Hypothalamic neuropeptides play a role in appetite and weight regulation. Food restriction for 2 weeks and food deprivation for 4 days were used as models to characterize the effects of weight loss on hypothalamic peptide gene expression in male and female rats. We used in situ hybridization to examine the mRNA levels of hypothalamic peptides which stimulate and inhibit food intake and found selective effects primarily in the arcuate nucleus. Neuropeptide Y (NPY) mRNA was increased and pro-opiomelanocortin (POMC) and galanin (GAL) mRNA were decreased in the hypothalamic arcuate nucleus and corticotropin-releasing hormone (CRH) mRNA was decreased in the hypothalamic paraventricular nucleus in male and female food-restricted and food-deprived rats. Food restriction produced larger changes in peptide mRNA expression than did food deprivation. Changes in NPY, POMC and CRH gene expression induced by food restriction were greater in male than female rats. Elevated NPY and reduced CRH gene expression may be a compensatory physiological response to restore food intake in food-restricted and food-deprived animals. The discrete changes in NPY, POMC, GAL and CRH gene expression in food-restricted and food-deprived animals suggest the involvement of these peptides in abnormal appetitive behavior and weight loss associated with human eating disorders.

Plasmodium falciparum malaria parasites invade human erythrocytes by means of a parasite receptor for erythrocytes, the 175-kD erythrocyte binding antigen (EBA-175). Similar to invasion efficiency, binding requires N-acetylneuraminic acid (Neu5Ac) on human erythrocytes, specifically the glycophorins. EBA-175 bound to erythrocytes with receptor-like specificity and was saturable. The specificity of EBA-175 binding was studied to determine if its binding is influenced either by simple electrostatic interaction with the negatively charged Neu5Ac (on the erythrocyte surface); or if Neu5Ac indirectly affected the conformation of an unknown ligand, or if Neu5Ac itself in specific linkage and carbohydrate composition was the primary ligand for EBA-175 as demonstrated for hemagglutinins of influenza viruses. Most Neu5Ac on human erythrocytes is linked to galactose by alpha 2-3 and alpha 2-6 linkages on glycophorin A. Soluble Neu5Ac by itself in solution did not competitively inhibit the binding of EBA-175 to erythrocytes, suggesting that linkage to an underlying sugar is required for binding in contrast to charge alone. Binding was competitively inhibited only by Neu5Ac(alpha 2-3)Gal-containing oligosaccharides. Similar oligosaccharides containing Neu5Ac(alpha 2-6)Gal-linkages had only slight inhibitory effects. Binding inhibition assays with modified sialic acids and other saccharides confirmed that oligosaccharide composition and linkage were primary factors for efficient binding. EBA-175 bound tightly enough to glycophorin A that the complex could be precipitated with an anti-glycophorin A monoclonal antibody. Selective cleavage of O-linked tetrasaccharides clustered at the NH2 terminus of glycophorin A markedly reduced binding in inhibition studies. We conclude that the Neu5Ac(a2,3)-Gal- determinant on O-linked tetrasaccharides of glycophorin A appear to be the preferential erythrocyte ligand for EBA-175.

Tumor angiogenesis is a key event in cancer progression. Here, we report that tumors can stimulate tumor angiogenesis by secretion of galectin-1. Tumor growth and tumor angiogenesis of different tumor models are hampered in galectin-1-null (gal-1(-/-)) mice. However, tumor angiogenesis is less affected when tumor cells express and secrete high levels of galectin-1. Furthermore, tumor endothelial cells in gal-1(-/-) mice take up galectin-1 that is secreted by tumor cells. Uptake of galectin-1 by cultured endothelial cells specifically promotes H-Ras signaling to the Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) kinase (Mek)/Erk cascade and stimulates endothelial cell proliferation and migration. Moreover, the activation can be blocked by galectin-1 inhibition as evidenced by hampered membrane translocation of H-Ras.GTP and impaired Raf/Mek/Erk phosphorylation after treatment with the galectin-1-targeting angiogenesis inhibitor anginex. Altogether, these data identify galectin-1 as a proangiogenic factor. These findings have direct implications for current efforts on galectin-1-targeted cancer therapies.

