The brown adipose tissue uncoupling protein 1 (UCP1) catalyzes proton reentry without ATP synthesis, thereby dissipating energy as heat. In contrast, the function(s) of the recently described homologs, UCP2 and UCP3, are less clear. The aim of the present study was to determine whether overexpressed UCP subtypes affect mitochondrial respiration and substrate oxidation in cultured insulin-secreting INS-1 insulinoma cells. Adenoviral overexpression of UCP2 significantly decreased the ADP/O ratio by 31% and 39% in comparison to beta-galactosidase (beta-gal) or the mitochondrial protein manganese superoxide dismutase (MnSOD), respectively, and increased state 4 respiration in the presence of succinate and oligomycin by 52% and 59% in comparison to beta-gal or MnSOD, respectively. Adenoviral overexpression of UCP3 also decreased the ADP/O ratio by 18% (nonsignificant) and increased state 4 respiration by 24% (nonsignificant) in comparison to ss-gal and significantly decreased the ADP/O ratio by 32% and increased state 4 respiration by 35% in comparison to MnSOD. Both UCP2 and UCP3 expression significantly increased whole cell lipid oxidation by 34% (P < 0.01) and 30% (P < 0.05), respectively, compared with cells expressing Ad5CMVlacZ. However, glucose oxidation was not significantly altered by UCP2 or UCP3 expression. Adenoviral UCP2 expression, but not UCP3 (compared with beta-gal), significantly inhibited insulin secretion in the presence of 15 mM glucose [6.17 +/- 0.42 ng/mg cell protein for beta-gal compared with 4.69 +/- 0.39 for UCP2 (P < 0.05) and 5.51 +/- 0.50 for UCP3]. Both overexpressed UCPs significantly reduced INS-1 cell ATP content. Within certain limitations, which are discussed, these data are the first to demonstrate increased respiration and impaired coupling of oxidative phosphorylation as a result of UCP homolog expression in isolated mammalian mitochondria. Our results also suggest an important role for UCP in lipid metabolism and, possibly, insulin secretion.

Targeted deletion of the connexin36 (Cx36) gene in the mouse genome leads to visual transmission defects, weakened synchrony of rhythmic inhibitory potentials in the neocortex, and disruption of gamma-frequency network oscillations. We have generated transgenic mice in which a reporter protein consisting of the exon1 coded N-terminal part of Cx36 fused to beta-galactosidase (N36-beta-gal) is expressed instead of Cx36. Here, we have used these mice for a detailed analysis of the reporter gene expression. By beta-gal staining of adult retina, we found expression of the lacZ reporter gene in the ganglion cell layer, in two rows of the inner nuclear layer, and in the photoreceptor layer. In the brain, beta-gal staining was present in gamma-aminobutyric acid (GABA)ergic neurons of the cerebellar nuclei, in non-GABAergic neurons of the inferior olive, in mitral cells of the olfactory bulb, and in parvalbumin-positive cells of the cerebral cortex. Outside the central nervous system, N36-beta-gal signals were detected in insulin producing beta-cells of the pancreas and in the medulla of the adrenal gland of adult Cx36(+/del[LacZ]) mice. This expression pattern suggests that Cx36 fulfills functional roles not only in several types of neurons in the retina and central nervous system but also in excitable cells of the pancreas and adrenal gland.

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.

To elucidate critical steps in the transcription initiation process, we have devised a protocol for obtaining information about DNA structure and DNA-protein interactions at nucleotide level resolution from intact yeast cells. Our procedure combines the ultraviolet light 'footprinting' method developed by Becker and Wang with the 'genomic sequencing' technique described by Church and Gilbert. Using this approach we were able to detect the binding of GAL 4 protein at sites within the upstream activating sequence (UASG) previously mapped using other in vivo and in vitro foot-printing procedures. We also observed transcription-dependent changes in sensitivity of DNA to ultraviolet-induced covalent modification at several positions between the upstream activating sequence and the transcription initiation sites of the GAL 1 and GAL 10 genes. The most prominent of these changes occurs at a common site within the putative 'TATA' boxes of the two genes. Ultraviolet modification at this site is enhanced only in transcriptionally active promoters.

