroscopic data are available on ionized PAHs. Here we present the near- and mid-infrared spectra of the polyacene cations anthracene, tetracene, and pentacene. We also report the vibrational frequencies and relative intensities of the pentacene anion. The cation bands corresponding to the CC modes are typically about 10-20 times more intense than those of the CH out-of-plane bending vibrations. For the cations the CC stretching and CH in-plane bending modes give rise to bands which are an order of magnitude stronger than for the neutral species, and the CH out-of-plane bends produce bands which are 3-20 times weaker than in the neutral species. This behavior is similar to that found for most other PAH cations. The most intense PAH cation bands fall within the envelopes of the most intense interstellar features. The strongest absorptions in the polyacenes anthracene, tetracene, and pentacene tend to group around 1400 cm-1 (between about 1340 and 1500 cm-1) and near 1180 cm-1, regions of only moderate interstellar emission. These very strong polyacene bands tend to fall in gaps in the spectra of the other PAH cations studied to date suggesting that while PAHs with polyacene structures may contribute to specific regions of the interstellar emission spectra, they are not dominant members of the interstellar PAH family.

Anthracenes↗
PubMed1993

Regional differences in the polypeptide composition of pig lens intracellular matrix and plasma membrane.

Analysis of pig lens urea-soluble fractions (USF) by gel filtration and SDS-PAGE revealed that the cortical USF is characterized by a high content of noncrystallin bands of 42, 44, 52, 55, 100 and 200 kD. These bands are markedly decreased in the nuclear USF though the 44-kD band to a lesser extent. These results suggested that pig lens nucleus contains much less cytoskeletal proteins than the cortex. Analysis of pig lens membrane showed that MP26 and MP17 are the major intrinsic membrane proteins and the relative amount of alpha A and beta B1b chains attached to the membrane are more abundant in the nucleus than in the cortex.

Animals↗
PubMed2003

Characterization of lipid in mature enamel using confocal laser scanning microscopy.

OBJECTIVE: To characterize the lipid components of organic matrix in mature human enamel using Confocal Laser Scanning Microscopy (CLSM) coupled with a hydrophobic fluorescent probe. METHODS: Twenty-four longitudinal sections of human enamel were fixed with 3.7% paraformaldehyde (PFA), partially decalcified with 0.5 M of EDTA, and labelled with a fluorescent probe (Nile red) before CLSM characterization. Based on the fluorescence spectra of Nile red in ethanol (1 microgram/ml), each enamel section was evaluated with 543 nm light source and with 590 nm long pass filter. Spectrophotometric analysis was carried out to characterize the autofluorescence and Nile red lipid fluorescence in mature enamel. Special optical parameters of the microscope were chosen to rule out the intrinsic fluorescence of the samples, and that induced by PFA. RESULTS: The intensity of autofluorescence and PFA-induced fluorescence were negligible above 565 nm; whereas the fluorescence of Nile red peaked at around 600 nm. Lipid material was identified in the cross-striations, the lines of Retzius, the Hunter-Schreger bands, inter-prismatic spaces, and inter-prismatic spaces in the mature human enamel. CONCLUSIONS: This technique successfully revealed the distribution of lipid components of organic matrix in mature human enamel and may be promising in assessing the changes of enamel organic elements in the developmental, pathological or experimental conditions.

Dental Enamel↗
PubMed1997

Identification of the components of simple protein mixtures by high-accuracy peptide mass mapping and database searching.

Peptide mass mapping by matrix-assisted laser desorption/ionization (MALDI) followed by database searching with the set of measured peptide masses is now a powerful method for the identification of pure proteins. Protein mixtures--such as frequently occur due to comigration in polyacrylamide gel bands--have hitherto required protein sequencing. Here we demonstrate that such protein bands can also be analyzed by peptide mass mapping alone. Database searching with the complete list of peptide masses determined by delayed-extraction MALDI mass spectrometry with a mass error of less than 30 ppm retrieves the most prominent protein in a mixture. In a second step, the protein identity is further confirmed by matching as many of the measured peptide masses as possible to the retrieved amino acid sequence. Peptide masses remaining after this "second pass search" are searched again to identify the next component in the protein mixture. This iterative process is repeated until all major ion signals are accounted for. Protein mixtures consisting of two or more individual components in a single gel band can be analyzed, further increasing the general applicability of MALDI peptide mapping for protein identification.

Amino Acid Sequence↗
PubMed2006

Exciton migration by ultrafast Förster transfer in highly doped matrixes.