Transcription repression by the basic region-helix-loop-helix-zipper (bHLHZip) protein Mad1 requires DNA binding as a ternary complex with Max and mSin3A or mSin3B, the mammalian orthologs of the Saccharomyces cerevisiae transcriptional corepressor SIN3. The interaction between Mad1 and mSin3 is mediated by three potential amphipathic alpha-helices: one in the N terminus of Mad (mSin interaction domain, or SID) and two within the second paired amphipathic helix domain (PAH2) of mSin3A. Mutations that alter the structure of the SID inhibit in vitro interaction between Mad and mSin3 and inactivate Mad's transcriptional repression activity. Here we show that a 35-residue region containing the SID represents a dominant repression domain whose activity can be transferred to a heterologous DNA binding region. A fusion protein comprising the Mad1 SID linked to a Ga14 DNA binding domain mediates repression of minimal as well as complex promoters dependent on Ga14 DNA binding sites. In addition, the SID represses the transcriptional activity of linked VP16 and c-Myc transactivation domains. When fused to a full-length c-Myc protein, the Mad1 SID specifically represses both c-Myc's transcriptional and transforming activities. Fusions between the GAL DNA binding domain and full-length mSin3 were also capable of repression. We show that the association between Mad1 and mSin3 is not only dependent on the helical SID but is also dependent on both putative helices of the mSin3 PAH2 region, suggesting that stable interaction requires all three helices. Our results indicate that the SID is necessary and sufficient for transcriptional repression mediated by the Mad protein family and that SID repression is dominant over several distinct transcriptional activators.

Aberrant expression of immature truncated O-glycans is a characteristic feature observed on virtually all epithelial cancer cells, and a very high frequency is observed in early epithelial premalignant lesions that precede the development of adenocarcinomas. Expression of the truncated O-glycan structures Tn and sialyl-Tn is strongly associated with poor prognosis and overall low survival. The genetic and biosynthetic mechanisms leading to accumulation of truncated O-glycans are not fully understood and include mutation or dysregulation of glycosyltransferases involved in elongation of O-glycans, as well as relocation of glycosyltransferases controlling initiation of O-glycosylation from Golgi to endoplasmic reticulum. Truncated O-glycans have been proposed to play functional roles for cancer-cell invasiveness, but our understanding of the biological functions of aberrant glycosylation in cancer is still highly limited. Here, we used exome sequencing of most glycosyltransferases in a large series of primary and metastatic pancreatic cancers to rule out somatic mutations as a cause of expression of truncated O-glycans. Instead, we found hypermethylation of core 1 β3-Gal-T-specific molecular chaperone, a key chaperone for O-glycan elongation, as the most prevalent cause. We next used gene editing to produce isogenic cell systems with and without homogenous truncated O-glycans that enabled, to our knowledge, the first polyomic and side-by-side evaluation of the cancer O-glycophenotype in an organotypic tissue model and in xenografts. The results strongly suggest that truncation of O-glycans directly induces oncogenic features of cell growth and invasion. The study provides support for targeting cancer-specific truncated O-glycans with immunotherapeutic measures.

In plants, the only known outer-chain elongation of complex N-glycans is the formation of Lewis a [Fuc alpha1-4(Gal beta1-3)GlcNAc-R] structures. This process involves the sequential attachment of beta1,3-galactose and alpha1,4-fucose residues by beta1,3-galactosyltransferase and alpha1,4-fucosyltransferase. However, the exact mechanism underlying the formation of Lewis a epitopes in plants is poorly understood, largely because one of the involved enzymes, beta1,3-galactosyltransferase, has not yet been identified and characterized. Here, we report the identification of an Arabidopsis thaliana beta1,3-galactosyltransferase involved in the biosynthesis of the Lewis a epitope using an expression cloning strategy. Overexpression of various candidates led to the identification of a single gene (named GALACTOSYLTRANSFERASE1 [GALT1]) that increased the originally very low Lewis a epitope levels in planta. Recombinant GALT1 protein produced in insect cells was capable of transferring beta1,3-linked galactose residues to various N-glycan acceptor substrates, and subsequent treatment of the reaction products with alpha1,4-fucosyltransferase resulted in the generation of Lewis a structures. Furthermore, transgenic Arabidopsis plants lacking a functional GALT1 mRNA did not show any detectable amounts of Lewis a epitopes on endogenous glycoproteins. Taken together, our results demonstrate that GALT1 is both sufficient and essential for the addition of beta1,3-linked galactose residues to N-glycans and thus is required for the biosynthesis of Lewis a structures in Arabidopsis. Moreover, cell biological characterization of a transiently expressed GALT1-fluorescent protein fusion using confocal laser scanning microscopy revealed the exclusive location of GALT1 within the Golgi apparatus, which is in good agreement with the proposed physiological action of the enzyme.

