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PubMed2003

Oxygen availability during orthotopic liver transplantation.

Studies have stressed the role of adequate tissue oxygenation in the light of an optimal patient outcome and allograft viability in liver transplantation. The practice of monitoring conventional hemodynamic parameters during liver transplantation could be complemented by parameters assessing real oxygen availability. In the present prospective study, real arterial available oxygen content (CavlO(2)) and its extraction ratio (O(2)ERavl) were calculated. These parameters include the effect of changes in oxyhemoglobin dissociation curve (ODC; expressed by P(50)) on oxygen availability, under the different circumstances occurring during liver transplantation. Sixteen adult cirrhotic patients were studied during orthotopic liver transplantation with the use of venovenous bypass. Classic hemodynamic measurements using a Swan-Ganz thermodilution catheter and arterial and mixed venous blood gas analysis were performed, and P(50), oxygen delivery index (DO(2 ind)), oxygen consumption index (VO(2 ind)), oxygen extraction ratio (O(2)ER), CavlO(2), and O(2)ERavl were calculated. Statistical analysis was performed using ANOVA for repeated measures and Spearman correlation coefficient matrix among the six variables (DO(2 ind), VO(2 ind), O(2)ER, P(50), CavlO(2), and O(2)ERavl) taken two at a time at every phase. Parameter P(50) changed from 25.98 +/- 1.10 to 23.15 +/- 2.24 (at the end of operation). A leftward shift of the ODC was observed. The results showed positive association between P(50) and CavlO(2) after the removal of the native liver, and a weak and inconsistent relation of DO(2 ind) with any of the other study variables. The intraoperative changes in P(50) values, which represent a shift of the ODC to the left, may reflect a more accurate estimation of O(2) release to the tissues, than the hemoglobin, Pao(2) and Sao(2) alone. Besides conventional hemodynamic parameters, P(50), which includes the effect of alterations in ODC on oxygen availability, could be of value in monitoring the systemic oxygenation during liver transplantation.

Adult↗
PubMed1998

Chemical modification of arginines by 2,3-butanedione and phenylglyoxal causes closure of the mitochondrial permeability transition pore.

We have investigated the role of arginine residues in the regulation of the mitochondrial permeability transition pore, a cyclosporin A-sensitive inner membrane channel. Isolated rat liver mitochondria were treated with the arginine-specific chemical reagent 2, 3-butanedione or phenylglyoxal, followed by removal of excess free reagent. After this treatment, mitochondria accumulated Ca2+ normally, but did not undergo permeability transition following depolarization, a condition that normally triggers opening of the permeability transition pore. Inhibition by 2,3-butanedione and phenylglyoxal correlated with matrix pH, suggesting that the relevant arginine(s) are exposed to the matrix aqueous phase. Inhibition by 2,3-butanedione was potentiated by borate and was reversed upon its removal, whereas inhibition by phenylglyoxal was irreversible. Treatment with 2,3-butanedione or phenylglyoxal after induction of the permeability transition by Ca2+ overload resulted in pore closure despite the presence of 0.5 mM Ca2+. At concentrations that were fully effective at inhibiting the permeability transition, these arginine reagents (i) had no effect on the isomerase activity of cyclophilin D and (ii) did not affect the rate of ATP translocation and hydrolysis, as measured by the production of a membrane potential upon ATP addition in the presence of rotenone. We conclude that reaction with 2,3-butanedione and phenylglyoxal results in a stable chemical modification of critical arginine residue(s) located on the matrix side of the inner membrane, which, in turn, strongly favors a closed state of the pore.

Animals↗
PubMed2000

Urinary-type plasminogen activator (uPA) expression and uPA receptor localization are regulated by alpha 3beta 1 integrin in oral keratinocytes.

Expression of urinary-type plasminogen activator (uPA) and its receptor (uPAR) is correlated with matrix proteolysis, cell adhesion, motility, and invasion. To evaluate the functional link between adhesion and proteolysis in gingival keratinocytes (pp126), cells were treated with immobilized integrin antibodies to induce integrin clustering. Clustering of alpha(3) and beta(1) integrin subunits, but not alpha(2), alpha(5), alpha(6), or beta(4), enhanced uPA secretion. Bead-immobilized laminin-5 and collagen I, two major alpha(3)beta(1) ligands, also induced uPA expression. Coordinate regulation of the serpin plasminogen activator inhibitor 1 was also apparent; however, a net increase in uPA activity was predominant. alpha(3)beta(1) integrin clustering induced extracellular signal-regulated kinase 1/2 phosphorylation, and both uPA induction and extracellular signal-regulated kinase activation were blocked by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. Integrin aggregation also promoted a dramatic redistribution of uPAR on the cell surface to sites of clustered alpha(3)beta(1) integrins. Co-immunoprecipitation of beta(1) integrin with uPAR provided further evidence that protein-protein interactions between uPAR and beta(1) integrin control uPAR distribution. As a functional consequence of uPA up-regulation and uPA-mediated plasminogen activation, the globular domain of the laminin-5 alpha(3) subunit, a major pp126 matrix protein, was proteolytically processed from a 190-kDa form to a 160-kDa species. Laminin-5 containing the 160-kDa alpha(3) subunit efficiently nucleates hemidesmosome formation and reduces cell motility. Together, these data suggest that multivalent aggregation of the alpha(3)beta(1) integrin regulates proteinase expression, matrix proteolysis, and subsequent cellular behavior.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed2002

Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli.

In Escherichia coli, the two-component Cpx system comprising the CpxA sensor kinase and the CpxR response regulator modulates gene expression in response to a variety of stresses including membrane-protein damage, starvation, and high osmolarity. To date, the few known CpxR-P target operons were mostly identified by genetic screens. To facilitate the discovery of all target operons, we derived a 15-bp weighted matrix for CpxR-P recognition that takes into account the relative base frequency at each nucleotide position. This matrix essentially consists of two tandem 5'-GTAAA-3' motifs separated by a 5-bp linker. All of the 15-bp stretches on both strands of the E. coli MG1655 genome were then scored for their degree of matching with the matrix and classified in statistical deviation groups. The effectiveness of this screening is indicated by the identification of eight new target operons (ung, ompC, psd, mviA, aroK, rpoErseABC, secA, and aer) among eleven candidates tested. Moreover, the matrix score correlates with the likelihood that a site is a true target and with the relative site affinity for CpxR-P in vitro. Our data indicate that some 100 operons are under direct CpxR-P control and that the signal transduction pathway interacts with several other control circuits in manners hitherto unanticipated.

Bacterial Proteins↗
PubMed1993

A correlation between DNA-nuclear matrix binding and relative radiosensitivity in two human squamous cell carcinoma cell lines.

Three aspects of DNA topology were examined in two human squamous cell carcinoma lines of differing radiosensitivity (SQ-9G, D0 = 1.46 Gy; and SQ-20B, D0 = 2.36 Gy). High-salt-extracted nuclei (nucleoids) were taken from gamma-irradiated cells, stained with ethidium bromide and examined by flow cytometry. After 5 Gy, nucleoids from SQ-9G cells became 30% less efficient at adopting positive DNA supercoils than were unirradiated controls. In contrast, only a 4% difference was found with the radioresistant SQ-20B line. Both lines produced positive supercoils more efficiently after irradiation if first exposed to the topoisomerase II inhibitor VP16. Ethidium bromide titration of nucleoids was consistent with each containing similar numbers and sizes of DNA loops. In each line approximately 30-35% of DNA was accessible to trioxsalen, as shown by inter-strand crosslinking after UV photo-activation. Exhaustive digestion of nuclear DNA by DNase I removed more DNA from the radiosensitive than from the radioresistant cell line (12% vs 28% remaining). This difference was thought to be due to the increased accessibility of SQ-9G DNA in vitro. We suggest that a looser association of SQ-9G DNA with the nuclear matrix both promotes DNase I digestion and affects the ability of SQ-9G nucleoids to maintain positive DNA supercoils after irradiation. These data implicate the DNA matrix attachment region in the expression of radiation sensitivity in the cell lines studied.

Carcinoma, Squamous Cell↗
PubMed2002

Inference of a genetic network by a combined approach of cluster analysis and graphical Gaussian modeling.

MOTIVATION: Recent advances in DNA microarray technologies have made it possible to measure the expression levels of thousands of genes simultaneously under different conditions. The data obtained by microarray analyses are called expression profile data. One type of important information underlying the expression profile data is the 'genetic network,' that is, the regulatory network among genes. Graphical Gaussian Modeling (GGM) is a widely utilized method to infer or test relationships among a plural of variables. RESULTS: In this study, we developed a method combining the cluster analysis with GGM for the inference of the genetic network from the expression profile data. The expression profile data of 2467 Saccharomyces cerevisiae genes measured under 79 different conditions (Eisen et al., PROC: Natl Acad. Sci. USA, 95, 14683-14868, 1998) were used for this study. At first, the 2467 genes were classified into 34 clusters by a cluster analysis, as a preprocessing for GGM. Then, the expression levels of the genes in each cluster were averaged for each condition. The averaged expression profile data of 34 clusters were subjected to GGM, and a partial correlation coefficient matrix was obtained as a model of the genetic network of S. cerevisiae. The accuracy of the inferred network was examined by the agreement of our results with the cumulative results of experimental studies.

