Premature senescence is a key process in the progression of diabetic nephropathy (DN). In our study, we hypothesized that receptors for advanced glycation end-products (RAGE) mediate endoplasmic reticulum (ER) stress to induce premature senescence via p21 signaling activation in diabetic nephropathy. Here, we demonstrated that elevated expression of RAGE, ER stress marker glucose-regulated protein 78 (GRP78), and cell-cycle regulator p21 was all positively correlated with enhanced senescence-associated-β-galactosidase (SA-β-gal) activity in DN patients. In addition, the fraction of SA-β-gal or cells in the G0G1 phase were enhanced in cultured mouse proximal tubular epithelial cells (PTECs) and the expression of RAGE, GRP78 and p21 was up-regulated by advanced glycation end-products (AGEs) in a dose- and time-dependent manner. Interestingly, ER stress inducers or RAGE overexpression mimicked AGEs induced-premature senescence, and this was significantly suppressed by p21 gene silencing. However, RAGE blocking successfully attenuated AGEs-induced ER stress and p21 expression, as well as premature senescence. Moreover, ER stress inducers directly caused p21 activation, premature senescence, and also enhanced RAGE expression by positive feedback. These observations suggest that RAGE promotes premature senescence of PTECs by activation of ER stress-dependent p21 signaling.

This study was performed to investigate the role of galectin-1 (Gal-1) in epithelial ovarian cancer (EOC) progression and chemoresistance. Tissue samples from patients with EOC were used to examine the correlation between Gal-1 expression and clinical stage of EOC. The role of Gal-1 in EOC progression and chemoresistance was evaluated in vitro by siRNA-mediated knockdown of Gal-1 or lentivirus-mediated overexpression of Gal-1 in EOC cell lines. To elucidate the molecular mechanisms underlying Gal-1-mediated tumor progression and chemoresistance, the expression and activities of some signaling molecules associated with Gal-1 were analyzed. We found overexpression of Gal-1 in advanced stages of EOC. Knockdown of endogenous Gal-1 in EOC cells resulted in the reduction in cell growth, migration, and invasion in vitro, which may be caused by Gal-1's interaction with H-Ras and activation of the Raf/extracellular signal-regulated kinase (ERK) pathway. Additionally, matrix metalloproteinase-9 (MMP-9) and c-Jun were downregulated in Gal-1-knockdown cells. Notably, Gal-1 overexpression could significantly decrease the sensitivities of EOC cells to cisplatin, which might be ascribed to Gal-1-induced activation of the H-Ras/Raf/ERK pathway and upregulation of p21 and Bcl-2. Taken together, the results suggest that Gal-1 contributes to both tumorigenesis and cisplatin resistance in EOC. Thus, Gal-1 is a potential therapeutic target for EOC.

We generated a sequential reporter-enzyme luminescence (SRL) technology for in vivo detection of beta-galactosidase (beta-gal) activity. The substrate, a caged D-luciferin-galactoside conjugate, must first be cleaved by beta-gal before it can be catalyzed by firefly luciferase (FLuc) to generate light. As a result, luminescence is dependent on beta-gal activity. Using this technology, constitutive beta-gal activity in engineered cells and inducible tissue-specific beta-gal expression in transgenic mice can now be visualized noninvasively over time. A substantial advantage of beta-gal as a bioluminescent probe is that the enzyme retains full activity outside of cells, unlike FLuc, which requires intracellular cofactors. As a result, antibodies conjugated to the recombinant beta-gal enzyme can be used to detect endogenous cells and extracellular antigens in vivo. Thus, coupling the properties of FLuc to the advantages of beta-gal permits bioluminescent imaging applications that previously were not possible.

