We have identified a novel nucleolar protein, Nop5p, that is essential for growth in Saccharomyces cerevisiae. Monoclonal antibodies B47 and 37C12 recognize Nop5p, which has a predicted size of 57 kDa and possesses a KKX repeat motif at its carboxyl terminus. Truncations that removed the KKX motif were functional and localized to the nucleolus, but conferred slow growth at 37 degreesC. Nop5p shows significant sequence homology with yeast Sik1p/Nop56p, and putative homologues in archaebacteria, plants, and human. Depletion of Nop5p in a GAL-NOP5 strain lengthened the doubling time about 5-fold, and selectively reduced steady-state levels of 40 S ribosomal subunits and 18 S rRNA relative to levels of free 60 S subunits and 25 S rRNA. Northern blotting and primer extension analyses showed that Nop5p depletion impairs processing of 35 S pre-rRNA at the A0 and A2 cleavage sites. Nop5p is associated with the small nucleolar RNAs U3, snR13, U14, and U18. Depletion of Nop5p caused the nucleolar protein Nop1p (yeast fibrillarin) to be localized to the nucleus and cytosol. Also, 37C12 co-immunoprecipitated Nop1p. These results suggest that Nop5p functions with Nop1p in the execution of early pre-rRNA processing steps that lead to formation of 18 S rRNA.

Embryonal carcinoma is a histologic subgroup of testicular germ cell tumors (TGCTs), and its cells may follow differentiation lineages in a manner similar to early embryogenesis. To acquire new knowledge about the transcriptional programs operating in this tumor development model, we used 22k oligo DNA microarrays to analyze normal and neoplastic tissue samples from human testis. Additionally, retinoic acid-induced in vitro differentiation was studied in relevant cell lines. We identified genes characterizing each of the known histologic subtypes, adding up to a total set of 687 differentially expressed genes. Among these, there was a significant overrepresentation of gene categories, such as genomic imprinting and gene transcripts associated to embryonic stem cells. Selection for genes highly expressed in the undifferentiated embryonal carcinomas resulted in the identification of 58 genes, including pluripotency markers, such as the homeobox genes NANOG and POU5F1 (OCT3/4), as well as GAL, DPPA4, and NALP7. Interestingly, abundant expression of several of the pluripotency genes was also detected in precursor lesions and seminomas. By use of tissue microarrays containing 510 clinical testicular samples, GAL and POU5F1 were up-regulated in TGCT also at the protein level and hence validated as diagnostic markers for undifferentiated tumor cells. The present study shows the unique gene expression profiles of each histologic subtype of TGCT from which we have identified deregulated components in selected processes operating in normal development, such as WNT signaling and DNA methylation.

Escherichia coli strains causing acute pyelonephritis often express multiple fimbrial types and haemolysin, which may contribute to their ability to adhere to, and interact with, kidney epithelial cells. Strain CFT073, a pap+, sfa+, pil+, hly+ pyelonephritis strain, previously established as virulent in the CBA mouse model of ascending urinary tract infection and cytotoxic for cultured human renal epithelial cells, was selected for construction of isogenic strains. From a gene bank of this strain, two distinct copies of the pap operon were isolated. The two P-fimbrial determinants were subcloned into pCVD442, a positive selection suicide vector containing the sacB gene of Bacillus subtilis. Deletion mutations were introduced into each of the two constructs, within papEFG of one operon and papDEFG of the other. Suicide vectors carrying pap deletions were mobilized from E. coli SM10 lambda pir into CFT073 (NalR) and cointegrates were passaged on non-selective medium. The first pap mutation was identified by screening a Southern blot of DNA from sucrose-resistant colonies using a papEFG probe. This mutant retained the MRHA+ phenotype since a second functional copy of pap was still present. A double pap-deletion mutant, UPEC76, confirmed by Southern blotting, was unable to agglutinate human type O erythrocytes or alpha Gal(1-4)beta Gal-coated latex beads. CBA mice (N = 100) were challenged transurethrally with 10(5), 10(6), 10(7), or 10(9) cfu of strains CFT073 or UPEC76. After one week, quantitative cultures of urine, bladder, and kidney were done and histologic changes were examined. No substantive differences in organism concentration or histological findings between parent and mutant were detected in urine, bladder, or kidney at any challenge concentration. We conclude that adherence by P fimbriae of uropathogenic E. coli strain CFT073 plays only a subtle role in the development of acute pyelonephritis in the CBA mouse model.

