Stem cells (SC) are able to self-renew and to differentiate into many types of committed cells, making SCs interesting for cellular therapy. However, the pool of SCs in vivo and in vitro consists of a mix of cells at several stages of differentiation, making it difficult to obtain a homogeneous population of SCs for research. Therefore, it is important to isolate and characterize unambiguous molecular markers that can be applied to SCs. Here, we review classical and new candidate molecular markers that have been established to show a molecular profile for human embryonic stem cells (hESCs), mesenchymal stem cells (MSCs), and hematopoietic stem cells (HSCs). The commonly cited markers for embryonic ESCs are Nanog, Oct-4, Sox-2, Rex-1, Dnmt3b, Lin-28, Tdgf1, FoxD3, Tert, Utf-1, Gal, Cx43, Gdf3, Gtcm1, Terf1, Terf2, Lefty A, and Lefty B. MSCs are primarily identified by the expression of CD13, CD29, CD44, CD49e, CD54, CD71, CD73, CD90, CD105, CD106, CD166, and HLA-ABC and lack CD14, CD31, CD34, CD45, CD62E, CD62L, CD62P, and HLA-DR expression. HSCs are mainly isolated based on the expression of CD34, but the combination of this marker with CD133 and CD90, together with a lack of CD38 and other lineage markers, provides the most homogeneous pool of SCs. Here, we present new and alternative markers for SCs, along with microRNA profiles, for these cells.

BACKGROUND

Damaged skeletal muscle is able to regenerate because of the presence of satellite cells, which are undifferentiated myoblasts. In contrast, destruction of cardiac myocytes is associated with an irreversible loss of myocardium and replacement with scar tissue, because it lacks stem cells. We tested the hypothesis that skeletal muscle satellite cells implanted into injured myocardium can differentiate into cardiac muscle fibers and thus repair damaged heart muscle.

METHODS

Two series of canine studies were performed. In the first series (n = 26), satellite cells were isolated from skeletal muscle, cultured, and labeled with tritiated thymidine. The cells were implanted into acutely cryoinjured myocardium and the specimens harvested 4 to 18 weeks later. In the second series (n = 20), satellite cells in culture were labeled with lacZ reporter gene, which encodes production of Escherichia coli beta-galactosidase. Four to 6 weeks later, beta-galactosidase activity was studied using X-Gal stain.

RESULTS

New striated muscles were found in the first series of experiments at the site of implantation, within a dense scar created by cryoinjury. These muscles showed histologic evidence of intercalated discs and centrally located nuclei, similar to those seen in cardiac muscle fibers. Tritiated thymidine radioactivity was not identified clearly, presumably due to dilutional effect as the stem cells replicated repeatedly. In the second series, histochemical studies of reporter gene-labeled and implanted satellite cells revealed the presence of beta-galactosidase within the cells at the implant site, which confirmed the survival of implanted cells.

CONCLUSIONS

Our data are consistent with the hypothesis of milieu-influenced differentiation of satellite cells into cardiac-like muscle cells. Confirmation of these findings and its functional capabilities could have important clinical implications.

OBJECTIVE

To compare replication competence and senescence in human corneal endothelial cells (HCECs) between the central and peripheral areas and between younger and older donors.

METHODS

Human corneas were obtained from the eye bank and separated into two groups: young (younger than 30 years) and old (older than 50 years). Corneas were cut in quarters and a 2-mm scrape wound was created in the endothelium from the periphery to the center. Unwounded endothelium acted as a negative control. Corneal pieces were incubated for 24, 36, 48, 60, 72, 84, and 96 hours in medium containing 8% fetal bovine serum (FBS) plus additional growth factors. Tissue was fixed, immunostained for minichromosome maintenance (MCM)-2, a marker of replication competence, and mounted in medium containing propidium iodide (PI) to visualize all nuclei. Fluorescence microscope images were used to count PI-stained and MCM2-positive HCECs in three 100-microm2 areas within the central and peripheral wound area. Results are expressed as mean number of cells/100 microm2. Senescent HCECs in ex vivo corneas were identified by staining for senescence-associated beta-galactosidase activity (SA-beta-Gal). Whole corneas were cut in quarters and incubated in staining solution containing SA-beta-Gal at pH 6.0. The number of cells stained for SA-beta-Gal and the grade of SA-beta-Gal intensity in three 100-microm2 areas were averaged for the central and peripheral areas from each donor. For all studies, results were compared between central and peripheral cornea and between younger and older donors.

