When galactose is added to logarithmically growing culture of the yeast Saccharomyces cerevisiae, a set of genes encoding galactose-metabolizing enzymes (<em>GAL</em> genes) starts to be transcribed within a few minutes. This rapid induction involves a serial interplay of Gal3p, Gal80p, and Gal4p. Recent experiments have indicated that a direct interaction between Gal3p and Gal80p plays a pivotal role in an early step of <em>GAL</em> induction. Here we demonstrate that complex of Gal3p and Gal80p, otherwise unstable, is stabilized in the presence of 0.1 mM galactose and 0.5 mM ATP. The requirement for galactose and ATP for stable complex formation is also observed by using highly purified Gal3p and Gal80p from yeast. We further show that thus formed Gal3p/Gal80p complex can easily be dissociated when it is washed with buffer lacking galactose. Finally, we show that mutant proteins encoded by <em>GAL</em>80S or <em>GAL</em>80DE21, which confer galactose-uninducible phenotype, fail to interact with Gal3p. These results strongly suggest that Gal3p functions as the sensor and transducer of galactose signal in the induction pathway of Gal4p-activated genes.

Cells adjust their transcriptional state to accommodate environmental and genetic perturbations. An open question is to what extent transcriptional response to perturbations has been specifically selected along evolution. To test the possibility that transcriptional reprogramming does not need to be 'pre-designed' to lead to an adaptive metabolic state on physiological timescales, we confronted yeast cells with a novel challenge they had not previously encountered. We rewired the genome by recruiting an essential gene, HIS3, from the histidine biosynthesis pathway to a foreign regulatory system, the GAL network responsible for galactose utilization. Switching medium to glucose in a chemostat caused repression of the essential gene and presented the cells with a severe challenge to which they adapted over approximately 10 generations. Using genome-wide expression arrays, we show here that a global transcriptional reprogramming (>1200 genes) underlies the adaptation. A large fraction of the responding genes is nonreproducible in repeated experiments. These results show that a nonspecific transcriptional response reflecting the natural plasticity of the regulatory network supports adaptation of cells to novel challenges.

This review provides an overview of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α-complementation, in which addition to the inactive dimers of peptides containing the "missing" N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of β-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.

Accumulation and deposition of beta-amyloid peptide, a major constituent in neuritic plaques are hallmarks of Alzheimer's disease (AD) and AD-related neurodegenerative diseases. beta-Amyloid (Abeta) is derived from the proteolytic cleavage of amyloid precursor protein (APP), a transmembrane protein present in three major isoforms in brain comprising 695, 751 and 770 amino acids, respectively. Among other post-translational modifications, APP is modified during maturation by N- and O-glycosylation, which are thought to be responsible for its expression and secretion. Unlike N-glycosylation, no sites of O-glycosylation of APP have previously been reported. We report here the identification of three specific O-glycosylation sites of the secreted APP695 (sAPP695) produced in CHO cells, using a combination of high-performance liquid chromatography and electrospray-tandem mass spectrometry. With the use of electron transfer dissociation and collision induced dissociation (ETD and CID), we identified type, composition and structures of the Core 1 type O-linked glycans attached at the residues Thr 291, Thr 292 and Thr 576 of the full-length APP695. The glycosylations comprise multiple short glycans, containing N-acetyl galactosamine (GalNAc), Gal-GalNAc and sialic acid terminated structures. The presence of the glycopeptides in the tryptic mixture was identified using the CID-generated sugar oxonium ions. ETD proved to be valuable for the unambiguous identification of the modified sites as ETD fragmentation occurred along the peptide backbone with little or no cleavage of the glycans. Thus, the combination of the CID and ETD techniques in LC-MS is shown here, as a powerful tool for de novo identification of O-glycosylations at unknown modification sites in proteins.

Galectin-3 (gal-3), a member of the beta-galactoside-binding proteins family, was identified as a binding partner of beta-catenin. Analysis of the human gal-3 sequence reveled a structural similarity to beta-catenin as it also contains the consensus sequence (S92XXXS96) for glycogen synthase kinase-3beta (GSK-3beta) phosphorylation and can serve as its substrate. In addition, Axin, a regulator protein of Wnt that complexes with beta-catenin, also binds gal-3 using the same sequence motif identified here by a deletion mutant analysis. The data presented here give credence to the suggestion that gal-3 is a key regulator in the Wnt/beta-catenin signaling pathway and highlight the functional similarities between gal-3 and beta-catenin.

