[Molecular and immunological detection of bacteria applied to bio-terrorism]. / Détection moléculaire et immunologique des bactéries dans le cadre du bioterrorisme.

Following the episode of letters containing anthrax in the USA in 2001, the fight against bio-terrorism became a priority for many countries (including France). The detection of bacteria in bio-terrorism settings is a major component of this fight. Indeed, the early detection of these bio-terrorism agents leads to an appropriate treatment and to a reduced transmission of the disease. Bacteria are important bio-terrorism agents, and the techniques used for their detection are constantly evolving. In this review, after describing the main bacteria that can be used for bio-terrorism, we also describe the techniques available for their detection: DNA or antigen detection.

Identification of protein candidates for the serodiagnosis of Q fever endocarditis by an immunoproteomic approach.

Q fever is a worldwide zoonosis caused by Coxiella burnetii bacterium. Two clinical forms are present: acute Q fever and chronic disease, including endocarditis. Currently, the diagnosis of Q fever endocarditis is based on the detection of anti-phase I antibodies. The objective of the study was to identify candidate proteins for the serological diagnosis of endocarditis due to C. burnetii. The immunoreactivities of sera from 12 patients with C. burnetii infections, including the sera from patients with endocarditis and with the acute clinical form of Q fever, were compared with those of three control subjects who did not have Q fever. We identified 29 candidate antigenic proteins by mass spectrometry. Two proteins, arginine repressor and OmpH, were recognised exclusively by the sera of patients with Q fever endocarditis. These proteins are promising candidates for the development of serodiagnostic assays for Q fever endocarditis.

Scenario modeling potential eco-efficiency gains from a transition to organic agriculture: life cycle perspectives on Canadian canola, corn, soy, and wheat production.

We used Life Cycle Assessment to scenario model the potential reductions in cumulative energy demand (both fossil and renewable) and global warming, acidifying, and ozone-depleting emissions associated with a hypothetical national transition from conventional to organic production of four major field crops [canola (Brassica rapa), corn (Zea mays), soy (Glycine max), and wheat (Triticum aestivum)] in Canada. Models of these systems were constructed using a combination of census data, published values, and the requirements for organic production described in the Canadian National Organic Standards in order to be broadly representative of the similarities and differences that characterize these disparate production technologies. Our results indicate that organic crop production would consume, on average, 39% as much energy and generate 77% of the global warming emissions, 17% of the ozone-depleting emissions, and 96% of the acidifying emissions associated with current national production of these crops. These differences were almost exclusively due to the differences in fertilizers used in conventional and organic farming and were most strongly influenced by the higher cumulative energy demand and emissions associated with producing conventional nitrogen fertilizers compared to the green manure production used for biological nitrogen fixation in organic agriculture. Overall, we estimate that a total transition to organic production of these crops in Canada would reduce national energy consumption by 0.8%, global warming emissions by 0.6%, and acidifying emissions by 1.0% but have a negligible influence on reducing ozone-depleting emissions.

Light therapy for managing sleep, behaviour, and mood disturbances in dementia.

BACKGROUND: Rest-activity and sleep-wake cycles are controlled by the endogenous circadian rhythm generated by the suprachiasmatic nuclei (SCN) of the hypothalamus. Degenerative changes in the SCN appear to be a biological basis for circadian disturbances in people with dementia, and might be reversed by stimulation of the SCN by light. OBJECTIVES: The review assesses the efficacy of bright light therapy (BLT) in managing sleep, behaviour, mood, and cognitive disturbances associated with dementia. SEARCH STRATEGY: The trials were identified from a search of the Specialized Register of the Cochrane Dementia and Cognitive Improvement Group on 27 January 2004 using the terms "bright light*", "light box*", "light visor*", "dawn-dusk*", phototherapy (MESH), phototherapy, "photo therapy", "light therapy" "light treatment", light*. SELECTION CRITERIA: All relevant, randomized controlled trials in which BLT, at any intensity and duration, was compared with a control group for the effect on managing sleep, behavioural, mood, and cognitive disturbances (as well as changes in institutionalization rates and cost of care) on people with dementia of any degree of severity. DATA COLLECTION AND ANALYSIS: Three reviewers independently assessed the retrieved articles for relevance, methodological quality, and extracted data from the selected studies. The statistically significant differences in changes in outcomes from baseline to end of treatment and from baseline to follow-up between the light therapy and control groups were examined. Each study was summarized using a measure of effect (e.g. mean difference). Owing to lack of homogeneity between studies, their results were not combined. MAIN RESULTS: Five studies met the inclusion criteria. However, only three were included in the analyses because of inappropriate analyses reported or inability to retrieve the required data from the investigators. This review revealed no adequate evidence of the effectiveness of BLT in managing sleep, behaviour, and mood disturbances associated with dementia. REVIEWERS' CONCLUSIONS: There is insufficient evidence to assess the value of BLT for people with dementia. The available studies are of poor quality and further research is required.

