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Aim: Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. Materials & Methods: An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. Results: A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. Conclusion: The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.

Chemotherapy is a major therapeutic strategy for patients with cancer. Owing to the severe inflammatory response of chemotherapy, patients experience extreme discomfort during treatment, and this may interrupt treatment completion. The vitamin D3 has a role in anti-inflammation, but no study has explored whether vitamin D3 has beneficial effects on patients undergoing chemotherapy. In this study, we investigated the effect of calcitriol (Vit-D) on inflammatory responses during 5-fluorouracil (5-FU) treatment. Rats were divided into five groups and treated with 1:1 dilution of 5-FU with equal amount of 0.9% saline, 1:3 dilution of 5-FU with 0.9% saline threefold dilution, 5-FU, Vit-D, or 5-FU + Vit-D. A single dose of 15 mg/kg of 5-FU was intravenously administered for 4 h, and the blood biochemical substances and inflammatory cytokines were assessed after the intervention. The 5-FU group had higher AST, ALT, LDH, and CPK levels than those in the 5-FU + Vit-D group. The 5-FU + Vit-D group had a lower TNF-α value than the 5-FU. The IL-6 levels in the 5-FU + Vit-D group were also significantly lower than those in 5-FU. Calcitriol administration during 5-FU therapy can alleviate the production of inflammatory cytokines and liver damage.

project grant (PTDC/OCE-ETA/31250/2017) with financial support from FCT/MCTES through national funds and co-financed by FEDER, under the Partnership Agreement PT2020 (UID/QUI/50006/2019-POCI/01/0145/FEDER/007265). Financial support was also obtained from the Spanish Ministry of Science, Innovation and Universities (CTQ2015-69021-R and RTI2018-102212-B-I00), the Xunta de Galicia (GRC2014/040, ED431C 2018/30, and Centro Singular de Investigacion de Galicia Accreditation 2016-2019, ED431G/09) and the European Union (European Regional Development Fund-ERDF). The interaction of two anthocyanins with a water-soluble polyanionic dendrimer was studied through UV/Vis, stopped-flow, and NMR spectroscopy. Cyanidin-3-glucoside (cy3glc) revealed a stronger interaction than malvidin-3-glucoside (mv3glc) at pH 1 according to their apparent association constants. A higher color increased was also obtained for cy3glc at pH 3.5 as a result of this stronger interaction. A high-frequency chemical shift of the cy3glc aromatic protons suggest the formation of ionic pairs. The interaction parameters (K≈700 m−1, n≈295) indicated the binding of approximately two anthocyanin molecules by each sulfate group. The equilibrium and rate constants of cy3glc in the presence of dendrimer showed an increased stability of the flavylium cation and a higher protection of this species from hydration (pK′a and pKh increased almost one pH unit). The tuning and color stabilization of anthocyanins by using this dendrimer allow novel applications as colorimetric sensors for food packaging. authorsversion published

Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well-explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N terminus into its “activation pocket,” indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein–protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*/Y75A/Y37bA, where C* is cysteinylated Cys-99) cannot form a dimer or tetramer, yet it is active but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity.

The well-studied B-domain from Staphylococcal protein A is a 59 amino acid three-helix bundle that binds the Fc portion of IgG with a dissociation constant of ~35 nM. The B-domain variant bearing a Gly to Ala mutation (=Z-domain) has been the subject of efforts to minimize a domain's size while retaining its function. We report X-ray crystallographic characterization of three steps in such a process using complexes with Fc: the full three-helix Z-domain, a 34 amino acid two-helix version called Z34C and a 13 amino acid single helix stabilized with an exo-helix tether, called LH1.

Traumatic and nontraumatic rhabdomyolysis can lead to acute renal failure (ARF), and acute alcohol intoxication can lead to multiple abnormalities of the renal tubules. We examined the effect of acute alcohol intoxication in a rat model of rhabdomyolysis and ARF. Intravenous injections of 5 g/kg ethanol were given to rats over 3 h, followed by glycerol-induced rhabdomyolysis. Biochemical parameters, including blood urea nitrogen (BUN), creatinine (Cre), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and creatine phosphokinase (CPK), were measured before and after induction of rhabdomyolysis. Renal tissue injury score, renal tubular cell expression of E-cadherin, nuclear factor-κB (NF-κB), and inducible nitric oxide synthase (iNOS) were determined. Relative to rats in the vehicle group, rats in the glycerol-induced rhabdomyolysis group had significantly increased serum levels of BUN, Cre, GOT, GPT, and CPK, elevated renal tissue injury scores, increased expression of NF-κB and iNOS, and decreased expression of E-cadherin. Ethanol exacerbated all of these pathological responses. Our results suggest that acute alcohol intoxication exacerbates rhabdomyolysis-induced ARF through its pro-oxidant and inflammatory effects.

Different infusion rates of doxorubicin (DOX) have been used for treating human malignancies. Organ toxicity after DOX infusion is a major issue in treatment disruption. However, whether different DOX infusion rates induce different toxicity is still unknown. In this study, we examined the toxicity effects of different DOX infusion rates in the early phase of organ toxicity. Thirty-six rats were randomly divided into 5-, 15-, and 30-min infusion rate groups. A single dose of DOX (8.3 mg/kg, I.V.) was administered at different infusion rates. Blood samples were collected from the femoral artery at 1, 3, 6, 9, 12, 18, 24, 36, and 48 h after DOX administration. The blood cell count and blood biochemistry were analyzed. The liver, kidney, and heart were removed for pathological examinations after the rats were sacrificed. Our findings show that the 30-min group had higher injury markers in the liver (glutamic oxaloacetic transaminase and glutamic pyruvic transaminase), kidneys (blood urea nitrogen and creatinine), and heart (creatine phosphokinase-MB and lactate dehydrogenase), and had higher tumor necrosis factor-alpha and interleukin 6 levels than did the other groups. The 30-min group also had more severe damage according to the pathological examinations. In conclusion, slower infusion of DOX induced a higher inflammatory response and greater organ damage.

Exhaustive exercise results in inflammation and oxidative stress, which can damage tissue. Previous studies have shown that vitamin D has both anti-inflammatory and antiperoxidative activity. Therefore, we aimed to test if vitamin D could reduce the damage caused by exhaustive exercise. Rats were randomized to one of four groups: control, vitamin D, exercise, and vitamin D+exercise. Exercised rats received an intravenous injection of vitamin D (1 ng/mL) or normal saline after exhaustive exercise. Blood pressure, heart rate, and blood samples were collected for biochemical testing. Histological examination and immunohistochemical (IHC) analyses were performed on lungs and kidneys after the animals were sacrificed. In comparison to the exercise group, blood markers of skeletal muscle damage, creatine kinase and lactate dehydrogenase, were significantly (P < 0.05) lower in the vitamin D+exercise group. The exercise group also had more severe tissue injury scores in the lungs (average of 2.4 ± 0.71) and kidneys (average of 3.3 ± 0.6) than the vitamin D-treated exercise group did (1.08 ± 0.57 and 1.16 ± 0.55). IHC staining showed that vitamin D reduced the oxidative product 4-Hydroxynonenal in exercised animals from 20.6% to 13.8% in the lungs and from 29.4% to 16.7% in the kidneys. In summary, postexercise intravenous injection of vitamin D can reduce the peroxidation induced by exhaustive exercise and ameliorate tissue damage, particularly in the kidneys and lungs.

Adipocyte fatty acid binding protein (A-FABP) is a key mediator of obesity-related metabolic syndrome (MetS). The aim of this study was to evaluate the relationship between A-FABP concentration and MetS in type 2 diabetes mellitus (DM) patients. Fasting blood samples were obtained from 165 type 2 DM volunteers. MetS and its components were defined using diagnostic criteria from the International Diabetes Federation. Among 165 DM patients, 113 patients (68.5%) had MetS. Diabetic persons who had MetS had significantly higher A-FABP levels (P<0.001) than those without MetS. Female DM persons had higher A-FABP level than man (P<0.001). No statistically significant differences in A-FABP levels were found in use of statin, fibrate, or antidiabetic drugs. Multivariate forward stepwise linear regression analysis revealed that body fat mass (P<0.001), logarithmically transformed creatinine (log-creatinine;P<0.001), female DM patients (P<0.001), and logarithmically transformed high sensitive C-reactive protein (log-hs-CRP;P=0.013) were positively correlated, while albumin (P=0.004) and glomerular filtration rate (GFR;P=0.043) were negatively correlated with serum A-FABP levels in type 2 DM patients. In this study, higher serum A-FABP level was positively associated with MetS in type 2 DM patients.