To compare features of the receptor-binding sites (RBSs) of different influenza virus hemagglutinins (HA), binding of a number of synthetic sialic acid (SA) analogs and natural sialosides by a panel of about 30 human influenza A and B virus strains was studied in a competitive ligand binding assay. For all the viruses tested, the N-acetyl group of Neu5Ac, as well as the natural orientation of the carboxylic group at C2 and the hydroxylic group at C4, was essential for binding. Significant type- and subtype-specific differences were observed in virus recognition of asialic parts of sialosides. H1 strains, unlike H3 and type B viruses, were found to bind alpha 2-6-sialyl-N-acetyllactosamine with about an order of magnitude higher affinity than alpha 2-6-sialyllactose (6'SL). The H1 viruses and the H3 strains with Gln in position 226 of HA, but not the H3 strains with Leu-226, bound 6'SL with a lower affinity than alpha 2-3-sialyllactose; this effect correlated clearly with the preferential binding by the former viruses of unsubstituted alpha Neu5Ac compared to methyl alpha-glycoside of Neu5Ac. Thus, differentiation between the types of the SA-Gal linkage by the A viruses appeared to depend, at least partially, upon the recognition by the HA of the first hydrocarbon group of the aglycon. Type B virus strains were distinct in having a lower affinity for the Neu5Ac moiety and in providing a higher contribution of the asialic portions of sialosides to the HA-ligand interactions. The last effects are presumably due to the amino acid insertions in the type B HA surrounding the RBS, which makes the receptor-binding pocket deeper. The results obtained in the present investigation indicate that while the functional groups of Neu5Ac studied are recognized by the RBSs of all influenza viruses, the magnitude of their contribution to the binding energy, as well as the contribution of the asialic portion of the receptor, may vary in dependence upon the virus type, subtype, and strain.

OBJECTIVE

Intestinal crypts constitute a niche in which epithelial progenitors respond to Wnt signals, replicate, and prepare to differentiate. Because mutations in Wnt pathway genes lead to intestinal cancer, the role of Wnt signaling in gut epithelial homeostasis is a subject of intense investigation. We studied how Wnt signaling is established during intestine development.

METHODS

We studied spatiotemporal features of Wnt signaling at formative stages in mouse embryos, when villous projections appear and crypt precursors occupy intervillus regions. We used TOP-GAL transgenic and Axin2(LacZ) mice, which report faithfully on canonical Wnt activity, relevant molecular markers, and embryos with aberrant beta-catenin activation.

RESULTS

Developing intestines first display evidence for Wnt signaling after appearance of villi. During villus morphogenesis, intervillus cells proliferate actively but lack signs of canonical Wnt signaling. Surprisingly, in late gestation and briefly thereafter, conspicuous Wnt activity is evident in differentiated, postmitotic villus epithelium. Neither Tcf4, a principal transcriptional effector of intestinal Wnt signals, nor candidate Wnt targets CD44 and cyclinD1 are expressed in late fetal villus cells that show high Wnt activity. Instead, those cells express the related factor Tcf3 and a different Wnt target, c-Myc. Premature and deregulated beta-catenin activation causes severe villus dysmorphogenesis in transgenic mice.

CONCLUSIONS

Relationships among Wnt signaling, epithelial proliferation, and tissue differentiation are reversed in the developing and adult gut. The canonical Wnt pathway has independent, albeit possibly overlapping, functions in early intestinal villi and adult crypts. These observations advance understanding of Wnt functions in intestinal development and disease.

Galectin-1 (Gal-1) is a beta-galactoside-binding protein endowed with anti-inflammatory properties. The purpose of this study was to investigate the effects of endogenous and exogenous Gal-1 on neutrophil recruitment onto TNF-treated endothelium. The effect of human recombinant (hr)Gal-1 on markers of neutrophil activation (CD11b expression, P-selectin glycoprotein ligand 1, and L-selectin shedding) was also assessed. Gal-1 inhibited the platelet-activating factor-induced increase in CD11b expression in a concentration-dependent manner, as assessed by flow cytometry. To determine the effects of Gal-1 on neutrophil recruitment, an in vitro flow chamber was used: Preincubation of neutrophils with hrGal-1 significantly decreased the extent of capture, rolling, and adhesion on activated endothelial monolayers. This inhibition was shared with the endogenous protein, as knockdown of endothelial Gal-1 using small interfering RNA resulted in a significant increase in the number of cells captured and rolling. To verify the effects of Gal-1 in an in vivo system, intravital microscopy of Gal-1 null mice and their wild-type counterparts was performed. Leukocyte adhesion and emigration were increased significantly in the cremasteric circulation of Gal-1 null mice inflamed with IL-1beta. These findings indicate that Gal-1 functions to limit neutrophil recruitment onto a TNF-treated endothelium, a property that may underline its inhibitory effects in acute inflammation.

