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PubMed2001

Comparing protein-ligand interactions in solution and single crystals by Raman spectroscopy.

By using a Raman microscope, we show that it is possible to probe the conformational states in protein crystals and crystal fragments under growth conditions (in hanging drops). The flavin cofactor in the enzyme para-hydroxybenzoate hydroxylase can assume two conformations: buried in the protein matrix ("in") or essentially solvent-exposed ("out"). By using Raman difference spectroscopy, we previously have identified characteristic flavin marker bands for the in and out conformers in the solution phase. Now we show that the flavin Raman bands can be used to probe these conformational states in crystals, permitting a comparison between solution and crystal environments. The in or out marker bands are similar for the respective conformers in the crystal and in solution; however, significant differences do exist, showing that the environments for the flavin's isoalloxazine ring are not identical in the two phases. Moreover, the Raman-band widths of the flavin modes are narrower for both in and out conformers in the crystals, indicating that the flavin exists in a more limited range of closely related conformational states in the crystal than in solution. In general, the ability to compare detailed Raman data for complexes in crystals and solution provides a means of bridging crystallographic and solution studies.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1979

Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.

We describe a rapid and very sensitive method for detecting proteins as antigens after their separation in polyacrylamide/agarose composite gels, with or without sodium dodecyl sulfate. The polyacrylamide matrix is crosslinked with a reagent that can be cleaved with periodate or alkali to facilitate transfer of the protein bands to diazobenzyloxymethyl-paper, where they are coupled covalently. Specific proteins are detected by autoradiography after sequential incubation with unfractionated, unlabeled specific antiserum and 125I-labeled protein A from Staphylococcus aureus. Antibody and protein A can be removed with urea and 2-mercaptoethanol, and the same paper can be probed again with a different antiserum. An antiserum specific for the simian virus 40 virion proteins VP3 and VP2 has been prepared; it does not crossreact with VP1, as demonstrated by this method. An antiserum raised in rabbits against simian virus 40-transformed rabbit kidney cells is shown to be directed primarily against a periodate-sensitive moiety present in tumor (T) antigen from infected or transformed cells, whereas an antiserum raised in rabbits against large T antigen purified from lytically infected monkey kidney cells by electrophoresis in the presence of sodium dodecyl sulfate [Lane, D.P. & Robbins, A.K. (1978) Virology 87, 182-193] is directed primarily against determinants that are not sensitive to periodate.

Antibody Specificity↗
PubMed2003

Yeast V1-ATPase: affinity purification and structural features by electron microscopy.

V1-ATPase from the yeast Saccharomyces cerevisiae was purified via a FLAG affinity tag introduced into the N terminus of the G subunit. The preparation migrated as a single band in native gel electrophoresis and contained subunits ABCDEFGH (with subunit C present at substoichiometric amounts) as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The initial specific Ca-ATPase activity was approximately 6 micromol/min/mg. The structure of the yeast V1-ATPase was studied by electron microscopy of negatively stained and frozen hydrated samples. A 25-A resolution three-dimensional model of the complex was calculated from two-dimensional projections by the angular reconstitution technique. The model shows six elongated densities arranged in pseudo-3-fold symmetry around a large central cavity. At the top of the molecule, various protrusions can be seen. At the bottom of the complex, two large masses are visible that are connected to the main body of the molecule. Comparison of the yeast V1 structure with the structure of the intact V1V0-ATPase from bovine brain clathrin-coated vesicles (Wilkens, S., Vasilyeva, E., and Forgac, M. (1999) J. Biol. Chem. 274, 31804-31810) indicates that the structure of the isolated V1 from yeast is very similar to the structure of the V1 domain in the intact V-ATPase complex.

Chromatography, Affinity↗
PubMed1993

Erythema chronicum migrans: an electron-microscopic study.

Spirochaetal organisms are found in skin specimens obtained by biopsy from a erythema chronicum migrans lesion. The histological picture shows a logical localization of a lymphohistiocytic cell infiltrate: deep dermal in the central papule and superficial in the erythematous border. The electron-microscopic characteristics of the micro-organisms in these specimens (regular waving appearance with coils every 0.8-1.2 microns, oblique striation of periplasmatic fibrils, cross-section of 0.3 micron, membranes) correspond to the spiral-like structure of Borreliae isolated from blood. The Borreliae seem to move freely through the matrix of the dermis and are not phagocytized. This brings on the typical clinical picture of the centrifugally spreading erythematous band.

