The Lex determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc-beta 1----R) has been implicated as having a role in mediating compaction of the mouse embryo at the morula stage (Fenderson, B., Zehavi, U., and Hakomori, S. (1984) J. Exp. Med. 160, 1591-1596). Here, we present evidence suggesting a role for Lex in F9 embryonal carcinoma cell adhesion and a mechanism for Lex recognition based on carbohydrate-carbohydrate interaction. Homotypic aggregation of F9 cells was inhibited by lacto-N-fucopentaose III, and F9 cells showed a preferential interaction with Lex liposomes. The following observations suggest that the structure capable of recognizing Lex per se on F9 cells is Lex: (i) Cell surface-labeled components solubilized in octylglucoside, affinity-bound on an Lex-octyl-Sepharose column, contained glycoproteins reactive with anti-Lex antibody. (ii) Liposomes containing Lex showed significant interaction with Lex glycolipid, but not other glycolipids, coated on a plastic surface. (iii) Liposomes containing Lex glycolipid were found to self-aggregate, whereas liposomes containing paragloboside (nLc4) or sialylparagloboside (IV3NeuAcnLc4) did not. (iv) The diffusibility of 3H-labeled lacto-N-fucopentaitol III (but not I or II), incubated with Lex liposome, from the lower to the upper Boyden chamber through a semipermeable membrane was inhibited. In all these experiments (i-iv), the interaction of Lex to Lex (or Lex to lacto-N-fucopentaose III) was clearly observed only in the presence of Ca2+ and Mg2+ and was enhanced by the presence of Mn2+. These interactions were inhibited by EDTA. The results suggest the novel hypothesis that carbohydrate-carbohydrate interactions may play an important role in controlling cell recognition during F9 cell aggregation and during embryonic development.

The priming of an immune response against a major histocompatibility complex class I-restricted antigen expressed by nonhematopoietic cells involves the transfer of that antigen to a host bone marrow-derived antigen presenting cell (APC) for presentation to CD8+ T lymphocytes. Dendritic cells (DC), as bone marrow-derived APC, are first candidates for presentation of tumor-associated antigens (TAA). The aim of this study was to see whether DC are able to prime in vivo antigen-specific cytotoxic T lymphocytes after exposure to a soluble protein antigen in vitro. Lacking a well-defined murine TAA, we took advantage of beta-galactosidase (beta-gal)-transduced tumor cell lines as a model in which beta-gal operationally functions as TAA. For in vivo priming both a DC line, transduced or not transduced with the gene coding for murine GM-CSF, and fresh bone marrow-derived DC (bm-DC), loaded in vitro with soluble beta-gal, were used. Priming with either granulocyte macrophage colony-stimulating factor-transduced DC line or fresh bm-DC but not with untransduced DC line generated CTL able to lyse beta-gal-transfected target cells. Furthermore, GM-CSF was necessary for the DC line to efficiently present soluble beta-gal as an H-2Ld-restricted peptide to a beta-gal-specific CTL clone. Data also show that a long-lasting immunity against tumor challenge can be induced using beta-gal-pulsed bm-DC as vaccine. These results indicate that effector cells can be recruited and activated in vivo by antigen-pulsed DC, providing an efficient immune reaction against tumors.

Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4(+) and CD8(+) T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca(2+)) influx, and that either the intracellular Ca(2+) chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca(2+)-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

The gal operon of Escherichia coli is transcribed from two overlapping promoters, PG1 and PG2. Cyclic AMP and its receptor protein (CRP) modulate the two promoters in opposite directions by binding to a single cat locus. Both the promoters are negatively regulated by a single repressor, the product of the galR gene. An operator site, defined by several mutations, has previously been located upstream from the cat locus. We have isolated and characterized a new set of cis-dominant constitutive mutations of the gal operon and determined their locations by DNA sequencing. From these studies, we propose the existence of a second functional gal operator element at an extraordinary site--within galE, the first structural gene. Both the operators, OE (exterior) and OI (interior), are involved in the repression of PG1 and PG2. This would be the first example of the presence of a functional operator element within a structural protein-coding region.