Fabry disease is an inborn error of glycosphingolipid catabolism, resulting from deficient activity of lysosomal alpha-galactosidase A (alpha-Gal A). A rare alternative splicing that introduces a 57-nucleotide (nt) intronic sequence to the alpha-Gal A transcript from intron 4 of the gene has been identified. In addition, a novel midintronic base substitution that results in substantially increased alternative splicing has been identified in a patient with Fabry disease who has the cardiac variant phenotype. The sequence of the patient's intron 4 contains a single G->>A transversion at genomic nt 9331 (IVS4+919 G->>A ), located at the minus sign4 position of the 3' end of the intronic insertion (nts 9278--9334 in the genomic sequence). Minigene constructs containing the entire intron 4 sequence with G, A, C, or T at nt 9331 within an alpha-Gal A complementary DNA expression vector were prepared and expressed in COS-1 cells. Whereas transfection of the G or T minigenes transcribed predominantly normal-sized transcripts, the transfection of the A or C minigenes produced a large amount of the alternatively spliced transcript. These results suggest that the G->>A mutation, within an A/C-rich domain, results in increased recognition of the alternative splicing by an A/C-rich enhancer-type exonic splicing enhancer. The intronic mutation was not observed in 100 unrelated unaffected men but was present in 6 unrelated patients with cardiac Fabry disease. Reverse-transcriptase polymerase chain reaction of total RNA of various normal human tissues revealed that the alternatively spliced transcript was present in all of the samples, and especially at a higher ratio in the lung and muscle. The normal transcript was present in the patients' lymphoblasts and resulted in approximately 10% residual enzyme activity, leading to a cardiac phenotype of Fabry disease.

Anti-Gal is the most abundant natural antibody in humans, constituting ~ 1% of immunoglobulins. Anti-Gal is naturally produced also in apes and Old World monkeys. The ligand of anti-Gal is a carbohydrate antigen called the 'α-gal epitope' with the structure Galα1-3Galβ1-4GlcNAc-R. The α-gal epitope is present as a major carbohydrate antigen in non-primate mammals, prosimians and New World monkeys. Anti-Gal can contributes to several immunological pathogeneses. Anti-Gal IgE produced in some individuals causes allergies to meat and to the therapeutic monoclonal antibody cetuximab, all presenting α-gal epitopes. Aberrant expression of the α-gal epitope or of antigens mimicking it in humans may result in autoimmune processes, as in Graves' disease. α-Gal epitopes produced by Trypanosoma cruzi interact with anti-Gal and induce 'autoimmune like' inflammatory reactions in Chagas' disease. Anti-Gal IgM and IgG further mediate rejection of xenografts expressing α-gal epitopes. Because of its abundance, anti-Gal may be exploited for various clinical uses. It increases immunogenicity of microbial vaccines (e.g. influenza vaccine) presenting α-gal epitopes by targeting them for effective uptake by antigen-presenting cells. Tumour lesions are converted into vaccines against autologous tumour-associated antigens by intra-tumoral injection of α-gal glycolipids, which insert into tumour cell membranes. Anti-Gal binding to α-gal epitopes on tumour cells targets them for uptake by antigen-presenting cells. Accelerated wound healing is achieved by application of α-gal nanoparticles, which bind anti-Gal, activate complement, and recruit and activate macrophages that induce tissue regeneration. This therapy may be of further significance in regeneration of internally injured tissues such as ischaemic myocardium and injured nerves.

