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PubMed1987

Effects of overproduction of single-stranded DNA-binding protein on RecA protein-dependent processes in Escherichia coli.

Overproduction of single-stranded DNA-binding protein (SSB) in Escherichia coli led to a decrease in the basal level of repressor LexA. Expression of the LexA-controlled genes was increased differentially, depending on the affinity of the LexA repressor for each promoter: expression of the recA and sfiA genes was increased 5-fold and 1.5-fold, respectively. Despite only a slight effect on expression of sfiA, which codes for an inhibitor of cell division, bacteria overproducing SSB produced elongated cells. In fact, the effect on cell shape appeared to be essentially independent of the expression of the sfiA and recA genes. Bacteria overproducing SSB were therefore phenotypically similar to bacteria partially starved of thymine, in which filamentation results from both sfiA-dependent and sfiA-recA-independent pathways. These data indicate that excess SSB acts primarily by perturbing DNA replication, thereby favoring gratuitous activation of RecA protein to promote cleavage of LexA protein. When bacteria overproducing SSB were exposed to a DNA-damaging agent such as ultraviolet light or mitomycin C, the recA and sfiA genes were fully induced. Induction of the sfiA gene occurred, however, at higher doses in bacteria overproducing SSB protein than in bacteria with normal levels of SSB. Whereas the efficiency of excision repair was apparently increased by excess SSB, the efficiency of post-replication recombinational repair was reduced as judged by a decrease in the recombination proficiency between a prophage and ultraviolet-irradiated heteroimmune infecting phage. Following induction of ssb+ bacteria with mitomycin C, the cellular content of SSB was slightly increased. These results provide evidence that SSB modulates RecA protein-dependent activities in vivo. It is proposed that SSB favors the formation of short complexes of RecA protein and single-stranded DNA that mediate cleavage of the LexA and lambda repressors, while it delays the formation of long nucleoprotein filaments, thereby slowing down RecA-promoted recombinational events in uninduced as well as in induced bacteria.

Bacterial Proteins↗
PubMed2005

Sensorimotor transduction of time information is preserved in subjects with cerebellar damage.

The cerebellar contribution to motor entrainment through rhythmic auditory stimuli was analyzed by comparing rhythmic motor responses in subjects with cerebellar pathologies and in healthy controls. Eleven patients with cerebellar lesions and eight healthy subjects tapped in synchrony with an auditory rhythmic stimulus using a hand-held pencil-shaped electrode connected to a PC. A 60-stimulus sequence was delivered with an ISI of 500 ms and changed at random to a new ISI value with either consciously perceived (+/-50 ms) or unperceived tempo changes (+/-10 ms). Synchronization patterns for both groups were computed based on the timing of inter-response intervals (IRIs) and synchronization errors (SE). Variability of IRI as well as the timing of adaptation patterns after the tempo changes were modeled and analyzed mathematically using a logistic/sigmoid function. Healthy subjects performed with significantly lower IRI variability than cerebellar patients. Patients with focal lesions performed with significantly lower IRI variability than patients with atrophic lesions. Asymptote parameters during isochronous synchronization as well as slope angles and symmetry points of the adaptation curves after tempo perturbation showed no significant differences between groups. Present data indicate that temporal variability of rhythmic motor responses is differentially affected by distinct cerebellar pathologies but that motor entrainment to auditory rhythms is not affected by lesion of the cerebellar circuits.

Acoustic Stimulation↗
PubMed2005

The effect of MCP-1 depletion on chemokine and chemokine-related gene expression: evidence for a complex network in acute inflammation.

The expression of chemokines has been suggested to involve an interdependent network, with the absence of a single chemokine affecting the expression of multiple other chemokines. Monocyte chemoattractant protein (MCP-1), a member of C-C chemokine superfamily, plays a critical role in the recruitment and activation of leukocytes during acute inflammation. To examine the effect of the loss of MCP-1 on expression of the chemokine network, we compared the mRNA expression profiles of MCP-1(-/-) and wild type mice during the acute inflammatory phase of excisional wounds. Utilizing a mouse cDNA array containing 514 chemokine and chemokine related genes, the loss of MCP-1 was observed to cause a significant upregulation of nine genes (Decorin, Persephin, IL-1beta, MIP-2, MSP, IL1ra, CCR5, CCR3, IL-11) and significant downregulation of two genes (CCR4 and CD3Z) in acute wounds. The array data was confirmed by semi-quantitative RT-PCR. The effect of MCP-1 deletion on chemokine expression was further examined in isolated macrophages. Compared to wild type, LPS-stimulated peritoneal macrophages from MCP-1(-/-) mice showed a significant increase in the expression of RANTES, MIP-1beta, MIP-1alpha and MIP-2 mRNA. The data suggest that loss of a single chemokine perturbs the chemokine network not only in the setting of acute inflammation but even in an isolated inflammatory cell, the macrophage.