To study the efficacy of neem cake, soil drenching of metalaxyl, post-emergence application of glyphosate at very low concentrations alone and in combination with 1% solution of (NH4)2SO4 and 125% of recommended fertility, field experiments were conducted at the villages Ganghala and Bidhwan in Bhiwani district of Haryana during Rabi seasons of 2014–15 and 2015–16, respectively. Feasibility of adoption of results of studies conducted earlier on use of glyphosate 25 g/ha at 30 DAS and 50 g/ha at 55 DAS is being demonstrated by multi location field trials through farmers participatory approach in different parts of state during 2010–2016. Neem cake 400 kg/ha at sowing fb soil drenching of metalaxyl MZ 0.2% at 25 DAS fb glyphosate at 40 g/ha at 45 DAS or neem cake 400 kg/ha fb pendimethalin (PPI) at 0.75 kg/ha fb metalaxyl 0.2% at 25 DAS did not prove effective in minimizing density of Orobanche. Post-emergence application of glyphosate at 25 and 50 g/ha with 1% solution of (NH4)2SO4 at 30 and 55 DAS showed promise with 85- 91% control of this weed not only in experimental fields but in large scale demonstrations on farmers’ fields.

The Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) generate bioactive lipid mediators that reduce inflammation. The present study evaluated the effect of SMOFlipid containing ω-3 PUFAs on wound healing. Rats were divided into a SMOFlipid (SMOF) group and a 0.9% saline (placebo) group, with eight rats in each group. Wound excision was performed on the dorsal surface of each rat. In the SMOF group, 1 gm/kg SMOFlipid was dissolved in 3 mL saline as a treatment; in the placebo group, 3 mL saline was prepared as a treatment. The treatments were administered intravenously at an initial rate of 0.2 mL/kg body weight/h immediately after wounding, for 72 h. Blood samples were collected for white blood cell, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 measurements at the baseline and at 1, 6, 12, 24, 48, and 72 h after intervention. Wound areas were measured over a 2-week period after excision, and a histological examination was performed. Compared with the placebo group, SMOFlipid supplementation engendered significant decreases in the wound area on day 3 (78.28 ± 5.25 vs. 105.86 ± 8.89%), day 5 (72.20 ± 4.31 vs. 96.39 ± 4.72%), day 10 (20.78 ± 1.28 vs. 39.80 ± 10.38%), and day 14 (7.56 ± 0.61 vs. 15.10 ± 2.42%). The placebo group had a higher TNF-α level than the SMOF group at 72 h. The IL-10 level was higher in the SMOF group than in the placebo group at 48 h. Histological analysis revealed a higher rate of fibroblast distribution and collagen fiber organization in the SMOF group (P = 0.01). SMOFlipid enriched in ω-3 PUFA accelerates wound healing.

Field experiments to study the efficacy of neem cake, soil drenching of metalaxyl, post-emergence application of glyphosate at very low concentrations alone and in combination with 1% solution of ammonium sulphate and 125% of recommended fertility, were conducted at village Ganghala (Bhiwani) during Rabi seasons of 2014–15 and village Bidhwan (Bhiwani) during 2015–16. Feasibility of adoption of results of studies conducted earlier on use of glyphosate 25 g/ha at 30 DAS and 50 g/ha at 55 DAS is being demonstrated by multi location field trials through farmers participatory approach in different parts of state during 2010–2016. Neem cake 400 kg/ha fb pendimethalin (PPI) at 0.75 kg/ha fb HW at 60 DAS did not prove effective in minimizing density of Orobanche. Post-emergence application of glyphosate at 25 and 50 g/ha at 25 and 55 DAS, respectively showed promising results with 7595% control of this weed in experimental field and 76.5% on large scale demonstrations at farmers’ fields. Biotype from Ganghala (Bhiwani) was found to be most robust biotype with 5840 seeds per capsule.

Alcohol intoxication and psychiatric medication overdoses, including antidepressants, are common emergency room events. Heavy alcohol and antidepressant exposure are able to induce changes in cytokines disturbing normal physiology. We examined the inflammatory and physiological effects of selective serotonin reuptake inhibitor (SSRI) medication after heavy alcohol exposure. Rats were randomly divided into Alc (EtOH 5g/kg, intravenous infusion for 3 h), SSRI (paroxetine oral intake) and Alc+SSRI groups. Serum samples were collected to measure blood ethanol, aspartate transferase, alanine transferase, creatine phosphokinase, lactate dehydrogenase, amylase, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) levels. Lactate dehydrogenase levels in bronchoalveolar lavage fluid were also examined. Liver, pancreas and lungs were removed after sacrifice and any pathological changes were catalogued. Ethanol infusion resulted in blood levels of ethanol of >100 mg/dL after ethanol infusion. Serum levels of aspartate transferase, alanine transferase, creatine phosphokinase, lactate dehydrogenase, amylase, TNF-α and IL-6 in the Alc+SSRI group were lower than in the Alc group. Moreover, pathological damages to the liver, pancreas and lungs were slightly lower in the Alc+SSRI group than in the Alc group. These findings suggested that SSRI is able to decrease the release of pro-inflammatory cytokines and thereby reduce liver and pancreas damage after heavy alcohol exposure.

The absorption kinetics of CO2 in aqueous solutions for low superficial loadings have been measured with a droplet train reactor. A well defined stream of uniform droplets was exposed to an atmosphere with exactly known CO2 concentration. Based on the short gas-liquid contact time within the reactor and a sensitive volumetric measurement of the CO2 flux the analysis of CO2 mass transfer in the liquid droplets was performed for small liquid loadings. © 2013 The Author.

The studies aim to develop detergents which show similar reaction kinetics to the reference sorbent 2-aminoethanol (MEA) 30 mass-%. Furthermore the solvent properties and the energy requirement for regeneration can be improved. Apart from the lower desorption enthalpy, it is necessary that the solutions show low corrosiveness, a low vapour pressure, no toxicity and a low degradation rate. These requirements can be achieved by employing carbonate solutions. The challenge is the improvement of the reaction kinetics by using additives which enhance the performance to or above the level of the reference sorbent. In addition to the detergent investigation a detailed understanding of the two-phase system of flue gas and wash solution is required also.

Background Hypothermia frequently occurs during fluid resuscitation of trauma victims, especially in patients with a major blood loss. Recent studies have suggested that mild hypothermia may ameliorate hemorrhagic shock (HS) induced splanchnic damage. Objective The aim of the present study is to compare the status of body temperature and splanchnic injury under different resuscitation speeds for HS in conscious rats. Methods Experimental study in an animal model of HS. Twenty-four male Wistar-Kyoto rats were used in the study. To mimic HS, 40% of the total blood volume was withdrawn. Fluid resuscitation was given 30 min after blood withdrawal. The rats were randomly divided into three groups; the control group, the 10-min rapid group, and the 12-h slow group. Results Levels of blood biochemical parameters, including aspartate transferase (GOT), and alanine transferase (GPT), were measured. Levels of serum tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) were measured and levels of bronchoalveolar lavage fluid (BALF) TNF-α and nitric oxide (NO) were measured by ELISA. The lung, liver and small intestine were examined for pathological changes 48 h after HS. Conclusions Initially slow rate resuscitation with limited-volume significantly decreased body temperature, serum GOT, GPT, TNF-α, and IL-6 levels, levels of TNF-α, and NO in BALF. Moreover, the slow group had lower injury scores in the lung, liver and small intestine than the rapid group after HS. This finding suggests that mild hypothermia induced by a slow fluid resuscitation rate with limited-volume ameliorates HS-induced splanchnic damage in conscious rats.