Animals↗
PubMed2005

Stokes-parameter analysis of the polarization of light transmitted through a chiral nematic liquid-crystal cell.

A spectroscopic Stokes polarimeter is used to directly measure the linearly, circularly, and randomly polarized components of light obtained on transmission of unpolarized light through thick chiral nematic liquid-crystal cells in the stop band. The Stokes parameters are simulated to fit the experimental data by use of the Berreman 4 x 4 transfer matrix by means of the Jones and Stokes vectors and taking into account multiple reflections at the interfaces of the cell. Excellent agreement is obtained. The transmitted light through a commercial cell is mainly circularly polarized at normal incidence, but a significant linearly polarized component is also observed. The model shows that this results from refractive-index mismatching at the liquid-crystal-alignment-layer interface, but a small linearly polarized component remains even with optimized index matching. An improved device configuration incorporating random defects at the exit boundary of the liquid crystal gives a highly circularly polarized output with virtually no linear or unpolarized components.

Journal Article↗
PubMed2003

[Gene expression of interstitial collagenase MMP-13 in progressive phase of rat liver fibrosis induced by ethanol].

OBJECTIVES: To demonstrate the gene expression of MMP-13 in the progressive phase of ethanol-induced experimental liver fibrosis in rats. METHODS: 34 SD rats were randomized into two groups. The rats in experimental group (n=24) were given ethanol (44%, 7g/kg) every day, and the rats in control group (n=10) were given equality normal saline. Liver samples were harvested from experimental rats at the 4th, 12th and 24th weeks respectively. The dynamic expression of MMP-13 mRNA was assayed by semi-quantity reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In normal rat liver, a faint band of MMP-13 mRNA was observed by RT-PCR (0.24+/-0.41). The gene expression of MMP-13 increased in the livers of rats treated with ethanol for 4 weeks (0.62+/-0.54), but it was not considered statistically, when compared with that in normal rats livers. And the livers from 12-week-treated rats showed a markedly MMP-13 mRNA expression (1.65+/-0.47, t=-4.363, P<0.01). Once the fibrosis became prominent (24 weeks), a faint band of MMP-13 mRNA was observed (0.39+/-0.25). CONCLUSION: MMP-13 participates in the degradation of newly-formed matrix in the early phase of rat liver fibrosis induced by ethanol, but it expresses in a distinct time frame

Animals↗
PubMed1992

[Ultrastructure of the long flexor and extensor tendons of the hand in rheumatic tenosynovitis].

Tendon ruptures are a frequent problem in patients affected by rheumatoid arthritis. The aim of the present study was to further characterize the known macroscopic changes of tendon tissue by micromorphological methods. Flexor and extensor tendon samples (n = 28) in rheumatoid arthritis were investigated by light- and transmission electron microscopy. In all cases, marked alterations in fibril architecture and structure were found. The fibril diameter showed a higher variability (range = 20-490 nm, x = 98.9 nm, s = 49.1 nm) in comparison with the control group of normal tendons (range = 20-290 nm, x = 119.0 nm, s = 34.9 nm). The average distance between the major bands in rheumatoid arthritis was reduced (from 57.3 nm down to 54.8 nm). Rare minor bands were detected. Oxytalan and mature elastic fibres were destroyed. These findings suggest that a causal correlation exists between altered matrix substructure and insufficient function of the tendon tissue in rheumatoid arthritis.

Adult↗
PubMed1986

An in vitro model of proliferation and differentiation of the chick retina: coaggregates of retinal and pigment epithelial cells.