The energy transfer between dye molecules and the mobility of the corresponding excitons are investigated in polymethyl methacrylate films highly doped with perylene bisimide dyes. The dynamics is measured by group delay corrected, femtosecond broad-band spectroscopy revealing the transfer route via absorption changes that are specific for the participating species. In films doped with 0.14 M perylene orange an ultrafast homotransfer between the dye molecules is found by analyzing the loss of the excitation-induced anisotropy. The process exhibits a stretched exponential time dependence which is characteristic for Förster energy transfer between immobilized molecules. The transfer time is 1.5 ps for an average transfer distance of 2.3 nm and results in a high mobility of the optically generated excitons. In addition, we find that the excitons move to perylene orange dimers, which have formed in low concentration during the sample preparation. The observed energy transfer time is slightly shorter than expected for a direct Förster transfer and indicates that exciton migration by multistep transfer between the monomers speeds up the transport to the dimers. In samples doped with perylene orange and perylene red heterotransfer to perylene red takes place with transfer times down to 600 fs. The mechanism is Förster transfer as demonstrated by the agreement with calculations assuming electric dipole interaction between immobilized and statistically distributed donor and acceptor units. The model predicts the correct time dependence and concentration scaling for highly doped as well as diluted samples. The results show that ultrafast exciton migration between dye molecules in highly doped matrixes is an attractive and efficient mechanism to transport and collect energy in molecular systems and organic electronic devices. Further optimization should lead to a loss-free transport over distances typical for the thickness of active layers in these systems.

Coloring Agents↗
PubMed2004

ADAM 12 cleaves extracellular matrix proteins and correlates with cancer status and stage.

ADAM 12 is a member of a family of disintegrin-containing metalloproteases that have been implicated in a variety of diseases including Alzheimer's disease, arthritis, and cancer. We purified ADAM 12 from the urine of breast cancer patients via Q-Sepharose anion exchange and gelatin-Sepharose affinity chromatography followed by protein identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Four peptides were identified that spanned the amino acid sequence of ADAM 12. Immunoblot analysis using ADAM 12-specific antibodies detected an approximately 68-kDa band identified as the mature form of ADAM 12. To characterize catalytic properties of ADAM 12, full-length ADAM 12-S was expressed in COS-7 cells and purified. Substrate specificity studies demonstrated that ADAM 12-S degrades gelatin, type IV collagen, and fibronectin but not type I collagen or casein. Gelatinase activity of ADAM 12 was completely abrogated by zinc chelators 1,10-phenanthroline and EDTA and was partially inhibited by the hydroxamate inhibitor Marimastat. Endogenous matrix metalloprotease inhibitor TIMP-3 inhibited activity. To validate our initial identification of this enzyme in human urine, 117 urine samples from breast cancer patients and controls were analyzed by immunoblot. The majority of samples from cancer patients were positive for ADAM 12 (67 of 71, sensitivity 0.94) compared with urine from controls in which ADAM 12 was detected with significantly lower frequency. Densitometric analyses of immunoblots demonstrated that ADAM 12 protein levels were higher in urine from breast cancer patients than in control urine. In addition, median levels of ADAM 12 in urine significantly increased with disease progression. These data demonstrate for the first time that ADAM 12 is a gelatinase, that it can be detected in breast cancer patient urine, and that increased urinary levels of this protein correlate with breast cancer progression. They further support the possibility that detection of urinary ADAM 12 may prove useful in the development of noninvasive diagnostic and prognostic tests for breast and perhaps other cancers.

ADAM Proteins↗
PubMed1993

A role for regulated secretion of apical extracellular matrix during epithelial invagination in the sea urchin.

Epithelial invagination, a basic morphogenetic process reiterated throughout embryonic development, generates tubular structures such as the neural tube, or pit-like structures such as the optic cup. The 'purse-string' hypothesis, which proposes that circumferential bands of actin microfilaments at the apical end of epithelial cells constrict to yield a curved epithelial sheet, has been widely invoked to explain invaginations during embryogenesis. We have reevaluated this hypothesis in two species of sea urchin by examining both natural invagination of the vegetal plate at the beginning of gastrulation and invagination induced precociously by Ca2+ ionophore. Neither type of invagination is prevented by cytochalasin D. In one species, treatment with A23187 three hours before the initiation of invagination resulted in the deposition of apical extracellular matrix at the vegetal plate, rather than invagination. This apical matrix contains chondroitin sulfate, as does the lumen of the archenteron in normal gastrulae. When the expansion of this secreted matrix was resisted by an agarose gel, the vegetal plate buckled inward, creating an archenteron that appeared 3-4 hours prematurely. Pretreatment with monensin, which blocks secretion, inhibits both Ca2+ ionophore-stimulated folding and natural invagination, demonstrating that secretion is probably required for this morphogenetic event. These results indicate that alternatives to the purse-string hypothesis must be considered, and that the directed deposition of extracellular matrix may be a key Ca(2+)-regulated event in some embryonic invaginations. A bending bilayer model for matrix-driven epithelial invagination is proposed in which the deposition of hygroscopic material into a complex, stratified extra-cellular matrix results in the folding of an epithelial sheet in a manner analagous to thermal bending in a bimetallic strip.

