Biomedical subjects
Publications and source records attributed to J Simon.
We report new precision measurements of the lifetimes of the Lambda(+)(c) and D0 from SELEX, the charm hadroproduction experiment at Fermilab. Based upon 1630 Lambda(+)(c) and 10 210 D0 decays we observe lifetimes of tau[Lambda(+)(c)] = 198.1+/-7.0+/-5.6 fs and tau[D0] = 407.9+/-6.0+/-4.3 fs.
The objective of this study was to examine the interaction of dopamine D2 receptor antagonists and dopamine uptake inhibitors on the regulation of extracellular dopamine release in the nucleus accumbens of Wistar rats employing in vivo microdialysis and in vitro dopamine uptake studies. Application of the D2 receptor antagonists raclopride (100 microm) or sulpiride (100 microm) alone through the microdialysis probe in the nucleus accumbens for 60 min increased the extracellular levels of dopamine in the nucleus accumbens to 150% and 200% of basal, respectively. Perfusion of the nucleus accumbens for 60 min with the dopamine uptake inhibitors, 1-[2-[bis(4-Fluorophenyl)methoxy]ethyl]-4-[3-phenylpropyl]piperazine dihydrochloride (GBR 12909; 100 microm) or 1-[2-(Diphenylmethoxy)ethyl]-4-(3-phenylpropyl)-piperazine dihydrochloride (GBR 12935; 100 microm) alone, increased the extracellular levels of dopamine in the nucleus accumbens to 400% and 350% of basal, respectively. Co-perfusion of 100 microM GBR 12909 or GBR 12935 with either 100 microM sulpiride or raclopride produced a significant reduction in the GBR 12909 or GBR 12935 induced increase in the extracellular levels of dopamine to basal levels. In vitro, GBR 12909 (1-9 nM) dose-dependently inhibited active uptake of [3H]dopamine in homogenates of the nucleus accumbens. Addition of 100 microm sulpiride had little effect on GBR 12909 inhibition of [3H] dopamine uptake, suggesting that dopamine D2 receptor antagonists are not blocking the actions of the GBR-type dopamine uptake inhibitors at the dopamine transporter. Overall, the data suggest that complex interactions occur in vivo between D2 antagonists and GBR-type dopamine uptake inhibitors, which negate their effects on elevating the extracellular levels of dopamine in the nucleus accumbens.
AIM: To develop non-invasive methods of measuring the quality of data recorded in general practice. METHODS: Laboratory and pharmaceutical claims data from fourteen practices (44 doctors) from the FirstHealth network of general practices were examined to determine the extent to which valid minimum bounds on expected rates of diagnosis coding could be established. These were compared with recorded rates in patient notes to measure completeness of diagnosis recording. Data completeness was measured for demographic data and a marker for the accuracy of gender coding was developed from diagnosis data. RESULTS: Minimum rates of diagnosis could be established for asthma, diabetes (NIDDM and IDDM), ischaemic heart disease, hypothyroidism, bipolar affective disorder and Parkinson's disease. Minimum bounds for the number of patients requiring monitoring of warfarin and digoxin levels were also established. These expected minimum rates were combined with measures of completeness of age, gender, ethnicity and smoking data, and a gender coding accuracy measure, to produce a set of fourteen data quality indicators. Pass/fail thresholds on each indicator were set and each of the fourteen practices was scored on the number of passes they achieved. The scores ranged from three to nine out of fourteen passses. CONCLUSIONS: Non-invasive data quality measures may be useful in providing feedback to general practitioners as part of a data quality improvement cycle. The sensitivity of this method will decline as data quality improves.