Pigment epithelial (PE) cells exert a pronounced organizing effect when added to embryonic day (E) 5-6 chick retinal cells in a reaggregation system such that after a period of 14-21 d of culture, the main layers of an intact E10-E14 retina are reconstructed (Vollmer et al., 1984). In the present study we investigated the time course of the formation of retina-like structures in retinal-pigment epithelial aggregates, in particular, the fate of the PE cells and their influence on processes of differentiation within the aggregates. PE cells first form a core in the center of the aggregates and migrate to the periphery at later stages. The PE core affects the organization of proliferation and differentiation, phenomena that were monitored using 3H-thymidine autoradiography and AChE histochemistry, respectively. A double-staining procedure combining both techniques on the same section is described. Soon after aggregation, a matrix zone and a zone of differentiated cells are formed. At later steps, proliferation becomes gradually restricted to a narrow band within the aggregates comparable to the in vivo situation. The spatiotemporal pattern of withdrawal from mitosis resembles that of the in vivo retina. Proliferation in aggregates is sustained over a longer period with PE, as compared with aggregates formed by retinal cells alone. AChE staining in aggregates in the presence of PE shows a layered appearance, while there is only crude sorting-out of labeled and unlabeled cells in aggregates composed of retinal cells only. The basis of PE cell action as well as the relevance of this in vitro system for understanding normal eye development are briefly discussed.

Animals↗
PubMed1994

Alpha 2-macroglobulin is present in and synthesized by the cornea.

PURPOSE: The purposes of this study were to determine whether the proteinase inhibitor alpha 2-macroglobulin is present in the cornea, and, if so, where it is located, and whether it is synthesized by the cornea, and, if so, where it is being synthesized. METHODS: alpha 2-Macroglobulin was immunolocalized using a double antibody technique and quantified by immunodot blot assays, and its identity was confirmed by Western blot analysis. Corneal synthesis of this inhibitor was determined by immunoprecipitation of extracts from corneas incubated in organ culture with 35S-methionine. mRNA was localized by in situ hybridization of 3H-labeled cDNA to the inhibitor. RESULTS: alpha 2-Macroglobulin was localized in the epithelial, endothelial, and stromal cells. It was also found in the stromal extracellular matrix. When extracts of the epithelium, stroma, and Descemet's membrane-endothelium were analyzed by Western blot, an immunoreactive band for this inhibitor was detected in all extracts. This band comigrated with the alpha 2-macroglobulin form isolated from plasma. Metabolically labeled inhibitor was immunoprecipitated from the stromal layer but not from the epithelial or endothelial layer. However, when examined by in situ hybridization, mRNA was localized to epithelial and endothelial cells in addition to stromal keratocytes. CONCLUSIONS: Because alpha 2-macroglobulin has the ability to inhibit a wide range of proteinases, it is probable that this inhibitor plays an important role in protecting the cornea from damage caused by proteinases. This includes proteinases synthesized by the cornea and those released from inflammatory cells and invading organisms.

Adolescent↗
PubMed1996

Matrix metalloproteinases in the neocortex and spinal cord of amyotrophic lateral sclerosis patients.

Matrix metalloproteinases (MMPs) were analyzed by immunohistochemistry and zymography in amyotrophic lateral sclerosis (ALS) and control brain and spinal cord specimens. Three major bands of enzyme activity (70, 100, and 130 kDa) were consistently observed and were subsequently identified as MMP-2 (70 kDa; also known as EC 3.4.24.24 or gelatinase A) and MMP-9 (100 and 130 kDa; also known as EC 3.4.24.35 or gelatinase B). Immunohistochemical studies established the presence of MMP-2 in astrocytes and MMP-9 in pyramidal neurons in the motor cortex and motor neurons in the spinal cord of ALS patients. Although a significant decrease in MMP-2 activity was noticed in the ALS motor cortex, statistically significant increases in MMP-9 (100-kDa) activity were observed in ALS frontal and occipital cortices (BA10 and 17) and all three spinal cord regions when compared with control specimens. The highest MMP-9 (100-kDa) activities in ALS were found in the motor cortex and thoracic and lumbar cord specimens. The abnormally high amount of MMP-9 and its possible release at the synapse may destroy the structural integrity of the surrounding matrix, thereby contributing to the pathogenesis of ALS.

Aged↗
PubMed1985

Double stranded RNA and the nuclear matrix--implications for the 2-5A system.