We have developed a number of broad-host-range plasmids that allow the expression of the Escherichia coli lac operon from any cloned promoter, and the creation of 'in phase' fusions between lacZ and other cloned genes. In a second series of constructions, the E. coli gal operon has been cloned into the broad-host-range vector and a plasmid carrying both the E. coli gal and lac genes is described. These plasmids have been transferred into Pseudomonas aeruginosa and Zymomonas mobilis and their effects on the utilisation of lactose and galactose have been investigated.

For many short-lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre-degradation step. The conjugated ubiquitin moieties function as a 'secondary' signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin--proline--beta-galactosidase (Ub-P-beta gal) is short-lived in the yeast Saccharomyces cerevisiae because its N-terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N-terminal ubiquitin in Ub-P-beta gal. The degradation of Ub-P-beta gal is shown to require Ubc4, one of at least seven ubiquitin-conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis-acting signal can be used to manipulate the in vivo half-lives of specific intracellular proteins.

BACKGROUND

Gastric cancer is the fourth most common cancer in the world and the second most prevalent cause of cancer related death. The development of gastric cancer is mainly associated with H. Pylori infection leading to a focus in pathology studies on bacterial and environmental factors, and to a lesser extent on the mechanistic development of the tumour. MicroRNAs are small non-coding RNA molecules involved in post-transcriptional gene regulation. They are found to regulate genes involved in diverse biological functions and alterations in microRNA expression have been linked to the pathogenesis of many malignancies. The current study is focused on identifying microRNAs involved in gastric carcinogenesis and to explore their mechanistic relevance by characterizing their targets.

RESULTS

Invitrogen NCode miRNA microarrays identified miR-449 to be decreased in 1-year-old Gastrin KO mice and in H. Pylori infected gastric tissues compared to tissues from wild type animals. Growth rate of gastric cell lines over-expressing miR-449 was inhibited by 60% compared to controls. FACS cell cycle analysis of miR-449 over-expressing cells showed a significant increase in the sub-G1 fraction indicative of apoptosis. ß-Gal assays indicated a senescent phenotype of gastric cell lines over-expressing miR-449. Affymetrix 133v2 arrays identified GMNN, MET, CCNE2, SIRT1 and CDK6 as miR-449 targets. Luciferase assays were used to confirm GMNN, MET, CCNE2 and SIRT1 as direct targets. We also show that miR-449 over-expression activated p53 and its downstream target p21 as well as the apoptosis markers cleaved CASP3 and PARP. Importantly, qPCR analyses showed a loss of miR-449 expression in human clinical gastric tumours compared to normal tissues.

CONCLUSIONS

In this study, we document a diminished expression of miR-449 in Gastrin KO mice and further confirmed its loss in human gastric tumours. We investigated the function of miR-449 by identifying its direct targets. Furthermore we show that miR-449 induces senescence and apoptosis by activating the p53 pathway.

Photosynthetically active chloroplasts retaining high rates of fatty acid synthesis from [1-(14)C]acetate were purified from leaves of both 16:3 (Solanum nodiflorum, Chenopodium album) and 18:3 plants (Amaranthus lividus, Pisum sativum). A comparison of lipids into which newly synthesized fatty acids were incorporated revealed that, in 18:3 chloroplasts, enzymic activities catalyzing the conversion of phosphatidate to diacylglycerol and of diacylglycerol to monogalactosyl diacylglycerol (MGD) were significantly less active than in 16:3 chloroplasts. In contrast, labeling rates of MGD from UDP-[(14)C]gal were similar for both types of chloroplasts.The composition and positional distribution of labeled fatty acids within the glycerides synthesized by isolated 16:3 and 18:3 chloroplasts were similar and in each case only a C18/C16 diacylglycerol backbone was synthesized. In nodiflorum chloroplasts, C18:1/C16:0 MGD assembled de novo was completely desaturated to the C18:3/C16:3 stage.Whereas newly synthesized C18/C18 MGD could not be detected in any of these chloroplasts if incubated with [(14)C]acetate after isolation, chloroplasts isolated from acetate-labeled leaves contained MGD with labeled C18 fatty acids at both sn-1 and sn-2 positions. Taken together, these results provide further evidence on an organellar level for the operation of pro- and eucaryotic pathways in the biosynthesis of MGD in different groups of plants.