Objectives Peritoneal fibrosis (PF) is a recognized complication of long term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The most commonly used method to create a PF rat model is an intraperitoneal injection of 0.1% chlorhexidine gluconate with 15% ethanol. The aim of this study was to use chlorhexidine digluconate without ethanol, given via a PD catheter, to create a PF rat model. Material and Methods PF was induced in Sprague-Dawley rats by 0.5 mL 0.1% chlorhexidine digluconate in normal saline administered daily for 1 or 2 weeks via a PD catheter. Conventional 4.25% Dianeal dialysate solution, 30 mL, was administered via a PD catheter and the dwell time was 4 hours. Dialysate samples (0.5 mL) and blood samples (0.5 mL) were taken at 0 and 4 hours after the dialysate fluid had been infused to measure glucose and urea. At the end of dialysis, the rats were sacrificed; the parietal peritonea of the liver and anterior abdominal wall muscle were harvested. The peritoneum was analyzed by microscopic examination and immunohistochemistry for transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF). Results After 4 hours of PD, the D4/D0 glucose level was increased, the D4/D4 urea level was decreased, the liver and muscle peritoneum was markedly thicker, and expression of TGF-β1, α-SMA, fibronectin, collagen, and VEGF-positive cells was increased in the PF groups compared with the vehicle groups, and also in the 2 week PF group compared with the 1 week PF group. Conclusion We developed a novel method of creating a PF rat model using chlorhexidine digluconate without ethanol via a PD catheter, which will be useful in the study of PF.

Background Hemorrhagic shock (HS) followed by resuscitation can result in production of several inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), leading to multiple organ dysfunction. Melatonin can attenuate organ damage with its anti-inflammation effects. The present study was designed to investigate the effects of melatonin on the physiopathology and cytokine levels after HS in rats. Methods HS was induced in rats by withdrawing 40% of the total blood volume (6mL/100 gm body weight) from a femoral artery catheter, immediately followed by intravenous injection of 10mg/kg melatonin. Mean arterial pressure and heart rate were monitored continuously for 48h after the start of blood withdrawal. Biochemical parameters, including levels of hemoglobulin, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and lactate, were determined 30min before and 0, 1, 3, 6, 12, 24, and 48h after induction of HS while an equal volume of normal saline was replaced as fluid resuscitation. Cytokine levels including TNF-α and IL-6 in the serum were measured at 1, 24, and 48h after HS. The kidney, liver, lung, and small intestine were removed for pathology assessment at 48h after HS. Results HS significantly increased the heart rate, blood GOT, GPT, BUN, Cre, LDH, CPK, lactate, TNF-α, and IL-6 levels, and decreased hemoglobulin and mean arterial pressure in rats. Treatment with melatonin preserved the mean arterial pressure, decreased tachycardia, and markers of organ injury, and suppressed the release of TNF-α and IL-6, with no change in hemoglobulin after HS in rats. Conclusion Treatment with melatonin suppresses the release of serum TNF-α and IL-6, and decreases the levels of markers of organ injury associated with HS, thus ameliorating HS-induced organ damage in rats.

Hepatocyte growth factor (HGF) binds to its target receptor tyrosine kinase, Met, as a single-chain form (pro-HGF) or as a cleaved two-chain disulfide-linked α/β-heterodimer. However, only two-chain HGF stimulates Met signaling. Proteolytic cleavage of the Arg(494)-Val(495) peptide bond in the zymogen-like pro-HGF results in allosteric activation of the serine protease-like β-chain (HGF β), which binds Met to initiate signaling. We use insights from the canonical trypsin-like serine protease activation mechanism to show that isolated peptides corresponding to the first 7-10 residues of the cleaved N terminus of the β-chain stimulate Met phosphorylation by pro-HGF to levels that are ∼25% of those stimulated by two-chain HGF. Biolayer interferometry data demonstrate that peptide VVNGIPTR (peptide V8) allosterically enhances pro-HGF β binding to Met, resulting in a K(D)(app) of 1.6 μm, only 8-fold weaker than the Met/HGF β-chain affinity. Most notably, in vitro cell stimulation with peptide V8 in the presence of pro-HGF leads to Akt phosphorylation, enhances cell survival, and facilitates cell migration between 75 and 100% of that found with two-chain HGF, thus revealing a novel approach for activation of Met signaling that bypasses proteolytic processing of pro-HGF. Peptide V8 is unable to enhance Met binding or signaling with HGF proteins having a mutated activation pocket (D672N). Furthermore, Gly substitution of the N-terminal Val residue in peptide V8 results in loss of all activity. Overall, these findings identify the activation pocket of the serine protease-like β-chain as a "hot spot" for allosteric regulation of pro-HGF and have broad implications for developing selective allosteric activators of serine proteases and pseudoproteases.

Objective Erythropoietin (EPO) has pleiotropic cytoprotective actions. We investigated the effects of EPO on the physiopathology and cytokine levels after haemorrhagic shock (HS) in conscious rats. Methods Rats received an intravenous injection of 300 U/kg EPO over 10 min followed by HS via withdrawal of 60% of total blood volume from a femoral arterial catheter (6 ml/100 g body weight) over 30 min. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 18 h after the start of blood withdrawal. Levels of biochemical parameters, including haemoglobin, GOT, GPT, BUN, creatinine (Cr), LDH, CPK, and lactate were measured at 30 min before the induction of HS and 0, 1, 3, 6, 9, 12, and 18 h after HS. Cytokine levels, including TNF-α and IL-6, in serum were measured at 1, 9, and 18 h after HS. The kidneys, liver, lungs, and small intestine were removed for pathology assessment at 48 h after HS. Results HS significantly increased HR, blood GOT, GPT, BUN, Cr, LDH, CPK, lactate, TNF-α, and IL-6 levels and decreased haemoglobin and MAP in rats. Pre-treatment with EPO improved survival rate, preserved the MAP, decreased the tachycardia and markers of organ injury, suppressed the release of TNF-α and IL-6 after HS in rats. Conclusion Pre-treatment with EPO suppresses the release of serum TNF-α and IL-6, along with decreasing the levels of markers of organ injury associated with HS, with such actions ameliorating HS-induced organ damage in rats.

The pathway for oxidative degradation of nicotine in Arthrobacter nicotinovorans includes two genetically and structurally unrelated flavoenzymes, 6-hydroxy-L-nicotine oxidase (6HLNO) and 6-hydroxy-D-nicotine oxidase, which act with absolute stereospecificity on the L- and D-forms, respectively, of 6-hydroxy-nicotine. We solved the crystal structure of 6HLNO at 1.95 A resolution by combined isomorphous/multiple-wavelength anomalous dispersion phasing. The overall structure of each subunit of the 6HLNO homodimer and the folds of the individual domains are closely similar as in eukaryotic monoamine oxidases. Unexpectedly, a diacylglycerophospholipid molecule was found to be non-covalently bound to each protomer of 6HLNO. The fatty acid chains occupy hydrophobic channels that penetrate deep into the interior of the substrate-binding domain of each subunit. The solvent-exposed glycerophosphate moiety is located at the subunit-subunit interface. We further solved the crystal structure of a complex of dithionite-reduced 6HLNO with the natural substrate 6-hydroxy-L-nicotine at 2.05 A resolution. The location of the substrate in a tight cavity suggests that the binding geometry of this unproductive complex may be closely similar as under oxidizing conditions. The observed orientation of the bound substrate relative to the isoalloxazine ring of the flavin adenine dinucleotide cofactor is suitable for hydride-transfer dehydrogenation at the carbon atom that forms the chiral center of the substrate molecule. A comparison of the substrate-binding modes of 6HLNO and 6-hydroxy-D-nicotine oxidase, based on models of complexes with the D-substrate, suggests an explanation for the stereospecificity of both enzymes. The two enzymes are proposed to orient the enantiomeric substrates in mirror symmetry with respect to the plane of the flavin.