Transcription from two overlapping gal promoters is repressed by Gal repressor binding to bipartite gal operators, O(E) and O(I), which flank the promoters. Concurrent repression of the gal promoters also requires the bacterial histone-like protein HU which acts as a co-factor. Footprinting experiments using iron-EDTA-coupled HU show that HU binding to gal DNA is orientation specific and is specifically dependent upon binding of GalR to both O(E) and O(I). We propose that HU, in concert with GalR, forms a specific nucleoprotein higher order complex containing a DNA loop. This way, HU deforms the promoter to make the latter inactive for transcription initiation while remaining sensitive to inducer. The example of gal repression provides a model for studying how a 'condensed' DNA becomes available for transcription.

Mutants that adsorb certain colicins without being killed, i.e., tolerant mutants (tol), were isolated from Escherichia coli K-12 strains. Selection was done either with colicin K or E2. Several groups of mutants showing different phenotypes were found, and some of them showed tolerance to both K and E colicins, which have different receptors. Many of these mutants mapped near gal. Typical mutants from group II, III, and IV were studied in more detail. The mutant loci were contransducible with gal by phage P1. The linkage order was deduced to be tol-gal-lambda. In partially diploid strains, these mutant loci are recessive to wild-type alleles. Temperature-dependent conditionally tolerant mutants were also isolated. Two groups were found: the first was tolerant to E2 and E3 at 40 C, but sensitive at 30 C; the second was tolerant to E2 at 30 C, but sensitive at 40 C. Experiments done with these mutants suggest that these mutations affect the heat lability of some protein that is necessary for the response of cells to colicins. Conditionally lethal tolerant mutants were isolated which at 40 C were tolerant to E2 and E3 and could not grow, but which at 30 C were fully sensitive and grew normally. The mutation mapped near malA. The tolerance at 40 C is not due to a consequence of an inactivation of general cellular metabolism, but presumably is a cause of the subsequent inhibition of cellular growth. The results suggest that some protein components involved in the response to colicin are also vital to normal cellular growth.

While it is well established that phenotypic modulation of vascular smooth muscle cells (VSMCs) contributes to the development and progression of vascular lesions, little is known regarding the molecular mechanisms of phenotypic modulation in vivo. Here we show that vascular injury reduces transcription of VSMC differentiation marker genes, and we identify cis regulatory elements that may mediate this decrease. Using a carotid wire-injury model in mice carrying transgenes for smooth muscle alpha-actin, smooth muscle myosin heavy chain, or a SM22alpha promoter-beta-gal reporter, we collected arteries 7 and 14 days after injury and assessed changes in endogenous protein and mRNA levels and in beta-gal activity. Endogenous levels for all markers were decreased 7 days after injury and returned to nearly control levels by 14 days. beta-gal staining in all lines followed a similar pattern, suggesting that transcriptional downregulation contributed to the injury-induced decreases. To begin to dissect this response, we mutated a putative G/C-rich repressor in the SM22alpha promoter transgene and found that this mutation significantly attenuated injury-induced downregulation. Hence, transcriptional downregulation contributes to injury-induced decreases in VSMC differentiation markers, an effect that may be partially mediated through a G/C-rich repressor element.

