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PubMed1995

Immunohistochemical evaluation of tenascin in benign eccrine gland tumors: comparison with laminin and fibronectin.

Surgically resected specimens of skin appendage tumors, spiradenoma, poroma of eccrine glands, syringoadenoma, syringoadenoma papilliferum and mixec tumor of skin were evaluated for tenascin, as well as the basement membrane-associated protein laminin, and the extracellular matrix protein fibronectin, using immunohistochemical methods. In normal sweat glands, tenascin was localized as a delicate, thin band in the immediate vicinity of the basement membrane and laminin was limited to the basement membrane. Tenascin in syringoadenoma was also expressed linearly adjacent to the basement membrane. Mixed tumors of skin showed a characteristic distribution of tenascin in the modified myoepithelial cell area adjacent to the tumor epithelia with a heterogeneous pattern, and particularly strong expression in the hyalinous and chondroid areas. In addition, the localization of laminin in the basement membranes of tumors, intracellular immunoreactivity was observed in syringoadenoma, syringocystadenoma papilliferum, and mixed tumor of skin. Fibronectin reactivity was visualized as a diffuse staining in the tumor stroma of eccrine poroma but its expression was restricted in eccrine spiradenoma. The present study suggests a heterogeneity in the distribution of basement membrane and extracellular matrix proteins in skin appendage tumors and elucidation of their functional roles would assist in understanding the changes in the supporting tumor stroma following neoplastic changes.

Cell Adhesion Molecules, Neuronal↗
PubMed1999

Extracellular matrix regulates apoptosis in human neutrophils.

BACKGROUND: During inflammation, polymorphonuclear neutrophils (PMNs) migrate into the affected tissue interacting with extracellular matrix (ECM) proteins. We tested the hypothesis that PMN-matrix interaction affects PMN apoptosis. METHODS: Apoptosis of human PMNs was detected by DNA-fragmentation assay and was quantitated by flow cytometry, ultraviolet and light microscopy. Cell adhesion was assessed by a toluidine blue assay, and cell spreading was detected by phase contrast microscopy. Protein tyrosine phosphorylation was studied using Western blotting and confocal microscopy. RESULTS: PMN apoptosis was not different in unstimulated cultures on either surface-adherent fibronectin or on PolyHema, a surface that prevents cell adherence. However, tumor necrosis factor-alpha (TNF alpha) treatment significantly increased apoptosis on fibronectin (37 +/- 4%) compared with PolyHema (20 +/- 3%). Tests on other matrix substances revealed that the percentage of apoptotic PMNs in the presence of TNF alpha was 8 +/- 1% on PolyHema, 26 +/- 4% on fibronectin, 17 +/- 2% on collagen I, 16 +/- 2% on collagen IV, and 16 +/- 3% on laminin (P < 0.05 for all matrices compared with PolyHema). Preincubation with genistein (50 microM) significantly inhibited TNF alpha-mediated apoptosis on fibronectin (39 +/- 4% to 21 +/- 4%) but not on PolyHema (21 +/- 4% to 16 +/- 4%). Genistein also reduced PMN spreading on fibronectin. In contrast, inhibitors of mitogen-activated protein kinase and protein kinase C showed no effect on PMN apoptosis. Fibronectin strongly increased tyrosine phosphorylation of three 102, 63, and 54 kDa proteins. Five newly tyrosine-phosphorylated 185, 85, 66, 56, and 42 kDa bands were also visible. Using confocal microscopy, highest tyrosine phosphorylation was localized to sites of cell-matrix interaction. CONCLUSIONS: ECM influences apoptosis in TNF alpha-activated, adherent, spreading PMNs. The process is regulated by tyrosine phosphorylation. Acceleration of apoptosis may shorten the PMN lifespan and thereby locally regulate inflammation.

Apoptosis↗
PubMed2001

Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles.

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

A novel concept for photovoltaic cells: clusters of titanium dioxide encapsulated within zeolites as photoactive semiconductors.