Developmental switching between life-cycle stages is a common feature among many pathogenic organisms. The protozoan parasite Entamoeba histolytica converts between cysts (essential for disease transmission) and trophozoites (responsible for tissue invasion). Identification of genes involved in the developmental pathway has been severely hindered by the inability to generate E. histolytica cysts in vitro. Using parasite strains derived from recent human infections and whole-genome transcriptional profiling, we determined that 1439 genes (approximately 15% of annotated genes) were potentially developmentally regulated. Genes enriched in cysts (672 in total) included cysteine proteinases and transmembrane protein kinases, which may be involved in signal transduction. Genes enriched in trophozoites (767 in total) included genes typically thought of as important in tissue invasion by trophozoites, including the Gal/GalNAc lectin light subunit and cysteine protease 1. Putative regulators of differentiation including possible G-protein coupled receptors, signal transduction proteins and transcription factors were identified. A number of E. histolytica stage-specific genes were also developmentally regulated in the reptilian parasite E. invadens, indicating that they likely have conserved functions in Entamoeba development. These advances lay the groundwork for dissection of the molecular signals that initiate stage conversion and development of novel diagnostic and therapeutic measures targeting E. histolytica cysts.

The localization of galanin (GAL) immunoreactive (IR) neuronal structures in the rat central nervous system has been investigated by using the indirect immunofluorescence technique. GAL-IR structures were seen in high concentrations in the hypothalamus, medulla oblongata, and spinal cord. Less extensive systems were detected in the telencephalon, thalamus, mesencephalon, and pons, while virtually no GAL-positive structures were seen in the olfactory bulb and cerebellum. Major populations of cell bodies staining for GAL-like material were seen in many areas. In the telencephalon somata were revealed in the bed nucleus of stria terminalis, in the nucleus of the diagonal band, medial septum, and in the medial aspects of the central amygdaloid nucleus, and in small numbers in cortical areas. The anterodorsal and periventricular nuclei of the thalamus contained positive cell bodies. In the hypothalamus GAL-IR somata were seen in the medial and lateral preoptic nuclei, arcuate nucleus, periventricular nucleus, in the dorsomedial nucleus, in the medial forebrain bundle area, in the tubular, caudal, accessory, supraoptic, and paraventricular magnocellular nuclei and lateral to the mammillary recess. The dorsal raphe nucleus hosted a large number of GAL-positive somata. Locus coeruleus of the pons contained a large number of GAL-IR perikarya. In the medulla oblongata positive somata were found in the caudal spinal trigeminal nucleus, the nucleus of the solitary tract, and in the ventral lateral area just rostral to area postrema. Small cell bodies were detected in the superficial layers of the dorsal horn of the spinal cord at all levels and in lamina X at lumbar levels. Analysis of GAL-positive fibers in the telencephalon revealed highly or medium-dense networks in the lateral septal nucleus, in the bed nucleus of stria terminalis, and in the central and medial amygdaloid nuclei. Positive fibers were found in the thalamus in and around the periventricular nucleus as well as in the lateral habenular nucleus and extending in a lateral, caudal direction from the third ventricle and fasciculus retroflexus to the lateral tip of the medial lemniscus. In the hypothalamus the external layer of the median eminence contained a very dense fiber network. Dense or medium-dense GAL-IR networks were detected in the periventricular nucleus, throughout the medial and lateral preoptic areas, in the medial forebrain bundle area, in the dorsomedial nucleus, and lateral to the mammillary recess. In the pons GAL-IR fibers were seen in the parabrachial nuclei, dorsal to the superior olive, and in the periaqueductal central gray.(ABSTRACT TRUNCATED AT 400 WORDS)

During the past two decades, the yeast Gal4 protein has been used as a model for studying transcriptional activation in eukaryotes. Many of the properties of transcriptional regulation first demonstrated for Gal4 have since been shown to be reiterated in the function of several other eukaryotic transcriptional regulators. Technological advances based on the transcriptional properties of this factor--such as the two-hybrid technology and Gal4-inducible systems for controlled gene expression--have had far-reaching influences in fields beyond transcription. In this review, we provide an updated account of Gal4 function, including data from new technologies that have been recently applied to the study of the GAL network.

