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Stanley N Cohen

Publications and source records attributed to Stanley N Cohen.

At least 19 recordsLinked to original sources
PubMed2006

Characterization of a large, stable, high-copy-number Streptomyces plasmid that requires stability and transfer functions for heterologous polyketide overproduction.

A major limitation to improving small-molecule pharmaceutical production in streptomycetes is the inability of high-copy-number plasmids to tolerate large biosynthetic gene cluster inserts. A recent finding has overcome this barrier. In 2003, Hu et al. discovered a stable, high-copy-number, 81-kb plasmid that significantly elevated production of the polyketide precursor to the antibiotic erythromycin in a heterologous Streptomyces host (J. Ind. Microbiol. Biotechnol. 30:516-522, 2003). Here, we have identified mechanisms by which this SCP2*-derived plasmid achieves increased levels of metabolite production and examined how the 45-bp deletion mutation in the plasmid replication origin increased plasmid copy number. A plasmid intramycelial transfer gene, spd, and a partition gene, parAB, enhance metabolite production by increasing the stable inheritance of large plasmids containing biosynthetic genes. Additionally, high product titers required both activator (actII-ORF4) and biosynthetic genes (eryA) at high copy numbers. DNA gel shift experiments revealed that the 45-bp deletion abolished replication protein (RepI) binding to a plasmid site which, in part, supports an iteron model for plasmid replication and copy number control. Using the new information, we constructed a large high-copy-number plasmid capable of overproducing the polyketide 6-deoxyerythronolide B. However, this plasmid was unstable over multiple culture generations, suggesting that other SCP2* genes may be required for long-term, stable plasmid inheritance.

Anti-Bacterial Agents↗
PubMed2006

Human MDM2 isoforms translated differentially on constitutive versus p53-regulated transcripts have distinct functions in the p53/MDM2 and TSG101/MDM2 feedback control loops.

Proteins encoded by the mdm2 gene, which has a pivotal role in the regulation of growth and differentiation, exist principally in human and murine cells as two isoforms that migrate in gels as 75-kDa and 90-kDa proteins. There is limited understanding of the respective biological roles of these isoforms, their molecular nature, and their mechanism of formation. We report here that human p75(MDM2) is an N-terminally truncated mixture of protein isoforms produced by the initiation of translation at two distinct internal AUG codons. The p75(MDM2) doublets and p90(MDM2), which is the full-length MDM2 protein, are expressed in approximately equal amounts from transcripts initiated at the constitutive P1 promoter of mdm2. Unlike murine transcripts initiated at the p53-activated P2 promoter, human cell transcripts initiated at the P2 promoter preferentially produce p90(MDM2). The ubiquitin enzyme variant protein TSG101, which interacts functionally with MDM2 in an autoregulatory loop that parallels the p53/MDM2 feedback control loop, interferes with degradation of both isoforms; however, only p90(MDM2) promotes proteolysis of TSG101 and p53. Our results reveal the mechanism of formation of the principal MDM2 isoforms, the differential effects of p53 on the production of these isoforms, and the differential abilities of human MDM2 isoforms as regulators of the MDM2/TSG101 and p53/MDM2 feedback control loops.

Animals↗
PubMed2006

Retention of core catalytic functions by a conserved minimal ribonuclease E peptide that lacks the domain required for tetramer formation.

Ribonuclease E (RNase E) is a multifunctional endoribonuclease that has been evolutionarily conserved in both Gram-positive and Gram-negative bacteria. X-ray crystallography and biochemical studies have concluded that the Escherichia coli RNase E protein functions as a homotetramer formed by Zn linkage of dimers within a region extending from amino acid residues 416 through 529 of the 116-kDa protein. Using fragments of RNase E proteins from E. coli and Haemophilus influenzae, we show here that RNase E derivatives that are as short as 395 amino acid residues and that lack the Zn-link region shown previously to be essential for tetramer formation (i.e. amino acid residues 400-415) are catalytically active enzymes that retain the 5' to 3' scanning ability and cleavage site specificity characteristic of full-length RNase E and that also confer colony forming ability on rne null mutant bacteria. Further truncation leads to loss of these properties. Our results, which identify a minimal catalytically active RNase E sequence, indicate that contrary to current models, a tetrameric quaternary structure is not required for RNase E to carry out its core enzymatic functions.

Catalytic Domain↗
PubMed2006

Txr1: a transcriptional regulator of thrombospondin-1 that modulates cellular sensitivity to taxanes.