RESULTS

In both age groups (n = 4/group), cells repopulated the wound area in a time-dependent manner. In corneas from older donors, significantly fewer HCECs migrated into the wound bed in the central cornea than in the periphery. At each time point, the density of cells in the central wound area was lower in corneas from older donors than from younger donors. In both age groups, the mean percentage of MCM2-positive cells increased with time until wound healing. In both age groups, more MCM2-positive cells were present in the wounded area of the peripheral than of the central cornea. At 36, 48, 60, and 72 hours after wounding, the percentage of MCM2-positive cells in the central or peripheral area of older corneas was significantly less than in the corresponding region in younger corneas. No MCM2-positive staining was observed in unwounded areas at any time point. HCECs in corneas from younger donors (n = 4) showed little to no SA-beta-Gal activity in either the central or peripheral area. SA-beta-Gal activity was easily detectable in corneas from older donors (n = 4) and a significantly higher percentage of central HCECs showed strong SA-beta-Gal activity compared with HCECs in the periphery.

CONCLUSIONS

In ex vivo corneas, HCECs from the peripheral area retain higher replication competence, regardless of donor age. HCECs in the central area of corneas from older donors retain replicative competence, but the relative percentage of cells that are competent to replicate is significantly lower than in the periphery or in the central area of corneas from younger donors. This reduction in replicative competence negatively correlates with the observed increase in the population of central HCECs exhibiting senescence-like characteristics.

Gal repressosome assembly and repression of the gal operon in Escherichia coli occurs when two dimeric GalR proteins and the histone-like HU protein bind to cognate sites causing DNA looping. Structure-based genetic analysis defined the GalR surfaces interacting to form a stacked, V-shaped, tetrameric structure. Stereochemical models of the four possible DNA loops compatible with the GalR tetramer configuration were constructed using the sequence-dependent structural parameters of the interoperator DNA and conformation changes caused by GalR and asymmetric HU binding. Evaluation of their DNA elastic energies gave unambiguous preference to a loop structure in which the two gal operators adopt an antiparallel orientation causing undertwisting of DNA.

Galectins, a family of glycan-binding proteins, influence tumor progression by modulating interactions between tumor, endothelial, stromal, and immune cells. Despite considerable progress in identifying the roles of individual galectins in tumor biology, an integrated portrait of the galectin network in different tumor microenvironments is still missing. We undertook this study to analyze the "galectin signature" of the human prostate cancer microenvironment with the overarching goal of selecting novel-molecular targets for prognostic and therapeutic purposes. In examining androgen-responsive and castration-resistant prostate cancer cells and primary tumors representing different stages of the disease, we found that galectin-1 (Gal-1) was the most abundantly expressed galectin in prostate cancer tissue and was markedly upregulated during disease progression. In contrast, all other galectins were expressed at lower levels: Gal-3, -4, -9, and -12 were downregulated during disease evolution, whereas expression of Gal-8 was unchanged. Given the prominent regulation of Gal-1 during prostate cancer progression and its predominant localization at the tumor-vascular interface, we analyzed the potential role of this endogenous lectin in prostate cancer angiogenesis. In human prostate cancer tissue arrays, Gal-1 expression correlated with the presence of blood vessels, particularly in advanced stages of the disease. Silencing Gal-1 in prostate cancer cells reduced tumor vascularization without altering expression of other angiogenesis-related genes. Collectively, our findings identify a dynamically regulated "galectin-specific signature" that accompanies disease evolution in prostate cancer, and they highlight a major role for Gal-1 as a tractable target for antiangiogenic therapy in advanced stages of the disease.

