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At least 991 records · Page 55Linked to original sources
PubMed2004

rpoB-PCR amplified gene and temporal temperature gradient gel electrophoresis: a rapid tool to analyse bacterial strains representative of cold-smoked salmon microflora.

AIM: To evaluate rpoB gene as a biomarker of microbial biodiversity associated to cold-smoked salmon by a novel nested-polymerase chain reaction/temporal temperature gradient gel electrophoresis (PCR/TTGE) technique applied on pure cultures of reference strains. METHODS AND RESULTS: DNA obtained from pure cultures of reference strains was used in a succession of a first PCR amplification of rpoB fragment with degenerated nonclamped primers and a nested-PCR with nondegenerated clamped primers. PCR products were then applied on a TTGE gel in order to analyse strains profile. High quantity of nested-PCR products were obtained for each tested strain and TTGE profiles showed a good separation between the different reference bacteria and an easy way to associate one band to one species. CONCLUSION: The nested-PCR/TTGE technique used in this study is a promising way of investigating bacterial community structure of cold-smoked salmon or other food matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its single copy state leading to single band profiles in TTGE, rpoB constitute a good potential molecular marker for further development of cold-smoked salmon biodiversity analysis.

Animals↗
PubMed1995

Thrombospondin co-localises with TGF beta and IGF-I in the extracellular matrix of human osteoblast-like cells and is modulated by 17 beta estradiol.

Thrombospondin (TSP) is a multifunctional glycoprotein which is synthesised by several cell types including osteoblasts, and incorporated into the extracellular matrix (ECM) of these cells. The function and regulation of TSP in bone is not clear. In this study, using a long term culture model of human osteoblast-like cells, we examined the distribution of TSP in the ECM and its modulation by added estradiol. In this model the osteoblast-like cells form a regular multilayer which continues to increase in depth up to 50 days post confluence. In the ECM of these cultures and in 19-week fetal bone, the bone markers osteocalcin and alkaline phosphatase were diffusely distributed in the matrix. In contrast, labelling for TSP was concentrated, confined to the banded collagen and its immediately adjacent ECM. This pattern of labelling resembled that of the growth factors transforming growth factor beta-I (TGF beta), and insulin-like growth factor-I (IGF-I), with which TSP label co-localised. Labelling intensities were comparable between fetal bone and the in vitro material for TSP, TGF beta and IGF-I. TSP label was present by 10 days post confluence, reached a maximum by 20 days, and declined slowly thereafter, a time course which was similar to that of IGF-I. Incubation of osteoblast-like cell cultures with 17 beta estradiol resulted in an increase in multilayer depth and a maximal 3-fold increase in TSP labeling at 30 days as well as approximately 2-fold increases for TGF beta and IGF-I. The dose-response relationship for these responses to estradiol treatment was biphasic with maximal increases at 10(-10) M-10(-11) M of added estradiol. Treatment with 17 alpha estradiol produced labelling intensities that were not significantly different from controls. Studies with other cell types have suggested that TSP may be involved in modulation of growth factor activity. The similarities between TSP, TGF beta and IGF-I, in terms of their distribution and regulation by 17 beta estradiol treatment, may indicate a role for TSP in modulating bone cell proliferation and function through interaction with local growth factors.

Alkaline Phosphatase↗
PubMed2003

An improved mechanically durable electrophoresis gel matrix that is fully compatible with fluorescence-based protein detection technologies.

Unfortunately, conventional large-format polyacrylamide gels are mechanically fragile, often tearing during the subsequent manipulations required for visualization of the proteins. This problem is compounded when large-format two-dimensional gels are subjected to multiple staining procedures in order to detect different classes of proteins, such as total protein, phosphoproteins, and glycoproteins. A mechanically durable liquid polyacrylamide-based matrix has been developed that, upon polymerization, facilitates the handling of one-dimensional and two-dimensional gels. The matrix, referred to as Rhinohide liquid acrylamide, is stable as a refrigerated solution for up to one year, and forms a polymer-reinforced polyacrylamide gel suitable for electrophoresis, upon addition of catalysts. The matrix is superior to previously reported durable gel matrices in that it does not cause distortion of high-molecular-weight bands and does not suffer from other spot morphology artifacts, such as doubling of protein spots in the molecular weight dimension. The matrix is particularly valuable for the analysis of proteins applying multiple applications of fluorescent dyes, as required with serial staining of proteins for phosphorylation, glycosylation, and total protein expression, using Pro-Q Diamond phosphoprotein stain, Pro-Q Emerald glycoprotein stain and SYPRO Ruby protein gel stain, respectively.