In this communication, we demonstrate that galectin (Gal)-9 acts to constrain CD8(+) T cell immunity to Herpes Simplex Virus (HSV) infection. In support of this, we show that animals unable to produce Gal-9, because of gene knockout, develop acute and memory responses to HSV that are of greater magnitude and better quality than those that occur in normal infected animals. Interestingly, infusion of normal infected mice with alpha-lactose, the sugar that binds to the carbohydrate-binding domain of Gal-9 limiting its engagement of T cell immunoglobulin and mucin (TIM-3) receptors, also caused a more elevated and higher quality CD8(+) T cell response to HSV particularly in the acute phase. Such sugar treated infected mice also had expanded populations of effector as well as memory CD8(+) T cells. The increased effector T cell responses led to significantly more efficient virus control. The mechanisms responsible for the outcome of the Gal-9/TIM-3 interaction in normal infected mice involved direct inhibitory effects on TIM-3(+) CD8(+) T effector cells as well as the promotion of Foxp3(+) regulatory T cell activity. Our results indicate that manipulating galectin signals, as can be achieved using appropriate sugars, may represent a convenient and inexpensive approach to enhance acute and memory responses to a virus infection.

Cellular senescence, the irreversible loss of replicative capacity, might be a tumour suppressor and a contributor to age-related loss of tissue function. The absence of quantitative tests for reliability of candidate markers for senescent cells is a major drawback in cell population studies. Fibroblasts in culture constitute mixed populations of proliferation-competent and senescent cells, with transition between these with increasing population doublings (PD). We estimated senescent fraction in human and mouse fibroblasts with high precision from easily observed growth curves using a dynamic simulation model. We also determined senescent fractions, at various PD (over a wide range of senescent cell frequencies) using candidate senescence markers: Ki67, p21 (CDKN1A), γH2AX, SAHF and Sen-β-Gal either alone or in combination, and compared with those derived from growth curves. This comparison allowed ranking of candidate markers. High rankings were obtained for Sen-β-Gal, SAHFs and the combination of Ki67 negativity with high (>5 per nucleus) γH2A.X foci density in MRC5 fibroblasts. We demonstrate that this latter marker combination, which can easily be performed in paraffin-embedded tissue, gives quantitative senescent cell frequency estimates in mouse embryonic fibroblast cultures and in mouse intestinal sections. The technique presented is a framework for quantitative assessment of markers for senescence.

BACKGROUND

Enzyme replacement therapy (ERT) has been shown to enhance microvascular endothelial globotriaosylceramide clearance in the hearts of patients with Fabry disease. Whether these results can be translated into an improvement of myocardial function has yet to be demonstrated.

RESULTS

Sixteen patients with Fabry disease who were treated in an open-label study with 1.0 mg/kg body weight of recombinant alpha-Gal A (agalsidase beta, Fabrazyme) were followed up for 12 months. Myocardial function was quantified by ultrasonic strain rate imaging to assess radial and longitudinal myocardial deformation. End-diastolic thickness of the left ventricular posterior wall and myocardial mass (assessed by magnetic resonance imaging, n=10) was measured at baseline and after 12 months of ERT. Data were compared with 16 age-matched healthy controls. At baseline, both peak systolic strain rate and systolic strain were significantly reduced in the radial and longitudinal direction in patients compared with controls. Peak systolic strain rate increased significantly in the posterior wall (radial function) after one year of treatment (baseline, 2.8+/-0.2 s(-1); 12 months, 3.7+/-0.3 s(-1); P<0.05). In addition, end-systolic strain of the posterior wall increased significantly (baseline, 34+/-3%; 12 months, 45+/-4%; P<0.05). This enhancement in radial function was accompanied by an improvement in longitudinal function. End-diastolic thickness of the posterior wall decreased significantly after 12 months of treatment (baseline, 13.8+/-0.6 mm; 12 months, 11.8+/-0.6 mm; P<0.05). In parallel, myocardial mass decreased significantly from 201+/-18 to 180+/-21 g (P<0.05).

CONCLUSIONS

These results suggest that ERT can decrease left ventricular hypertrophy and improve regional myocardial function.

