Search PubMed⌕ Search

Biomedical subjects

S L Marcus

Publications and source records attributed to S L Marcus.

At least 19 recordsLinked to original sources
PubMed2002

Photodynamic therapy for Barrett's esophagus: a review.

Barrett's esophagus is the major risk factor for the development of esophageal adenocarcinoma, which is increasing in incidence faster than any other cancer in the Western world. Barrett's esophagus has previously been considered an irreversible lesion that required life-long surveillance to detect malignant transformation. However, endoscopic ablative techniques to destroy the abnormal mucosa and allow squamous regeneration have now been developed. Photodynamic therapy (PDT) is a non-thermal technique where the interaction of a photosensitizer in the tissues and light of a known wavelength results in tissue destruction. It appears to be an effective tool for ablating dysplasia and superficial cancers in Barrett's esophagus. The status of PDT for this disease is reviewed.

Aminolevulinic Acid↗
PubMed2001

A synaptojanin-homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation.

The Ser-Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5' phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a DeltasigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.

Amino Acid Sequence↗
PubMed2001

Structural and biochemical characterization of the type III secretion chaperones CesT and SigE.

Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease. This specialized secretion process requires customized chaperones specific for particular effector proteins. The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers. Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein. Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones.

Amino Acid Sequence↗
PubMed2001

SifA permits survival and replication of Salmonella typhimurium in murine macrophages.

SifA was originally identified as a virulence factor required for formation of Salmonella-induced filaments (Sifs), elongated tubules rich in lysosomal glycoproteins that extend from the Salmonella-containing vacuole in infected epithelial cells. Here, we demonstrate that deletion mutants of ssaR, a component of the SPI-2 type III secretion system, do not form Sifs in HeLa epithelial cells. This suggests that SifA is a translocated effector of this system, acting within host cells to form Sifs. In support of this hypothesis, transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused extensive vacuolation of LAMP-1-positive compartments. Filamentous tubules that closely resembled Sifs were also observed in transfected cells, demonstrating that SifA is sufficient to initiate alteration of host cell endosomal structures. deltasifA mutants were impaired in their ability to survive/replicate in RAW 264.7 murine macrophages, a phenotype similar to ssaR mutants. Our findings suggest that SifA is an effector of the SPI-2 type III secretion system and allows colonization of murine macrophages, the host niche exploited during systemic phases of disease in these animals. A family of SifA-related proteins and their importance to Salmonella pathogenesis is also discussed.

Amino Acid Sequence↗
PubMed2000

Activation of Akt/protein kinase B in epithelial cells by the Salmonella typhimurium effector sigD.

The serine-threonine kinase Akt is a protooncogene involved in the regulation of cell proliferation and survival. Activation of Akt is initiated by binding to the phospholipid products of phosphoinositide 3-kinase at the inner leaflet of the plasma membranes followed by phosphorylation at Ser(473) and Thr(308). We have found that Akt is activated by Salmonella enterica serovar Typhimurium in epithelial cells. A bacterial effector protein, SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. In HeLa cells, wild type S. typhimurium induced translocation of Akt to membrane ruffles and phosphorylation at residues Thr(308) and Ser(473) and increased kinase activity. In contrast, infection with a SigD deletion mutant did not induce phosphorylation or activity although Akt was translocated to membrane ruffles. Complementation of the SigD deletion strain with a mutant containing a single Cys to Ser mutation (C462S), did not restore the Akt activation phenotype. This residue has previously been shown to be essential for inositol phosphatase activity of the SigD homologue, SopB. Our data indicate a novel mechanism of Akt activation in which the endogenous cellular pathway does not convert membrane-associated Akt into its active form. SigD is also the first bacterial effector to be identified as an activator of Akt.

Base Sequence↗
PubMed2000

Salmonella pathogenicity islands: big virulence in small packages.

Reflecting a complex set of interactions with its host, Salmonella spp. require multiple genes for full virulence. Many of these genes are found in 'pathogenicity islands' in the chromosome. Salmonella typhimurium possesses at least five such pathogenicity islands (SPI), which confer specific virulence traits and may have been acquired by horizontal transfer from other organisms. We highlight recent progress in characterizing these SPIs and the function of some of their genes. The role of virulence genes found on a highly conserved plasmid is also discussed. Collectively, these packages of virulence cassettes are essential for Salmonella pathogenesis.