Endotoxin shock can induce the production of several inflammatory mediators such as TNF-alpha, IL-6, and IL-1beta, leading to multiple organ dysfunction and death. Erythropoietin (EPO) has been found to interact with its receptor (EPO-R), expressed in a wide variety of non-hematopoietic tissues, to induce a range of pleiotropic cytoprotective actions. We investigated the effects of low doses of EPO (300U/kg, intravenous administration) on the physiopathology and cytokine levels in endotoxin shock in conscious rats. Endotoxin shock was induced by intravenous injection of Escherichia coli lipopolysaccharide (20mg/kg) in conscious rats. Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored for 48h after LPS administration. Levels of biochemical and cytokine parameters, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), and creatine phosphokinase (CPK) were measured at 0, 1, 3, 6, 9, 12, 18, 24, and 48h after sepsis. Serum TNF-alpha, IL-6, and IL-1beta level was measured at 1h after sepsis. Endotoxin shock significantly increased blood GOT, GPT, BUN, Cre, LDH, CPK, TNF-alpha, IL-6, IL-1beta levels, and HR, while it decreased MAP. EPO further increased the markers of organ injury (GOT, GPT, BUN, Cre, LDH, and CPK), inflammatory biomarkers (TNF-alpha, IL-6, and IL-1beta) and did not affect MAP and HR after LPS. EPO disserved endotoxin shock-induced liver, kidney, lung, and small intestine damage in conscious rats. In conclusion, pre-treatment with low doses of EPO increased the release of TNF-alpha, IL-6, and IL-1beta, along with aggravating endotoxin shock-induced markers of organ injury in conscious rats.

Rosiglitazone is a peroxisome proliferator-activated receptor (PPAR)-γ agonist. By inhibiting nuclear factor κB (NF-κB), it decreases tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) and has an anti-inflammatory effect. Endotoxin shock can induce the production of several inflammatory mediators such as TNF-α and IL-6, leading to multiple organ dysfunction and death. We investigated the effects of rosiglitazone (.3 mg/kg, intravenous administration) on the physiologic attributes and cytokine levels in endotoxin shock in conscious rats. Endotoxin shock was induced by intravenous injection of Klebsiella pneumoniae lipopolysaccharides (LPSs; 10 mg/kg) in conscious rats. Mean arterial pressure (MAP) and heart rate (HR) were continuously monitored for 24 hr after LPS administration. Levels of biochemical and cytokine parameters, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), glucose, TNF-α, and IL-6 were measured at 0, 1, 3, 6, 12, and 24 hr after sepsis. Endotoxin shock significantly increased blood GOT, GPT, BUN, Cre, LDH, CPK, glucose, TNF-α, and IL-6 levels and HR, while also decreasing MAP. Rosiglitazone diminished the increase in HR, decreased the markers of organ injury (GOT, GPT, BUN, Cre, LDH, CPK, glucose) and inflammatory biomarkers (TNF-α, IL-6), and did not affect MAP after LPS. In conclusion, rosiglitazone ameliorated endotoxin shock-induced markers of organ injury and suppressed the release of TNF-α and IL-6 in conscious rats.

BACKGROUND Rhabdomyolysis is one of the causes of acute renal failure. Pentobarbital enhances the action of gamma-aminobutyric acid and suppresses the activities of nuclear factor (NF)-kappaB pathways. In this study, we used pentobarbital to study the effects on the glycerol-induced rhabdomyolysis with acute renal failure in conscious rats. METHODS Rhabdomyolysis was induced by intramuscular injection of 10 mL/kg of 50% glycerol in conscious rats. Ten minutes later, the rats received an intravenous injection of pentobarbital (10 mg/kg in 0.5 mL/h normal saline) or normal saline (0.5 mL/h). Biochemical substances, including blood urea nitrogen (BUN), creatinine (Cre), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and creatine phosphokinase (CPK) were measured at 0 hour, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and 48 hours. Rats were killed by decapitation at 48 hours after glycerol administration, and the kidneys were removed immediately for pathological findings and immunohistochemistry. RESULTS Intramuscular injection of glycerol significantly increased blood BUN, Cre, GOT, GPT, CPK levels and induced severe histopathologic damage in the kidneys. NF-kappaB and inducible nitric oxide synthase (iNOS) were increased, and E-cadherin was decreased after glycerol administration, as detected by immunohistochemistry in the kidneys. Posttreatment with pentobarbital decreased blood BUN, Cre, GOT, GPT, CPK levels, decreased the markers of kidney injury, and suppressed the release of NF-kappaB and iNOS after rhabdomyolysis. CONCLUSION Posttreatment with pentobarbital suppressed the activities of NF-kappaB and iNOS, decreased BUN, Cre, GOT, GPT, CPK levels, and decreased the markers of kidney injury after rhabdomyolysis. These actions ameliorated rhabdomyolysis-induced acute renal failure in conscious rats.

Objectives Multiple organ dysfunction resulting from hemorrhagic shock (HS) and subsequent resuscitation was mediated by several inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10). The present study was designed to investigate the protective effects of fluvastatin on these mediators after HS in rats. Methods The experimental rats were randomly divided into three groups. The vehicle group received only vitamin K without HS, the HS-control group received vitamin K and HS, and the HS-experimental group received both vitamin K and fluvastatin (1 mg/kg) before HS. HS was produced by bleeding from a femoral arterial catheter to remove 60% of total blood volume (6 ml/100 g BW) over 30 min. The mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 12 h after the start of blood withdrawal. The biochemical parameters, including arterial blood gas, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and lactate were obtained at 30 min before induction of HS and at 0, 1, 3, 6, 9 and 12 h after HS. Equal volume of normal saline was given to replace blood volume loss. Cytokine levels including TNF-α and IL-10 in serum were measured at 1 h after HS. Kidney, liver, lung and small intestine were removed for pathology examination at 48 h after HS. Results HS significantly increased HR, blood GOT, GPT, BUN, Cre, LDH, CPK, lactate, TNF-α and IL-10 levels, and also induced metabolic acidosis and decreased MAP in rats. Pre-treatment with fluvastatin was found to improve survival rate, preserved MAP, decreased the markers of organ injury, suppressed the release of TNF-α and increased IL-10 after HS in rats. Conclusion Pre-treatment with fluvastatin can suppress the release of serum TNF-α and can also increase serum IL-10 level to protect HS-induced multi-organ damage in rats.

⋄ Background Serum ghrelin levels are elevated in uremic patients. However, no data are available on the relationship between metabolic syndrome and serum ghrelin levels in peritoneal dialysis (PD) patients. ⋄ Methods Metabolic syndrome and its components were defined using diagnostic criteria recommended by the International Diabetes Federation. Fasting serum samples were taken from 30 PD patients. Serum ghrelin levels were measured by using a commercial enzyme-linked immunosorbent assay kit. ⋄ Results Of the 30 PD patients, 53.3% (16/30) had metabolic syndrome. Fasting serum ghrelin corrected inversely with metabolic syndrome among these PDs patients ( p = 0.002). By odds ratio (OR) analysis of metabolic syndrome and metabolic syndrome diagnostic criteria, the predictors for metabolic syndrome are fasting glucose [OR: 39.00; 95% confidence interval (CI): 3.80 to 399.85; p < 0.001], triglycerides (OR: 37.50; 95% CI: 3.64 to 386.51; p < 0.001), and waist circumference (OR: 4.20; 95% CI: 1.95 to 9.03; p < 0.001). Univariate linear regression analysis showed that body weight ( r = –0.461, p = 0.010), waist circumference ( r = –0.390, p = 0.033), and body mass index ( r = –0.438, p = 0.016) were negatively correlated with serum ghrelin, and serum high density lipoprotein [HDL ( r = 0.626, p < 0.001)] was positively correlated with serum ghrelin. Multivariate forward stepwise linear regression analysis of the significant variables showed that HDL ( R2 change = 0.392; p < 0.001) was the independent predictor of serum ghrelin in PD patients and explained 39.2% of the variance. ⋄ Conclusions These results indicate an inverse association between circulating fasting ghrelin and metabolic syndrome in PD patients. Serum HDL was associated with serum ghrelin among PD patients.

