bluuebunny/crossref_metadata_2025 huggingface dataset:
Murine renal organic anion transporters mOAT1 and mOAT3 facilitate the transport of neuroactive tryptophan metabolites
Authors: Bahn Andrew , Ljubojević Marija , Lorenz Heiko , Schultz Christian , Ghebremedhin Estifanos , Ugele Bernhard , Sabolić Ivan , Burckhardt Gerhard , Hagos Yohannes
Published: June 2005

Tryptophan metabolites such as kynurenate (KYNA), xanthurenate (XA), and quinolinate are considered to have an important impact on many physiological processes, especially brain function. Many of these metabolites are secreted with the urine. Because organic anion transporters (OATs) facilitate the renal secretion of weak organic acids, we investigated whether the secretion of bioactive tryptophan metabolites is mediated by OAT1 and OAT3, two prominent members of the OAT family. Immunohistochemical analyses of the mouse kidneys revealed the expression of OAT1 to be restricted to the proximal convoluted tubule (representing S1 and S2 segments), whereas OAT3 was detected in almost all parts of the nephron, including macula densa cells. In the mouse brain, OAT1 was found to be expressed in neurons of the cortex cerebri and hippocampus as well as in the ependymal cell layer of the choroid plexus. Six tryptophan metabolites, including the bioactive substances KYNA, XA, and the serotonin metabolite 5-hydroxyindol acetate inhibited [3H] p-aminohippurate (PAH) or 6-carboxyfluorescein (6-CF) uptake by 50–85%, demonstrating that these compounds interact with OAT1 as well as with OAT3. Half-maximal inhibition of mOAT1 occurred at 34 μM KYNA and 15 μM XA, and it occurred at 8 μM KYNA and 11.5 μM XA for mOAT3. Quinolinate showed a slight but significant inhibition of [3H]PAH uptake by mOAT1 and no alteration of 6-CF uptake by mOAT3. [14C]-Glutarate (GA) uptake was examined for both transporters and demonstrated differences in the transport rate for this substrate by a factor of 4. Trans-stimulation experiments with GA revealed that KYNA and XA are substrates for mOAT1. Our results support the idea that OAT1 and OAT3 are involved in the secretion of bioactive tryptophan metabolites from the body. Consequently, they are crucial for the regulation of central nervous system tryptophan metabolite concentration.
Metabolic and cellular plasticity in white adipose tissue I: effects of β3-adrenergic receptor activation
Authors: Granneman James G. , Li Pipeng , Zhu Zhengxian , Lu Yuyan
Published: June 2005

Selective agonists of β3-adrenergic receptors (Adrb3) exhibit potent anti-diabetes properties in rodent models when given chronically, yet the mechanisms involved are poorly understood. A salient feature of chronic Adrb3 activation is pronounced remodeling of white adipose tissue (WAT), which includes mitochondrial biogenesis and elevation of metabolic rate. To gain insights into potential mechanisms underlying WAT remodeling, the time course of remodeling induced by the Adrb3 agonist CL-316,243 (CL) was analyzed using histological, physiological, and global gene profiling approaches. The results indicate that continuous CL treatment induced a transient proinflammatory response that was followed by cellular proliferation among stromal cells and multilocular adipoctyes. CL treatment strongly fragmented the central lipid storage droplet of mature adipocytes and induced mitochondrial biogenesis within these cells. Mitochondrial biogenesis was correlated with the upregulation of genes involved in fatty acid oxidation and mitochondrial electron transport activity. The elevated catabolic activity of WAT was temporally correlated with upregulation of peroxisome proliferator-activated receptor-α and its target genes, suggesting involvement of this transcription factor in coordinating the gene program that elevates WAT catabolic activity.
A proinflammatory tumor that activates protein degradation sensitizes rats to catabolic effects of endotoxin
Authors: Mackenzie Michelle L. , Bedard Nathalie , Wing Simon S. , Baracos Vickie E.
Published: June 2005

Chronic or acute inflammation may participate in the etiology of cancer cachexia. To investigate the interaction between tumor and a secondary inflammatory stimulus on muscle wasting, rats with and without tumors (Yoshida ascites hepatoma) received low doses of endotoxin (LPS, 400 μg/kg sc) or saline. Nitrogen balance was measured 24 h before and after LPS/saline. Epitrochlearis muscle was used to measure in vitro protein metabolism, and gastrocnemius muscle was used for quantification of the mRNA for components of the ubiquitin proteolytic pathway. The YAH reduced muscle mass ( P = 0.002), increased muscle protein degradation ( P = 0.042), and elevated mRNA expression of components of the ubiquitin proteolytic pathway ( P < 0.01) including ubiquitin, ubiquitin-conjugating enzyme E214k, and ubiquitin ligases muscle RING Finger 1 and atrogin-1. Although the selected low dose of LPS had no impact on protein metabolism in control rats, LPS in rats bearing YAH caused weight loss ( P = 0.0007), lowered nitrogen balance ( P = <0.0001), and increased muscle protein degradation ( P = 0.0336). In conclusion, the presence of a tumor can potentiate whole body and muscle-specific catabolic losses of protein in response to a stimulus that is not catabolic in healthy animals. This effect might be dependent on the inflammatory nature of the tumor.
Differential effects of pharmacological liver X receptor activation on hepatic and peripheral insulin sensitivity in lean andob/obmice
Authors: Grefhorst Aldo , van Dijk Theo H. , Hammer Anke , van der Sluijs Fjodor H. , Havinga Rick , Havekes Louis M. , Romijn Johannes A. , Groot Pieter H. , Reijngoud Dirk-Jan , Kuipers Folkert
Published: June 2005