Animals↗
PubMed2006

Pro-oxidative diesel exhaust particle chemicals inhibit LPS-induced dendritic cell responses involved in T-helper differentiation.

BACKGROUND: Epidemiologic studies show that exposure to ambient particulate matter leads to asthma exacerbation. Diesel exhaust particles (DEPs), a model pollutant, act as an adjuvant for allergic sensitization. Increasing evidence shows that this effect could be mediated by an effect on dendritic cells (DCs). OBJECTIVE: Our aim was to elucidate the mechanism by which pro-oxidative DEP chemicals change DC function so that these antigen-presenting cells strengthen the immune response to an experimental allergen. METHODS: We exposed murine bone marrow-derived DCs and a homogeneous myeloid DC line, BC1, to DEPs and organic extracts made from these particles to determine how the induction of oxidative stress affects cellular maturation, cytokine production, and activation of antigen-specific T cells. RESULTS: DEP extracts induced oxidative stress in DCs. This change in redox equilibrium interfered in the ability of Toll-like receptor agonists to induce the expression of maturation receptors (eg, CD86, CD54, and I-A(d)) and IL-12 production. This perturbation of DC function was accompanied by decreased IFN-gamma and increased IL-10 induction in antigen-specific T cells. The molecular basis for the perturbation of DC function is the activation of a nuclear factor-erythroid 2 (NF-E2)-related factor 2-mediated signaling pathway that suppresses IL-12 production. NF-E2-related factor 2 deficiency abrogates the perturbation of DC function by DEPs. CONCLUSION: These data provide the first report that pro-oxidative DEP chemicals can interfere in T(H)1-promoting response pathways in a homogeneous DC population and provide a novel explanation for the adjuvant effect of DEPs on allergic inflammation. CLINICAL IMPLICATIONS: These data clarify the adjuvant effect of particulate air pollutants in allergic inflammatory disease.

Adjuvants, Immunologic↗
PubMed2005

The influence of different cultivation conditions on the metabolome of Fusarium oxysporum.

The two most widespread pentose sugars found in the biosphere are d-xylose and l-arabinose. They are both potential substrates for ethanol production. The purpose of this study was to better understand the redox constraints imposed to Fusarium oxysporum during utilization of pentoses. In order to increase ethanol yield and decrease by-product formation, nitrate was used as nitrogen source. The use of NADH, the cofactor in denitrification process when using nitrate as a nitrogen source, improved the ethanol yield on xylose to 0.89 mol mol(-1) compared to the ethanol yield achieved using ammonium as nitrogen source 0.44 mol mol(-1). The improved ethanol yield was followed by a 28% decrease in yield of the by-product xylitol. In order to investigate the metabolic pathway of arabinose and the metabolic limitations for the efficient ethanol production from this sugar, the extracellular and intracellular metabolite profiles were determined under aerobic and anaerobic cultivation conditions. The results of this study clearly show difficulties in channelling of glucose-1-P (G1P) to pentose phosphate pathway (PPP) and reduced NADPH regeneration, suggesting that NADPH becomes a limiting factor for arabinose conversion, resulting in excessive acetate production. Variations of the fungus intracellular amino and non-amino acid pool, under different culture conditions, were evaluated using principal component analysis (PCA). PCA projection of the metabolome data collected from F. oxysporum subjected to environmental perturbations succeeded to visualize different physiological states and the conclusions of this study were that the metabolite profile is unique according to: (1) the carbon source and (2) the oxygen supply, and to a lesser extent to the cultivation phase.

Adaptation, Physiological↗
PubMed2004

Nitric oxide acts on the mitochondria and protects human endothelial cells from apoptosis.