Animals↗
PubMed2000

Photodynamic therapy for dysplastic Barrett's oesophagus: a prospective, double blind, randomised, placebo controlled trial.

BACKGROUND AND AIMS: Photodynamic therapy (PDT) is a treatment in which cell damage is achieved by the action of light on a photosensitizing agent. We have assessed the potential use of PDT in the ablation of Barrett's oesophagus. METHODS: Thirty six patients with dysplastic Barrett's oesophagus receiving acid suppression medication with omeprazole were randomised to receive oral 5-aminolaevulinic acid (ALA) 30 mg/kg or placebo, followed four hours later by laser endoscopy. Follow up endoscopy was performed at one, six, 12, and 24 months. RESULTS: Of 18 patients in the ALA group, a response was seen in 16 (median decrease in area in the treated region 30%; range 0-60%). In the placebo group, a decrease in area of 10% was observed in two patients with no change in 16 (median 0%; range 0-10%; treatment v placebo, p<0.001). No dysplasia was seen in the columnar epithelium within the treatment area of any patient in the PDT group. However, in the placebo group, persistent low grade dysplasia was found in 12 patients (p<0.001). There were no short or long term major side effects. The effects of treatment were maintained for up to 24 months. CONCLUSIONS: This is the first randomised controlled trial of PDT for Barrett's oesophagus. It demonstrates that ALA induced PDT can provide safe and effective ablation of low grade dysplastic epithelium.

Adult↗
PubMed1999

The p56(lck)-interacting protein p62 stimulates transcription via the SV40 enhancer.

p62 is a recently identified ubiquitin-binding, cytosolic phosphoprotein that interacts with several signal transduction molecules including the tyrosine kinase p56(lck) and the protein kinase C-zeta. p62 is therefore suggested to serve an important role in signal transduction in the cell, although the physiological function of p62 remains undefined. Here we demonstrate by transient transfection assays that p62 stimulates the transcription of reporter genes linked to the simian virus 40 (SV40) enhancer. A putative p62-responsive element was localized to the B domain of the distal 72-base pair repeat of the SV40 enhancer. p62 was unable to bind this element in vitro, nor was it able to activate transcription when directly tethered to a promoter, suggesting that p62 stimulates transcription via an indirect mechanism. Stimulation of transcription mediated by p62 was dependent on its amino-terminal region, which is also necessary for interaction with cell surface signaling molecules. These findings indicate that p62 may link extracellular signals directly to transcriptional responses, and identify the SV40 enhancer as a downstream target for signal transduction pathways in which p62 participates.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Salmonella typhimurium virulence genes are induced upon bacterial invasion into phagocytic and nonphagocytic cells.

Survival and growth of salmonellae within host cells are important aspects of bacterial virulence. We have developed an assay to identify Salmonella typhimurium genes that are induced inside Salmonella-containing vacuoles within macrophage and epithelial cells. A promoterless luciferase gene cassette was inserted randomly into the Salmonella chromosome, and the resulting mutants were screened for genes upregulated in intracellular bacteria compared to extracellular bacteria. We identified four genes in S. typhimurium that were upregulated upon bacterial invasion of both phagocytic and nonphagocytic cells. Expression of these genes was not induced by factors secreted by host cells or media alone. All four genes were induced at early time points (2 to 4 h) postinvasion and continued to be upregulated within host cells at later times (5 to 7 h). One mutant contained an insertion in the ssaR gene, within Salmonella pathogenicity island 2 (SPI-2), which abolished bacterial virulence in a murine typhoid model. Two other mutants contained insertions within SPI-5, one in the sopB/sigD gene and the other in a downstream gene, pipB. The insertions within SPI-5 resulted in the attenuation of S. typhimurium in the mouse model. The fourth mutant contained an insertion within a previously undescribed region of the S. typhimurium chromosome, iicA (induced intracellularly A). We detected no effect on virulence as a result of this insertion. In conclusion, all but one of the genes identified in this study were virulence factors within pathogenicity islands, illustrating the requirement for specific gene expression inside mammalian cells and indicating the key role that virulence factor regulation plays in Salmonella pathogenesis.

Animals↗
PubMed1999

Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7.