Liver X receptor (LXR) agonists have been proposed to act as anti-diabetic drugs. However, pharmacological LXR activation leads to severe hepatic steatosis, a condition usually associated with insulin resistance and type 2 diabetes mellitus. To address this apparent contradiction, lean and ob/ob mice were treated with the LXR agonist GW-3965 for 10 days. Insulin sensitivity was assessed by hyperinsulinemic-euglycemic clamp studies. Hepatic glucose production (HGP) and metabolic clearance rate (MCR) of glucose were determined with stable isotope techniques. Blood glucose and hepatic and whole body insulin sensitivity remained unaffected upon treatment in lean mice, despite increased hepatic triglyceride contents (61.7 ± 7.2 vs. 12.1 ± 2.0 nmol/mg liver, P < 0.05). In ob/ob mice, LXR activation resulted in lower blood glucose levels and significantly improved whole body insulin sensitivity. GW-3965 treatment did not affect HGP under normo- and hyperinsulinemic conditions, despite increased hepatic triglyceride contents (221 ± 13 vs. 176 ± 19 nmol/mg liver, P < 0.05). Clamped MCR increased upon GW-3965 treatment (18.2 ± 1.0 vs. 14.3 ± 1.4 ml·kg−1·min−1, P = 0.05). LXR activation increased white adipose tissue mRNA levels of Glut4, Acc1 and Fasin ob/ob mice only. In conclusion, LXR-induced blood glucose lowering in ob/ob mice was attributable to increased peripheral glucose uptake and metabolism, physiologically reflected in a slightly improved insulin sensitivity. Remarkably, steatosis associated with LXR activation did not affect hepatic insulin sensitivity.
ClC-5: role in endocytosis in the proximal tubule
Authors: Wang Yinghong , Cai Hui , Cebotaru Liudmila , Hryciw Deanne H. , Weinman Edward J. , Donowitz Mark , Guggino Sandra E. , Guggino William B.
Published: June 2005

The proper functioning of the Cl−channel, ClC-5, is essential for the uptake of low molecular mass proteins through receptor-mediated endocytosis in the proximal tubule. Dent’s disease patients with mutant ClC-5 channels and ClC-5 knockout (KO) mice both have low molecular mass proteinuria. To further understand the function of ClC-5, endocytosis was studied in LLC-PK1cells and primary cultures of proximal tubule cells from wild-type (WT) and ClC-5 KO kidneys. Endocytosis in the proximal tubule cells from KO mice was reduced compared with that in WT animals. Endocytosis in WT but not in KO cells was inhibited by bafilomycin A-1 and Cl−depletion, whereas endocytosis in both WT and KO cells was inhibited by the NHE3 blocker, S3226. Infection with adenovirus containing WT ClC-5 rescued receptor-mediated endocytosis in KO cells, whereas infection with any of the three disease-causing mutants, myc-W22G-ClC-5, myc-S520P-ClC-5, or myc-R704X-ClC-5, did not. WT and the three mutants all trafficked to the apical surface, as assessed by surface biotinylation. WT-ClC-5 and the W22G mutant were internalized similarly, whereas neither the S520P nor the R704X mutants was. These data indicate that ClC-5 is important for Cl−and proton pump-mediated endocytosis. However, not all receptor-mediated endocytosis in the proximal tubule is dependent on ClC-5. There is a significant fraction that can be inhibited by an NHE3 blocker. Our data from the mutants suggest that defective targeting and trafficking of mutant ClC-5 to the endosomes are a major determinant in the lack of normal endocytosis in Dent’s disease.
Oxygen availability limits renal NADPH-dependent superoxide production
Authors: Chen Yifan , Gill Pritmohinder S. , Welch William J.
Published: June 2005

Renal oxygen tension is substantially lower in the medulla than in the cortex and is reduced in hypertensive rats, a model of oxidative stress. Expression of NADPH oxidase, the primary source for superoxide anion (O2−·) in the kidney, is elevated in hypertension. Because molecular oxygen (O2) is required for O2−· formation, we tested the hypothesis that renal NADPH oxidase activity is limited by low O2. O2−· production by rat kidney tissue or cultured cells exposed to levels of Po2that mimics those in the kidney was assessed by lucigenin-enhanced chemiluminescence. NADPH-dependent O2−· production by kidney homogenates decreased reversibly by 60–90% after graded reductions of ambient O2from 10 to 0% (76 to 2 mmHg Po2). The NADPH-dependent O2−· production by the kidney homogenate was reduced by decreasing Po2below ∼30 mmHg. The response of tissue homogenates to low Po2was not different between normotensive and hypertensive rats. Similarly, NADPH-dependent O2−· production was lower during 2% O2compared with 10% O2in rat proximal tubule cells (−57 ± 1%), vascular smooth muscle (−42 ± 5%), cardiomyocytes (−57 ± 1%), and mouse inner medulla collecting duct cells (−58 ± 3%). We conclude that O2−· production by NADPH oxidase is dependent on availability of O2. Therefore, O2−· generation may be limited in the kidney, both in the normal renal medulla and in the cortex of hypertensive kidneys.
Angiotensin II, reactive oxygen species, and Ca2+signaling in afferent arterioles
Authors: Fellner Susan K. , Arendshorst William J.
Published: June 2005

