OsbZIP52/RISBZ5 is a member of the basic leucine zipper (bZIP) transcription factor (TF) family in rice (Oryza sativa) isolated from rice (Zhonghua11) panicles. Expression of the OsbZIP52 gene was strongly induced by low temperature (4°C) but not by drought, PEG, salt, or ABA. The subcellular localization of OsbZIP52-GFP in onion (Allium cepa) epidermis cells revealed that OsbZIP52 is a nuclear localized protein. A transactivation assay in yeast demonstrated that OsbZIP52 functions as a transcriptional activator and can specifically bind to the G-box promoter motif. In a yeast two-hybrid (Y-2-H) experiment, OsbZIP52 was able to form homodimeric complexes. Rice plants overexpressing OsbZIP52 showed significantly increased sensitivity to cold and drought stress. Real-time PCR analysis revealed that some abiotic stress-related genes, such as OsLEA3, OsTPP1, Rab25, gp1 precursor, β-gal, LOC_Os05g11910 and LOC_Os05g39250, were down-regulated in OsbZIP52 overexpression lines. These results suggest that OsbZIP52/RISBZ5 could function as a negative regulator in cold and drought stress environments.

Normal cell growth and division in the yeast Saccharomyces cerevisiae involve dramatic and frequent changes in the organization of the actin cytoskeleton. Previous studies have suggested that the reorganization of the actin cytoskeleton in accordance with cell cycle progression is controlled, directly or indirectly, by the cyclin-dependent kinase Cdc28. Here we report that by isolating rapid-death mutants in the background of the Start-deficient cdc28-4 mutation, the essential yeast gene PAN1, previously thought to encode the yeast poly(A) nuclease, is identified as a new factor required for normal organization of the actin cytoskeleton. We show that at restrictive temperature, the pan1 mutant exhibited abnormal bud growth, failed to maintain a proper distribution of the actin cytoskeleton, was unable to reorganize actin the cytoskeleton during cell cycle, and was defective in cytokinesis. The mutant also displayed a random pattern of budding even at permissive temperature. Ectopic expression of PAN1 by the GAL promoter caused abnormal distribution of the actin cytoskeleton when a single-copy vector was used. Immunofluorescence staining revealed that the Pan1 protein colocalized with the cortical actin patches, suggesting that it may be a filamentous actin-binding protein. The Pan1 protein contains an EF-hand calcium-binding domain, a putative Src homology 3 (SH3)-binding domain, a region similar to the actin cytoskeleton assembly control protein Sla1, and two repeats of a newly identified protein motif known as the EH domain. These findings suggest that Pan1, recently recognized as not responsible for the poly(A) nuclease activity (A. B. Sachs and J. A. Deardorff, erratum, Cell 83:1059, 1995; R. Boeck, S. Tarun, Jr., M. Rieger, J. A. Deardorff, S. Muller-Auer, and A. B. Sachs, J. Biol. Chem. 271:432-438, 1996), plays an important role in the organization of the actin cytoskeleton in S. cerevisiae.

Glucose metabolism and glucose-stimulated insulin secretion are thought to be controlled at the level of glucose phosphorylation in pancreatic islet beta-cells. In the current study we have investigated the importance of glucose phosphorylation by using recombinant adenovirus as a gene delivery system for isolated rat islets. Treatment of islets with a virus containing the cDNA encoding the Escherichia coli beta-galactosidase gene (AdCMV-beta GAL) resulted in efficiencies of gene transfer of 70.3 +/- 2.5 and 61.2 +/- 2.2% in two independent experiments. Treatment of islets with a virus containing the cDNA encoding rat hexokinase I (AdCMV-HKI) resulted in a 10.7-fold increase in immunodetectable hexokinase protein and a similar increase in enzyme activity. A large percentage of the overexpressed hexokinase activity was associated with a cell fraction enriched in mitochondria. These changes in enzyme level were accompanied by a 2-fold increase in insulin release and [5-3H]glucose usage at basal glucose concentrations (3 mM). The rate of glucose usage at 20 mM glucose and the magnitude of the insulin secretory response to this stimulatory level of the sugar were unchanged relative to control islets. Overexpression of hexokinase I in isolated islets therefore creates a phenotype of elevated basal insulin release similar to that seen in islets from obese and insulin-resistant mammals. The discrepancy between the large increase in hexokinase activity and the small increase in glucose usage and insulin release may indicate, however, that other steps in glucose metabolism become rate-limiting after only modest increases in glucose-phosphorylating activity.

Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.

In view of recent serious adverse events and advances in gene therapy technologies, the use of regulatable expression systems is becoming recognized as indispensable adjuncts to successful clinical gene therapy. In the present work we optimized high-capacity adenoviral (HC-Ad) vectors encoding the novel tetracycline-dependent (TetOn)-regulatory elements for efficient and regulatable gene expression in the rat brain in vivo. We constructed two HC-Ad vectors encoding beta-galactosidase (beta-gal) driven by a TetOn system containing the rtTAS(s)M2 transactivator and the tTS(Kid) repressor under the control of the murine cytomegalovirus (mCMV) (HC-Ad-mTetON-beta-Gal) or the human CMV (hCMV) promoter (HC-Ad-hTetON-beta-Gal). Expression was tightly regulatable by doxycycline (Dox), reaching maximum expression in vivo at 6 days and returning to basal levels at 10 days following the addition or removal of Dox, respectively. Both vectors achieved higher transgene expression levels compared to the expression from vectors encoding the constitutive mCMV or hCMV promoter. HC-Ad-mTetON-beta-Gal yielded the highest transgene expression levels and expressed in both neurons and astrocytes. Antivector immune responses continue to limit the clinical use of vectors. We thus tested the inducibility and longevity of HC-Ad-mediated transgene expression in the brain of rats immunized against adenovirus by prior intradermal injections of RAds. Regulated transgene expression from HC-Ad-mTetON-beta-Gal remained active even in the presence of a significant systemic immune response. Therefore, these vectors display two coveted characteristics of clinically useful vectors, namely their regulation and effectiveness even in the presence of prior immunization against adenovirus.

Intracisternal A-particle (IAP) sequences are endogenous retrovirus-like mobile elements, or retrotransposons, present at 1,000 copies in the mouse genome. These elements transpose in a replicative manner via an RNA intermediate and its reverse transcription, and their transposition should therefore be tightly controlled by their transcription level. To analyze the in vivo pattern of expression of these retrovirus-like elements, we constructed several independent transgenic mice with either a complete IAP element marked with an intron or with the IAP promoter, or long terminal repeat (LTR), alone controlling the expression of a lacZ reporter gene with a nuclear localization signal. For all transgenic lines analyzed, IAP expression as determined by reverse transcription-PCR analysis was found to be essentially restricted to the male germ line. Furthermore, in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of all organs disclosed specific beta-galactosidase-positive blue cells only within the testis, found as patches along the seminiferous tubules and often organized as assemblies of 2, 4, 8, or 16 cells. Histochemical analyses of tissues from 13.5-day-old embryos to adults demonstrated that this LTR activity is restricted to gonocytes and premeiotic undifferentiated spermatogonia. Finally, analysis of the methylation status of both transgenes and endogenous IAP LTRs demonstrated identical patterns, with methylation in somatic tissues and hypomethylation in the testis. Transgenic mice therefore reveal an intrinsic, highly restricted IAP expression which had escaped detection in previous global Northern (RNA) blot analyses and with possible strong biological relevance, as IAP activation specifically within the germ line might be a way to generate diversity at the evolutionary level without being deleterious to individuals.

