Immunohistochemical profile of galectin-8 expression in benign and malignant tumors of epithelial, mesenchymatous and adipous origins, and of the nervous system.

This study aims to investigate whether the immunohistochemical expression of galectin-8 could be used as a diagnostic marker in tumor tissues of various histogenetic origins including specimens from epithelial (n=145), mesenchymatous (n=16), adipous (n=10) and central and peripheral nervous system (n=25) tissue, and 4 mesotheliomas. Immunohistochemical reactions were carried out with a polyclonal anti-galectin-8 antibody and histological slides from tissues derived from the files of the Laboratory of Anatomopathology of University Erasmus Hospital, Brussels. Formalin-fixed paraffin-embedded tissues of 45 normal cases as well as 41 benign and 114 malignant tumors were studied. Marked decreases in immunohistochemical galectin-8 expression were observed in colon (p=0.001), pancreas (p=0.007), liver (p=0.0008), skin (p=0.002) and larynx (p=0.02) tissue when comparing malignant tissue to normal tissue and/or benign tumors. The reverse relationship was observed for breast tissue (p=0.007). No statistically significant differences (p>0.05) were detected when comparing normal tissue and/or benign to malignant tumors in lung, bladder, kidney, prostate and stomach tissue. Significant galectin-8 expression was also measured in non-epithelial tissue including tumors of the central and peripheral nervous system as well as in skeletal muscle and mesotheliomas. Immunohistochemical monitoring of galectin-8 thus reveals an organ-type-dependent regulation of expression upon malignant transformation of various tissue types of epithelial origin. This observation will prompt further studies to delineate any relationship with prognosis.

Galectin-8 functions as a matricellular modulator of cell adhesion.

The interaction of cells with the extracellular matrix regulates cell adhesion and motility. Here we demonstrate that different cell types adhere and spread when cultured in serum-free medium on immobilized galectin-8, a mammalian beta-galactoside-binding protein. At maximal doses, galectin-8 is equipotent to fibronectin in promoting cell adhesion and spreading. Cell adhesion to immobilized galectin-8 is mediated by sugar-protein interactions with integrins, and galectin-8 triggers integrin-mediated signaling cascades including Tyr phosphorylation of focal adhesion kinase and paxillin. Cell adhesion is potentiated in the presence of Mn(2+), whereas it is interrupted in the presence of soluble galectin-8, integrin beta(1) inhibitory antibodies, EDTA, or thiodigalactoside but not by RGD peptides. Furthermore, cells readily adhere onto immobilized monoclonal galectin-8 antibodies, which are equipotent to integrin antibodies in promoting cell adhesion. Cell adhesion to immobilized galectin-8 is partially inhibited by serum proteins, suggesting that complex formation between immobilized galectin-8 and serum components generates a matrix that is less supportive of cell adhesion. Accordingly, cell motility on immobilized galectin-8 readily takes place in the presence of serum. Truncation of the C-terminal half of galectin-8, including one of its two carbohydrate recognition domains, largely abolishes its ability to modulate cell adhesion, indicating that both carbohydrate recognition domains are required to maintain a functional form of galectin-8. Collectively, our findings implicate galectin-8 as a physiological modulator of cell adhesion. When immobilized, it functions as a matrix protein equipotent to fibronectin in promoting cell adhesion by ligation and clustering of cell surface integrin receptors. In contrast, when present in excess as a soluble ligand, galectin-8 (like fibronectin) forms a complex with integrins that negatively regulates cell adhesion. Because of its dual effects on the adhesive properties of the cells and its association with fibronectin, galectin-8 might be considered a novel type of matricellular protein.

Galectin-8 binding to integrins inhibits cell adhesion and induces apoptosis.