In afferent arteriolar vascular smooth muscle cells, ANG II induces a rise in cytosolic Ca2+([Ca2+]i) via inositol trisphosphate receptor (IP3R) stimulation and by activation of the adenine diphosphate ribose (ADPR) cyclase to form cyclic ADPR, which sensitizes the ryanodine receptor (RyR) to Ca2+. We hypothesize that ANG II stimulation of NAD(P)H oxidases leads to the formation of superoxide anion (O2−·), which, in turn, activates ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2+]i. ANG II rapidly increased [Ca2+]iby 124 ± 12 nM. In the presence of apocynin, a specific inhibitor of NAD(P)H oxidase assembly, the [Ca2+]iresponse was reduced to 35 ± 5 nM ( P < 0.01). Tempol, a superoxide dismutase mimetic, did not alter the [Ca2+]iresponse to ANG II at a concentration of 10−4M (99 ± 12 nM), but 10−3M tempol reduced the response to 32 ± 3 nM ( P < 0.01). The addition of nicotinamide, an inhibitor of ADPR cyclase, to apocynin or tempol (10−3M) resulted in no further inhibition. Measurement of superoxide production with the fluorescent probe tempo 9-AC showed that ANG II caused an increase of 48 ± 20 arbitrary units; apocynin or diphenyl iodonium (an inhibitor of flavoprotein oxidases) inhibited the response by 94%. Hydrogen peroxide (H2O2) was studied at physiological (10−7M) and higher concentrations. In the presence of H2O2(10−7M), neither baseline [Ca2+]inor the response to ANG II was altered (125 ± 15 nM), whereas H2O2(10−6and 10−5M) inhibited the [Ca2+]iresponse to ANG II by 35 and 46%, respectively. We conclude that ANG II rapidly activates NAD(P)H oxidases of afferent arterioles, leading to the formation of O2−·, which then stimulates ADPR cyclase to form cADPR. cADPR, by sensitizing the RyR to Ca2+, augments the Ca2+response (calcium-induced calcium release) initiated by activation of the IP3R.
Zinc protects renal function during cadmium intoxication in the rat
Authors: Jacquillet G. , Barbier O. , Cougnon M. , Tauc M. , Namorado M. C. , Martin D. , Reyes J. L. , Poujeol P.
Published: June 2005

This study investigates the effect in the rat of chronic CdCl2intoxication (500 μg Cd2+/kg, daily ip injection for 5 days) on renal function and the changes in tight junction proteins claudin-2, claudin-3, and claudin-5 present in rat kidney. We also studied the effect of coadministration of ZnCl2(500 μg Zn2+/kg) during chronic CdCl2intoxication. Our results indicate that 1) most of the filtered Cd2+is reabsorbed within the kidney; 2) chronic Cd2+intoxication can induce a change in renal handling of ions without altering glomerular filtration rate; 3) a delayed nephropathy, showing Fanconi-like features, appears more than 5 days after the end of CdCl2exposure; 4) epithelial integrity is altered by chronic Cd2+intoxication affecting the expression and localization of claudin tight junction proteins; and 5) cotreatment with Zn2+protects against the renal toxic effects of Cd2+, preventing altered claudin expression and inhibiting apoptosis. In conclusion, these results show that Cd2+toxicity and cellular toxic mechanisms are complex, probably affecting both membrane transporters and tight junction proteins. Finally, Zn2+supplementation may provide a basis for future treatments.
Competitive Dynamics in Cortical Responses to Visual Stimuli
Authors: Moldakarimov Samat , Rollenhagen Julianne E. , Olson Carl R. , Chow Carson C.
Published: June 2005

Neurons in the visual cortex of the macaque monkey exhibit a variety of competitive behaviors, including normalization and oscillation, when presented with multiple visual stimuli. Here we argue that a biophysically plausible cortical circuit with opponent inhibition, spike-frequency adaptation, and synaptic depression can account for the full range of behaviors. The governing parameter is the strength of inhibition between competing neuronal pools. As the strength of inhibition is increased, the pattern of network behavior shifts from normalization mode to oscillatory mode, with oscillations occurring at progressively lower frequency until, at the extreme, winner-take-all behavior appears.
Evidence That Long-Term Hyperexcitability of the Sensory Neuron Soma Induced by Nerve Injury inAplysiaIs Adaptive
Authors: Gasull Xavier , Liao Xiaogang , Dulin Michael F. , Phelps Cynthia , Walters Edgar T.
Published: June 2005

Peripheral axotomy induces long-term hyperexcitability (LTH) of centrally located sensory neuron (SN) somata in diverse species. In mammals this LTH can promote spontaneous activity of pain-related SNs, and such activity may contribute to neuropathic pain and hyperalgesia. However, few axotomized SN somata begin to fire spontaneously in any species, and why so many SNs display soma LTH after axotomy remains a mystery. Is soma LTH a side effect of injury with pathological but no adaptive consequences, or was this response selected during evolution for particular functions? A hypothesis for one function of soma LTH in nociceptive SNs in Aplysia californica is proposed: after peripheral injury that produces partial axotomy of some SNs, compensation for sensory deficits and protective sensitization are achieved by facilitating afterdischarge near the soma, which amplifies sensory input from injured peripheral fields. Four predictions of this hypothesis were confirmed in SNs that innervate the tail. First, LTH of SN somata was induced by a relatively natural axotomizing event—a small cut across part of the tail in the absence of anesthesia. Second, soma LTH was selectively expressed in SNs having axons in cut or crushed nerves rather than nearby, uninjured nerves. Third, after several weeks soma LTH began to reverse when functional recovery of the interrupted afferent pathway was shown by reestablishment of a centrally mediated siphon reflex. Fourth, axotomized SNs developed central afterdischarge that amplified sensory discharge coming from the periphery, and the afterdepolarization underlying this afterdischarge was enhanced by previous axotomy.
Mouse Chromaffin Cells Have Two Populations of Dense Core Vesicles
Authors: Grabner Chad P. , Price Steven D. , Lysakowski Anna , Fox Aaron P.
Published: June 2005