Colicin E-resistant mutants were isolated in Escherichia coli K-12 which, although still apparently possessing the E receptor and adsorbing colicin, were nevertheless insensitive (refractory) to its effect. Eight phenotypic groups were obtained, but some mutants from three of these groups were all shown to map at gal, whereas a second refractory locus, giving resistance to E1 alone, mapped close to thy. It is suggested that the successful fixation of any of the three distinct colicins of group E may involve a dual role for the cell surface "receptor," the first for the binding of the protein and the second for the correct orientation of the bound molecule relative to the cytoplasmic membrane. The majority of the refractory mutants isolated may derive from changes in components concerned with the second of these receptor functions. Two groups of mutants, however, refractory to only E1 or E2, probably reflect changes in the intracellular transmission systems which specifically mediate the effects of these two colicins, the changes not allowing transmission through the cytoplasmic membrane to the respective targets of the colicins. The E1 adsorption site was shown to be distinct from that for E2 and E3, indicating an early separation of the colicin E transmission systems.

Anaphylaxis is a severe allergic reaction that can be rapidly progressing and fatal, and therefore establishing its cause is pivotal to long-term risk management. Our recent work has identified a novel IgE antibody response to a mammalian oligosaccharide epitope, galactose-alpha-1,3-galactose (alpha-gal). IgE to alpha-gal has been associated with 2 distinct forms of anaphylaxis: (1) immediate-onset anaphylaxis during first exposure to intravenous cetuximab and (2) delayed-onset anaphylaxis 3 to 6 hours after ingestion of mammalian food products (eg, beef and pork). Results of our studies and those of others strongly suggest that tick bites are a cause, if not the only significant cause, of IgE antibody responses to alpha-gal in the southern, eastern, and central United States; Europe; Australia; and parts of Asia. Typical immune responses to carbohydrates are considered to be T-cell independent, whereas IgE antibody production is thought to involve sequential class-switching that requires input from T cells. Therefore, establishing the mechanism of the specific IgE antibody response to alpha-gal will be an important aspect to address as this area of research continues.

OBJECTIVE

Galectin-3 (Gal-3) is a 26-kDa lectin known to regulate many aspects of inflammatory cell behavior. We assessed the hypothesis that increased levels of Gal-3 contribute to atherosclerotic plaque progression by enhancing monocyte chemoattraction through macrophage activation.

RESULTS

Gal-3 was found to be upregulated in unstable plaque regions of carotid endarterectomy (CEA) specimens compared with stable regions from the same patient (3.2-fold, P<0.05) at the mRNA (n=12) and (2.3-fold, P<0.01) at the protein level (n=9). Analysis of aortic tissue from ApoE-/- mice on a high fat diet (n=14) and wild-type controls (n=9) showed that Gal-3 mRNA and protein levels are elevated by 16.3-fold (P<0.001) and 12.2-fold (P<0.01) and that Gal-3 staining colocalizes with macrophages. In vitro, conditioned media from Gal-3-treated human macrophages induced an up to 6-fold increase in human monocyte chemotaxis (P<0.01, ANOVA), an effect that was reduced by 66 and 60% by Pertussis Toxin (PTX) and the Vaccinia virus protein 35K, respectively. Microarray analysis of human macrophages and subsequent qPCR validation confirmed the upregulation of CC chemokines in response to Gal-3 treatment.

CONCLUSIONS

Our data suggest that Gal-3 is both a marker of atherosclerotic plaque progression and a central contributor to the pathology by amplification of key proinflammatory molecules.

OBJECTIVE

Carcinoma-associated fibroblasts (CAFs) in tumor stroma play an important role in tumor progression and have been associated with a poor prognosis in oral squamous cell carcinoma (OSCC). However, how CAFs influence OSCC malignancy and whether normalizing CAFs inhibits cancer progression remain unclear.

METHODS

The relationship between the expression of Galectin-1 (Gal-1) and alpha-smooth muscle actin (α-SMA, a CAF marker) in OSCC patient samples and primary cultured CAFs was examined by quantitative real-time PCR, Western blotting, and immunofluorescence. To examine the effect of Gal-1 on CAF activation and CAF-mediated tumor invasion and migration in vitro, Gal-1 expression was knocked down by small hairpin RNA. Finally, cancer cells and CAFs were coimplanted into SCID mice to evaluate the effect of Gal-1 on CAF-modulated tumor progression in vivo.

