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At least 919 records · Page 51Linked to original sources
PubMed1979

Mechanisms of Listeria monocytogenes-mediated modulation of tumor growth.

Studies using Listeria monocytogenes as an antitumor agent were initiated to determine the requirements for Listeria-mediated tumor inhibition to occur. When Strain 13 guinea pigs were injected with an admixture of viable Listeria and a methylcholanthrene-induced fibrosarcoma in a ratio of 1 bacterium to 100 tumor cells, Listeria had a marked capacity to inhibit tumor growth. This confirms an earlier study in our laboratory (M. M. Dustoor, A. Fulton, W. Croft, and A. A. Blazkovec, Infect. Immun. 23:54-60, 1979). At no time did animals exhibit overt symptoms of disease as a result of Listeria infection. Animals treated with antilymphocyte serum, which had previously been shown to abrogate T-cell functions, were no longer able to suppress Listeria-tumor cell mixtures. Treatment in vivo with carrageenan, a macrophage-inhibitory agent, also abrogated Listeria-mediated tumor inhibition. These results suggest that Listeria-mediated inhibition requires intact T-lymphocyte and macrophage function. Experiments in which Listeria was given in admixture with the tumor cells or in the opposite flank demonstrated that the antitumor effects require intimate association of the Listeria and tumor cells. Histopathological studies, showing that macrophages and lymphocytes are the predominant inflammatory cells present at sites of tumor destruction, further suggest a role for these cells in Listeria-mediated inhibition. Animals which had rejected prior Listeria-tumor cell inocula were resistant to rechallenge with the homologous tumor for more than 1 year. This work thus confirms in vitro studies demonstrating that both lymphocytes and macrophages are required for Listeria-mediated tumor inhibition to occur. This study demonstrates that viable Listeria can have potent antitumor effects without causing overt disease as a result of Listeria infection.

Animals↗
PubMed2005

Gene silencing and overexpression of porcine peptidoglycan recognition protein long isoforms: involvement in beta-defensin-1 expression.

Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased by Salmonella infection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 have N-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide beta-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged with Listeria monocytogenes results in downregulation of beta-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of beta-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.

Amino Acid Sequence↗
PubMed1998

DdlN from vancomycin-producing Amycolatopsis orientalis C329.2 is a VanA homologue with D-alanyl-D-lactate ligase activity.

Vancomycin-resistant enterococci acquire high-level resistance to glycopeptide antibiotics through the synthesis of peptidoglycan terminating in D-alanyl-D-lactate. A key enzyme in this process is a D-alanyl-D-alanine ligase homologue, VanA or VanB, which preferentially catalyzes the synthesis of the depsipeptide D-alanyl-D-lactate. We report the overexpression, purification, and enzymatic characterization of DdlN, a VanA and VanB homologue encoded by a gene of the vancomycin-producing organism Amycolatopsis orientalis C329.2. Evaluation of kinetic parameters for the synthesis of peptides and depsipeptides revealed a close relationship between VanA and DdlN in that depsipeptide formation was kinetically preferred at physiologic pH; however, the DdlN enzyme demonstrated a narrower substrate specificity and commensurately increased affinity for D-lactate in the C-terminal position over VanA. The results of these functional experiments also reinforce the results of previous studies that demonstrated that glycopeptide resistance enzymes from glycopeptide-producing bacteria are potential sources of resistance enzymes in clinically relevant bacteria.

Actinomycetaceae↗
PubMed1995

Host cellular annexin II is associated with cytomegalovirus particles isolated from cultured human fibroblasts.

