Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1. families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1 L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c-both tagged and detagged-from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies. Crown copyright (C) 2003 Published by Elsevier Inc. All rights reserved.

"What do other people think?" - The role of prejudice and social norms in the expression of opinions against gay men

It is generally acknowledged that it is no longer socially acceptable to espouse prejudiced beliefs, yet prejudiced attitudes and discriminatory behaviours still occur. The present study sought to determine when and by whom prejudiced attitudes would be expressed. Specifically, an experiment was conducted to examine the impact of injunctive social norms emanating from a social group with which participants identified and participants' level of homophobia on the expression of opinions about gay men. Participants were presented with information indicating that the majority of group members agreed with a number of prejudiced injunctive statements (pro-prejudice norm), that the majority disagreed with the statements (anti-prejudice norm), or they were given no information about other group members' opinions (control). Participants then reported their own responses to the same injunctive statements. Participants' levels of homophobia were assessed either before or after they were given the normative information. The results indicated that activation of a pro-prejudice injunctive norm for those higher in homophobia resulted in more prejudiced opinions being expressed in comparison to those who received no normative information or those who had a nonprejudiced norm activated. Those lower in homophobia expressed less prejudiced opinions than those higher in homophobia and this did not differ as a function of social norm. The results demonstrate how prejudice can come to be expressed even in the presence of a broad societal norm that suggests that is it wrong to express such opinions.

Expression of the CD44v2-10 isoform confers a metastatic phenotype: Importance of the heparan sulfate attachment site CD44v3

We expressed the full-length CD44v2-10 isoform in SKHep1 cells, a nonmetastatic human hepatocellular carcinoma cell line that does not express any endogenous CD44v isoforms. In SCID mice, expression of CD44v2-10 by SKHep1 cells had no effect on s.c. primary tumor development but caused pulmonary metastases in 41% (7 of 17) of animals compared with control SKHep1 cells (0 of 16; P < 0.01). CD44v2-10 expression by SKHep1 cells resulted in enhanced heparan sulfate (HS) attachment and an enhanced capacity to bind heparin-binding growth factors. Mutation of the v3 domain to prevent HS attachment and growth factor binding abolished the metastatic phenotype, demonstrating that HS modification of CD44v2-10 plays a critical role in the development of metastases in this model. However, in vitro proliferation, motility, and invasion were not altered by CD44v2-10 expression.

Steatosis in chronic hepatitis C: Association with increased messenger RNA expression of collagen I, tumor necrosis factor-α and cytochrome P450 2E1

Background: Increased levels of tumor necrosis factor (TNF)-alpha and oxidative stress have been implicated as factors contributing to hepatic injury in fatty liver diseases. As steatosis is associated with an accelerated progression of fibrosis in chronic hepatitis C (HCV), we hypothesized that the messenger (m)RNA expression of genes involved with the production of reactive oxygen species, inflammation and cellular injury would be increased in liver tissue from subjects with steatosis and chronic HCV. Methods: Real-time polymerase chain reaction was performed to determine relative mRNA expression levels of collagen I, TNF-alpha, cytochrome P450 2E1 (CYP 2E1), transforming growth factor-beta1 and CD14 in liver biopsies from 38 patients with chronic HCV. The mRNA expression levels were compared between subjects with and without steatosis, fibrosis, and inflammation. Results: Multivariate analysis demonstrated that collagen I mRNA expression was increased by 199% in steatosis (P = 0.02), 85% in moderate to severe fibrosis (P = 0.02) and 157% in inflammation (P = 0.03). Livers of patients with steatosis also had an increase in TNF-alpha mRNA expression by 50% (P = 0.03) and CYP 2E1 expression by 37% (P = 0.04) compared with non-steatotic livers. Tumor necrosis factor-alpha protein was localized to Kupffer cells, bile ducts and portal inflammatory cells by immunohistochemistry. Conclusion: Increased expression of TNF-alpha may be involved in the pathogenesis of liver injury and progression of fibrosis in individuals who have steatosis in association with chronic HCV. (C) 2003 Blackwell Publishing Asia Pty Ltd.