Avian influenza viruses are considered to be key contributors to the emergence of human influenza pandemics. A major determinant of infection is the presence of virus receptors on susceptible cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. While ducks are the major reservoir for influenza viruses, they are typically resistant to the effects of viral infection, in contrast to the frequently severe disease observed in chickens. In order to understand whether differences in receptors might contribute to this observation, we studied the distribution of influenza receptors in organs of ducks and chickens using lectin histochemistry with linkage specific lectins and receptor binding assays with swine and avian influenza viruses. Although the intestinal epithelial cells of both species expressed only SAalpha2,3-Gal receptors, we found widespread presence of both SAalpha2,6-Gal and SAalpha2,3-Gal receptors in many organs of both chickens and ducks. Co-expression of both receptors may allow infection of cells with both avian and human viruses and so present a route to genetic reassortment. There was a marked difference in the primary receptor type in the trachea of chickens and ducks. In chicken trachea, SAalpha2,6-Gal was the dominant receptor type whereas in ducks SAalpha2,3-Gal receptors were most abundant. This suggests that chickens could be more important as an intermediate host for the generation of influenza viruses with increased ability to bind to SAalpha2,6-Gal receptors and thus greater potential for infection of humans. Chicken tracheal and intestinal epithelial cells also expressed a broader range of SAalpha2,3-Gal receptors (both beta(1-4)GlcNAc and beta(1-3)GalNAc subtypes) in contrast to ducks, which suggests that they may be able to support infection with a broader range of avian influenza viruses.

The O-linked oligosaccharides (O-glycans) in mammalian glycoproteins are classified according to their core structures. Among the most common is the core 1 disaccharide structure consisting of Galbeta1-->3GalNAcalpha1->>Ser/Thr, which is also the precursor for many extended O-glycan structures. The key enzyme for biosynthesis of core 1 O-glycan from the precursor GalNAc-alpha-Ser/Thr is UDP-Gal:GalNAc-alpha-Ser/Thr beta3-galactosyltransferase (core1 beta3-Gal-T). Core 1 beta3-Gal-T activity, which requires Mn2+, was solubilized from rat liver membranes and purified 71,034-fold to apparent homogeneity (>90% purity) in 5.7% yield by ion exchange chromatography on SP-Sepharose, affinity chromatography on immobilized asialo-bovine submaxillary mucin, and gel filtration chromatography on Superose 12. The purified enzyme is free of contaminating glycosyltransferases. Two peaks of core 1 beta3-Gal-T activity were identified in the final step on Superose 12. One peak of activity contained protein bands on non-reducing SDS-PAGE of approximately 84- and approximately 86-kDa disulfide-linked dimers, whereas the second peak of activity contained monomers of approximately 43 kDa. Reducing SDS-PAGE of these proteins gave approximately 42- and approximately 43-kDa monomers. Both the 84/86-kDa dimers and the 42/43-kDa monomers have the same novel N-terminal sequence. The purified enzyme, which is remarkably stable, has an apparent Km for UDP-Gal of 630 microm and an apparent Vmax of 206 micromol/mg/h protein using GalNAcalpha1-O-phenyl as the acceptor. The reaction product was generated using asialo-bovine submaxillary mucin as an acceptor; treatment with O-glycosidase generated the expected disaccharide Galbeta1-->3GalNAc. These studies demonstrate that activity of the core 1 beta1,3-Gal-T from rat liver is contained within a single, novel, disulfide-bonded, dimeric enzyme.

OBJECTIVE

To determine whether a reporter gene can be introduced into the adult mammalian retina in vivo through means of a recombinant replication-deficient adenovirus.

METHODS

A dilution series of purified Ad.CMVlacZ ranging from 10(5) to 10(11) pfu/ml was prepared and microinjected into the subretinal space of adult CD-1 mice. This virus contained the cytomegalovirus (CMV)-promoted Escherichia coli reporter gene, lacZ. LacZ expression was assessed in enucleated eyes from 0 to 95 days after injection by beta-galactosidase (beta-Gal) assay.

RESULTS

The efficiency of transfection increased as a function of concentration of recombinant virus injected. Eyes injected with greater than 10(7) pfu of Ad.CMVlacZ demonstrated beta-Gal activity lasting at least 95 days. LacZ expression was apparent only in those cells directly exposed to the adenovirus. LacZ expression was observed in the retinal pigment epithelium (RPE) at high efficiency at 48 hours after exposure. By 2 weeks after injection of>> 10(7) pfu, lacZ was also expressed in photoreceptors, but at lower density.

