Magnesium deficiency during pregnancy in mice impairs placental size and function.

OBJECTIVE: Maternal magnesium (Mg) deficiency has been associated with fetal growth restriction. Using a mouse model of maternal Mg deficiency-induced fetal growth restriction, we sought to investigate the effect of Mg deficiency on placental physiology and function. METHODS: In vivo: Pregnant Swiss Webster mice were fed either 100% of the recommended amount of Mg (control) or 10%Mg (Mg-deficient) (8 per group). Dams were euthanized on gestational day 17 and placentas were collected, weighed and assessed for Mg concentrations, as well as nutrient transporter mRNA expression. For nutrient transfer studies, control and Mg-deficient dams (6 per group) were injected with (14)C-amino acids and (3)H-glucose and trans-placental passage was determined. In vitro: BeWo placental cells were grown in media containing 10%Mg to 100%Mg and the effects of Mg status on cell proliferation, oxidative stress and nutrient uptake were measured. Data were analyzed by Student's t-tests comparing controls vs. Mg-deficient animals or cells. For multiple comparisons, data were analyzed by ANOVA followed by Dunnett's post hoc testing. RESULTS: In vivo: Maternal Mg deficiency decreased placental Mg content, placental and fetal weights, ratio of fetal:placental weight (P < 0.05), placental Slc7a5 transporter mRNA expression and transplacental nutrient transport (P < 0.05). In vitro: Mg deficiency reduced BeWo nutrient uptake (P < 0.01) and cell proliferation (P < 0.01), and increased oxidative stress (P < 0.01). CONCLUSION: These findings highlight the adverse effects of maternal Mg deficiency on fetal weight and placental function, including transport and proliferation and may explain the fetal growth restriction observed with moderate Mg deficiency in mice.

Obesity shifts house dust mite-induced airway cellular infiltration from eosinophils to macrophages: effects of glucocorticoid treatment.

Although classically characterized by chronic airway inflammation with eosinophil infiltration, asthma is a complex and multifactorial condition with numerous clinical phenotypes. Epidemiological studies strongly support the link between obesity and asthma and suggest that obesity precedes and promotes asthma development, increases asthma severity, and reduces steroid responsivity. Using a house dust mite (HDM) model of airway hyperresponsiveness in C57BL/6 mice, we examined the effects of diet-induced obesity on allergic airway inflammation and its treatment with dexamethasone. When compared to lean mice treated with HDM, obese-HDM mice had reduced plasma adiponectin, an anti-inflammatory adipokine, lower eosinophil and higher macrophage infiltration into the lungs and bronchoalveolar lavage (BAL) fluid, increased expression of total, M1, and M2 macrophage markers in the lungs, and enhanced Th2 and non-Th2 cytokine expression in the lungs. While Th2-associated responses in obese-HDM mice were suppressed by systemic dexamethasone, several Th2-independent responses, including total and M1 macrophage markers in the lungs, and lung CXC-motif ligand 1 (CXCL1) levels, were not improved following dexamethasone treatment. Thus, HDM combined with obesity promotes mixed localized inflammatory responses (e.g., M1, M2, Th1, and Th2) and shifts the cellular infiltration from eosinophils to macrophages, which are less sensitive to dexamethasone regulation. Because obese asthmatics exhibit more severe symptoms, lack a predominance of Th2 biomarkers, and are predicted to experience more steroid resistance when compared to lean asthmatics, this model could be used to study blunted steroid responses in obese-HDM mice and to define the macrophages found in the lungs.

Magnesium sulfate reduces bacterial LPS-induced inflammation at the maternal-fetal interface.

