Lead in the marine environment: concentrations and effects on invertebrates.

Lead (Pb) is a non-essential metal naturally present in the environment and often complexed with other elements (e.g., copper, selenium, zinc). This metal has been used since ancient Egypt and its extraction has grown in the last centuries. It has been used until recently as a fuel additive and is currently used in the production of vehicle batteries, paint, and plumbing. Marine ecosystems are sinks of terrestrial contaminations; consequently, lead is detected in oceans and seas. Furthermore, lead is not biodegradable. It remains in soil, atmosphere, and water inducing multiple negative impacts on marine invertebrates (key species in trophic chain) disturbing ecological ecosystems. This review established our knowledge on lead accumulation and its effects on marine invertebrates (Annelida, Cnidaria, Crustacea, Echinodermata, and Mollusca). Lead may affect different stages of development from fertilization to larval development and can also lead to disturbance in reproduction and mortality. Furthermore, we discussed changes in the seawater chemistry due to Ocean Acidification, which can affect the solubility, speciation, and distribution of the lead, increasing potentially its toxicity to marine invertebrates.

Spatiotemporal and functional characterisation of transient receptor potential vanilloid 4 (TRPV4) in the murine intervertebral disc.

The molecular regulators of mechano-transduction in intervertebral disc (IVD) cells are not well understood. The aim of the present study was to characterise the expression and function of the mechano-sensitive ion channel TRPV4 in the IVD. A novel transgenic reporter mouse, in which the endogenous Trpv4 locus drove the expression of LacZ, was used to localise Trpv4 expression at specific stages of spine development [embryonic day (E) 8.5, 12.5, 17.5, postnatal day 1] and time points following skeletal maturity (2.5, 6, 9 and 12 months). The TRPV4-specific agonist GSK1016790A and antagonist GSK2193874 were used to assess the functional response of annulus fibrosus (AF) cells using epifluorescence imaging with Ca2+-sensitive Fura-2 dye and F-actin staining. The effects of TRPV4 agonism and antagonism in mechanically stimulated AF cells were quantified by gene expression analysis. Trpv4 expression was specific to the developing notochord and intervertebral mesenchyme at E12.5. At 2.5, 6 and 9 months, Trpv4 expression was detected in the nucleus pulposus, inner AF, cartilage endplate and vertebral growth plate. AF cells treated with GSK1016790A demonstrated heterogeneity in TRPV4-dependent Ca2+ responses (no response, calcium oscillation or sustained response). TRPV4-induced Ca2+ signalling was associated with Rho/ROCK-dependent actin cytoskeleton remodelling and stress-fibre formation. In AF cells, cyclic-tensile-strain-induced changes in Acan and Prg4 expression were mediated by TRPV4 channel activation. These data establish TRPV4 as an important mechano- sensor regulating IVD mechano-biology.

Economic analysis of healthcare-associated infection prevention and control interventions in medical and surgical units: systematic review using a discounting approach.

Nosocomial or healthcare-associated infections (HCAIs) are associated with a financial burden that affects both patients and healthcare institutions worldwide. The clinical best care practices (CBPs) of hand hygiene, hygiene and sanitation, screening, and basic and additional precautions aim to reduce this burden. The COVID-19 pandemic has confirmed these four CBPs are critically important prevention practices that limit the spread of HCAIs. This paper conducted a systematic review of economic evaluations related to these four CBPs using a discounting approach. We searched for articles published between 2000 and 2019. We included economic evaluations of infection prevention and control of Clostridioides difficile-associated diarrhoea, meticillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, and carbapenem-resistant Gram-negative bacilli. Results were analysed with cost-minimization, cost-effectiveness, cost-utility, cost-benefit and cost-consequence analyses. Articles were assessed for quality. A total of 11,898 articles were screened and seven were included. Most studies (4/7) were of overall moderate quality. All studies demonstrated cost effectiveness of CBPs. The average yearly net cost savings from the CBPs ranged from $252,847 (2019 Canadian dollars) to $1,691,823, depending on the rate of discount (3% and 8%). The average incremental benefit cost ratio of CBPs varied from 2.48 to 7.66. In order to make efficient use of resources and maximize health benefits, ongoing research in the economic evaluation of infection control should be carried out to support evidence-based healthcare policy decisions.

C57BL/6 mice are resistant to joint degeneration induced by whole-body vibration.