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For the widely distributed P2Y receptors for nucleotides, the transductional and functional responses downstream of their coupling to G proteins are poorly characterized. Here we describe apoptotic induction and the associated differential stimulation of mitogen-activated protein (MAP) kinase family members by the human P2Y(1) receptor. The potent P2Y(1) receptor agonist, 2-methylthio-ADP (2-MeSADP), stimulated the extracellular-signal regulated kinases (ERK1/2) (EC(50) approximately 5 nm) as well as several, but not all isoforms detected, of the stress-activated protein kinase (SAPK) family. Phospho-isoforms of p38 were unaffected. The induced kinase activity was blocked by the P2Y(1) receptor-selective antagonist, adenosine-2'-phosphate-5'-phosphate, but unaffected by pertussis toxin. In addition, the endogenous ligand ADP, and significantly also 2-MeSATP, induced concentration-dependent phosphorylation changes in the same MAP kinase family members. The sustained activation of ERK1/2 was associated with Elk-1 phosphorylation that was abolished by the MEK1 inhibitor, PD 98059. However, the concomitant transient activation of the SAPKs was not sufficient to induce c-Jun or ATF-2 phosphorylation. The transient phase of the ERK activity was partially inhibited either by the phosphatidylinositol 3-kinase inhibitor, LY 294002, or the PKC inhibitor, Gö 6976. In addition, the Src inhibitor, PP1, or expression of dominant negative Ras also attenuated the transient phase of ERK phosphorylation. In contrast, inhibition of Ras or Src had no effect on the sustained ERK activity, which was critically dependent on phosphatidylinositol 3-kinase. The transient SAPK activity was suppressed by expression of a dominant negative form of MKK4. Furthermore, this kinase-deficient mutant inhibited 2-MeSADP-induced caspase-3 stimulation and the associated decrease in cell number. In conclusion, adenosine di- and triphosphate stimulation of the human P2Y(1) receptor can transiently activate the Ras-ERK cascade via the cooperative effects of phosphatidylinositol 3-kinase, Src and PKC. The sustained ERK stimulation, via a Ras-insensitive pathway, culminates in Elk-1 activation without inducing a proliferation effect. The transient SAPK activity did not evoke transcription factor phosphorylation but was required for the P2Y(1) receptor-mediated apoptotic function.
The expression of the P2 receptors and their functional responses were studied in rat thyroid FRTL-5 cells. RT-PCR analysis revealed transcripts for the G protein-coupled P2Y(2), P2Y(4) and P2Y(6) receptors, and for the transmitter-gated ion channel P2X(3), P2X(4) and P2X(5) subunits. In Fura-2-loaded cells, UTP, ATP, ATPgammaS or UDP increased [Ca(2+)](i), and behaved as potent full agonists, while 2-Methylthio-ATP (2-MeSATP), alpha,beta-methylene-ATP (alpha,beta-meATP) and pure ADP were weak agonists. The agonist-mediated [Ca(2+) ](i) increases were diminished in Ca(2+) -free buffer, and by pertussis toxin (PTX) or suramin treatments. ATP, UTP, UDP and ATPgammaS increased (3)H-thymidine incorporation into DNA and expression of the protooncogenes c-Fos and c-Jun, while 2-MeSATP was ineffective, and alpha,beta-meATP gave a response only at 100-microM dose. The ATP-stimulated expression of c-Fos and c-Jun was dependent on Ca(2+), and protein kinase C, but not on calmodulin or Ca(2+)/calmodulin-dependent protein kinase II. Extracellular signal-regulated kinases (ERK1 and ERK2) are also involved as the MEK inhibitor, PD98059, reduced both ATP-evoked (3)H-thymidine incorporation and c-Fos and c-Jun expression. These results indicate that multiple P2Y receptor subtypes and at least the P2X(5) subtype are functionally expressed in FRTL-5 cells, and that nucleotides acting via P2 receptors are involved in the regulation of DNA-synthesis.
Local and systemic macrophage activation was examined during the course of monoarticular murine antigen-induced arthritis (AIA), induced by systemic immunization and subsequent local induction. The levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-12p70, and nitric oxide (NO) were determined in joints, sera, and supernatants of peritoneal macrophages (the latter unstimulated or stimulated ex vivo with LPS/IFN-gamma). In comparison with normal mice, systemic immunization (day 0) was associated to significant rise of TNF-alpha in serum, IL-1beta in the joints, IL-6 in unstimulated macrophages and IL-12p70 in stimulated macrophages. Local induction led to a further significant increase of: (i) TNF-alpha, IL-1beta, and IL-6 in the joints; and (ii) IL-1beta, and IL-6 in sera and stimulated macrophages during acute and/or early chronic AIA (days 1 to 7). Unstimulated macrophages showed increased NO release (day 3), while stimulated macrophages significantly increased secretion of IL-12p70 (day 1). In late chronic AIA (day 21), cytokine/NO expression returned to immunization levels or below at all sites; solely IL-1beta in the joints remained significantly above normal levels. Therefore, the prevalently local AIA model is characterized by a mixture of local and systemic activation of the mononuclear phagocyte system (MPS). While systemic MPS activation preceding arthritis induction can be attributed to systemic immunization, further systemic activation during arthritis appears an integral pathogenetic component of AIA.