Bayes Theorem↗
PubMed2000

Cell cycle-dependent phosphorylation of the ATRX protein correlates with changes in nuclear matrix and chromatin association.

Mutations in the ATRX gene are associated with an X-linked mental retardation (XLMR) syndrome most often accompanied by alpha-thalassaemia (ATR-X syndrome). The ATRX gene encodes a predicted protein of 280 kDa featuring a PHD zinc finger motif and an ATPase/helicase domain of the SWI/SNF type; the vast majority of mutations in the ATRX gene fall within these two motifs. Although these domains are suggestive of a role for ATRX in transcriptional regulation by affecting chromatin structure and/or function, the precise cellular role of the ATRX protein remains undefined. Using indirect immunofluorescence and biochemical fractionation, we demonstrate that the ATRX protein has a punctate nuclear staining pattern and that it is tightly associated with the nuclear matrix at interphase. At the onset of M phase, the ATRX protein was associated mainly with condensed chromatin. The association of the ATRX protein with chromosomes at mitosis is concomitant with phosphorylation of the protein and its association with heterochromatin protein 1alpha (HP1alpha). The phosphorylation-dependent changes in localization between the nuclear matrix and condensed chromatin are consistent with a dual role for ATRX, possibly involving gene regulation at interphase and chromosomal segregation at mitosis.

Animals↗
PubMed2005

ASIAN: a web server for inferring a regulatory network framework from gene expression profiles.

The standard workflow in gene expression profile analysis to identify gene function is the clustering by various metrics and techniques, and the following analyses, such as sequence analyses of upstream regions. A further challenging analysis is the inference of a gene regulatory network, and some computational methods have been intensively developed to deduce the gene regulatory network. Here, we describe our web server for inferring a framework of regulatory networks from a large number of gene expression profiles, based on graphical Gaussian modeling (GGM) in combination with hierarchical clustering (http://eureka.ims.u-tokyo.ac.jp/asian). GGM is based on a simple mathematical structure, which is the calculation of the inverse of the correlation coefficient matrix between variables, and therefore, our server can analyze a wide variety of data within a reasonable computational time. The server allows users to input the expression profiles, and it outputs the dendrogram of genes by several hierarchical clustering techniques, the cluster number estimated by a stopping rule for hierarchical clustering and the network between the clusters by GGM, with the respective graphical presentations. Thus, the ASIAN (Automatic System for Inferring A Network) web server provides an initial basis for inferring regulatory relationships, in that the clustering serves as the first step toward identifying the gene function.

Cluster Analysis↗
PubMed2006

Elevated glucose induction of thrombospondin-1 up-regulates fibronectin synthesis in proximal renal tubular epithelial cells through TGF-beta1 dependent and TGF-beta1 independent pathways.

BACKGROUND: TGF-beta1 bioactivation, consequent to the interaction of latent TGF-beta1 with thrombospondin-1 (TSP-1), correlates with matrix accumulation in mesangial cells. Tubulointerstitial damage predicts poor renal survival. There is little data on TGF-beta1 bioactivation and matrix synthesis in human proximal renal tubular epithelial cells under the influence of high glucose concentrations. This study thus investigates the role of TSP-1 in mediating elevated glucose-induction of TGF-beta1 bioactivation and fibronectin (FN) synthesis in human proximal tubular epithelial cells. METHODS: Human proximal renal tubular epithelial cells (HK-2 cells) were incubated with 5, 10, 20 or 30 mM D-glucose for up to 3 weeks either in the presence or absence of TSP-1 blocking peptide. In separate studies HK-2 cells were incubated with exogenous TSP-1 (0-10 ng/ml) or TGF-beta1 (0-10 ng/ml) for 24 h. Cell proliferation was assessed by [(3)H]-thymidine incorporation. TGF-beta1 transcript, secretion and bioactivity were investigated by quantitative real-time PCR, ELISA and the MLEC bioassay respectively. TSP-1 and FN synthesis were assessed by quantitative real-time PCR, ELISAs and Western blot analysis. RESULTS: Elevated glucose concentrations increased TSP-1 synthesis, which was associated with reduced cell proliferation, increased TGF-beta1 bioactivity, and stimulation of FN synthesis. The inclusion of TSP-1 blocking peptide to cells stimulated with elevated glucose concentration abrogated activation of TGF-beta1 and induction of FN secretion. Exogenous TSP-1 increased bioactive TGF-beta1 in HK-2 cells to initiate FN accumulation. Of interest is our observation that TSP-1 also increased matrix synthesis through a mechanism independent of TGF-beta1. TGF-beta1 in turn modulated TSP-1 synthesis, indicative of an autocrine loop between TSP-1 and TGF-beta1. CONCLUSIONS: TSP-1 plays an important role in the induction of matrix synthesis by high glucose concentrations in human proximal renal tubular epithelial cells, through TGF-beta1 dependent and TGF-beta1 independent pathways. Pharmacological intervention targeting increased TSP-1 expression may interrupt the pathogenesis of diabetic nephropathy.