The monocytic leukemia zinc finger protein MOZ is a histone acetyltransferase.

The monocytic leukemia zinc finger protein (MOZ) gene is rearranged in t(8;16)(p11;p13), t(8;22)(p11;q13) and inv(8)(p11q13) associated with acute myeloid leukemia. The other fusion partners involved are CBP, p300 and TIF2, transcriptional coactivators with known or potential histone acetyltransferase (HAT) activity. MOZ itself is a 2004-residue protein containing a putative acetyl CoA-binding motif, so it was hypothesized that MOZ is a HAT. Here we present direct evidence that MOZ has intrinsic HAT activity. Moreover, MOZ possesses a transcriptional repression domain at its N-terminal part and an activation domain at its C-terminal part. The activation domain does not show sequence similarity to any yeast proteins, but when tethered, it is able to activate transcription in yeast. Therefore, MOZ is a HAT with characteristics of a transcriptional coregulator, supporting the hypothesis that aberrant acetylation by abnormal MOZ proteins leads to leukemogenesis.

Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants.

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.

Forensic issues in pain: review of current practice.

Forensic activity in pain practice is reviewed with reference to the differing roles of the pain clinician and the independent expert. Ethical guidelines and recommendations for assessment, documentation, record review, and court testimony are discussed. Specific issues include the assessment of disability and impairment, malingering, and application of the Daubert standard in forensic pain practice. Examples of case law are reviewed for civil liability and CRPS, malpractice with opioid prescribing, and practice issues in a correctional setting.

HDAC4, a human histone deacetylase related to yeast HDA1, is a transcriptional corepressor.

Histone acetylation plays an important role in regulating chromatin structure and thus gene expression. Here we describe the functional characterization of HDAC4, a human histone deacetylase whose C-terminal part displays significant sequence similarity to the deacetylase domain of yeast HDA1. HDAC4 is expressed in various adult human tissues, and its gene is located at chromosome band 2q37. HDAC4 possesses histone deacetylase activity intrinsic to its C-terminal domain. When tethered to a promoter, HDAC4 represses transcription through two independent repression domains, with repression domain 1 consisting of the N-terminal 208 residues and repression domain 2 containing the deacetylase domain. Through a small region located at its N-terminal domain, HDAC4 interacts with the MADS-box transcription factor MEF2C. Furthermore, HDAC4 and MEF2C individually upregulate but together downmodulate c-jun promoter activity. These results suggest that HDAC4 interacts with transcription factors such as MEF2C to negatively regulate gene expression.

Identification of a human histone acetyltransferase related to monocytic leukemia zinc finger protein.

We describe here the identification and functional characterization of a novel human histone acetyltransferase, termed MORF (monocytic leukemia zinc finger protein-related factor). MORF is a 1781-residue protein displaying significant sequence similarity to MOZ (monocytic leukemia zinc finger protein). MORF is ubiquitously expressed in adult human tissues, and its gene is located at human chromosome band 10q22. MORF has intrinsic histone acetyltransferase activity. In addition to its histone acetyltransferase domain, MORF possesses a strong transcriptional repression domain at its N terminus and a highly potent activation domain at its C terminus. Therefore, MORF is a novel histone acetyltransferase that contains multiple functional domains and may be involved in both positive and negative regulation of transcription.

Negative regulation of glucocorticoid-dependent induction of c-fos by ras in intestinal epithelial cells.