The quantal hypothesis states that neurotransmitter is released in discrete packages, quanta, thought to represent the neurotransmitter content of individual vesicles. If true, then vesicle size should influence quantal size. Although chromaffin cells are generally thought to have a single population of secretory vesicles, our electron microscopy analysis suggested two populations as the size distribution was best described as the sum of two Gaussians. The average volume difference was fivefold. To test whether this difference in volume affected quantal size, neurotransmitter release from permeabilized cells exposed to 100 μM Ca2+was measured with amperometry. Quantal content was bimodally distributed with both large and small events; the distribution of vesicle sizes predicted by amperometry was extremely similar to those measured with electron microscopy. In addition, each population of events exhibited distinct release kinetics. These results suggest that chromaffin cells have two populations of dense core vesicles (DCV) with unique secretory properties and which may represent two distinct synthetic pathways for DCV biogenesis or alternatively they may represent different stages of biosynthesis.
GABAergic Control of the Ascending Input From the Median Raphe Nucleus to the Limbic System
Authors: Li Shaomin , Varga Viktor , Sik Attila , Kocsis Bernat
Published: June 2005

The median raphe nucleus (MRN) is the primary source of serotonergic afferents to the limbic system that are generally considered to suppress hippocampal theta oscillations. GABA receptors are expressed in the MRN by serotonergic and nonserotonergic cells, including GABAergic and glutamatergic neurons. This study investigated the mechanisms by which the fluctuating GABA tone in the MRN leads to induction or suppression of hippocampal theta rhythm. We found that MRN application of the GABAA agonist muscimol (0.05–1.0 mM) or GABAB agonist baclofen (0.2 mM) by reverse microdialysis had strong theta promoting effects. The GABAA antagonist bicuculline infused in low concentrations (0.1, 0.2 mM) eliminated theta rhythm. A short period of theta activity of higher than normal frequency preceded hippocampal desynchronization in 46% of rats. Bicuculline in larger concentrations (0.5, 1.0, 2.0 mM) resulted in a biphasic response of an initial short (<10 min) hippocampal desynchronization followed by stable theta rhythm that lasted as long as the infusion continued. The frequency and amplitude of theta waves were higher than in control recordings and the oscillations showed a conspicuous intermittent character. Hippocampal theta rhythm elicited by MRN administration of bicuculline could be completely (0.5 mM bicuculline) or partially (1.0 mM bicuculline) blocked by simultaneous infusion of the GABAB antagonist CGP35348 . Our findings suggest that the GABAergic network may have two opposing functions in the MRN: relieving the theta-generators from serotonergic inhibition and regulating the activity of a theta-promoting circuitry by the fluctuating GABA tone. The two mechanisms may be involved in different functions.
Temperature Compensation of Neuromuscular Modulation inAplysia
Authors: Zhurov Yuriy , Brezina Vladimir
Published: June 2005

Physiological systems that must operate over a range of temperatures often incorporate temperature-compensatory mechanisms to maintain their output within a relatively narrow, functional range of values. We analyze here an example in the accessory radula closer (ARC) neuromuscular system, a representative part of the feeding neuromusculature of the sea slug Aplysia. The ARC muscle's two motor neurons, B15 and B16, release, in addition to ACh that contracts the muscle, modulatory peptide cotransmitters that, through a complex network of effects in the muscle, shape the ACh-induced contractions. It is believed that this modulation is critical in optimizing the performance of the muscle for successful, efficient feeding behavior. However, previous work has shown that the release of the modulatory peptides from the motor neurons decreases dramatically with increasing temperature. From 15 to 25°C, for example, release decreases 20-fold. Yet Aplysia live and feed successfully not only at 15°C, but at 25°C and probably at higher temperatures. Here, working with reduced B15/B16–ARC preparations in vitro as well as a mathematical model of the system, we have found a resolution of this apparent paradox. Although modulator release decreases 20-fold when the temperature is raised from 15 to 25°C, the observed modulation of contraction shape does not decrease at all. Two mechanisms are responsible. First, further downstream within the modulatory network, the modulatory effects themselves—experimentally dissected by exogenous modulator application—have temperature dependencies opposite to that of modulator release, increasing with temperature. Second, the saturating curvature of the dose–response relations within the network diminishes the downstream impact of the decrease of modulator release. Thus two quite distinct mechanisms, one depending on the characteristics of the individual components of the network and the other emerging from the network's structure, combine to compensate for temperature changes to maintain the output of this physiological system.
Met‐Enkephalin Involvement in Morphine‐Modulated Peritonitis in Swiss Mice
Authors: Chadzinska Magdalena , Scislowska-Czarnecka Anna , Pierzchala-Koziec Krystyna , Plytycz Barbara
Published: June 2005