In vivo gene expression: DNA electrotransfer

The use of electric pulses to deliver therapeutic molecules to tissues and organs in vivo is a rapidly growing field of research. Electrotransfer can be used to deliver a wide range of potentially therapeutic agents, including drugs, proteins, oligonucleotides, RNA and DNA. Optimization of this approach depends upon a number of parameters such as target organ accessibility, cell turnover, microelectrode design, electric pulsing protocols and the physiological response to the therapeutic agent. Many organs have been successfully transfected by electroporation, including skin, liver, skeletal and cardiac muscle, male and female germ cells, artery, gut, kidney, retinal ganglion cells, cornea, spinal cord, joint synovium and brain. Electrotransfer technology is relevant in a variety of research and clinical settings including cancer therapy, modulation of pathogenic immune reactions, delivery of therapeutic proteins and drugs, and the identification of drug targets by the modulation of normal gene expression. This, together with the capacity to deliver very large DNA constructs, greatly expands the research and clinical applications of in vivo DNA electrotransfer.

Rabbit CYP4B1 engineered for high-level expression in Escherichia coli: ligand stabilization and processing of the N-terminus and heme prosthetic group

Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hernoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme. (C) 2003 Elsevier Science (USA). All rights reserved.

Polymorphisms in the 5 '-untranslated region of the human serotonin receptor 1B (HTR1B) gene affect gene expression

We present evidence of complex balancing regulation of HTR1B transcription by common polymorphisms in its promoter. Computational analysis of the HTR1B gene predicted that a 50 segment, spanning common DNA sequence variations, T-261G, A-161T, and -182INS/DEL-181, contained a putative functional promoter. Using a secreted alkaline phosphatase (SEAP) reporter gene system, we found that the haplotype -261G_-182INS-181_A-161 enhanced transcriptional activity 2.3-fold compared with the haplotype T-261_-182INS-181_A-161. Conversely, -161T reversed this, and the net effect when -261G and -161T were in the same haplotype (-261G_-182INS-181_-161T) was equivalent to the major haplotype (T-261_-182INS-181_A-161). Electrophoretic mobility shift experiments showed that -261G and -161T modify the binding of transcription factors (TFs): -261G generates a new AP2 binding site, while alleles A-161 and -161T exhibit different binding characteristics to AP1. T-261G and A-161T were found to be in linkage disequilibrium (LD) with G861C in a European ancestry population. Interestingly, G861C has been reported to be associated with several psychiatric disorders. Our results indicate that HTR1B is the target of substantial transcriptional genetic regulation by common haplotypes, which are in LD with the HTR1B single-nucleotide polymorphism (SNP) most commonly used in association studies.

Expression of the HPV16E7 oncoprotein by thymic epithelium is accompanied by disrupted T cell maturation and a failure of the thymus to involute with age

Transgenic mice expressing the E7 protein of HPV16 from the keratin 14 promoter demonstrate increasing thymic hypertrophy with age. This hypertrophy is associated with increased absolute numbers of all thymocyte types, and with increased cortical and medullary cellularity. In the thymic medulla, increased compartmentalization of the major thymic stromal cell types and expansion of thymic epithelial cell population is observed. Neither an increased rate of immature thymocyte division nor a decreased rate of immature thymocyte death was able to account for the observed hypertrophy. Thymocytes with reduced levels of expression of CD4 and/or CD8 were more abundant in transgenic (tg) mice and became increasingly more so with age. These thymic SP and DP populations with reduced levels of CD4 and/or CD8 markers had a lower rate of apoptosis in the tg than in the non-tg mice. The rate of export of mature thymocytes to peripheral lymphoid organs was less in tg animals relative to the pool of available mature cells, particularly for the increasingly abundant CD4lo population. We therefore suggest that mature thymocytes that would normally die in the thymus gradually accumulated in E7 transgenic animals, perhaps as a consequence of exposure to a hypertrophied E7-expressing thymic epithelium or to factors secreted by this expanded thymic stromal cell population. The K14E7 transgenic mouse thus provides a unique model to study effects of the thymic epithelial cell compartment on thymus development and involution.