A well-defined antigen/antibody system was used to evaluate the effect of immune tolerance on the spectrum of specificities of natural antibodies. The antibody used in this study, anti-Gal, is a naturally occurring, polyclonal IgG that constitutes 1% of the circulating IgG in humans. We have previously shown that anti-Gal, purified from AB sera, specifically interacts with glycosphingolipids bearing a Gal alpha 1----3Gal epitope, but not with the closely related B antigen in which the penultimate galactose of the Gal alpha 1----3Gal epitope is fucosylated Gal alpha 1----3(Fuc alpha 1----2)Gal. This narrow specificity was assumed to be the result of an effective immune tolerance mechanism that prevents the expression of antibody clones that can recognize both the Gal alpha 1----3Gal and the self B epitopes. If the assumption that immune tolerance determines the range of anti-Gal specificity is correct, then anti-Gal from individuals lacking the B antigen (A and O blood types) would be expected to interact with both Gal alpha 1----3Gal and Gal alpha 1----3(Fuc alpha 1----2)Gal epitopes. In this study, anti-Gal from the serum of individuals of various blood types was purified by affinity chromatography on Gal alpha 1----3Gal adsorbent and tested for its reaction with the B antigen. Whereas anti-Gal from AB and B individuals only reacted with Gal alpha 1----3Gal epitopes, anti-Gal from A and O individuals reacted with both Gal alpha 1----3Gal and B epitopes. Furthermore, it was determined that the majority of anti-B reactivity in A and O individuals is in fact anti-Gal antibodies capable of recognizing both Gal alpha 1----3Gal and B epitopes. It can be concluded from these results that immune tolerance accurately controls the spectrum of natural antibody specificities by preventing the production of antibody clones that can interact with self antigens.

The primary step in the pathogenesis of bacillary dysentery is the penetration of intestinal epithelial cells by shigellae. Lacking this capacity, Shigella flexneri becomes avirulent. By means of intergeneric conjugation between various Escherichia coli K-12 Hfr strains and S. flexneri 2a virulent recipients and by reciprocal transduction analysis with phage P1 vir, we established a locus on the genome of S. flexneri 2a which is necessary for the ability of this strain to penetrate epithelial cells as measured by the Sereney test for keratoconjunctivitis. This locus, termed kcpA (in reference to its involvement in provoking keratoconjunctivitis), has been positioned between the lac and gal chromosomal markers and is contransducible with the purE allele.

The region of the Autographa californica nuclear polyhedrosis virus (AcMNPV) encompassing the EcoRI T fragment (29.0 to 30.1 map units) was characterized by DNA sequencing, transcriptional mapping, and site-directed mutagenesis. The largest transcript from this region, an early 1.7-kilobase (kb) poly(A)+ RNA, encompassed three tandem, nonoverlapping open reading frames (ORFs). The largest of these ORFs, ETL, was proximal to the 5' end of the transcript and had the capacity to encode a 28-kilodalton (kDa) polypeptide. A recombinant virus, vETL beta gal, containing the Escherichia coli beta-galactosidase (beta gal) gene fused to the N-terminal two-thirds of the ETL ORF, produced blue plaques in the presence of a chromogenic indicator of beta gal and wild-type levels of polyhedra in cell culture. This recombinant was also infectious in insect larvae by oral administration of occluded virus. Comparison of vETL beta gal and wild-type viral proteins pulse-labeled at various times postinfection (p.i.) revealed (i) absence of a virus-induced 28-kDa polypeptide, (ii) early expression of a large (approximately 130-kDa) polypeptide which may be the ETL-beta gal fusion protein, (iii) a delay in expression of early 35 and 40-kDa polypeptides, and (iv) a 4- to 6-h delay in the expression of late proteins in vETL beta gal-infected cells. Cycloheximide did not inhibit synthesis of the 1.7-kb RNA but did inhibit its shutoff, which occurs at 12 h p.i. in the absence of inhibitors. Thus, the ETL gene product is apparently an early 28-kDa protein which is necessary, directly or indirectly, for timely expression of many other AcMNPV genes. The promoter-leader regions of the 1.7-kDa transcript showed significant sequence similarities to the leader of the AcMNPV IE-1 gene. The middle ORF within the 1.7-kb transcript, ETM, would encode a hydrophobic polypeptide of 113 amino acid residues. ETS, a small ORF within and proximal to the 3' end of the 1.7-kb transcript, was also transcribed as a set of smaller (approximately 0.5-kb) RNAs initiated heterogeneously in the region between ETL and ETS and persisting throughout infection.