RNA species present on rat liver nuclear matrices were investigated. Nuclear matrices prepared by extensive digestion of isolated nucleii with DNase and RNaseA followed by low and high (2M NaC1) salt washes were labelled in vitro with T4 RNA ligase and [5'-32P]pCp and the labelled RNA analysed by gel electrophoresis. Despite the extensive RNaseA treatment, a prominent RNA species migrating as a heterodisperse band of 220-300 nucleotides (termed MX220-300), was observed--only minor amounts of other RNA molecules were seen. A comparison of RNA isolated from in-vitro labelled nuclear matrices with isolated matrix RNA that was subsequently labelled, indicated that part of MX220-300 was preferentially exposed on the nuclear matrix structure. Analysis of MX220-300 indicated that it was composed of a polyadenylic acid moiety hydrogen bonded to a smaller molecule of polyuridylic acid. No evidence was found for the presence of guanosine or cytosine residues. Control experiments in which labelled polyuridylic acid was added to nucleii prior to the preparation of MX220-300, virtually excluded the possibility that the partial double stranded RNA structure was an artefact of matrix preparation. An analysis of proteins in the nuclear matrix structure that interact with double stranded (ds)RNA showed at least 2 proteins having molecular weights of 62K and 66K daltons that recognized and bound polyadenylic/polyuridylic acid. Competition experiments with unlabelled polyinosinic/polycytidylic acid indicated that these proteins specifically recognized the dsRNA structure. The 62K and 66K dalton matrix proteins that specifically bound dsRNA were observed in nuclear matrices prepared from HeLa, Ehrlich ascites tumor and rat liver cells. It is not known whether these matrix located dsRNA binding proteins have 2-5A synthetase activity. The relevance of the above findings to the 2-5A system will be discussed.

Adenine Nucleotides↗
PubMed2005

Resolution of three fluorescence components in the spectra of all-trans-1,6-diphenyl-1,3,5-hexatriene under isopolarizability conditions.

all-trans-1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence in solution consists of emissions from the S1 (2(1)A(g)) and S2 (1(1)B(u)) states of the s-trans,s-trans conformer (s-t-DPH) and emission from the S1 state of the s-cis,s-trans conformer (s-c-DPH). The contribution of s-c-DPH fluorescence increases upon excitation at longer wavelengths, and both minor emissions, s-c-DPH and 1(1)B(u) s-t-DPH fluorescence, contribute more at higher temperatures (Ts). Resolution of a spectrothermal matrix of DPH fluorescence spectra by principal component analysis with self-modeling (PCA-SM) is hampered by T-dependent changes in the spectra of the individual components. We avoided differential polarizability-dependent spectral shifts by measuring the spectra in n-alkanes (Cn, C8 to C16 with n even) at T values selected to keep the index of refraction constant, hence under isopolarizability conditions. Compensation of the spectra for T-induced broadening allowed resolution of the spectral matrix into its three components. The optimum van't Hoff plot gives Delta H = 2.83 kcal/mol for s-c-DPH/s-t-DPH equilibration, somewhat smaller than the 3.4 kcal/mol calculated value, and the optimum Boltzmann distribution law plot gives Delta E(ab) = 4.09 kcal/mol for 1(1)B(u)/2(1)A(g) equilibration. The 1(1)B(u) fluorescence spectrum bears mirror-image symmetry with the DPH absorption spectrum, and the energy gap, 1431 cm(-1), is consistent with the 1615 cm(-1) difference between the lowest energy bands in the 1(1)B(u) and 2(1)A(g) fluorescence spectra. The results give V(ab) = 198 +/- 12 cm(-1) for the vibronic matrix coupling element between the 2(1)A(g) and 1(1)B(u) states. Fluorescence quantum yields and lifetimes under isopolarizability conditions reveal an increase in the effective radiative rate constant of s-t-DPH with increasing T.

Journal Article↗
PubMed1996

Characteristics of single-stranded DNA separation by capillary gel electrophoresis.