Animals↗
PubMed1987

Immunoelectron microscopic localization of fibronectin in the smooth muscle layer of mouse small intestine.

We studied the ultrastructural distribution of fibronectin in the smooth muscle layer of mouse small intestine with affinity-purified antibodies using the immunogold technique. Fibronectin was present over the pericellular area extending from the cell membrane to the extracellular matrix beyond the basal lamina. Distribution of the glycoprotein over the pericellular area was heterogeneous, i.e., it was localized more abundantly in the narrow space between smooth muscle cells, the gaps having a width of 60-80 nm where the two dense bands in adjacent cells matched each other. Such localization suggests that fibronectin contributes to cell adhesion. Within the basement membrane, gold label was localized both in lamina lucida and lamina densa, more densely in the latter than in the former. Fibronectin was also co-distributed with collagen fibers in the extracellular matrix. Within smooth muscle cells, gold particles were observed on rough endoplasmic reticulum and secretory vesicle-like structures. These results suggest that smooth muscle cells synthesize fibronectin and secrete it as a component of the basal lamina and extracellular matrix.

Animals↗
PubMedDate not supplied

Effects of pressure overload on the passive mechanics of the rat left ventricle.

Both myocyte growth and changes in the extracellular matrix may affect the passive mechanics of the left ventricle (LV). Pressure-volume (PV) relationships and midwall two-dimensional strains versus passive loading were measured in isolated rat hearts 2 and 6 weeks after ascending aortic banding. Collagen area fractions and perimysial fibril orientations were determined with picrosirius-polarization microscopy, and the equatorial region of the LV was modeled with finite element analysis of a transversely isotropic cylinder with the same material properties in hypertrophy and control. Compared with weight-matched shams, heart weight increased at 2 (19%) and 6 (22%) weeks, as did LV wall thickness (6% and 31%, respectively). The PV curve became less compliant with hypertrophy; only circumferential strain decreased after hypertrophy. Collagen area fractions were not different at either subendocardium or subepicardium (3.37 +/- 1.06 versus 3.96 +/- 0.76 at 2 weeks and 3.61 +/- 1.30 versus 4.22 +/- 1.50 at 6 weeks for banded and sham, respectively; subendocardium). Collagen and muscle fiber orientations also did not change with hypertrophy. The finite element model predicted trends in the strains similar to those found experimentally. Thus, in this model of pressure-overload hypertrophy, the decreases in compliance and circumferential strain of the passive LV are not due to changes in the percentage of extracellular matrix, but rather to global geometric changes.

Analysis of Variance↗
PubMed1988

The extracellular matrix of Xenopus laevis eggs: a quick-freeze, deep-etch analysis of its modification at fertilization.

Eggs of the amphibian, Xenopus laevis, were quick-frozen, deep-etched, and rotary-shadowed. The structure of the extracellular matrix surrounding these eggs, including the perivitelline space and the vitelline envelope (VE), was visualized in platinum replicas by electron microscopy. The perivitelline space contains an elaborate filamentous glycocalyx which connects microvillar tips to the plasma membrane, to adjacent microvilli, and to the overlying VE. The VE is comprised of two layers, the innermost of which is a thin network of horizontal fibrils lying on the tips of the microvilli. The outermost is a thicker layer of large, cable-like fibers which twist and turn throughout the envelope. Upon fertilization, three dramatic modifications of the matrix occur. A thin sheet of smooth material, termed the smooth layer, is deposited on the tips of the microvilli and separates the egg from the overlying envelopes. The VE above is transformed from a thick band of cable-like fibers to concentric fibrous sheets, the altered VE. Finally, an ornate band of particles, corresponding to the fertilization layer in previous studies, is deposited at the altered VE/jelly interface. The altered VE and the fertilization layer comprise the fertilization envelope, which effects the structural block to polyspermy.

Animals↗
PubMed1998

Matrilin-3 forms disulfide-linked oligomers with matrilin-1 in bovine epiphyseal cartilage.