To probe the potential contribution of beta-galactoside-contributing epitopes and receptor proteins (gal-1 and gal-3) to colon malignancy, we first examined the expression of galectins and binding sites in clinical specimens by lectin and immunohistochemistry. Sixty-seven colonic surgical resections were studied, including 10 normal, 10 mild dysplasias, 10 severe dysplasias and 37 cancers. gal-1 and gal-3 were expressed in variable amounts in the epithelial cells and the connective tissue of normal colon. Their expression significantly increased with the degree of dysplasia, suggesting that gal-1 and gal-3 and their binding sites are related to malignant progression, while gal-8 has been associated with suppressor activity. To study the functional aspects, the influence of these galectins on the migration of 4 human colorectal cancer cell lines (HCT-15, LoVo, DLD-1, CoLo201) was studied. In agreement with histopathologic monitoring, these tumor cells were found to produce gal-3, while only CoLo201 was positive for gal-1. Except for DLD-1 and gal-1, the lines exhibited gal-1 binding sites on the surface, prompting study by computer-assisted videomicroscopy of the effect on cell migration of the presence of galectin on the culture substrate. The level of cell migration for HCT-15, LoVo and CoLo201 cells was significantly reduced by 0.15 microg/cm(2) gal-1, and the presence of a blocking antibody at least reduced this effect. gal-3 significantly reduced cell migration in all 4 of the in vitro cell lines.

We report the isolation of a new homeobox gene from Xenopus laevis genomic DNA. The homeodomain sequence is highly diverged from the prototype Antennapedia sequence, and contains a unique histidine residue in the helix that binds to DNA. The homeodomain is followed by a 65 amino acid carboxyterminal domain, the longest found to date in any vertebrate homeobox gene. We have raised specific antibodies against an XlHbox 8-beta-gal fusion protein to determine the spatial and temporal expression of this gene. The nuclear protein first appears in a narrow band of the endoderm at stage 33 and develops into expression within the epithelial cells of the pancreatic anlagen and duodenum. Expression within the pancreatic epithelium persists into the adult frog. This unprecedented restriction to an anteroposterior band of the endoderm suggests that vertebrate homeobox genes might be involved in specifying positional information not only in the neuroectoderm and mesoderm, but also in the endoderm. Our data suggest that XlHbox 8 may therefore represent the first member of a new class of position-dependent transcription factors affecting endodermal differentiation.

A new approach to measuring the slowing of growth due to the manufacture of proteins not needed by a bacterium is presented. An entire single colony of Escherichia coli was used to start a chemostat culture that was then given a selective pressure by the addition of phenylgalactoside (phi-gal). This enriched the population for constitutive mutants that produced beta-galactosidase without induction and could split phi-gal, consume the galactose, and grow faster. When the phi-gal was removed, the constitutives grew slower than the parental strain and were gradually lost. This procedure allows competition experiments to be carried out with minimum effects due to genetic drift. Experiments with both strains having wild-type and mutant permease genes were conducted. With the former the selective disadvantage was initially much greater than expected from the simplest hypothesis that extra unused proteins would slow growth in proportion to their fraction of the total protein synthesis. This phase was followed by a second phase where the selective disadvantage was smaller than predicted by this simple hypothesis. With a very slowly reverting permease negative strain the selective disadvantage, and therefore the protein burden, was found to be much smaller and not statistically different from zero. Thus, while one would expect under carbon and energy limitation in the chemostat the protein burden to be larger than under unlimited conditions, it is so small that even the refined technique used here could not measure it accurately. It is certainly less than the fraction of 'waste' protein synthesis; but it could be between zero and the fraction of the cells' energy and carbon budget spent on manufacture of the proteins of the lac operon.

OBJECTIVE

T-cell immunoglobulin mucin (TIM) genes are expressed by T cells and regulate host immunity and tolerance. CD4(+) T cells mediate innate immunity-dominated liver ischemia-reperfusion injury (IRI) by unknown mechanisms. TIM-1 is involved in liver IRI, which is activated in part by the Toll-like receptor (TLR)4; we investigated the role of TIM-3 and TLR4 in IRI.

METHODS

Using an antibody against TIM-3 (anti-TIM-3), we studied TIM-3 signaling in mice following partial warm liver ischemia and reperfusion.

RESULTS

Mice given anti-TIM-3 had more liver damage than controls. Histological studies revealed that anti-TIM-3 increased hepatocellular damage and local neutrophil infiltration, facilitated local accumulation of T cells and macrophages, and promoted liver cell apoptosis. Intrahepatic neutrophil activity; induction of proinflammatory cytokines and chemokines; and expression of cleaved caspase-3, nuclear factor-κB, and TLR4 all increased in mice given anti-TIM-3. Administration of anti-TIM-3 followed by anti-galectin-9 (Gal-9 is a TIM-3 ligand) increased production of interferon-γ by concanavalin A (ConA)-stimulated spleen T cells and expression of tumor necrosis factor-α and interleukin-6 in ConA-stimulated macrophages co-cultured with T cells. Anti-TIM-3 did not affect liver IRI in TLR4-deficient mice.