The interaction of cells with the extracellular matrix regulates cell adhesion, motility, growth, survival and differentiation through integrin-mediated signal transduction. Here we demonstrate that galectin-8, a secreted mammalian (beta)-galactoside binding protein, inhibits adhesion of human carcinoma (1299) cells to plates coated with integrin ligands, and induces cell apoptosis. Pretreatment of the cells with Mn(2+), which increases the affinity of integrins for their ligands, abolished the inhibitory effects of galectin-8. The inhibitory effects of galectin-8 were specific and were not mimicked by plant lectins or other galectins (galectin-1 and galectin-3). In accordance with its anti-adhesive effects, transfection of galectin-8 cDNA into 1299 cells significantly reduced (by 75%) colony formation, when compared to the number of colonies formed by cells transfected with an empty vector. Affinity chromatography over immobilized galectin-8 indicated that few membrane proteins interacted with galectin-8 in a sugar-dependent manner. Microsequencing and western immunoblotting revealed that (alpha)(3)(beta)(1 )integrin derived from 1299 as well as other cells (e.g. HeLa and human endothelial cells) is a major galectin-8 binding-protein. Furthermore, immunoprecipitation and immunohistochemical studies suggested that endogenous galectin-8, secreted from 1299 cells, forms complexes with (alpha)(3)(beta)(1) integrins expressed on the surface of 1299 cells. Galectin-8 also interacts with other members of the integrin family, like (alpha)(6)(beta)(1 )integrins. In contrast, galectin-8 only minimally interacts with (alpha)(4 )or (beta)(3 )integrins. We propose that galectin-8 is an integrin binding-protein that interacts to a different extent with several, but not all members of the integrin family. Binding of galectin-8 modulates integrin interactions with the extracellular matrix and thus regulates cell adhesion and cell survival.

Competitive hybridization: theory and application in isolation and quantification of differentially regulated genes.

Competitive hybridization is a simple yet powerful method that was developed to screen cDNA libraries for differentially regulated genes. The method is based on competition between unlabeled cDNA from the mRNA of one sample and labeled cDNA from another sample. By manipulating the amount of competing unlabeled cDNA, background signals from the nonregulated genes can be increased or reduced, enabling the signals from differentially regulated genes to be contrasted and to be identified in a quantitative manner. To demonstrate the feasibility of the method, we screened a citrus cDNA library for ethylene-induced genes and identified three genes with different levels of ethylene induction. The mathematical basis of the method and its possible application in gene chip technology are discussed.

Effect of buffer solutions on activation of Shamouti orange pyrophosphate-dependent phosphofructokinase by fructose 2,6-bisphosphate.

Shamouti phosphofructokinase (PFP) activation depends on the presence of fructose 2,6-bisphosphate (Fru-2,6-P2) in the glycolytic reaction. The effect of activation by Fru-2,6-P2 differs considerably, however, according to the buffer (pH 8.0) in which the reaction is performed: Ka = 2.77 +/- 0.3 nM in Hepes-NaOH and 7.75 +/- 1.49 nM in Tris-HCl. The presence of chloride ions (39 mM) in the Tris-HCl buffer inhibits PFP. Indeed, when using a Hepes-NaOH buffer and then adding 39 mM NaCl, Ka = 8.12 +/- 0.52 nM. The Ki for chloride ions is approximately 21.7 mM. In the gluconeogenic reaction, Shamouti PFP generally showed a high endogenous activity. Addition of Fru-2,6-P2 did not modify the velocity and the Vmax of the enzyme; however, its presence increased the affinity of the enzyme for Fru-1,6-P2 from 200 +/- 15.6 microM in absence of Fru-2,6-P2 to 89 +/- 10.3 microM in its presence (10 microM). In the presence of chloride (39 mM), the affinity for the substrate decreased with K(m) = 150 +/- 14 microM. The calculated Ki for chloride ions equals 56.9 mM. In both the glycolytic and the gluconeogenic reactions, Vmax is not affected; therefore, the inhibition mode of chloride is competitive.

Kinetic properties of ATP-dependent phosphofructokinase from grapefruit juice sacs: effect of TCA cycle intermediates.

Grapefruit juice sac ATP-PFK was studied kinetically for its substrates ATP and Fru-6-P at pH = 7.5. The Km for ATP is equal to 39.8 +/- 4.6 microM. ATP becomes inhibitory at concentrations above 80 microM. The Km for ATP is not affected by the addition of citrate (10 mM). For Fru-6-P, the saturation curve is sigmoidal, with an S0.5 equal to 0.17 +/- 0.03 mM, in the presence of Mg++ (2.5 mM) and ATP (1 mM). ATP-PFK shows a negative cooperativity at lower concentrations of Fru-6-P (h = 0.5), while higher concentrations of the substrate induce a positive cooperation (h = 1.5). The presence of citrate affects the S0.5 affinity value, but not the Vmax. The presence of citrate (10 mM) removes the cooperative effect at higher concentrations of the substrate, as h = 1.0. A theoretical Ki for citrate was calculated and equals 1.30 mM.

Citrate uptake into tonoplast vesicles from acid lime (Citrus aurantifolia) juice cells.