Discrete clusters of TiO(2) (of only a few titanium atoms) are prepared within the internal micropore space of zeolite Y (4.8 wt % Ti loading) and characterized by Raman spectroscopy (rutile- and anatase-like structures), electron microscopy combined with elemental analyses (coincident Si and Ti spatial distribution), and X-ray diffraction (minor zeolite crystallinity decrease). The parent TiO(2)@Y sample is modified either by adsorption of acid-organic compounds (benzoic and 4-aminobenzoic acids or catechol) or by nitrogen doping. After modification, the optical UV/Vis spectrum of the parent TiO(2)@Y (onset of the absorption band at wavelengths <300 nm and bandgap of 4.2 eV) changes, and the appearance of new bands expanding to the visible region is observed. In contrast to the inactive zeolite Y matrix, all the zeolite-encapsulated TiO(2) species exhibit a photovoltaic response. The influence of the I(2)/I(3) (-) concentration in the electrolyte solution on the temporal profile of the photovoltage clearly shows that I(2)/I(3) (-) is also a suitable carrier for the positive charge in zeolite-based photovoltaic devices. The photocurrent response and the efficiency of the photovoltaic cell based on zeolite-encapsulated TiO(2) materials depend on the nature of the organic modifier and on the N-doping. The most efficient photovoltaic cell is that based on N-doped TiO(2)@Y, which exhibits a V(OC) (voltage at open circuit) of 270 mV, an I(SC) of 5.8 muA (current at short circuit), and a fill factor (FF) of 0.4. Although these values are low compared to current dye-sensitized TiO(2) solar cells, our findings could open up a promise for a stimulating research on the photovoltaic activity of zeolite-based host-guest solids.

Journal Article↗
PubMed1991

Microfilamentous type VI collagen in the hyalinized stroma of the hypertrophied ligamentum flavum.

Thickened ligamenta flava obtained from 14 patients with spinal canal stenosis were examined with special reference to type VI collagen. The characteristic histological finding in the thickened area was rupture or normal elastic fibre meshwork with resultant fibrosis which usually appeared hyaline. Using an immunohistological method, collagen types VI, I and III were found to be present in the hyaline matrix. Ultrastructural study revealed many microfilamentous structures of type VI collagen admixed in loosely packed, banded collagen fibres. With differential salt precipitation of pepsin-extracted collagen the existence of type VI collagen was confirmed by SDS-polyacrylamide gel electrophoresis analysis and Western blotting analysis using anti-type VI collagen antibody. Quantification of type VI collagen in pepsin-extracted crude collagen samples by an inhibition enzyme-linked immunosorbent assay showed an increasing amount of type VI collagen in the thickened ligamenta flava compared to the normal ligaments. Thus, increase of type VI collagen is the main contribution to the thickening of the ligamentum flavum. This may represent an adaptational and reparative process associated with disruption of elastic fibres.

Collagen↗
PubMed1980

Further studies on absorption changes arising in dye-stained nerves during excitation.

Changes in light absorption during nerve excitation (absorption responses) were detected from the crab leg nerve, the rabbit vagus, and the rat superior cervical ganglion (SCG) stained with a merocyanine-rhodanine. Dependences of the responses on the wavelength and polarization of the incident light (absorption spectra) showed characteristic features with the respective nerves. In the crab nerve, the pattern of response spectra was precisely analyzed based on the previously proposed scheme, which included the shift of absorption bands and the statistical reorientation of absorption oscillators of the dye molecules in the membrane matrix during nerve excitation. Different patterns of the response spectra between the crab nerve and the rabbit vagus suggested that distinct physicochemical environments of the dye occurred in these two classes of membranes. On the other hand, the characteristic pattern that arose in the rat SCG was explained by its morphological form, that is, unlike those in a bundle of axons, the membrane elements in the ganglion were randomly oriented with respect to the direction of the light polarization.

Animals↗
PubMed1985

The alkali burned cornea: electron microscopical, enzyme histochemical, and biochemical observations.

The early phase of wound healing after small central alkali burns of the guinea pig cornea was studied using electron microscopical, enzyme histochemical, and biochemical techniques. In the first phase, which was morphologically characterized by the destruction of the epithelium and keratocytes and by the infiltration of the cornea with polymorphonuclear leukocytes, an increase in the activity of lysosomal phosphatases and glycosidases (beta-D-glucuronidase, acid beta-D-galactosidase, beta-D-N-acetylglucosaminidase) was noticed. In the second phase, the cornea was invaded by capillaries and fibroblasts. In this phase, the activity of proteases (aminopeptidase M, dipeptidyl peptidase IV) increased intra- and extracellularly, suggesting that these enzymes may be involved in the turnover of the collagenous matrix and the ground substance. Using synthetic 4-methoxy-2-naphthylamine substrates and fluorescence-band detection techniques after isoelectric focusing, an increase in the activity of endopeptidases was demonstrated. The decreased activity of gamma-glutamyl transpeptidase may be linked with the activation of latent collagenase.

Alkalies↗
PubMed2000

Development of the gerbil inner ear observed in the hemicochlea.

A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.