Using transcripts initiated at a chromosomally integrated retrovirus-based promoter to perturb gene expression randomly in human prostate cancer cells, we isolated cell clones resistant to taxane lethality and discovered the role of a previously uncharacterized gene, txr1, in this phenotype. We show that txr1 impedes taxane-induced apoptosis in tumor cells by transcriptionally down-regulating the production of thrombospondin-1 (TSP-1)--known earlier for both its anti-angiogenic and proapoptotic actions. Decrease of Txr1 or treatment with TSP-1 or TSP-1 mimetic peptide sensitized cells to taxane cytotoxicity by activating signaling through the CD47 receptor (also known as the integrin-associated protein), whereas interference with CD47 function reduced taxane-induced cell death. Cellular abundance of Txr1 and TSP-1 varied inversely, and alteration of the level of both proteins correlated highly with taxol resistance in 13 of 19 NCI-60 cancer cell lines. Our results reveal a hitherto unsuspected mechanism of taxane resistance, elucidate the role of txr1 in this resistance, and identify txr1 as a regulator of TSP-1 production and an agent for its chemotherapeutic modulation.

Antineoplastic Agents↗
PubMed2006

Differential modulation of E. coli mRNA abundance by inhibitory proteins that alter the composition of the degradosome.

In Escherichia coli the initial step in the processing or decay of many messenger and structural RNAs is mediated by the endonuclease RNase E, which forms the core of a large RNA-catalysis machine termed the degradosome. Previous experiments have identified a protein that globally modulates RNA abundance by binding to RNase E and regulating its endonucleolytic activity. Here we report the discovery of RraB, which interacts with a different site on RNase E and interferes with cleavage of a different set of transcripts. We show that expression of RraA or RraB in vivo is accompanied by dramatic, distinct, and inhibitor-specific changes in degradosome composition--and that these are in turn associated with alterations in RNA decay and global transcript abundance profiles that are dissimilar to the profile observed during simple RNase E deficiency. Our results reveal the existence of endonuclease binding proteins that modulate the remodelling of degradosome composition in bacteria and argue that such degradosome remodelling is a mechanism for the differential regulation of RNA cleavages in E. coli.

Endoribonucleases↗
PubMed2006

The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin.

Toxins produced by Bacillus anthracis and other microbial pathogens require functions of host cell genes to yield toxic effects. Here we show that low density lipoprotein receptor-related protein 6 (LRP6), previously known to be a coreceptor for the Wnt signaling pathway, is required for anthrax toxin lethality in mammalian cells. Downregulation of LRP6 or coexpression of a truncated LRP6 dominant-negative peptide inhibited cellular uptake of complexes containing the protective antigen (PA) carrier of anthrax toxin moieties and protected targeted cells from death, as did antibodies against epitopes in the LRP6 extracellular domain. Fluorescence microscopy and biochemical analyses showed that LRP6 enables toxin internalization by interacting at the cell surface with PA receptors TEM8/ATR and/or CMG2 to form a multicomponent complex that enters cells upon PA binding. Our results, which reveal a previously unsuspected biological role for LRP6, identify LRP6 as a potential target for countermeasures against anthrax toxin lethality.

Animals↗
PubMed2006

Comparison of secondary prevention care after myocardial infarction and stroke.

BACKGROUND: Whether secondary prevention of atherosclerosis is performed as frequently after cerebrovascular events (stroke or transient ischemic attack) as after cardiac events (myocardial infarction or angina) is unknown. METHODS: We compared the receipt of six secondary preventive care processes among 943 persons with a prior cardiac event to that among 523 persons with a prior cerebrovascular event using a representative sample of the US population. RESULTS: The cardiac event group had higher rates for three care processes: antithrombotic medication use in the past year (83-77%, p = 0.01), ever advised to exercise more (66-52%, p < 0.001), and ever advised to eat fewer high-fat or high-cholesterol foods (70-54%, p < 0.001). CONCLUSIONS: Compared to the cardiac event group, the quality of care of the cerebrovascular event group is lower and should be improved.

Aged↗
PubMed2006

Ischemic stroke in a postpartum patient: understanding the epidemiology, pathogenesis, and outcome of Moyamoya disease.