RESULTS

Gal-1 expression is positively associated with α-SMA in the stroma of OSCC specimens. Gal-1 knockdown decreases activated CAF characteristics, resulting in a decrease in α-SMA expression and extracellular matrix protein production. Notably, blocking Gal-1 expression significantly inhibits CAF-conditioned medium-induced tumor cell migration and invasion, possibly by reducing the production of monocyte chemotactic protein-1 (MCP-1/CCL2). MCP-1 induces the migration of OSCC cells by binding to the receptor CCR2; adding an MCP-1 antibody to CAF-conditioned medium that inhibits the interaction between MCP-1 and CCR2 abolishes migration. Finally, we found that Gal-1 knockdown in CAFs significantly reduces CAF-augmented tumor growth and metastasis in vivo.

CONCLUSIONS

Our findings demonstrate that Gal-1 regulates CAF activation and indicate that targeting Gal-1 in CAFs inhibits OSCC metastasis by modulating MCP-1 expression.

We have examined the carbohydrate binding specificity of the B4 lectin from Vicia villosa seeds. The B4 lectin agglutinates Tn-exposed erythrocytes specifically and binds to these erythrocytes (1.4 X 10(6) sites/cell) with an association constant of 4.2 X 10(7) M-1. The concentrations of saccharides and glycopeptides of defined structure which cause 50% inhibition of B4 lectin binding to Tn-exposed erythrocytes were determined. N-Acetylgalactosamine is the best monosaccharide inhibitor, causing 50% inhibition of binding at a concentration of 0.04 mM. Other monosaccharides inhibit lectin binding in the following order of decreasing potency: N-acetylgalactosamine greater than methyl-alpha-galactopyranoside greater than p-nitrophenyl-alpha- or beta-galactopyranoside greater than methyl-beta-galactopyranoside, galactose greater than galactosamine greater than mannose, N-acetylglucosamine. The disaccharide Gal beta 1,3GalNAc causes 50% inhibition of binding at a concentration of 2.8 mM, a concentration similar to that of the p-nitrophenyl-alpha- or beta-galactopyranosides. Glycopeptides containing O-glycosidically linked oligosaccharide units are significantly more potent inhibitors of lectin binding than the oligosaccharide units alone. The most potent glycopeptide inhibitor is a fetuin glycopeptide containing two alpha-linked N-acetylgalactosamine units. This glycopeptide causes 50% inhibition of lectin binding at a concentration of 0.00034 mM and probably closely resembles the B4 lectin binding site on Tn-exposed erythrocytes.

Multiple studies indicate that mRNA processing defects cause mRNAs to accumulate in discrete nuclear foci or dots, in mammalian cells as well as yeast. To investigate this phenomenon, we have studied a series of GAL reporter constructs integrated into the yeast genome adjacent to an array of TetR-GFP-bound TetO sites. mRNA within dots is predominantly post-transcriptional, and dots are adjacent to but distinct from their transcription site. These reporter genes also localize to the nuclear periphery upon gene induction, like their endogenous GAL counterparts. Surprisingly, this peripheral localization persists long after transcriptional shutoff, and there is a comparable persistence of the RNA in the dots. Moreover, dot disappearance and gene delocalization from the nuclear periphery occur with similar kinetics after transcriptional shutoff. Both kinetics depend in turn on reporter gene 3'-end formation signals. Our experiments indicate that gene association with the nuclear periphery does not require ongoing transcription and suggest that the mRNPs within dots may make a major contribution to the gene-nuclear periphery tether.