Tissue-specific gene expression in soybean (Glycine max) detected by cDNA microarray analysis

We have constructed cDNA microarrays for soybean (Glycine max L. Merrill), containing approximately 4,100 Unigene ESTs derived from axenic roots, to evaluate their application and utility for functional genomics of organ differentiation in legumes. We assessed microarray technology by conducting studies to evaluate the accuracy of microarray data and have found them to be both reliable and reproducible in repeat hybridisations. Several ESTs showed high levels (>50 fold) of differential expression in either root or shoot tissue of soybean. A small number of physiologically interesting, and differentially expressed sequences found by microarray analysis were verified by both quantitative real-time RT-PCR and Northern blot analysis. There was a linear correlation (r(2) = 0.99, over 5 orders of magnitude) between microarray and quantitative real-time RT-PCR data. Microarray analysis of soybean has enormous potential not only for the discovery of new genes involved in tissue differentiation and function, but also to study the expression of previously characterised genes, gene networks and gene interactions in wild-type, mutant or transgenic; plants.

Systemic gene expression in arabidopsis during an incompatible interaction with Alternaria brassicicola

Pathogen challenge can trigger an integrated set of signal transduction pathways, which ultimately leads to a state of high alert, otherwise known as systemic or induced resistance in tissue remote to the initial infection. Although large-scale gene expression during systemic acquired resistance, which is induced by salicylic acid or necrotizing pathogens has been previously reported using a bacterial pathogen, the nature of systemic defense responses triggered by an incompatible necrotrophic fungal pathogen is not known. We examined transcriptional changes that occur during systemic defense responses in Arabidopsis plants inoculated with the incompatible fungal pathogen Alternaria brassicicola. Substantial changes (2.00-fold and statistically significant) were demonstrated in distal tissue of inoculated plants for 35 genes (25 up-regulated and 10 down-regulated), and expression of a selected subset of systemically expressed genes was confirmed using real-time quantitative polymerase chain reaction. Genes with altered expression in distal tissue included those with putative functions in cellular housekeeping, indicating that plants modify these vital processes to facilitate a coordinated response to pathogen attack. Transcriptional up-regulation of genes encoding enzymes functioning in the beta-oxidation pathway of fatty acids was particularly interesting. Transcriptional up-regulation was also observed for genes involved in cell wall synthesis and modification and genes putatively involved in signal transduction. The results of this study, therefore, confirm the notion that distal tissue of a pathogen-challenged plant has a heightened preparedness for subsequent pathogen attacks.

Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in arabidopsis

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1. function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.

Granulocyte-colony stimulating factor increases CD123hi blood dendritic cells with altered CD62L and CCR7 expression

Changes in blood dendritic cell (BDC) counts (CD123(hi)BDC and CD11c(+)BDC) and expression of CD62L, CCR7, and CD49d were analyzed in healthy donors, multiple myeloma (MM), and non-Hodgkin lymphoma (NHL) patients, who received granulocyte-colony stimulating factor (G-CSF) containing peripheral blood stem cell (PBSC) mobilization protocols. Low-dose G-CSF in healthy donors (8-10 mug/ kg/d subcutaneously) and high-dose G-CSF in patients (30 mug/kg/d) increased CD123(hi)BDC (2- to 22-fold, mean 3.7 x 10(6)/ L-17.7 x 10(6)/L and 1.9 x 10(6)/L-12.0 x 10(6)/ L) in healthy donors and MM but decreased CD11c(+)BDC (2- to 10-fold, mean 5.7 x 10(6)/L-1.6 x 10(6)/L) in NHL patients, on the day of apheresis, compared with steady state. After apheresis, CD123(hi)BDC counts remained high, whereas low CD11c(+)BDC counts tended to recover in the following 2-5 days. Down-regulation of CD62L and up-regulation of CCR7 on CD123(hi)BDC were found in most healthy donors and MM patients. CD49d expression was unchanged. Thus, PBSC mobilization may change BDC counts by altering molecules necessary for BDC homing from blood into tissues.