Galactose-inducible genes (GAL genes) in yeast Saccharomyces cerevisiae are efficiently transcribed only when the sequence-specific transcription activator Gal4p is activated. Activation of Gal4p requires the interaction between the Gal4p inhibitory protein Gal80p and the galactokinase paralog, Gal3p. It has been proposed that Gal3p binds to a Gal80p-Gal4p complex in the nucleus to activate Gal4p. Here, we present evidence that the Gal3p-Gal80p interaction occurs in the cytoplasm, and concurrently, Gal80p is removed from Gal4p at the GAL gene promoter. We also show that GAL gene expression can be activated by heterologous protein-protein interaction in the cytoplasm that is independent of galactose and Gal3p function. These results indicate that galactose-triggered Gal3p-Gal80p association in the cytoplasm activates Gal4p in the nucleus.

The ability of two negative regulatory proteins, Gal and Lac repressors, to induce DNA bending was tested by the respective cloning of gal and lac operator DNA sequences into a sensitive vector, pBend2. pBend2 can generate a large number of DNA fragments in each of which a cloned operator is present in circularly permuted positions along the length of the DNA. Gel electrophoresis of these DNA fragments individually complexed to a repressor shows that the Gal repressor bends both of the gal operators, OE and OI. Similarly, the Lac repressor induces a bend to a lac operator DNA. In each case, the center of the average bent segment is located at or close to the dyad symmetry axis of the operator sequence. In view of these findings, we discuss how these negative regulatory proteins may function by a more dynamic mechanism than was perceived previously.

BACKGROUND

Transcription initiation from the two overlapping promoters of the gal operon in Escherichia coli is negatively regulated by binding of Gal repressor (GalR) to bipartite operators, which encompass the promoters. Coordinated repression of the two promoters requires GalR binding to both operators. In a purified system, GalR, nevertheless, fails to show the coordinated repression, predicting the participation of an additional factor(s) in the regulation in vivo.

RESULTS

We have purified a protein that restored the expected GalR-mediated repression for the in vitro system and have identified this factor to be the bacterial histone-like protein HU. In vitro transcription assays in the presence of GalR and HU show that, just as in vivo, the coordinated repression of the two gal promoters requires GalR binding to both operators and is sensitive to the inducer, D-galactose. The GalR and HU dependent repression also requires supercoiled DNA template and prevents open complex formation.

CONCLUSIONS

We propose that HU, acting as a co-factor, brings about the GalR-mediated repression by forming a distinct nucleoprotein complex of higher order structure. Although how HU participates in the assembly process is unknown, there may be a cooperative effect in the formation of the repression complex.

Social transmission of food preference (STFP) is a test of olfactory memory that can be used in mice. Confounds in STFP that can lead to misinterpretation of an STFP deficit as a memory impairment include changes in social interaction and olfaction. The issue of changes in social interaction was addressed by evaluating an observer-centric and a demonstrator-centric method for scoring the interaction phase of STFP in mice. The demonstrator-centric method was applied to a line of STFP-impaired, galanin-overexpressing transgenic (GAL-tg). GAL-tg mice were impaired in STFP without deficits in social interaction. In tests of olfactory ability, GAL-tg mice were unimpaired on buried-food and habituation-dishabituation tasks. The current studies describe an expanded method for using STFP in mice and confirm a deficit in olfactory memory in GAL-tg mice.

Mammalian C-type retroviruses are inactivated by human serum, following triggering of the classical complement cascade. This may have inhibited transmission to humans of C-type oncoviruses from other mammals. Indeed, the retroviruses human immunodeficiency virus and human T-cell leukaemia virus are resistant to human complement. Antibody-independent activation of human C1q, the first component of the classical pathway, by retroviral envelope proteins has been described. However, retroviruses produced from human cells are resistant to inactivation by human complement and human serum is known to contain antibodies directed against carbohydrates on retroviral envelopes. Gal(alpha 1-3)Gal terminal carbohydrates are expressed by most mammals but are absent in humans, which lack a functional (alpha 1-3)galactosyltransferase gene. Here, we demonstrate that anti-Gal(alpha 1-3)Gal antibodies in human serum inactivate retroviruses produced from animal cells. Expression of porcine (alpha 1-3)galactosyltransferase in human cells renders the cells and the retroviruses they produce sensitive to human serum.