OBJECTIVE: Whole-body vibration (WBV) platforms are commercially available devices that are used clinically to treat numerous musculoskeletal conditions based on their reported ability to increase bone mineral density and muscle strength. Despite widespread use, there is an alarming lack of understanding of the direct effects of WBV on joint health. Previous work by our lab demonstrated that repeated exposure to WBV using protocols that model those used clinically, induces intervertebral disc (IVD) degeneration and osteoarthritis-like damage in the knee of skeletally mature, male mice of a single outbred strain (CD-1). The present study examined whether exposure to WBV induces similar deleterious effects in a genetically different strain of mouse (C57BL/6). DESIGN: Male 10-week-old C57BL/6 mice were exposed to vertical sinusoidal WBV for 30 min/day, 5 days/week, for 4 or 8 weeks using previously reported protocols (45 Hz, 0.3 g peak acceleration). Following WBV, joint tissues were examined using histological analysis and gene expression was quantified using real-time PCR (qPCR). RESULTS: Our analyses show a lack of WBV-induced degeneration in either the knee or IVDs of C57BL/6 mice exposed to WBV for 4 or 8 weeks, in direct contrast to the WBV-induced damage previously reported by our lab in CD-1 mice. CONCLUSIONS: Together with previous studies from our group, the present study demonstrates that the effects of WBV on joint tissues vary in a strain-specific manner. These findings highlight the need to examine genetic or physiological differences that may underlie susceptibility to the deleterious effects of WBV on joint tissues.

Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone.

OBJECTIVE: Low-amplitude, high-frequency whole-body vibration (WBV) has been adopted for the treatment of musculoskeletal diseases including osteoarthritis (OA); however, there is limited knowledge of the direct effects of vibration on joint tissues. Our recent studies revealed striking damage to the knee joint following exposure of mice to WBV. The current study examined the effects of WBV on specific compartments of the murine tibiofemoral joint over 8 weeks, including microarchitecture of the tibia, to understand the mechanisms associated with WBV-induced joint damage. DESIGN: Ten-week-old male CD-1 mice were exposed to WBV (45 Hz, 0.3 g peak acceleration; 30 min/day, 5 days/week) for 4 weeks, 8 weeks, or 4 weeks WBV followed by 4 weeks recovery. The knee joint was evaluated histologically for tissue damage. Architecture of the subchondral bone plate, subchondral trabecular bone, primary and secondary spongiosa of the tibia was assessed using micro-CT. RESULTS: Meniscal tears and focal articular cartilage damage were induced by WBV; the extent of damage increased between 4 and 8-week exposures to WBV. WBV did not alter the subchondral bone plate, or trabecular bone of the tibial spongiosa; however, a transient increase was detected in the subchondral trabecular bone volume and density. CONCLUSIONS: The lack of WBV-induced changes in the underlying subchondral bone suggests that damage to the articular cartilage may be secondary to the meniscal injury we detected. Our findings underscore the need for further studies to assess the safety of WBV in the human population to avoid long-term joint damage.

The effect of exercise mode on the acute response of satellite cells in old men.

AIM: A dysregulation of satellite cells may contribute to the progressive loss of muscle mass that occurs with age; however, older adults retain the ability to activate and expand their satellite cell pool in response to exercise. The modality of exercise capable of inducing the greatest acute response is unknown. We sought to characterize the acute satellite cell response following different modes of exercise in older adults. METHODS: Sedentary older men (n = 22; 67 ± 4 years; 27 ± 2.6 kg*m(-2) ) were randomly assigned to complete an acute bout of either resistance exercise, high-intensity interval exercise on a cycle ergometer or moderate-intensity aerobic exercise. Muscle biopsies were obtained before, 24 and 48 h following each exercise bout. The satellite cell response was analysed using immunofluorescent microscopy of muscle cross sections. RESULTS: Satellite cell expansion associated with type I fibres was observed 24 and 48 h following resistance exercise only (P ˂ 0.05), while no expansion of type II-associated satellite cells was observed in any group. There was a greater number of activated satellite cells 24 h following resistance exercise (pre: 1.3 ± 0.1, 24 h: 4.8 ± 0.5 Pax7 + /MyoD+cells/100 fibres) and high-intensity interval exercise (pre: 0.7 ± 0.3, 24 h: 3.1 ± 0.3 Pax7 + /MyoD+cells/100 fibres) (P ˂ 0.05). The percentage of type I-associated SC co-expressing MSTN was reduced only in the RE group 24 h following exercise (pre: 87 ± 4, 24 h: 57 ± 5%MSTN+ type I SC) (P < 0.001). CONCLUSION: Although resistance exercise is the most potent exercise type to induce satellite cell pool expansion, high-intensity interval exercise was also more potent than moderate-intensity aerobic exercise in inducing satellite cell activity.