The observation that the glycolipid alpha-galactosylceramide (alpha-GalCer) is a potent stimulator of natural killer T (NKT) cells has provided an important means for investigating NKT cell biology. alpha-GalCer is presented on CD1d to the invariant NKT receptor, leading to interleukin-12 (IL-12) production by dendritic cells (DCs) and to NK cell activation. We review our research on the tumor-protective properties of alpha-GalCer, particularly the major role played by DCs. We compared administration of alpha-GalCer on mature DCs with soluble glycolipid and found that DCs induced more prolonged interferon-gamma (IFN-gamma) production by NKT cells and better protection against B16 melanoma. Human alpha-GalCer-loaded DCs also expanded NKT cell numbers in cancer patients. alpha-GalCer-activated NKT cells were then found to induce DC maturation in vivo. The maturing DCs produced IL-12, upregulated co-stimulatory molecules, and induced adaptive immunity to captured cellular antigens, including prolonged, combined CD4(+)/CD8(+) T-cell immunity to dying tumor cells. Surprisingly, co-stimulator-poor tumor cells, if directly loaded with alpha-GalCer ('tumor/Gal') and injected intravenously, also induced strong NKT- and NK-cell responses. The latter killed the tumor/Gal, which were subsequently cross presented by CD1d on DCs to elicit DC maturation and prolonged adaptive T-cell immunity, which lasted 6-12 months. These findings help explain tumor protection via alpha-GalCer and urge development of the DC-NKT axis to provide innate and adaptive immunity to human cancers.

The sequence of the glycosyl residues and the anomeric configurations of the glycosyl linkages of the acidic polysaccharides secreted by Rhizobium leguminosarum 128c53, Rhizobium leguminosarum 128c63, Rhizobium trifolii NA30, and Rhizobium trifolii 0403 have been determined. All four polysaccharides were found to have the following glycosyl repeating-unit structure, where galactosyl is Gal, glucosyl is Glc, glucuronosyl is GlcA, and pyruvyl is Pyr: [Formula: see text] Each of the glycosyl residues of these polysaccharides was determined to be in the d configuration and in the pyranose ring form. These results add support to the proposal that R. leguminosarum and R. trifolii have a particularly close genetic relationship. The significance of these results with regard to the possible function of these polysaccharides in the nodulation process is discussed.

Mucin-type O-glycosylation is initiated by UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc-transferases). The role each GalNAc-transferase plays in O-glycosylation is unclear. In this report we characterized the specificity and kinetic properties of three purified recombinant GalNAc-transferases. GalNAc-T1, -T2, and -T3 were expressed as soluble proteins in insect cells and purified to near homogeneity. The enzymes have distinct but partly overlapping specificities with short peptide acceptor substrates. Peptides specifically utilized by GalNAc-T2 or -T3, or preferentially by GalNAc-T1 were identified. GalNAc-T1 and -T3 showed strict donor substrate specificities for UDP-GalNAc, whereas GalNAc-T2 also utilized UDP-Gal with one peptide acceptor substrate. Glycosylation of peptides based on MUC1 tandem repeat showed that three of five potential sites in the tandem repeat were glycosylated by all three enzymes when one or five repeat peptides were analyzed. However, analysis of enzyme kinetics by capillary electrophoresis and mass spectrometry demonstrated that the three enzymes react at different rates with individual sites in the MUC1 repeat. The results demonstrate that individual GalNAc-transferases have distinct activities and the initiation of O-glycosylation in a cell is regulated by a repertoire of GalNAc-transferases.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an important marker of gene expression. However, the sensitivity of wild-type GFP has been below that of standard reporter proteins, such as beta-galactosidase, which utilize enzymatic amplification. To improve the detection of GFP in transfected mammalian cells, we have constructed a unique GFP variant which contains chromophore mutations that make the protein 35 times brighter than wild-type GFP, and is codon-optimized for higher expression in mammalian cells. These changes in the GFP coding sequence provide an enhanced GFP (EGFP) that greatly increases the sensitivity of the reporter protein. We show that the EGFP expression vector delivered into mammalian cells gives rise to bright fluorescence that is readily detectable following a 16-24 hr transfection interval. Visual detection of transfected cells with EGFP appears to be more sensitive than equivalent measurements with beta-galactosidase catalyzed conversion of the X-gal substrate. We conclude that EGFP allows sensitive and convenient detection of gene transfer in mammalian cells.