CONCLUSIONS

These results demonstrate that high efficiency stable transfer of functional genes can be achieved in vivo in post-mitotic mammalian retina using recombinant adenoviral vectors. Adenovirus vectors appear to be a promising means for delivering therapeutic genes in vivo to the mammalian neural retina and particularly to the RPE.

Hypoxia initiates an adaptive physiological response in all organisms and plays a role in the pathogenesis of several human diseases. The hypoxia/HIF-inducible factor-1 (HIF-1) transcription factor mediates transcriptional responses to hypoxia by binding to a cis-acting hypoxia-responsive element (HRE) present within target genes. The use of the HIF-1/HRE system of gene regulation can be utilized as a mechanism to target expression of therapeutic genes to hypoxic cells or cells that have a constitutively active HIF-1/HRE pathway due to cell transformation. Given the rapid resistance of tumors to single therapeutic strategies, new vector systems need to be developed that can deliver multimodal therapy. Here we show that HREs function as classical enhancer elements and function bidirectionally to co-regulate the expression of two genes. We designed a large series of novel bidirectional hypoxia/HIF-responsive expression vectors using HREs derived from the human vascular endothelial growth factor (VEGF) and erythropoietin (EPO) genes. We measured the ability of these constructs to express the luciferase and LacZ/beta-galactosidase (beta-gal) reporter genes bidirectionally under normoxic (21% O(2)) versus hypoxic (1, 3, 5, and 10% O(2)) conditions by transient transfection in three human glioma cell lines (LN229, U251MG and U138MG). Nine constructs were identified that exhibited moderate to high inducibility at 1% O(2) while maintaining tight regulation under normoxic conditions. Moreover, the level of activation was a function of O(2) concentration and was exponential at O(2) levels below 5%. These vectors will be valuable tools in a variety of gene therapy applications targeting pathological activation of the HIF-1/HRE pathway.

Prostate cancer is one of the malignant tumors which exhibit resistance to anticancer drugs, at least in part due to enhanced antiapoptotic mechanisms. Therefore, the understanding of such mechanisms should improve the design of chemotherapy against prostate cancer. Galectin-3 (Gal-3), a multifunctional oncogenic protein involved in the regulation of tumor proliferation, angiogenesis, and apoptosis has shown antiapoptotic effects in certain cell types. Here, we show that the expression of exogenous Gal-3 in human prostate cancer LNCaP cells, which do not express Gal-3 constitutively, inhibits anticancer drug-induced apoptosis by stabilizing the mitochondria. Thus, Gal-3-negative cells showed 66.31% apoptosis after treatment with 50 micromol/L cis-diammine-dichloroplatinum for 48 hours, whereas two clones of Gal-3-expressing cells show only 2.92% and 1.42% apoptotic cells. Similarly, Gal-3-negative cells showed 43.8% apoptosis after treatment with 300 micromol/L etoposide for 48 hours, whereas only 15.38% and 14.51% of Gal-3-expressing LNCaP cells were apoptotic. The expression of Gal-3 stimulated the phosphorylation of Ser(112) of Bcl-2-associated death (Bad) protein and down-regulated Bad expression after treatment with cis-diammine-dichloroplatinum. Gal-3 also inhibited mitochondrial depolarization and damage after translocation from the nuclei to the cytoplasm, resulting in inhibition of cytochrome c release and caspase-3 activation. These findings indicate that Gal-3 inhibits anticancer drug-induced apoptosis through regulation of Bad protein and suppression of the mitochondrial apoptosis pathway. Therefore, targeting Gal-3 could improve the efficacy of anticancer drug chemotherapy in prostate cancer.