In order to investigate the regulatory mechanisms involved in the expression of fos and jun family members by glucocorticoids, and the effect of ras transformation in intestinal epithelial cells, we used the rat cell line IEC-6. Dexamethasone treatment induced transiently c-jun mRNAs, in contrast to the sustained expression of c-fos, whereas its effect on junB expression resulted in a later increase. Dexamethasone-dependent stimulation of c-fos and c-jun was modulated predominantly at the level of transcription. Sustained levels of induced c-fos and c-jun proteins were observed after dexamethasone treatment. AP-1 DNA-binding capacity of c-fos, and to a smaller extent c-jun, was increased by glucocorticoids later than after serum treatment. To analyse the effect of ras on the glucocorticoid response of AP-1 components, we studied several IEC-6 cell clones transformed by the Ha-ras oncogene. In comparison to normal cells, these transformants displayed increased AP-1 DNA-binding activity with higher levels of junB and variable levels of c-jun in the AP-1 complex. Ras transformation repressed the growth-inhibitory properties of glucocorticoids. Furthermore, ras inhibited the glucocorticoid-dependent induction of c-fos protein and mRNA, leading to changes in AP-1 composition as compared to normal cells. As assessed by transient transfection luciferase assays, glucocorticoids induced significantly a minimal promoter containing 3 copies of an AP-1 DNA-binding site as well as the murine c-fos -276 to +112 promoter in non-transformed cells. In contrast, glucocorticoid addition did not induce these constructs in two ras transformed cell lines. These results suggest that ras negatively modulates specific responses of intestinal epithelial cells to glucocorticoids.

Activation of haptoglobin gene expression by cAMP involves CCAAT/enhancer-binding protein isoforms in intestinal epithelial cells.

CCAAT/enhancer-binding protein (C/EBP) isoforms are expressed in rodent intestine and in the rat intestinal epithelial cell line IEC-6 but their role remains to be determined. Treatment of IEC-6 cells with the adenylate cyclase activator forskolin led to coordinate induction of C/EBP isoforms alpha, beta and delta at the mRNA and protein levels. Transient transfection assays showed that their expression is controlled at the transcriptional level. Forskolin treatment induced haptoglobin mRNA levels. Electrophoretic mobility shift and supershift assays demonstrated an increase in DNA-binding activities of the three C/EBP isoforms to the haptoA and haptoC C/EBP DNA-binding sites of the proximal haptoglobin promoter. Site-specific mutations of both sites led to a decrease in transcriptional induction by forskolin, suggesting that C/EBP isoforms are involved in the cAMP-dependent regulation of the acute-phase protein gene haptoglobin in intestinal epithelial cells.

Regional differences in endothelial function in horse lungs: possible role in blood flow distribution?

We investigated regional differences of in vitro responses of pulmonary arteries (6-mm OD) from the dorsocaudal (top) and cranioventral (bottom) lung regions to endothelium-dependent vasodilators (methacholine, bradykinin, and calcium ionophore A-23187). Methacholine relaxed endothelium-intact top vessels; however, in bottom vessels, a small relaxation preceded a profound contraction. In top vessels, removal of endothelial cells converted relaxation to contraction, and in bottom vessels it abolished relaxation and enhanced contraction. Bradykinin and A-23187 were more potent and caused greater endothelium-mediated relaxation in top than in bottom arteries. The endothelium-independent vasodilator sodium nitroprusside caused similar relaxations in all rings. Nomega-nitro-L-arginine and NG-monomethyl-L-arginine and methylene blue abolished relaxation of top and bottom arteries to methacholine; meclofenamate had little effect. We conclude that regional differences in endothelium-mediated relaxation are caused by differences in the magnitude of the endothelial release of nitric oxide. Similar differences in endothelium-dependent flow-mediated vasodilation and endothelial nitric oxide release may result in preferential perfusion of caudodorsal lung regions.

NF-kappaB protects HIV-1-infected myeloid cells from apoptosis.