To study the efficiency of RNA-based vaccines, RNA coding for the model antigen beta-galactosidase (beta-gal) was transcribed in vitro from a lacZ gene flanked by stabilizing Xenopus laevis beta-globin 5' and 3' sequences and was protected from RNase degradation by condensation with the polycationic peptide protamine. The liposome-encapsulated condensed RNA-peptide complex, the condensed RNA-peptide complex without liposome or naked, unprotected RNA, was injected into BALB/c (H-2(d)) mice. All preparations led to protein expression in the local tissue, activation of L(d)-restricted specific cytotoxic T lymphocytes (CTL) and production of IgG antibodies reactive against beta-gal. RNA-triggered CTL were as efficient in the lysis of lacZ-transfected target cells as CTL triggered by a lacZ-DNA eukaryotic expression vector. Immunization with RNA transcribed from a cDNA library from the beta-gal-expressing cell line P13.1 again led to beta-gal-specific CTL and IgG induction. Thus, both naked and protected RNA can be used to elicit a specific immune response in vivo, whereby the protected RNA is stable in vitro for a longer period of time. RNA vaccines can be produced in high amounts and have the same major advantages as DNA vaccines but lack the potentially harmful effect of DNA integration into the genome.

Galectin-1 (GAL-1), a member of a family of conserved beta-galactoside-binding proteins, has been shown to induce in vitro apoptosis of activated T cells and immature thymocytes. We assessed the therapeutic effects and mechanisms of action of delivery of GAL-1 in a collagen-induced arthritis model. A single injection of syngeneic DBA/1 fibroblasts engineered to secrete GAL-1 at the day of disease onset was able to abrogate clinical and histopathological manifestations of arthritis. This effect was reproduced by daily administration of recombinant GAL-1. GAL-1 treatment resulted in reduction in anticollagen immunoglobulin (Ig)G levels. The cytokine profile in draining lymph node cells and the anticollagen IgG isotypes in mice sera at the end of the treatment clearly showed inhibition of the proinflammatory response and skewing towards a type 2-polarized immune reaction. Lymph node cells from mice engaged in the gene therapy protocol increased their susceptibility to antigen-induced apoptosis. Moreover, GAL-1-expressing fibroblasts and recombinant GAL-1 revealed a specific dose-dependent inhibitory effect in vitro in antigen-dependent interleukin 2 production to an A(q)-restricted, collagen type 2-specific T cell hybridoma clone. Thus, a correlation between the apoptotic properties of GAL-1 in vitro and its immunomodulatory properties in vivo supports its therapeutic potential in the treatment of T helper cell type 1-mediated autoimmune disorders.

Fabry disease is an X-linked lysosomal storage disease afflicting 1 in 40,000 males with chronic pain, vascular degeneration, cardiac impairment, and other symptoms. Deficiency in the lysosomal enzyme alpha-galactosidase (alpha-GAL) causes an accumulation of its substrate, which ultimately leads to Fabry disease symptoms. Here, we present the structure of the human alpha-GAL glycoprotein determined by X-ray crystallography. The structure is a homodimer with each monomer containing a (beta/alpha)8 domain with the active site and an antiparallel beta domain. N-linked carbohydrate appears at six sites in the glycoprotein dimer, revealing the basis for lysosomal transport via the mannose-6-phosphate receptor. To understand how the enzyme cleaves galactose from glycoproteins and glycolipids, we also determined the structure of the complex of alpha-GAL with its catalytic product. The catalytic mechanism of the enzyme is revealed by the location of two aspartic acid residues (D170 and D231), which act as a nucleophile and an acid/base, respectively. As a point mutation in alpha-GAL can lead to Fabry disease, we have catalogued and plotted the locations of 245 missense and nonsense mutations in the three-dimensional structure. The structure of human alpha-GAL brings Fabry disease into the realm of molecular diseases, where insights into the structural basis of the disease phenotypes might help guide the clinical treatment of patients.