This report highlights the advantages of low-affinity, multivalent interactions to recognize one cell type over another. Our goal was to devise a strategy to mediate selective killing of tumor cells, which are often distinguished from normal cells by their higher levels of particular cell surface receptors. To test whether multivalent interactions could lead to highly specific cell targeting, we used a chemically synthesized small-molecule ligand composed of two distinct motifs: (1) an Arg-Gly-Asp (RGD) peptidomimetic that binds tightly (Kd approximately 10(-9)M) to alphavbeta3 integrins and (2) the galactosyl-alpha(1-3)galactose (alpha-Gal epitope), which is recognized by human anti-alpha-galactosyl antibodies (anti-Gal). Importantly, anti-Gal binding requires a multivalent presentation of carbohydrate residues; anti-Gal antibodies interact weakly with the monovalent oligosaccharide (Kd approximately 10(-5)M) but bind tightly (Kd approximately 10(-11) M) to multivalent displays of alpha-Gal epitopes. Such a display is generated when the bifunctional conjugate decorates a cell possessing a high level of alphavbeta3 integrin; the resulting cell surface, which presents many alpha-Gal epitopes, can recruit anti-Gal, thereby triggering complement-mediated lysis. Only those cells with high levels of the integrin receptor are killed. In contrast, doxorubicin tethered to the RGD-based ligand affords indiscriminate cell death. These results highlight the advantages of exploiting the type of the multivalent recognition processes used by physiological systems to discriminate between cells. The selectivity of this strategy is superior to traditional, abiotic, high-affinity targeting methods. Our results have implications for the treatment of cancer and other diseases characterized by the presence of deleterious cells.

Polarization of the microtubule cytoskeleton is an early event in establishment of anterior-posterior polarity for the Drosophila oocyte. During stages 8-9 of oogenesis, when oskar mRNA is transported to the posterior pole of the oocyte, a fusion protein consisting of the plus-end-directed microtubule motor kinesin and beta-galactosidase (Kin:beta gal) similarly localizes to the posterior pole, thereby suggesting that plus ends of microtubules are pointed to the posterior. In this paper, we have substituted the motor domain of Kin:beta gal with the putative motor domain (head) from the kinesin-related protein Nod. In cells with defined microtubule polarity, the Nod:beta gal fusion protein is an in vivo minus-end reporter for microtubules. Nod:beta gal localizes to apical cytoplasm in epithelial cells and to the poles of mitotic spindles in dividing cells. In stage 8-10 oocytes, the Nod fusion localizes to the anterior margin, thus supporting the hypothesis that minus ends of microtubules at these stages are primarily at the anterior margin of the oocyte. The fusion protein also suggests a polarity to the microtubule cytoskeleton of dendrites and muscle fibers, as it accumulates at the ends of dendrites in the embryonic PNS and is excluded from terminal cytoplasm in embryonic muscle. Finally, the reciprocal in vivo localization of Nod:beta gal and Kin:beta gal suggests that the head of Nod may be a minus-end-directed motor.

PADGEM (platelet activation-dependent granule-external membrane protein) is a leukocyte receptor of activated platelets that mediates cellular adhesion of platelets to neutrophils and monocytes. To identify the natural ligand on neutrophils and monocytes that interacts with PADGEM, we have evaluated anti-leukocyte antibodies for their ability to block leukocyte-PADGEM binding. Only anti-CD15 antibodies were able to inhibit the binding of neutrophils, monocytes, HL60 cells, and U937 cells to platelets. Anti-CD15 antibodies inhibited the binding of U937 cells to PADGEM-expressing COS cells and to purified PADGEM incorporated into phospholipid vesicles. The CD15 antigen, lacto-N-fucopentaose III (Gal beta 1----4[Fuc alpha 1----3]NAcGlc beta 1----3Gal-beta 1----4Glc), inhibited the interaction of neutrophils or HL60 cells with platelets, whereas lacto-N-fucopentaose I did not; lacto-N-fucopentaose II demonstrated minimal inhibition. Lacto-N-fucopentaose III, and to a lesser extent lacto-N-fucopentaose II, but not lacto-N-fucopentaose I, inhibited the interaction of HL60 cells with COS cells transfected with PADGEM cDNA. CD15, lacto-N-fucopentaose III or Lex, is a component of the PADGEM ligand on neutrophils and monocytes.