OBJECTIVES: Maternal magnesium sulfate (MgSO4) administration exerts anti-inflammatory and fetal neuroprotective effects. Based on the link between placental inflammation and fetal immune responses, we examined the effect of MgSO4 on LPS-induced inflammation at the maternal-fetal interface. STUDY DESIGN: In vivo model: Pregnant rats (GD19) were injected intraperitoneally with saline, LPS, or MgSO4 plus LPS (n = 6 per group). Rats were euthanized; placentas were assayed for CCL2, IL6, and TNFα and placentas were screened for gene expression. Ex vivo model: Human placental cultures were treated with vehicle, LPS, or MgSO4 plus LPS. Supernatants were assayed for CCL2, IL6, and TNFα. In addition, placental cultures were analyzed for inflammation-related gene expression and NFκ B activation. RESULTS: In vivo model: Maternal LPS administration resulted in pro-inflammatory mediator production within the placenta; maternal MgSO4 treatment significantly attenuated LPS-induced inflammation. Several placental transcripts (APOE, CCL4, CXCL1, and NFκBIZ) differentially expressed following maternal LPS challenge were counter-regulated by MgSO4 treatment. Ex vivo model: LPS promoted human placental inflammation and MgSO4 significantly reduced inflammation induced by LPS. MgSO4 treatment of human placental explants significantly reversed the expression of numerous genes sensitive to LPS regulation and suppressed LPS-induced NFκB activation. CONCLUSIONS: MgSO4 administration inhibited placental inflammation during LPS-mediated maternal infection. Several placental inflammatory genes whose expression was regulated by LPS were reversed by MgSO4 treatment. Our data support the hypothesis that MgSO4 attenuates excessive inflammation at the maternal-fetal interface, which when uncontrolled may compromise neonatal health, including neurologic outcomes.

Photodynamic therapy for circumscribed choroidal haemangioma: a case report.

Choroidal haemangioma is a benign tumour with visual acuity diminution due to subretinal fluid accumulation. There are many modalities of treatment of this visually disabling syndrome, some of them being argon laser photocoagulation, cryotherapy, external beam irradiation, proton beam radiotherapy, episcleral plaque radiotherapy and transpupillary thermotherapy. Another new modality of treatment with remarkable success rate is photodynamic therapy. In this modality a photosensitiser is injected intravenously followed by irradiation of a specific wave length for a specified time period. The photosensitiser concentrates within the vascular channels and after irradiation these channels are irreversibly obliterated. A 62 years old female patient of choroidal haemangioma, who presented in eye outpatient department was treated with the standard protocol used for photodynamic therapy. On follow-up of this patient it was found that there was improvement in the visual acuity from 6/12 in the left eye (affected eye) to 6/9. Not only was there an improvement in the visual acuity but there was anatomical improvement too as was evident by regressed cystoid macular oedema and circumscribed choroidal haemangioma. After six months of follow-up there was no leakage of dye with digital fluorescein angiography and indocyanine green.

Physiological activities of carbon monoxide-releasing molecules: Ca ira.

In this issue of British Journal of Pharmacology, Megías and colleagues demonstrate how preincubation of human colonic Caco-2 cells with CORM-2, a carbon monoxide releasing molecule (CO-RM), reduces the expression of inducible nitric oxide synthase, interleukin (IL)-6 and IL-8 caused by proinflammatory cytokines. A role for IL-6 in the regulation of metalloproteinase (MMP)-7 expression by CORM-2 is described. However, it is the demonstration that CORM-2 inhibits MMP-7 or matrilysin expression, which is most intriguing as this small MMP has been implicated in carcinogenesis. Thus, CO-RMs appear to now possess chemoprotective properties and, in this particular case, may influence inflammation-induced colon carcinogenesis via modulation of nuclear factors participating in the transcription of genes implicated in the development of intestinal inflammation and cancer. This report opens yet another door for research involving these exciting molecules and it is now clear that further discoveries of the beneficial properties of CO-RMs will go on.

Cross-sectional study of consumption, compliance and awareness about antibiotic utilisation amongst the urban community in Kolkata.