A natural IgG antibody (anti-Gal) with alpha-galactosyl binding specificity has been found in large amounts (0.5 - 1.0% of serum IgG) in all individuals tested. It has been purified by affinity chromatography on a column of melibiose-Sepharose. In addition to its affinity for normal and pathological senescent human red cells, the antibody readily interacts with rabbit red blood cell (RRBC) glycolipids with alpha-galactosyl terminal residues. Two types (glycosidic linkages of 1----3 vs. 1----4) of rabbit red cells glycolipids with terminal alpha-galactosyl residues were tested for antibody binding. The antibody specifically bound to glycolipids with Gal alpha 1----3 terminal residues, and treatment of these glycolipids with alpha-galactosidase abolished binding. Hemagglutination inhibition studies with oligosaccharides of known structure also showed that the antibody binds specifically to glycoconjugates with an alpha 1----3 terminal galactose residue. Anti-Gal did not bind to a human B-active glycolipid, indicating that fucose-linked alpha 1----2 to the penultimate galactose prevents anti-Gal binding. The anti-Gal specificity for RRBC glycolipids also paralleled that of the alpha-galactosyl-specific Bandeiraea simplicifolia lectin. The possible reasons for the occurrence of this unique antibody in human serum are discussed.

After a limited number of population doublings (PDs), cultures of normal mammalian diploid cells undergo an irreversible growth arrest known as replicative senescence [1]. As well as contributing to cellular ageing, senescence is viewed as an important mechanism of tumour suppression by preventing the emergence of immortal cell clones [2-4]. Senescent cells have a number of characteristics that distinguish them from cycling or quiescent cells including elevated levels of two cyclin-dependent kinase (Cdk) inhibitors, p16INK4a and p21CIP1 [5-11]. Here, we demonstrate that both of these Cdk inhibitors, as well as other members of their protein families (the INK4 and CIP/KIP families, respectively [12]), induce several facets of the senescent phenotype when ectopically expressed in young human diploid fibroblasts. These include a reduced proliferative capacity, an altered size and shape, the presence of underphosphorylated retinoblastoma protein (pRb), increased expression of plasminogen activator inhibitor (PAI-1) and the appearance of senescence-associated beta-galactosidase (SA-beta-gal) activity [2,3,13-15]. A 20 amino acid peptide from p16INK4a that inhibits Cdks active in the G1 phase of the cell cycle [16] produces similar effects in a dose-dependent manner suggesting that, in primary fibroblasts, inhibition of G1-specific Cdk activity is sufficient to induce phenotypic changes that normally occur at the end of their finite lifespan.

T cell immunoglobulin-3 (Tim-3) has been identified as a marker of differentiated interferon-γ-producing CD4(+) T helper type 1 and CD8(+) T cytotoxic type 1 cells. The interaction of Tim-3 with its ligand, galectin-9 (Gal-9), induces cell death, and in vivo blockade of this interaction results in exacerbated autoimmunity and abrogation of tolerance in experimental models, establishing Tim-3 as a negative regulatory molecule. Recent studies have uncovered additional mechanisms by which Tim-3 negatively regulates T cell responses, such as promoting the development of CD8(+) T cell exhaustion and inducing expansion of myeloid-derived suppressor cells. In contrast to this inhibitory effect on T cells, Tim-3-Gal-9 interaction promotes macrophage clearance of intracellular pathogens. Here, we focus on the emerging role for Tim-3 in tumor and antimicrobial immunity.

The complete sequence of the transposable DNA element IS2 in gal OP-308:: IS2 (I) has been determined. This element is 1.327 bp long. The integrated element is flanked by a five base pair long sequence duplication. The termini of IS2 are not perfect inverted repeats, but a close approximation.