Thioacetamide (TAA) has been used extensively in the development of animal models of acute liver injury. Frequently, TAA is administered intraperitoneally to induce liver damage under anaesthesia. However, it is rarely administered by intravenous injection in conscious rats. The experiments in this study were designed to induce acute liver damage by single intravenous injection of TAA (0, 70 and 280 mg/kg) in unrestrained rats. Biochemical parameters and cytokines measured during the 60-h period following TAA administration, included white blood cells (WBC), haemoglobulin (Hb), platelet, aspartate transferase (GOT), alanine transferase (GPT), total bilirubin (TBIL), direct bilirubin (DBI), albumin, ammonia (NH3), r-glutamyl transpeptidase (r-GT), tumour necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Rats were sacrificed by decapitation 60 h after TAA administration and livers were removed immediately for pathology and immunohistochemical (IHC) examination. Another group of rats were sacrificed by decapitation 1, 6 and 24 h after TAA administration and livers were removed immediately for time course change of pathology and IHC examination. TAA significantly increased blood WBC, GOT, GPT, TBIL, DBIL, NH3, r-GT, TNF-alpha and IL-6 levels but decreased the blood Hb, platelet and albumin level. The levels of histopathological damage in the liver after intravenous TAA administration were also increased with a dose-dependent trend and more increased at 60 h after TAA administration. The levels of inducible nitric oxide synthase (iNOS) and nuclear factor-kappaB (NF-kappaB) detected by IHC in the liver after intravenous TAA administration were also increased with a dose-dependent trend and more increased at 1 h after TAA administration. Single intravenous TAA administration without anaesthesia is a restorable animal model which may be used to investigate acute liver damage.

The freshwater clam is a widely-consumed shellfish and is used as a remedy for chronic hepatitis in Asia. However, its contribution to acute liver injury (ALI) remains unclear. The aim of this study is to assess the protective effects of freshwater clam extract (CE) in ALI induced by hemorrhage in rats. Rats were randomly divided into 5 groups, (1) blood loss (BL) 40%, (2) CE 150 mg/kg plus BL 40%, (3) CE 75 mg/kg plus BL 40%, (4) CE 150 mg/kg, and (5) CE 75 mg/kg groups. CE was given by femoral vein catheter in Groups 2 to 5. Initial hemorrhage was induced by withdrawing blood (loss 40% of total blood volume) from a femoral arterial catheter after CE administration in Groups 2 and 3. The levels of blood tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) were measured at several time points during the experimental period. Rats were sacrificed after 48 hours, and the liver was harvested for hematoxylin and eosin (HE) stain to show liver tissue injury. The results indicated that hemorrhage significantly decreased mean arterial pressure (MAP), increased blood AST, ALT and LDH levels and induced liver injury. Pre-treatment with the CE increased MAP and IL-10 levels and decreased AST, ALT, LDH and TNF-α levels after hemorrhage. The HE stains showed diminished organ injury in the CE groups. In conclusion, freshwater clam extract is a potential immunomodulating agent and ameliorates acute liver injury.

As assuring cell quality is an essential parameter for the success of stem cell therapy, the impact of various senescence-inducing stress signals, and strategies to circumvent them, has been an important area of focus in stem cell research. The aim of this study was to demonstrate the capacity of trans-cinnamaldehyde (TC) in reversing stress-induced senescence and maintaining the quality of stem cells in a chemically (H2O2)-induced cell senescence model. Because of the availability and the promising application potential in regenerative medicine, adipose-derived stem cells (ADSCs) were chosen for the study. We found that H2O2 treatment resulted in the expression of senescence characteristics in the ADSCs, including decreased proliferation rate, increased senescence-associated β-galactosidase (SA-β-gal) activity, decreased silent mating type information regulation 2 homolog ( SIRT1) expression, and decreased telomerase activity. However, TC treatment was sufficient to rescue or reduce the effects of H2O2 induction, ultimately leading to an increased proliferation rate, a decrease in the percentage of SA-β-gal-positive cells, upregulation of SIRT1 expression, and increased telomerase activity of the senescent ADSCs at the cellular level. Moreover, a chemically induced liver fibrosis animal model was used to evaluate the functionality of these rescued cells in vivo. Liver dysfunction was established by injecting 200 mg/kg thioacetamide (TAA) intraperitoneally into Wistar rats every third day for 60 days. The experimental rats were separated into groups: normal group (rats without TAA induction), sham group (without ADSC transplantation), positive control group (transplanted with normal ADSCs), H2O2 group (transplanted with H2O2-induced senescent ADSCs), and H2O2 + TC group (transplanted with ADSCs pretreated with H2O2 and then further treated with TC). In the transplantation group, 1 × 106 human ADSCs were introduced into each rat via direct liver injection. Based on the biochemical analysis and immunohistochemical staining results, it was determined that the therapeutic effects on liver fibrosis by the induced senescent ADSCs (H2O2 group) were not as significant as those exerted by the normal ADSCs (the positive control group). However, the H2O2 + TC group showed significant reversal of liver damage when compared to the H2O2 group 1 week posttransplantation. These data confirmed that the TC treatment had the potential to reduce the effects of H2O2-induced senescence and to restore in vivo functionality of the induced senescent ADSCs. It is therefore suggested that TC has potential applications in maintaining the quality of stem cells and could aid in treating senescence-related disorders.

Field, greenhouse, and growth chamber experiments were conducted to determine the effects of different burial treatments on photosynthesis (carbon dioxide exchange rate), chlorophyll-a fluorescence, leaf area, biomass, leaf thickness, total chlorophyll content and chlorophyll a/b ratio of ten sand dune species: Agropyron psammophilum, Cakile edentula, Cirsium pitcheri, Corispermum hyssopifolium, Elymus canadensis, Oenothera biennis, Panicum virgatum, Strophostyles helvola, Tusilago farfara, and Xanthium strumarium. Although there were significant differences between species, all of them exhibited stimulation in growth following burial in sand. Generally, buried plants of these species showed an increase in biomass, photosynthetic efficiency, and chlorophyll-a fluorescence because of higher energy content in their roots, rhizomes, and underground stems. The main reasons for the stimulation in growth were an increase in leaf area, leaf thickness, and root biomass. The total chlorophyll content of leaves of buried plants of A. psammophilum, E. canadensis, and P. virgatum was higher than controls, but there were no significant differences for Cirsium pitcheri, O. biennis, and S. helvola. The similarities and differences exhibited by the test species in their responses to burial would be ecologically adaptive to survive the harsh environmental conditions of foredunes. All species showed a clear compensatory response following recovery from the burial episode and surpassed control by enhancing the vital physiological, morphological and growth functions.

Proteolytic processing of zymogen Factor VII to Factor VIIa (FVIIa) is necessary but not sufficient for maximal proteolytic activity, which requires an additional allosteric influence induced upon binding to its cofactor tissue factor (TF). A key conformational change affecting the zymogenicity of FVIIa involves a unique three-residue shift in the position of beta-strand B2 in their zymogen and protease forms. By selectively introducing new disulfide bonds, we locked the conformation of these strands into an active TF*FVIIa-like state. FVIIa mutants designated 136:160, 137:159, 138:160, and 139:157, reflecting the position of the new disulfide bond (chymotypsinogen numbering), were expressed and purified by TF affinity chromatography. Mass spectrometric analysis of tryptic peptides from the FVIIa mutants confirmed the new disulfide bond formation. Kinetic analysis of amidolytic activity revealed that all FVIIa variants alone had increased specific activity compared to wild type, the largest being for variants 136:160 and 138:160 with substrate S-2765, having 670- and 330-fold increases, respectively. Notably, FVIIa disulfide-locked variants no longer required TF as a cofactor for maximal activity in amidolytic assays. In the presence of soluble TF, activity was enhanced 20- and 12-fold for variants 136:160 and 138:160, respectively, compared to wild type. With relipidated TF, mutants 136:160 and 137:159 also had an approximate threefold increase in their V(max)/K(m) values for FX activation but no significant improvement in TF-dependent clotting assays. Thus, while large rate enhancements were obtained for amidolytic substrates binding at the active site, macro-molecular substrates that bind to FVIIa exosites entail more complex catalytic requirements.