Disinfection byproduct formation during biofiltration cycle: Implications for drinking water production.

The goal of this study was to investigate the potential of biofiltration to reduce the formation potential of disinfection byproducts (DBPs). Particularly, the work investigates the effect of the duration of the filter cycle on the formation potential of total trihalomethanes (TTHM) and five species of haloacetic acids (HAA5), dissolved oxygen (DO), organic carbon, nitrogen and total phosphorous concentrations along with biofilm coverage of the filter media and biomass viability of the attached cells. The study was conducted on a full-scale biologically active filter, with anthracite and sand media, at the Britannia water treatment plant (WTP), located in Ottawa, Ontario, Canada. The formation potential of both TTHMs and HAA5s decreased due to biofiltration. However the lowest formation potentials for both groups of DBPs and or their precursors were observed immediately following a backwash event. Hence, the highest percent removal of DBPs was observed during the early stages of the biofiltration cycle, which suggests that a higher frequency of backwashing will reduce the formation of DBPs. Variable pressure scanning electron microscopy (VPSEM) analysis shows that biofilm coverage of anthracite and sand media increases as the filtration cycle progressed, while biomass viability analysis demonstrates that the percentage of cells attached to the anthracite and sand media also increases as the filtration cycle progresses. These results suggest that the development and growth of biofilm on the filters increases the DPB formation potential.

Vascular endothelial cell function in catastrophic antiphospholipid syndrome: a case report and review of the literature.

Catastrophic antiphospholipid syndrome (CAPS) is a rare autoimmune condition, which has been associated with a high mortality rate. However, with current management that includes a combination of anticoagulation, glucocorticoid administration, and plasma exchange, mortality rate has declined. Despite survival improvement with new generation immunosuppressive agents, their mechanisms of action are poorly defined, and CAPS is still considered a high-risk complication in patients known with antiphospholipid antibody syndrome. Herein, we present a case of a 79-year-old male who presented with a myocardial infarct and renal failure secondary to CAPS following a splenectomy for immune thrombocytopenia. Regardless of rapid combination of first-line treatment and rituximab therapy, the patient developed lethal cardiogenic shock secondary to mitral valve papillary muscle necrosis. Discussion of the pathophysiology and avenues of future therapies in CAPS are reported.

Non-traumatic necrosis of bone (osteonecrosis) is associated with endothelial cell activation but not thrombophilia.

OBJECTIVE: The pathophysiology of non-traumatic osteonecrosis (ON) or avascular necrosis (AVN) of the femoral head remains poorly understood. Some studies have suggested the contribution of underlying thrombophilia as a mechanism; however, no specific thrombophilic factor has been consistently found in association with the disease. We are presenting data suggesting a role for endothelial cell activation rather than thrombophilia in ON. METHODS: A prospective consecutive cohort of 49 patients with a diagnosis of ON. The disease was considered idiopathic in five and secondary in 44 patients. The investigation included a coagulation and thrombophilia profile, endothelial cell activation and non-specific inflammatory markers as well as a biochemical profile. Statistical analysis using Fisher's exact test was obtained to assess correlation between endothelial cell markers and variables of inflammation. RESULTS: Patients with non-traumatic ON were not found to have a specific underlying thrombophilic factor compared with the general population. Out of 49 patients,19 had elevation of at least one endothelial cell markers. We found that activation of endothelial cell markers was independently correlated to ON but not correlated to the presence of inflammation (P = 1.0000). CONCLUSION: These results suggest that non-traumatic ON is not associated with a specific thrombophilic abnormality in those affected. This study demonstrates a potential association between regional endothelial dysfunction and ON. More studies are needed at a molecular level to further investigate the specific role of endothelium in the physiopathology of ON. A better understanding of the underlying mechanism could lead to potential preventive and therapeutic strategies of this devastating disease.

Preliminary results of the laparoscopic adjustable gastric banding procedure by a new generation of silicone band: MIDBAND.