The optimization of electrophoretic conditions for fast separation of single stranded DNA by capillary gel electrophoresis was investigated. Mobilities and band broadening of the DNA fragments were measured for a 4% T, 5% C gel at 100-300 V/cm. The resolution of DNA peaks was evaluated for band spacing and band width. Band spacing decreased with increasing electric field strengths as well as DNA fragment size; resolution for large DNA fragments also decreased at higher electric field strengths. The measured mobilities in a 4% T, 5% C gel were analyzed by the reptation theory and compared with those in a 9% T, 0% C matrix. The reptation plots indicate that the onset of reptation in a 4% T, 5% C gel occurs at a DNA size of about 200 bases and about 150 bases in a 9% T, 0% C matrix. The 4% T, 5% C gel proved superior to the 9% T, 0% C matrix with respect to resolution and analysis time. The optimum electric field strength and migration distance for specific DNA fragment length (200, 400 and 700 bases) in the shortest time were calculated from band spacing and the measured band width for the 4% T, 5% C gel at Rs = 0.5. Under optimized conditions, analysis of DNA fragments up to 200, 400 and 700 bases could be carried out in about 5, 50, and 230 min, respectively.

Acrylic Resins↗
PubMed1995

Age-dependent changes in proteoglycan biosynthesis in human intervertebral discs.

Proteoglycans from annulus fibrosus and nucleus pulposus of human intervertebral disc were investigated by electrophoresis in a composite agarose-polyacrylamide gel and immunohistochemically using various monoclonal antibodies against components of extracellular matrix. There were at least five different populations of proteoglycans in both annulus fibrosus and nucleus pulposus. Proteoglycans represented by individual electrophoretic bands differed from each other in hydrodynamic size but all of them contained epitopes present in keratan sulphate. Chondroitin sulphate could be detected in populations with molecular weight above 200,000 daltons. We could postulate that the cleavage of proteoglycan chains starts at the C-terminal end. This is supported by the finding that the globular region G2 on the protein core was detected in the same populations as keratan sulphate. We could confirm these results using tissue cultures of nucleus pulposus, inner and external part of annulus fibrosus. Of interest is the finding that there is de novo synthesis of globular domain G1 only in structures of intervertebral disc from a 16-year-old male and not in the tissue of a 69-year-old male. This might contribute to an explanation of decreased aggregation of proteoglycans during the aging process.

Adolescent↗
PubMed1992

Is a pyrene-like molecular ion the cause of the 4,430-angstroms diffuse interstellar absorption band?

The diffuse interstellar bands (DIBs), ubiquitous absorption features in astronomical spectra, have been known since early this century and now number more than a hundred. Ranging from 4,400 angstroms to the near infrared, they differ markedly in depth, width and shape, making the concept of a single carrier unlikely. Whether they are due to gas or grains is not settled, but recent results suggest that the DIB carriers are quite separate from the grains that cause visual extinction. Among molecular candidates the polycyclic aromatic hydrocarbons (PAHs) have been proposed as the possible carriers of some of the DIBs, and we present here laboratory measurements of the optical spectrum of the pyrene cation C16H10+ in neon and argon matrices. The strongest absorption feature falls at 4,435 +/- 5 angstroms in the argon matrix and 4,395 +/- 5 angstroms in the neon matrix, both close to the strong 4,430-angstroms DIB. If this or a related pyrene-like species is responsible for this particular band, it must account for 0.2% of all cosmic carbon. The ion also shows an intense but puzzling broad continuum, extending from the ultraviolet to the visible, similar to what is seen in the naphthalene cation and perhaps therefore a common feature of all PAH cations. This may provide an explanation of how PAHs convert a large fraction of interstellar radiation from ultraviolet and visible wavelengths down to the infrared.

Argon↗
PubMed1995

A 25.7 x 10(3) M(r) hydra metalloproteinase (HMP1), a member of the astacin family, localizes to the extracellular matrix of Hydra vulgaris in a head-specific manner and has a developmental function.