Animals↗
PubMed1991

Inhibition of gastric mucosal laminin receptor by Helicobacter pylori lipopolysaccharide.

Laminin receptor was isolated from gastric epithelial cell membrane by the procedure involving membrane solubilization with octylglucoside followed by affinity chromatography on laminin-coupled Sepharose. The receptor protein, eluted from the matrix with cation-free EDTA buffer, yielded on SDS-PAGE a single 67kDa band. After radioiodination, the protein was incorporated into liposomes which displayed specific affinity toward the laminin-coated surface. The binding of liposomal receptor to the laminin-coated surface was inhibited by lipopolysaccharide from H.pylori. The inhibitory effect was proportional to the concentration of lipopolysaccharide up to 50 micrograms/ml at which point a 96% decrease in the receptor binding occurred. It is suggested that a similar process may account for the loss of mucosal integrity in the pathogenesis of H. pylori associated gastric disease.

Animals↗
PubMed1987

Structural proteins of bovine parainfluenza-3 virus.

Six structural proteins of bovine parainfluenza-3 virus (PI-3V) labeled with [35S]-methionine could be resolved by polyacrylamide gel electrophoresis (PAGE). Five structural proteins of this virus had been previously reported. The 6 proteins found in this study were: L, a 180,000 (180 kD) molecular weight (MW) large protein; P, 83 kD phosphoprotein; HN, 69 kD hemagglutinin-neuraminidase glycoprotein; NP, 66 kD nucleocapsid protein; F, 55 kD fusion glycoprotein; and M, 38 kD matrix protein. Selective labeling with [2-3H]-mannose revealed only HN and F glycoprotein bands. A cellular actin protein (43 kD), associated with many enveloped viruses, was also found as a seventh protein in bovine PI-3V.

Animals↗
PubMed2000

Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques.

Lysozyme distribution and conformation in poly(lactic-co-glycolic acid)(PLGA) microspheres was determined using various infrared spectroscopic techniques. Infrared microscopy and confocal laser scanning microscopy indicated that the protein was homogeneously distributed inside the microspheres in small cavities resulting from the water-in-oil emulsification step. Part of the protein was observed at or near the cavity walls, while the rest was located within these cavities. Attenuated total reflectance (ATR) and photoacoustic spectroscopy (PAS) also showed that there is hardly any protein at the surface of the microspheres. Since this microsphere formulation gave a large burst release (ca. 50%), this burst release can not be caused by protein at the surface of the particles. Probably, the protein is rapidly released through pores in the PLGA matrix. Conformational analysis of lysozyme in the PLGA microspheres by KBr pellet transmission suffered from band shape distortion and baseline slope. Despite incomplete subtraction of the PLGA background, a characteristic band of non-covalent aggregates at 1625 cm(-1) was observed in the second derivative spectrum of the protein Amide I region. The other Fourier-transform infrared (FTIR) methods yielded similar results, indicating that the sample preparation procedure did not introduce artifacts. The observed aggregation signal may correspond to the protein adsorbed to the cavity walls inside the microspheres.

Anti-Infective Agents↗
PubMedDate not supplied

Stratification of α ganglion cells and ON/OFF directionally selective ganglion cells in the rabbit retina.

The correlation between cholinergic sensitivity and the level of stratification for ganglion cells was examined in the rabbit retina. As examples, we have used ON or OFF alpha ganglion cells and ON/OFF directionally selective (DS) ganglion cells. Nicotine, a cholinergic agonist, depolarized ON/OFF DS ganglion cells and greatly enhanced their firing rates but it had modest excitatory effects on ON or OFF alpha ganglion cells. As previously reported, we conclude that DS ganglion cells are the most sensitive to cholinergic drugs. Confocal imaging showed that ON/OFF DS ganglion cells ramify precisely at the level of the cholinergic amacrine cell dendrites, and co-fasciculate with the cholinergic matrix of starburst amacrine cells. However, neither ON or OFF alpha ganglion cells have more than a chance association with the cholinergic matrix. Z -axis reconstruction showed that OFF alpha ganglion cells stratify just below the cholinergic band in sublamina a while ON alpha ganglion cells stratify just below cholinergic b . The latter is at the same level as the terminals of calbindin bipolar cells. Thus, the calbindin bipolar cell appears to be a prime candidate to provide the bipolar cell input to ON alpha ganglion cells in the rabbit retina. We conclude that the precise level of stratification is correlated with the strength of cholinergic input. Alpha ganglion cells receive a weak cholinergic input and they are narrowly stratified just below the cholinergic bands.