Chitosan-DNA nanoparticles were synthesized from the complexation of the cationic polymer with a ss-gal DNA plasmid, in order to study the efficacy of chitosan to develop a non-viral gene delivery system that can be optimized for efficient gene therapy. The optimal binding conditions were determined with the fluorescamine and PicoGreen assays. DNA distribution within the nanoparticle was visualized by electron transmission microscopy, while the size and morphology were assessed by atomic force microscopy. The transfection potential was evaluated for the first time on human mesenchymal stem cells (MSCs), on human osteosarcoma cells (MG63) and on human embryonic kidney cells (HEK293). The LipofectAMINE(TM) 2000 (LF) reagent was used in comparison. The effect of chitosan-DNA nanoparticles on cell viability was illustrated with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay. The nanoparticles formed are of a diameter inferior to 100nm with a homogenous distribution of DNA. The transfection of HEK293 cells is superior to that seen with MG63 cells and MSCs, however not surpassing that seen with LF. Minimal cytotoxicity is seen with the polyplexes compared to greater than 50% toxicity with LF. These results suggest that chitosan-DNA nanoparticles have favorable characteristics for non-viral gene delivery, are cell type dependent and not cytotoxic.

Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein.

Erythroid Krüppel-like factor (EKLF) is an erythroid cell-specific DNA-binding protein that activates transcription from the beta-globin CACCC element, a functionally important and evolutionarily conserved component of globin as well as other erythroid cell-specific promoters and enhancers. We have attempted to elucidate the molecular role of EKLF in erythrocyte-specific transcriptional activation. First, in vivo and in vitro analyses have been used to demonstrate that the level of activation by EKLF is dependent on the orientation and number of CACCC elements, that EKLF contains separable activation and DNA-binding domains, and that the EKLF proline-rich region is a potent activator in CV-1 cells when fused to a nonrelated DNA-binding module. Second, we have established a transient assay in murine erythroleukemia cells in which reproducible levels of a reporter can be induced when linked to a locus control region enhancer-beta-globin promoter and in which induction is abolished when the promoter CAC site is mutated to a GAL site. Third, we demonstrate that the EKLF transactivation region, when fused to the GAL DNA-binding domain, can restore inducibility to this mutated construct and that this inducibility exhibits activator-, promoter-, and cell-type specificity. These results demonstrate that EKLF provides a crucial transactivation function for globin expression and further reinforce the idea that EKLF is an important regulator of CACCC element-directed transcription in erythroid cells.

Free-living bacteria are expert in adapting to variations in nutrient availability, often using an array of transport systems of different affinities to scavenge for particular substrates (multiport). This review concentrates on the regulation of expression of different transporters contributing to multiport in response to varying nutrient levels. A novel mechanism of controlling bacterial transport affinity under sugar limitation is described. In particular, switching from glucose-rich to glucose-limited conditions results in Escherichia coli orchestrating outer membrane changes as well as the induction of a periplasmic binding protein-dependent (ABC-type) transport system. The changes leading to the high affinity transport pathway are directed towards uptake of rapidly utilisable concentrations and are optimal close to 10-6 M medium glucose. High affinity transport is absent under both glucose-rich 'feast' and glucose-starved 'famine' conditions hence high affinity transporters are not simply repressed by excess nutrient. Rather, the improvement in glucose scavenging involves induction of genes in 2 distinct regulons (mgl/gal and mal/lamB) through synthesis of 2 different endogenous inducer molecules (galactose, maltotriose). Endoinducer levels are tightly controlled by extracellular glucose concentration at different glucose-limited growth rates. Aside from endoinducers, the elevated intracellular level of cAMP plays a role in induction of the high-affinity pathway but cAMP-mediated relief from catabolite repression is not itself sufficient for high affinity transport. In contrast to the repressive role of glucose when present at millimolar concentrations, micromolar glucose also leads to the induction of transport systems for other sugars, further broadening the scavenging potential of nutrient-limited bacteria for other substrates.