A cross-sectional survey was conducted upon 500 respondents, comprising of 250 adults and 250 children who did consume antibiotics in the previous three months. Data were analysed to determine the patterns of utilisation, compliance and awareness regarding antibiotic medication amongst a selected urban population at Kolkata. Antibiotic consumption without prescription was evident amongst 41.2% of adults in comparison to that of 8.4% in children (P < 0.01). Compliance to daily dosage was observed in 40.8% of adults in comparison to 82.8% in children (P < 0.01). Awareness pattern regarding antibiotics were reported to have been more in the children group (16.4%) while compared to the adults (8%). The knowledge regarding antibiotic resistance remained more or less similar in both the groups. The study concludes that high over the counter (OTC) sale and inadequate compliance to antibitotic medication needs further intervention approach towards information, education and communication (IEC) to all concerned.

The tyrosine kinase inhibitor tyrphostin AG 126 reduces the multiple organ failure induced by zymosan in the rat.

OBJECTIVE: To investigate the effects of tyrphostin AG 126, a tyrosine kinase inhibitor, on the multiple organ failure (MOF) caused by zymosan in the rat. DESIGN: Zymosan (500 mg/kg, suspended in saline solution, i.p.) causes an enhanced formation of reactive oxygen species, which contribute to the pathophysiology of MOF. After zymosan or saline administration, animals were monitored for 12 days. MEASUREMENTS AND RESULTS: Treatment of rats with tyrphostin AG 126 (10 mg/kg, 3 mg/kg or 1 mg/kg intraperitoneally, 1 h and 6 h after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan in a dose-dependent fashion. Tyrphostin AG 126 also attenuated the lung, liver, and intestinal injury (histology) as well as the increase in the levels of myeloperoxidase and malondialdehyde caused by zymosan in the lung, liver, and intestine. Immunohistochemical analysis for nitrotyrosine, poly (ADP-ribose) polymerase (PAR), iNOS, and COX-2 revealed a positive staining in lung, liver and intestine from zymosan-treated rats. The degree of staining for nitrotyrosine, PAR, iNOS, and COX-2 were markedly reduced in tissue sections obtained from zymosan-treated rats which had received tyrphostin AG 126. Furthermore, treatment of rats with tyrphostin AG 126 significantly reduced the production of peroxynitrite and of pro-inflammatory cytokines TNF-alpha and IL-1beta. CONCLUSIONS: This study provides the first evidence that the protein kinase inhibitor tyrphostin AG 126 attenuates the degree of MOF associated with zymosan-induced peritonitis in the rat.

Using PAC nested deletions to order contigs and microsatellite markers at the high repetitive sequence containing Npr3 gene locus.

Highly polymorphic di- and tetranucleotide repeats in and around Npr3, a potential candidate gene for hypertension, have been identified using a novel approach. Because this chromosomal site is rich in repetitive DNA and difficult to sequence, P1 artificial chromosomes were retrofitted with a loxP transposon to map the gene sequence within a clone using a series of nested deletions. Sequences from ends of deletions 1-3 kb apart identified a (CA)(20) and a (TA)(18)-(CA)(8) repeat 8 kb upstream and within an intron of Npr3, respectively. DNA from 17 individuals was analyzed for length polymorphisms in these and eight additional repeats identified in 200 kb of working draft sequence from this region in GenBank. The sequence contigs and microsatellite repeats from GenBank were ordered using the P1-derived artificial chromosome deletion series. Several of these repeats were found to vary considerably in length in the set of genomic DNA tested. Since this site in chromosome 5p has recently been implicated in disease in studies with genetically hypertensive rats, the microsatellite markers reported here will be useful for genetic analysis and may even be implicated in the disease process in humans. We discuss how these types of data are useful for interpreting draft DNA sequence coming out of the genome projects, and the utility of deletion clones as a resource for ordering contigs and gap filling.

The selective guanylate cyclase inhibitor ODQ reduces multiple organ injury in rodent models of Gram-positive and Gram-negative shock.