Morphine coinjection with zymosan inhibits pain and leukocyte
accumulation during peritonitis in several strains of mice, and affects systems of endogenous opioids. Present investigations focus on Met‐enkephalin (Met‐ENK) in the inflamed peritoneal cavity and brain centers of Swiss mice. Males of Swiss mice were
IP injected with zymosan or zymosan supplemented with morphine. At the selected time points the peritoneal leukocytes were counted and the Met‐ENK level was measured in exudatory fluid and leukocytes, striatum, hypothalamus, and pituitary gland. The
Met‐ENK content in peritoneal fluid rised sharply after zymosan injection, which corresponded with its decline in exudatory leukocytes, hypothalamus, and striatum. Morphine coinjection enhanced intraperitoneal accumulation of Met‐ENK and its release
from exudatory leukocytes, but inhibited its early fluctuations in hypothalamus and striatum. Effects of morphine‐modulated inflammation on the Met‐ENK system lasted longer than 7 days.
Peptidoglycan and Bacterial DNA Induce Inflammation and Coagulation Markers in Synergy
Authors: Amoureux Marie-Claude , Rajapakse Nandani , Stipkovits Lazlo , Szathmary Susan
Published: June 2005

Bacterial compounds signal the presence of foreign pathogens in the innate immune system. These microbial components are key players in infectious diseases and implicate toll‐like receptors in the activation of inflammation and coagulation. Nevertheless, the existence of a synergistic relationship between peptidoglycan and bacterial DNA on these two physiological responses has not been investigated. The present study reports new
findings on the regulation of tumor necrosis factor alpha and tissue factor in peripheral blood mononuclear cells by peptidoglycan and bacterial DNA. These were found to induce tumor necrosis factor alpha and tissue factor simultaneously and in a synergistic manner. These findings provide a new proinflammatory and procoagulant mechanism likely to play a role in sepsis pathogenesis.
Antiinflammatory and Immunomodulating Properties of FungalMetabolites
Authors: Lull Cristina , Wichers Harry J. , Savelkoul Huub F. J.
Published: June 2005

We discuss current information on the ability of extracts and
isolated metabolites from mushrooms to modulate immune responses. This can
result in a more enhanced innate and acquired disease resistance. The major
immunomodulating effects of these active substances derived from mushrooms
include mitogenicity and activation of immune effector cells, such as
lymphocytes, macrophages, and natural killer cells, resulting in the
production of cytokines, including interleukins (ILs), tumor necrosis factor
alpha (TNF)‐α, and interferon gamma (INF)‐γ. In particular, the ability of selective mushroom extracts to modulate the differentiation capacity of CD4 + T cells to mature into T H1 and/or T H2 subsets will be discussed. As a consequence these extracts will have profound effects in particular diseases, like chronic autoimmune T H1‐mediated or allergic T H2‐mediated diseases. Immunosuppressive effects by mushroom components have
also been observed. The therapeutic effects of mushrooms, such as anticancer activity, suppression of autoimmune diseases, and allergy have been associated with their immunomodulating effects. However, further studies are needed to determine the
molecular mechanisms of the immunomodulating effects of mushrooms metabolites both individually and in complex mixtures, for example, extracts.
Flow Cytometric Evaluation of Human Neutrophil Apoptosis During Nitric Oxide Generation In Vitro: The Role of Exogenous Antioxidants
Authors: Sulowska Zofia , Majewska Ewa , Klink Magdalena , Banasik Malgorzata , Tchórzewski Henryk
Published: June 2005

Among numerous inflammatory mediators a nitric oxide molecule is supposed to be important in the modulation of neutrophil survival in vivo and
in vitro. The effect of exogenous supply of NO donors such as SNP, SIN‐1, and GEA‐3162 on the course of human neutrophil apoptosis and the role of extracellular antioxidants in this process was investigated. Isolated from peripheral blood, neutrophils were cultured in the presence or absence of NO donor compounds and antioxidants for 8, 12, and 20 hours. Apoptosis of neutrophils was determined in vitro by flow cytometric analysis of cellular DNA content and Annexin V protein binding to the cell surface. Exposure of human neutrophils to GEA‐3162 and SIN‐1 significantly accelerates and enhances their apoptosis in vitro in a time‐dependent fashion. In the presence of SNP, intensification of apoptosis has not been revealed until 12 hours after the culture. The inhibition of GEA‐3162‐ and SIN‐1‐mediated neutrophil apoptosis by superoxide dismutase (SOD) but not by catalase (CAT) was observed. Our results show that SOD and CAT can protect neutrophils against NO‐donors‐induced apoptosis and suggest that the interaction of NO and oxygen metabolites signals may determine the destructive or protective role of NO donor compounds during apoptotic neutrophil death.
Proinflammatory Cytokines in AntilipopolysaccharideImmunity Against Klebsiella Infections
Authors: Rukavina Tomislav , Vasiljev Vanja , Ticac Brigita
Published: June 2005

This study was undertaken in order to determine whether proinflammatory cytokines are involved in a previously described protection against Klebsiella infection mediated by antilipopolysaccharide antibodies. BALB/c mice were
infected intraperitoneally with a lethal challenge of
Klebsiella pneumoniae Caroli. One group of mice was protected with monoclonal antibodies against lipopolysaccharide prior to infection and the second was not. We determined the number of colony‐forming units at different time points in the blood of infected animals and paralleled them with plasma levels of five proinflammatory cytokines measured by enzyme immunoassays. Our results show that the two groups of animals tested expressed
different plasma concentrations for all cytokines. The greatest difference was detected 24 hours after infection, with a higher production in the unprotected group. We concluded that a reduced cytokine production is partially responsible for the survival of protected animals.
Maternal and Cord Blood Levels of Serum Amyloid A, C‐Reactive Protein, Tumor Necrosis Factor‐α, Interleukin ‐1β, and Interleukin‐8 During and After Delivery
Authors: Cicarelli Luciane Marzzullo , Perroni Adriana Guimarães , Zugaib Marcelo , de Albuquerque Paulo Basto , Campa Ana
Published: June 2005