Drug targeting to a highly expressed, noninternalizable determinant up-regulated on the perturbed endothelium may help to manage inflammation and thrombosis. We tested whether inter-cellular adhesion molecule-1 (ICAM-1) targeting is suitable to deliver antithrombotic drugs to the pulmonary vascular lumen. ICAM-1 antibodies bind to the surface of endothelial cells in culture, in perfused lungs, and in vivo. Proinflammatory cytokines enhance anti-ICAM binding to the endothelium without inducing internalization. (125)I-labeled anti-ICAM and a reporter enzyme (beta-Gal) conjugated to anti-ICAM bind to endothelium and accumulate in the lungs after intravenous administration in rats and mice. Anti-ICAM is seen to localize predominantly on the luminal surface of the pulmonary endothelium by electron microscopy. We studied the pharmacological effect of ICAM-directed targeting of tissue-type plasminogen activator (tPA). Anti-ICAM/tPA, but not control IgG/tPA, conjugate accumulates in the rat lungs, where it exerts plasminogen activator activity and dissolves fibrin microemboli. Therefore, ICAM may serve as a target for drug delivery to endothelium, for example, for pulmonary thromboprophylaxis. Enhanced drug delivery to sites of inflammation and the potential anti-inflammatory effect of blocking ICAM-1 may enhance the benefit of this targeting strategy.

A new Clostridium perfringens-Escherichia coli shuttle plasmid has been constructed and its complete DNA sequence compiled. The vector, pJIR418, contains the replication regions from the C. perfringens replicon pIP404 and the E. coli vector pUC18. The multiple cloning site and lacZ' gene from pUC18 are also present, which means that X-gal screening can be used to select recombinants in E. coli. Both chloramphenicol and erythromycin resistance can be selected in C. perfringens and E. coli since pJIR418 carries the C. perfringens catP and ermBP genes. Insertional inactivation of either the catP or ermBP genes can also be used to directly screen recombinants in both organisms. The versatility of pJIR418 and its applicability for the cloning of toxin genes from C. perfringens have been demonstrated by the manipulation of a cloned gene encoding the production of phospholipase C.

We have investigated the question of whether the gel mobility-shift assay can provide data that are useful to the demonstration of cooperativity in the site-specific binding of proteins to DNA. Three common patterns of protein-DNA interaction were considered: (i) the cooperative binding of a protein to two sites (illustrated by the Escherichia coli Gal repressor); (ii) the cooperative binding of a bidentate protein to two sites (illustrated by the E. coli Lac repressor); and (iii) the cooperative binding of a protein to three sites (illustrated by the lambda cI repressor). A simple, rigorous, and easily extendable statistical mechanical approach to the derivation of the binding equations for the different patterns is presented. Both simulated and experimental data for each case are analyzed. The mobility-shift assay provides estimates of the macroscopic binding constants for each step of ligation based on its separation of liganded species by the number of ligands bound. Resolution of the binding constants depends on the precision with which the equilibrium distribution of liganded species is determined over the entire range of titration of each of the sites. However, the evaluation of cooperativity from the macroscopic binding constants is meaningful only for data that are also accurate. Some criteria that are useful in evaluating accuracy are introduced and illustrated. Resolution of cooperative effects is robust only for the simplest case, in which there are two identical protein binding sites. In this case, cooperative effects of up to 1,000-fold are precisely determined. For heterogeneous sites, cooperative effects of greater than 1,000-fold are resolvable, but weak cooperativity is masked by the heterogeneity. For three-site systems, only averaged pair-wise cooperative effects are resolvable.