A comparison of noncollagenous matrix proteins from different types of bovine cartilage by SDS-polyacrylamide gel electrophoresis showed a prominent 240-kDa component in extracts of epiphyseal but not tracheal tissue. On amino-terminal sequence analysis, it gave two sequences. One matched the NH2 terminus of cartilage matrix protein (CMP) as reported for tracheal cartilage. The other did not match any known protein sequence. Further analysis of the 240-kDa protein after reduction of disulfides resolved two bands on SDS-polyacrylamide gel electrophoresis. Isolation and sequence analysis of tryptic peptides confirmed that one was bovine CMP and the other a CMP homolog. A data base search identified the latter as matrilin-3, a molecule recently predicted from human and mouse cDNA sequences (Wagener, R., Kobbe, B., and Paulsson, M. (1997) FEBS Lett. 413, 129-134). Matrilin-3 and CMP (matrilin-1) were prominent in equimolar amounts in fetal bovine epiphyseal cartilage and absent from adult articular cartilage. Adult tracheal cartilage contained almost exclusively CMP. Although the mechanism of polymeric assembly is unknown, the matrilin-3 chain appears to function in the matrix linked to matrilin-1 in the form of disulfide-bonded heteromeric molecules. The results indicate a molecular stoichiometry of (matrilin-1)2(matrilin-3)2.

Amino Acid Sequence↗
PubMed1992

Abnormal extracellular matrix and excessive growth of human adult polycystic kidney disease epithelia.

Human autosomal dominant polycystic kidney disease (ADPKD) epithelia were grown in primary monolayer cultures and their properties compared with intact kidney epithelial cultures derived from individually microdissected normal human kidney proximal convoluted tubules (PCT), proximal straight tubules (PST), and cortical collecting tubules (CCT). In vivo, ADPKD cyst epithelia exhibited a thickened basement membrane, and immunofluorescence demonstrated the presence of laminin, fibronectin, type IV collagen, and heparan sulfate proteoglycan in basement membranes and type I collagen in the interstitium. ADPKD epithelia grown in culture synthesized and secreted basally a unique, extracellular matrix that took the form of proteinaceous spheroids when the cells were grown on dried, type I collagen. Incorporation of H2[S35O4] into basement membrane extracts was increased more than ten-fold in ADPKD epithelia by comparison to normal PST and CCT. In addition to incorporation into the normal tubular basement membrane 220 kD band, radioactivity was also seen at 175 kD and 150 kD in ADPKD extracts. Growth in culture of cyst-lining ADPKD epithelia was more rapid than normal tubules, and was abnormal since there was no absolute requirement for added extracellular matrix. However, when ADPKD epithelia were grown on different, exogenous matrix protein components, a profound influence on both structure and epithelial cell proliferation was seen. Growth on a complete basement membrane three-dimensional gel derived from the Engelbreth-Holm-Swarm (EHS) sarcoma led to a reduction in the numbers of spheroids and increase in amorphous filaments. Incorporation of [3H]-thymidine into ADPKD epithelia was greater than into normal PCT, PST, and CCT and was also greatly modified by the type of extracellular matrix components provided. In studies using single matrix components, the strongest proliferative response was seen when ADPKD epithelia were plated on type I collagen greater than type IV collagen greater than fibronectin greater than laminin. These findings suggest that the excessive growth of cyst-lining epithelia may be, at least in part, a result of abnormal basement membrane and extracellular matrix production by ADPKD cells.

Basement Membrane↗
PubMed2000

Participation of macrophages in glomerular sclerosis through the expression and activation of matrix metalloproteinases.

In order to investigate the role of macrophages in glomeruli in the progression of glomerular sclerosis, methyl-cellulose (MC) was administered intraperitoneally to Wistar rats, in addition to intravenous injection of anti-thy1-1 antibody. In this group of rats (Thy-1 + MC group), many macrophages infiltrated in the lytic mesangium accompanied by rupture of capillary loops at an early stage and stayed with abundant deposition of mesangial matrices until day 35, whereas the proliferative lesions following mesangiolysis almost vanished in the rats treated with anti-thy1-1 antibody alone (Thy-1 group). In immunostaining, matrix metalloproteinase (MMP)-9 was expressed along regenerating capillaries of the Thy-1 group and in extracapillary lesions of the Thy-1 + MC group after day 7. In gelatin zymography, the gelatinolytic band for MMP-9 was expressed much more strongly in the Thy-1 + MC group than in the Thy-1 group at day 3, but it was expressed a little more strongly in the Thy-1 group than in the Thy-1 + MC group at day 7. The bands for an active form of MMP-2 were more strongly expressed in the Thy-1 + MC group than in the Thy-1 group throughout the experimental period. These results suggest that persistent accumulation of macrophages in mesangium induces glomerular sclerosis through expression and activation of MMP.