CONCLUSIONS

TIM-3 blockade exacerbated local inflammation and liver damage, indicating the importance of TIM-3-Gal-9 signaling in maintaining hepatic homeostasis. TIM-3-TLR4 cross-regulation determined the severity of liver IRI in TLR4-dependent manner; these findings provide important information about the modulation of innate vs adaptive responses in patients that received liver transplants. Negative co-stimulation signaling by hepatic T-cells might be developed to minimize innate immunity-mediated liver tissue damage.

Two monospecific human antibodies (anti-OFA-I-1 and anti-OFA-I-2) produced in vitro by lymphoblast cell lines originating from melanoma patients have been shown previously to recognize cell surface antigens (OFA-I-1 and OFA-I-2) on human tumors and fetal brain: OFA-I-1 is expressed on a variety of human tumors, while OFA-I-2 has been detected only on tumors of neuroectodermal origin. Evidence presented in this report suggests that the two antigens expressed by a cultured human melanoma cell line (M14) are chemically distinct and that OFA-I-2 is a cell surface glycolipid, ganglioside GD2: GalNAc beta 1 leads to 4 NeuAc alpha 2 leads to 8NeuAc alpha 2 leads to 3 Gal beta 1 leads to 4Glc-ceramide.

Proteins which recognize specific sequences of DNA play a fundamental role in the regulation of protein synthesis in all organisms. A particular helix of the bacterial protein lac repressor recognizes the bases in the major groove of the lac operator. We show that the first two residues of this recognition helix interact independently with two base pairs. This allows us in many cases to predict repression as an indicator of strength of the repressor-operator complex. Rules of recognition can be derived for 16 symmetric operators. They also apply to the gal repressor and possibly to other bacterial repressors.

The liver is an attractive target tissue for gene therapy. Current approaches for hepatic gene delivery include retroviral and adenoviral vectors, liposome/DNA, and peptide/DNA complexes. This study describes a technique for direct injection of DNA into liver that led to significant gene expression. Gene expression was characterized in both rats and cats following injection of plasmid DNA encoding several different proteins. Luciferase activity was measured after injection of plasmid DNA encoding the luciferase gene (pCMVL), beta-galactosidase (beta-Gal) activity was evaluated in situ using plasmid DNA encoding Lac Z (pCMV beta), and serum concentration of secreted human alpha-1-antitrypsin was measured following injection of plasmid DNA encoding this protein (pRC/CMV-sHAT). Several variables, including injection technique, DNA dose, and DNA diluent, were investigated. Direct injection of pCMVL resulted in maximal luciferase expression at 24-48 hr. beta-Gal staining demonstrated that the majority of transfected hepatocytes were located near the injection site. Significant concentrations of human alpha-1-antitrypsin were detected in the serum of animals injected with pRC/CMV-sHAT. These findings demonstrate the general principle that direct injection of plasmid DNA into liver can lead to significant gene expression.

Oligosaccharides released from the lipooligosaccharides (LOS) of Haemophilus influenzae nontypable strain 2019 by mild acid hydrolysis were fractionated by size exclusion chromatography and analyzed by liquid secondary ion mass spectrometry. The major component of the heterogeneous mixture was found to be a hexasaccharide of Mr 1366, which lost two phosphoethanolamine groups upon treatment with 48% aqueous HF. The dephosphorylated hexasaccharide was purified and shown by tandem mass spectrometry, composition analysis, methylation analysis, and two-dimensional nuclear magnetic resonance studies to be Gal beta 1----4Glc beta 1----(Hep alpha 1----2Hep alpha 1----3) 4Hep alpha 1----5anhydro-KDO, where Hep is L-glycero-D-manno-heptose and KDO is 3-deoxy-D-manno-octulosonic acid. An analogous structure containing authentic KDO was generated from LOS that had been HF-treated prior to acetic acid hydrolysis, suggesting that the reducing terminal anhydro-KDO moiety is produced as an artifact of the hydrolysis procedure by beta-elimination of a phosphate substituent from C-4 of KDO. Mass spectral analyses of O-deacylated LOS and free lipid A confirmed that, in addition to the two phosphoethanolamines on the oligosaccharide and two phosphates on the lipid A, another phosphate group exists on the KDO. This KDO does not appear to be further substituted with additional KDO residues in intact H. influenzae 2019 LOS. The terminal disaccharide epitope, Gal beta 1----4Glc beta 1----, of the hexasaccharide is also present on lactosylceramide, a precursor to human blood group antigens. It is postulated that the presence of this structure on H. influenzae LOS may represent a form of host mimicry by the pathogen.