Involvement of DNA looping between two spatially separated gal operators, OE and OI, in repression of the gal operon has been demonstrated in vivo. An in vitro transcription assay using a minicircle DNA containing the gal promoter region with lac operators was employed to elucidate the molecular mechanism of repression. Wild-type lac repressors (LacI+ protein molecules), which are capable of associating into a tetramer and forming a DNA loop, repressed transcription from promoter sites P1 and P2, whereas a non-looping lac repressor mutant (LacI(adi)) failed to show normal repression of both of the gal promoters. Thus a DNA loop is also required for repression of transcription in vitro. Repression mediated by DNA looping resulted in the inhibition of the synthesis of complete as well as aborted transcripts, demonstrating that the repressive action was on the formation or activity of the initial transcribing complex. Under similar conditions, the gal repressor (GalR protein) did not repress the gal promoters effectively, apparently because it failed to loop DNA containing gal operators in the purified system. The component(s) or conditions that aid GalR in DNA looping remain to be identified.

To regulate gene expression following adenovirus-mediated gene transfer, a strategy was devised utilizing co-infection with two separate adenovirus vectors designed such that the product of one vector modulated the promoter of the second vector. To evaluate this strategy, AdEGR1.TNF, an adenovirus expressing tumor necrosis factor-alpha (TNF) under the control of the early growth response 1 (EGR1) promoter, was used to regulate a transcription unit in AdIL8.beta gal, an adenovirus vector in which the TNF sensitive interleukin-8 (IL-8) promoter drives the expression of beta-galactosidase (beta-gal). Following infection of HS24 cells with AdIL8.beta gal, addition of TNF to the culture induced the expression of beta-gal. Infection of HS24 cells with AdEGR1.TNF resulted in a dose-dependent secretion of TNF. Little beta-gal was produced following co-infection of the cells with the control vector AdCMV.Null (expressing no specific gene) and AdIL8.beta gal. In contrast, co-infection with AdIL8.beta gal and AdEGR1.TNF demonstrated, for a given dose of AdIL8.beta gal, increasing amounts of beta-gal expression dependent on the dose of AdEGR1.TNF. This model suggests control of gene expression in adenovirus-mediated gene transfer can be regulated by utilizing a promoter-gene expression cassette in one vector that modulates the expression of a promoter-gene expression cassette in a second vector.

We have used the human Lewis blood group fucosyltransferase cDNA and cross-hybridization procedures to isolate a human gene that encodes a distinct fucosyltransferase. Its DNA sequence predicts a type II transmembrane protein whose sequence is identical to 133 of 231 amino acids at corresponding positions within the catalytic domain of the Lewis fucosyltransferase. When expressed by transfection in cultured cell lines, this gene determines expression of a fucosyltransferase capable of efficiently utilizing N-acetyllactosamine to form the Lewis x determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc). By contrast, biochemical and flow cytometry analyses suggest that the enzyme cannot efficiently utilize the type II acceptor NeuNAc alpha 2----3Gal beta 1----4GlcNAc, to form the sialyl Lewis x determinant. In Chinese hamster ovary cells, however, the enzyme can determine expression of the alpha 2----3-sialylated, alpha 1----3-fucosylated structure known as VIM-2, a putative oligosaccharide ligand for ELAM-1. Cell adhesion assays using VIM-2-positive, sialyl Lewis x-negative transfected Chinese hamster ovary cells indicate that surface expression of the VIM-2 determinant is not sufficient to confer ELAM-1-dependent adhesive properties upon the cells. These results demonstrate that substantial structural similarities can exist between mammalian glycosyltransferases with closely related enzymatic properties, thus facilitating isolation of their cognate genes by cross-hybridization methods. The results further suggest that cell surface expression of the VIM-2 determinant is not necessarily sufficient to mediate ELAM-1-dependent cell adhesion.