C‐reactive protein (CRP) and serum amyloid A (SAA) are acute‐phase
proteins mainly synthesized by the liver in response to some cytokines. They are potentially useful to diagnosing infection and monitoring different clinical conditions. The aim of this study was to measure SAA and CRP in maternal and cord blood during and
after delivery and try to correlate these proteins with tumor necrosis factor‐α, interleukin ‐1β, and interleukin‐8. Acute‐phase proteins and cytokines were measured by ELISA in 24 healthy pregnant women undergoing vaginal delivery or Cesarean section. Cord blood samples in addition to maternal blood were collected. SAA and CRP reached the maximum maternal serum levels 24 hours after delivery, while cytokines remained constant over time. SAA and CRP were significantly higher in maternal serum than in newborn′s (P < .001) at the moment of delivery. SAA and CRP, regardless of the type of delivery, reproduce the common pattern observed in most inflammatory conditions. Proinflammatory cytokine serum levels do not mirror the increase in SAA and CRP levels.
Serious Consequences to the Use of Cephalosporins as the First Line of Antimicrobial Therapy Administered in Hemodialysis Units
Authors: Marchaim Dror , Lazarovitch Zipora , Efrati Shai , Dishy Victor , Weissgarten Joshua , Boldur Ida , Zaidenstein Ronit
Published: June 2005

Background: The dramatic spread of vancomycin-resistant enterococci (VRE) among hemodialysis (HD) patients led to the replacement of vancomycin with cephalosporins as part of the primary empiric therapy for bacterial infections in HD units. The aim of the study was to examine the effects of this new regimen on the colonization rate of resistant bacteria among HD patients. Methods: Rectal swabs were taken from 105 HD patients and 91 control hospitalized patients. Groups were matched for age, sex, nursing home residency and background diseases. Enterococci were tested for vancomycin resistance, Staphylococcus aureus isolates were tested for methicillin resistance (MRSA), and Enterobacteriaceae were tested for extended-spectrum β-lactamase (ESBL) activity. Results: In the HD group 1 VRE, 1 MRSA and 9 ESBL-producing organisms were isolated compared to 1 MRSA and 1 ESBL organism in the control group (p = 0.018 for ESBL). In the year prior to the study, the use of cephalosporins had been enhanced in the HD group compared to the control group (p < 0.001), and in the HD ESBL-positive patients compared to the HD ESBL-negative ones (p = 0.007). The overall use of antibiotics in the control group was the same as in the HD group. In a subanalysis of the HD group alone, the ESBL carriers were older, sicker, used more antibiotics, were hospitalized frequently and had a higher mortality rate, compared to noncarriers. Conclusions: The use of cephalosporins as first-line therapy in HD patients in central Israel reduced the prevalence of VRE colonization but may have contributed to the emergence of ESBL-producing organisms through induction of selection pressure. This may lead to serious complications in the management of these patients.
Transforming Growth Factor-β1 SNPs: Genetic and Phenotypic Correlations in Progressive Kidney Insufficiency
Authors: Khalil M. Salah , El Nahas A.M. , Blakemore A.I.F.
Published: June 2005

Associations have been described between polymorphisms of cytokine and growth factor genes and susceptibility to, or progression of, an increasing number of diseases. TGF-β1 plays an important role in the pathogenesis of experimental and clinical glomerulosclerosis and tubulointerstitial fibrosis. In this study, single nucleotide polymorphisms (SNPs) in the TGFβ1 gene were investigated as possible markers for the progression of chronic kidney failure (CKF). 145 Caucasian patients with CKF were screened for four TGFβ1 SNPs: T-509C in the promoter region; Arg25Pro and Leu10Pro in exon 1 and Thr263Ile in exon 5. There were significant differences between CKF patients and controls in allele frequencies of two of the SNPs, Leu10Pro (p = 0.038) and C-509T (p = 0.02) and in haplotype distributions (p = 0.0175), indicating an association with susceptibility to CKF. We also observed a significant association between progression of CKF and homozygosity for Arg25 (odds ratio 3.77, 95% confidence interval 1.57–9.04, p = 0.002). Homozygosity for Arg25 was also associated with severity of proteinuria at diagnosis (p = 0.038), plasma TGF-β1 protein levels (p = 0.01), and severity of glomerulosclerosis (p = 0.04). Homozygosity for -509T was associated with severity of proteinuria at diagnosis (p = 0.0017), level of renal tubular TGF-β1 immunostaining (p = 0.0006) and with severity of renal interstitial inflammatory cellular infiltration (p = 0.01). Tubular TGF-β1 immunostaining was significantly higher in biopsies with inflammatory cellular infiltration compared those without inflammation (p = 0.0048). There was a significant difference in haplotype distributions between CKF patients with progressive, as opposed to non-progressive disease (p = 0.0484). TGFβ1 SNPs may be useful prognostic indicators for the progression of CKF.
Effect of Olopatadine and Other Histamine H1 Receptor Antagonists on the Skin Inflammation Induced by Repeated Topical Application of Oxazolone in Mice
Authors: Tamura Tadafumi , Masaki Shigehiro , Ohmori Kenji , Karasawa Akira
Published: June 2005