Cell Line↗
PubMed1983

Nutritional status in intensive care patients: evaluation in 84 unselected patients.

A prospective nutritional evaluation of 84 unselected patients admitted to a multidisciplinary ICU was performed using anthropometric measurements, creatinine height index (CHI), and serum protein assays. All values tested were significantly lower than those of 40 healthy controls. A matrix of correlation coefficients showed many similarities among the variables studied. Fatal outcome and poor prognosis indices, such as sepsis and renal failure, were analyzed separately. Anthropometric measurements and CHI were not different in the separate groups. Albumin (Alb) and transferrin (Tr) were not different in patients with or without sepsis. Retinol binding prealbumin was significantly higher in patients with renal failure. Thyroxin-binding prealbumin (TBPA) was significantly lower in all the groups. We emphasize the interest of this rapid turnover protein in evaluating nutritional status. We suggest: (1) a systematic nutritional assessment, and (2) an aggressive nutritional support in the ICU patient.

Acute Kidney Injury↗
PubMed2003

Identification of the relative distribution of rare-earth ions in phosphate glasses.

The relative distribution of rare-earth ions R3+ (Dy3+ or Ho3+) in the phosphate glass RAl(0.30)P(3.05)O(9.62) was measured by employing the method of isomorphic substitution in neutron diffraction. It is found that 7.9(7) R-R nearest neighbors reside at 5.62(6) A in a network made from interlinked PO4 tetrahedra. Provided that the role of Al is explicitly considered, a self-consistent account of the local matrix atom correlations can be developed in which there are 1.68(9) bridging and 2.32(9) terminal oxygen atoms per phosphorus.

Journal Article↗
PubMed1997

Localization of brain electrical activity via linearly constrained minimum variance spatial filtering.

A spatial filtering method for localizing sources of brain electrical activity from surface recordings is described and analyzed. The spatial filters are implemented as a weighted sum of the data recorded at different sites. The weights are chosen to minimize the filter output power subject to a linear constraint. The linear constraint forces the filter to pass brain electrical activity from a specified location, while the power minimization attenuates activity originating at other locations. The estimated output power as a function of location is normalized by the estimated noise power as a function of location to obtain a neural activity index map. Locations of source activity correspond to maxima in the neural activity index map. The method does not require any prior assumptions about the number of active sources of their geometry because it exploits the spatial covariance of the source electrical activity. This paper presents a development and analysis of the method and explores its sensitivity to deviations between actual and assumed data models. The effect on the algorithm of covariance matrix estimation, correlation between sources, and choice of reference is discussed. Simulated and measured data is used to illustrate the efficacy of the approach.

Algorithms↗
PubMed1987

Classification of normal colorectal mucosa and adenocarcinoma by morphometry.

Semi-automatic image analysis was used to make a morphometrical assessment of 15 nuclear and cellular variables in normal (n = 20) and malignant (n = 30) colorectal epithelium. Principal components analysis on the matrix of correlations between variables identified four main sources of variation within the dataset. These were, in decreasing order of importance: (1) nuclear size, nuclear cytoplasmic ratio and nuclear position within the cell; (2) the variability of nuclear size; (3) nuclear elongation and polarity; (4) nuclear shape and its variation. Discriminant analysis was conducted between histologically normal mucosa (n = 10) and adenocarcinoma in ulcerative colitis (n = 20). Using stepwise variable selection, the mean nuclear cytoplasmic ratio (normal, mean 20.4 (s.d. +/- 2.0); tumour, mean 39.7 (s.d. +/- 7.0)) and the coefficient of variation of nucleus to cell apex distance (normal, mean 19.2 (s.d. +/- 7.5); tumour, mean 47.8 (s.d. +/- 9.1)) were chosen as discriminating features. They were used to derive a discriminant function which gave perfect discrimination between the two groups. Scatter plots of these two variables confirmed complete separation of normal mucosa from adenocarcinoma and provided a simple method of applying the discriminant function. Discriminatory performance did not deteriorate when the function was applied to further normals (n = 10) and adenocarcinoma (n = 10). This study highlights the descriptive differences between normal and malignant colorectal epithelium and shows that case allocation may be made to these two lesion categories using a morphometrically-derived classification rule.