Inhibition of mTOR by rapamycin prevents cellular senescence. Here we investigated the effects of MEK and PI-3K on cellular senescence. Unlike LY294002 (PI-3K inhibitor), both U0126 and PD98059 (MEK inhibitors) did not significantly decrease beta-Gal staining in aging human fibroblasts and fibrosarcoma cells. However, using a sensitive, functional method, we identified that not only LY294002 but also U0126 prevented irreversible loss of proliferative potential associated with cellular senescence. At concentrations that blocked S6 phosphorylation, rapamycin, U0126 and LY294002 equally prevented senescence. Furthermore, there was no additive effect by combining of rapamycin with either U0126 or LY294002. Taken together this suggests that (a) simultaneous activation of PI-3K and MEK is required to ensure cellular senescence and (b) U0126 and LY294002 suppress senescence via the rapamycin-sensitive pathway.

High-sensitivity glycan profiling providing detailed structural information is very important in the search for glycan disease markers. By combining a straight-forward and fast preparation protocol of mucins with high-throughput nanoLC/MS, we have been able to study the O-glycosylation of the colon MUC2 mucin from one single biopsy (approximately 5 mg wet tissue as starting material) collected from the sigmoid colon during routine colonoscopy of 25 normal control patients. This large mucin glycoprotein was recovered from the guanidinium chloride-extracted insoluble pellet, reduced and alkylated, separated by SDS-agarose polyacrylamide composite gel electrophoresis, and transferred to a PVDF membrane. The O-linked oligosaccharides of the major MUC2 monomer band were released by reductive beta-elimination and analyzed by nanoLC/mass spectrometry and MS(n). The aim was to identify the MUC2 O-glycans of the sigmoid colon and provide a comprehensive catalog of the O-glycan repertoire. More than 100 complex O-linked oligosaccharides were identified, of which some had not been described before. Most of the oligosaccharides were based on the core 3 structure with sialic acid at the 6-position of the GalNAc and the substructure Gal beta 1-3/4-GlcNAc beta 1-3(NeuAc-6)GalNAcol was found in most glycans. The most abundant components were -Gal-(Fuc)GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-(NeuAc-)Gal-4/3GlcNAc-3(NeuAc-6)GalNAcol, GalNAc-3(NeuAc-6) GalNAcol, and GlcNAc-3(NeuAc-6)GalNAcol. In contrast to the O-glycans of other mucins, the sigmoid MUC2 O-glycan repertoire and relative amounts in normal individuals were relatively constant.

Fibroblast growth factor 14 (FGF14) belongs to a distinct subclass of FGFs that is expressed in the developing and adult CNS. We disrupted the Fgf14 gene and introduced an Fgf14(N-beta-Gal) allele that abolished Fgf14 expression and generated a fusion protein (FGF14N-beta-gal) containing the first exon of FGF14 and beta-galactosidase. Fgf14-deficient mice were viable, fertile, and anatomically normal, but developed ataxia and a paroxysmal hyperkinetic movement disorder. Neuropharmacological studies showed that Fgf14-deficient mice have reduced responses to dopamine agonists. The paroxysmal hyperkinetic movement disorder phenocopies a form of dystonia, a disease often associated with dysfunction of the putamen. Strikingly, the FGF14N-beta-gal chimeric protein was efficiently transported into neuronal processes in the basal ganglia and cerebellum. Together, these studies identify a novel function for FGF14 in neuronal signaling and implicate FGF14 in axonal trafficking and synaptosomal function.

Several plant lectins were used to characterize the cell-surface carbohydrates expressed on sensory ganglion cells and their central terminals in the spinal cord dorsal horn. In the rat, galactose-terminal glycoconjugates on a large subpopulation of small neurons whose central axons project to the substantia gelatinosa were demonstrated with the alpha-D-galactose-specific Griffonia Simplicifolia I-B4 (GSA) lectin. This neuron subset was labelled by alternative D-galactose-, N-Acetylgalactosamine-, and beta Gal(1,3)NAcGal-binding lectins. Similar GSA lectin reactivity was also illustrated in selected peripheral autonomic, gustatory and visceral sensory and enteric neurons, and the accessory olfactory bulb. The sensory neuron-specific isoenzyme, fluoride-resistant acid phosphatase (FRAP) co-localized with the GSA lectin, as did the monoclonal antibody (MAb) 2C5, which is directed against a lactoseries carbohydrate constituting a backbone structure of ABH human blood group antigens. In contrast, calcitonin gene-related peptide-immunoreactivity (CGRP-IR), used as a representative marker of peptidergic neurons, exhibited limited co-localization with GSA. A polyclonal anti-rat red blood cell (RBC) antibody co-localized with GSA, suggesting that lectin-reactive carbohydrates on rat sensory neurons are related to rat RBC antigens. In the human spinal cord, the L-fucose-binding Ulex europaeus-I (UEA) lectin also labelled the substantia gelatinosa; in rabbit, a small sensory ganglion cell subset and the spinal cord substantial gelatinosa was co-labelled by both the GSA and UEA lectins. These studies illustrate significant lectin-reactive cell surface carbohydrate expression by non-peptidergic, FRAP(+) sensory ganglion cells in the rat, and provide a means for visualizing the extensive, non-peptidergic, small sensory ganglion cell subpopulations, probably including a substantial proportion of nociceptive and unmyelinated peripheral axons.