Acrylamide↗
PubMed1996

Luminescence Properties of Platinum(II) Dithiooxamide Compounds.

Tight contact ion pairs of general formula {Pt(H(2)-R(2)-dto)(2)(2+),(X(-))(2)} have been prepared, and their absorption spectra and luminescence properties (at room temperature in dichloromethane fluid solution and at 77 K in butyronitrile rigid matrix) have been studied (dto = dithiooxamide; R = methyl, X = Cl (1); R = butyl, X = Cl (2); R = benzyl, X = Cl (3); R = cyclohexyl, X = Cl (4); R = cyclohexyl, X = Br (5); R = cyclohexyl, X = I (6)). The absorption spectra of all the compounds are dominated by moderately strong Pt(dpi)/S(p) to dithiooxamide (pi) charge transfer (Pt/S --> dto CT) bands in the visible region (epsilon in the 10(4)-10(5) M(-)(1) cm(-)(1) range). Absorption features are also present at higher energies, due to pi-pi transitions centered in the dto ligands (ligand centered, LC). All the compounds exhibit a unstructured luminescence band in fluid solution at room temperature, with the maximum centered in the 700-730 nm range. The luminescence bands are blue-shifted about 4000 cm(-)(1) on passing to the rigid matrix at 77 K. Luminescence lifetimes are on the 10(-)(8)-10(-)(7) s time scale at room temperature and 1 order of magnitude longer at 77 K. Luminescence is assigned to triplet Pt/S --> dto CT excited states in all cases. Compounds 3-6 also exhibit a second higher-energy luminescence band at room temperature, centered at about 610 nm, attributed to a LC excited state. Charge transfer interactions between halides and dto ligands destabilize dto-centered orbitals, affecting the energy of Pt/S --> dto CT transitions and states. The X counterions and X --> dto CT levels are proposed to play a role in promoting excited state conversion between LC and Pt/S --> dto CT levels. The R substituents on the nitrogen atoms of the dto ligands influence the absorption and photophysical properties of the compounds, by affecting proximity of the ion pairs. The possibility to functionalize the R substituents may open the way to interface these luminescent compounds with desired substrates and to construct supramolecular assemblies.

Journal Article↗
PubMed1990

Interactions of glycolytic enzymes with erythrocyte membranes.

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.

Animals↗
PubMed2006

Temperature scanning FTIR analysis of hydrogen bonding states of various saccharides in amorphous matrixes below and above their glass transition temperatures.

Temperature scanning Fourier transform infrared, TS-FTIR, spectroscopy of various amorphous sugar matrixes was conducted to investigate the relationship between the glass transition temperature, T(g), of an amorphous sugar matrix and the nature of the hydrogen bonds in the matrix. An amorphous sugar matrix was prepared by air-drying an aqueous solution of sugar, and the degree of formation of hydrogen bonds in the matrix was evaluated at different temperatures using the peak positions of the IR band corresponding to the O-H stretching vibration at around 3400 cm(-1). The T(g) value increased with increasing peak position of the O-H stretching vibration at T(g) and were correlated reasonably well with the magnitude of the peak shift by the temperature increase (from 25 degrees C) to the T(g) value. This demonstrates that the amorphous sugar matrix, in which the segments are fixed by fewer hydrogen bonds, has a higher thermal resistance. The glycosidic linkage largely contributes to the restriction of the segments, pyranose ring, rather than a hydrogen bond. As the degree of polymerization of pyranose rings increases, the degree of hydrogen bond formation needed to hold the matrix in a fixed position decreases. However, the magnitude of the restriction of pyranose rings by a glycosidic linkage changes depending on the type: the restrictions imposed by alpha-1,1 and -1,6 glycosidic linkages are the tightest and most flexible of all of the types of glycosidic linkages, respectively.

Hydrogen Bonding↗
PubMed2005

Internal rotation in propionic acid: near-infrared-induced isomerization in solid argon.