BACKGROUND: The surgical treatment of morbid obesity by laparoscopic adjustable gastric banding has become a "gold standard" in Europe. Currently, five types of silicone bands are used in the majority of countries performing bariatric surgery. METHODS: The MIDBAND was introduced to the European market in 2000. It is placed around the stomach using the Pars Flaccida technique described by Forsell. A prospective multicentric study on 113 cases was carried out to evaluate technical feasibility, complications, and the midterm weight loss outcomes (2 years). RESULTS: The percentage of excess body weight loss was 52.58% at 2 years. Perioperative mortality was nil and the complication rate was low (slippage <2%). CONCLUSION: These encouraging results require longer-term studies to validate this procedure.

An in vitro tissue model to study the effect of age on nucleus pulposus cells.

Differentiation between age (physiological) and disease-induced changes in the nucleus pulposus will facilitate our understanding of the mechanism(s) leading to the development of degenerative disc disease. The aim of this study was to develop an in vitro model that would allow the study of age-induced alterations of cell function in nucleus pulposus. Nucleus pulposus (NP) cells were isolated from intervertebral discs obtained from either calves (<9 months) or cows (>18 months). The cells were placed in culture and grown for 19 days. Although nucleus pulposus tissue was formed by the cells of the two different ages the more mature (older) cells formed less tissue as determined histologically by light microscopy. This was confirmed biochemically as the wet weight and proteoglycan content of the tissue formed by the older cells were significantly less than that of the younger tissue. The older cells accumulated less proteoglycans as determined by quantifying radioisotope incorporation. The older cells showed lower constitutive gene expression of collagen type II and aggrecan whereas collagen type I and link protein levels were similar to those of the younger cells. Metalloprotease (MMP) 13 gene and protein expression increased with age. There was no change in the levels of gene expression of MMP 2 and TIMP 1, 2, or 3 with age. Cells obtained from NP tissue harvested from younger or mature animals showed both genotypic and phenotypic differences in vitro that resulted in the inability of the older cells to reconstitute their extracellular matrix to the same extent as the younger cells. This suggests that this in vitro NP tissue model will be suitable to determine the mechanism(s) regulating age-induced changes.

Retinoic acid and lipopolysaccharide act synergistically to increase prostanoid concentrations in rats in vivo.

Vitamin A and its active metabolite retinoic acid (RA) modulate host-pathogen interactions by interfering with the host immune and inflammatory response including prostaglandin (PG) biosynthesis. The effects of RA on phospholipase A(2) (PLA(2)) and cyclooxygenase (COX) isoforms in vitro are controversial, and few in vivo studies exist. We investigated the in vivo effects of RA on PG biosynthesis in the presence or absence of lipopolysaccharide (LPS) in rats. RA alone [10 mg/(kg. d) for 5 d] increased plasma and liver PG concentrations by increasing COX-1 protein expression (twofold that of control rats). RA acted synergistically with LPS to increase plasma (400-fold) and liver (15-fold) concentrations of prostaglandin E(2) (PGE(2)) and significantly, but to a lesser extent, other PG compared with RA rats, in the absence of major differences in PLA(2) expression or activity or COX-1 and COX-2 mRNA or protein expression. The RA + LPS-mediated increase in PGE(2) was significantly attenuated (97%) by aminoguanidine (AG), a relatively specific inhibitor of the inducible nitric oxide synthase (NOS2), consistent with the previously reported synergistic effect of RA and LPS on NOS2 expression and activity. In addition, RA and LPS induced the expression of the microsomal isoform of PGE synthase (mPGES). In conclusion, in vivo, RA and LPS increased PG and especially PGE(2) concentrations. The PGE(2) increase was associated with NOS2-mediated activation of COX and induction of mPGES. These results contribute to the characterization of the effects of vitamin A on the host inflammatory response.

Evidence of functional myocardial ischemia associated with myocardial dysfunction in brain-dead pigs.