Recently we described the isolation of spontaneous bacteriophage K139-resistant Vibrio cholerae O1 El Tor mutants. In this study, we identified phage-resistant isolates with intact O antigen but altered core oligosaccharide which were also affected in galactose catabolism; this strains have mutations in the galU gene. We inactivated another gal gene, galE, and the mutant was also found to be defective in the catabolism of exogenous galactose but synthesized an apparently normal lipopolysaccharide (LPS). Both gal mutants as well as a rough LPS (R-LPS) mutant were investigated for the ability to colonize the mouse small intestine. The galU and R-LPS mutants, but not the galE mutant, were defective in colonization, a phenotype also associated with O-antigen-negative mutants. By investigating several parameters in vitro, we could show that galU and R-LPS mutants were more sensitive to short-chain organic acids, cationic antimicrobial peptides, the complement system, and bile salts as well as other hydrophobic agents, indicating that their outer membrane no longer provides an effective barrier function. O-antigen-negative strains were found to be sensitive to complement and cationic peptides, but they displayed significant resistance to bile salts and short-chain organic acids. Furthermore, we found that galU and galE are essential for the formation of a biofilm in a spontaneous phage-resistant rugose variant, suggesting that the synthesis of UDP-galactose via UDP-glucose is necessary for biosynthesis of the exopolysaccharide. In addition, we provide evidence that the production of exopolysaccharide limits the access of phage K139 to its receptor, the O antigen. In conclusion, our results indicate involvement of galU in V. cholerae virulence, correlated with the observed change in LPS structure, and a role for galU and galE in environmental survival of V. cholerae.

The susceptibility of hematopoietic progenitor cells to infection by human cytomegalovirus (HCMV) was investigated using several strains of HCMV, including the recombinant strain RC256. RC256 is derived from the laboratory strain Towne and contains the Escherichia coli LacZ gene coding for beta-galactosidase (beta-gal) regulated by an early HCMV promoter. Expression of LacZ allowed the detection of HCMV in individual hematopoietic cells. Clonogeneic bone marrow (BM) progenitors, including CD34+ cells, could be infected with HCMV and would then form normal hematopoietic colonies. By polymerase chain reaction (PCR) amplification of DNA, HCMV could be detected in both erythroid and myeloid colonies. LacZ activity was observed predominantly in cells of myelomonocytic lineage. When cells derived from HCMV-infected progenitors were cocultivated with permissive human fibroblasts, infectious virus expressing LacZ was recovered. Although no characteristic HCMV cytopathology was observed in BM colonies, high virus to cell ratios resulted in a moderate inhibition of colony formation. Since infected hematopoietic progenitors can harbor HCMV for weeks and through several differentiation steps in culture, we postulate that in vivo these cells may serve as a reservoir of latent virus and contribute to HCMV dissemination.

The only identified component of the scrapie prion is PrPSc, a glycosylinositol phospholipid (GPI)-linked protein that is derived from the cellular isoform (PrPC) by an as yet unknown posttranslational event. Analysis of the PrPSc GPI has revealed six different glycoforms, three of which are unprecedented. Two of the glycoforms contain N-acetylneuraminic acid, which has not been previously reported as a component of any GPI. The largest form of the GPI is proposed to have a glycan core consisting of Man alpha-Man alpha-Man-(NeuAc-Gal-GalNAc-)Man-GlcN-Ino. Identical PrPSc GPI structures were found for two distinct isolates or "strains" of prions which specify different incubation times, neuropathology, and PrPSc distribution in brains of Syrian hamsters. Limited analysis of the PrPC GPI reveals that it also has sialylated glycoforms, arguing that the presence of this monosaccharide does not distinguish PrPC from PrPSc.