Sialyltransferases are a family of 10-12 enzymes that catalyze the transfer of sialic acid to carbohydrate groups of glycoproteins and glycolipids. Three sialyltransferase cDNAs have been cloned, revealing a highly conserved sialylmotif in the catalytic domain of these enzymes. Using a polymerase chain reaction-based approach, we cloned a 150-base pair fragment of a new sialymotif from human placenta mRNA, which was then used as a probe to clone the complete coding sequence of the corresponding gene from a cDNA library. Like the other members of the sialyltransferase gene family cloned to date, the new cDNA coded for a protein predicted to have an NH2-terminal signal-anchor sequence and had the sialylmotif located in the center of the molecule. Comparison with the three other cloned sialyltransferases revealed extensive sequence homology that was not recognized earlier. Expression of a soluble recombinant form of the protein in COS-1 cells produced an active sialyltransferase, which used oligosaccharide, glycoprotein, and glycolipid acceptor substrates with terminal galactose in the Gal beta 1,3GalNAc and Gal beta 1, 4GlcNAc sequences but not the Gal beta 1,3GlcNAc sequence. The sialylated products were sensitive to digestion with the Newcastle disease virus sialidase, which is specific for sialic acid-galactose linkages in the alpha 2,3 linkage. The results suggest that this new member of the sialyltransferase gene family is the enzyme previously described as a glycolipid sialyltransferase activity (SAT-3), which forms the terminal sequences NeuAc alpha-2,3Gal beta 1,3GalNAc-R and NeuAc alpha 2,3Gal beta 1, 4GlcNAc-R.

Gal or Lac repressor binding to an upstream DNA segment, in the absence of DNA looping, represses the P1 promoter located on the same face and activates the P2 promoter situated on the opposite face of the DNA helix in the gal operon. Both inhibition and stimulation of transcription requires the physical presence of the C-terminal domain of the alpha subunit of RNA polymerase although the latter is not required for transcription itself. We propose that Gal and Lac repressors inhibit or stimulate transcription initiation by disabling or stimulating RNA polymerase activity at a post-binding step by directly or indirectly altering the C-terminal alpha domain to an unfavorable state at P1 or a more favorable state at P2, respectively.

The synthesis of glycoproteins containing N-linked complex oligosaccharides is blocked by swainsonine at the step catalyzed by Golgi mannosidase II (Tulsiani, D. R. P., Harris, T. M., and Touster, O. (1982) J. Biol Chem. 257, 7936-7939). Accordingly, hybrid glycoproteins might be produced in the presence of swainsonine. In this report, we demonstrate that swainsonine causes human skin fibroblasts to synthesize such glycoproteins. In control fibroblasts, there were approximately equal amounts of complex and high mannose glycoproteins. In the presence of swainsonine (10 micrograms/ml), most of the complex glycoproteins were replaced by hybrid types. The principal oligosaccharide had the following structure: (formula; see text) A smaller amount of the asialo hybrid was also produced. The structure of the hybrid was established by Bio-Gel P-4 fractionation of oligosaccharides produced by endoglycosidase H treatment of pronase-derived glycopeptides, followed by examination of the susceptibility of the oligosaccharide to glycohydrolases and by its adsorbability to serotonin-Sepharose 4B. The same hybrid oligosaccharide was produced efficiently by rat liver Golgi membranes in the presence of ([3H] Man)5GlcNAc, UDP-GlcNAc, UDP-Gal, CMP-NeuAc, and swainsonine. Golgi mannosidase II had no action on the hybrid oligosaccharide, and little action on asialo hybrid, but both were converted to the mannosidase II substrate, GlcNAcMan5GlcNAc, by appropriate treatment with neuraminidase and beta-galactosidase. Jack bean alpha-D-mannosidase gave the expected yields of free mannose from the various oligosaccharides studied in this work. Swainsonine should be useful in investigating the role of oligosaccharide structure of glycoproteins because of its ability to alter the oligosaccharide.