Galectin-1 (Gal-1), a carbohydrate-binding protein whose secretion is enhanced by hypoxia, promotes tumor aggressiveness by promoting angiogenesis and T-cell apoptosis. However, the importance of tumor versus host Gal-1 in tumor progression is undefined. Here we offer evidence that implicates tumor Gal-1 and its modulation of T-cell immunity in progression. Comparing Gal-1-deficient mice as hosts for Lewis lung carcinoma cells where Gal-1 levels were preserved or knocked down, we found that tumor Gal-1 was more critical than host Gal-1 in promoting tumor growth and spontaneous metastasis. Enhanced growth and metastasis associated with Gal-1 related to its immunomodulatory function, insofar as the benefits of Gal-1 expression to Lewis lung carcinoma growth were abolished in immunodeficient mice. In contrast, angiogenesis, as assessed by microvessel density count, was similar between tumors with divergent Gal-1 levels when examined at a comparable size. Our findings establish that tumor rather than host Gal-1 is responsible for mediating tumor progression through intratumoral immunomodulation, with broad implications in developing novel targeting strategies for Gal-1 in cancer.

The polypeptide chain of human lactotransferrin possesses two glycoslyation sites to which glycans are linked through an N-(beta-aspartyl)-N-acetylglucosaminylamine bond and which are structurally heterogenous. After chymotryptic or pronase digestions, glycopeptides with five different glycan structures could be isolated. For three of them, the structure has been determined by the application of methanolysis, methylation analysis, hydrazinolysis/nitrous deamination, enzymatic cleavage and 1H-NMR spectroscopy at 360 MHz: Glycopeptides A and B: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)GlcNAc(beta-1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-)Asn; Glycopeptide C: NeuAc(alpha 2-6)(Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)(Gal(beta 1-4)[Fuc(alpha 1-3)]GlcNAc(beta 1-2)Man(alpha 1-6))Man(beta 1-4)GlcNAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-)Asn. Glycopeptide D: NeuAc(alpha 2-6)Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-3)[Gal(beta 1-4)GlcNAc(beta 1-2)Man(alpha 1-6)]Man(beta 1-4)Glc-NAc(beta 1-4)[Fuc(alpha 1-6)]GlcNAc(beta 1-)Asn. Two other glycopeptides were obtained in very low amount and possess more complex structures.

The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection--mediated by natural antibodies in humans to pig antigens, complement fixation to endothelial cells, and the rapid onset of intravascular coagulation. It is now clear that the major target of the natural IgM and IgG antibodies is the terminal carbohydrate epitope Gal alpha(1,3)Gal, formed by the alpha 1,3galactosyl transferase, which places a terminal galactose residue in an alpha-linkage to another galactose. The alpha 1,3galactosyl transferase in the pig gives rise to very high endothelial cell expression of Gal alpha(1,3)Gal, a ready explanation for the hyperacute rejection of vascularized organs. In addition the parenchuma of liver and kidneys have high levels of Gal alpha-(1,3)Gal. These tissues will all fail in a pig-to-human transplant in what can now be precisely defined in terms of antigen and antibody. We have already made some suggestions for removal of anti-Gal alpha(1,3)Gal antibodies and if the procedure were technically feasible xenotransplantation could be attempted now, especially in patients doomed to a certain death because of the absence of a donor (especially for liver where ex vivo perfusion could be performed). However, the immune system is far from simple, as is shown by the healthy status of mice lacking MHC Class I, Class II or both Class I & II molecules. Perhaps the curtain is about to go up to reveal a new scene! Islets differ from the other tissues and may well not undergo acute antibody-mediated hyperacute rejection--it will be of interest to see how these fare in xenotransplantation models or even in patients. Again, normal individuals do not have anti-islet antibodies; but a proportion of diabetic patients do have such antibodies--whether these will cause hyperacute or acute rejection or are markers of immunity of T-cell type, remains to be seen. Whatever, the area is exciting, is progressing rapidly and, as indicated elsewhere, within a few years we should know whether modified pig tissue can be grafted to some patients. The isolation of the cDNA clone encoding the pig alpha 1,3 galactosyl transferase is an essential first step in the production of a transgenic pig lacking the alpha 1,3Galactosyltransferase and therefore the Gal alpha(1,3)Gal epitope, and such animals could serve as donor for human transplantation.

