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Abstract Background Sequence analysis of the orangutan genome revealed that recent proliferative activity of Alu elements has been uncharacteristically quiescent in the Pongo (orangutan) lineage, compared with all previously studied primate genomes. With relatively few young polymorphic insertions, the genomic landscape of the orangutan seemed like the ideal place to search for a driver, or source element, of Alu retrotransposition. Results Here we report the identification of a nearly pristine insertion possessing all the known putative hallmarks of a retrotranspositionally competent Alu element. It is located in an intronic sequence of the DGKB gene on chromosome 7 and is highly conserved in Hominidae (the great apes), but absent from Hylobatidae (gibbon and siamang). We provide evidence for the evolution of a lineage-specific subfamily of this shared Alu insertion in orangutans and possibly the lineage leading to humans. In the orangutan genome, this insertion contains three orangutan-specific diagnostic mutations which are characteristic of the youngest polymorphic Alu subfamily, AluYe5b5_Pongo. In the Homininae lineage (human, chimpanzee and gorilla), this insertion has acquired three different mutations which are also found in a single human-specific Alu insertion. Conclusions This seemingly stealth-like amplification, ongoing at a very low rate over millions of years of evolution, suggests that this shared insertion may represent an ancient backseat driver of Alu element expansion.

Abstract Background The human genome contains approximately one million Alu elements which comprise more than 10% of human DNA by mass. Alu elements possess direction, and are distributed almost equally in positive and negative strand orientations throughout the genome. Previously, it has been shown that closely spaced Alu pairs in opposing orientation (inverted pairs) are found less frequently than Alu pairs having the same orientation (direct pairs). However, this imbalance has only been investigated for Alu pairs separated by 650 or fewer base pairs (bp) in a study conducted prior to the completion of the draft human genome sequence. Results We performed a comprehensive analysis of all (> 800,000) full-length Alu elements in the human genome. This large sample size permits detection of small differences in the ratio between inverted and direct Alu pairs (I:D). We have discovered a significant depression in the full-length Alu pair I:D ratio that extends to repeat pairs separated by ≤ 350,000 bp. Within this imbalance bubble (those Alu pairs separated by ≤ 350,000 bp), direct pairs outnumber inverted pairs. Using PCR, we experimentally verified several examples of inverted Alu pair exclusions that were caused by deletions. Conclusions Over 50 million full-length Alu pairs reside within the I:D imbalance bubble. Their collective impact may represent one source of Alu element-related human genomic instability that has not been previously characterized.

Background: Medial patello-femoral ligament (MPFL) reconstruction is one of the therapeutic options to treat patellofemoral instability. Classically, a à la carte treatment of skeletal and ligament abnormalities is described. This option is difficult to achieve in children because bony procedures can damage the femoral and/or tibial growth plate. The objective was to evaluate a strategy for isolated reconstruction of the MPFL in the treatment of objective patellar instabilities in children, in a large cohort. The return to sport, knee function and pain or discomfort were studied as secondary endpoints., Methods: This French multicenter retrospective study included 54 pediatric patients with objective patellofemoral instability. Patients were included if they had presented at least 2 episodes of objective patella dislocation. A Deie-like technique with gracilis tendon graft, soft tissue femoral fixation and patellar bone tunnels for patellar fixation was used. Recurrence of dislocation was studied as the primary endpoint, and the recurrence rate was compared with the literature. A comparison of functional scores (Kujala, Lille femoro-patellar instability score or LFPI Score and Tegner activity score) and NRS between pre- and postoperative was studied as a secondary objective., Results: A recurrence of femoro-patellar instability was observed for five patients within 2 years follow up (9%). A significant improvement of the Kujala, LFPI score, Tegner and NRS scores was observed (p<0.001)., Conclusion: Isolated reconstruction of the MPFL presents a risk of recurrence of 9% at 2years follow-up. This technique significantly improves the functional scores of the knee. This modified Deie technique provides good clinical and functional results, allowing return to sports with an acceptable risk of recurrence of patellar dislocation, similar to those observed in the literature. Isolated MPFL reconstruction as a first-line treatment appears to be a reliable and effective technique in terms of recurrence of dislocation and functional scores. It allows early recovery and rehabilitation and has lower morbidity than procedures requiring bone gestures., Level of Evidence: III, retrospective comparative study., (Copyright © 2022. Published by Elsevier Masson SAS.)

Diabetes through adenosine A1 receptor (A1R) and P2 receptors (P2Rs) may lead to disturbances in renal microvasculature. We investigated the renal microvascular response to Ap4A, an agonist of P2Rs, in streptozotocin-induced diabetic rats. Using laser Doppler flowmetry, renal blood perfusion (RBP) was measured during infusion of Ap4A alone or in the presence of A1R antagonist, either DPCPX (8-cyclopentyl-1,3-dipropylxanthine) or 8-cyclopentyltheophylline (CPT). Ap4A induced a biphasic response in RBP: a phase of rapid decrease was followed by a rapid increase, which was transient in diabetic rats but extended for 30 min in nondiabetic rats. Phase of decreased RBP was not affected by DPCPX or CPT in either group. Early and extended increases in RBP were prevented by DPCPX and CPT in nondiabetic rats, while in diabetic rats, the early increase in RBP was not affected by these antagonists. A1R mRNA and protein levels were increased in isolated glomeruli of diabetic rats, but no changes were detected in P2Y1R and P2Y2R mRNA. Presence of unblocked A1R is a prerequisite for the P2R-mediated relaxing effect of Ap4A in nondiabetic conditions, but influence of A1R on P2R-mediated renal vasorelaxation is abolished under diabetic conditions., (© 2020 S. Karger AG, Basel.)