BACKGROUND: Moyamoya disease (MMD), or spontaneous occlusion of blood vessels in the circle of Willis, is a noninflammatory, nonatheromatous vasculopathy that often presents with ischemia or hemorrhage and has a tendency to affect young women. We discuss a case of a woman 7 days postpartum with new onset strokes, suspected initially to have benign angiopathy of the central nervous system (BACNS)/postpartum angiopathy versus primary CNS vasculitis. Cerebral angiography was suspicious for MMD and the patient underwent surgical revascularization. OBJECTIVES: To review the epidemiology, presentation, diagnosis, differential diagnosis, treatment, prognosis, and postpartum maternal outcomes of MMD. METHODS: The authors reviewed the English-language literature published between 1996 and 2004 using Medline databases. Additional articles were obtained from references described in literature obtained by the primary search. RESULTS: MMD is characterized by severe bilateral stenosis of the distal internal carotid arteries and their first branches, with prominent collateralization. Autoantibodies relating to endothelial cell dysfunction have been studied as possible pathogenetic mechanisms. The gold standard for diagnosing MMD is digital subtraction contrast angiography (DSA). MMD-related findings on imaging studies may, at times, be mistaken for those of vasculitis. A surgical approach may need to be considered. Despite treatment, the overall prognosis of MMD is worse in adults compared with children. CONCLUSION: Awareness of the specific features of MMD is crucial for clinical decision-making to avoid unnecessary exposure to aggressive immunosuppressive therapy. It is important for rheumatologists to consider MMD in the differential diagnosis of suspected CNS vasculitis.

Adult↗
PubMed2006

rag genes: novel components of the RamR regulon that trigger morphological differentiation in Streptomyces coelicolor.

The filamentous bacterium, Streptomyces coelicolor, undergoes a complex cycle of growth and development in which morphological differentiation coincides with the activation of the orphan response regulator RamR and the biosynthesis of a morphogenic peptide called SapB. SapB is a lantibiotic-like molecule derived from the product of the ramS gene that promotes formation of aerial hyphae by breaking the aqueous tension on the surface of the substrate mycelium. A ramR-disrupted mutant is delayed in aerial hyphae formation while constitutive overexpression of ramR accelerates aerial hyphae formation in the wild-type strain and restores SapB biosynthesis and aerial hyphae formation in all developmental mutants (bld) tested. Using DNA microarrays to globally identify S. coelicolor genes whose transcription was affected by ramR mutation or overexpression, we discovered a ramR-activated locus of contiguous cotranscribed developmental genes that modulate both aerial hyphae formation and sporulation. The genes of this cluster of ramR-activated genes (rag), which are chromosomally distant from previously known RamR-regulated genes, include: ragA (sco4075) and ragB (sco4074), which encode two subunits of an ABC transporter, ragK (sco4073), a putative histidine kinase, and ragR (sco4072), a ramR paralogue. Promoter mapping and protein-DNA binding experiments indicate that RamR activates ragABKR transcription directly, by binding to three sequence motifs in the ragABKR promoter region. A constructed ragABKR null mutant was able to synthesize SapB and erect aerial hyphae; however, these hyphae were unusually branched, reminiscent of substrate hyphae. Subsequent stages of differentiation, septation and sporogenesis were delayed. The role of ragABKR in aerial hyphae formation was shown both by epistasis (ragR-activated aerial hyphae formation in bld mutants) and extracellular complementation (ragR-induced synthesis of an activity allowing aerial hyphae formation in bld mutants) experiments. In conclusion, the ragABKR locus activates a SapB-independent developmental pathway that is involved in both aerial hyphae formation and sporulation, serving to integrate sequential morphogenic changes.

Bacterial Proteins↗
PubMed2006

RNase E maintenance of proper FtsZ/FtsA ratio required for nonfilamentous growth of Escherichia coli cells but not for colony-forming ability.

Inactivation or deletion of the RNase E-encoding rne gene of Escherichia coli results in the growth of bacterial cells as filamentous chains in liquid culture (K. Goldblum and D. Apirion, J. Bacteriol. 146:128-132, 1981) and the loss of colony-forming ability (CFA) on solid media. RNase E dysfunction is also associated with abnormal processing of ftsQAZ transcripts (K. Cam, G. Rome, H. M. Krisch, and J.-P. Bouché, Nucleic Acids Res. 24:3065-3070, 1996), which encode proteins having a central role in septum formation during cell division. We show here that RNase E regulates the relative abundances of FtsZ and FtsA proteins and that RNase E depletion results in decreased FtsZ, increased FtsA, and consequently an altered FtsZ/FtsA ratio. However, while restoration of the level of FtsZ to normal in rne null mutant bacteria reverses the filamentation phenotype, it does not restore CFA. Conversely, overexpression of a related RNase, RNase G, in rne-deleted bacteria restores CFA, as previously reported, without affecting FtsZ abundance. Our results demonstrate that RNase E activity is required to maintain a proper cellular ratio of the FtsZ and FtsA proteins in E. coli but that FtsZ deficiency does not account for the nonviability of cells lacking RNase E.