Platelet glycoprotein IV (GPIV, Mr 88,000), which is immunologically related to the leukocyte differentiation antigen CD36, has been isolated from both intact and trypsinized platelet membranes by a series of steps involving (i) phase partitioning in Triton X-114, (ii) ion exchange chromatography on DEAE-cellulose, (iii) lectin affinity chromatography on wheat germ agglutinin-Sepharose, and (iv) size exclusion chromatography on Ultrogel AcA-44. The homogenous product contained 26% carbohydrate (sialic acid, Gal, Man, GalNAc, GlcNAc), of which approximately two-thirds were in alkali-labile O-glycosidic linkages. A rabbit polyclonal antibody raised against purified GPIV gave a single band on immunoblot and on immunoprecipitation from solubilized, 3H-labeled platelet membranes indicating its monospecificity. The antibody gave a strongly positive reaction with platelets on flow cytofluorimetry further confirming the surface location of GPIV. Immunoblotting and flow cytometry also identified GPIV-like molecules on the surface of U937, HEL, and C32 cells but not on HT1080 fibroblasts. Amino acid analysis showed values comparable with those deduced from the cloning data for GPIb, GPIIb, and GPIIIa. Automated Edman degradation allowed the identification of the sequence of the first 36 residues of the NH2-terminal domain. G-X-D-R-N-X-G-L-I-A-G-A-V-I-G-A-V- L-A-V-F-G-G-I-L-M-P-V-G-D-L-P-X-Q-K-F. There are no identifiable homologies between the NH2-terminal domain and other known protein sequences. Following a hydrophilic hexapeptide, there is a hydrophobic sequence of 23 amino acids (underlined) that is of the size and composition expected for a transmembrane domain. Since the NH2-terminal domain lacks tyrosine, but GPIV may be readily iodinated in intact platelets, this suggests that GPIV may have a configuration expressing other extramembranous domains.

Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6-related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and beta-galactosidase (Adbeta-gal). Compared with cardiomyocytes infected with Adbeta-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Adbeta-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal alpha-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal-regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytes infected with Adbeta-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.

The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.

Cellular senescence is a stress-response phenomenon in which cells lose the ability to proliferate; it is induced by telomere shortening, activation of oncogenes or tumor suppressor genes, or exposure to a sub-lethal dose of DNA damaging agents or oxidative stresses. cDNA microarray analysis reveals that the levels of interferons (IFNs) and IFN-inducible genes were altered during replicative senescence in human umbilical vascular endothelial cells (HUVECs). However, the role of IFNs in cellular senescence of HUVECs remains unidentified. This study demonstrated that prolonged treatment with IFN-gamma induced cellular senescence in HUVECs, as confirmed by G0/G1 cell cycle arrest, up-regulation of p53 and p21 protein levels, increased SA-beta-gal staining, and the accumulation of phospho-H(2)AX foci. IFN-gamma-induced cellular senescence was observed only in p16-knockdown cells or p16-null mouse embryonic fibroblasts (MEFs), but not in p53-knockdown cells or p53-null MEFs. IFN-gamma treatment increased ROS production, and an antioxidant, N-acetylcysteine, inhibited IFN-gamma-induced cellular senescence. Knockdown of ATM kinase or IFI16 rescued IFN-gamma-induced cellular senescence. Therefore, these results suggest that IFN-gamma might play an important role in cellular senescence through a p53-dependent DNA damage pathway and contribute to the pathogenesis of atherosclerosis via its pro-senescent activity.