The conformational system of propionic acid (CH3CH2COOH) is studied in solid argon. It is predicted by the ab initio calculations that this molecule has four stable conformers. These four structures are denoted Tt, Tg+/-, Ct, and Cg+/-, and they differ by the arrangement around the C-O and Calpha-C bonds. The ground-state Tt conformer is the only form present at 8 K after deposition of an argon matrix containing propionic acid. For the CH3CH2COOH and CH3CH2COOD isotopologues, narrow-band excitation of the first hydroxyl stretching overtone of the conformational ground state promotes the Calpha-C and C-O internal rotations producing the Tg+/- and Ct conformers, respectively. A subsequent vibrational excitation of the produced Tg+/- form induces its conversion to the Cg+/- conformer by rotation around the C-O bond. In the dark, all of the produced conformers decay to the conformational ground state at different rates. The decay kinetics and its temperature dependence allow the identification of the conformers by IR absorption spectroscopy, which is supported by ab initio calculations of their vibrational spectra. For the CH3CH2COOD isotopologue, the excitation of molecules isolated in different matrix sites results in site-dependent photoisomerization rates for the Calpha-C and C-O internal rotations, which also confirm the identification of the photoproducts.

Journal Article↗
PubMed1997

Direct osteolysis induced by metastatic murine melanoma cells: role of matrix metalloproteinases.

We examined the osteolytic ability of metastatic cells and the role of tumour matrix metalloproteinases (MMPs) in bone degradation. The histomorphometry of experimental bone metastases of B16/F1 melanoma cells showed that osteolysis was associated with a 90% decrease in osteoclast number and predominance of cancer cells overlaying resorption pits. In vitro, B16/F1 cells and their conditioned medium (CM) degraded 3H-proline-labelled extracellular matrices from osteoblast-like cells and 45Ca-labelled calvariae. Using bone slices, we observed morphological evidence of degradation by B16/F1 cells. A role for tumour MMPs in bone degradation was supported by inhibition of degradation by 1,10-phenanthroline, collagen I degradation by tumour cells and the presence of TPA-inducible M(r) 90,000, 84,000 and 64,000 gelatinolytic, and 54,000 caseinolytic bands in B16/F1-CM. These studies indicate that metastatic cancer cells degrade bone matrix directly and that this is partially mediated by MMPs.

Animals↗
PubMed2002

Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466.

White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV.

Amino Acid Sequence↗
PubMed1985

The morphological features of bovine meshwork cells in vitro and their synthetic activities.

The morphological characteristics of 3rd-passage cultured bovine meshwork cells were investigated, as were some of their synthetic activities. Growing meshwork cells had the ultrastructural characteristics of metabolically active cells whereas postconfluent cells formed gap junctions at their lateral borders and were closer in their fine structure to meshwork cells in vivo. The incorporation of 3H proline and 3H glucosamine was significantly greater in growing than postconfluent cultures. The normal bovine outflow system was rich in type I collagen but type V collagen and fibronectin was also evident. The 3rd-passage cells were shown to synthesize type I collagen, type V collagen and fibronectin. However, ultrastructural studies of the extracellular matrix which was produced in vitro demonstrated the presence of small fibrils with no distinctive banding pattern, unlike the larger collagen fibrils with a 640 A banding pattern seen in vivo.

Animals↗
PubMed1999

Selective and transient expression of a native chondroitin sulfate epitope in Deiters' cells, pillar cells, and the developing tectorial membrane.

The tectorial membrane (TM) is an acellular connective tissue overlying the sensory hair cells of the organ of Corti. Association of the tectorial membrane with the stereocilia of the sensory hair cells is necessary for proper auditory function. During development, the mature tectorial membrane is thought to arise by fusion of a "major" and "minor" tectorial membrane (Lim, Hear Res 1986;22:117-146). Several proteins and glycoconjugates have been detected in the developing TM; however, the specific molecules which mediate fusion of the two components of the TM have not been identified. In the present study, a novel monoclonal antibody (TC2) that recognizes a native epitope on glycosaminoglycans enriched in chondroitin-4-sulfate revealed a transient and restricted expression in the developing gerbil TM. The localization patterns suggest that Deiters' and pillar cells secrete a TC2-positive matrix prior to birth that later becomes incorporated into the marginal band and superior layer (cover net) of the TM. The developmental timecourse and patterns of TC2 reactivity suggest that this molecule may play a critical role in the fusion of the minor TM with the major TM.

Animals↗
PubMed1995

Characterization of gelatinases in human placenta.

Matrix metalloproteinases were extracted from human placenta. Gelatin zymograms showed four bands with gelatinase activity. These four bands were detected at Mr 72,000, 92,000, 130,000, and 210,000 respectively. Reduced and alkylated samples were detected at Mr 72,000 and 92,000. Immunoblotting analysis showed that the enzymes of Mr 130,000 and 210,000 were derived from gelatinase B (EC 3.4.24.35). Also, reduced gelatinase was activated by 4-aminophenylmercuric acetate more readily than a nonreduced gelatinase. This indicates that human placental gelatinases are stabilized by the tissue inhibitor of metalloproteinases.