Histamine H1 receptor antagonists have long been prescribed for atopic dermatitis as an adjuvant therapy with topical therapy by local applied steroids. Olopatadine is one of the second-generation histamine H1 receptor antagonists that are treated for allergic disorders. We investigated that the effect of olopatadine on oxazolone-induced chronic contact hypersensitivity response in BALB/c mice compared with other histamine H1 receptor antagonists loratadine, cetirizine and fexofenadine. The chronic contact hypersensitivity induced by repeated application of oxazolone was treated with olopatadine and other histamine H1 receptor antagonists at the effective doses on histamine-induced paw edema in mice. The effects of these drugs in the oxazolone-induced model were quantified by measurements of ear swelling, and levels of cytokines in the lesioned ear. Olopatadine significantly inhibited the ear swelling and the increased production of IL-4, IL-1β, IL-6, GM-CSF and NGF in the lesioned ear. On the other hand, the other histamine H1 receptor antagonists did not significantly suppress the increase in ear thickness. Moreover, they did not affect the production of cytokines in the lesioned ear. These results indicate that olopatadine appears to exert additional biological effects besides its blockade of the histamine H1 receptor.
Chlamydia pneumoniae induces nitric oxide synthase and lipoxygenase- dependent production of reactive oxygen species in platelets
Authors: Bylin Helena , Leanderson Per , Richter Arina , Grenegård Magnus , Bengtsson Torbjörn , Kälvegren Hanna
Published: June 2005

There is increasing evidence that Chlamydia pneumoniae is linked to atherosclerosis and thrombosis. In this regard, we have recently shown that C. pneumoniae stimulates platelet aggregation and secretion, which may play an important role in the progress of atherosclerosis and in thrombotic vascular occlusion. The aims of the present study were to investigate the effects of C. pneumoniae on platelet-mediated formation of reactive oxygen species (ROS) and oxidation of low-density lipoprotein (LDL) in vitro. ROS production was registered as changes in 2‘,7’-dichlorofluorescin- fluorescence in platelets with flow cytometry. LDL-oxidation was determined by measuring thiobarbituric acid reactive substances (TBARs). We found that C. pneumoniae stimulated platelet production of ROS. Polymyxin B treatment of C. pneumoniae, but not elevated temperature, abolished the stimulatory effects on platelet ROS- production, which suggests that chlamydial lipopolysaccharide has an important role. Inhibition of nitric oxide synthase with nitro-L-arginine, lipoxygenase with 5,8,11-eicosatriynoic acid and protein kinase C with GF 109203X significantly lowered the production of radicals. In contrast, inhibition of NADPH-oxidase with di-phenyleneiodonium (DPI) did not affect the C. pneumoniae induced ROS-production. These findings suggest that the activities of nitric oxide synthase and lipoxygenase are the sources for ROS and that the generation is dependent of the activity of protein kinase C. The C. pneumoniae-induced ROS-production in platelets was associated with an extensive oxidation of LDL, which was significantly higher compared to the effect obtained by separate exposure of LDL to C. pneumoniae or platelets. In conclusion, C. pneumoniae interaction with platelets leading to aggregation, ROS-production and oxidative damage on LDL, may play a crucial role in the development of atherosclerotic cardiovascular disease.
Transcriptional regulation of ADAMTS13
Authors: Bockmeyer Clemens L. , Kentouche Karim , Sieber Matthias W. , Oberle Volker , Kaufmann Roland , Deigner Hans-Peter , Lösche Wolfgang , Claus Ralf A.
Published: June 2005

The metalloproteinase ADAMTS13 cleaves VWF multimers instantaneously when they are released from endothelial cells. Absent or manifestly diminished proteolytic activity of ADAMTS13 results in the appearance and accumulation of ultralarge VWF multimers (ULVWFM) in plasma, characterised by the manifestation of Thrombotic Thrombocytopenic Purpura (TTP). Despite congenital defects, infections and the actions of drugs such as cyclosporine A, doxycycline and corticosteroids apparently are involved in its development. To examine the possibility of transcriptional regulation of ADAMTS13 activity, we analyzed RNA levels in various cell culture systems and the response to known and assumed modulators of gene expression. We demonstrate the expression of ADAMTS13 in liver homogenates and a parenchyma liver cell culture system Hep3B, supporting the hypothesis that liver is an important source of plasma ADAMTS13, whereas there was no alteration in gene expression after stimulation of liver cells with proinflammatory stimuli such as endotoxin, TNF-α, IL-6, IL-1β as well as immuno-suppressive agents, such as cyclosporine A, a variety of steroids as well as doxycycline. Therefore, we analysed the ADAMTS13 gene for binding sites of transcription factors in silico and compared the data with those found in two sets of 24 genes considered either as differentially regulated by prototypic inflammatory regulation or as unvaried under various conditions. On the basis of these data, the promotor of ADAMTS13 features the characteristics of a gene, which remains unvaried under a variety of conditions. To our knowledge, the current data demonstrate for the first time, that an alteration in transcriptional activity is negligible in accounting for diminished proteolytic activity as observed under various experimental and, in particular, clinical conditions.
Tick saliva is a potent inhibitor of endothelial cell proliferation and angiogenesis
Authors: Mather Thomas N. , Ribeiro José M. C. , Francischetti Ivo M. B.
Published: June 2005