The goal of this study was to enhance gene delivery and tumor cell transfection in vivo by using a combination of ultrasonication with complex nanoparticles consisting of two types of nanoparticles: PEI/DNA beta-gal plasmid with highly positive zeta-potential and air-filled poly (lactic-co-glycolic acid) (PLGA) particles (with negative zeta-potential) manufactured in our laboratory. The PLGA/PEI/DNA nanoparticles were a colloid with positive zeta-potential and injected i.v. in nude mice with DU145 human prostate tumors. We found that the combination of PLGA/PEI/DNA nanoparticles with ultrasonication substantially enhanced tumor cell transfection in vivo. The overexpression of beta-gal gene was evaluated histochemically and by Western blot analysis. At least an 8-fold increase of the cell transfection efficacy was obtained in irradiated tumors compared to non-irradiated controls, while little to no cell death was produced by ultrasonication.

BACKGROUND

Vascular cells have a finite cell lifespan and eventually enter an irreversible growth arrest, cellular senescence. The functional changes associated with cellular senescence are thought to contribute to human aging and age-related vascular disorders. Ras, an important signaling molecule involved in atherogenic stimuli, is known to promote aging in yeast and cellular senescence in primary human fibroblasts. The aim of this study was to investigate the role of Ras-induced vascular smooth muscle cell (VSMC) senescence in atherogenesis.

RESULTS

We introduced an activated ras allele (H-rasV12) into human VSMCs using retroviral infection. Introduction of H-rasV12 induced a growth arrest with phenotypic characteristics of cellular senescence, such as enlarged cell shapes and increases in expression of cyclin-dependent kinase inhibitors and senescence-associated beta-galactosidase (SA-beta-gal) activity. Activation of Ras drastically increased expression of proinflammatory cytokines, in part through extracellular signal-regulated kinase activation. To determine whether Ras activation induces cellular senescence in vivo, we transduced the adenoviral vector encoding H-rasV12 into rat carotid arteries injured by a balloon catheter. Introduction of Ras into the arteries enhanced vascular inflammation and senescence compared with mock-infected injured arteries. Moreover, SA-beta-gal-positive VSMCs were detected in the intima of advanced human atherosclerotic lesions and exhibited increased levels of extracellular signal-regulated kinase activity and proinflammatory cytokine expression.

CONCLUSIONS

Our results suggest that atherogenic stimuli mediated by Ras induce VSMC senescence and vascular inflammation, thereby contributing to atherogenesis. This novel mechanism of atherogenesis may provide insights into a new antisenescence treatment for atherosclerosis.

Metastasis-suppressing gene product CD82 and its analogue CD9 are considered to suppress the malignancy of various human cancers, although the rationale for this effect is unknown. The present study addresses phenotypic changes in Chinese hamster ovary mutant cell line ldlD deficient in UDP-Glc 4-epimerase and expressing CD82 or CD9 by cDNA transfection. Only CD82- or CD9-expressing cells grown in Gal-supplemented medium showed reduced motility and massive cell death, which are characteristic of apoptosis, after a latent period. Under this condition, endogenous GM3 synthesis was observed as a common factor, and N-glycosylation occurred at a high level in CD82 and to a lesser extent in CD9. Thus, the malignancy-suppressing effect of CD82 or CD9 is based partially on cell motility inhibition and apoptosis induction promoted by concurrent GM3 synthesis and N-glycosylation.

OBJECTIVE

To compare the expression of galectin 9 (Gal-9) in synovial tissue (ST) from rheumatoid arthritis (RA) patients and osteoarthritis (OA) patients and to evaluate the effects of Gal-9 on fibroblast-like synoviocytes (FLS) in these patients.

METHODS

The expression of Gal-9 in ST and FLS was compared using immunohistochemical techniques. Apoptotic cells in RA and OA ST samples were detected by TUNEL assay. Apoptosis of FLS was analyzed by the sub-G(1) method in vitro. The in vivo suppressive effects of Gal-9 on collagen-induced arthritis (CIA) in a mouse model were also elucidated.