Purpose: Remote ischemic preconditioning protects peripheral organs against prolonged ischemia/reperfusion injury via circulating protective factors. Preconditioning with helium protected healthy volunteers against postischemic endothelial dysfunction. We investigated whether plasma from helium-treated volunteers can protect human umbilical vein endothelial cells (HUVECs) against hypoxia in vitro through release of circulating of factors., Methods: Healthy male volunteers inhaled heliox (79% helium, 21% oxygen) or air for 30 min. Plasma was collected at baseline, directly after inhalation, 6 h and 24 h after start of the experiment. HUVECs were incubated with either 5% or 10% of the plasma for 1 or 2 h and subjected to enzymatically induced hypoxia. Cell damage was measured by LDH content. Furthermore, caveolin 1 (Cav-1), hypoxia-inducible factor (HIF1α), extracellular signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription (STAT3) and endothelial nitric oxide synthase (eNOS) were determined., Results: Prehypoxic exposure to 10% plasma obtained 6 h after helium inhalation decreased hypoxia-induced cell damage in HUVEC. Cav-1 knockdown in HUVEC abolished this effect., Conclusions: Plasma of healthy volunteers breathing helium protects HUVEC against hypoxic cell damage, possibly involving circulating Cav-1.

Caveolins are involved in anaesthetic-induced cardioprotection. Actin filaments are located in close connection to Caveolins in the plasma membrane. We hypothesised that helium might affect the cytoskeleton and induce secretion of Caveolin. HCAEC, HUVEC and Cav-1 siRNA transfected HUVEC were exposed for 20 minutes to either helium (5% CO 2 , 25% O 2 , 70% He) or control gas (5% CO 2 , 25% O 2 , 70% N 2 ). Cells and supernatants were collected for infrared Western blot analysis, immunofluorescence staining, nanoparticle tracking analysis and permeability measurements. Helium treatment increased the cortical localisation of F-actin fibers in HUVEC. After 6 hours, helium decreased cellular Caveolin-1 (Cav-1) levels and increased Cav-1 levels in the supernatant. Cell permeability was decreased 6 and 12 hours after helium treatment, and increased levels of Vascular Endothelial - Cadherin (VE-Cadherin) and Connexin 43 (Cx43) were observed. Transfection with Cav-1 siRNA abolished the effects of helium treatment on VE-Cadherin, Cx43 levels and permeability. Supernatant obtained after helium treatment reduced cellular permeability in remote HUVEC, indicating that increased levels of Cav-1 are responsible for the observed alterations. These findings suggest that Cav-1 is secreted after helium exposure in vitro, altering the cytoskeleton and increasing VE-Cadherin and Cx43 expression resulting in decreased permeability in HUVEC.

Ischemia/reperfusion (I/R) of the heart becomes injurious when duration of the ischemic insult exceeds a certain threshold (approximately ≥20 min). Mitochondrial bound hexokinase II (mtHKII) protects against I/R injury, with the amount of mtHKII correlating with injury. Here, we examine whether mtHKII can induce the transition from non-injurious to injurious I/R, by detaching HKII from mitochondria during a non-injurious I/R interval. Additionally, we examine possible underlying mechanisms (increased reactive oxygen species (ROS), increased oxygen consumption (MVO 2 ) and decreased cardiac energetics) associated with this transition. Langendorff perfused rat hearts were treated for 20 min with saline, TAT-only or 200 nM TAT-HKII, a peptide that translocates HKII from mitochondria. Then, hearts were exposed to non-injurious 15-min ischemia, followed by 30-min reperfusion. I/R injury was determined by necrosis (LDH release) and cardiac mechanical recovery. ROS were measured by DHE fluorescence. Changes in cardiac respiratory activity (cardiac MVO 2 and efficiency and mitochondrial oxygen tension (mitoPO 2 ) using protoporphyrin IX) and cardiac energetics (ATP, PCr, ∆G ATP ) were determined following peptide treatment. When exposed to 15-min ischemia, control hearts had no necrosis and 85% recovery of function. Conversely, TAT-HKII treatment resulted in significant LDH release and reduced cardiac recovery (25%), indicating injurious I/R. This was associated with increased ROS during ischemia and reperfusion. TAT-HKII treatment reduced MVO 2 and improved energetics (increased PCr) before ischemia, without affecting MVO 2 /RPP ratio or mitoPO 2 . In conclusion, a reduction in mtHKII turns non-injurious I/R into injurious I/R. Loss of mtHKII was associated with increased ROS during ischemia and reperfusion, but not with increased MVO 2 or decreased cardiac energetics before damage occurs.

Purinoceptors (adrengeric receptors and P2 receptors) are expressed on the cellular components of the glomerular filtration barrier, and their activation may affect glomerular permeability to albumin, which may ultimately lead to albuminuria, a well-established risk factor for the progression of chronic kidney disease and development of cardiovascular diseases. We investigated the mechanisms underlying the in vitro and in vivo purinergic actions on glomerular filter permeability to albumin by measuring convectional albumin permeability (Palb) in a single isolated rat glomerulus based on the video microscopy method. Primary cultured rat podocytes were used for the analysis of Palb, cGMP accumulation, PKG-Iα dimerization, and immunofluorescence. In vitro, natural nucleotides (ATP, ADP, UTP, and UDP) and nonmetabolized ATP analogs (2-meSATP and ATP-γ-S) increased Palb in a time- and concentration-dependent manner. The effects were dependent on P2 receptor activation, nitric oxide synthase, and cytoplasmic guanylate cyclase. ATP analogs significantly increased Palb, cGMP accumulation, and subcortical actin reorganization in a PKG-dependent but nondimer-mediated route in cultured podocytes. In vivo, 2-meSATP and ATP-γ-S increased Palb but did not significantly affect urinary albumin excretion. Both agonists enhanced the clathrin-mediated endocytosis of albumin in podocytes. A product of adenine nucleotides hydrolysis, adenosine, increased the permeability of the glomerular barrier via adrenergic receptors in a dependent and independent manner. Our results suggest that the extracellular nucleotides that stimulate an increase of glomerular Palb involve nitric oxide synthase and cytoplasmic guanylate cyclase with actin reorganization in podocytes., (Copyright © 2016 the American Physiological Society.)