gpL115 is a lymphocyte surface component that is deficient in patients with the X-chromosome-linked immune deficiency Wiskott-Aldrich syndrome (6). The glycoprotein nature of gpL115 is demonstrated through labeling in carbohydrate moieties by [3H]NaBH4 and its synthesis by lymphocytes through labeling with [35S]methionine. Native gpL115 adheres to wheat germ lectin-Sepharose and sialidase-treated gpL115 does not adhere, indicating that native gpL115 adheres via clusters of sialic acid residues. When tested on peanut lectin, which shows specificity for the disaccharide Gal beta 1-3GalNAc, gpL115 is nonadherent and sialidase-treated gpL115 is adherent, indicating the presence of the sequence sialic acid-Gal beta 1-3GalNAc, which is characteristic for O-linked (mucin-type, acidic-type) carbohydrates. A surface glycoprotein with all the above characteristics was found on the lymphoblastoid cell line CEM. CEM cells were used as immunogen to generate the monoclonal antibody L10, an IgG1, which binds native and sialidase-treated gpL115 . Sialidase-treatment of gpL115 significantly alters its physical properties, reducing its electrophoretic mobility and changing its behavior on isoelectrofocusing. Cumulatively, these findings indicate that gpL115 , like glycophorin of erythrocytes and GPIb of platelets, is a sialoglyco protein with significant quantities of O-linked carbohydrate. On treatment with limiting sialidase concentrations, gpL115 of normal lymphocytes is transformed into a series of partially desialylated species of decreasing electrophoretic mobility. This finding resembles the situation with lymphocytes of some Wiskott-Aldrich syndrome patients. Lymphocytes of eight Wiskott-Aldrich syndrome patients were found to be deficient in 125I-labeled gpL115 . Lymphocytes from three of these patients displayed an abnormal 125I-component of apparent mol wt 135,000.

The galactoxylomannan (GalXM) obtained from the culture supernatant of an acapsular mutant of Cryptococcus neoformans Cap67 was purified by Concanavalin A affinity, ion-exchange, and gel-filtration chromatographies. The structure of GalXM was determined by methylation analysis and by 1D and 2D NMR spectroscopic studies of the intact polysaccharide and of the oligosaccharide fragments generated by Smith degradation and by acetolysis. GalXM is a complex polysaccharide with an alpha-(1-->6) -galactan backbone. The polysaccharide is branched at c-3 of alternate Gal units of the backbone. C-3 is the point of attachment of the oligosaccharide side chains comprised of alpha-D-Man- (1-->3)-alpha-D-Man-(1-->4)- beta-D-Gal-substituted with zero to three terminal beta-Xyl residues as shown in the following structure: [formula: see text].

BACKGROUND

Galectin-3 (Gal-3) is a recently discovered marker for myocardial fibrosis and elevated levels are associated with an impaired outcome after short-term follow-up in heart failure (HF) patients. However, whether Gal-3 is related to cardiac remodeling and outcome after long-term follow-up is unknown. Therefore, we determined the utility of Gal-3 as a novel biomarker for left ventricular remodeling and long-term outcome in patients with severe chronic HF.

RESULTS

A total of 240 HF patients with New York Heart Association (NYHA) Class III and IV were included. Patients were followed for 8.7 ± 1 years, had a mean age of 71 ± 0.6 years and 73 % of the study population was male. Circulating levels of NT-proBNP and Gal-3 were measured. Serial echocardiography was performed at baseline and at 3 months. At baseline median left ventricular end-diastolic volume (LVEDV) was 267 mL [interquartile range 232-322]. Patients were divided into three groups according to the change in LVEDV. Patients in whom the LVEDV decreased over time had significant lower levels of Gal-3 at entry compared to patients in whom the LVEDV was stable or increased (14.7 vs. 17.9 vs. 19.0 ng/mL; p = 0.004 for trend), whereas no significant differences were seen in levels of NT-proBNP (p = 0.33). Multivariate linear regression analyses revealed that Gal-3 levels were positively correlated to change in LVEDV (p = 0.007). In addition, Gal-3 was a significant predictor of mortality after long-term follow-up (p = 0.001).

CONCLUSIONS

Gal-3 is associated with left ventricular remodeling determined by serial echocardiography and predicts long-term mortality in patients with severe chronic HF.