Proliferation of small blood vessels in synovial tissues is one of the pathologic features of rheumatoid arthritis. In this study we tested the hypothesis that nitric oxide (NO) protects endothelial cells (ECs) against apoptogenic agents in vitro. Human umbilical-vein endothelial cells (HUVECs) were cultured with and without NO donor S -nitro- N -acetylpenicillamine (SNAP) and further incubated in the presence or absence of Z-leucine-leucine-leucine-aldehyde (LLL-CHO), etoposide, or C2-ceramide. After cultivation, apoptosis of HUVECs was quantified on the basis of disruption of mitochondrial transmembrane potential (DeltaPsim), activation of caspases, and the presence of hypodiploid DNA-positive cells. Treatment of HUVECs with LLL-CHO, etoposide, or C2-ceramide induced DeltaPsim, activation of caspase-3, caspase-8, and caspase-9 and the appearance of hypodiploid DNA-positive cells. NO production in HUVECs was clearly increased by SNAP. Apoptotic cell death in HUVECs induced by LLL-CHO, etoposide, and C2-ceramide was significantly suppressed by SNAP treatment. HUVECs in vitro expressed Bcl-2, Bcl-xL, and Bax; however, expression was not changed by SNAP treatment in the presence or absence of LLL-CHO, etoposide, or C2-ceramide. Although the molecule(s) responsible for the protective effects of NO remains to be identified, our data imply that NO protects HUVECs against mitochondrial perturbation caused by apoptogenic agents. These results suggest that NO promotes endothelial-cell proliferation and angiogenesis in the synovial tissues of patients with rheumatoid arthritis and that NO may be a therapeutic target for rheumatoid arthritis.

Apoptosis↗
PubMed2006

The effect of uremia on platelet contractile force, clot elastic modulus and bleeding time in hemodialysis patients.

INTRODUCTION: Uremic bleeding frequently occurs in dialysis patients. Although its mechanism is not well characterized, acquired platelet dysfunction has been implicated in its pathogenesis. Skin bleeding time has been used to characterize platelet dysfunction in this population. However, the bleeding time is prone to error. The goal of this study was to compare the bleeding time to the novel platelet function parameters platelet contractile force and clot elastic modulus as well as platelet aggregation studies in controls and patients receiving maintenance hemodialysis. MATERIALS AND METHODS: Forty-five subjects completed this study (25 controls, 20 dialysis). All subjects had the Ivy skin bleeding time procedure performed, as well as the collection of whole blood samples for the determination of platelet contractile force, clot elastic modulus, % von Willebrand Factor antigen, and platelet aggregation studies. Pearson's correlation determined the relationships between skin bleeding time and platelet function and clot structure parameters and markers of renal dysfunction. RESULTS: Bleeding time was significantly prolonged in the dialysis group relative to controls. The platelet function parameters were not significantly different between groups. There was a significant relationship between bleeding time and creatinine concentration, however, no relationship existed between bleeding time and platelet function parameters. CONCLUSIONS: Skin bleeding time poorly correlates with measurements of platelet function. There were no significant differences noted in platelet function between the groups despite the prolongations in bleeding time in the dialysis group. These data may suggest that the bleeding time reflects perturbations in platelet adhesion or secretion, and not aggregation. Further study is needed to characterize platelet function in dialysis patients.

Adult↗
PubMed2006

Xenopus fibrillin regulates directed convergence and extension.

Fibrillin-based human diseases such as Marfan syndrome and congenital contractural arachnodactyly implicate fibrillins in the function and homeostasis of multiple adult tissues. Fibrillins are also expressed in embryos, but no early developmental role has been described for these proteins. We use three independent methods to reveal a role for Xenopus fibrillin (XF) at gastrulation. First, expressing truncated forms of XF in the embryo leads to failure of gastrulation concomitant with a dominant-negative effect on native fibrillin fibril assembly. Expressing truncated XF also inhibits normal progression of the patterned, polarized cell motility that drives convergence and extension at gastrulation and perturbs directed extension in cultured explants of dorsal mesoderm. Second, injection of a synthetic peptide encoding a cell-binding domain of XF into midgastrula embryos causes acute failure of gastrulation associated with defective fibrillin fibril assembly. These injections also reveal a critical role for this peptide in the fibril assembly process. Third, morpholino-mediated knockdown of translation of XF in the embryo also perturbs normal gastrulation and directed extension. Together, these data show that native Xenopus fibrillin is essential for the process of directed convergent extension in presumptive notochord at gastrulation.

Amino Acid Sequence↗
PubMedDate not supplied

Control of endocardial cushion and cardiac valve maturation by BMP signaling pathways.