Background: Diabetic nephropathy is characterized by the dysfunction of renal microvasculature. The involvement of the P2X7 receptor, being a part of the purinergic system, is presumable in this process. The aim of our study was to investigate the P2X7 receptor-mediated renal microvasculature response and renal metabolism of extracellular adenine nucleotides in diabetic rats., Methods: Study was performed on streptozotocin-induced diabetic Wistar rats. The vascular response to BzATP, an agonist of the P2X7 receptor, was monitored based on the changes of cortical blood flow (CBF), glomerular filtration rate (GFR) and glomerular inulin space (GIS). The renal interstitial fluid (RIF) was probed by microdialysis technique and concentrations of ATP and adenosine were measured. Activity on NTDPase and 5'-nucleotidases was measured on renal membranes., Results: Diabetic kidneys were characterized by decreased ATP RIF and increased adenosine RIF concentrations with accompanied enhancement of NTDPase and 5'-nucleotidase activities. BzATP induced a more pronounced increase of CBF and decrease of GFR and GIS in diabetes rats. These effects were abolished by A438079, an antagonist of the P2X7 receptor., Conclusions: It is possible that increased P2X7 receptor reactivity may be involved in the pathogenesis of diabetic nephropathy., (Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Vagal activation can reduce inflammation and disease activity in various animal models of intestinal inflammation via the cholinergic anti-inflammatory pathway. In the current model of this pathway, activation of descending vagal efferents is dependent on a signal initiated by stimulation of vagal afferents. However, little is known about how vagal afferents are activated, especially in the context of subclinical or clinical pathogenic bacterial infection. To address this question, we first determined if selective lesions of capsaicin-sensitive vagal afferents altered c-Fos expression in the nucleus of the solitary tract (nTS) after mice were inoculated with either Campylobacter jejuni or Salmonella typhimurium. Our results demonstrate that the activation of nTS neurons by intraluminal pathogenic bacteria is dependent on intact, capsaicin sensitive vagal afferents. We next determined if inflammatory mediators could cause the observed increase in c-Fos expression in the nTS by a direct action on vagal afferents. This was tested by the use of single-cell calcium measurements in cultured vagal afferent neurons. We found that tumor necrosis factor alpha (TNFα) and lipopolysaccharide (LPS) directly activate cultured vagal afferent neurons and that almost all TNFα and LPS responsive neurons were sensitive to capsaicin. We conclude that activation of the afferent arm of the parasympathetic neuroimmune reflex by pathogenic bacteria in the gut is dependent on capsaicin sensitive vagal afferent neurons and that the release of inflammatory mediators into intestinal tissue can be directly sensed by these neurons., (Copyright © 2013. Published by Elsevier B.V.)

Background: Kidney noradrenergic innervation regulates tubular function. Adenosine triphosphate (ATP)-a co-transmitter of norepinephrine-acts on purinoceptors, including ion channel receptor, P2X. P2X receptor agonists, α,β-methylene ATP (α,β-meATP) and β,γ-methylene ATP (β,γ-meATP), induce natriuresis. Regarding the functional co-localization of adrenoceptors and P2X receptors, we evaluated rat renal tubular system sensitivity to natriuretic action of P2X receptor agonists in chronically denervated kidney., Methods: Clearance studies with α,β-meATP and β,γ-meATP (intravenous infusion rate, 2 µmol/kg + 20 nmol/kg/min) were performed after bilateral surgical kidney denervation (DNx) and sham-operation (Sham). Na/K-ATPase activity was measured in isolated rat renal proximal tubules., Results: In DNx compared with Sham, saline infusion significantly increased renal sodium and urine excretion and P2X receptor agonist infusion was significantly more natriuretic and diuretic. In DNx and Sham, respectively, α,β-meATP increased fractional excretion of sodium (FE(Na)) by 2 ± 0.3 and 0.6 ± 0.1% and urine (FE(V)) by 1.6 ± 0.3 and 0.9 ± 0.2%; β,γ-meATP had similar effects. In both groups of rats, natriuretic and diuretic actions were abolished by P2 receptor blocker (pyridoxal-phosphate-6-azophenyl-2',4'-disulphonate, PPADS), mean arterial blood pressure and glomerular filtration rate remained unchanged during infusion of P2X receptor agonists and antagonist and basal Na/K-ATPase activities in isolated proximal tubules were similar. Both α,β-meATP and β,γ-me-ATP decreased the Na/K-ATPase activity, with 20% inhibition (P < 0.05) in denervated and innervated rats; these inhibitory effects were abolished in the presence of PPADS., Conclusions: Decreased renal sympathetic activity enhances the natriuretic effect of P2X receptor stimulation. This effect is probably not related to altered Na/K-ATPase activity in renal proximal tubules.