Culture Media↗
PubMed2006

Phenotype-based identification of host genes required for replication of African swine fever virus.

African swine fever virus (ASFV) produces a fatal acute hemorrhagic fever in domesticated pigs that potentially is a worldwide economic threat. Using an expressed sequence tag (EST) library-based antisense method of random gene inactivation and a phenotypic screen for limitation of ASFV replication in cultured human cells, we identified six host genes whose cellular functions are required by ASFV. These included three loci, BAT3 (HLA-B-associated transcript 3), C1qTNF (C1q and tumor necrosis factor-related protein 6), and TOM40 (translocase of outer mitochondrial membrane 40), for which antisense expression from a tetracycline-regulated promoter resulted in reversible inhibition of ASFV production by >99%. The effects of antisense transcription of the BAT3 EST and also of expression in the sense orientation of this EST, which encodes amino acid residues 450 to 518 of the mature BAT3 protein, were investigated more extensively. Sense expression of the BAT3 peptide, which appears to reversibly interfere with BAT3 function by a dominant negative mechanism, resulted in decreased synthesis of viral DNA and proteins early after ASFV infection, altered transcription of apoptosis-related genes as determined by cDNA microarray analysis, and increased cellular sensitivity to staurosporine-induced apoptosis. Antisense transcription of BAT3 reduced ASFV production without affecting abundance of the virus macromolecules we assayed. Our results, which demonstrate the utility of EST-based functional screens for the detection of host genes exploited by pathogenic viruses, reveal a novel collection of cellular genes previously not known to be required for ASFV infection.

African Swine Fever Virus↗
PubMed2005

Recurrent embolic strokes and cardiac valvular disease in a patient with non-small cell adenocarcinoma of lung.

The etiology and mechanisms of stroke could differ in cancer compared to non-cancer patients due to altered blood coagulability and/or non-bacterial thrombotic endocarditis (NBTE). These conditions could be either missed by using inappropriate diagnostic methods or misinterpreted. For instance, certain techniques (transthoracic echocardiography, TTE) may provide false-negative results and delay appropriate therapy. On the other hand, these patients, by having atypical findings, may challenge the differential between bacterial and sterile valvular heart disease. Cerebrovascular disease in cancer patients is often aggressive with tendency to recurrent events and rapid neurological devastation. Timely diagnosis is crucial. Current treatment approach to NBTE includes anticoagulation. We report a case of multiple embolic strokes in a patient diagnosed with lung cancer. Primary and secondary stroke prevention is discussed with relevant review of the literature.

Aged↗
PubMed2005

Regional organization of gene expression in Streptomyces coelicolor.

Based on the chromosomal locations of genes inferred from sequence analysis to be essential for the viability of Streptomyces coelicolor, Bentley et al. [Bentley, S.D., et al. 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2), Nature 417, 141-147.] have suggested that a 4.9 Mb central region of the linear S. coelicolor chromosome encodes 'core' functions expressed during vegetative growth of this species, while 1.5 Mb and 2.3 Mb chromosomal DNA segments lateral to this core encode auxiliary functions proposed to be required under other growth conditions. To examine this hypothesis and experimentally identify genes expressed during vegetative growth of S. coelicolor cultures, we used DNA microarrays to measure globally the abundance of S. coelicolor transcripts in cells growing in liquid medium. We found that, overall, genes corresponding to the 4.9 Mb core region of the S. coelicolor M145 chromosome were more highly expressed under non-limiting growth conditions than genes in the 1.5 Mb left and 2.3 Mb right chromosome arms, supporting the notion of the core versus auxiliary organization of genes on the chromosome. To examine how this chromosomal distribution of transcripts changes under other growth conditions, we also measured gene expression changes during stationary phase and several stress conditions. During stationary phase, the composition of S. coelicolor transcripts appears to shift from large quantities of growth-related transcripts encoded in the core region to those of less characterized genes, which may be essential for differentiation and other physiological responses, encoded throughout the chromosome. After temperature and osmotic upshifts, we found that S. coelicolor transiently induces a set of several hundred genes located throughout the chromosome, which may function in response mechanisms common to the two stress conditions.