BACKGROUND: Cardiac dysfunction after brain death has been documented, but its mechanisms remain unclear. Myocardial ischemia has been suggested as a possible cause. The aim of the present study was to investigate the existence of an imbalance between myocardial oxygen delivery and demand as a possible cause of myocardial dysfunction in brain-dead pigs. METHODS AND RESULTS: Interstitial myocardial lactate and adenosine concentrations were assessed with cardiac microdialysis in 2 groups of animals: brain-dead pigs (n=7) and brain-dead pigs treated with labetalol (10+/-3 mg/kg) (n=7). Heart rate (HR), left ventricular (LV) dP/dt(max), rate-pressure product (RPP), cardiac output (CO), and left anterior descending coronary artery blood flow (QLAD) were continuously monitored. Brain-dead pigs exhibited a transient significant increase in HR, LV dP/dt(max), RPP, and CO and a limited increase in QLAD. This resulted in functional myocardial ischemia attested to by the significantly increased adenosine and lactate microdialysate concentrations. In brain-dead pigs treated with labetalol, there was a moderate increase in HR, QLAD, and adenosine microdialysate concentrations; LV dP/dt(max), RPP, CO, and myocardial lactate concentrations remained stable, confirming the preservation of aerobic metabolism. CONCLUSIONS: Brain death was associated with an increase in myocardial interstitial adenosine and lactate concentrations, as well as with myocardial dysfunction; all were attenuated by labetalol, suggesting an imbalance between oxygen consumption and oxygen delivery as a possible cause of myocardial dysfunction after brain death.

Lipopolysaccharide-induced increase of prostaglandin E(2) is mediated by inducible nitric oxide synthase activation of the constitutive cyclooxygenase and induction of membrane-associated prostaglandin E synthase.

NO produced by the inducible NO synthase (NOS2) and prostanoids generated by the cyclooxygenase (COX) isoforms and terminal prostanoid synthases are major components of the host innate immune and inflammatory response. Evidence exists that pharmacological manipulation of one pathway could result in cross-modulation of the other, but the sense, amplitude, and relevance of these interactions are controversial, especially in vivo. Administration of 6 mg/kg LPS to rats i.p. resulted 6 h later in induction of NOS2 and the membrane-associated PGE synthase (mPGES) expression, and decreased constitutive COX (COX-1) expression. Low level inducible COX (COX-2) mRNA with absent COX-2 protein expression was observed. The NOS2 inhibitor aminoguanidine (50 and 100 mg/kg i.p.) dose dependently decreased both NO and prostanoid production. The LPS-induced increase in PGE(2) concentration was mediated by NOS2-derived NO-dependent activation of COX-1 pathway and by induction of mPGES. Despite absent COX-2 protein, SC-236, a putative COX-2-specific inhibitor, decreased mPGES RNA expression and PGE(2) concentration. Ketoprofen, a nonspecific COX inhibitor, and SC-236 had no effect on the NOS2 pathway. Our results suggest that in a model of systemic inflammation characterized by the absence of COX-2 protein expression, NOS2-derived NO activates COX-1 pathway, and inhibitors of COX isoforms have no effect on NOS2 or NOS3 (endothelial NOS) pathways. These results could explain, at least in part, the deleterious effects of NOS2 inhibitors in some experimental and clinical settings, and could imply that there is a major conceptual limitation to the use of NOS2 inhibitors during systemic inflammation.

Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions.

We have studied the role of phosphorylation in the activation of metal-regulatory transcription factor-1 (MTF-1) and metallothionein (MT) gene expression. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulates MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to examine the possible involvement of kinase cascades in the activation of MTF-1. Metal-induced MT gene expression was abrogated by protein kinase C (PKC), c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase, and tyrosine-specific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase reporter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation of MT gene transcription by metals. By using dominant-negative mutants of PKC, JNK, mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evidence supporting a role for PKC and JNK in the activation of MTF-1 in response to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated gene expression. This suggests that phosphorylation is essential for MTF-1 transactivation function. We hypothesize that metal-induced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Thus, metal ions such as cadmium could activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 signal transduction pathway.

The impact of pre-existing end-stage renal disease on survival in acutely injured trauma patients.