Adenocarcinoma↗
PubMed1997

Multitryptophan-fluorescence-emission decay of beta-glycosidase from the extremely thermophilic archaeon Sulfolobus solfataricus.

The emission decay of intrinsic fluorescence of the extremely thermophilic beta-glycosidase from Sulfolobus solfataricus has been investigated as functions of temperature and of iodide-quencher concentration by frequency-domain fluorometry. This protein contains 68 tryptophans and provides a matrix for correlation of the average spectroscopic behaviour with solvent exposure and local dynamics. At each temperature, the emission is very heterogeneous and interpretable in terms of quasicontinuous bimodal distribution of fluorescence lifetimes. We associate the component of the bimodal distribution to two distinct classes of tryptophanyl residues that differ in microenvironmental characteristics. Temperature and quenching experiments show that the long-lived component includes tryptophanyl residues located in buried regions with high rigidity; the short distributional component corresponds to tryptophans embedded in more flexible and exposed regions. This proposal has been confirmed by examination of the crystallographic structure. The data suggest that, at least for this protein, there is a good correlation between residue exposure and lifetime distributional components. The conformational dynamics of the two classes of tryptophanyl residues is affected differently by temperature, suggesting that the protein regions in which they are located give different contributions to enzyme properties, such as flexibility, stability and function.

Circular Dichroism↗
PubMed1976

Computerized tomography in the diagnosis of cerebral atrophy.

Computerized tomograms were studied of 67 adults whose lumbar pneumoencephalograms were normal or showed ventricular dilatation with or without widening of the sulci. The maximum ventricular area, measured from 80 x 80 matrix printouts, correlates well with measures of ventricular size on the pneumoencephalogram. An area of 10 cm2 is suggested as the upper limit of normal. The correlation between measures of sulcal width on computerized tomography and pneumoencephalography is less precise, but normal sulci and gross degrees of cortical atrophy can be identified.

Adolescent↗
PubMed1997

Proteoglycan synthesis in the intervertebral disk nucleus: the role of extracellular osmolality.

Proteoglycans, through their polyelectrolyte properties, regulate the ionic composition and hence the osmotic pressure of the extracellular matrix. We measured the change in [35S]sulfate incorporation, a marker of proteoglycan synthesis, in explants of bovine nucleus pulposus. During incubation, nucleus slices swelled 200% and proteoglycans leached from the matrix, so that extracellular osmolality fell from 420-450 to approximately 300 mosmol/kg H2O. When in vivo extracellular osmolality was maintained either by adding 80 mM NaCl or 150 mM sucrose to the swollen tissue or by preventing swelling, synthesis rates were 260-280% greater than in swollen tissue. Synthesis rates also increased 200% in cells isolated from the nucleus pulposus by enzyme digestion when medium osmolality was raised from 280 to 430 mosmol/ kgH2O by sucrose addition. The cells, either in the tissue or isolated from it, swelled by more than 20% as osmolality fell from 430 to 280 mosmol/kgH2O and showed little regulatory volume decrease over 150 min. Synthesis rates thus appear to be regulated by extracellular osmolality rather than by the macromolecular composition of the matrix and correlated well with measured changes in cell volume.

Animals↗
PubMed1998

Calcification of human vascular cells in vitro is correlated with high levels of matrix Gla protein and low levels of osteopontin expression.

The cellular and molecular events leading to calcification in atherosclerotic lesions are unknown. We and others have shown that bone-associated proteins, particularly matrix Gla protein (MGP) and osteopontin (OP), can be detected in atherosclerotic lesions, thus suggesting an active calcification process. In the present study, we aimed to determine whether human vascular smooth muscle cells (VSMCs) could calcify in vitro and to determine whether MGP and OP have a role in vascular calcification. We established that human aortic VSMCs and placental microvascular pericytes spontaneously form nodules in cell culture and induce calcification, as detected by von Kossa's method, Alizarin red S staining, and electron microscopy. The cells in calcifying nodules differed from those in monolayer cultures by expressing higher levels of the SMC markers alpha-SM actin, SM22alpha, and calponin. In addition, Northern blot analysis revealed that in human VSMCs, calcification was associated with increased levels of MGP mRNA. In contrast, OP mRNA was barely detectable in calcified human VSMCs and pericyte nodules, nor was OP protein detected, suggesting that OP was not necessary for calcification to occur. These studies reveal that human VSMCs are capable of inducing calcification and that MGP may have a role in human vascular calcification.

Adolescent↗