A significant amount of host cellular annexin II was found to be associated with human cytomegalovirus isolated from cultured human fibroblasts (approximately 1,160 molecules per virion). This composition was established by four different analytical approaches that included (i) Western blot (immunoblot) analysis of gradient-purified virions with a monoclonal antibody specific for annexin II, (ii) peptide mapping and sequence analysis of virus-associated proteins and proteins dissociated from virus following EDTA treatment, (iii) electron microscopic immunocytochemistry of gradient-purified virions, and (iv) labeling of virus-associated proteins by lactoperoxidase-catalyzed radioiodination. These results indicated that annexin II was primarily localized to the viral surface, where it bound in a divalent cation-dependent manner. In functional experiments, a rabbit antiserum raised against annexin II inhibited cytomegalovirus plaque formation in human foreskin fibroblast monolayers in a concentration-dependent manner. Cumulatively, these studies demonstrate an association of host annexin II with cytomegalovirus particles and provide evidence for the involvement of this cellular protein in virus infectivity.

Animals↗
PubMed1998

An endoplasmic reticulum-targeting signal sequence enhances the immunogenicity of an immunorecessive simian virus 40 large T antigen cytotoxic T-lymphocyte epitope.

An immunological hierarchy among three H-2Db-restricted cytotoxic T lymphocyte (CTL) determinants in simian virus 40 (SV40) large T antigen (Tag) was described previously: determinants I and II/III are immunodominant, whereas determinant V is immunorecessive. To assess the immunogenicity of each determinant individually and define mechanisms that contribute to the immunorecessive nature of determinant V, we constructed a panel of recombinant vaccinia viruses (rVVs) expressing minigenes encoding these determinants in various polypeptide contexts. We found the following. (i) Immunization of mice with an rVV encoding full-length SV40 Tag resulted in priming for CTL responses to determinants I and II/III but not determinant V. (ii) rVVs encoding peptide I or II/III in the cytosol or targeted to the endoplasmic reticulum (ER) were highly antigenic and immunogenic. (iii) rVVs encoding peptide V minigenes were antigenic and immunogenic if the peptide was targeted to the ER, expressed in the cytosol with short flanking sequences, or expressed from within a self-protein, murine dihydrofolate reductase. (iv) Presentation of the nonflanked peptide V (preceded by a Met codon only) could be enhanced by using a potent inhibitor of the proteasome. (v) H-2Db-epitope V peptide complexes decayed more rapidly than complexes containing epitope I or II/III peptides. In brefeldin A blocking experiments, functional epitope V complexes were detected longer on targets expressing ER-targeted epitope V than on targets expressing forms of epitope V dependent on the transporter associated with antigen processing. Therefore, limited formation of relatively unstable cell surface H-2Db complexes most likely contributes to the immunorecessive nature of epitope V within SV40 Tag. Increasing the delivery of epitope V peptide to the major histocompatibility complex class I presentation pathway by ER targeting dramatically enhanced the immunogenicity of epitope V.

Animals↗
PubMed2000

Lentivirus vector gene expression during ES cell-derived hematopoietic development in vitro.

The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.

Animals↗
PubMed1995

The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1.

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

Anaerobiosis↗
PubMed1999

ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.

The transcription of genes encoding proteins involved in the hepatic synthesis of lipids from glucose is strongly stimulated by carbohydrate feeding. It is now well established that in the liver, glucose is the main activator of the expression of this group of genes, with insulin having only a permissive role. While ADD1/SREBP-1 has been implicated in lipogenic gene expression through temporal association with food intake and ectopic gain-of-function experiments, no genetic evidence for a requirement for this factor in glucose-mediated gene expression has been established. We show here that the transcription of ADD1/SREBP-1c in primary cultures of hepatocytes is controlled positively by insulin and negatively by glucagon and cyclic AMP, establishing a link between this transcription factor and carbohydrate availability. Using adenovirus-mediated transfection of a powerful dominant negative form of ADD1/SREBP-1c in rat hepatocytes, we demonstrate that this factor is absolutely necessary for the stimulation by glucose of L-pyruvate kinase, fatty acid synthase, S14, and acetyl coenzyme A carboxylase gene expression. These results demonstrate that ADD1/SREBP-1c plays a crucial role in mediating the expression of lipogenic genes induced by glucose and insulin.

Adenoviridae↗
PubMed2000

Potentiation of GATA-2 activity through interactions with the promyelocytic leukemia protein (PML) and the t(15;17)-generated PML-retinoic acid receptor alpha oncoprotein.