Several lines of investigation suggest that granulocyte colony-stimulating factor (G-CSF) augments all-trans retinoic acid (ATRA)–induced neutrophil differentiation in acute promyelocytic leukemia (APL). We sought to characterize the relationship between G-CSF– and ATRA-mediated neutrophil differentiation. We established a G-CSF receptor-transduced promyelocytic cell line, EPRO-Gr, derived from the granulocyte-macrophage colony-stimulating factor (GM-CSF)–dependent EPRO cell line harboring a dominant-negative retinoic acid receptor alpha (RARα). In EPRO-Gr, neutrophil differentiation occurs either in GM-CSF upon addition of ATRA or upon induction with G-CSF alone. Transient transfection of EPRO-Gr cells with a RARE-containing reporter plasmid demonstrates increased activity in the presence of ATRA, but not G-CSF, while STAT3 phosphorylation occurs only in response to G-CSF. This suggests that ATRA-mediated differentiation of EPRO-Gr cells occurs via a RARE-dependent, STAT3-independent pathway, while G-CSF–mediated differentiation occurs via a RARE-independent, STAT3-dependent pathway. ATRA and G-CSF thus regulate differentiation by divergent pathways. We characterized these pathways in the APL cell line, NB4. ATRA induction of NB4 cells resulted in morphologic differentiation and up-regulation of C/EBPϵ and G-CSFR, but not in STAT3 phosphorylation. The addition of G-CSF with ATRA during NB4 induction resulted in STAT3 phosphorylation but did not enhance differentiation. These results may elucidate how G-CSF and ATRA affect the differentiation of primary and ATRA-resistant APL cells.

Atmospheric nitrogen input of about 1.622 g m-2 year-1 is a major source of nitrogen for plants in the foredunes of Lake Huron at Pinery Provincial Park. The concentration of total nitrogen and nit...

Background. Angiogenesis has been proposed as a potential mechanism whereby transmyocardial laser revascularization (TMLR) has provided clinical relief of angina. Experimental work has found histologic evidence supporting this, as well as an improved response when angiogenic growth factors have been added to TMLR. The purpose of this study was to demonstrate that the molecular response to TMLR was an increase in the production of endogenous vascular endothelial growth factor to promote angiogenesis. Methods. Ameroid constrictors were placed on the proximal circumflex artery in 12 domestic pigs. After a chronic ischemic zone was established the animals were randomly divided into two groups. In the TMLR group the ischemic zone was treated with carbon dioxide laser. In the control group the ischemic zone was untreated. Six weeks later the animals were sacrificed, and sections from the ischemic zone and the nonischemic zone were submitted for immunohistochemical, histologic, and molecular analysis. Messenger RNA was obtained from northern blot analysis after being probed with vascular endothelial growth factor. Results. There was a twofold increase in the vascular endothelial growth factor messenger RNA in the ischemic zone of the TMLR group compared with the control group. Additionally, there was a threefold increase in the number of new blood vessels in the ischemic zone of the TMLR group compared with the control group. Conclusions. Transmyocardial laser revascularization promotes angiogenesis by upregulation of vascular endothelial growth factor. The resulting angiogenesis could be the principle mechanism for the clinical efficacy of TMLR.

A greenhouse study was conducted to determine the effects of increasing concentrations of salt (NaCl) solutions applied to the soil containing mycorrhizal and non-mycorrhizal plants of the leguminous species Strophostyles helvola (L.) Ell. The dry weight of roots and shoots and leaf chlorophyll content of S. helvola plants decreased with an increase in the concentrations of salt solutions. Although increasing salt concentrations promoted hyphal growth in mycorrhizal plants, the percent uncolonized root surface, arbuscules and vesicles decreased. An increase in the concentration of salt solutions did not decrease the number of root nodules. The data clearly showed evidence of significantly higher chlorophyll content, shoot dry weight and number of root nodules in mycorrhizal than non-mycorrhizal plants. High salt concentrations had a negative effect on the growth of S. helvola plants but the negative effects were partially mitigated by the presence of mycorrhizal fungi.

Hemorrhagic shock (HS) following acute alcohol intoxication can increase proinflammatory cytokine production and induce marked immunosuppression. We investigated the effects of ethanol on physiopathology and cytokine levels following HS in acutely alcohol-intoxicated rats. Rats received an intravenous injection of 5 g/kg ethanol over 3 h followed by HS induced by withdrawal of 40% of total blood volume from a femoral arterial catheter over 30 min. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 48 h after the start of blood withdrawal. Biochemical parameters, including hemoglobin, ethanol, glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), and creatine phosphokinase (CPK), were measured at 30 min before induction of HS and 0, 1, 3, 6, 9, 12, 18, 24, and 48 h after HS. Serum tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) levels were measured at 1 and 12 h after HS. The liver, kidneys, and lungs were removed for pathology at 48 h later. HS significantly increased HR, blood GOT, GPT, BUN, Cre, LDH, CPK, TNF-α, and IL-6 levels and decreased hemoglobin and MAP in rats. Acute ethanol intoxication further increased serum levels of GOT, GPT, BUN, Cre, LDH, CPK, TNF-αand IL-6 elevation following HS. Acutely intoxicated rats exacerbated the histopathologic changes in the liver, kidneys, and lungs following HS.

Background Large-volume, rapid crystalloid infusion may increase endothelial cell damage and induce shear stress, potentially leading to multiple-organ dysfunction syndrome. Limited guideline data for fluid administration are currently available, especially for the aging population. The aim of the present study was to compare the degree of organ damage in conscious aging rats when different resuscitation speeds were used during the treatment of hemorrhagic shock (HS). Methods Eighteen aging male Wistar-Kyoto rats were randomly divided into the following three groups: the control group, 30-min rapid resuscitation group, and 12-h slow resuscitation group. To mimic HS, 40% of the total blood volume was withdrawn. Fluid resuscitation (1:3) was given at 30 min after the blood withdrawal. Blood biochemical parameters including glucose, lactic acid, and lactate dehydrogenase (LDH) were measured along with the levels of serum and bronchoalveolar lavage fluid, tumor necrosis factor alpha (TNF-α), and interleukin 10 by enzyme-linked immunosorbent assay. The lungs were examined for pathologic changes, and the injury score at 24 h after HS was calculated. Results Compared with slow-rate resuscitation, initially rapid and immediate resuscitation significantly increased the serum levels of glucose, LDH, and proinflammatory cytokines (TNF-α and interleukin 10), and bronchoalveolar lavage fluid levels of white blood cells, TNF-α, and LDH as well as produced pathologic changes in the organ. The lung injury scores were higher after induced HS in aging rats. Conclusions The slow and continuous (12 h) fluid resuscitation rate ameliorated HS-induced organ damage in conscious aging rats.

Peritoneal fibrosis is a major complication of peritoneal dialysis that can lead to ultrafiltration failure. This study investigates the protective effects of calcitriol on chlorhexidine digluconate-induced peritoneal fibrosis in rats. Peritoneal fibrosis was induced in Sprague-Dawley rats by daily administration of 0.5mL 0.1% chlorhexidine digluconate in normal saline via peritoneal dialysis for 1week. Rats received daily intravenous injections of calcitriol (low-dose, 10ng/kg; or high-dose, 100ng/kg) for 1week. After 7days, conventional 4.25% Dianeal (30mL) was administered via peritoneal dialysis over 4h. Peritoneal solute transport was calculated from the dialysate concentration relative to its concentration in the initial infused dialysis solution (D4/D0 glucose) for glucose, and the dialysate-to-plasma concentration ratio (D4/P4 urea) at 4h for urea. Rats were then sacrificed and the liver peritoneum was harvested for immunohistochemical analysis via microscopy. After dialysis, the D4/P4 Urea level was reduced; increases were observed in the D4/D0 glucose level and the levels of active transforming growth factor-β1 and angiotensin II in serum and dialysate; the liver peritoneum and muscle peritoneum was markedly thickened, and the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and phosphorylated Smad2/3 (P-Smad2/3)-positive cells in the liver peritoneum was elevated in the peritoneal fibrosis group compared with the vehicle group. Calcitriol decreased the serum and dialysate active transforming growth factor-β1 and angiotensin II level, decreased the thickness of the liver peritoneum and muscle peritoneum, and decreased the expression of α-SMA, fibronectin, collagen, vascular endothelial growth factor, angiotensin II, transforming growth factor-β1, and P-Smad2/3-positive cells in liver peritoneum cells. High-dose calcitriol exhibited better protective effects against peritoneal fibrosis than did the lower dose. Calcitriol protected against chlorhexidine digluconate-induced peritoneal fibrosis in rats by decreasing transforming growth factor-β1 and angiotensin II production.