Congenital heart defects, the leading cause of deaths from birth defects, are estimated to occur in close to 1% of live newborns. Among these, abnormal septation of the heart and valve anomalies are the most frequent forms. Despite progress defining several genes involved in normal heart development, we still have a limited understanding of the signaling pathways involved in morphogenesis of the outflow tract (OFT) and, to date, very few genes have been identified that are responsible for defects in humans. Bone Morphogenetic Protein (BMP) signaling pathways are emerging as vital regulators of multiple aspects of cardiogenesis, including the septation of the OFT and valve maturation. Genetic and other in vivo evidence is now supporting the role for BMPs as inducers of endocardial cushion epithelial-to-mesenchymal transformation that was suggested by in vitro explant studies as well as by their patterns of expression in the developing heart. Here, we review briefly the in vitro data, and detail the novel mouse models where perturbed BMP signaling pathways result in impaired OFT septation and semilunar valvulogenesis. We propose that growth of the OFT valve cushions is regulated by the level of BMP signaling, under the control of other signaling pathways.

Animals↗
PubMed1992

Ion channels in icosahedral virus: a comparative analysis of the structures and binding sites at their fivefold axes.

An analysis of the crystallographically determined structures of the icosahedral protein coats of Tomato Bushy Stunt Virus, Southern Bean Mosaic Virus, Satellite Tobacco Necrosis Virus, Human Rhinovirus 14 and Mengovirus around their fivefold axes is presented. Accessibilities surfaces, electrostatic energy profile calculations, ion-protein interaction energy calculations, free energy perturbation methods and comparisons with structures of chelating agents are used in this study. It is concluded that the structures built around the viral fivefold axes would be adequate for ion binding and transport. Relative ion preferences are derived for the binding sites, using free energy perturbation methods, which are consistent with the experimental data when available. In the cases where crystallographic studies determined the existence of ions on the fivefold axes, our results indicate that they would correspond to ions in crystallization or purification buffers. The environment of the fivefold axes are rich in polar residues in all icosahedral viral structures whose atomic coordinates are available, including some that are not being analyzed in detail in this work. The fivefold channel-like structures have most of the basic properties expected for real ion channels including a funnel at the entrance, a polar internal environment with frequent alternation of acidic and basic residues, ion binding sites, the capability to induce ion dehydration and ion transit from the external viral surface to the binding sites.

Capsid↗
PubMed1983

Interaction of bleomycin A2 with poly(deoxyadenylylthymidylic acid). A proton nuclear magnetic resonance study of the influence of temperature, pH, and ionic strength.

The binding of bleomycin A2 to poly(deoxyadenylylthymidylic acid) [poly(dA-dT)] has been monitored by proton nuclear magnetic resonance spectroscopy. This study includes an analysis of the effects of temperature, ionic strength, and pH. Sites of drug-nucleic acid interaction have been delineated on the basis of chemical shift perturbations of drug and nucleic acid resonances. The data indicate that the binding of the antibiotic occurs with partial intercalation of the aromatic bithiazole group and immobilization of the cationic dimethylsulfonium group. This complex dissociates as the nucleic acid is denatured to the single-stranded form. The absence of significant pH effects suggests that the N terminus of bleomycin A2, which contains the titratable groups, does not contribute to the interaction of the drug molecule with poly(dA-dT). The problems associated with assigning a specific geometry to the drug-nucleic acid complex are discussed.

Bleomycin↗
PubMed2003

Avian sulfhydryl oxidase is not a metalloenzyme: adventitious binding of divalent metal ions to the enzyme.

Metal- and flavin-dependent sulfhydryl oxidases catalyze the generation of disulfide bonds with reduction of oxygen to hydrogen peroxide. The mammalian skin enzyme has been reported to be copper-dependent, but a recent protein sequence shows it belongs to the Quiescin/sulfhydryl oxidase (QSOX) flavoprotein family. This work demonstrates that avian QSOX is not a metalloenzyme, and that copper and zinc ions inhibit the oxidation of reduced pancreatic ribonuclease by the enzyme. Studies with Zn(2+), as a redox inactive surrogate for copper, show that one Zn(2+) binds to four-electron-reduced QSOX by diverting electrons away from the flavin and into two of the three redox active disulfide bridges in the enzyme. The resulting zinc complex is modestly air-stable, reverting to a spectrum of the native protein with a t(1/2) of 40 min, whereas the four-electron-reduced native QSOX is reoxidized in less than a second under comparable conditions. Using tris(2-carboxyethyl)phosphine hydrochloride (TCEP), an alternate substrate of QSOX that binds Zn(2+) relatively weakly (unlike dithiothreitol), allows rapid inhibition of oxidase activity to be demonstrated at low micromolar metal levels. Zinc binding was followed by rapid-scanning spectrophotometry. Copper also binds the four-electron-reduced form of QSOX with a visible spectrum suggestive of active site occupancy. In addition to interactions with the reduced enzyme, dialysis experiments show that multiple copper and zinc ions can bind to the oxidized enzyme without the perturbation of the flavin spectrum seen earlier. These data suggest that a reinvestigation of the metal content of skin sulfhydryl oxidases is warranted. The redox-modulated binding of zinc to QSOX is considered in light of evidence for a role of zinc-thiolate interactions in redox signaling and zinc mobilization.