Previous studies from our laboratory have reported a marked reduction in glomerular filtration rate (GFR) and sodium reabsorption in renal proximal tubule during intravenous infusion of P(1),P(4)-diadenosine tetraphosphate (Ap(4)A) at dose of 1.0 micromol/kg + 10 nmol/kg/min (i.v., injection followed by infusion) in anaesthetized Wistar rats. In the present study, the changes of GFR and urine sodium excretion were investigated in response to systemic infusion of Ap(4)A at different doses. Ap(4)A at dose of 0.1 micromol/kg + 1.0 nmol/kg/min did not change GFR and sodium urinary excretion whereas 2-fold higher dose produced significant (3.4-fold) increase in sodium excretion without changes in GFR. Significant but transient reduction in GFR by approximately 21% was observed during infusion of Ap(4)A at dose of 0.5 micromol/kg + 5.0 nmol/kg/min. Higher doses of Ap(4)A (1.0 micromol/kg + 10 nmol/kg/min and 2.0 micromol/kg + 20 nmol/kg/min) reduction in GFR and marked natriuresis. Our results suggest that tubular sodium transport systems are more sensitive to Ap(4)A than systems involved in GFR regulation.

Background/aims: P1,P4-diadenosine tetraphosphate (Ap4A) is a vasoactive dinucleotide possessing natriuretic activity. It is unclear, however, which part of the nephron is the target site of action for Ap4A., Methods: We evaluated the tubular sites of Ap4A action using the lithium clearance technique., Results: Ap4A at a priming dose of 2 micromol/kg with subsequent infusion at 20 nmol/kg/min increased fractional water and sodium excretion 2.5- and 5.6-fold, respectively. Moreover, Ap4A increased lithium clearance 1.9-fold and fractional lithium excretion 2.8-fold. Fractional water and sodium excretion from distal nephron segments was not significantly affected by Ap4A., Conclusion: These results suggest that Ap4A induces natriuresis mainly through inhibition of proximal tubular reabsorption of sodium., (Copyright 2007 S. Karger AG, Basel.)

1. Diadenosine polyphosphates (P(1),P(3)-diadenosine triphosphate, Ap(3)A; P(1),P(4)-diadenosine tetraphosphate, Ap(4)A; and P(1),P(5)-diadenosine pentaphosphate, Ap(5)A) are vasoactive molecules. The experimental model of isolated rat renal glomeruli was used to investigate their effects on glomerular vasculature. We measured the changes of glomerular inulin space (GIS) as a marker of glomeruli contractility. 2. Ap(4)A and Ap(5)A induced concentration- and time-dependent reduction of GIS whereas Ap(3)A had no effect. The effects of Ap(4)A and Ap(5)A (both at 1 microM) were prevented by a nonselective P2 receptor antagonist, that is, suramin (10 microM) and P2Y receptor antagonist - reactive blue 2 (50 microM). However, the antagonist of P1 receptor, that is, theophylline (1 microM) and A(1) receptor 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 10 microM) did not affect the responses of glomeruli to Ap(4)A or Ap(5)A. 3. Ap(3)A, in contrast to Ap(4)A and Ap(5)A, prevented angiotensin II-induced reduction of GIS in a concentration- and time-dependent manner. This effect was partially prevented by suramin and markedly reduced by reactive blue 2 and the specific antagonist of P2Y(1) receptor - MRS 2179 (10 microM). However, theophylline and the specific antagonist of A(2) receptor - 3,7-dimethyl-1-propargylxanthine (DMPX; 10 microM) - did not affect Ap(3)A action. 4. We indicate that diadenosine polyphosphates changed the glomerular volume via activation of P2 receptors. We suggest that extracellular Ap(4)A and Ap(5)A via P2X and P2Y receptors may decrease and Ap(3)A via, at least in part, P2Y(1) receptors may increase filtration surface, which in turn may modify glomerular filtration rate.

Cyclic AMP has been generally recognised as activator of cAMP-dependent protein kinases. However, there is little evidence about role of cAMP-dependent protein kinase (PKA), in particular izoenzymes PKA-I and PKA-II, in glomeruli contractility. We measured changes of glomerular inulin space (GIS) as a marker of its contractility in the presence of phosphodiesterase resistance cAMP analogues; activators and inhibitors of PKA. Activator of PKA i.e. (Sp) 8-Cl-cAMPS (0.1-100 microM) decreased GIS. (Rp) 8-Cl-cAMPS (0.1-100 microM), inhibitor of PKA, was ineffective but shifted concentration-response curve of (Sp) 8-Cl-cAMPS to right at 50 microM. Specific A site activation by N6-benzoyl-cAMP decreased GIS with maximum at 0.1 microM. Activation of B site by 8-aminobutyloamino-cAMP (0.1-100 microM) had no effect. However, specific activation of both sites on PKA-I or PKA-II by site-selective analogue pairs e.g. 8-aminobutyloamino-cAMP plus 8-piperidino-cAMP or N6-benzoyl-cAMP plus 8-piperidino-cAMP respectively, significantly increased sensitivity of glomeruli to analogues. Our data suggest that activation of PKA-I or PKA-II might have an important role in the regulation of glomerular contractility.

Background: Nebivolol and carvedilol are third-generation beta-adrenoreceptor antagonists, which unlike classic beta-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors., Methods and Results: Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [3H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or reactive blue 2 inhibited both beta-blocker-induced glomerular vasorelaxations and beta-blocker-stimulated NO release from GECs. Both beta-blocker-induced vasorelaxations were in the micromolar concentration range identical to that required for the beta-blocker stimulation of ATP and NO release from GECs. The maximum of NO release for nebivolol and carvedilol was very similar (188+/-14 and 226+/-17, respectively). Blockade of ATP release by a mechanosensitive ion channel blocker, Gd3+, inhibited the beta-blocker-dependent release of ATP and NO from GECs., Conclusions: These results demonstrate for the first time that nebivolol and carvedilol induce relaxation of renal glomerular microvasculature through ATP efflux with consequent stimulation of P2Y-purinoceptor-mediated NO release from GECs.