Action Potentials↗
PubMed2001

Direct microextraction and analysis of rough-type lipopolysaccharides by combined thin-layer chromatography and MALDI mass spectrometry.

A rapid method for the microscale extraction of lipopolysaccharides (endotoxins, LPSs) from rough-type Gram-negative bacteria was developed using thin-layer chromatography (TLC) combined with improved conditions for LPS analysis by mass spectrometry. TLC of intact bacteria on silica gel plates in an appropriate solvent selectively extracted and separated their LPS components. The bands of molecular species were scraped from the plates after nondestructive visualization, directly mixed with matrix, and analyzed by laser desorption time-of-flight mass spectrometry. Lipids A and Re-type LPSs were analyzed after transfer to a membrane. Adding citric acid to the matrix gave greatly improved mass spectra. The system allows characterization of bacterial LPS at the microscale level and is equally well applicable to heterogeneous LPS and lipid A preparations (Escherichia coli lipid A and Bordetella lipopolysaccharides were used). The technique provides a rapid determination of the heterogeneity of unmodified preparations and the determination of the molecular weight of each separated component.

Chromatography, Thin Layer↗
PubMed2006

Detection of phosphopeptides by localized surface plasma resonance of titania-coated gold nanoparticles immobilized on glass substrates.

We herein demonstrate a new sensing method for phosphopeptides by localized surface plasmon resonance (LSPR) using titania-coated gold nanoparticles immobilized on the surface of a glass slide as the sensing substrate and using UV-visible spectrophotometry as the detection tool. Titania has been known to be an effective substrate for binding with phosphorylated species. The detection principle is the shift of wavelength of optical absorption due to SPR of the gold nanoparticles induced by binding of phosphorylated species with titania on the surface of the gold nanoparticles. The feasibility of the approach is demonstrated by detection of tryptic digest products of beta-casein and milk. Gold nanoparticles coated with thin films of titania, immobilized on a glass slide, can selectively bind traces of phosphopeptides from complex samples, resulting in a wavelength shift of the absorption band in the SPR spectrum with good reproducibility. The LSPR results are confirmed by matrix-assisted laser desorption/ionization mass spectrometry. The detection limit for the tryptic digest product of beta-casein is 50 nM.

Glass↗
PubMed2001

Ovine lentivirus is aetiologically associated with chronic respiratory disease of sheep on the Laikipia Plateau in Kenya.

A study was undertaken to investigate the occurrence of ovine lentivirus (OvLV) infection in sheep with chronic respiratory disease on the Laikipia Plateau, Kenya. All seven Merino crossbred sheep with chronic dyspnoea and emaciation examined for gross and microscopic lesions had lymphoid interstitial pneumonia (LIP), and one also had pulmonary abscesses. Two of the sheep with LIP also had lesions of ovine pulmonary carcinoma (OPC, jaagsiekte). Using in situ hybridization, OvLV DNA localized to a high proportion of pulmonary macrophages in lungs with lesions of LIP. Lung tissue samples from six of these sheep were positive for a syncytium-inducing virus in cultures of lamb testis cells. Thin-section electron microscopy of infected cells showed virions with morphogenesis typical of lentiviruses. In a western blotting assay, monoclonal antibodies to the OvLV capsid (CA, p27) and matrix (MA, p15) proteins of a North American OvLV isolate reacted with similar-sized bands of the virus, and serum from six of the sheep were reactive with CA from the Kenyan viral isolate. Using an OvLV agar gel immunodiffusion (AGID) test, all seven sheep were positive for serum antiviral antibody, as were 29% of 63 clinically normal sheep from Laikipia District. However, when sera from the healthy sheep were tested in a western blot assay, only 52% had IgG reactive to the OvLV CA, indicating a high rate of false negative reactions with the AGID test. Serum samples from 87 Red Maasai or Dorper crossbred sheep from two farms in other parts of Kenya were OvLV seronegative by both the AGID test and the western blot assay. These results document the first identification of OvLV as a cause of chronic respiratory disease in sheep in Kenya and show a high rate of infection in sheep flocks, with a high prevalence of chronic respiratory disease.

Animals↗
PubMed2000

Purification and partial characterization of the hydroxylase component of the methanesulfonic acid mono-oxygenase from methylosulfonomonas methylovora strain M2.