To confirm whether human cancer-induced stromal cells are derived from bone marrow, bone marrow (BM) cells obtained from beta-galactosidase transgenic and recombination activating gene 1 (RAG-1) deficient double-mutant mice (H-2b) were transplanted into sublethally irradiated severe combined immunodeficient (SCID) mice (H-2d). The human pancreatic cancer cell line Capan-1 was subcutaneously xenotransplanted into SCID recipients and stromal formation was analyzed on day 14 and on day 28. Immunohistochemical and immunofluorescence studies revealed that BM-derived endothelial cells (X-gal/CD31 or H-2b/CD31 double-positive cells) and myofibroblasts (X-gal/alpha-smooth muscle actin or H-2b/alpha-smooth muscle actin double-positive cells) were present within and around the cancer nests. On day 14, the frequencies of BM-derived endothelial cells and BM-derived myofibroblasts were 25.3+/-4.4% and 12.7+/-9.6%, respectively. On day 28, the frequency of BM-derived endothelial cells was 26.7+/-9.7%, which was similar to the value on day 14. However, the frequency of BM-derived myofibroblasts was significantly higher (39.8+/-17.1%) on day 28 than on day 14 (P<0.05). The topoisomerase IIalpha-positive ratio was 2.2+/-1.2% for the H-2b-positive myofibroblasts, as opposed to only 0.3+/-0.4% for the H-2b-negative myofibroblasts, significant proliferative activity was observed in the BM-derived myofibroblasts (P<0.05). Our results indicate that BM-derived myofibroblasts become a major component of cancer-induced stromal cells in the later stage of tumor development.

DNA molecules created by fusing a 365-base-pair segment of yeast DNA encoding the galactose-regulated upstream promoter element (gal) to a set of derivatives that systematically delete sequences upstream from the his3 gene are introduced in single copy back into the yeast genome precisely at the his3 locus and then assayed for transcription. Fusions of the gal regulatory element to his3 derivatives containing all normal mRNA coding sequences but lacking essentially the entire promoter region fail to express his3 under any growth conditions. Fusions to derivatives lacking the his3 upstream promoter element but containing the "TATA box" place his3 expression under gal control--i.e., extremely high RNA levels in galactose-containing medium and essentially no his3 RNA in glucose-containing medium. However, of the two normal his3 initiation sites, only the downstream one is activated by the gal element. In fusions of this type, neither the orientation of the gal element nor the distance between the element and the his3 TATA box affects the level or the initiation points of transcription. However, the gal element does not influence transcription when placed 100 or 300 base pairs downstream from the normal mRNA start sites. Fusions to derivatives containing the entire his3 promoter region restore the basal level of his3 transcription in glucose-grown cells, and both transcriptional initiation sites are used. Furthermore, RNA levels in galactose-grown cells, although somewhat higher than in glucose-grown cells, are significantly below the fully induced level. The distance from his3 coding sequences does not affect RNA levels, suggesting that specific sequences, possibly corresponding to the his3 upstream promoter element, reduce the ability of the gal element to activate transcription. Analysis of chromatin from some of these strains indicates a DNase I-hypersensitive site(s) in the middle of the gal element. However, this structural feature is not correlated with transcriptional initiation because it is found when cells are grown in glucose medium and also in derivatives lacking a TATA box. Thus, the gal upstream element possesses most, but not all, of the properties of viral and cellular enhancer sequences of higher eukaryotes. In addition, it appears that the his3 and gal upstream sequences represent two distinct classes of promoter elements, which activate transcription from different initiation sites.