The cell homogenate and the soluble cell fraction of Wolinella succinogenes grown with formate and fumarate catalyzed the oxidation of benzyl viologen radical by methacrylate [apparent Km=0.23 mM, Vmax=1.0 U (mg cell protein) -1] or acrylate [apparent Km=0.50 mM, Vmax=0.77 U (mg cell protein) -1]. Crotonate did not serve as an oxidant. A mutant of W. succinogenes lacking the fccABC operon was unable to catalyze methacrylate or acrylate reduction. In contrast, the inactivation of fccC alone had no effect on these activities. Methacrylate reduction by benzyl viologen radical was not catalyzed by fumarate reductase isolated from the membrane of W. succinogenes. Cells grown with formate and fumarate did not catalyze methacrylate reduction by formate, and W. succinogenes did not grow with formate and methacrylate as catabolic substrates. The results suggest that the reduction of methacrylate or acrylate by benzyl viologen radical is most likely catalyzed either by the periplasmic flavoprotein FccA or by a complex consisting of FccA and the predicted c-type cytochrome FccB. The metabolic function of the fccABC operon remains unknown.
In this study we have expressed recombinant P2X7 receptors in HEK293 cells and examined the reasons for the species- and agonist-dependent differences in the time taken for the closure of the P2X7 receptor ion-channels after agonist removal. Channel closure times, measured in electrophysiological studies or by measuring cellular permeability to ethidium cations, were slower at rat than at human or mouse P2X7 channels following washout of the P2X7 agonist 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (BzATP). In contrast, there were no species differences in channel closure times when ATP was the agonist. BzATP was more potent than ATP at the three species homologues and exhibited highest potency for rat P2X7 receptors suggesting that channel closure time was related to agonist potency. Furthermore, BzATP potency for the P2X7 receptor could be modified by changing extracellular ionic concentrations or by mutating the receptor and modifications which increased agonist potency also increased the time taken for channel closure. The dependence of channel closure time on agonist potency suggests it reflects agonist dissociation from the P2X7 receptor rather being an intrinsic property of the ion-channel. Consistent with this, our previous studies have shown that agonist potency increases after repeated agonist applications and in this study channel closure time at rat P2X7 receptors increased after repeated agonist applications. Overall these results suggest that the species differences in channel closure times reflect differences in agonist dissociation rates which arise as a consequence of the marked species differences in agonist potency.
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Despite intensive research, the nucleotide P2 receptor that is involved in the aggregation and activation of platelets by ADP has remained elusive. However, now two research groups have independently identified a new platelet receptor of unexpected structure, P2Y(12), that acts with the P2Y(1) receptor to form the site of ADP activation and explains the multiple transduction mechanisms observed in response to ADP in platelets. Recent evidence also suggests that a third component, ATP action on the P2X(1) receptor ion channel, contributes to platelet activation.
The aim of this study was to examine the efficacy and safety of both oxaliplatin as a single agent and oxaliplatin in combination with dailyx5 bolus 5-fluorouracil and folinic acid (5-FU/FA, Mayo clinic regimen) in the first-line treatment of metastatic colorectal cancer (CRC) patients. 73 advanced CRC patients were randomised to receive either oxaliplatin 85 mg/m(2) every 2 weeks (35 patients), or the same treatment combined with 5-FU 425 mg/m(2)/day and FA 20 mg/m(2)/dayx5 days every 4 weeks (38 patients). Treatment was continued until disease progression or unacceptable toxicity. All patients had documented inoperable disease and no previous chemotherapy for advanced disease. Based on the investigators' assessment of best response, objective response rate was 9% (95% confidence interval (CI) 2-24%) in the oxaliplatin arm, and 45% (95% CI 27-64%) in the oxaliplatin+5-FU/FA arm. Median progression-free survival (PFS) was 2 months (95% CI 1.7-2.4 months) in the oxaliplatin arm and 3.9 months (95% CI 2.9-5 months) in the oxaliplatin+5-FU/FA arm. Severe neutropenia was seen in 23% of patients in the oxaliplatin+5-FU/FA arm, and none in the oxaliplatin arm. There were two treatment-related deaths, both in the oxaliplatin+5-FU/FA arm. In the oxaliplatin+5-FU/FA arm, severe diarrhoea, vomiting and stomatitis were seen in 34, 14 and 14% of the patients, respectively. In conclusion, oxaliplatin at a dose of 85 mg/m(2) given every 2 weeks was well tolerated and has limited activity in metastatic CRC, while the combination of this treatment with the full-dose Mayo clinic regimen (5-FU bolus 425 mg/m(2)/day+FA 20 mg/m(2)/dayx5 days every 4 weeks), although active, was unfeasible due to a high level of myelosuppression and gastrointestinal toxicity. Alternative lower dosing or other regimens are to be explored to ascertain the value of bolus 5-FU/FA combined with oxaliplatin.