Twelve 14C-acetylated glycopeptides have been subjected to affinity chromatography on concanvalin A (Con A)--Sepharose at pH 7.5. The elution profiles could be classified into four distinct patterns. The first pattern showed no retardation of glycopeptide on the column and was elicited with a glycopeptide having three peripheral oligosaccharide chains: (abstract:see text). Such glycopeptides have only a single mannose residue capable of interacting with Con A--Sepharose; an interacting mannose residue is either an alpha-linked nonreducing terminal residue or an alpha-linked 2-O-substituted residue. The second type of profile showed a retarded elution of glycopeptide with buffer lacking methyl alpha-D-glucopyranoside (indicative of weak interaction with the column) and was given by glycopeptides with the structures: (abstract: see text) where R1 is either H or a sialyl residue. The third profile type showed tight binding of glycopeptide to Con A--Sepharose and elution as a sharp peak with 0.1 M methyl alpha-D-glucopyranoside; glycopeptides giving this pattern had the structures: (abstract: see text) where R2 is either H, glcNAc, Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. These glycopeptides all have two interacting mannose residues, the mimimum required for binding to the column; one of these mannose residues must, however, be a terminal residue to obtain tight binding and sharp elution. The fourth profile type showed tight binding of glycopeptide to the column but elution with 0.1 M methyl alpha-D-glucopyranoside resulted in a broad peak indicating very tight binding; glycopeptides showing this behaviour had the structures: (abstract: see text) where R3 is either GlcNAc,Gal-beta 1,4-GlcNAc, or sialyl-Gal-beta 1,4-GlcNAc. Therefore it can be concluded that although a minimum of two interacting mannose residues is required for binding to Con A--Sepharose, the residues linked to these mannoses can either strengthen or weaken binding to the column.

Epidermis is renewed by a population of stem cells that have been defined in vivo by slow turnover, label retention, position in the epidermis, and enrichment in beta1 integrin, and in vitro by clonogenic growth, prolonged serial passage, and rapid adherence to extracellular matrix. The goal of this study is to determine whether clonogenic cells with long-term growth potential in vitro persist in vivo and give rise to a fully differentiated epidermis. Human keratinocytes were genetically labeled in culture by transduction with a retrovirus encoding the lacZ gene and grafted to athymic mice. Analysis of the cultures before grafting showed that 21.1-27.8% of clonogenic cells with the capacity for >30 generations were successfully transduced. In vivo, beta-galactosidase (beta-gal) positive cells participated in the formation of a fully differentiated epithelium and were detected throughout the 40-week postgraft period, initially as loosely scattered clusters and later as distinct vertical columns. Viable cells recovered from excised grafts were seeded at clonal densities and 23.3-33.3% of the colonies thus formed were beta-gal positive. In addition, no evidence of transgene inactivation was obtained: all keratinocyte colonies recovered from grafted tissue that were beta-gal negative also lacked the lacZ transgene. These results show that cells with long-term growth properties in vitro do indeed persist in vivo and form a fully differentiated epidermis, thereby exhibiting the properties of stem cells.

Expression of xylose isomerase was repressed in Bacillus subtilis strains W23, 168, and BR151 and could be induced in the presence of xylose. The expression was also glucose repressed in strains 168 and BR151, although this effect was not observed with W23. A xyl-cat fusion gene was constructed on a multicopy plasmid, from which the xyl promoter located on a 366-base-pair (bp) DNA fragment derived from W23 directed the expression of chloramphenicol resistance. The regulation of expression was not very pronounced in this multicopy situation. The xyl promoter is a strong signal for transcription initiation. The 5' sequence of the xyl mRNA was identified by nuclease S1 mapping. The promoter consisted of the -10 sequence TAAGAT, the -35 sequence TTGAAA spaced by 17 bp, and an upstream poly(A) block with 14 As out of 17 bp. To study the regulation, a xyl-lacZ fusion gene was constructed and integrated as a single copy into the amygene of B. subtilis 168. This strain grows blue on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) indicator plates in the presence of xylose and white in the presence of glucose. Quantitatively, the induction of beta-galactosidase by xylose was 100-fold. In the presence of xylose plus glucose, the expression of the indicator gene was repressed to 30% of the fully induced level. About 25 to 60% of the maximal lacZ expression was obtained with this strain when the 366-bp xyl DNA fragment was provided in trans on a multicopy plasmid. This result indicates that repression in the absence of xylose is mediated in trans by a soluble factor which is expressed at a low level in B. subtilis 168. The xylose effect depended on negative regulation. The estimations of mRNA amounts by dot blot analysis showed unambiguously that the induction by xylose occurs at the level of transcription. The possible molecular mechanisms are discussed with respect to the nucleotide sequence of the 366-bp xyl regulatory DNA.