Hydra extracellular matrix (ECM) is composed of a number of components seen in vertebrate ECM such as laminin, type IV collagen, fibronectin, and heparan sulfate proteoglycan. A number of functional studies have shown that hydra ECM plays an important role in pattern formation and morphogenesis of this simple metazoan. The present study was designed to identify matrix degrading proteinases in hydra and determine their potential function in hydra morphogenesis. Using SDS-PAGE gelatin-zymography, five gelatinolytic bands were identified with relative molecular masses of 67 x 10(3), 51-58 x 10(3) (a triplet) and 25-29 x 10(3), respectively. Inhibition studies indicated that all of these gelatinases were metalloproteinases. Gelatin-zymography indicated that there was a differential distribution of these gelatinases along the longitudinal axis of hydra, with the 67 x 10(3) M(r) gelatinase being concentrated in the body column, while the 51-58 x 10(3) M(r) gelatinase triplet and the 25-29 x 10(3) M(r) gelatinase concentrated in the head region. Purification procedures were successfully developed for the 25-29 x 10(3) M(r) metalloproteinase which has been termed hydra metalloproteinase 1 (HMP1) and which appeared as a single band with a SDS-PAGE mobility of 25.7 x 10(3) M(r). The N-terminal sequence of purified HMP1 indicated that it has structural homology with metalloproteinases that belong to the astacin family. Subsequent cloning and sequencing of cDNA clones confirmed the identification of HMP1 as an astacin-like metalloproteinase. Immunocytochemical studies with antibodies generated against the purified enzyme and to a synthetic peptide indicated that HMP1 was localized to the ECM of tentacles. Functional studies were performed in which purified HMP1, anti-HMP1 IgG, or suspected substrates of HMP1 (e.g. growth factors such as TGF-beta 1) were introduced into the interepithelial compartment of hydra using a 'DMSO loading' procedure. These studies indicated that HMP1 has a functional role during a number of developmental processes such as head regeneration and cell differentiation/transdifferentiation of tentacle battery cells.

Amino Acid Sequence↗
PubMed2006

Quadrature orthogonal signal corrected two-dimensional correlation spectroscopy.

Orthogonal signal correction (OSC) removes a substantial part of the spectral response that is orthogonal to the selected external variable. The combined use of OSC filtering and two-dimensional (2D) correlation analysis was proposed in the previous study (Wu, Noda, Meersman, and Ozaki, J. Mol. Struct., 2006, paper in press) to enable one to obtain high-quality 2D correlation spectra by eliminating any information unrelated to the external variables. However, the direct application of OSC to two-dimensional (2D) correlation analysis will result in the loss of the component that is significantly perpendicular to the external variable but also is the portion significant to the asynchronous 2D correlation analysis. Therefore, in order to avoid the problem of losing the valuable asynchronous 2D correlation information, the present study proposes a modified OSC filtering method, which is called quadrature OSC (QOSC) filtering. By replacing the external variable vector y used for OSC filtering with a two-column Y matrix consisting of y and its Hilbert-Noda transformation, the component of the spectral data asynchronously correlated to the external variable y is preserved. The performance of this technique on two simulated spectra data sets with a strong contaminant band and systematic noises has demonstrated that QOSC filtering 2D correlation analysis enables not only the elimination of the influence of signals that are unrelated to the external variable but also the preservation of the portions of information in the data matrix that are 90 degrees out of phase with y. It enables OSC 2D to deal with the problems of losing the portion of information that is perpendicular to the external variable y but is quite significant to the 2D correlation analysis.

Journal Article↗
PubMed1979

Purification and characterization of a major component from the cytoplasmic matrix of cultured murine L cells.

This study describes the purification and preliminary characterization of a 94 000 molecular weight protein (S-94) previously known only as a major band in SDS-polyacrylamide gels of total proteins from cultured murine cells. Native S-94 is a soluble constituent of the cytoplasm and sediments at 5.0 S in sucrose gradients, behavior compatible with that of a 94 000 molecular weight monomer. S-94 is purified more than 20-fold from the 100 000 X g supernatant of murine L or L1210 lymphoma cells by DEAE-Sephadex (A-50) chromatography in the presence of 2-mercaptoethanol followed by Sephadex G-200 chromatography in 8 M urea. The protein prepared by this procedure is extensively aggregated, but is essentially homogeneous by SDS-polyacrylamide gel electrophoresis. S-94 preparations from the two types of murine cells behave identically during the purification procedure and are presumed to be the same protein. It appears that these cells contain only one major protein of 94 000 molecular weight. Purified S-94 yields a distinctive pattern of fragments following CNBr degradation, including one peptide of 36 100 molecular weight. The amino acid composition is distinguished by being relatively rich in threonine, glycine and lysine, but poor in valine, leucine and tyrosine.

Amino Acids↗