It has previously been observed that Escherichia coli lon mutations increase the levels of enzymes involved in the synthesis of colanic acid capsular polysaccharide (A. Markovitz, p. 415-462, in I. Sutherland, ed., Surface Carbohydrates of the Prokaryotic Cell, 1977). To determine how lon regulates these enzymes, we have isolated, mapped, and characterized lac operon and lac protein fusions to genes necessary for capsule synthesis by the Mu d(lac Amp) in vivo fusion technique of Casadaban and Cohen (M. J. Casadaban and S. N. Cohen, Proc. Natl. Acad. Sci. U.S.A. 76:4530-4533, 1979). At least five genes have been identified which share a common pattern of regulation: they are transcribed at low levels in lon+ strains and at significantly higher levels in lon strains. These genes are located in a cluster close to udk at 45 min on the E. coli map; we have named these genes cpsA, B, C, D, and E. An additional locus, cpsF, located at 90 min, is regulated in a similar manner to cpsA to E but is not essential for colanic acid synthesis. Similar studies on the transcriptional regulation of fusions in the gal and manA operons, also necessary for colanic acid synthesis, do not show significant regulation by the lon locus. Therefore, the regulatory system described here does not extend to all genes in the colanic acid synthesis pathway.

Human influenza viruses are more efficiently isolated by inoculating patient samples into the amniotic rather than the allantoic cavity of embryonated chicken eggs. This type of cultivation selects virus variants with mutations around the hemagglutinin (HA) receptor binding site. To understand the molecular basis of these phenomena, we investigated the abundances of sialic acid (SA) linked to galactose (Gal) by the alpha-2,3 linkage (SA alpha2,3Gal) and SA alpha2,6Gal in egg amniotic and allantoic cells and in Madin-Darby canine kidney (MDCK) cells. Using SA-Gal linkage-specific lectins (Maackia amurensis agglutinin specific for SA alpha2,6Gal and Sambucus nigra agglutinin specific for SA alpha2,3Gal), we found SA alpha2,3Gal in both allantoic and amniotic cells and SA alpha2,6Gal in only the amniotic cells. MDCK cells contained both linkages. To investigate how this difference in abundances of SA alpha2,3Gal and SA alpha2,6Gal in allantoic and amniotic cells affects the appearance of host cell variants in eggs, we determined the receptor specificities and HA amino acid sequences of two different patient viruses which were isolated and passaged in the amnion or in the allantois and which were compared with MDCK cell-grown viruses. We found that the viruses maintained high SA alpha2,6Gal specificities when grown in MDCK cells or following up to two amniotic passages; however, further passages in either the amnion or allantois resulted in the acquisition of, or a complete shift to, SA alpha2,3Gal specificity, depending on the virus strain. This change in receptor specificity was accompanied by the appearance of variants in the population with Leu-to-Gln mutations at position 226 in their HA. These findings suggest that lack of SA alpha2,6Gal linkages in the allantois of chicken eggs is a selective pressure for the appearance of host cell variants with altered receptor specificities and amino acid changes at position 226.

Escherichia coli strains bind to Gal alpha 1-4Gal-containing glycolipids via P pili-associated G-adhesins. Three functional classes of adhesins with different binding specificities are encoded by conserved G-alleles. We suggest that the Class I papG-allele of strain J96 is a novel acquisition possibly introduced via horizontal gene transfer into one of the two P pili gene clusters carried by this strain. Closely related strains in the ECOR collection of natural E. coli isolates carry either a Class II or a Class III G-adhesin. Data indicate that genetic exchanges involving either entire pap or prs gene clusters or individual pap/prs genes have occurred. We propose that the retention and spread of pap/prs DNA among E. coli is the result of selection pressure exerted by mammalian intestinal isoreceptors.

Ischemic stroke is caused by the interruption of cerebral blood flow that leads to brain damage with long-term sensorimotor deficits. Stem cell transplantation may recover functional deficit by replacing damaged brain. In this study, we attempted to test whether the human neural stem cells (NSCs) can improve the outcome in the rat brain with intravenous injection and also determine the migration, differentiation and the long-term viabilities of human NSCs in the rat brain. Focal cerebral ischemia was induced by intraluminal thread occlusion of middle cerebral artery (MCA). One day after surgery, the rats were randomly divided into two groups: NSCs-ischemia vs. Ischemia-only. Human NSCs infected with retroviral vector encoding beta galactosidase were intravenously injected in NSCs-ischemia group (5 x 10(6) cells) and the same amount of saline was injected in Ischemia-only group for control. The animals were evaluated for 4 weeks using turning in an alley (TIA) test, modified limb placing test (MLPT) and rotarod test. Transplanted cells were detected by X gal cytohistochemistry or beta gal immunohistochemistry with double labeling of other cell markers. The NSCs-ischemia group showed better performance on TIA test at 2 weeks, and MLPT and rotarod test from 3 weeks after ischemia compared with the Ischemia-only group. Human NSCs were detected in the lesion side and labeled with marker for neurons or astrocytes. Postischemic hemispheric atrophy was noted but reduced in NSCs-ischemia group. X gal+ cells were detected in the rat brain as long as 540 days after transplantation. Our data suggest intravenously transplanted human NSCs can migrate and differentiate in the rat brain with focal ischemia and improve functional recovery.