Chinese hamster ovary (CHO) cells cluster in the presence of pertussis toxin, a response that is correlated with the ADP-ribosylation of a Mr = 41,000 membrane protein by the toxin. A ricin-resistant line of CHO cells (CHO-15B) which specifically lacks the terminal NeuAc----Gal beta 4GlcNAc oligosaccharide sequence on glycoproteins did not cluster in response to pertussis toxin. These cells do contain the Mr = 41,000 protein substrate for the enzymatic activity of the toxin which suggests that pertussis toxin, like certain plant lectins, does not bind to or is not internalized by the CHO-15B cells. There was no evidence of pertussis toxin binding to gangliosides or neutral glycolipids isolated from CHO cells but the toxin bound to a Mr = 165,000 component in N-octyglucoside extracts of CHO cells that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted to nitrocellulose. Plant lectins from Ricinus communis and Erythina cristagalli detected a similar size band in CHO cells and also did not react with CHO-15B cells. Unlike pertussis toxin, these plant lectins recognized two other major bands in CHO cell extracts and reacted best after sialidase treatment of nitrocellulose transfers containing CHO cell extracts. Conversely, sialidase treatment abolished binding a pertussis toxin and wheat germ agglutinin, a plant lectin that reacts with multivalent sialic acid residues on glycoproteins, to the Mr = 165,000 band. Purified B oligomer of pertussis toxin also uniquely detected a Mr = 165,000 component in CHO cell extracts while the A subunit of pertussis toxin was unreactive. These results indicate that pertussis toxin binds to a CHO cell glycoprotein with N-linked oligosaccharides and that sialic acid contributes to the complementary receptor site for the toxin. In addition, they suggest that a glycoprotein may serve as a cell surface receptor for pertussis toxin and that this interaction is mediated by a lectin-like binding site located on the B oligomer.

HEM13 of Saccharomyces cerevisiae encodes coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway. Expression of HEM13 is repressed by oxygen and heme. This study investigated the regulatory pathway responsible for the regulation of HEM13 expression. The transcriptional activator HAP1 is demonstrated to be required for the full-level expression of HEM13 in the absence of heme. It is also shown that the repression of HEM13 transcription caused by heme involves the HAP1 and ROX1 gene products; a mutation in either gene results in derepression of HEM13 expression. The heme-dependent expression of ROX1 was found to require functional HAP1, leading one to propose that repression of HEM13 results from a pathway involving HAP1-mediated regulation of ROX1 transcription in response to heme levels followed by ROX1-mediated repression of HEM13 transcription. In support of this model, expression of ROX1 under control of the GAL promoter was found to result in repression of HEM13 transcription in a hap1 mutant strain. The ability of ROX1 encoded by the galactose-inducible ROX1 construct to function in the absence of HAP1 indicates that the only role of HAP1 in repression of HEM13 is to activate ROX1 transcription.

In this study the sugar composition of podocalyxin was determined in puromycin aminonucleoside-treated (PAN) rats and controls. Podocalyxin from both control and PAN rats bound 125I-WGA and 125I-peanut lectin (the latter only after neuraminidase treatment) on nitrocellulose transfers. Purified podocalyxin from both control and PAN rats was found to contain sialic acid, Gal, GlcNac, and Man but lacked Fuc and GalNac by gas-liquid chromatography. In PAN rats the sialic acid content of podocalyxin was reduced from 4.5% to 1.5%, whereas the concentration of the other sugars (with the possible exception of Gal) was similar to that of controls. The density of podocalyxin on the epithelial cell surface was estimated after immunogold labeling with anti-podocalyxin IgG, and no differences were found between PAN rats and controls. These data indicate that the reduced total glomerular sialic acid content found in PAN is due to the combined effects of the decreased podocyte plasmalemmal surface area and the reduced sialic acid content of podocalyxin.