Alkylation↗
PubMed2002

Synergistic induction of the senescence-associated genes by 5-bromodeoxyuridine and AT-binding ligands in HeLa cells.

5-Bromodeoxyuridine induces a senescence-like phenomenon in mammalian cells. This effect was dramatically potentiated by AT-binding ligands such as distamycin A, netropsin, and Hoechst 33258. The genes most remarkably affected by these ligands include the widely used senescence-associated genes and were located on or nearby Giemsa-dark bands of human chromosomes. We hypothesize that AT-rich scaffold/nuclear matrix attachment region sequences are involved in this phenomenon. In fact, upon substitution of thymine with 5-bromouracil, a rat S/MAR sequence reduced its degree of bending and became insensitive to cancellation of the bending by distamycin A. The S/MAR sequence containing 5-bromouracil also bound more tightly to nuclear scaffold proteins in vitro and this binding was not inhibited by distamycin A. Under the same conditions, the S/MAR sequence containing thymine easily dissociated from the nuclear scaffold proteins. Taken together, the synergistic induction of the genes may be explained not only by opening of condensed chromatin by distamycin A but also by increase in the binding of 5-bromouracil-containing S/MAR sequences to the nuclear scaffolds.

AT Rich Sequence↗
PubMed1999

Large-magnitude high-spin nuclear parameters in a Ti3+ center from X-band EPR measurements at 10 K.

Precise 10 K X-band EPR measurements and subsequent spin-Hamiltonian analysis by direct matrix diagonalization methods are reported for a Ti3+ (S = 1/2) center in tetragonal zircon (zirconium silicate, ZrSiO4). A special, and previously unobserved, feature of the supposedly uniaxial spectrum is the marked angular dependence of the titanium hyperfine lines in the perpendicular, ab crystal plane. As discussed, this can only arise from the presence of high-spin nuclear terms of dimension BIk, SIk (k = 3, 5) in the spin Hamiltonian. Parameters arising from these terms were determined to have magnitudes very much larger than observed previously in first-row transition ions. The consequences of precise determination of these high-spin parameters are significant and several: a precise determination of the nuclear quadrupole tensor leading to a ratio 47P/49P in excellent agreement with the ratio derived from the corresponding nuclear quadrupole moments; an apparent anisotropy in the nuclear Zeeman interaction which can be identified with anisotropy in the chemical shielding "tensor"; a marked hyperfine anomaly. The origin and significance of these observations are discussed.

Anisotropy↗
PubMed1989

Establishment of human osteoblastic cells derived from periosteum in culture.

We isolated osteoblastic cells derived from human periosteum and established them in culture. Their growth depended on the presence of ascorbic acid, and the doubling time was 40 to 60 h. The requirement for ascorbic acid was used to high production of collagen. These cells produced mainly type I collagen and only small amounts of type III collagen determined by reducing sodium dodecyl sulfate SDS-polyacrylamide gel electrophoresis. The total collagen yield was about 10 mg from 2 X 10(7) cells. The cells could be continuously cultured in alpha-minimum essential medium supplemented with 10% fetal bovine serum for 18 to 40 population doubling levels, depending on the age of the donated periosteum. These cells have the ability to calcify when incubated with 2 mM alpha-glycerophosphate-Na2. Calcification as viewed by the naked eye appeared from Day 15 after treatment. Treatment with the active formed vitamin D3, 1, 25 dihydroxyvitamin D3 enhanced calcification significantly and stimulated osteocalcin production. By electron microscopy, cells with many projections on their surfaces showed well-developed rough endoplasmic reticulum and actinlike fibers, and larger numbers of lysosomes, mitochondria, and secretion granules. Many matrix vesicles, in which minerals were initially localized, and well-banded collagen fibrils were seen in the intercellular spaces. These observations demonstrate typical osteoblastic morphology. The above results indicate that cultured cells from human periostem are osteoblastic cells that have the capacity to differentiate into osteocytes and to deposit calcified minerals in response to 1,25 dihydroxyvitamin D3.

Alkaline Phosphatase↗
PubMed1996

scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis.