Duodenal expression of iron transport molecules in untreated haemochromatosis subjects

Background and aims: In HFE associated hereditary haemochromatosis, the duodenal enterocyte behaves as if iron deficient and previous reports have shown increased duodenal expression of divalent metal transporter 1 (DMT1) and iron regulated gene 1 (Ireg1) in affected subjects. In those studies, many patients had undergone venesection, which is a potent stimulus of iron absorption. Our study investigated duodenal expression of DMT1 ( IRE and non-IRE), Ireg1, hephaestin, and duodenal cytochrome-b (Dyctb) in untreated C282Y homozygous haemochromatosis patients, iron deficient patients, and iron replete subjects. Methods: Total RNA was extracted from duodenal biopsies and expression of the iron transport genes was assessed by ribonuclease protection assay. Results: Expression of DMT1 ( IRE) and Ireg1 was increased 3 - 5-fold in iron deficient subjects compared with iron replete subjects. Duodenal expression of DMT1 ( IRE) and Ireg1 was similar in haemochromatosis patients and iron replete subjects but in haemochromatosis patients with elevated serum ferritin concentrations, both DMT1 ( IRE) and Ireg1 expression were inappropriately increased relative to serum ferritin concentration. Hephaestin and Dcytb levels were not upregulated in haemochromatosis. DMT1 ( IRE) and Ireg1 levels showed significant inverse correlations with serum ferritin concentration in each group of patients. Conclusions: These findings are consistent with DMT1 ( IRE) and Ireg1 playing primary roles in the adaptive response to iron deficiency. Untreated haemochromatosis patients showed inappropriate increases in DMT1 ( IRE) and Ireg1 expression for a given level of serum ferritin concentration, although the actual level of expression of these iron transport genes was not significantly different from that of normal subjects.

Effects of A1 adenosine receptor overexpression on normoxic and post-ischemic gene expression

Objectives: To identify potential molecular genetic determinants of cardiovascular ischemic tolerance in wild-type and transgenic hearts overexpressing A(1) adenosine receptors (A(1)ARs). Methods: cDNA microarrays were used to explore expression of 1824 genes ill wild-type hearts and ischemia-tolerant mouse hearts overexpressing A(1)ARs. Results: Overexpression of A(1)ARs reduced post-ischemic contractile dysfunction, limited arrhythmogenesis, and reduced necrosis by similar to80% in hearts subjected to 30 min global ischemia 60 mill reperfusion. Cardioprotection was abrogated by acute A(1)AR antagonism, and only a small number (19) of genes were modified by A(1)AR overexpression in normoxic hearts. Ischemia-reperfusion significantly altered expression of 75 genes in wild-type hearts (14 induced, 61 down-regulated), including genes for metabolic enzymes, structural/motility proteins, cell signaling proteins, defense/growth proteins, and regulators of transcription and translation. A(1)AR overexpression reversed the majority of gene down-regulation whereas gene induction was generally unaltered. Additionally, genes involved in cell defence, signaling and gene expression were selectively modified by ischemia in transgenic hearts (33 induced, 10 down-regulated), possibly contributing to the protected phenotype. Real-time PCR verified changes in nine selected genes, revealing concordance with array data. Transcription of the A(1)AR gene was also modestly reduced post-ischemia, consistent with impaired functional sensitivity to A(1)AR stimulation Conclusions: Data are presented regarding the early post-ischemic gene profile of intact heart. Reduced A(1)AR transcription is observed which may contribute to poor outcome from ischemia. A(1)AR overexpression selectively modifies post-ischemic gene expression, potentially contributing to ischemic-tolerance. (C) 2003 European Society of Cardiology. Published by Elsevier Science B.V. All rights reserved.