The reductase enzyme and the hydroxylase enzyme of the three-component methanesulfonic acid mono-oxygenase (MSAMO) from Methylosulfonomonas methylovora were purified. Purification of the reductase from M. methylovora using a range of chromatographic techniques was accompanied by complete loss of activity. Expression of the reductase as a glutathionine S-transferase fusion protein in Escherichia coli cells was successful as judged from the size of the polypeptide band obtained on induction with isopropyl thio-beta-D-galactoside. Subsequent affinity purification of the fusion protein, however, led to a protein extract containing only glutathionine S-transferase protein, indicating that the fusion protein was unstable in vitro. The hydroxylase component of the MSAMO was purified from M. methylovora to near electrophoretic homogeneity using Q-Sepharose, hydroxyapatite and Mono Q chromatography. SDS/PAGE of the purified hydroxylase showed a single band at approximately 43.7 kDa for the alpha-subunit and a double band at approximately 23 kDa for the beta-subunit. MS scans obtained with matrix-assisted laser desorption/ionization and electrospray ionization showed single peaks for both subunits, with a mass of 48 145.4 Da for alpha, 20 479.1 Da for beta, and 68 624.5 for the alphabeta-monomer. Gel filtration revealed a mass of 209 kDa, suggesting an alpha3beta3 structure for the native enzyme. Purified hydroxylase enzyme exhibited absorbance maxima at 330 nm, 460 nm and 570 nm, indicating the presence of iron-sulfur centres. The protein preparations contained 1 mol sulfide and 3-4 mol iron per mol alphabeta-monomer. Chromium, cobalt, copper, lead, nickel, molybdenum, tungsten and vanadium were not found. Flavins were also absent. Antibodies raised against the native hydroxylase enzyme cross-reacted with cell-free extract from M. methylovora cells grown with methanesulfonate, but not with extract from cells grown with methanol, confirming that MSAMO was specifically induced during growth on methanesulfonate.

Chromatography, Agarose↗
PubMed1996

Arrest of neuronal migration by excitatory amino acids in hamster developing brain.

The influence of the excitotoxic cascade on the developing brain was investigated using ibotenate, a glutamatergic agonist of both N-methyl-D-aspartate (NMDA) ionotropic receptors and metabotropic receptors. Injected in the neopallium of the golden hamster at the time of production of neurons normally destined for layers IV, III, and II, ibotenate induces arrests of migrating neurons at different distances from the germinative zone within the radial migratory corridors. The resulting cytoarchitectonic patterns include periventricular nodular heterotopias, subcortical band heterotopias, and intracortical arrests of migrating neurons. The radial glial cells and the extracellular matrix are free of detectable damage that could suggest a defect in their guiding role. The migration disorders are prevented by coinjection of DL-2-amino-7-phosphoheptanoic acid, an NMDA ionotropic antagonist, but are not prevented by coinjection of L(+)-2-amino-3-phosphonopropionic acid, a metabotropic antagonist. This implies that an excess of ionic influx through the NMDA channels of neurons alters the metabolic pathways supporting neuronal migration. Ibotenate, a unique molecular trigger of the excitotoxic cascade, produces a wide spectrum of abnormal neuronal migration patterns recognized in mammals, including the neocortical deviations encountered in the human brain.

2-Amino-5-phosphonovalerate↗
PubMed1999

Molecular cloning of mouse glycolate oxidase. High evolutionary conservation and presence of an iron-responsive element-like sequence in the mRNA.

Iron regulatory proteins (IRPs) control the synthesis of several proteins in iron metabolism by binding to iron-responsive elements (IREs), a hairpin structure in the untranslated region (UTR) of corresponding mRNAs. Binding of IRPs to IREs in the 5' UTR inhibits translation of ferritin heavy and light chain, erythroid aminolevulinic acid synthase, mitochondrial aconitase, and Drosophila succinate dehydrogenase b, whereas IRP binding to IREs in the 3' UTR of transferrin receptor mRNA prolongs mRNA half-life. To identify new targets of IRPs, we devised a method to enrich IRE-containing mRNAs by using recombinant IRP-1 as an affinity matrix. A cDNA library established from enriched mRNA was screened by an RNA-protein band shift assay. This revealed a novel IRE-like sequence in the 3' UTR of a liver-specific mouse mRNA. The newly identified cDNA codes for a protein with high homology to plant glycolate oxidase (GOX). Recombinant protein expressed in bacteria displayed enzymatic GOX activity. Therefore, this cDNA represents the first vertebrate GOX homologue. The IRE-like sequence in mouse GOX exhibited strong binding to IRPs at room temperature. However, it differs from functional IREs by a mismatch in the middle of its upper stem and did not confer iron-dependent regulation in cells.

Alcohol Oxidoreductases↗