Cell lines expressing varying levels of ganglioside GM3 at the cell surface show different degrees of adhesion and spreading on solid phase coated with such glycosphingolipids (GSLs) as Gg3 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1Cer), LacCer (Gal beta 1----4Glc beta 1----1Cer), or Gb4 (GalNAc beta 1----3Gal alpha 1----4Gal beta 1----4Glc beta 1----1Cer) (where Cer is ceramide), which may have structures complementary to GM3, but not on solid phase coated with various other GSLs. The degree of cell adhesion and spreading on Gg3 was correlated with the degree of cell-surface GM3 expression, as defined by reactivity with anti-GM3 monoclonal antibody (mAb) DH2. Only cells with high GM3 expression adhered on solid phase coated with LacCer or Gb4. Adhesion of GM3-expressing cells on Gg3-, LacCer-, and Gb4-coated solid phase is based on interaction of GM3 with Gg3 and, to a lesser extent, with LacCer and Gb4, as demonstrated by: (i) the interaction of the GM3 liposome with solid phase coated with Gg3, LacCer, and Gb4, respectively; (ii) the abolition of cell adhesion on each GSL-coated solid phase by treatment of cells with mAb DH2 or sialidase; and (iii) the inhibition of cell adhesion by treatment of GSL-coated solid phase with mAb specific to each GSL. Sialosyllactosyl-lysyllysine conjugate was bound to Gg3 adsorbed on a C18 silica gel column in the presence of bivalent cation, suggesting that the carbohydrate moiety of GM3 is involved in GM3-Gg3 interaction. Not only the adhesion and spreading of GM3-expressing cells, but also their cell motility was greatly enhanced on Gg3-coated solid phase, as determined by Transwell assay and phagokinetic track assay on a gold sol-coated surface. Spreading and motility of GM3-expressing cells on Gg3-coated solid phase were both inhibited by treatment of cells with mAb DH2 or sialidase. These results provide evidence that not only cell adhesion, but also spreading and motility in these cell lines are controlled by complementary GSL-GSL interaction.

The asparagine-linked sugar chains of the membrane of baby hamster kidney cells and their polyoma transformant were quantitatively released as oligosaccharides by hydrazinolysis and labeled by NaB3H4 reduction. The radioactive oligosaccharides thus obtained were fractionated by paper electrophoresis. The neutral oligosaccharides of both cells were exclusively of high mannose type. The acidic oligosaccharides were bi-, tri-, and tetraantennary complex-type sugar chains with Man alpha 1----6 (Man alpha 1----3) Man beta 1----4 GlcNAc beta 1----4 (+/- Fuc alpha 1----6) GlcNAc as their cores and Gal beta 1----4 GlcNAc and various lengths of Gal beta 1----4 GlcNAc repeating chains in their outer-chain moieties. Prominent features of these acidic oligosaccharides are that all sialic acid residues were N-acetylneuraminic acid and were linked exclusively at C-3 of the nonreducing terminal galactose residues of the outer chains. Comparative study of oligosaccharides of the two cells by Bio-Gel P-4 column chromatography revealed that transformation of baby hamster kidney cells leads to a reduction in high mannose-type oligosaccharides and an increase in tetraantennary oligosaccharides. Increase of the outer chains linked at C-6 of the Man alpha 1----6 residue of the core is the cause of increase in the relative amount of highly branched oligosaccharides in the polyoma transformant.

The CDK inhibitor p21waf is a principal mediator of p53 function but can also be transactivated by many p53-independent stimuli leading to cell growth arrest or differentiation. In order to study the function of p21waf in a p53-deficient environment, we established an inducible expression of p21waf in the p53-null lung cancer cell line H1299, based on the muristerone-regulated system. Overexpression of p21waf led cells to growth arrest which after several days became irreversible and the arrested cells acquired a senescent phenotype as judged by cell shape, the senescence-associated beta-gal marker and inhibition of colony formation. The effect of p21waf overexpression, in the absence of p53, on the cytotoxicity caused by irradiation, doxorubicin and taxol was studied. Expression of p21waf provided protection against the cytotoxic effect of radiation and doxorubicin but not of taxol. These results are relevant to treatment of cancer when p53 is inactive.