Body fluids are frequently submitted to the clinical laboratory. Standard chemistry analyzers are generally not FDA approved for body fluid analysis and regulatory authorities require individual laboratories to validate their performance characteristics.Precision, linearity, accuracy, and sample stability were assessed for 10 different chemistry analytes using ascites, pleural, peritoneal dialysate, and synovial fluids on the Beckman Coulter (BC) AU5431. The effect of altered protein concentration and pH was investigated. Chemistry results from the AU5431 were compared with results from the BC AU5832 and the BC AU680. Additionally, 22 paired serum/pleural fluids were evaluated for their biochemical ability to distinguish a transudate from an exudate.Precision for each assay and body fluid type had a CV14.5%. Recovery for the various analytes in all fluid types was 94.9-112%. CAP survey samples were within one SDI of the peer group. Sample pH, but not protein concentration, affected chemistry results. Inter-instrument comparison of results using the AU5431, AU5800, and AU680 was excellent. A combinatorial approach for discriminating between transudates and exudates had a sensitivity and specificity of 94% and 50%, respectively.Body fluid samples with pH values within the acceptable range can be analyzed on the BC AU instruments.

The technical performance of serum fructosamine assay as a short-term index of diabetic control was evaluated by using the BM/Hitachi 747-200 Automatic Analyzer (Boehringer Mannheim Corp). Intra- and interassay precisions (CV) were 15.5 micromol/L, and bilirubin interfered substantially at concentrations >68.4 micromol/L. A normal reference range of up to 282 micromol/L (mean +/- 2SD = 242 +/- 40) was derived from results obtained from 228 nondiabetic subjects. In 222 diabetic subjects, serum fructosamine concentration correlated well with fasting glucose concentration (r = 0.74) and with Hb A1c concentration (r = 0.80). This automated fructosamine assay has the advantages of technical simplicity, low cost, and reduced analytical time compared with the Hb A1c method.

Greenhouse experiments were conducted to deter- mine the effects of sand burial on survival and growth of seedlings of Cirsium pitcheri. In 1992-1993, seedlings were buried to depths of 0, 25, 50, 75, and 100% of their height while in 1993-1994, the seedlings were buried to depths of 0, 4 cm (single burial), 4 cm (repeated burial of 1 cm every 8 days), 8 cm (single burial) and 8 cm (repeated burial of 2 cm every 8 days). Several physiological traits, net photosynthetic rate, chlorophyll a:b ratio, leaf area, number and length of leaves, number of internodes, amount of tillering, and biomass, were measured. The results showed that all seedlings died in the complete (100%) burial, 20 % died in the 75% burial and none died in the 0, 25 and 50 % burial treatments. Burial of seedlings to a depth of 25% stimulated their growth but 75% burial significantly decreased the total dry weight. Repeated burial treatments exhibited significantly greater stimulation of growth than single burial. Surviving seedlings grew through the sand deposit by elongating the stem and leaf petioles, increasing the number of nodes and the length of internodes. This elongation occurred at the expense of development of the root system indicating that available energy was re-allocated to above-ground parts.

Freshwater clam ( Corbicula fluminea), a popular edible shellfish in Asia, is said to have beneficial effects on liver function. However, scientific evidence for such benefit is limited. In this study, the authors aimed to assess the treatment effects of freshwater clam extract (FCE) administration after hemorrhagic shock (HS) in rats. The authors randomly divided animals into three groups. After inducing HS in rats in the HS + FCE ( n = 12) and HS groups, the authors fed 20 mg/kg FCE orally to rats in the HS group only. The authors neither induced HS in nor fed FCE to rats ( n = 8) in the vehicle group. The authors measured the blood levels of white blood cells (WBC), aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), and tumor necrosis factor-alpha (TNF-α) at several time points during the experiment. After 48 hr, the authors sacrificed the rats and harvested the livers for hematoxylin and eosin (HE) staining. The HS significantly decreased mean arterial pressure (MAP), increased blood AST, ALT, and LDH levels and induced liver injury in rats. Treatment with FCE increased MAP level and decreased AST, ALT, LDH, and TNF-α levels after hemorrhage. The HE staining showed diminished organ injury in the FCE-treated group. In conclusion, the administration of posttreatment FCE suppressed the release of pro-inflammatory TNF-α production after HS and decreased the levels of markers of liver injury associated with HS in rats. These beneficial effects suggest that FCE is a potential immunomodulator.

Eur J Clin Invest 2010; 40 (4): 301–309 Abstract Background Peritoneal fibrosis (PF) is a recognized complication of long-term peritoneal dialysis (PD) and can lead to ultrafiltration failure. The present study was designed to investigate the protective effects of aliskiren on chlorhexidine digluconate-induced PF in rats. Materials and methods The PF was induced in Sprague–Dawley rats by daily administration of 0·5 mL 0·1% chlorhexidine digluconate in normal saline via PD tube for 1 week. Rats received daily intravenous injections of low-dose aliskiren (1 mg kg−1) or high-dose aliskiren (10 mg kg−1) for 1 week. After 7 days, conventional 4·25% Dianeal (30 mL) was administered via a PD catheter with a dwell time of 4 h and assessed of peritoneal function. At the end of dialysis, rats were sacrificed and the liver peritoneum was harvested for microscopically and immunohistochemistry. Results There was no significant difference in mean arterial pressure and heart rate between groups. After 4 h of PD, the D4/P4 urea level was reduced, the D4/D0 glucose level, serum and dialysate transforming growth factor-β1 (TGF-β1) level was increased, the liver peritoneum was markedly thicker, and the expression of TGF-β1, alpha-smooth muscle actin (α-SMA), fibronectin, collagen, and vascular endothelial growth factor (VEGF) were elevated in the PS group compared with the vehicle group. Aliskiren decreased the serum and dialysate TGF-β1 level, decreased the thickness of the liver peritoneum, and decreased the expression of TGF-β1, α-SMA, fibronectin, collagen, and VEGF-positive cells in liver peritoneum. Moreover, high-dose aliskiren had better protective effects against PF than low dose in rats. Conclusions Aliskiren protected against chlorhexidine digluconate-induced PF in rats by decreasing TGF-β1 production.

Patients with hypertension have higher mortality rates from hemorrhagic shock (HS) than normotensive patients. Several inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha) and interleukin 10 (IL-10) can be produced by HS and lead to multiple organ dysfunction and death. We investigated the effects of high dose (10 mg/kg/hr) and low dose (1 mg/kg/hr) propofol treatment after HS in conscious spontaneously hypertensive rats (SHRs). By withdrawing 40% of total blood volume from a femoral arterial catheter (6 ml/100 g body weight [BW]) for more than 30 min, HS was induced. The mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 24 hr after the start of blood withdrawal. Levels of biochemical parameters, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine (Cre), creatine phosphokinase (CPK), and lactic dehydrogenase (LDH) were measured 30 min before and 0, 1, 3, 6, 9, 12, 18, and 24 hr after the 30-min blood withdrawal period. Cytokine levels, including TNF-alpha and IL-10 in the serum, were measured 1 hr after HS. The kidney, liver, and lung were removed for pathology assessment at 48 hr after HS. HS significantly increased blood GOT, GPT, BUN, LDH, CPK, TNF-alpha, and IL-10 levels in conscious SHRs. Posttreatment propofol decreased serum TNF-alpha level, increased serum IL-10 level, attenuated the severity of organ damage, and improved survival rate after HS. This treatment protected SHRs against HS-induced organ damage. Moreover, high-dose propofol had a more protective effect than low-dose propofol against HS in conscious SHRs.