Chromosomes, Bacterial↗
PubMed2005

Effects of threshold choice on biological conclusions reached during analysis of gene expression by DNA microarrays.

Global analysis of gene expression by using DNA microarrays is employed increasingly to search for differences in biological properties between normal and diseased tissue. In such studies, expression that deviates from defined thresholds commonly is used for creating genetic signatures that characterize disease vs. normality. Although it is axiomatic that the threshold parameters applied to microarray analysis will alter the contents of such genetic signatures, the extent to which threshold choice can affect the fundamental conclusions made from microarray-based studies has not been elucidated. We used GABRIEL (Genetic Analysis By Rules Incorporating Expert Logic), a platform of knowledge-based algorithms for the global analysis of gene expression, together with conventional statistical approaches, to examine the sensitivity of conclusions to threshold choice in recently published microarray-based studies. An analysis of the effects of threshold decisions in one of these studies [Ramaswamy, S., Ross, K. N., Lander, E. S. & Golub, T. R. (2003) Nat. Genet. 33, 49-54], which arrived at the important conclusion that the metastatic potential of primary tumors is encoded by the bulk of cells in the tumor, is the focus of this article. We discovered that support for this conclusion highly depends on the threshold used to create gene expression signatures. We also found that threshold choice dramatically affected the gene function categories represented nonrandomly in signatures. Our results suggest that the robustness of biological conclusions made by using microarray analysis should be routinely assessed by examining the validity of the conclusions by using a range of threshold parameters.

Adenocarcinoma↗
PubMed2005

An integrated data analysis approach to characterize genes highly expressed in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer deaths worldwide. New diagnostic and therapeutic options are needed for more effective and early detection and treatment of this malignancy. We identified 703 genes that are highly expressed in HCC using DNA microarrays, and further characterized them in order to uncover novel tumor markers, oncogenes, and therapeutic targets for HCC. Using Gene Ontology annotations, genes with functions related to cell proliferation and cell cycle, chromatin, repair, and transcription were found to be significantly enriched in this list of highly expressed genes. We also identified a set of genes that encode secreted (e.g. GPC3, LCN2, and DKK1) or membrane-bound proteins (e.g. GPC3, IGSF1, and PSK-1), which may be attractive candidates for the diagnosis of HCC. A significant enrichment of genes highly expressed in HCC was found on chromosomes 1q, 6p, 8q, and 20q, and we also identified chromosomal clusters of genes highly expressed in HCC. The microarray analyses were validated by RT-PCR and PCR. This approach of integrating other biological information with gene expression in the analysis helps select aberrantly expressed genes in HCC that may be further studied for their diagnostic or therapeutic utility.

Biomarkers, Tumor↗
PubMed2005

Withdrawal of warfarin prior to a surgical procedure: time to follow the guidelines?

BACKGROUND AND OBJECTIVE: Patients with cardiogenic sources of embolism may be at increased risk of cerebral infarction when anticoagulation therapy is suspended for surgical procedures. The purpose of this study was to determine frequency of cardioembolic cerebral infarction during periprocedural warfarin withdrawal. METHODS: Retrospective analysis of prospective cerebral infarction registry data from two tertiary medical centers. RESULTS: Over a 12-month period, 14 cases of cardioembolic cerebral infarction occurring during the period of warfarin withdrawal for a medical procedure were observed, accounting for 7.1% of the 197 cardioembolic cerebral infarctions encountered. Across all patients, cerebral infarctions developed an average of 5.4 days after the last dose of warfarin (range 3-8). Among the 14 patients (8 males and 6 females) with warfarin cessation-related infarcts, age ranged from 54 to 91 years. Each had been on chronic anticoagulation with warfarin for more than 1 year. Retrospective analysis suggested that all these cerebral infarctions had been potentially preventable. In each case, either the planned procedure did not require discontinuation of warfarin or, when withdrawal was required, no bridging, parenteral anticoagulation was provided to lessen the risk during the warfarin-free period. CONCLUSION: Patients at high risk of cardioembolic cerebral infarction may benefit from more intensive management strategies to reduce cerebral infarction risk during periprocedural periods.

Aged↗