Effects of Ap4A and NAD--precursor of adenosine, on renal plasma flow (RPF), glomerular filtration rate (GFR) and urine excretion were determined in the anaesthetised rats. Infusion of Ap4A or NAD (i.v., bolus--1 micromol/kg followed by 10 nmol/min/kg) decreased RPF and GFR (by 30 and 40%, respectively). In spite of GFR reduction during Ap4A infusion, the significant increase in sodium excretion and urine flow was noticed: fractional sodium (FENa) and urine excretion (FEurine) rose 15-fold and 2.5-fold in comparison with the control value, respectively. In contrast to Ap4A, NAD-induced decrease in GFR was associated with parallel decrease in sodium and urine excretion, thus the FENa and FEurine did not significantly change. Pretreatment with adenosine deaminase (adenosine degrading enzyme, 2 U/min/kg) or theophylline (P1-receptors antagonist, 0.2 mmol/min/kg) ceased responses to NAD, whereas Ap4A-induced changes were not affected. Pre-treatment with suramin (P2-receptors antagonist, (i.v., bolus--12 mg/kg followed by 1.2 mg/min/kg) completely abolished the renal effects of Ap4A. We conclude that Ap4A may exert specific action on renal function. It acts different from NAD that modified renal function through its hydrolysis product--adenosine. Ap4A might reduce glomerular filtration rate and evoke natriuresis and diuresis, and its effects are probably mediated through stimulation of P2-receptors.

The activity of adenosine kinase (AK) was significantly impaired in splenocytes isolated from diabetic rats. Administration of insulin to diabetic animals restored AK activity, protein, and mRNA levels in diabetic splenocytes. Experiments performed on cultured rat lymphocytes demonstrated that insulin did not change the stability of AK mRNA. Insulin induced AK gene expression in a dose- and time-dependent manner. Maximal increases in AK mRNA (3.9-fold) and activity level (3.7-fold) were observed at the fourth and fifth hours of cell incubation with 10 nM insulin, respectively. The insulin effect on AK expression was not influenced by dibutyryl cAMP (dcAMP). On the other hand dcAMP weakly increased (1.7-fold) basal expression of AK. Exposure of rat lymphocytes to wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3K), or rapamycin, an inhibitor of mTOR, did not affect the ability of insulin to stimulate expression of AK. Prior treatment of the cells with 10 microM PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) kinase (MEK) completely blocked insulin-stimulated expression of AK gene. Insulin produced a significant transient increase in the tyrosine phosphorylation of ERK1/2, and PD98059 inhibited this phosphorylation. Furthermore exposure of cells to insulin has resulted in transient phosphorylation of Elk-1 on Ser-383 and sustained elevation of c-Jun and c-Fos protein. The maximal phosphorylation of Elk-1 was observed at 15 min, and was blocked by PD98059. We concluded that insulin stimulates AK gene expression through a series of events occurring sequentially. This includes activation of the MAPK cascade and subsequent phosphorylation of Elk-1 followed by increased expression of c-fos and c-jun genes.

Glomerular filtration rate (GFR) in response to adenosine precursor, NAD, and glomeruli contractility in response to adenosine were evaluated in streptozotocin-induced diabetic rats with severe (blood glucose 27.8 +/- 1.2 mmol/L) and moderate hyperglycaemia (18.2 +/- 0.9 mmol/L) compared with nondiabetic (ND)-rats. In anaesthetised rats, basal GFR was greater in moderately diabetic rats compared with severely diabetic rats (p < 0.05) and ND-rats (p < 0.02). Intravenous infusion of 5 nmol x min(-1) x kg(-1) NAD reduced GFR and renal plasma flow (RPF) in diabetic rats but had no effect on these parameters in ND-rats. Moreover, NAD-induced reduction of GFR and RPF was greater in rats with severe diabetes (41% and 30%, respectively) than in with moderate diabetes (25% and 26%, respectively). Theophylline (0.2 micromol x min(-1) x kg(-1) ) abolished renal response to NAD. Isolated glomeruli contraction in response to adenosine, assessed by glomerular 3H-inulin space reduction, was lowered in moderately diabetic-group and enhanced in severely diabetic-group. compared with ND-group (p < 0.05). Adenosine A1-receptor antagonist DPCPX inhibited adenosine-induced glomeruli contraction. This differential response of diabetic renal glomeruli to adenosine suggests that impaired glomerular contractility in response to adenosine could be responsible for hyperfiltration in moderate diabets, whereas, the increased adenosine-dependent contractility of glomeruli in severe diabetes may increase the risk of acute renal failure in this condition.

Campylobacter jejuni, a gram-negative spiral shaped bacterium, is a frequent cause of gastrointestinal food-borne illness in humans throughout the world. Illness with C. jejuni ranges from mild to severe diarrheal disease. This article focuses on Campylobacter virulence determinants and their potential role in the development of C. jejuni-mediated enteritis. A model is presented that diagrams the interactions of C. jejuni with the intestinal epithelium. Additional work to identify and characterize C. jejuni virulence determinants is certain to provide novel insights into the diversity of strategies employed by bacterial pathogens to cause disease.