A clone containing a Neocallimastix frontalis cDNA assumed to encode the beta subunit of succinyl-CoA synthetase (SCSB) was identified by sequence homology with prokaryotic and eukaryotic counter-parts. An open reading frame of 1311 bp was found. The deduced 437 amino acid sequence showed a high degree of identity to the beta-succinyl-CoA synthetase of Escherichia coli (46%), the mitochondrial beta-succinyl-CoA synthetase from pig (48%) and the hydrogenosomal beta-succinyl-CoA synthetase from Trichomonas vaginalis (49%). The G + C content of the succinyl-CoA synthetase coding sequence (43.8%) was considerably higher than that of the 5' (14.8%) and 3' (13.3%) non-translated flanking sequences, as has been observed for other genes from N. frontalis. The codon usage pattern was biased, with only 34 codons used and a strong preference for a pyrimidine (T) in the third positions of the codons. The coding sequence of the beta-succinyl-CoA synthetase cDNA was cloned in an E. coli expression vector encoding a 6(His) tag. The recombinant protein was purified by affinity binding and used to produce polyclonal antibodies. The anti-succinyl-CoA synthetase serum recognized a 45 kDa protein from a N. frontalis fraction enriched for hydrogenosomes and similar polypeptides in two related anaerobic fungi, Piromyces rhizinflata (45 kDa) and Caecomyces communis (47 kDa). Immunocytochemical experiments suggest that succinyl-CoA synthetase is located in the hydrogenosomal matrix. Staining for SCS activity in native electrophoretic gels revealed a band with an apparent molecular weight of approximately 330 kDa. The C-terminus of the succinyl-CoA synthetase sequence was devoid of the typical targeting signals identified so far in microbody proteins, indicating that N. frontalis uses a different signal for sorting SCSB into hydrogenosomes. Based on comparisons with other proteins we propose a putative N-terminal targeting signal for succinyl-CoA synthetase of N. frontalis that shows some of the features of mitochondrial targeting sequences.

Amino Acid Sequence↗
PubMed2006

Fast chemical shift mapping with multiecho balanced SSFP.

OBJECT: A method is proposed that provides spectroscopic images with high spatial resolution and moderate spectral resolution at very short total data acquisition times. MATERIALS AND METHODS: Balanced steady-state free precession (bSSFP, TrueFISP, FIESTA, b-FFE) is combined with a multiecho readout gradient and frequency-sensitive reconstruction such as Fourier reconstruction known from echo-planar spectroscopic imaging (EPSI) or matrix inversion. Balanced SSFP imaging requires short repetition times to minimize banding artefacts, thereby restricting the achievable frequency resolution. RESULTS: Two-dimensional (2D) high-resolution spectroscopic images were produced of three 1H resonances (water, acetone and fat) on phantoms and water/fat separation in vivo within 1-2 s. Additionally, fast 31P spectroscopic images were acquired from a phantom consisting of two resonances within 195 ms. CONCLUSION: Frequency-sensitive reconstruction of multiecho bSSFP data can provide spectroscopic images with high spatial and temporal resolution while the frequency resolution is moderate at around 100 Hz. The method can also separate more than three resonances, allowing for hetero-nuclei metabolite mapping, for example 13C and 31P.

Carbon Radioisotopes↗
PubMed1983

Cross-linking of mitochondrial matrix proteins in situ.

Different cross-linkers (10 mM) of varying specificity and arm length were found to cross-link mitochondria matrix proteins in situ in 2 min at pH 7.4. As seen by SDS-polyacrylamide electrophoresis, the disappearance of individual protein bands was accompanied by concomitant appearance of polymeric aggregates that failed to enter the 4% spacer gel. The disorganization of the mitochondrial matrix infrastructure either by swelling or sonication of the mitochondria resulted in a decrease in the rate of cross-linking. Leakage of citrate synthase, malate dehydrogenase and fumarase was found to be reduced when cross-linked mitochondria were made permeable with toluene. On lysing the cross-linked mitochondria, a major part of the matrix protein (75%) was found to sediment with the membrane fraction. The activities of citrate synthase malate dehydrogenase and fumarase in rat liver mitochondria were also found to increase in the percipitates with concomitant decrease in their activities in the soluble matrix fraction. These results indicate that the cross-linkers enters the mitochondria and cross-links matrix proteins including Krebs cycle enzymes either to the mitochondrial membranes, or to themselves resulting in very large molecular weight complexes. These results are interpreted to mean that in liver mitochondria, the Krebs cycle enzyme are preferentially located near the membrane.

Animals↗