We report for the first time that saliva of the hard tick and Lyme disease vector,Ixodes scapularis,is a potent inhibitor of angiogenesis. Saliva (≤ 1:500 dilutions) or salivary gland (0.1–0.5 pairs/assay) dose-dependently inhibits microvascular endothelial cell (MVEC) proliferation. Inhibition was also detected with the saliva of the cattle tick Boophilus microplus but not with the salivary gland of Anopheles gambiae, An. stephensi, Lutzomyia longipalpis, Phlebotomus papatasi, Aedes aegypti, Culex quinquefasciatus, and Cimex lectularius. Inhibition of MVEC proliferation by I. scapularis saliva was accompanied by a change in cell shape (shrinkage of the cytoplasm with loss of cell-cell interactions) and apoptosis which was estimated by expression of phosphatidylserine using the Apopercentage dye, and by a typical pattern of chromatin margination, condensation, and fragmentation as revealed by nuclear staining with Hoechst 33258.The effect of saliva appears to be mediated by endothelial cell α5β1 integrin, because monoclonal antibodies against this but not αvβ3, αvβ5, α9β1, or α2β1 integrins remarkably block its effect. In addition, SDS/PAGE shows that saliva specifically degrades purified α5β1 but not αvβ5 or αvβ3 integrins. Incubation of saliva with EDTA and 1,10-phenanthroline, but not phenylmethylsulfonyl fluoride (PMSF), inhibits saliva-dependent degradation of purified α5β1 integrin, suggesting that a metalloprotease is responsible for the activity. Finally, saliva at ≤ 1:1,000 dilutions blocks sprouting formation from chick embryo aorta implanted in Matrigel, an in vitro model of angiogenesis. These findings introduce the concept that tick saliva is a negative modulator of angiogenesis-dependent wound healing and tissue repair, therefore allowing ticks to feed for days. Inhibition of angiogenesis was hitherto an unidentified biologic property of the saliva of any blood-sucking arthropod studied so far. Its presence in tick saliva may be regarded as an additional source of angiogenesis inhibitors with potential applications for the study of both vector and vascular biology.
Inhibition of lipopolysaccharide-induced tissue factor expression in monocytes by urinary trypsin inhibitor in vitro and in vivo
Authors: Molor-Erdene Perenlei , Isobe Hirotaka , Uchiba Mitsuhiro , Harada Naoaki , Shimozawa Nobuhiko , Okabe Hiroaki , Okajima Kenji
Published: June 2005

Tissue factor (TF) plays a critical role in the pathogenesis of disseminated intravascular coagulation (DIC) observed in patients with septic shock. Urinary trypsin inhibitor (UTI), a multivalent protease inhibitor, is currently used for treatment of patients with septic shock. This study was undertaken to determine whether UTI reduces LPS-induced coagulation abnormalities by inhibiting lipopolysaccharide (LPS)-induced expression of TF by monocytes. UTI inhibited LPS-induced increases in both TF activities andTF mRNA expression in monocytes without affecting the viability. Although activation of nuclear factor-κB (NF-κB), activator protein-1 (AP-1) and extracellular signal-regulated kinase (ERK)1/2 were shown to be critically involved in LPS-induced increases in TF activities in isolated monocytes, UTI inhibited phosphorylation of ERK1/2 and decreased expression of early growth response factor-1 (Egr-1) induced by LPS without affecting the activation of NF-κB and AP-1. UTI inhibited both the expression of TF mRNA in whole blood, increases in TF activities in mononuclear cells, and increases in serum levels of fibrin and fibrinogen degradation products (E) in rats given LPS without affecting the number of monocytes in the peripheral blood. Taken together these results strongly suggested that UTI might reduce LPS-induced coagulation abnormalities in rats by inhibiting TF expression in monocytes through inhibition of Egr-1 expression.
Pharmacological properties of YM-254890, a specific Gαq/11 inhibitor, on thrombosis and neointima formation in mice
Authors: Taniguchi Masatoshi , Moritani Yumiko , Uemura Toshio , Shigenaga Takeshi , Takamatsu Hajime , Hayashi Kazumi , Takasaki Jun , Saito Tetsu , Nagai Koji , Kawasaki Tomihisa
Published: June 2005

The pharmacological properties of YM-254890,a specific Gαq/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of Gαq/11.
Glycoprotein IIb/IIIa inhibition reduces prothrombotic events under conditions of deep hypothermic circulatory arrest
Authors: Azevedo Ruben , Beierlein Wolfram , Wendel Hans P. , Dietz Klaus , Ziemer Gerhard , Straub Andreas
Published: June 2005

Deep Hypothermic Circulatory Arrest (DHCA) is employed during thoracic aortic and congenital heart surgery, and can induce postoperative neurological damage probably caused by microthrombembolism. Hypothermia has been reported to induce platelet activation and aggregation. The platelet activation marker P-selectin mediates binding of platelets to leukocytes. Tirofiban and eptifibatide, short-acting inhibitors of the platelet fibrinogen receptor GP IIb/IIIa, have recently been shown to protect platelet function without increasing bleeding during heart surgery using cardiopulmonary bypass. The aim of this study was to investigate the effect of tirofiban and eptifibatide on platelets and platelet-leukocyte interaction under DHCA conditions in vitro.Platelet aggregation, binding of the GP IIb/IIIa activation specific antibody PAC-1, P-selectin expression as well as monocyte and granulocyte content of aggregates were investigated in un-stimulated and ADP-stimulated samples using flow cytometry. Tirofiban and eptifibatide inhibited massive platelet aggregation and PAC-1 binding which were induced by DHCA conditions. P-selectin expression was inhibited by tirofiban but increased by eptifibatide at hypothermia. Platelet-bound leukocytes were present in all samples. Eptifibatide increased granulocyte content of aggregates at hypothermia in ADP-stimulated samples. We conclude that under conditions of DHCA both tirofiban and eptifibatide inhibit platelet aggregation but have different effects on platelet P-selectin expression and platelet-leukocyte interaction. Application of a short-acting and non-activating GP IIb/IIIa inhibitor should be considered during DHCA in vivo to prevent occlusion of the microvasculature and subsequent organ damage.