The hematopoietically expressed GATA family of transcription factors function as key regulators of blood cell fate. Among these, GATA-2 is implicated in the survival and growth of multipotential progenitors. Here we report that the promyelocytic leukemia protein (PML) can complex with GATA-2 and potentiate its transactivation capacity. The binding is mediated through interaction of the zinc finger region of GATA-2 and the B-box domain of PML. The B-box region of PML is retained in the PML-RARalpha (retinoic acid receptor alpha) fusion protein generated by the t(15;17) translocation characteristic of acute promyelocytic leukemia (APL). Consistent with this, we provide evidence that GATA-2 can physically associate with PML-RARalpha. Functional experiments further demonstrated that this interaction has the capacity to render GATA-dependent transcription inducible by retinoic acid, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.

Animals↗
PubMed2002

Generation of mice deficient for macrophage galactose- and N-acetylgalactosamine-specific lectin: limited role in lymphoid and erythroid homeostasis and evidence for multiple lectins.

Macrophage receptors function in pattern recognition for the induction of innate immunity, in cellular communication to mediate the regulation of adaptive immune responses, and in the clearance of some glycosylated cells or glycoproteins from the circulation. They also function in homeostasis by initiating the engulfment of apoptotic cells. Evidence has suggested that macrophage receptors function to recognize cells that are destined for programmed cell death but not yet overtly apoptotic. We have examined the function of a macrophage receptor specific for unsialylated glycoproteins, known as the mouse macrophage galactose- and N-acetylgalactosamine-specific lectin (mMGL) (Ii et al., J. Biol. Chem. 265:11295-11298, 1990; Sato et al., J. Biochem. [Tokyo] 111:331-336, 1992; Yamamoto et al., Biochemistry 33:8159-8166, 1994). With targeted disruption, we tested whether mMGL is necessary for macrophage function, controlled thymic development, the loss of activated CD8 T cells, and the turnover of red blood cells. Evidence indicates that mMGL may play a nonessential role in several of these macrophage functions. Experiments are presented that indicate the existence of another galactose- and N-acetylgalactosamine-recognizing lectin distinct from mMGL. This may explain the absence of a strong phenotype in mMGL-deficient mice.

Animals↗
PubMed2002

Normal p53 function in primary cells deficient for Siah genes.

Overexpression studies have suggested that Siah1 proteins may act as effectors of p53-mediated cellular responses and as regulators of mitotic progression. We have tested these hypotheses using Siah gene knockout mice. Siah1a and Siah1b were not induced by activation of endogenous p53 in tissues, primary murine embryonic fibroblasts (MEFs) or thymocytes. Furthermore, primary MEFs lacking Siah1a, Siah1b, Siah2, or both Siah2 and Siah1a displayed normal cell cycle progression, proliferation, p53-mediated senescence, and G(1) phase cell cycle arrest. Primary thymocytes deficient for Siah1a, Siah2, or both Siah2 and Siah1a, E1A-transformed MEFs lacking Siah1a, Siah1b, or Siah2, and Siah1b-null ES cells all underwent normal p53-mediated apoptosis. Finally, inhibition of Siah1b expression in Siah2 Siah1a double-mutant cells failed to inhibit cell division, p53-mediated induction of p21 expression, or cell cycle arrest. Our loss-of-function experiments do not support a general role for Siah genes in p53-mediated responses or mitosis.

Animals↗
PubMed2003

Progesterone and glucocorticoid receptors recruit distinct coactivator complexes and promote distinct patterns of local chromatin modification.