There is shortage of extensive clinicopathologic studies of cellular senescence because the most reliable senescence biomarker, the detection of Senescence-Associated-beta-galactosidase activity (SA-β-gal), is inapplicable in archival material and requires snap-frozen tissues. We validated the histochemical Sudan-Black-B (SBB) specific stain of lipofuscin, an aggregate of oxidized proteins, lipids and metals, known to accumulate in aged tissues, as an additional reliable approach to detect senescent cells independently of sample preparation. We analyzed cellular systems in which senescence was triggered by replicative exhaustion or stressful stimuli, conditional knock-in mice producing precancerous lesions exhibiting senescence, and human preneoplastic lesions known to contain senescent cells. In the above settings we demonstrated co-localization of lipofuscin and SA-β-gal in senescent cells in vitro and in vivo (cryo-preserved tissue), strongly supporting the candidacy of lipofuscin for a biomarker of cellular senescence. Furthermore, cryo-preserved tissues positive for SA-β-gal were formalin-fixed, paraffin-embedded, and stained with SBB. The corresponding SA-β-gal positive tissue areas stained specifically for lipofuscin by SBB, whereas tissues negative for SA-β-gal were lipofuscin negative, validating the sensitivity and specificity of the SBB staining to visualize senescent cells in archival material. The latter unique property of SBB could be exploited in research on widely available retrospective tissue material.

Haemophilus influenzae lipopolysaccharide (LPS) contains structures, defined by monoclonal antibodies, which undergo phase variation. This investigation reports the nucleotide sequence of lic2A, which is required for the expression of at least three phase-variable LPS epitopes, one of which has the structure alpha Gal(1-4)beta Gal. lic2A contains multiple tandem repeats of the tetramer 5'-CAAT-3'. Previous studies have correlated changes in the number of 5'-CAAT-3' repeats with the phase-variable expression of the alpha Gal(1-4)beta Gal epitope. To obtain direct evidence for this, the 5'-CAAT-3' repeat region from lic2A was amplified directly from immunostained colonies and sequenced. This demonstrated that the variable expression of LPS epitopes, including alpha Gal(1-4)beta Gal, is in part directly dependent upon the number of copies of 5'-CAAT-3' within lic2A.

The precise origins of postnatal subventricular zone (SVZ) cells are not known. Furthermore, the gliogenic potential of progenitors expressing Dlx genes that migrate ventrodorsally from the ganglionic eminences has not been explored in vivo. Here, we identify the embryonic origins of two distinct populations of postnatal SVZ cells: SVZ border cells, which express Zebrin II, and migratory cells in the central SVZ, which are generally devoid of Zebrin II expression (Staugaitis et al., 2001). Zebrin II is expressed by all cells of the telencephalic primordium, with its expression becoming restricted to astrocytes in the mature telencephalon. As the neuroepithelium folds during corticostriatal sulcus formation (embryonic day 13-15), a wedge of Zebrin II+ cells is created at the presumptive site of the dorsolateral SVZ. At this time, Dlx2-expressing cells and their progeny begin to migrate ventrodorsally along a medial path from the ganglionic eminences. These migratory subpallial cells invade the wedge of Zebrin II+ cells to form the central region of the SVZ. We used a Dlx2/tauLacZ knock-in to perform a short-term lineage analysis of Dlx2-expressing cells throughout SVZ formation and the postnatal peak of gliogenesis. Dlx2/tauLacZ [beta-galactosidase (beta-gal)]-expressing cells populate the central SVZ, whereas Zebrin II-expressing cells form its borders. Furthermore, beta-gal expression demonstrates a lineage relationship between Dlx2-expressing cells and glia residing in the dorsal telencephalon. We propose a model for the formation of the postnatal SVZ and demonstrate that subpallium-derived Dlx2-expressing cells give rise to astrocytes and oligodendrocytes in the white matter and cerebral cortex.