OBJECTIVE: An enhanced formation of endogenous nitric oxide contributes to the circulatory failure caused by endotoxin (lipopolysaccharide). Many of the biological actions of nitric oxide are mediated by the guanylate cyclase/cyclic guanosine 3prime;,5'-monophosphate system. We recently discovered that two cell wall components, namely lipoteichoic acid and peptidoglycan of the Gram-positive bacterium Staphylococcus aureus, synergize to cause shock and multiple organ dysfunction syndrome in the rat. Here we investigate the effects of a selective guanylate cyclase inhibitor, 1H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ), on the circulatory failure and multiple organ dysfunction syndrome (kidney, liver, lung) caused by a) coadministration of lipoteichoic acid and peptidoglycan (Gram-positive shock) or b) lipopolysaccharide (Gram-negative shock) in the anesthetized rat. Furthermore, we investigated whether ODQ scavenges superoxide anions and/or hydroxyl radicals. DESIGN: The in vivo portion of the study was a prospective, randomized, controlled animal study. The in vitro portion included a) cultured ventricular myoblasts of the rat, H9c2(2-1) cells, and b) a cell free superoxide anion assay system. SETTING: University-based research laboratory. SUBJECTS: Seventy-five anesthetized, male Wistar rats were used for the in vivo study. INTERVENTIONS: For the in vivo portion of the study, after surgical preparation, anesthetized rats were observed for 6 hrs. All rats were pretreated and received an intravenous infusion of saline (1.5 mL.kg-1.hr-1), which was maintained throughout the experiment. The rats were assigned to nine groups. Group 1 contained control rats (sham) subjected to 2 mL/kg saline intraperitoneally, 2 hrs before the experiment (n = 7). Group 2 contained control rats (sham) that received 2 mg/kg ODQ intraperitoneally, 2 hrs before the experiment (n = 9). Group 3 contained control rats (sham) that received 2 mL/kg dimethyl sulfoxide, 30% v/v in saline intraperitoneally, as a vehicle for ODQ, 2 hrs before the experiment (n = 6). In group 4 rats, Gram-positive shock was induced by coadministration of lipoteichoic acid (3 mg/kg intravenously) and peptidoglycan (10 mg/kg intravenously) (n = 10). In group 5, rats were pretreated with ODQ (as described previously) before lipoteichoic acid/peptidoglycan (n = 9). In group 6, rats were pretreated with dimethyl sulfoxide (as described previously) before lipoteichoic acid/peptidoglycan (n = 7). In group 7, Gram-negative shock was induced by lipopolysaccharide (6 mg/kg intravenously) (n = 11). In group 8, rats were pretreated with ODQ (as described previously) before lipopolysaccharide (n = 8). In group 9, rats were pretreated with dimethyl sulfoxide (as described previously) before lipopolysaccharide (n = 8). For the in vitro portion of the study, rat cells were preincubated with vehicle (saline and/or dimethyl sulfoxide) and ODQ (0.1 microM to 1 mM) for 2 hrs. The cells then were exposed to H2O2 (1 mM) for 4 hrs at 37 degrees C, after which time cell viability was determined by measuring the mitochondrial-dependent reduction of 3-(4,5-di-methyliazol-2-yl)-2,5-diphenyltetrazolium bromide to blue formazan. Next, an aqueous solution was incubated with ODQ (as described previously), and superoxide anions were produced by using a hypoxanthine/xanthine-oxidase assay. The chemiluminescence assay was used to evaluate any potential antioxidative effects of ODQ. MEASUREMENTS AND MAIN RESULTS: In vivo, administration of lipoteichoic acid/peptidoglycan or lipopolysaccharide resulted within 6 hrs in hypotension, acute renal dysfunction, hepatocellular injury, and lung injury. Pretreatment of rats with ODQ attenuated the renal dysfunction, lung injury, and hepatocellular injury caused by lipoteichoic acid/peptidoglycan or lipopolysaccharide. In vitro, administration of H2O2 (for 4 hrs) to rat cardiomyoblasts decreased mitochondrial respiration attributable to generation of hydroxyl radicals. Pretreatment of cells with ODQ did not abolish this cell injury. In addition, ODQ did not scavenge superoxide anions. CONCLUSIONS: These results imply that ODQ, an inhibitor of guanylate cyclase, reduces the multiple organ injury and dysfunction caused by wall fragments of Gram-positive or Gram-negative bacteria in the anesthetized rat. The observed protective effects of ODQ are not attributable to the ability of ODQ to reduce the formation or the effects of superoxide anions or hydroxyl radicals.