It is well established that steroid receptor function requires interaction with coactivators. However, the mechanisms through which steroid receptors elicit precise assembly of coactivator complexes and the way the steroid activation signal is transduced remain elusive. Using a T47D cell line stably integrated with a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter, we demonstrate that specific steroid receptors exhibit preferential recruitment of SRC-1 family coactivators, which determines the subsequent recruitment of specific downstream coregulator molecules. Upon ligand treatment, progesterone receptor (PR) interacted preferentially with SRC-1, which recruited CBP and significantly enhanced acetylation at K5 of histone H4. In contrast, activated glucocorticoid receptor (GR) preferentially associated with SRC-2 (TIF-2/GRIP-1), which subsequently recruited pCAF and led to specific modification of histone H3, suggesting that specific coactivators recruit distinct histone acetyltransferases to modulate the transcription of steroid-responsive genes. Loss-of-function experiments further support the predicted roles of SRC-1 and SRC-2 in, respectively, PR- and GR-mediated transcription on the MMTV promoter. This study indicates that differential recruitment of coactivators by nuclear receptors determines the assembly of coactivator complexes on target promoters to mediate specific transcription signals.

Animals↗
PubMed1995

Subjective perception of additional support requirements of elderly patients discharged from accident and emergency departments.

Fifty per cent of elderly patients discharged from accident and emergency (A&E) departments experience functional deterioration and increased dependence; 5.6% require readmission within 14 days. Discharge is often to inadequate community support. Functional assessment by A&E department staff may identify patients at greatest risk. The predictive ability of 25 patients aged 75 years or over to identify their additional support requirements following discharge from the A&E department was evaluated retrospectively using interview questionnaires 10-20 days after discharge. A total of 23 patients, of mean age 79.3 years, were interviewed. Six patients expressed concern both about coping at home and about needing extra support; four of the nine patients who recruited additional help were in this group (P = 0.239; n.s.). There was no correlation between additional support needed and patients living alone (P = 0.302; n.s.), dependent relative (P = 0.325; n.s.) or existing domiciliary support (P = 0.197; n.s.). All patients were satisfied with their management, and of the six who expressed concern about being able to cope at home, none informed A&E staff. Patients' perception is an unreliable indicator of their need for additional help and their ability to manage at home after discharge from A&E departments. Additional support requirements appear to be unrelated to domestic circumstances.

Aftercare↗
PubMed1988

Gene duplication in haloaromatic degradative plasmids pJP4 and pJP2.

pJP2 and pJP4 are 2,4-dichlorophenoxyacetic acid catabolic plasmids, and they show DNA sequence homology. Most of the pJP2 molecules (80% or more) isolated from 2,4-dichlorophenoxyacetic acid grown cells of Alcaligenes eutrophus harbor a tandem duplication of a 25-kilobase (kb) segment encoding the catabolic functions. Unlike plasmid pJP4, pJP2 in A. eutrophus gives rise to a 3-chlorobenzoate phenotype without further genetic rearrangement. pJP4 under 3-chlorobenzoate selection contains an inverted duplication of 24.5 kb. Absence of selective pressure results in the prompt loss of one copy of the duplication in pJP4, but not of the tandem duplication in pJP2. In both pJP4 and pJP2, mutation of the duplicated copy, rather than gene dosage, is likely to be the basis of phenotypic change of catabolic functions. Experiments using the cloned DNA suggest that a tandem duplication is more stable than an inverted duplication.

Alcaligenes↗
PubMed2004

Genetics of atherosclerosis.

Atherosclerosis, the primary cause of coronary artery disease (CAD) and stroke, is a disorder with multiple genetic and environmental contributions. Genetic-epidemiologic studies have identified a surprisingly long list of genetic and nongenetic risk factors for CAD. However, such studies indicate that family history is the most significant independent risk factor (15, 52, 77). Many Mendelian disorders associated with atherosclerosis, such as familial hypercholesterolemia (FH), have been characterized, but they explain only a small percentage of disease susceptibility (although a substantial fraction of early CAD). Most cases of myocardial infarction (MI) and stroke result from the interactions of multiple genetic and environmental factors, none of which can cause disease by itself. Successful discovery of these genetic factors will require using complementary approaches with animal models, large-scale human genetic studies, and functional experiments. This review emphasizes the common, complex forms of CAD.

Arteriosclerosis↗
PubMed1998

Brain plasticity and behavior.