To distinguish between mechanisms of eukaryotic transcriptional activation, we tested whether yeast upstream promoter elements can stimulate transcription by a heterologous transcription machinery, bacteriophage T7 RNA polymerase. The gal enhancer-like element recognized by GAL4 protein or the ded1 poly(dA-dT) element was placed upstream of the T7 promoter and his3 structural gene, and T7 RNA polymerase was produced in yeast cells. Under conditions where the gal element would normally be either activating or nonactivating, his3 transcription by T7 RNA polymerase was not stimulated above the level observed in the absence of any upstream element. In contrast, the ded1 poly(dA-dT) element stimulated transcription 7-fold, similar to the enhancement observed on the native ded1 promoter. Activation by the ded1 element thus may involve effects on the chromatin template that facilitate entry of the transcription machinery, whereas activation by the gal element may involve specific contacts between GAL4 and the transcriptional machinery.

Despite its importance, the molecular basis of mammalian gamete recognition has remained unclear. The enzyme beta-1,4-galactosyltransferase (Gal-transferase) has been viewed traditionally as a biosynthetic component of the Golgi complex, but is also found on the surface of many cells where it can bind its specific glycoside substrate on adjacent cell surfaces or in the extracellular matrix. In mouse it has been suggested that Gal-transferase on the sperm head mediates fertilization by binding oligosaccharide residues in the egg coat, or zona pellucida, and that the ability of the zona pellucida to bind sperm is conferred by oligosaccharides of the ZP3 glycoprotein. However, it has not been confirmed that Gal-transferase and ZP3 are in fact complementary gamete receptors whose interaction mediates sperm-egg binding. Here we show that mouse sperm Gal-transferase specifically recognizes those oligosaccharides on ZP3 that have sperm-binding activity, but does not interact with other zona pellucida glycoproteins. In contrast, all zona pellucida glycoproteins are recognized by non-sperm Gal-transferase, demonstrating a more stringent substrate specificity for the sperm enzyme. This interaction is required for sperm-egg binding because blocking or removing the binding site for Gal-transferase on ZP3 inhibits its ability to bind sperm. After the release of the sperm acrosome, the transferase relocalizes to a new membrane domain where it can no longer bind to ZP3, which is consistent with the inability of acrosome-reacted sperm to bind ZP3 or to initiate binding to the zona pellucida. Following fertilization, ZP3 is modified by egg cortical granule secretions so that it loses sperm receptor activity, which can be accounted for by a selective loss of its binding site for sperm Gal-transferase. These results show that sperm surface beta-1,4-galactosyltransferase and the egg-coat glycoprotein ZP3 are complementary adhesion molecules that mediate primary gamete binding in the mouse.

The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and co-immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.

There is growing evidence that some terrestrial avian species may play a role in the genesis of influenza viruses with pandemic potential. In the present investigation, we examined whether quail, a widespread-farmed poultry, possess the proper characteristics for serving as an intermediate host for the zoonotic transmission of influenza viruses. Using a lectin-based staining based on specific agglutinins, we found that, in addition to the presence of sialic acid alpha2,3-galactose (SAalpha2,3-gal) linked receptors, there are abundant sialic acid alpha2,6-galactose (SAalpha2,6-gal) linked receptors in quail trachea and intestine. The presence of abundant SAalpha2,6-gal-linked receptors explains, at least in part, the circulation of avian influenza viruses with human-like receptor specificity in quail. In quail trachea, SAalpha2,3-gal linked receptors are present primarily in non-ciliated cells, while SAalpha2,6-gal linked receptors are localized predominantly on the surface of ciliated cells. In quail intestine, both types of receptors were found on epithelial cells as well as in crypts. In a solid-phase overlay binding assay, both avian and human influenza viruses bind to plasma membranes prepared from epithelial cells of quail trachea and intestine, strongly suggesting that these receptors are functional for binding of influenza viruses from different species. Together with previous observations, these results are consistent with the notion that quail could provide an environment for the spread of reassortants between avian and human influenza viruses, thus acting as a potential intermediate host.