The transcriptional activation function of the Saccharomyces cerevisiae <em>GAL</em>4 protein is modulated by the <em>GAL</em>80 and <em>GAL</em>3 proteins. In the absence of galactose, <em>GAL</em>80 inhibits the function of <em>GAL</em>4, presumably by direct binding to the <em>GAL</em>4 protein. The presence of galactose triggers the relief of the <em>GAL</em>80 block. The key to this relief is the <em>GAL</em>3 protein. How <em>GAL</em>3 and galactose activate <em>GAL</em>4 is not understood, but the long-standing notion has been that a galactose derivative formed by catalytic activity of <em>GAL</em>3 is the inducer that interacts with <em>GAL</em>80 or the <em>GAL</em>80-<em>GAL</em>4 complex. Here we report that overproduction of the <em>GAL</em>3 protein causes constitutive expression of <em>GAL</em>/MEL genes in the absence of exogenous galactose. Overproduction of the <em>GAL</em>1 protein (galactokinase) also causes constitutivity, consistent with the observations that <em>GAL</em>1 is strikingly similar in amino acid sequence to <em>GAL</em>3 and has <em>GAL</em>3-like induction activity. Cells lacking the <em>GAL</em>10-encoded UDP-galactose-UDP-glucose epimerase retained the constitutivity response to overproduction of <em>GAL</em>3, making it unlikely that constitutivity is due to endogenously produced galactose. A galactose-independent mechanism of constitutivity is further indicated by the inducing properties of two newly created galactokinaseless alleles of <em>GAL</em>1. On the basis of these data, we propose a new model for galactose-induced activation of the <em>GAL</em>4 protein. This model invokes galactose-activation of the <em>GAL</em>3 and <em>GAL</em>1 proteins which in turn elicit an alteration of the <em>GAL</em>80-<em>GAL</em>4 complex to activate <em>GAL</em>4. This model is consistent with all the known features of the system and has important implications for manipulating <em>GAL</em>4-dependent transcriptional activation in vitro.

The protective functions of saliva are attributed, in part, to its serous and mucous glycoproteins. We have studied, as representative molecules, the proline-rich glycoprotein (PRG) from human parotid saliva and the high (MG1) and low (MG2) molecular weight mucins from submandibular-sublingual saliva. PRG (38.9 kDa) contains 40% carbohydrate consisting of 6 triantennary N-linked units and a single peptide chain of 231 amino acids, 75% of which = PRO + GLY + GLN. PRG's secondary structure is comprised of 70% random coil (naked regions) and 30% beta-turns (glycosylated domains). MG1 (greater than 10(3) kDa) contains 15% protein (several disulfide linked subunits), 78% carbohydrate (290 units of 4-16 residues), 7% sulfate, and small amounts of covalently linked fatty acids. MG2 (200-250 kDa) contains 30% protein (single peptide chain), 68% carbohydrate (170 units of 2-7 residues), and 2% sulfate. The major carbohydrate units of MG2 are: NeuAc alpha 2,3Gal beta 1,3GalNAc,Gal beta 1,3GalNAc, and Fuc alpha 1,2Gal beta 1,3GalNAc. MG1 contains hydrophobic domains, as evidenced by its ability to bind fluorescent hydrophobic probes; MG2 does not. Collectively, the biochemical and biophysical comparisons between MG1 and MG2 indicate that these two mucins are structurally different. Several functional properties of MG1, MG2, and PRG have been examined, including their presence in two-hour in vivo enamel pellicle, binding to synthetic hydroxyapatite, lubricating properties, and interactions with oral streptococci. The data presented suggest that these glycoproteins may have multiple functions which are predicated, in part on their carbohydrate units. The potential significance of the structure-function relationships of these glycoproteins to the oral ecology is discussed.

The carbohydrate deficient glycoprotein (CDG) syndromes are a family of genetic multisystemic disorders with severe nervous system involvement. This report is on a child with a CDG syndrome that differs from the classical picture but is very similar to a patient reported in 1991. Both these patients are therefore designated CDG syndrome type II. Compared with type I patients they have a more severe psychomotor retardation but no peripheral neuropathy nor cerebellar hypoplasia. The serum transferrin isoform pattern obtained by isoelectric focusing showed disialotransferrin as the major fraction. The serum disialotransferrin, studied in the present patient, contained two moles of truncated monoantennary Sialyl-Gal-GlcNAc-Man(alpha 1-->3)[Man(alpha 1-->6)]Man(beta 1-->4)GlcNAc (beta 1-->4)GlcNAc-Asn per mole of transferrin. A profoundly deficient activity of the Golgi enzyme N-acetylglucosaminyltransferase II (EC 2.4.1.143) was demonstrated in fibroblasts.