Animals↗
PubMed2003

Beta-amyloid induces the production of active, matrix-degrading proteases in cultured rat astrocytes.

The senile and neuritic plaque neuropathology of Alzheimer's disease (AD) is accompanied by an inflammatory response that includes activated astrocytes and microglia. Activated mononuclear phagocytes and reactive astrocytes, in response to inflammatory cytokines, secrete a set of extracellular matrix (ECM)-degrading enzymes that include the matrix metalloproteinases (MMPs). The major peptide component of senile plaques of AD, beta-amyloid (Abeta), stimulates the production of several MMPs from cultured rat astrocytes and microglia. The purpose of this study was two-fold: (1) to compare the pattern of MMP induction in rat astrocytes on treatment with 'soluble' and 'fibrillar' Abeta(1-40) and Abeta(1-42), and (2) to examine whether treatment of astrocytes with Abeta results in degraded fragments of ECM. Abeta aggregation differentially affected the production of MMP-2 and MMP-9 in astrocyte cultures. Activation experiments with amino phenyl mercuric acetate suggested that the 52-54 kDa gelatin-degrading activity was an activated form of MMP-2. In addition, Abeta peptide induced both MMP-3 and plasminogen activator-like activity from astrocytes. When medium from Abeta-treated, astrocyte cultures was immunoblotted for fibronectin, several immunopositive, lower molecular weight bands were observed as compared to untreated conditioned medium, suggestive of the presence of an active fibronectin-degrading protease. Thus, Abeta induces the secretion of several matrix-degrading proteases and stimulates matrix degradation in rat astrocytes. Since matrix-degrading proteases are elevated in AD brain, these proteases may influence the stability of ECM or other MMP substrates and thus may play a role in the neurotrophic/neurotoxic events associated with AD.

Amyloid beta-Peptides↗
PubMed2004

Immunolocalization of enamelysin (matrix metalloproteinase-20) in the forming rat incisor.

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.

Amelogenin↗
PubMed1999

Matrix metalloproteinases 2, 9, and 13, and tissue inhibitors of metalloproteinases 1 and 2 in experimental lung silicosis.

Exposure to silica induces granulomatous lung inflammation evolving to fibrosis through yet unclear pathogenic mechanisms. We examined the expression of extracellular matrix remodeling molecules: collagenase 3, gelatinases A and B, and TIMP-1 and TIMP-2 in experimental lung silicosis. Rats were instilled with 50 mg of silica and sacrificed after 15 and 60 d. At 60 d a significant increase in lung collagen content was found (170.2 +/- 34.4 versus 88.2 +/- 20.8 microgram/mg in controls, p = 0.01). Gelatin zymography of bronchoalveolar lavage fluid (BALF) from 15 and 60 d revealed bands of progelatinase A and progelatinase B, and lung tissue zymograms showed in addition, the active gelatinase A form at 15 d. By in situ hybridization and immunohistochemistry, early silicotic granulomas exhibited intense staining for all matrix metalloproteinases (MMPs) and TIMPs assayed. Labeling was restricted inside granulomas and surrounding areas. Late silicotic granulomas at 60 d showed lower MMP expression than did early lesions, and in highly fibrotic nodules scarce signal was usually found. TIMP-1 and TIMP-2 showed a moderate reduction in 60-d silicotic nodules. These findings suggest that an imbalance in the expression of MMPs and TIMPs may be implicated in extracellular matrix remodeling and basement membrane disruption during experimental lung silicosis.

Animals↗
PubMed1992

High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography.

The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS-methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS-5% methanol (v/v) at 50 degrees C (pH 6.9). With this mobile phase the determination of bendroflumethiazide and chlorthalidone was adequate. Acetazolamide, ethacrynic acid, furosemide, hydrochlorothiazide and probenecid were overlapped by the urine matrix, and the retention of amiloride and triamterene was too long.

Acetazolamide↗
PubMed1996

Application of chemometric methods in Auger electron microanalysis of composites.

Principal component analysis and cluster analysis have been used in AES investigations of fibre-matrix interactions in alumina fibre reinforced MgLi-alloys prepared by high-pressure infiltration. The samples have been fractured in ultra high vacuum to expose surfaces and interfaces without contamination. All main components exhibit Auger valence band transitions which change their shape with the chemical state. Chemometric methods have been utilized to identify characteristic peak shapes and to classify the investigated areas by composition. Fibre fracture surfaces are characterized by Al, magnesia and Li oxide formed by a redox reaction of alumina with Mg and Li penetrating along grain boundaries. For samples with high silica content a thin interfacial oxide layer on matrix grain boundaries and matrix-fibre interfaces has been found.

Journal Article↗