Brain-derived neurotrophic factor (BDNF), one of the members of the nerve growth factor family of neurotrophins, is expressed in developing gustatory papillae and is thought to be the neurotrophin that supports gustatory innervation during development. BDNF expression does not cease after development but continues in some taste cells of adult mice. To determine which types of taste cells produce BDNF, we undertook an immunohistochemical study of taste cells in BDNF(LacZ) gene targeted "knock-in" adult mice. In these mice, beta-galactosidase (beta-gal) immunoreactivity is an indicator of cells that produce BDNF transcripts. In the tongues of adult BDNF(LacZ) mice, beta-gal (BDNF) is present in long slender taste cells, as well as pyriform taste cells. Bromodeoxyuridine labeling experiments in BDNF(LacZ) mice indicate that BDNF is not present in taste cells that are younger than 3 days postmitotic. BDNF mainly colocalizes with markers of type II and type III taste cells: ubiquitin carboxyl terminal hydrolase (PGP 9.5), serotonin (5-HT), neural cell adhesion molecule (N-CAM), synaptic associated protein of 25 kDa (SNAP-25), and to a lesser extent with alpha-gustducin. beta-Gal immunoreactivity is not associated with blood group H antigen, a marker of type I taste cells. We conclude that BDNF is absent from basal cells and type I (blood group H antigen immunoreactive) taste cells but is present in differentiated type II and type III taste cells. The presence of SNAP-25 in BDNF-expressing cells suggests a role for BDNF in synaptic formation and transmission.

BACKGROUND

The pygopus gene of Drosophila encodes an essential component of the Armadillo (beta-catenin) transcription factor complex of canonical Wnt signaling. To better understand the functions of Pygopus-mediated canonical Wnt signaling in kidney development, targeted mutations were made in the two mammalian orthologs, Pygo1 and Pygo2.

RESULTS

Each mutation deleted >80% of the coding sequence, including the critical PHD domain, and almost certainly resulted in null function. Pygo2 homozygous mutants, with rare exception, died shortly after birth, with a phenotype including lens agenesis, growth retardation, altered kidney development, and in some cases exencephaly and cleft palate. Pygo1 homozygous mutants, however, were viable and fertile, with no detectable developmental defects. Double Pygo1/Pygo2 homozygous mutants showed no apparent synergy in phenotype severity. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced levels of expression in Pygo1-/-/Pygo2-/- mutants, with tissue-specific variation in degree of diminution. The Pygo1 and Pygo2 genes both showed widespread expression in the developing kidney, with raised levels in the stromal cell compartment. Confocal analysis of the double mutant kidneys showed disturbance of both the ureteric bud and metanephric mesenchyme-derived compartments. Branching morphogenesis of the ureteric bud was altered, with expanded tips and reduced tip density, probably contributing to the smaller size of the mutant kidney. In addition, there was an expansion of the zone of condensed mesenchyme capping the ureteric bud. Nephron formation, however, proceeded normally. Microarray analysis showed changed expression of several genes, including Cxcl13, Slc5a2, Klk5, Ren2 and Timeless, which represent candidate Wnt targets in kidney development.

CONCLUSIONS

The mammalian Pygopus genes are required for normal branching morphogenesis of the ureteric bud during kidney development. Nevertheless, the relatively mild phenotype observed in the kidney, as well as other organ systems, indicates a striking evolutionary divergence of Pygopus function between mammals and Drosophila. In mammals, the Pygo1/Pygo2 genes are not absolutely required for canonical Wnt signaling in most developing systems, but rather function as quantitative transducers, or modulators, of Wnt signal intensity.