Calpain inhibitor-1 reduces renal ischemia/reperfusion injury in the rat.

BACKGROUND: Activation of the cysteine protease calpain has been implicated in renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of calpain inhibitor-1 (Cal I-1) in an in vivo model of renal I/R injury. METHODS: Male Wistar rats were administered Cal I-1 (10 mg/kg, IP) 30 minutes before undergoing bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Plasma concentrations of urea, creatinine, Na(+), gamma-glutamyl transferase (gamma GT), aspartate aminotransferase (AST) and urinary Na(+), glutathione S-transferase (GST), and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal dysfunction and I/R injury. Creatinine clearance (C(Cr)) and fractional excretion of Na(+) (FE(Na)) were used as indicators of glomerular and tubular function, respectively. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of neutrophil infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). RESULTS: Cal I-1 significantly reduced I/R-mediated increases in urea, creatinine, gamma GT, AST, NAG, and FE(Na) and significantly improved C(Cr). Cal I-1 also significantly reduced kidney MPO activity and MDA levels. Cal I-1 also reduced histologic evidence of I/R-mediated renal damage and caused a substantial reduction in the expression of iNOS and COX-2, both of which involve activation of nuclear factor-kappa B (NF-kappa B). CONCLUSIONS: : These results suggest that Cal I-1 reduces the renal dysfunction and injury associated with I/R of the kidney. We suggest that the mechanism could involve the inhibition of I/R-mediated activation of NF-kappa B.

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF-kappaB (kidney), expression of iNOS and COX-2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF-kappaB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF-kappaB to DNA and the degradation of IkappaBalpha, IkappaBbeta, and IkappaBvarepsilon. Selective inhibition of iNOS activity with L-NIL reduced the circulatory failure and liver injury, while selective inhibition of COX-2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF-kappaB and thus prevention of the expression of NFkappaB-dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX-2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.

Analysis of a clonal selection event during transposon-mediated nested-deletion formation in rare BAC and PAC clones.

Nested deletions from one end of the genomic DNA in bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) are readily generated by inserting a loxP site-containing Tn10 minitransposon into the recombinant clone and transducing with P1 phage. Although the size of clones in the deletion series is largely random, in about 5% of BACs and PACs the distribution appears skewed to a certain length, and in rare cases (<1%) is definitely skewed to a particular size. Here we investigate this relatively rare phenomenon and validate that sequence-specific transposon insertions are not the cause of such skewed nested-deletion libraries. Instead, a detailed analysis of our experiments with a BAC clone demonstrating this unusual feature indicates that deletions of a certain size arise from clonal expansion of a transposon insertion as a result of transient derepression of the transposase gene prior to IPTG induction. Transposition itself shows no bias to any particular region of insert DNA in the clone. We suggest a simple modification to the procedure for generating nested-deletions that allows all BACs and PACs to produce nested-deletions of random size. These findings should provide additional insight into the causes of site selectivity in genomic clones with other inducible transposon systems.

Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus.

Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric alpha-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.

Tempol, a membrane-permeable radical scavenger, reduces oxidant stress-mediated renal dysfunction and injury in the rat.