Most human pyelonephritis Escherichia coli isolates express both mannose (MS)- and globoside (Gal-Gal)-binding pili. An ascending E. coli urinary tract infection model was established in the 16-wk-old female BALB/c mouse to compare the pathogenic significance of MS and Gal-Gal pili and their efficacy as vaccines for the prevention of pyelonephritis. The distribution and density of pilus receptor compounds in urogenital tissues and as soluble compounds in urine were determined with antibodies to the synthetic receptor analogues, alpha D-Gal(1----4) beta D-Gal and alpha D-Man(1----2) alpha D-Man. Both carbohydrates were detected in vagina, bladder, ureter, and renal pelvis epithelium and in collecting duct and tubular cells. A pilus receptor compound also was detected in urine. It competitively inhibited the binding capacity of MS pili and was found to be physically, chemically, and immunologically related to Tamm-Horsfall uromucoid. Infectivity and invasiveness were quantitatively and histologically characterized for four E. coli strains: J96, a human pyelonephritis strain that expresses both MS and Gal-Gal pili; two recombinant strains prepared from J96 chromosomal DNA encoding MS pili or Gal-Gal pili; and the nonpiliated K12 recipient. Intravesicular administration of J96 (10(6) colony-forming units [CFU]) resulted in renal colonization and invasion in each of nine mice. The Gal-Gal clone (10(6) CFU) colonized the kidneys in each of 10 mice but did not invade. In contrast, the MS clone (10(6) CFU) did not colonize renal epithelium or invade. This effect was superceded when larger doses (greater than or equal to 10(10) CFU) of the MS clone were administered in volumes that cause acute vesicoureteric reflux. The efficacy was determined of vaccines composed of pure MS or Gal-Gal pili or the lipopolysaccharide containing O somatic antigen of the challenge strain, J96. The Gal-Gal pilus vaccine blocked renal colonization in 19 of 22 mice and renal invasion in 10 of 11 mice. Gal-Gal pili may be useful immunogens for the prevention of pyelonephritis in anatomically normal urinary tracts.

The coexistence of galanin (GAL)-like immunoreactivity (LI) with markers for catecholamines, 5-hydroxytryptamine (5-HT), GABA, or some neuropeptides was mapped in the rat CNS by using adjacent sections, as well as by elution-restaining and double-labeling immunocytochemistry. Many instances of coexistence were observed, but there were also numerous GAL-positive cell body populations displaying distributions similar to those of these markers but without apparent coexistence. In the hypothalamic arcuate nucleus GAL-LI was found in a large proportion of tyrosine hydroxylase (TH)-positive cell bodies (A12 cells), both in the dorsomedial and ventrolateral subdivisions, with a higher number in the latter. GAL-LI coexisted in glutamic acid decarboxylase (GAD)-positive somata in the posterior aspects of the arcuate nucleus and at all rostrocaudal levels in fibers in the external layer of the median eminence. In the anterior hypothalamus, a large population of the cells of the parvocellular and magnocellular paraventricular nuclei contained both GAL-LI and vasopressin-LI. Moreover, somata containing both GAD- and GAL-LI were seen lateral to the mammillary recess in the tuberal and caudal magnocellular nuclei. Some of the neurons of the caudal group were shown to project to the occipital cortex using combined retrograde tracing and immunofluorescence. With regard to mesencephalic and medullary catecholamine neurons, GAL-LI coexisted in a large proportion of the noradrenergic locus coeruleus somata (A6 cell group) and in the A4 group dorsolateral to the fourth ventricle, as well as in the caudal parts of the A2 group in the dorsal vagal complex. However, in more rostral parts of the latter, especially in the medial subdivision of the solitary tract nucleus, a very large population of GAL-IR small cell bodies was seen intermingling with catecholamine neurons, but they did not contain TH-LI. Furthermore, GAL-IR cell bodies coextensive with, but not coexisting in, TH-IR somata were seen in the C1 (epinephrine) horea in the ventrolateral medulla at the level of area postrema and in the most rostral aspects of the C1 group. Finally, 5-HT-positive cell bodies of the mesencephalic and medullary raphe nuclei and a subpopulation of coarse 5-HT nerve fibers in the hippocampus co-contained GAL-LI. The present results demonstrate that a GAL-like peptide is present in many systems containing other neuroactive compounds, including dopamine, norepinephrine, 5-HT, GABA, and vasopressin.(ABSTRACT TRUNCATED AT 400 WORDS)