Citrate transport into the vacuoles of acid lime juice cells was investigated using isolated tonoplast vesicles. ATP stimulated citrate uptake in the presence or in the absence of a Delta mu H+. Energization of the vesicles only by an artificial K+ gradient (establishing an inside-positive Delta psi) also resulted in citrate uptake as was the case of a Delta pH dominated Delta mu H+. Addition of inhibitors to endomembrane ATPases showed no direct correlation between the inhibition to the tonoplast bound H+/ATPase and citrate uptake. The data indicated that, although some citrate uptake can be accounted for by Delta psi and by a direct primary active transport mechanism involving ATP, under in vivo conditions of vacuolar pH of 2.0, citrate uptake is driven by Delta pH.

Bacterial cellulose-binding domain modulates in vitro elongation of different plant cells

Recombinant cellulose-binding domain (CBD) derived from the cellulolytic bacterium Clostridium cellulovorans was found to modulate the elongation of different plant cells in vitro. In peach (Prunus persica L.) pollen tubes, maximum elongation was observed at 50 &mgr;g mL-1 CBD. Pollen tube staining with calcofluor showed a loss of crystallinity in the tip zone of CBD-treated pollen tubes. At low concentrations CBD enhanced elongation of Arabidopsis roots. At high concentrations CBD dramatically inhibited root elongation in a dose-responsive manner. Maximum effect on root hair elongation was at 100 &mgr;g mL-1, whereas root elongation was inhibited at that concentration. CBD was found to compete with xyloglucan for binding to cellulose when CBD was added first to the cellulose, before the addition of xyloglucan. When Acetobacter xylinum L. was used as a model system, CBD was found to increase the rate of cellulose synthase in a dose-responsive manner, up to 5-fold compared with the control. Electron microscopy examination of the cellulose ribbons produced by A. xylinum showed that CBD treatment resulted in a splayed ribbon composed of separate fibrillar subunits, compared with a thin, uniform ribbon in the control.

Fru 2,6-P2 and citrate: intracellular distribution in citrus tissues and effect on grapefruit leaf PFP.

Grapefruit leaf PFP was studied for its activation by fructose 2,6-bisphosphate (Fru 2,6-P2) in the forward and reverse reactions. In the glycolytic reaction, a Ka of 4.0 +/- 0.12 nM was obtained. This constant is affected by the presence of increasing concentrations of citrate (1, 5 and 20 nM) with a Ka(citrate) of 4.5 +/- 0.09, 6.9 +/- 0.05 and 38.2 +/- 1.4 respectively. The inhibition mode of citrate is competitive with Fru 2,6-P2, but non-linear in relation of increasing concentrations of the inhibitor. The intracellular distribution and concentration of the key regulatory metabolite Fru 2,6-P2 was further investigated in citrus leaves and juice cells. Fru 2,6-P2 was only found in the cytosol of juice cells. Fru 2,6-P2 was detected under both conditions with higher concentrations found under aerobiosis.

Characterization of abscisic Acid-induced ethylene production in citrus leaf and tomato fruit tissues.

Abscisic acid (ABA) significantly stimulated ethylene production in citrus (Citrus sinensis [L.] Osbeck, cv Shamouti orange) leaf discs. The extent of stimulation was dependent upon the concentration of ABA (0.1-1 milimolar) and the duration of treatment (15-300 minutes). Aging the discs before applying ABA increased ABA-induced ethylene production due to enhancement of both ethylene-forming enzyme activity and the responsiveness of ABA. Discs excised from mature leaves were much more responsive to ABA than discs excised from young or senescing leaves. ABA stimulated ethylene production shortly after application, suggesting that ABA does not enhance ethylene production via the acceleration of senescence. The stimulating effect of ABA on ethylene production resulted mainly from the enhancement of 1-aminocylopropane-1-carboxylic acid synthesis. Stimulation of ethylene production by ABA in intact citrus leaves and tomato (Lycopersicon esculentum Mill., cv Castlemart) fruit was small but could be increased by various forms of wounding.

Ethylene-enhanced catabolism of [C]indole-3-acetic Acid to indole-3-carboxylic Acid in citrus leaf tissues.

Exogenous [(14)C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole-3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [(14)C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGIu). Increased formation of ICGIu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGIu were identified by combined gas chromatography-mass spectrometry. Formation of ICGIu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene reduces endogenous IAA levels.