Background: Extracellular ATP signaling affects the cells of renal glomeruli via activation of P2-purinoceptors, denoted as P2X and P2Y. Through either of these purinoceptors, ATP is able to stimulate an increase in intracellular [Ca2+]. P2Y-receptors are expressed on mesangial and endothelial cells, thus may participate in contraction and relaxation of glomeruli, respectively. Moreover, P2Y-receptors possess activity of ecto-ATPase which may lead to dephosphorylation of ATP and generation of adenosine. The aim of the present study was to investigate the involvement of P2Y-receptors in responses of renal glomeruli to extracellular ATP., Material and Methods: Renal glomeruli were isolated from rats by sieving technique. [3H]-inulin was used to measure the intracapillary volume of isolated glomeruli. Changes of intracapillary volume reflect contraction and relaxation of the glomeruli. ATP and adenosine concentration in the incubation mixture were measured using luminometric methods., Results: Extracellular ATP (1 microM) induced relaxation of Ang II-precontracted glomeruli in time-dependent manner. The glomeruli relaxed completely at 2nd minute of incubation. The relaxation was considerably diminished at 5th minute of incubation as compared to 2nd minute. Relaxing effect was completely prevented by an antagonist of P2Y-receptors i.e. reactive blue 2. The decrease in ATP concentration with time was accompanied by a rise in adenosine concentration which led to contraction of glomeruli. Non-metabolised analogue of ATP, an agonist of P2Y-receptors i.e. 2-methylthio-ATP (1 microM) induced complete relaxation at 2nd minute of incubation but there was no effect at 5th minute of incubation., Conclusions: The extracellular ATP through activation of P2Y-receptors may regulate the volume of renal glomeruli, which in turn influences on the glomerular filtration rate, through at least two mechanisms: one is ATP-dependent glomerular relaxation in the initiate phase and the other is glomerular contraction caused by either ATP itself or adenosine formed from ATP hydrolysis in maintenance phase.

Campylobacter jejuni, a common commensal in chickens, is one of the leading causes of bacterial gastroenteritis in humans worldwide. The aims of this investigation were twofold. First, we sought to determine whether mutations in the C. jejuni ciaB and pldA virulence-associated genes impaired the organism's ability to colonize chickens. Second, we sought to determine if inoculation of chicks with C. jejuni mutants could confer protection from subsequent challenge with the C. jejuni wild-type strain. The C. jejuni ciaB gene encodes a secreted protein necessary for the maximal invasion of C. jejuni into cultured epithelial cells, and the pldA gene encodes a protein with phospholipase activity. Also included in this study were two additional C. jejuni mutants, one harboring a mutation in cadF and the other in dnaJ, with which we have previously performed colonization studies. In contrast to results with the parental C. jejuni strain, viable organisms were not recovered from any of the chicks inoculated with the C. jejuni mutants. To determine if chicks inoculated with the C. jejuni mutants become resistant to colonization by the C. jejuni parental strain upon subsequent challenge, chicks were inoculated either intraperitoneally (i.p.) or both orally and i.p. with the C. jejuni mutants. Inoculated birds were then orally challenged with the parental strain. Inoculation with the C. jejuni mutants did not provide protection from subsequent challenge with the wild-type strain. In addition, neither the C. jejuni parental nor the mutant strains caused any apparent morbidity or mortality of the chicks. We conclude that mutations in genes cadF, dnaJ, pldA, and ciaB impair the ability of C. jejuni to colonize the cecum, that chicks tolerate massive inoculation with these mutant strains, and that such inoculations do not provide biologically significant protection against colonization by the parental strain.

Background: Atrial natriuretic factor (ANF)-induced increase in glomerular filtration rate (GFR) is inhibited on low sodium intake. It has been shown that activation of renin-angiotensin system on low sodium intake antagonizes the biological effect of ANF by interfering in the intracellular metabolism of cGMP. We have previously indicated that the renin-angiotensin system increases activity of Ca2+/calmodulin dependent-cyclic GMP phosphodiesterase (cGMP-PDE) in glomeruli and thereby inhibits the ANF-induced increase in GFR in low sodium-treated rats. The aim of the present study was to investigate whether low sodium intake might change glomerular cGMP metabolism by the alternative branch of the signal transduction pathway, namely protein kinase-C (PKC) activation., Material and Methods: cGMP formation and PKC activity were examined in isolated glomeruli from the rats maintained for five days on a normal or a low sodium diet. Renal hemodynamic parameters in clearance experiments during infusion of ANF (0.5 Kg/min/kg body weight) in both groups of rats were also evaluated., Results: Low sodium intake inhibited ANF-dependent increase in GFR and nephrogenous cGMP excretion, whereas urinary sodium excretion did not differ appreciably in rats on either diet. The basal and ANF-stimulated cGMP formation in isolated glomeruli was significantly inhibited in low sodium-treated rats as compared to normal sodium-treated rats. The inhibitory effect of low sodium intake on basal and ANF-stimulated glomerular cGMP formation was completely prevented by a selective cGMP-PDE inhibitor, zaprinast, but not affected by PKC activator, PMA, or PKC inhibitor, H-7. The activity of PKC in glomeruli neither in membrane fraction nor in cytosol fraction did not differ significantly between normal and low sodium-treated rats., Conclusions: These results demonstrate that the blunted glomerular response to ANF in rats on low sodium intake is due to decrease ability of cGMP formation in glomeruli by increasing activity of cGMP-PDE without altering activity of PKC.

Campylobacter jejuni are a common cause of human diarrheal illness. Previous work has demonstrated that C. jejuni synthesize a novel set of proteins upon coculturing with epithelial cells, some of which are secreted. The secreted proteins have been collectively referred to as Campylobacter invasion antigens (Cia proteins). Metabolic labeling experiments revealed that Cia protein synthesis and secretion are separable and that secretion is the rate-limiting step of these processes. Additional work indicated that Cia protein synthesis is induced in response to bile salts and various eukaryotic host cell components. Host cell components also can induce Cia protein secretion. Culturing C. jejuni on plates supplemented with the bile salt deoxycholate retarded the inhibitory effect of chloramphenicol on C. jejuni invasion, as judged by the gentamicin-protection assay. These data suggest that the coordinate expression of the genes encoding the Cia proteins is subject to environmental regulation.