Salt spray is an important abiotic stress that affects plant and other biotic communities in the vicinity of sea coasts. Salt stress refers only to excess of ions in the environment, but along sea coasts it specifically involves increased amounts of Na+ and Cl− ions. Anyone who has visited a sea coast on windy days has experienced the landward movement of salt in the form of salt spray. Salisbury (1805) reported details of a marine storm in England following gale force winds from the east for one week. He writes: ‘On the 14th of January 1803, I observed an east window of my house, which had been cleaned a few days before, covered on the outside with an apparent hoar frost.’ Chemical analysis showed that it was salt from salt spray that had been deposited on plants, buildings and other objects. In spring of that year he made two observations: (i) plant taxa showed differential tolerance to salt spray and (ii) injury was more pronounced on the windward than the leeward sides of plants. Salt spray acts as a strong environmental stress and populations of biotic organisms have evolved traits that allow them to tolerate the effects of salt. The salt crystals also act as condensation nuclei in the air and damage plants by abrasion during wind storms. However, salt spray may also be beneficial because it improves plant growth by providing some essential nutrients. Many researchers have examined the role of salt spray on survival, distribution and growth form of plant species. In this chapter the effects of salt spray and soil salinity on seed germination, seedling survival and plant growth will be examined. Symptoms of injury to plants, mechanisms of salt tolerance and comparisons between plant species native to the coastal dunes will also be discussed. Three factors—wind speed, wave amplitude and wind direction—influence the formation of salt spray. Waves of high amplitude produce four basic types of breakers: surging, collapsing, plunging and spilling. The steepest waves with high turbulence create spilling breakers with a bore accompanied by large quantities of small foam bubbles.

Proinflammatory cytokines and hyperhomocysteinemia are associated with clinically relevant restenosis in coronary artery disease. N-acetylcysteine (NAC) can decrease proinflammatory cytokines and plasma homocystine as well as reduce contrast-induced nephropathy. The aim of this study, therefore, was to compare normal saline hydration with and without intravenous NAC in terms of changes in renal function, proinflammatory cytokines, inflammatory markers, and plasma total homocysteine during coronary angiography. Forty-six patients who underwent coronary angiography and/or stent implantation for unstable angina were enrolled and assigned to NAC or NS treatment groups based on normal saline hydration with or without intravenous NAC, respectively. The NS group had lower serum creatinine (Cre: p = 0.02) and plasma total homocysteine (tHcy; p0.001) and increased glomerular filtration rate (GFR; p = 0.003) after angiography. In the NAC group, the serum blood urea nitrogen (BUN; p = 0.001), Cre (p0.001), and plasma tHcy (p0.001) were lower, and the GFR (P = 0.013) was increased after angiography. There were no statistically significant differences in serum high-sensitivity C reactive protein (hs-CRP), tumor necrosis factor-alpha (TNF-alpha), or interleukin-10 (IL-10) before and after angiography in the NS and NAC groups. Intergroup comparison revealed that plasma tHcy level was lower for the NAC-treated patients (p = 0.002), with lower plasma tHcy level before and after treatment in this group (p0.001). Normal saline hydration can improve renal function and decrease plasma tHcy after coronary angiography with or without NAC; however, the combination of the two decreases plasma tHcy more than normal saline hydration alone.

SUMMARY 1 Propofol is an anti-inflammatory agent commonly used for general anaesthesia and sedation in intensive care unit patients. Haemorrhagic shock (HS) followed by resuscitation can induce the production of several inflammatory mediators, such as tumour necrosis factor (TNF)-a and interleukin (IL)-10, leading to multiple organ dysfunction and death. 2 In the present study, we investigated the effects of treatment with high-dose (10 mg/kg per h) and low-dose (1 mg/kg per h) propofol after HS on the physiopathology and cytokine levels in conscious rats. 3 The experiments were designed to induce HS by withdrawing 40% of total blood volume from a femoral artery catheter (6 mL/100 g bodyweight) over 30 min. Mean arterial pressure (MAP) and heart rate (HR) were monitored continuously for 48 h after the start of blood withdrawal. Levels of biochemical parameters, including glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), blood urea nitrogen (BUN), creatinine, creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), were measured 30 min before and 0, 1, 3, 6, 9, 12, 18, 24 and 48 h after the end of the 30 min blood withdrawal period. Serum cytokine levels, including TNF-a and IL-10, were measured at 1, 6, 12, 24 and 48 h after HS. The kidneys, liver, lungs and small intestine were removed for pathological assessment 48 h after HS. 4 In the present study, HS significantly increased blood GOT, GPT, BUN, LDH, CPK, TNF-a and IL-10 levels in conscious rats. Post-treatment with high-dose propofol accentuated systemic hypotension, increased serum TNF-a and IL-10 levels and increased the severity of organ damage after HS. In contrast, post-treatment with low-dose propofol did not affect MAP, but did suppress increased serum levels of the inflammatory cytokines and improved the survival rate after HS, thus protecting rats against HS-induced organ damage. 5 In conclusion, post-treatment with low-dose propofol ameliorated HS-induced markers of organ injury, suppressed the release of TNF-a and IL-10 and protected against HS-induced liver, kidney, lung and small intestine damage in conscious rats. These findings suggest the need to investigate whether low-dose propofol may be beneficial for patients with HS-induced organ damage.

Limitations of current anticoagulant therapies have led us to develop two distinct classes of exosite peptide inhibitors for the initiator of the clotting process, the tissue factor-factor VIIa (TF.FVIIa) complex (Roberge, M., Santell, L., Dennis, M. S., Eigenbrot, C., Dwyer, M. A., and Lazarus, R. A. (2001) Biochemistry 40, 9522-9531). Although both peptide classes are potent and selective inhibitors of TF.FVIIa, neither showed 100% inhibition at saturating concentrations. Crystal structures of these peptides in complex with the FVII/FVIIa protease domain revealed their distinct binding sites and close proximity to the active site. The favorable orientation of the 15-mer A-site peptide A-183 (EEWEVLCWTWETCER) suggested that a C-terminal extension into the FVIIa active site could yield a chimeric inhibitor that was not only potent and selective but complete as well. A novel two-step "protease switch" approach using substrate phage display was developed by first binding all phage containing A-183 and C-terminal extension libraries to immobilized and inactive FVIIa. Upon altering pH and adding TF to switch on FVIIa enzymatic activity, only those phage released by proteolytic cleavage within the extension were propagated. This process selected for both preferred sequence and length in the extension, leading to a 27-mer peptide A-183X (EEWEVLCWTWETCERGEGVEEELWEWR) with a C-terminal 12-mer extension containing an Arg in the P1 position. A-183X was a more potent and complete inhibitor of FX activation, having a maximal extent of inhibition of approximately 99% with an IC50 of 230 pm versus A-183 which maximally inhibited to 74% with an IC50 of 1.5 nm. A-183X also had a maximal prolongation of the prothrombin time of 7.6- versus 1.9-fold for A-183, making it a more effective anticoagulant.

Because of the inverse correlation that exists between serum HDL-C concentration and risk of atherosclerotic disease (1), monitoring of high-density lipoprotein cholesterol (HDL-C) in serum is clinically important. In 1993, the National Cholesterol Education Program Adult Treatment Panel II (NCEP ATP II) revised its guidelines for diagnosis and treatment of hypercholesterolemia in adults to include HDL-C measurement at the initial screening stage when total cholesterol is measured (2). The enhanced role of HDL-C in clinical practice increases the need for reliable and readily performable HDL-C measurement. Most routine laboratories use a two-step method: chemical precipitation of lipoproteins containing apoprotein B, then quantification of HDL as cholesterol remaining in the supernate. Such precipitation-based methods are time-consuming, labor-intensive, require relatively large volumes of serum, and cannot be fully automated. Recently, several methods of direct HDL-C measurement have become commercially available, including the use of magnetically responsive particles with polyanion-metal combinations (3), the use of polyethylene glycol (PEG) with antibodies against apoprotein B and apoprotein CIII (4)(5), and the use of PEG-modified enzymes and sulfated α-cyclodextrin (6)(7). Okazaki et al. (8) also recently proposed the use of high-performance liquid chromatography (HPLC) as a tool to compare different methods for HDL-C. The goal of this study was to evaluate three assays for homogeneous HDL-C on BM/Hitachi 747–200 Automatic Analyzer (Boehringer Mannheim Corp.): the direct HDL-cholesterol reagent kit (Boehringer Mannheim Corp.), the N-geneousTM HDL cholesterol reagent kit (Genzyme Diagnostics), and EZ-HDLTM cholesterol reagent kit (Sigma Diagnostics). For each HDL-C assay, the reagent kit contains Reagent 1 and Reagent 2; Reagent 1 is liquid, and Reagent 2 is lyophilized. Reaction principles of these three methods are quite different; the direct-HDL uses PEG-modified cholesterol esterase and cholesterol oxidase as well as sulfated α-cyclodextrin to provide selective determination of HDL-C in serum; …