BACKGROUND: The generation of reactive oxygen species (ROS) contributes to the pathogenesis of renal ischemia-reperfusion injury. The aim of this study was to investigate the effects of tempol in (1) an in vivo rat model of renal ischemia/reperfusion injury and on (2) cellular injury and death of rat renal proximal tubular (PT) cells exposed to oxidant stress in the form of hydrogen peroxide (H2O2). METHODS: Male Wistar rats underwent bilateral renal pedicle clamping for 45 minutes followed by reperfusion for six hours. Tempol (30 mg/kg/h), desferrioxamine (DEF; 40 mg/kg/h), or a combination of tempol (30 mg/kg/h) and DEF (40 mg/kg/h) were administered prior to and throughout reperfusion. Plasma concentrations of urea, creatinine, Na+, gamma-glutamyl transferase (gammaGT), aspartate aminotransferase (AST), and urinary Na+ and N-acetyl-beta-D-glucosaminidase (NAG) were measured for the assessment of renal function and reperfusion injury. Kidney myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured for assessment of polymorphonuclear (PMN) cell infiltration and lipid peroxidation, respectively. Renal sections were used for histologic grading of renal injury and for immunohistochemical localization of nitrotyrosine and poly(ADP-ribose) synthetase (PARS). Primary cultures of rat PT cells were incubated with H2O2 (1 mmol/L for 4 h) either in the absence or presence of increasing concentrations of tempol (0.03 to 10 mmol/L), DEF (0.03 to 10 mmol/L), or a combination of tempol (3 mmol/L) or DEF (3 mmol/L). PT cell injury and death were determined by evaluating mitochondrial respiration and lactate dehydrogenase (LDH) release, respectively. RESULTS: In vivo, tempol significantly reduced the increase in urea, creatinine, gammaGT, AST, NAG, and FENa produced by renal ischemia/reperfusion, suggesting an improvement in both renal function and injury. Tempol also significantly reduced kidney MPO activity and MDA levels, indicating a reduction in PMN infiltration and lipid peroxidation, respectively. Tempol reduced the histologic evidence of renal damage associated with ischemia/reperfusion and caused a substantial reduction in the staining for nitrotyrosine and PARS, suggesting reduced nitrosative and oxidative stress. In vitro, tempol significantly attenuated H2O2-mediated decrease in mitochondrial respiration and increase in LDH release from rat PT cells, indicating a reduction in cell injury and death. Both in vivo and in vitro, the beneficial actions of tempol were similar to those obtained using the Fe2+ chelator DEF. However, coadministration of DEF and tempol did not produce any additional beneficial actions against renal ischemia/reperfusion injury or against oxidative stress-mediated PT cell injury/death. CONCLUSION: Our results suggest that the membrane-permeable radical scavenger, tempol, reduces the renal dysfunction and injury associated with ischemia/reperfusion of the kidney.

Lipoteichoic acid induces delayed protection in the rat heart: A comparison with endotoxin.

Classic ischemic preconditioning transiently (30 to 120 minutes) protects the myocardium against subsequent lethal ischemia/reperfusion injury. After dissipation of this acute protection, a second window of protection (SWOP) appears 12 to 24 hours later; this SWOP lasts up to 3 days. Several triggers induce a SWOP, including brief repetitive cycles of coronary artery occlusion, rapid ventricular pacing, stimulation of adenosine A(1) receptors, and administration of wall fragments of Gram-negative bacteria, such as lipopolysaccharide (LPS). The aim of this study was to investigate whether lipoteichoic acid (LTA), a cell wall fragment of Gram-positive bacteria, can induce a SWOP in a rat model of left anterior descending coronary artery (LAD) occlusion (25 minutes) and reperfusion (2 hours). Thus, 166 male Wistar rats were pretreated (2 to 24 hours) with saline, LTA (1 mg/kg IP), or LPS (1 mg/kg IP) and subjected to LAD occlusion/reperfusion. Pretreatment with LTA or LPS for 16 hours led to a substantial, approximately 65%, reduction in infarct size and a reduction in the release of cardiac troponin T into the plasma. The dose of LTA used had no toxic effect (on any of the parameters studied), whereas the same dose of LPS caused a time-dependent activation of the coagulation system and liver injury. By use of RNase protection assays, it was determined that LPS caused a time-dependent induction of tumor necrosis factor-alpha, interleukin-1beta, and manganese superoxide dismutase mRNA content in the heart, whereas LTA failed to induce manganese superoxide dismutase. LPS also caused an upregulation of the expression of intercellular adhesion molecule-1 and P-selectin, whereas LTA downregulated these molecules and attenuated the accumulation of polymorphonuclear granulocytes caused by myocardial ischemia/reperfusion. This study demonstrates for the first time that pretreatment with LTA at 8 to 24 hours before myocardial ischemia significantly reduces (1) infarct size, (2) cardiac troponin T, and (3) the histological signs of tissue injury in rats subjected to LAD occlusion and reperfusion. The mechanism(s) underlying the observed cardioprotective effects of LTA warrants further investigation but is likely to be related to its ability to inhibit the interactions between the coronary vascular endothelium and polymorphonuclear granulocytes. Therefore, LTA represents a novel and promising agent capable of enhancing myocardial tolerance to ischemia/reperfusion injury.