Amino Acid Sequence↗
PubMed1999

Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase.

Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P. denitrificans oxidase). Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase. The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase. Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center. The data suggest that E89II (E. coli numbering) is critical for the function of the heme copper oxidases. The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant. Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.

Alanine↗
PubMed2006

Hydrophobicity, shape, and pi-electron contributions during translesion DNA synthesis.

Translesion DNA synthesis, the ability of a DNA polymerase to misinsert a nucleotide opposite a damaged DNA template, represents a common route toward mutagenesis and possibly disease development. To further define the mechanism of this promutagenic process, we synthesized and tested the enzymatic incorporation of two isosteric 5-substituted indolyl-2'deoxyriboside triphosphates opposite an abasic site. The catalytic efficiency for the incorporation of the 5-cyclohexene-indole derivative opposite an abasic site is 75-fold greater than that for the 5-cyclohexyl-indole derivative. The higher efficiency reflects a substantial increase in the k(pol) value (compare 25 versus 0.5 s(-1), respectively) as opposed to an influence on ground-state binding of either non-natural nucleotide. The faster k(pol) value for the 5-cyclohexene-indole derivative indicates that pi-electron density enhances the rate of the enzymatic conformational change step required for insertion opposite the abasic site. However, the kinetic dissociation constants for the non-natural nucleotides are identical and indicate that pi-electron density does not directly influence ground-state binding opposite the DNA lesion. Surprisingly, each non-natural nucleotide can be incorporated opposite natural templating bases, albeit with a greatly reduced catalytic efficiency. In this instance, the lower catalytic efficiency is caused by a substantial decrease in the k(pol) value rather than perturbations in ground-state binding. Collectively, these data indicate that the rate of the conformational change during translesion DNA synthesis depends on pi-electron density, while the enhancement in ground-state binding appears related to the size and shape of the non-natural nucleotide.

DNA↗
PubMed2006

Molecular recognition between protein and nicotinamide dinucleotide in intact, proton-translocating transhydrogenase studied by ATR-FTIR Spectroscopy.

Nicotinamide dinucleotide binding to transhydrogenase purified from Escherichia coli was investigated by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Detergent-free transhydrogenase was deposited as a thin film on an ATR prism, and spectra were recorded during perfusion with buffers in the presence and absence of dinucleotide (NADP(+), NADPH, NAD(+), or NADH) in both H(2)O and D(2)O media. IR spectral changes were attributable to the bound dinucleotides and to changes in the protein itself. The dissociation constant of NADPH was estimated to be approximately 5 muM from a titration of the magnitude of the IR changes against the nucleotide concentration. IR spectra of related model compounds were used to assign principle bands of the dinucleotides. This information was combined with IR data on amino acids and with protein crystallographic data to identify interactions between specific parts of the dinucleotides and their binding sites in the protein. Several IR bands of bound nucleotide were sharpened and/or shifted relative to those in aqueous solution, reflecting a restriction to motion and a change in environment upon binding. Alterations in the protein secondary structure indicated by amide I/II changes were distinctly different for NADP(H) and for NAD(H) binding. The data suggest that NADP(H) binding leads to perturbation of a deeply buried part of the polypeptide backbone and to protonation of a carboxylic acid residue.

Deuterium Exchange Measurement↗
PubMed1996

An approach to rapid estimation of relative binding affinities of enzyme inhibitors: application to peptidomimetic inhibitors of the human immunodeficiency virus type 1 protease.