Quail (Coturnix japonica) protamine, full-length cDNA sequence, and the function and evolution of vertebrate protamines.

Using the chicken protamine gene as a probe, we have isolated and sequenced several positive clones from a quail testis cDNA library which reveal the complete sequence for the quail protamine cDNA. The predicted amino acid sequence for the quail protamine contains the N-terminal tetrapeptide ARYR present in the N-terminal region of the mammalian protamines as well as several conserved motifs and arginine clusters. In addition the size of the quail protamine (56 amino acids) is closer to that of mammals (50 amino acids) than that of the chicken (61 amino acids). Altogether this data strongly suggests the existence of an avian-mammalian protamine gene line during evolution. Southern blot analysis suggests a small number of copies (2) per haploid genome (similar to that of chicken). The reported quail protamine cDNA sequence is the second avian protamine for which the amino acid sequence is available so far and provides new insights into vertebrate protamine function and evolution.

The tube esophagram: a technique for obtaining a detailed double-contrast examination of the esophagus.

Although double-contrast esophagography is capable of delineating fine surface morphologic detail in the esophagus, it is not possible to obtain an optimal examination on all patients. Tube esophagography is a complementary technique that can provide a more detailed double-contrast examination of the esophagus. This procedure was performed on 45 patients in whom the routine double-contrast study was inconclusive. The tube esophagram contributed significantly to the radiologic evaluation in 33 cases, providing additional information in 23 and actually altering the final radiologic diagnosis in 10. The tube esophagram was particularly useful in depicting the distal esophagus when the initial double-contrast study was suboptimal due to inadequate distension and/or barium pooling that obscured mucosal detail in this region. The tube esophagram is a valuable adjunctive procedure that can lead to a more definitive radiologic diagnosis when the routine double-contrast examination is inconclusive.

Ethylene binding during leaf development and senescence and its inhibition by silver nitrate.

Ethylene binding in situ was tested in leaves of tobacco (Nicotiana tabacum L. var. Xanthi) and of gynoecious and monoecious genotypes of cucumber (Cucumis sativus L.) during their development and senescence. Ethylene binding per gm fresh leaf remained constant during early stages of tobacco leaf development. It decreased in fully expanded leaves and during senescence of detached tobacco leaves. On a per leaf basis, ethylene binding increased as the leaf developed. The pattern for changes in ethylene binding by gynoecious and monoecious cucumber leaves were different from each other. Short-term treatment of detached tobacco and cucumber leaves with a solution of AgNO(3) (50 µM) markedly decreased their ability to bind ethylene concomitant to an inhibition in chlorophyll breakdown. The data support the hypothesis that ethylene-induced chlorophyll breakdown during leaf senescence may require the binding of ethylene to its receptor in vivo.

Non-specific normal immunosuppressive protein (Nip): purification and further biochemical characterization.

The non-specific normal immunosuppressive protein (Nip) has been described in our laboratory and its biological activity was extensively studied. In the present study, further purification analyses of Nip were conducted. Fractionation of Nip by Ultrogel AcA 34 column resulted in peak (I) that displayed Nip activity in that it exhibited marked inhibition of in-vitro blastogenic responses of human lymphocytes to mitogenic stimulation, in vitro it inhibited EL4 tumour-cell proliferation. Partial dissociation of Ultrogel peak I on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) resulted chiefly in three bands: one of high molecular weight, which is considered to be the carrier, an intermediate band of 50,000-60,000 (under non-reducing conditions) and a band of high mobility and low molecular weight approximately 6,000-14,000. Further fractionation of peak I on Sepharose-6B in 6 M guanidine-HCl resulted in two main peaks. The biological activity resided in the second peak, which corresponded to the low molecular weight fraction of 6,000-14,000. Nip is suggested to be a complex molecule comprised of a low molecular weight peptide or glycopeptide, which displays the biological activity, and a macromolecular glycoprotein carrier, which conserves its stability.

Drug-induced esophagitis detected by double-contrast radiography.

Patients with esophageal symptoms following drug ingestion underwent double-contrast upper gastrointestinal studies, and radiographic findings are described. Superficial esophageal ulceration and subtle mucosal abnormalities, which have not been seen on single-contrast radiographs, were confirmed on double-contrast radiographs. Erosions or ulcers usually occur in the region of the aortic arch and occasionally lower in the esophagus. Repeat esophagrams after withdrawal of the medication indicate resolution of the symptoms.

Effect of ethylene on [C]indole-3-acetic Acid metabolism in leaf tissues of woody plants.