HIV-1 infection of primary monocytic cells and myeloid cell lines results in sustained NF-kappaB activation. Recently, NF-kappaB induction has been shown to play a role in protecting cells from programmed cell death. In the present study, we sought to investigate whether constitutive NF-kappaB activity in chronically HIV-1-infected promonocytic U937 (U9-IIIB) and myeloblastic PLB-985 (PLB-IIIB) cells affects apoptotic signaling. TNFalpha and cycloheximide caused infected cells to undergo apoptosis more rapidly than parental U937 and PLB-985 cells. Inhibition of TNFalpha-induced NF-kappaB activation using the antioxidant N-acetylcysteine (NAC) resulted in increased apoptosis in both U937 and U9-IIIB cells, while preactivation of NF-kappaB with the non-apoptotic inducer IL-1beta caused a relative decrease in apoptosis. Inhibition of constitutive NF-kappaB activity in U9-IIIB and PLB-IIIB cells also induced apoptosis, suggesting that NF-kappaB protects cells from a persistent apoptotic signal. TNFalpha plus NAC treatment resulted in a marked decrease in Bcl-2 protein levels in HIV-1-infected cells, coupled with an increase in Bax protein compared to uninfected cells, suggesting that the difference in susceptibility to TNFalpha-induced apoptosis may relate to the differences in relative levels of Bcl-2 and Bax. The protective role of NF-kappaB in blocking TNFalpha- and HIV-1-induced apoptosis was supported by studies in Jurkat T cells engineered to express IkappaB alpha repressor mutants (TD-IkappaB) under the control of a tetracycline-responsive promoter. Cells underwent apoptosis in response to TNFalpha only when NF-kappaB activation was inhibited by TD-IkappaB expression. As was observed for the U9-IIIB cells, TNFalpha treatment also induced a marked decrease in Bcl-2 protein levels in TD-IkappaB expressing cells. These experiments demonstrate that apoptotic signaling is perturbed in HIV-1-infected U9-IIIB cells and indicate that NF-kappaB activation may play an additional protective role against HIV-1-induced apoptosis in myeloid cells.

Inducible expression of IkappaBalpha repressor mutants interferes with NF-kappaB activity and HIV-1 replication in Jurkat T cells.

Human immunodeficiency virus (HIV-1) utilizes the NF-kappaB/Rel proteins to regulate transcription through NF-kappaB binding sites in the HIV-1 long terminal repeat (LTR). Normally, NF-kappaB is retained in the cytoplasm by inhibitory IkappaB proteins; after stimulation by multiple activators including viruses, IkappaBalpha is phosphorylated and degraded, resulting in NF-kappaB release. In the present study, we examined the effect of tetracycline-inducible expression of transdominant repressors of IkappaBalpha (TD-IkappaBalpha) on HIV-1 multiplication using stably selected Jurkat T cells. TD-IkappaBalpha was inducibly expressed as early as 3 h after doxycycline addition and dramatically reduced both NF-kappaB DNA binding activity and LTR-directed gene activity. Interestingly, induced TD-IkappaBalpha expression also decreased endogenous IkappaBalpha expression to undetectable levels by 24 h after induction, demonstrating that TD-IkappaBalpha repressed endogenous NF-kappaB-dependent gene transcription. TD-IkappaBalpha expression also sensitized Jurkat cells to tumor necrosis factor-induced apoptosis. De novo HIV-1 infection of Jurkat cells was dramatically altered by TD-IkappaBalpha induction, resulting in inhibition of HIV-1 multiplication, as measured by p24 antigen, reverse transcriptase, and viral RNA. Given the multiple functions of the NF-kappaB/IkappaB pathway, TD-IkappaBalpha expression may interfere with HIV-1 multiplication at several levels: LTR-mediated transcription, Rev-mediated export of viral RNA, inhibition of HIV-1-induced pro-inflammatory cytokines, and increased sensitivity of HIV-1-infected cells to apoptosis.

Quantitative culture of HIV-1 from bronchoalveolar lavage cells.

Determination of the infectious virus burden at the organ level is critical for understanding the pathophysiology of human immunodeficiency virus type 1 (HIV-1) infection. To evaluate the burden of HIV-1 in the lung, quantitative cultures were performed on bronchoalveolar lavage (BAL) cells from 11 HIV-1 seropositive subjects without respiratory infections and compared with peripheral blood mononuclear cells (PBMC) obtained from the same subjects. Fifty percent (50%) of subjects had positive BAL cell cultures while 82% had positive PBMC cultures. There was much less virus cultured from BAL cells than from PBMCs, whether using phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte (PBL) targets (p < 0.05) or adherent monocyte targets (p < 0.02). There was no significant difference between the HIV-1 titers obtained for BAL cells whether using PHA-stimulated PBL or adherent monocyte targets (p = 0.13). These studies demonstrate that BAL cell cultures for HIV-1 in subjects without respiratory infections are less frequently positive than PBMC cultures, that less virus can be recovered from BAL cells than from PBMC, and that HIV-1 isolates from BAL cells replicate in both PHA-stimulated PBL targets and adherent monocyte targets. Quantitative assessment of virus burden in the lung is important for future studies of HIV-1 pathogenesis and for evaluating potential antiretroviral therapies aimed at altering the natural history of organ dysfunction associated with retroviral replication.

Ventilation and carbon dioxide exchange in exercising horses: effect of inspired oxygen fraction.