Human adenoviruses have been developed as an attractive vehicle for in vivo liver-directed gene therapy. Problems with the application of first generation recombinant adenoviruses to liver-directed gene therapy have been transient expression of the recombinant gene and development of hepatitis. Previous studies in mouse models of gene transfer to liver and lung suggested that MHC class I-restricted cytotoxic T lymphocytes (CTLs) to viral antigens may be effectors in the elimination of transgene expression. The goal of this study was to evaluate the importance of viral antigens versus transgene product in inducing CTL mediated hepatocyte destruction in vivo. Immunization of C57BL/6 mice with a lacZ-expressing adenovirus elicited CTL responses to both viral antigens and the transgene product, beta-galactosidase (beta-gal). Adoptive transfer experiments, as well as studies involving lacZ-transgenic mice (ROSA-26) revealed that CTLs to viral antigens are sufficient to destroy virus-infected hepatocytes, indicating that CTLs to beta-gal can not solely account for the observed hepatocyte destruction that has characterized the use of first generation viruses. In addition, we confirmed that B cell-mediated events do not participate in destruction of hepatocytes in vivo, despite the production of virus- and beta-gal-specific antibodies. These data confirm the hypothesis that viral gene expression elicits host responses that contribute to the problem of transgene instability. Recombinant adenoviruses must be redesigned to diminish viral gene expression if they are to be used in the treatment of chronic diseases.

AGA and AGG codons for arginine are the least used codons in Escherichia coli, which are encoded by a rare tRNA, the product of the dnaY gene. We examined the positions of arginine residues encoded by AGA/AGG codons in 678 E. coli proteins. It was found that AGA/AGG codons appear much more frequently within the first 25 codons. This tendency becomes more significant in those proteins containing only one AGA or AGG codon. Other minor codons such as CUA, UCA, AGU, ACA, GGA, CCC and AUA are also found to be preferentially used within the first 25 codons. The effects of the AGG codon on gene expression were examined by inserting one to five AGG codons after the 10th codon from the initiation codon of the lacZ gene. The production of beta-galactosidase decreased as more AGG codons were inserted. With five AGG codons, the production of beta-galactosidase (Gal-AGG5) completely ceased after a mid-log phase of cell growth. After 22 hr induction of the lacZ gene, the overall production of Gal-AGG5 was 11% of the control production (no insertion of arginine codons). When five CGU codons, the major arginine codon were inserted instead of AGG, the production of beta-galactosidase (Gal-CGU5) continued even after stationary phase and the overall production was 66% of the control. The negative effect of the AGG codons on the Gal-AGG5 production was found to be dependent upon the distance between the site of the AGG codons and the initiation codon. As the distance was increased by inserting extra sequences between the two codons, the production of Gal-AGG5 increased almost linearly up to 8 fold. From these results, we propose that the position of the minor codons in an mRNA plays an important role in the regulation of gene expression possibly by modulating the stability of the initiation complex for protein synthesis.

IFN-gamma plays a central role in antitumor immunity. T cell Ig and mucin domain (Tim-3) is expressed on IFN-gamma-producing Th1 cells; on interaction with its ligand, galectin-9, Th1 immunity is terminated. In this study, we show that transgenic overexpression of Tim-3 on T cells results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Molecular characterization of CD11b(+)Ly-6G(+) cells reveals a phenotype consistent with granulocytic myeloid-derived suppressor cells. Accordingly, we find that modulation of the Tim-3/galectin-9 (Gal-9) pathway impacts on tumor growth. Similarly, overexpression of Tim-3 ligand, Gal-9, results in an increase in CD11b(+)Ly-6G(+) cells and inhibition of immune responses. Loss of Tim-3 restores normal levels of CD11b(+)Ly-6G(+) cells and normal immune responses in Gal-9 transgenic mice. Our data uncover a novel mechanism by which the Tim-3/Gal-9 pathway regulates immune responses and identifies this pathway as a therapeutic target in diseases where myeloid-derived suppressor cells are disadvantageous.