Underivatized neutral oligosaccharides from human milk were analyzed by nano-electrospray ionization (ESI) using a quadrupole ion trap mass spectrometer (QIT-MS) in the negative-ion mode. Under these conditions neutral oligosaccharides are observed as deprotonated molecules [M-H]- with high intensity. CID-experiments of these species with the charge localized at the reducing end lead to C-type fragment ions forming a "new" reducing end. Fragmentations are accompanied by cross-ring cleavages that yield information about linkages of internal monosaccharides. Several isomeric compounds with distinct structural features, such as different glycosidic linkages, fucosylation and branching sites were investigated. The rules governing the fragmentation behavior of this class of oligosaccharides were elucidated and tested for a representative number of certain isomeric glycoforms using the MS/MS and MS(n) capabilities of the QIT. On the basis of the specific fragmentation behavior of deprotonated molecules, the position of fucoses and the linkage type (Gal beta-->3 GlcNAc or Gal beta1-->4 GlcNAc) could be determined and linear and branched could be differentiated. Rules could be established which can be applied in further investigations of these types of oligosaccharides even from heterogenous mixtures.

BACKGROUND

Recent evidence has demonstrated that ethanol intake can stimulate the expression and production of the feeding-stimulatory peptide, galanin (GAL), in the hypothalamic paraventricular nucleus (PVN), and that PVN injection of this peptide, in turn, can increase the consumption of ethanol. To test the hypothesis that other feeding-related systems are involved in ethanol intake, this study examined the effect of ethanol on the hypothalamic opioid peptides, enkephalin (ENK), and dynorphin (DYN).

METHODS

Adult, male Sprague-Dawley rats were trained to voluntarily drink increasing concentrations of ethanol, up to 9% v/v, on a 12-hour access schedule or were given a single injection of ethanol (10% v/v) versus saline vehicle. The effect of ethanol on GAL, ENK, and DYN mRNA was measured using real-time quantitative polymerase chain reaction and radiolabeled in situ hybridization, while radioimmunoassay was used to measure peptide levels. In addition to blood alcohol, circulating levels of triglycerides (TG), leptin, and insulin were also measured.

RESULTS

The data demonstrated that: (1) rats voluntarily drinking 9% v/v ethanol (approximately 2.0 g/kg/d) show a significant increase in GAL, ENK, and DYN mRNA in the PVN compared with water-drinking rats; (2) voluntary consumption of ethanol also increases peptide levels of ENK and DYN in the PVN; (3) acute injection of 10% ethanol (1.0 g/kg of 10% v/v) similarly increases the expression of GAL, ENK, and DYN in the PVN; and (4) ethanol consumption and injection, while having little effect on leptin and insulin, consistently increase circulating levels of TG as well as alcohol, both of which are strongly, positively correlated with peptide expression in the PVN.

CONCLUSIONS

These findings, together with published studies, suggest a possible role for hypothalamic opioid peptides in the drinking of ethanol. Based on evidence that dietary fat and lipid injections stimulate the PVN peptides and injection of the opiates and GAL increase ethanol intake, it is proposed that both TG and alcohol in the circulation, which are elevated by the ingestion or injection of ethanol, are involved in stimulating these peptides in the PVN, which in turn promote further consumption of ethanol.

In the yeast Saccharomyces cerevisiae, the interplay between Gal3p, Gal80p and Gal4p determines the transcriptional status of the genes needed for galactose utilization. The interaction between Gal80p and Gal4p has been studied in great detail; however, our understanding of the mechanism of Gal3p in transducing the signal from galactose to Gal4p has only begun to emerge recently. Historically, Gal3p was believed to be an enzyme (catalytic model) that converts galactose to an inducer or co-inducer, which was thought to interact with GALGAL genes with the energy status of the cell.

We have analyzed a GAL1 mutant (gal1-r strain) of the yeast Kluyveromyces lactis which lacks the induction of beta-galactosidase and the enzymes of the Leloir pathway in the presence of galactose. The data show that the K. lactis GAL1 gene product has, in addition to galactokinase activity, a function required for induction of the lactose system. This regulatory function is not dependent on galactokinase activity, as it is still present in a galactokinase-negative mutant (gal1-209). Complementation studies in Saccharomyces cervisiae show that K. lactis GAL1 and gal1-209, but not gal1-r, complement the gal3 mutation. We conclude that the regulatory function of GAL1 in K. lactis soon after induction is similar to the function of GAL3 in S. cerevisiae.