End-stage renal disease and associated dialysis procedures alter homeostatic mechanisms and adversely affect the respiratory, cardiac, and central nervous systems. Currently outcomes research in acutely injured trauma patients utilizes Trauma and Injury Severity Score methodology with the Injury Severity Score and Revised Trauma Score, which do not account for comorbidities. Literature has yet to emerge that analyzes the effects of end-stage renal disease on acutely injured trauma patients. A retrospective review at an urban Level I trauma center was performed of all end-stage renal disease patients' medical records who were admitted for acute traumatic injury from 1994 through 1997. The charts were abstracted for age, sex, race, method of dialysis, specific injury, need for operation, etiology of trauma, length of stay, disposition from hospital, morbidity, and mortality. The Injury Severity Score; probability of survival; and W, M, and Z statistics were then calculated. The data collected were then compared with the overall data for the trauma center including patients with and those without end-stage renal disease during this time period. Mortality for patients with end-stage renal disease after suffering an acute traumatic injury is 2.45 that of the general population. Increased mortality was most prevalent in operative patients and those with Injury Severity Score >15. The average length of stay in the hospital was 55.3 per cent longer for patients with end-stage renal disease. Pre-existing end-stage renal disease negatively impacts survival after traumatic injury. A prospective multicentered study comparing renal patients with nonrenal patients is warranted. This would confirm the need for databases to account for the increased morbidity and mortality associated with end-stage renal disease when calculating probability of survival values for acutely injured trauma patients. Similarly future studies analyzing the affects of other comorbidities such as diabetes, chronic obstructive pulmonary disease, and hypertension on acutely injured trauma patients would help develop a more accurate method of predicting outcomes.

The value of clonality in the diagnosis and follow-up of patients with cutaneous T-cell infiltrates.

The diagnosis of early stages of cutaneous T-cell lymphoma (CTCL) is often difficult, especially for lesions that are at the borderline between reactive and neoplastic skin T-cell infiltrates. T-cell monoclonality in these lesions is considered by some to be an important prognostic factor of neoplastic evolution, whereas others claim that clonality can also be found in benign skin infiltrates and is therefore of limited diagnostic value. To address this controversy, the authors analyzed retrospectively eight patients with lymphocytic skin lesions who progressed to CTCL, and three patients with recurrent T-cell lymphocytic infiltrates who had not developed CTCL. From a total of 65 biopsies of eight progressing patients, 32 were diagnosed as histologically malignant and 33 were diagnosed as benign or borderline. The authors found clonality by either polymerase chain reaction or Southern blot analysis in 88% of malignant and in 79% of nonmalignant lesions. None of the 37 biopsies of non-progressing patients was clonal. These results indicate strongly that the presence of monoclonality in T-cell skin infiltrates is related closely to the risk of developing CTCL. The value of clonality as a marker of malignancy is supported by the absence of T-cell clonal populations in all infiltrates from patients who had not progressed to lymphoma.

Retinoic acid attenuates inducible nitric oxide synthase (NOS2) activation in cultured rat cardiac myocytes and microvascular endothelial cells.

S. Grosjean, Y. Devaux, C. Seguin, C. Meistelman, F. Zannad, P.-M. Mertes, R. A. Kelly and D. Ungureanu-Longrois. Retinoic Acid Attenuates Inducible Nitric Oxide Synthase (NOS2) Activation in Cultured Rat Cardiac Myocytes and Microvascular Endothelial Cells. Journal of Molecular and Cellular Cardiology (2001) 33, 933-945. The inducible NO synthase (NOS2) in cardiac tissue contributes to myocardial and coronary inflammation and dysfunction. Several natural (endogenous) hormones such as retinoic acid, the active metabolite of vitamin A, have the ability to attenuate NOS2 activation in inflammatory cells. The aim of this study was to investigate the effect of RA on NOS2 activation in cultured cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). CMEC were stimulated either with a combination of 10 microg/ml lipopolysaccharide (LPS) and 50 IU/ml interferon- gamma (IFN- gamma) or with a combination of 1 ng/ml interleukin-1 beta (IL-1 beta)+IFN- gamma whereas ARVM were stimulated with 1 ng/ml IL-1 beta and 50 IU/ml IFN- gamma in the absence or presence of all-trans retinoic acid (atRA). Activation of the NOS2 pathway was estimated by measurement of mRNA (Northern blot) and protein (Western blot) expression, enzyme activity by conversion of [(3)H]L -arginine to [(3)H]L -citrulline, and nitrite accumulation. NOS2 mRNA half-life was studied in CMEC and ARVM in the presence of actinomycin D. In CMEC and ARVM stimulated with a combination of LPS and/or cytokines, atRA (10(-6), 10(-5)M) significantly (P<0.05) attenuated NOS2 mRNA and protein expression, enzymatic activity and reduced supernatant nitrite concentration. Upon stimulation with LPS/IFN- gamma, atRA significantly decreased NOS2 mRNA half-life. This was not seen after stimulation with IL-1 beta/IFN- gamma. These results document for the first time an effect of RA on NOS2 activation in cardiac cells. They may contribute to the characterization of the immunomodulatory effects of retinoids in myocardial and coronary inflammatory disorders.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1.

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.