OBJECTIVE

Aldosterone (Aldo) is involved in arterial stiffness and heart failure, but the mechanisms have remained unclear. Galectin-3 (Gal-3), a β-galactoside-binding lectin, plays an important role in inflammation, fibrosis, and heart failure. We investigated here whether Gal-3 is involved in Aldo-induced vascular fibrosis.

RESULTS

In rat vascular smooth muscle cells Gal-3 overexpression enhanced specifically collagen type I synthesis. Moreover Gal-3 inhibition by modified citrus pectin or small interfering RNA blocked Aldo-induced collagen type I synthesis. Rats were treated with Aldo-salt combined with spironolactone or modified citrus pectin for 3 weeks. Hypertensive Aldo-treated rats presented vascular hypertrophy, inflammation, fibrosis, and increased aortic Gal-3 expression. Spironolactone or modified citrus pectin treatment reversed all the above effects. Wild-type and Gal-3 knock-out mice were treated with Aldo for 6 hours or 3 weeks. Aldo increased aortic Gal-3 expression, inflammation, and collagen type I in wild-type mice at both the short- and the long-term, whereas no changes occurred in Gal-3 knock-out mice.

CONCLUSIONS

Our data indicate that Gal-3 is required for inflammatory and fibrotic responses to Aldo in vascular smooth muscle cells in vitro and in vivo, suggesting a key role for Gal-3 in vascular fibrosis.

METHODS

Exosomes, small vesicles participating in intercellular communication, have been extensively studied recently; however, the role of edible plant derived exosomes in interspecies communication has not been investigated. Here, we investigate the biological effects of edible plant derived exosome-like nanoparticles (EPDENs) on mammalian cells.

RESULTS

In this study, exosome-like nanoparticles from four edible plants were isolated and characterized. We show that these EPDENs contain proteins, lipids, and microRNA. EPDENs are taken up by intestinal macrophages and stem cells. The results generated from EPDEN-transfected macrophages indicate that ginger EPDENs preferentially induce the expression of the antioxidation gene, heme oxygenase-1 and the anti-inflammatory cytokine, IL-10; whereas grapefruit, ginger, and carrot EPDENs promote activation of nuclear factor like (erythroid-derived 2). Furthermore, analysis of the intestines of canonical Wnt-reporter mice, i.e. B6.Cg-Tg(BAT-lacZ)3Picc/J mice, revealed that the numbers of β-galactosidase(+) (β-Gal) intestinal crypts are increased, suggesting that EPDEN treatment of mice leads to Wnt-mediated activation of the TCF4 transcription machinery in the crypts.

CONCLUSIONS

The data suggest a role for EPDEN-mediated interspecies communication by inducing expression of genes for anti-inflammation cytokines, antioxidation, and activation of Wnt signaling, which are crucial for maintaining intestinal homeostasis.

OBJECTIVE

Vascular endothelial growth factor (VEGF) plays an important role in inducing angiogenesis. Mesenchymal stem cells (MSCs) may have potential for differentiation to several types of cells, including myocytes. We hypothesized that transplantation of VEGF-expressing MSCs could effectively treat acute myocardial infarction (MI) by providing enhanced cardioprotection, followed by angiogenic effects in salvaging ischemic myocardium.

RESULTS

The human VEGF165 gene was transfected to cultured MSCs of Lewis rats using an adenoviral vector. Six million VEGF-transfected and LacZ-transfected MSCs (VEGF group), LacZ-transfected MSCs (control group), or serum-free medium only (medium group) were injected into syngeneic rat hearts 1 hour after left coronary artery occlusion. At 1 week after MI, MSCs were detected by X-gal staining in infarcted region. High expression of VEGF was immunostained in the VEGF group. At 28 days after MI, infarct size, left ventricular dimensions, ejection fraction, E wave/A wave ratio and capillary density of the infarcted region were most improved in the VEGF group, compared with the medium group. Immunohistochemically, alpha-smooth muscle actin-positive cells were most increased in the VEGF group.