Receptors for extracellular nucleotides (P2-purinoceptors) are expressed in renal glomerulus; both on mesangial and endothelial cells. In the present study we have evaluated the potential role of ATP in the regulation of glomerular contraction and relaxation. Using [3H]-inulin we measured the Glomerular Inulin Space (GIS), (that reflects mainly glomerular intracapillary volume), in the presence of ATP and its analogues e.g. 2-methylthio-ATP (P2Y-receptor agonist) and beta,gamma-methylene-ATP (P2X-receptor agonist). Incubation of the intact glomeruli with ATP or 2-methylthio-ATP or beta,gamma-methylene-ATP induced a decrease of GIS in similar magnitude as angiotensin II e.g.: about 10% of the basal value. When glomeruli were precontracted with angiotensin II it was observed that both ATP and 2-methylthio-ATP induced an increase of GIS to the basal value, similarly to atrial natriuretic factor. Furthermore, there was no relaxing effect with beta,gamma-methylene-ATP. We suggest that, these bidirectional changes of the intracapillary volume induced by the extracellular ATP may contribute to regulation of glomerular dynamics.

A DNA sequence was identified in isolates of Salmonella enterica serotype Typhimurium definitive type 104 (DT104). The PCR amplification of an internal segment of this sequence identified DT104 and the closely related U302 phage type among 146 isolates of S. enterica serotype Typhimurium tested, thus providing a tool for rapid identification of DT104 and related isolates.

Adenosine has been implicated as an important endogenous regulator of various tissue functions. In diabetes, the responsiveness of several tissues to adenosine is altered. The aim of this study was to investigate the activities of enzymes metabolizing adenosine in tissues of diabetic rats. The cytosolic activity (V(max)) of adenosine kinase (AK) was decreased by 50% in the kidney and by 40% in the heart and liver of diabetic rats. A decrease in the V(max) of AK in diabetic tissues was not associated with a change in the K(m) for adenosine. Evaluation of AK gene transcript status showed significantly lower levels of AK mRNA in diabetic tissues as compared to normal tissues. In diabetic kidneys, the level of AK gene transcript was lowered by 50% on first day after streptozotocin administration, and these reduced levels were sustained declined during the next 10 days. Smaller changes in AK gene transcript levels were observed in the heart and liver than in the kidney. The cytosolic activities of 5'-nucleotidase, AMP deaminase, and adenosine deaminase were unchanged in kidney, heart, and liver of diabetic rats. These results suggest that the turnover of the AMP-adenosine metabolic cycle might be impaired in diabetic tissues due to the reduced activity of adenosine kinase., (Copyright 2000 Academic Press.)

Virulence gene expression in most bacteria is a highly regulated phenomenon, affected by a variety of parameters including osmolarity, pH, ion concentration, iron levels, growth phase, and population density. Virulence genes are also regulated by temperature, which acts as an 'on-off' switch in a manner distinct from the more general heat-shock response. Here, we review temperature-responsive expression of virulence genes in four diverse pathogens.

We detail two methods for detection of cell death induced by infection of a human monocytic cell line with invasive Campylobacter bacteria. Staining with a natural ligand for exposed phosphatidylserine residues coupled with propidum iodide discriminated between apoptosis and necrosis. Additionally, cells infected with a bacterial strain expressing green fluorescent protein stained with dye sensitive to mitochondrial membrane potential demonstrated a direct association of bacteria with dying cells. Analyses of cells stained by these methods employing flow cytometry enumerated proportions of cell populations undergoing either apoptosis or necrosis after bacterial infection in vitro.

Campylobacter jejuni is a common cause of human gastrointestinal illness throughout the world. Infections with C. jejuni and Campylobacter coli are frequently acquired by eating undercooked chicken. The ability of C. jejuni to become established in the gastrointestinal tract of chickens is believed to involve binding of the bacterium to the gastrointestinal surface. A 37-kD outer membrane protein, termed CadF, has been described that facilitates the binding of Campylobacter to fibronectin. This study was conducted to determine whether the CadF protein is required for C. jejuni to colonize the cecum of newly hatched chicks. Day-of-hatch chicks were orally challenged with C. jejuni F38011, a human clinical isolate, or challenged with a mutant in which the cadF gene was disrupted via homologous recombination with a suicide vector. This method of mutagenesis targets a predetermined DNA sequence and does not produce random mutations in unrelated genes. The parental C. jejuni F38011 readily colonized the cecum of newly hatched chicks. In contrast, the cadF mutant was not recovered from any of 60 chicks challenged, indicating that disruption of the cadF gene renders C. jejuni incapable of colonizing the cecum. CadF protein appears to be required for the colonization of newly hatched leghorn chickens.

Cicletanine ((+/-)3-(4-chlorophenyl)-1,3-dihydro-7-hydroxy-6-methylfuro-[3,4-c ] pyridine) 3-(4-chlorophenyl)-1,3-dihydro-7-hydroxy-6-methylfuro-[3,4-c] pyridine) is a novel antihypertensive agent that has been shown to possess vasorelaxant, natriuretic, and diuretic properties in preclinical and clinical studies. The mechanism(s) by which cicletanine induces these biological effects has not been definitely established, although it appears to differ from that of other classes of antihypertensive drugs. The salidiuretic activity appears to be the result of an action of the sulfoconjugated metabolite of cicletanine, which inhibits the apical Na+-dependent Cl-/HCO3- anion exchanger in the distal convoluted tubule. The mechanism of the vasodilating effect of cicletanine seems to be complex; it may include stimulation of vascular prostaglandin synthesis, inhibition of the low Km cyclic GMP phosphodiesterases, and blockade of Ca2+ channels either directly or indirectly through a K+-channel opening effect. The drug has also been shown to interact with alpha-adrenergic, vascular histamine, and muscarinic receptors. We have also reviewed the other vascular effects of the drug, such as stimulation of nitric oxide synthesis and inhibition of both myosin light chain kinase and protein kinase C. Cicletanine protects cardiovascular and renal systems against the injuries induced by hypertension, in addition to its lowering of arterial pressure. Similarly to the vasorelaxant action of cicletanine, the various properties of the drug likely contribute to its protective effect against injury in hypertension.