Thoroughbred horses (TB) have no ventilatory response to added CO2 during near-maximal exercise. To see whether that reflects mechanical limits to ventilation or the control of breathing, we examined the effects of varying inspired O2 fraction (0.16, 0.21, or 0.30) in five normal TB standing quietly and galloping at 10 and 14 m/s on a level treadmill. We measured gas exchange (O2 consumption and CO2 production) and ventilation with a flow-through mask system. We also measured PO2, PCO2, and O2 contents in arterial and mixed venous blood and calculated cardiac output by using the Fick equation. Low inspired O2 fraction (0.16 vs. 0.21) had significant effects in TB galloping at 14 m/s. Arterial PO2 then was 38 Torr compared with 56 Torr for horses on air. Tidal volume and minute ventilation were 20% greater than their corresponding values on air, which were 12 liters and 1,475 l/min, respectively, whereas respiratory frequency did not change. O2 consumption and CO2 production were unchanged, but alveolar ventilation was 6% greater, despite increased alveolar and physiological dead spaces, so arterial PCO2 was lower (45 vs. 50 Torr on air). Thus, hypoxia was an effective stimulus to breathing, and minute ventilation was not mechanically limited in TB breathing air at the speeds studied.

Cardiac output but not high pulmonary artery pressure varies with FIO2 in exercising horses.

Horses have high mean pulmonary artery pressure (Ppa) both at rest and during exercise (approximately 30 and > or = 80 mmHg, respectively). The mechanisms are unknown. To see if hypoxic pulmonary vasoconstriction (HPV) plays a role, we compared pulmonary artery pressure-flow (Ppa-Q) curves when inspired O2 fraction (FIO2) was 0.16, 0.21, and 0.30, in 5 normal Thoroughbred horses standing quietly and while galloping at 10 and 14 m/sec on a level treadmill. We calculated O2 consumption (VO2) from measurements of respired gas composition and flow, and calculated Q from VO2 and measurements of oxygen content in arterial and mixed venous blood (CaO2 and CVO2). VO2 was 3.8, 74 and 128 ml.min-1.kg-1, at rest and at 10 and 14 m/sec, and did not vary with FIO2 at any speed. At 14 m/sec only, when FIO2 was lowered to 0.16, CaO2 fell (to 14.7 from 20 ml/dl on air), Q increased (to 0.86 from 0.66 L.min-1.kg-1 on air), and stroke volume increased (to 4.1 from 3.2 ml.kg-1 on air). Slopes and intercepts of Ppa-Q curves did not vary with FIO2. We conclude that HPV does not contribute to the high Ppa of exercising horses breathing air near sea level.

Influence of cyclooxygenase inhibitors on furosemide-induced hemodynamic effects during exercise in horses.

Furosemide, which commonly is used as a prophylactic treatment for exercise-induced pulmonary hemorrhage in horses, may mediate hemodynamic changes during exercise by altering prostaglandin metabolism. To determine if furosemide's hemodynamic effects during exercise in horses could be reversed, cyclooxygenase inhibitors were administered with furosemide. Four treatments were administered 4 hours prior to treadmill exercise at 9 and 13 m/s. They included a control treatment (10 ml of 0.9% NaCl solution, IV), furosemide (1 mg/kg of body weight, IV) administered alone, and furosemide in combination with phenylbutazone (4 mg/kg, IV, q 12 h for 2 days) or with flunixin meglumine (1.1 mg/kg, IV, on the day of experiment). Five horses were randomly assigned to complete all treatments. Physiologic variables at rest prior to exercise were not influenced by treatments. Furosemide, administered alone, reduced mean right atrial pressure and mean pulmonary artery pressure during exercise. The combinations of furosemide and flunixin meglumine or furosemide and phenylbutazone, at both levels of exercise intensity, returned mean right atrial pressure and mean pulmonary artery pressure to the value of the control treatment. During rest and exercise, plasma lactate concentration, PCV, heart rate, mean carotid artery pressure, oxygen consumption, carbon dioxide elimination, and cardiac output were not altered by any of the treatments. At 5 minutes after exercise, the administration of furosemide, alone or with phenylbutazone, reduced mean right atrial pressure. Other measured variables were not significantly influenced by treatments during recovery from exercise. These results suggested that cyclooxygenase inhibition partially reverses the decrease in mean right atrial pressure or pulmonary artery pressure induced by furosemide during exercise.(ABSTRACT TRUNCATED AT 250 WORDS)