Inhibitors of poly (ADP-ribose) synthetase reduce renal ischemia-reperfusion injury in the anesthetized rat in vivo.

The activation of poly (ADP-ribose) synthetase (PARS) subsequent to DNA damage caused by reactive oxygen or nitrogen species has been implicated in several pathophysiological conditions, including ischemia-reperfusion injury and shock. The aim of this study was to investigate whether PARS inhibitors could provide protection against renal ischemia-reperfusion injury in the rat in vivo. Male Wistar rats were subjected to 45 min bilateral clamping of the renal pedicles, followed by 6 h reperfusion (control animals). Animals were administered the PARS inhibitors 3-aminobenzamide, 1, 5-dihydroxyisoquinoline, or nicotinamide during the reperfusion period. Ischemia, followed by reperfusion, produced significant increases in plasma concentrations of urea, creatinine, and fractional excretion of Na(+) (FE(Na)) and produced a significant reduction in glomerular filtration rate (GFR). However, administration of the PARS inhibitors significantly reduced urea and creatinine concentrations, suggesting improved renal function. The PARS inhibitors also significantly increased GFR and reduced FE(Na), suggesting the recovery of both glomerular and tubular function, respectively, with a more pronounced recovery of tubular function. In kidneys from control animals, histological examination revealed severe renal damage and immunohistochemical localization demonstrated PARS activation in the proximal tubule. Both renal damage and PARS activation were attenuated by administration of PARS inhibitors during reperfusion. Therefore, we propose that PARS activation contributes to renal reperfusion injury and that PARS inhibitors may be beneficial in renal disorders associated with oxidative stress-mediated injury.

Biochemical consequences of a mutation that controls the cholesterol dependence of Semliki Forest virus fusion.

The enveloped alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-triggered membrane fusion reaction that requires cholesterol and sphingolipid in the target membrane. Cholesterol-depleted insect cells are highly resistant to alphavirus infection and were used to select srf-3, an SFV mutant that is approximately 100-fold less cholesterol dependent for infection due to a single amino acid change in the E1 spike subunit, proline 226 to serine. Sensitive lipid-mixing assays here demonstrated that the in vitro fusion of srf-3 and wild-type (wt) virus with cholesterol-containing liposomes had comparable kinetics, activation energies, and sphingolipid dependence. In contrast, srf-3 fusion with sterol-free liposomes was significantly more efficient than that of wt virus. Thus, the srf-3 mutation does not affect its general fusion properties with purified lipid bilayers but causes a marked and specific reduction in cholesterol dependence. Upon exposure to low pH, the E1 spike subunit undergoes distinct conformational changes, resulting in the exposure of an acid conformation-specific epitope and formation of an E1 homotrimer. These conformational changes were strongly cholesterol and sphingolipid dependent for wt SFV and strikingly less cholesterol dependent for srf-3. Our results thus demonstrate the functional importance of fusogenic E1 conformational changes in the control of SFV cholesterol dependence.

Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms.

A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

An epitope of the Semliki Forest virus fusion protein exposed during virus-membrane fusion.

Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.