Mutations at the Nramp1 locus in vivo cause susceptibility to infection by unrelated intracellular microbes. Nramp1 encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-type Nramp1(G169) cDNA into RAW 264.7 macrophages (which bear a homozygous mutant Nramp1(D169) allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane of Salmonella typhimurium and Yersinia enterocolitica containing phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication of S. typhimurium. Studies with a replication-defective S. typhimurium mutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant of S. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection of Nramp1 cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.

Alleles↗
PubMed1996

A p56(lck) ligand serves as a coactivator of an orphan nuclear hormone receptor.

Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily, acts as a transcriptional repressor by antagonizing the functions of other nuclear hormone receptors and by actively silencing transcription. However, in certain contexts, COUP-TFII stimulates transcription directly. A cellular factor, isolated by interaction cloning, bound COUP-TFII in vitro and allowed COUP-TFII to function as a transcriptional activator in mammalian cells. This factor is identical to a recently described ligand of the tyrosine kinase signaling molecule p56(lck), suggesting that it mediates cross-talk between mitogenic and nuclear hormone receptor signal transduction pathways.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Identification of COUP-TFII as a peroxisome proliferator response element binding factor using genetic selection in yeast: COUP-TFII activates transcription in yeast but antagonizes PPAR signaling in mammalian cells.

Peroxisome proliferator-response elements (PPRE) are cis-acting regulatory elements that confer responsiveness to peroxisome proliferators and various fatty acids by serving as target sites for ligand-activated peroxisome proliferator-activated receptor (PPAR)/retinoid X receptor (RXR) heterodimers. Other cellular factors, including additional nuclear hormone receptors, also interact with PPREs and modulate PPAR function. We have developed a positive selection strategy in yeast to identify mammalian factors that functionally interact with PPREs. Saccharomyces cerevisiae containing an integrated copy of the HIS3 gene under transcriptional control of a minimal CYC1 promoter and two copies of the rat enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase PPRE was constructed and transformed with a rat liver cDNA yeast expression library. Plasmids were isolated from his + transformants. One plasmid contained a cDNA encoding the complete rat chicken ovalbumin upstream promoter transcription factor II (COUP-TFII), an orphan member of the nuclear hormone receptor superfamily. COUP-TFII potently activated PPRE-linked reporter gene expression in yeast, and COUP-TFII synthesized in yeast or in vitro formed specific protein/DNA complexes with this PPRE. Significantly, COUP-TFII did not activate transcription of PPRE-linked reporter genes in mammalian cells but rather strongly inhibited induction mediated by PPAR/RXR. Our findings demonstrate the utility of using genetic screening in yeast to identify sequence-specific DNA binding transcription factors.

Animals↗
PubMed1996

Photodynamic therapy (PDT) and photodiagnosis (PD) using endogenous photosensitization induced by 5-aminolevulinic acid (ALA): mechanisms and clinical results.

5-Aminolevulinic acid (ALA), when added to many tissues, results in the accumulation of sufficient quantities of the endogenous photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway, to produce a photodynamic effect when exposed to activating light. Therefore, ALA is the only photodynamic therapy (PDT) agent in current clinical development that is a biochemical precursor of a photosensitizer. Topical ALA application, followed by exposure to activating light (ALA PDT), has been reported effective for the treatment of a variety of dermatologic diseases including cutaneous superficial and nodular basal cell carcinoma, Bowen's disease, and actinic (solar) keratoses. Local internal application of ALA has also been used for selective endometrial ablation in animal model systems and in human clinical studies has shown selective formation of PpIX within the endometrium. PpIX induced by ALA application has also been used as a fluorescence detection marker for photodiagnosis (PD) of cancer and dysplastic conditions of the urinary bladder and other organs. Systemic, oral administration of ALA has been used for ALA PDT of superficial head and neck cancer, various gastrointestinal cancers, and the condition known as Barrett's esophagus. The current state of knowledge of the mechanisms of endogenous topical and systemic photosensitization using ALA, the results of published clinical trials, and possible methods of increasing the efficacy of endogenous photosensitization for ALA PDT are reviewed in this paper.

Aminolevulinic Acid↗
PubMed1996

Photodynamic therapy (PDT) and photodiagnosis (PD) using endogenous photosensitization induced by 5-aminolevulinic acid (ALA): current clinical and development status.