Immunoglobulins (IgG) are soluble serum glycoproteins in which the oligosaccharides play significant roles in the bioactivity and pharmacokinetics. Recombinant immuno-globulins (rIgG) produced in different host cells by recombinant DNA technology are becoming major therapeutic agents to treat life threatening diseases such as cancer. Since glycosylation is cell type specific, rIgGs produced in different host cells contain different patterns of oligosaccharides which could affect the biological functions. In order to determine the extent of this variation N-linked oligosaccharide structures present in the IgGs of different animal species were characterized. IgGs of human, rhesus, dog, cow, guinea pig, sheep, goat, horse, rat, mouse, rabbit, cat, and chicken were treated with peptide-N-glycosidase-F (PNGase F) and the oligosaccharides analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for neutral and acidic oligosaccharides, in positive and negative ion modes, respectively. The data show that for neutral oligosaccharides, the proportions of terminal Gal, core Fuc and/or bisecting GlcNAc containing oligosaccharides vary from species to species; for sialylated oligosaccharides in the negative mode MALDI-TOF-MS show that human and chicken IgG contain oligosaccharides with N-acetylneuraminic acid (NANA), whereas rhesus, cow, sheep, goat, horse, and mouse IgGs contain oligosaccharides with N-glycolylneuraminic acid (NGNA). In contrast, IgGs from dog, guinea pig, rat, and rabbit contain both NANA and NGNA. Further, the PNGase F released oligosaccharides were derivatized with 9-aminopyrene 1,4,6-trisulfonic acid (APTS) and analyzed by capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-LIF results indicate that the proportion of the two isomers of monogalactosylated, biantennary, complex oligosaccharides vary significantly, suggesting that the branch specificity of beta1, 4-galactosyltransferase might be different in different species. These results show that the glycosylation of IgGs is species-specific, and reveal the necessity for appropriate cell line selection to express rIgGs for human therapy. The results of this study are useful for people working in the transgenic area.

Eight cDNAs encoding galectin 4 (Gal-4), UGT2B4 (UDP-glucuronosyltransferase), ribosomal phosphoprotein P0 (rpP0), dek, insulin-like growth factor binding protein (IGFBP) 1, vitronectin, retinoic acid-induced gene E (RIG-E), and CYP3A4 (cytochrome P450 nifedipine oxidase) were identified as differentially expressed genes between human hepatocellular carcinoma (HCC) and matched nontumorous liver tissues. Higher levels of UGT2B4, rpP0, dek, vitronectin, Gal-4, and IGFBP-1 mRNAs combined with a lower level of RIG-E mRNA were observed in at least four of five primary HCCs compared to matched nontumorous liver tissues. Furthermore, a pathological study suggested that the levels of UGT2B4, rpP0, dek, and vitronectin increased and the level of RIG-E decreased with the histological grading. On the other hand, the expression of CYP3A4 mRNA and CYP3A7 (P-450 Fla) mRNA, a transcript found in the fetus and highly homologous to CYP3A4, was higher in all nontumorous liver and some of the carcinoma tissues from five HCC patients, whereas it was significantly lower in normal liver tissues from two non-HCC patients. The examination using HCC cell lines HuH-7 and HepG2 under different growth conditions suggested that the expression of dek mRNA was growth-associated. In contrast, the expression of Gal-4, UGT2B4, IGFBP-1, and RIG-E mRNAs was regulated in a cell density-dependent manner: the levels of Gal-4, UGT2B4, and IGFBP-1 were undetectably low, whereas the level of RIG-E was high in rapidly proliferating, subconfluent HCC cells in 10% serum; however, the expression levels were reversed in dense, overcrowded cultures. In addition, IGFBP-1 and Gal-4 mRNAs were also induced by reducing the serum concentration to 0.1%. We also demonstrated that sodium butyrate, an inducer of differentiation, up-regulated and down-regulated RIG-E and dek mRNAs, respectively, in a dose-dependent manner in HuH-7 cells, supporting, in part, our pathological observation. In summary, therefore, high expression of Gal-4, UGT2B4, rpP0, dek, IGFBP-1, and vitronectin, together with low expression of RIG-E, was correlated with the malignant potential of HCC. CYP3A4 and CYP3A7 could be induced in HCC-bearing livers. These transcripts are differentially regulated depending on cell-cell contact, serum growth factors, growth and differentiation status, and/or other mechanisms in premalignant and malignant liver cells.