Induction of transcription of the GAL genes of yeast by galactose is a multistep process: Galactose frees the activator Gal4 of its inhibitor, Gal80, allowing Gal4 to recruit proteins required to transcribe the GAL genes. Here, we show that deletion of components of either the HSP90 or the HSP70 chaperone machinery delays this induction. This delay remains when the galactose-signaling pathway is bypassed, and it cannot be explained by a chaperone requirement for DNA binding by Gal4. Removal of promoter-bound nucleosomes is delayed in a chaperone mutant, and our findings suggest an involvement of HSP90 and HSP70 in this early step in gene induction.

The Saccharomyces cerevisiae <em>GAL</em>/MEL regulon genes are normally induced within minutes of galactose addition, but gal3 mutants exhibit a 3-5-day induction lag. We have discovered that this long-term adaptation (LTA) phenotype conferred by gal3 is complemented by multiple copies of the <em>GAL</em>1 gene. Based on this result and the striking similarity between the <em>GAL</em>3 and <em>GAL</em>1 protein sequences we attempted to detect galactokinase activity that might be associated with the <em>GAL</em>3 protein. By both in vivo and in vitro tests the <em>GAL</em>3 gene product does not appear to catalyze a galactokinase-like reaction. In complementary experiments, Escherichia coli galactokinase expressed in yeast was shown to complement the gal1 but not the gal3 mutation. Thus, the complementation activity provided by <em>GAL</em>1 is not likely due to galactokinase activity, but rather due to a distinct <em>GAL</em>3-like activity. Overall, the results indicate that <em>GAL</em>1 encodes a bifunctional protein. In related experiments we tested for function of the LTA induction pathway in gal3 cells deficient for other gene functions. It has been known for some time that gal3gal1, gal3gal7, gal3gal10, and gal3 rho- are incapable of induction. We constructed isogenic haploid strains bearing the gal3 mutation in combination with either gal15 or pgi1 mutations: the gal15 and pgi1 blocks are not specific for the galactose pathway in contrast to the gal1, gal7 and gal10 blocks. The gal3gal5 and gal3pgi1 double mutants were not inducible, whereas both the gal5 and pgi1 single mutants were inducible. We conclude that, in addition to the <em>GAL</em>3-like activity of <em>GAL</em>1, functions beyond the galactose-specific <em>GAL</em>1, <em>GAL</em>7 and <em>GAL</em>10 enzymes are required for the LTA induction pathway.

Dendritic cells (DCs) loaded with alpha-galactosylceramide (alpha-GalCer) are known to be active APCs for the stimulation of innate NKT and NK cell responses in vivo. In this study, we evaluated the capacity of non-DCs to present alpha-GalCer in vitro and in vivo, particularly tumor cells loaded with alpha-GalCer (tumor/Gal). Even though the tumor cells lacked expression of CD40, CD80, and CD86 costimulatory molecules, the i.v. injection of tumor/Gal resulted in IFN-gamma secretion by NKT and NK cells. These innate responses to tumor/Gal, including the induction of IL-12p70, were comparable to or better than alpha-GalCer-loaded DCs. B16 melanoma cells that were stably transduced to express higher levels of CD1d showed an increased capacity relative to wild-type B16 cells to present alpha-GalCer in vivo. Three different tumor cell lines, when loaded with alpha-GalCer, failed to establish tumors upon i.v. injection, and the mice survived for at least 6 mo. The resistance against tumor cells was independent of CD4 and CD8 T cells but dependent upon NKT and NK cells. Mice were protected from the development of metastases if the administration of live B16 tumor cells was followed 3 h or 3 days later by the injection of CD1d(high)-alpha-GalCer-loaded B16 tumor cells with or without irradiation. Taken together, these results indicate that tumor/Gal are effective APCs for innate NKT and NK cell responses, and that these innate immune responses are able to resist the establishment of metastases in vivo.