Exogenous provision of 5-aminolevulinic acid (ALA) to many tissues results in the accumulation of sufficient quantities of the endogenous photosensitizer protoporphyrin IX (PpIX) via the heme biosynthetic pathway, to produce a photodynamic effect when exposed to activating light. Therefore, ALA may be considered the only current photodynamic therapy (PDT) agent in clinical development that is a biochemical precursor of a photosensitizer. Topical ALA application, followed by exposure to activating light (ALA PDT), has been reported effective for the treatment of a variety of dermatologic diseases including cutaneous superficial and nodular basal cell carcinoma, Bowen's disease, actinic (solar) keratoses, and T cell lymphoma. Local internal application of ALA has also been used for selective endometrial ablation in animal model systems and, in human clinical studies, it has shown selective formation of PpIX within the endometrium. PpIX induced by ALA application has also been used as a fluorescence detection marker for photodiagnosis (PD) of cancer and dysplastic conditions of the urinary bladder and other organs. Systemic, oral administration of ALA has been used for ALA PDT of superficial head and neck cancer, various gastrointestinal cancers, and the condition known as Barrett's esophagus. This paper reviews the current clinical and development status of ALA PDT and PD.

Aminolevulinic Acid↗
PubMed1995

Calreticulin modulates the in vitro DNA binding but not the in vivo transcriptional activation by peroxisome proliferator-activated receptor/retinoid X receptor heterodimers.

Calreticulin is a ubiquitous calcium binding/storage protein found primarily in the endoplasmic reticulum. Calreticulin has been shown to inhibit DNA binding and transcriptional activation by glucocorticoid and androgen hormone receptors by binding to the conserved sequence KXFF(K/R)R, present in the DNA-binding domains of all known members of the steroid/nuclear hormone receptor superfamily. To determine whether calreticulin might be a general regulator of hormone-responsive pathways, we examined its effect on DNA binding in vitro and transcriptional activation in vivo by heterodimers of the peroxisome proliferator-activated receptor (PPAR) and the 9-cis retinoic acid receptor (RXR alpha). We show here that purified calreticulin inhibits the binding of PPAR/RXR alpha heterodimers and of other nuclear hormone receptors, to peroxisome proliferator-responsive DNA elements in vitro. However, overexpression of calreticulin in transiently transfected cultured cells had little or no effect on transactivation mediated by PPAR/RXR alpha. Therefore, while calreticulin inhibits the binding of both nuclear and steroid hormone receptors to cognate response elements in vitro, our findings suggest that calreticulin does not necessarily play an important role in the regulation of all classes of hormone receptors in vivo.

Amino Acid Sequence↗
PubMed1995

Transactivation by PPAR/RXR heterodimers in yeast is potentiated by exogenous fatty acid via a pathway requiring intact peroxisomes.

Peroxisome proliferator-activated receptors (PPARs) are orphan members of the nuclear hormone receptor superfamily. PPARs bind to cognate response elements through heterodimerization with retinoid X receptors (RXRs). Together PPAR/RXR regulate the transcription of genes for which products are involved in lipid homeostasis, cell growth, and differentiation. PPARs are activated by fatty acids and by nongenotoxic rodent hepatocarcinogens called peroxisome proliferators through as of yet undefined signal transduction pathways. In an effort to elucidate the requirements for PPAR function and the pathways of its activation, we expressed mouse PPAR alpha and human RXR alpha in the yeast Saccharomyces cerevisiae. Mouse PPAR alpha and human RXR alpha had little activity individually in yeast; however, when cosynthesized, they were able to synergistically activate transcription via cognate response elements. Transactivation was independent of exogenously added activators of either receptor but was potentiated by the addition of petroselinic acid, a fatty acid shown to activate PPARs in mammalian cells. Similar experiments were carried out in a mutant yeast strain lacking peroxisomes entirely or in a mutant strain deficient for 3-ketoacyl-CoA thiolase, the final enzyme of the peroxisomal beta-oxidation cascade. The findings showed that constitutive transactivation by PPAR/RXR did not require the complete beta-oxidation pathway or intact peroxisomes but required intact peroxisomes for potentiation by exogenously added petroselinic acid. This study demonstrates that at least part of the mammalian peroxisome proliferator-signaling pathway can be faithfully reconstituted in yeast and that activation of PPAR by at least one particular fatty acid requires the integrity of peroxisomes.

Animals↗