CONCLUSIONS

This combined strategy of cell transplantation with gene therapy could be a useful therapy for the treatment of acute MI.

Phosphorylation of the yeast transcription factor <em>GAL</em>4 at S699 is required for efficient galactose-inducible transcription. We demonstrate that this site is a substrate for the RNA polymerase holoenzyme-associated CDK SRB10. S699 phosphorylation requires SRB10 in vivo, and this site is phosphorylated by purified SRB10/ SRB11 CDK/cyclin in vitro. RNA Pol II holoenzymes purified from WT yeast phosphorylate <em>GAL</em>4 at sites observed in vivo whereas holoenzymes from srb10 yeast are incapable of phosphorylating <em>GAL</em>4 at S699. Mutations at <em>GAL</em>4 S699 and srb10 are epistatic for <em>GAL</em> induction, demonstrating that SRB10 regulates <em>GAL</em>4 activity through this phosphorylation in vivo. These results demonstrate a function for the SRB10/ CDK8 holoenzyme-associated CDK that involves regulation of transactivators by phosphorylation during transcriptional activation.

BACKGROUND

A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid alpha2,3-galactose (SAalpha2,3-Gal) linked receptors, whereas human strains bind to sialic acid alpha2,6-galactose (SAalpha2,6-Gal) linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAalpha2,3-GalG(1-3)GalNAc and SAalpha2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II) and Sambucus nigra agglutinin (SNA) respectively.

RESULTS

Both SAalpha2,3-Gal and SAalpha2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon). Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors from duodenum to colon in the pig.

CONCLUSIONS

The extensive presence of SAalpha2,3-Gal and SAalpha2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors in the pig intestines highlights a need for clarification on the presence of SA receptors in the human intestinal tract.

The human JC polyomavirus (JCV) causes a fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML), in immunocompromised individuals. Current treatment options for PML are inadequate. Sialylated oligosaccharides and the serotonin receptor are known to be necessary for JCV entry, but the molecular interactions underlying JCV attachment remain unknown. Using glycan array screening and viral infectivity assays, we identify a linear sialylated pentasaccharide with the sequence NeuNAc-α2,6-Gal-β1,4-GlcNAc-β1,3-Gal-β1,4-Glc (LSTc) present on host glycoproteins and glycolipids as a specific JCV recognition motif. The crystal structure of the JCV capsid protein VP1 was solved alone and in complex with LSTc. It reveals extensive interactions with the terminal sialic acid of the LSTc motif and specific recognition of an extended conformation of LSTc. Mutations in the JCV oligosaccharide-binding sites abolish cell attachment, viral spread, and infectivity, further validating the importance of this interaction. Our findings provide a powerful platform for the development of antiviral compounds.

In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, <em>GAL</em>4, <em>GAL</em>80, and <em>GAL</em>3. We have isolated the <em>GAL</em>80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL1 and <em>GAL</em> cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type <em>GAL</em>80 allele with an in vitro-created <em>GAL</em>80 deletion-disruption mutation. Enzyme activities and RNA levels for the <em>GAL</em> cluster and MEL1 genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the <em>GAL</em> cluster and MEL1 genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the <em>GAL</em>80 gene in a gal4 cell does not restore <em>GAL</em> cluster and MEL1 gene expression. The data demonstrate that (i) the <em>GAL</em>80 protein is a purely negative regulator, (ii) the <em>GAL</em>80 protein does not mediate carbon catabolite repression, and (iii) the <em>GAL</em>4 protein is not simply an antagonizer of <em>GAL</em>80-mediated repression.

NMU2, a nondifferentiating mutant myogenic cell line, gives rise to rhabdomyosarcomas in mice. We show that constitutive expression of RNA from 0.2 kb of the alpha-tropomyosin (Tm) 3' untranslated region (UTR), but not control 3'UTRs, suppresses anchorage-independent growth and tumor formation by NMU2 cells. When beta-galactosidase (beta-gal)-labeled cells were implanted into muscles of adult mouse hindlimbs, Tm 3'UTR expression suppressed the proliferation, invasion, and destruction of muscle tissues characteristic of NMU2. In the rare tumors that developed from Tm 3'UTR transfectants, RNA expression was extinguished. These results suggest that suppression of tumorigenicity is dependent on the continued expression of Tm transcripts lacking a coding region. We conclude that untranslated RNAs can function as regulators (riboregulators) that suppress tumor formation.