Presented here is the first evidence that Campylobacter jejuni secrete proteins upon co-cultivation with host cells and in INT 407 cell-conditioned medium. A C. jejuni gene designated ciaB for Campylobacter invasion antigen B was identified, using a differential screening technique, which is required for this secretion process and the efficient entry of this bacterium into a host cell. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73 154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins associated with the invasion of host cells from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Confocal microscopic examination of C. jejuni-infected cells revealed that CiaB was translocated into the cytoplasm of the host cells. Culturing C. jejuni with INT 407 cells or in INT 407-conditioned medium resulted in the secretion of at least eight proteins, ranging in size from 12.8 to 108 kDa, into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all eight proteins, indicating that CiaB is required for the secretion process. The identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization and the pathogenesis of C. jejuni-mediated enteritis.

Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.

Campylobacter jejuni is a Gram negative, microaerophilic pathogen that causes gastroenteritis in humans. The genome of C. jejuni is AT-rich, with a mol% G + C of 30.4. This high AT content was hypothesized to result in unique codon usage. In the present study, we analyzed the codon usage of sixty-seven C. jejuni genes and generated a codon frequency table. As predicted, the codon usage of C. jejuni revealed a strong bias towards codons ending in A or U. In addition to determining codon usage frequencies, the relative synonymous codon usage values were calculated to identify rare and optimal codons. Seventeen codons were identified as optimal and twelve codons as rare. Thirty-two codons exhibited little or no bias. A plot of the effective number of codons versus the third position %G + C values for the sixty-seven genes revealed that C. jejuni uses an average of 39 of the 61 codons to encode proteins. These data will be useful for various molecular analyses including selection of degenerate primers to screen C. jejuni-genomic DNA libraries.

Clinical and in vitro experimental data suggest that invasion of intestinal epithelial cells is an essential step in the pathogenesis of Campylobacter jejuni-mediated enteritis. However, the molecular mechanism of C. jejuni internalization remains poorly defined. The goal of this study was to identify a gene that encodes a protein required for the internalization of C. jejuni into host cells. A C. jejuni gene, designated ciaB, was identified upon immunoscreening C. jejuni genomic DNA-phage libraries with an antiserum generated against C. jejuni co-cultivated with INT 407 cells. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73,154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins, associated with invasion of host cells, from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Immunoblot analysis using an anti-CiaB specific antibody revealed that CiaB is secreted into the supernatant fluids upon co-cultivation of C. jejuni with INT 407 cell conditioned medium. Metabolic labeling experiments revealed that at least eight C. jejuni proteins, ranging in size from 12.8 to 108 kDa, are secreted into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all proteins, indicating that CiaB is required for the secretion process. Identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization.

Campylobacter jejuni is a common cause of human gastrointestinal disease worldwide. Despite the prevalence of C. jejuni infections, the mechanisms of C. jejuni pathogenesis remain ill-defined. Invasion of the cells lining the intestinal tract is hypothesized to be essential for the development of C. jejuni-mediated enteritis. Recent studies in our laboratory have revealed that C. jejuni secrete proteins, termed Cia for Campylobacter invasion antigens, upon incubation with human intestinal cells. A mutation in one of the genes encoding a secreted protein resulted in an invasion-deficient phenotype. The purpose of this study was to identify a component capable of stimulating the synthesis and secretion of the Cia proteins from C. jejuni. Here, we report that these processes can be induced upon incubating C. jejuni in medium supplemented with fetal bovine serum. The synthesis and secretion of the Cia proteins were not affected by heat-treatment of the fetal bovine serum, indicating that the stimulating molecule in serum is heat stable. The stimulatory molecule was not unique to fetal bovine serum as sera from other sources including human, pig, sheep, goat, rabbit, mouse, and chicken also induced the synthesis and release of the Cia proteins. These findings indicate that the synthesis and secretion of the Cia proteins can be induced in a cell-free system by incubating C. jejuni in serum-supplemented tissue culture medium.

Campylobacter jejuni and Campylobacter coli are common causes of gastrointestinal disease and a proportion of C. jejuni infections have been shown to be associated with the Guillain-Barré syndrome. The waaC gene from Campylobacter coli, involved in lipopolysaccharide core biosynthesis, was cloned by complementation of a heptose-deficient strain of Salmonella typhimurium, as judged by novobiocin sensitivity, lipopolysaccharide (LPS)-specific phage sensitivity, and polyacrylamide-resolved lipopolysaccharide profiles. The C. jejuni waaC gene was subsequently cloned using the waaC gene isolated from C. coli as a probe. The C. jejuni and C. coli waaC genes are capable of encoding proteins of 342 amino acids with calculated molecular masses of 39381Da and 39317Da, respectively. Sequence and in-vitro analyses suggested that the C. coli waaC gene may be transcribed from its own promoter. Translation of the C. coli waaC gene in a cell-free system yielded a protein with a Mr of 39000. The waaC gene was detected in every C. jejuni and C. coli isolate tested as judged by dot-blot hybridization analysis. Southern hybridization analysis indicated that both Campylobacter species contain a single copy of the waaC gene. Unlike Escherichia coli and S. typhimurium isolates, the waaC gene in C. jejuni and C. coli isolates does not appear to be linked to the waaF (rfaF) gene.

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.