The effect of ethylene on [(14)C]indole-3-acetic acid (IAA) metabolism was investigated in defoliation sensitive leaf tissues of citrus (Citrus sinensis) and resistant leaf tissues of eucalyptus (Eucalyptus camaldulensis). IAA metabolites were fractionated into 80% ethanol-soluble, H(2)O-soluble, NaOH-soluble, and insoluble components. In citrus, pretreatment with 25 microliters per liter ethylene for 24 hours significantly increased the amount of ethanol- and H(2)O-extractable conjugates during the first hour of incubation in [(14)C]IAA and increased 3- to 4-fold the formation of NaOH-extractable conjugates during the entire 6-hour incubation period. However, induction of the IAA-aspartate conjugation system was inhibited by ethylene. In eucalyptus, ethylene pretreatment only slightly stimulated the formation of IAA metabolites. Increased formation of ethanol-extractable conjugates in ethylene-pretreated eucalyptus tissues was observed only after 6 hours of incubation. Chromatographic analysis indicated that the ethanol and H(2)O extracts of both species contained various low molecular weight conjugates, whereas in citrus leaf tissues high molecular weight conjugates accounted for most of the greater radioactivity detected in the NaOH extracts as a result of ethylene-pretreatment. It is suggested that ethylene may reduce the level of endogenous IAA in citrus leaf tissues by stimulating IAA conjugation.

Cefotaxime in the treatment of infections of the skin and skin structure.

In a series of open, noncomparative studies, cefotaxime was given to 360 hospitalized patients with bacterial wound infections such as cellulitis, abscesses, or necrotizing ulcers of the skin or subcutaneous tissues. The drug was administered intramuscularly or intravenously in a mean dosage of 4 g per day (range, 1.4-12.0 g per day) in three or four equal doses for at least five days. Clinical response to therapy could not be evaluated for 100 patients, and bacterologic response could not be evaluated for 145 patients. Clinical response was satisfactory in 93.5% of the 260 patients for whom therapy could be evaluated, and bacterial response was satisfactory in 84% of the 225 patients for whom therapy could be evaluated. These rates of response include both single- and multiple-pathogen infections. There were nine instances of superinfection.

Inhibition of ethylene biosynthesis by aminoethoxyvinylglycine and by polyamines shunts label from 3,4-[C]methionine into spermidine in aged orange peel discs.

The flux of radioactivity from 3,4-[(14)C]methionine into S-adenosyl-l-methionine (SAM), 1-aminocyclopropane-1-carboxylic acid (ACC), spermine, and spermidine while inhibiting conversion of ACC to ethylene by 100 millimolar phosphate and 2 millimolar Co(2+) was studied in aged peel discs of orange (Citrus sinensis L. Osbeck) fruit. Inhibition up to 80% of ethylene production by phosphate and cobalt was accompanied by a 3.3 times increase of label in ACC while the radioactivity in SAM was only slightly reduced. Aminoethoxyvinylglycine (AVG) increased the label in SAM by 61% and reduced it in ACC by 47%. Different combinations of standard solution, in which putrescine or spermidine were administered alone or with AVG, demonstrated clearly that inhibition of ethylene biosynthesis-at the conversion of SAM to ACC-by AVG, exogenous putrescine or exogenous spermidine, stimulated the incorporation of 3,4-[(14)C]methionine into spermidine.

Growth and metabolic activity of lemon juice vesicle explants in vitro.

Actively growing juice-vesicle explants, established from small lemon (Citrus limon L. Burm. f. var. ;Eureka') fruits, were cultured in vitro on defined media. The juice vesicles continued to enlarge in culture for several months, without callus proliferation, and their initial growth was promoted by indoleacetic acid, gibberellic acid, and N(6)-benzyladenine.The metabolic competence of the explants, and its relevance to whole fruit development, was further investigated. Juice vesicles continued to accumulate protein on all media, for at least 16 days, but sugars did not change much during culture. Acid invertase increased dramatically during the first days of culture, and its specific activity was markedly promoted by indoleacetic acid, and to a lesser degree by gibberellic acid. Total peroxidase of juice vesicles increased progressively up to the 11th day of culture, and its specific activity was promoted by all three hormones, especially by N(6)-benzyladenine. Explant growth was accompanied by appearance of several cathodic and anodic isoperoxidases.The results suggest that the study of this unique juice vesicle system may contribute to a better understanding of fruit development.