Brain plasticity refers to the brain's ability to change structure and function. Experience is a major stimulant of brain plasticity in animal species as diverse as insects and humans. It is now clear that experience produces multiple, dissociable changes in the brain including increases in dendritic length, increases (or decreases) in spine density, synapse formation, increased glial activity, and altered metabolic activity. These anatomical changes are correlated with behavioral differences between subjects with and without the changes. Experience-dependent changes in neurons are affected by various factors including aging, gonadal hormones, trophic factors, stress, and brain pathology. We discuss the important role that changes in dendritic arborization play in brain plasticity and behavior, and we consider these changes in the context of changing intrinsic circuitry of the cortex in processes such as learning.

Animals↗
PubMed2005

Association between Hsp90 and the ClC-2 chloride channel upregulates channel function.

The voltage-dependent ClC-2 chloride channel has been implicated in a variety of physiological functions, including fluid transport across specific epithelia. ClC-2 is activated by hyperpolarization, weakly acidic external pH, intracellular Cl-, and cell swelling. To add more insight into the mechanisms involved in ClC-2 regulation, we searched for associated proteins that may influence ClC-2 activity. With the use of immunoprecipitation of ClC-2 from human embryonic kidney-293 cells stably expressing the channel, followed by electrophoretic separation of coimmunoprecipitated proteins and mass spectrometry identification, Hsp70 and Hsp90 were unmasked as possible ClC-2 interacting partners. Association of Hsp90 with ClC-2 was confirmed in mouse brain. Inhibition of Hsp90 by two specific inhibitors, geldanamycin or radicicol, did not affect total amounts of ClC-2 but did reduce plasma membrane channel abundance. Functional experiments using the whole cell configuration of the patch-clamp technique showed that inhibition of Hsp90 reduced ClC-2 current amplitude and impaired the intracellular Cl- concentration [Cl-]-dependent rightward shift of the fractional conductance. Geldanamycin and radicicol increased both the slow and fast activation time constants in a chloride-dependent manner. Heat shock treatment had the opposite effect. These results indicate that association of Hsp90 with ClC-2 results in greater channel activity due to increased cell surface channel expression, facilitation of channel opening, and enhanced channel sensitivity to intracellular [Cl-]. This association may have important pathophysiological consequences, enabling increased ClC-2 activity in response to cellular stresses such as elevated temperature, ischemia, or oxidative reagents.

Animals↗
PubMed2006

Platelet-derived growth factor-BB represses smooth muscle cell marker genes via changes in binding of MKL factors and histone deacetylases to their promoters.

A hallmark of smooth muscle cell (SMC) phenotypic switching is suppression of SMC marker gene expression. Although myocardin has been shown to be a key regulator of this process, the role of its related factors, MKL1 and MKL2, in SMC phenotypic switching remains unknown. The present studies were aimed at determining if: 1) MKL factors contribute to the expression of SMC marker genes in cultured SMCs; and 2) platelet-derived growth factor-BB (PDGF-BB)-induced repression of SMC marker genes is mediated by suppression of MKL factors. Results of gain- and loss-of-function experiments showed that MKL factors regulated the expression of single and multiple CArG [CC(AT-rich)(6)GG]-containing SMC marker genes, such as smooth muscle (SM) alpha-actin and telokin, but not CArG-independent SMC marker genes such as smoothelin-B. Treatment with PDGF-BB reduced the expression of CArG-containing SMC marker genes, as well as myocardin expression in cultured SMCs, while it had no effect on expression of MKL1 and MKL2. However, of interest, PDGF-BB induced the dissociation of MKL factors from the CArG-containing region of SMC marker genes, as determined by chromatin immunoprecipitation assays. This dissociation was caused by the competition between MKL factors and phosphorylated Elk-1 at early time points, but subsequently by the reduction in acetylated histone H4 levels at these promoter regions mediated by histone deacetylases, HDAC2, HDAC4, and HDAC5. Results provide novel evidence that PDGF-BB-induced repression of SMC marker genes is mediated through combinatorial mechanisms, including downregulation of myocardin expression and inhibition of the association of myocardin/MKL factors with CArG-containing SMC marker gene promoters.

Actins↗