RESULTS

The percentage of Gal-9-positive cells in ST samples and the amount of Gal-9 in synovial fluid samples were significantly higher in patients with RA than in patients with OA, suggesting the involvement of Gal-9 in the development of RA. Compared with the 2 wild-type Gal-9 forms, stable Gal-9, a mutant protein resistant to proteolysis, significantly induced apoptosis of FLS from RA patients. In contrast, other galectins, such as Gal-1, Gal-3, and Gal-8, did not induce apoptosis or suppress the proliferation of human RA FLS. Stable Gal-9 preferentially induced apoptosis and suppressed the proliferation of RA FLS in vitro. It also induced apoptosis of cells in RA ST implanted into SCID mice in vivo. In a mouse model of CIA, apoptotic cells were detected in the joints of stable Gal-9-treated mice, but not phosphate buffered saline-treated mice, and suppressed CIA characterized by pannus formation with inflammatory cell infiltration and bone/cartilage destruction.

CONCLUSIONS

Gal-9-induced apoptosis of hyperproliferative RA FLS may play a critical role in the suppression of RA.

Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O-demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease.

Adeno-associated virus (AAV) is a promising vector for central nervous system (CNS) gene transfer, but a number of issues must be addressed if AAV is to be used for widespread delivery throughout the CNS. Our aim was to test the effect of dose, route of delivery, and hydroxyurea treatment on brain expression of beta-galactosidase activity after cerebral inoculation with an rAAV-lacZ vector (rAAV-beta-gal). We also wished to test whether an immune response appeared against the vector and the transgene product. We found in BALB/c mice that beta-Gal expression increased during the first 2 months after inoculation, then decreased slightly by 4 months, and continued out to 6, 12, and 15 months in single animals. Cerebral injection produced localized beta-Gal expression that did not diffuse to other regions despite a fivefold increase in injection volume. Intraventricular injection resulted in negligible transduction. Antibodies to AAV capsid protein and beta-Gal appeared at low levels at 2 and 4 months, but correlated poorly with beta-Gal expression and did not prevent readministration of rAAV-beta-gal. Hydroxyurea treatment did not result in increased transduction in vivo. We conclude that our study confirms rAAV vectors as having considerable potential for CNS gene transfer; however, several important problems must be addressed if this vector system is to be used for long-term transduction of the entire brain. Sustained, regulatable expression will be needed if rAAV is to be used in the treatment of chronic CNS disease. The difficulty in delivering AAV to diverse regions of the brain is an important problem that must be overcome if these vectors are to be used for anything beyond localized transduction.

The establishment of the floor plate at the ventral midline of the CNS is dependent on an inductive signaling process mediated by the secreted protein Sonic hedgehog (Shh). To understand molecularly how floor plate induction proceeds we identified a Shh-responsive regulatory element that directs transgene reporter expression to the ventral midline of the CNS and notochord in a Shh-like manner and characterized critical cis-acting sequences regulating this element. Cross-species comparisons narrowed the activity of the Shh floor plate enhancer to an 88-bp sequence within intron 2 of Shh that included highly conserved binding sites matching the consensus for homeodomain, Tbx and Foxa transcription factors. Mutational analysis revealed that the homeodomain and Foxa binding sites are each required for activation of the Shh floor plate enhancer, whereas the Tbx site was required for repression in regions of the CNS where Shh is not normally expressed. We further show that Shh enhancer activity was detected in the mouse node from where the floor plate and notochord precursors derive. Shh reporter expression was restricted to the ventral (mesodermal) layer of the node in a pattern similar to endogenous Shh. X-gal-positive cells emerging from the node were only detected in the notochord lineage, suggesting that the floor plate and notochord arise from distinct precursors in the mouse node.

BACKGROUND

Recent evidence has accumulated that MicroRNA (miRNA) dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML) and may contribute to onco-/leukemo-genesis.

METHODS

The expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML patients in 1st complete remission (CR) and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and β-Galactosidase ( SA-β-Gal) staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1.

RESULTS

We found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2'-deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells.

CONCLUSIONS

Taken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression.