We and others have previously shown that a 67-kD cell surface elastin/laminin-binding protein (EBP) is responsible for cell adhesion to elastin and laminin and for mediating the process of elastin fiber assembly, but the nature of this protein was unknown. In this report we provide evidence that a 67-kD catalytically inactive form of beta-galactosidase produced by alternative splicing demonstrates immunological and functional similarity and sequence homology to the 67-kD EBP, suggesting that the two might be the same. Antibody prepared to a synthetic peptide, N-Ac-GSPSAQDEASPL, corresponding to a frame-shift-generated sequence unique to the alternatively spliced form of human beta-galactosidase, also recognized sheep EBP both on Western blotting and in aortic tissue. Furthermore, this synthetic peptide (S-GAL) binds to elastin and laminin, but not to fibronectin, collagen I, or collagen III. Moreover, both tropoelastin and laminin which bind to S-GAL peptide affinity columns can be specifically eluted from them with an excess of free S-GAL peptides. In addition, sequence homology among this splice variant of human beta-galactosidase, sheep EBP, and NH2-terminal sequences of some elastases suggests that these proteins share a common ligand-binding motif that has not been previously recognized.

Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations.

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.

Fabry disease is a lysosomal storage disorder caused by deficient lysosomal alpha-galactosidase A (alpha-Gal A) activity. Deficiency of the enzyme activity results in progressive deposition of neutral glycosphingolipids with terminal alpha-galactosyl residue in vascular endothelial cells. We recently proposed a chemical chaperone therapy for this disease by administration of 1-deoxygalactonojirimycin, a potent inhibitor of the enzyme, at subinhibitory intracellular concentrations [Fan, J.-Q., Ishii, S., Asano, N. and Suzuki, Y. (1999) Nat. Med. 5, 112-115]. 1-Deoxygalactonojirimycin served as a specific chaperone for those mutant enzymes that failed to maintain their proper conformation to avoid excessive degradation. In order to establish a correlation between in vitro inhibitory activity and intracellular enhancement activity of the specific chemical chaperone, a series of 1-deoxygalactonojirimycin derivatives were tested for activity with both alpha-Gal A and Fabry lymphoblasts. 1-Deoxygalactonojirimycin was the most potent inhibitor of alpha-Gal A with an IC50 value of 0.04 microM. alpha-Galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin were effective inhibitors with IC50 values of 0.21, 4.3 and 16 microM, respectively. N-Alkylation, deoxygenation at C-2 and epimerization at C-3 of 1-deoxygalactonojirimycin markedly lowered or abolished its inhibition toward alpha-Gal A. Inclusion of 1-deoxygalactonojirimycin, alpha-galacto-homonojirimycin, alpha-allo-homonojirimycin and beta-1-C-butyl-deoxygalactonojirimycin at 100 microM in culture medium of Fabry lymphoblasts increased the intracellular alpha-Gal A activity by 14-fold, 5.2-fold, 2.4-fold and 2.3-fold, respectively. Weaker inhibitors showed only a minimum enhancement effect. These results suggest that more potent inhibitors act as more effective specific chemical chaperones for the mutant enzyme, and the potent competitive inhibitors of alpha-Gal A are effective specific chemical chaperones for Fabry disease.

Galectin-1 (Gal-1) is involved in several pathological activities associated with tumor progression and chemoresistance, however, the role and molecular mechanism of Gal-1 activity in hepatocellular carcinoma (HCC) epithelial-mesenchymal transition (EMT) and sorafenib resistance remain enigmatic. In the present study, forced Gal-1 expression promoted HCC progression and sorafenib resistance. Gal-1 elevated αvβ3-integrin expression, leading to AKT activation. Moreover, Gal-1 overexpression induced HCC cell EMT via PI3K/AKT cascade activation. Clinically, our data revealed that Gal-1 overexpression is correlated with poor HCC survival outcomes and sorafenib response. These data suggest that Gal-1 may be a potential therapeutic target for HCC and a biomarker for predicting response to sorafenib treatment.