This report describes a method for rapid assessment of the binding affinities of a series of analogous ligands to an enzyme. This approach is based on two variables (scores), representing (i) the enthalpy of binding and (ii) the strength of hydrophobic interaction. The method is then used to evaluate the binding of 11 different peptidomimetic inhibitors to the HIV-1 protease. Three-dimensional structures of these enzyme-inhibitor complexes are modeled based on the crystal structures of HIV-1 protease complexes with the known inhibitors. These structures are minimized using the AMBER force field, and the scores of binding enthalpy for each of the ligands are calculated. A second score to represent the hydrophobic interaction between a pair of atoms uses an exponential function of distance between the atoms and the product of their atomic hydrophobicity constants. This exponential function is used to assess the hydrophobic interaction energy between an enzyme and its inhibitor and also to compute and display a 'molecular hydrophobicity map' as a 3D visualization tool. These methods are then applied to obtain trends in relative binding affinities of pairs of analogous inhibitors. Calculated scores agree well with corresponding results from thermodynamic cycle perturbation (TCP) simulations as well as experimental binding data. Since the proposed calculations are computationally cheaper and faster than TCP calculations, it is suggested that these scores can form the basis for rapid, preliminary theoretical screening of proposed derivatives of an inhibitor prior to TCP analysis, synthesis, and testing.

HIV Protease↗
PubMed2006

Quantitative proteomic and genomic profiling reveals metastasis-related protein expression patterns in gastric cancer cells.

Gastric cancer is a leading cause of death worldwide, and patients have an overall 5-year survival rate of less than 10%. Using quantitative proteomic techniques together with microarray chips, we have established comprehensive proteome and transcriptome profiles of the metastatic gastric cancer TMC-1 cells and the noninvasive gastric cancer SC-M1 cell. Our qualitative protein profiling strategy offers the first comprehensive analysis of the gastric cancer cell proteome, identifying 926 and 909 proteins from SC-M1 and TMC-1 cells, respectively. Cleavable isotope-coded affinity tagging analysis allows quantitation of a total of 559 proteins (with a protein false-positive rate of <0.005), and 240 proteins were differentially expressed (>1.3-fold) between the SC-M1 and TMC-1 cells. We identified numerous proteins not previously associated with gastric cancer. Notably, a large subset of differentially expressed proteins was associated with tumor metastasis, including proteins functioning in cell-cell and cell-extracellular matrix (cell-ECM) adhesion, cell motility, proliferation, and tumor immunity. Gene expression profiling by DNA microarray revealed differential expression (of >2-fold) of about 1000 genes. The weak correlation observed between protein and mRNA profiles highlights the important complementarities of DNA microarray and proteomics approaches. These comparative data enabled us to map the disease-perturbed cell-cell and cell-ECM adhesion and Rho GTPase-mediated cytoskeletal pathways. Further validation of a subset of genes suggests the potential use of vimentin and galectin 1 as markers for metastasis. We demonstrate that combining proteomic and genomic approaches not only provides a rapid, robust, and sensitive platform to elucidate the molecular mechanisms underlying gastric cancer metastasis but also may identify candidate diagnostic markers and therapeutic targets.

Biomarkers, Tumor↗
PubMed1998

Expression of fission yeast cdc25 alters the frequency of lateral root formation in transgenic tobacco.

Lateral root formation was examined following the expression of a fission yeast mitotic regulator gene, cdc25, under the control of a tetracycline-inducible promoter in cultured roots of tobacco. Over expression of cdc25 in fission yeast results in premature cell division at a reduced cell size. Our aim was to examine whether cdc25 expression would affect cell size in the tobacco roots, and what effect this would have on lateral root morphogenesis. Transgene integration was confirmed by Southern blotting; it was inherited as a dominant Mendelian trait. Conditions for optimal expression, determined using plants transformed with gus under the control of the same promoter, were: addition of tetracycline (5 micrograms/ml) every 72 h, to cultured roots in Murashige-Skoog liquid medium in darkness at 27 degrees C. After the addition of tetracycline, cdc25 transcripts were detected using RT-PCR, initially after 48 h, and more strongly after 72 h. Appearance of cdc25 transcripts was followed by major changes in the roots. Compared with controls, lateral root primordia were initiated more frequently, were significantly smaller and comprised smaller cells at mitosis. However, cdc25 expression did not perturb normal development of the lateral roots. The data are consistent with cdc25 expression leading to a greater frequency of lateral root primordium formation and establishing a new threshold size for cell division in the primordia which was then maintained throughout subsequent development.

Cell Cycle Proteins↗