Galectin (Gal)-9 was first described as an eosinophil chemoattractant. With the progress in research, Gal-9 has come to be known as a versatile immunomodulator that is involved in various aspects of immune regulations, and the entire picture of the function still remains elusive. To uncover as-yet unknown activity of Gal-9, we have been examining the effect of the protein in various disease animal models. Here we show that Gal-9 attenuated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mast cell stabilizing effect of Gal-9. In vitro studies of mast cell degranulation involving RBL-2H3 cells demonstrated that Gal-9 suppressed degranulation from the cells stimulated by IgE plus antigen and that the inhibitory effect was completely abrogated in the presence of lactose, indicating lectin activity of Gal-9 is critical. We found that Gal-9 strongly and specifically bound IgE, which is a heavily glycosylated immunoglobulin, and that the interaction prevented IgE-antigen complex formation, clarifying the mode of action of the anti-degranulation effect. Gal-9 is expressed by several mast cells including mouse mast cell line MC/9. The fact that immunological stimuli of MC/9 cells augmented Gal-9 secretion from the cells implies that Gal-9 is an autocrine regulator of mast cell function to suppress excessive degranulation. Collectively, these findings shed light on a novel function of Gal-9 in mast cells and suggest a beneficial utility of Gal-9 for the treatment of allergic disorders including asthma.

Fra1 is an immediate-early gene encoding a member of the AP-1 transcription factor family, which has diverse roles in development and oncogenesis. To determine the function of Fra1 in mouse development, the gene was inactivated by gene targeting. Foetuses lacking Fra1 were severely growth retarded and died between E10.0 and E10.5, owing to defects in extra-embryonic tissues. The placental labyrinth layer, where X-gal staining revealed expression of Fra1, was reduced in size and largely avascular, owing to a marked decrease in the number of vascular endothelial cells, as shown by the lack of Flk1 expression. In contrast, the spongiotrophoblast layer was unaffected and expressed the marker genes 4311 (Tpbp) and Flt1. Furthermore, mutant foetuses exhibited yolk-sac defects that may contribute to their growth retardation and lethality. Importantly, when the placental defect was rescued by injection of Fra1(-)(/)(-) ES cells into tetraploid wild-type blastocysts, Fra1(-)(/)(-) pups were obtained that were no longer growth retarded and survived up to 2 days after birth without apparent phenotypic defects. These data indicate that a defect in the extra-embryonic compartment is causal to the observed lethality, and suggest that Fra1 plays a crucial role in establishing normal vascularisation of the placenta.

The neuropeptide galanin mediates its effects through the receptor subtypes Gal(1), Gal(2), and Gal(3) and has been implicated in anxiety- and depression-related behaviors. Nevertheless, the receptor subtypes relevant to these behaviors are not known because of the lack of available galanin-selective ligands. In this article, we use behavioral, neurochemical, and electrophysiological approaches to investigate the anxiolytic- and antidepressant-like effects of two potent small-molecule, Gal(3)-selective antagonists, SNAP 37889 and the more soluble analog SNAP 398299. Acute administration of SNAP 37889 or SNAP 398299 enhanced rat social interaction. Furthermore, acute SNAP 37889 was also shown to reduce guinea pig vocalizations after maternal separation, to attenuate stress-induced hyperthermia in mice, to increase punished drinking in rats, and to decrease immobility and increase swimming time during forced swim tests with rats. Moreover, SNAP 37889 increased the social interaction time after 14 days of treatment and maintained its antidepressant effects during forced swim tests with rats after 21 days of treatment. In microdialysis studies, SNAP 37889 partially antagonized the galanin-evoked reduction in hippocampal serotonin (5-hydroxytryptamine, 5-HT), as did the 5-HT(1A) receptor antagonist WAY100635. Their combination produced a complete reversal of the effect of galanin. SNAP 398299 partially reversed the galanin-evoked inhibition of dorsal raphe cell firing and galanin-evoked hyperpolarizing currents. These results indicate that Gal(3)-selective antagonists produce anxiolytic- and antidepressant-like effects, possibly by attenuating the inhibitory influence of galanin on 5-HT transmission at the level of the dorsal raphe nucleus.