This study investigates the presence of sensory modulation dysfunction (SMD) among children with attention-deficit-hyperactivity disorder (ADHD). Twenty-six children with ADHD (mean age 8.3 years, 18 males, 8 females), and 30 typically developing children (mean age 8.2 years, 21 males, 9 females) were tested using a laboratory procedure that gauges responses to repeated sensory stimulation by measuring electrodermal reactivity (EDR). Parental report measures of limitations in sensory, emotional, and attentional dimensions were administered using the Short Sensory Profile, the Leiter International Performance Scale-Revised, Parent Rating subscales, and the Child Behavior Checklist (CBCL). Compared to the typical sample, the children with ADHD displayed greater abnormalities in sensory modulation on both physiological and parent-report measures. The children with ADHD also displayed more variability in responses. Within the group with ADHD, levels of SMD were highly correlated with measures of psychopathology on the CBCL. Implications of findings relate to the importance of considering sensory processing abilities in a subgroup of children with ADHD.
The Bcl-2-related survival proteins confer cellular resistance to a wide range of agents. Bcl-xL-expressing hepatocyte cell lines are resistant to tumour necrosis factor and anti-cancer drugs, but are more sensitive than isogenic control cells to antimycin A, an inhibitor of mitochondrial electron transfer. Computational molecular docking analysis predicted that antimycin A interacts with the Bcl-2 homology domain 3 (BH3)-binding hydrophobic groove of Bcl-xL. We demonstrate that antimycin A and a Bak BH3 peptide bind competitively to recombinant Bcl-2. Antimycin A and BH3 peptide both induce mitochondrial swelling and loss of DeltaPsim on addition to mitochondria expressing Bcl-xL. The 2-methoxy derivative of antimycin A3 is inactive as an inhibitor of cellular respiration but still retains toxicity for Bcl-xL+ cells and mitochondria. Finally, antimycin A inhibits the pore-forming activity of Bcl-x L in synthetic liposomes, demonstrating that a small non-peptide ligand can directly inhibit the function of Bcl-2-related proteins.
1. A P2Y (nucleotide) receptor activity in a clonal population (B10) of rat brain capillary endothelial cells is coupled to inhibition of adenylyl cyclase and has functional similarities to the P2Y(T) (previously designated 'P2T') receptor for ADP of blood platelets. However, the only P2Y receptor which was detectable in a previous study of B10 cells by mRNA analysis was the P2Y(1) receptor, which elsewhere shows no transduction via cyclic nucleotides. We have sought here to clarify these issues. 2. The inhibition of forskolin-stimulated adenylyl cyclase induced by purified nucleotides was measured on B10 cells. The EC(50) value for 2-methylthioADP (2-MeSADP) was 2.2 nM and, surprisingly, 2-MeSATP was an almost equally strong agonist (EC(50)=3.5 nM). ATP and 2-ClATP were weak partial agonists (EC(50)=26 microM and 10 microM respectively) and under appropriate conditions could antagonise the activity on 2-MeSADP. 3. A known selective antagonist of the platelet P2Y(T) receptor, 2-propylthioadenosine-5'-(beta,gamma)-difluoromethylene) triphosphonate (AR-C 66096), was a competitive antagonist of this B10 cell receptor, with pK(B)=7.6. That ligand is inactive at the P2Y(1) receptor in the same cells. Conversely, the competitive P2Y(1) receptor antagonists, the 3', 5'- and 2', 5'-adenosine bis-monophosphates, are, instead, weak agonists at the adenylyl cyclase-inhibitory receptor. 4. The inhibition of adenylyl cyclase by 2-MeSADP was completely abolished by pertussis toxin. 5. In summary, these brain endothelial cells possess a P2Y(T)-type receptor in addition to the P2Y(1) receptor. The two have similarities in agonist profiles but are clearly distinguishable by antagonists and by their second messenger activations. The possible relationships between the B10 and platelet P2Y(T) receptors are discussed.
OBJECTIVE: Mild hyperhomocysteinemia is a significant and independent risk factor for vascular diseases. Blood total homocysteine concentration (tHcy) is considered to be the product of an interaction between genetic and nutritional factors notably intake of folate, vitamin B12 and pyridoxine. The aim of the study was to determine whether regular intake of beer containing large amount of folate and other vitamins influences the tHcy blood concentrations. DESIGN: Cross-sectional population-based survey. SETTING: Adult population, residents of Pilsen (Czech Republic) and vicinity. SUBJECTS: Population series included 292 males and 251 females aged 35-65 y, mean age 53.4 y. All subjects were examined by a standard protocol for clinical, anthropometrical and laboratory estimations. MAIN OUTCOME MEASURES: tHcy was measured by high-pressure liquid chromatography with fluorescent detection, blood folate and B12 levels immunochemically using commercial kits. RESULTS: Beer intake was associated with blood folate and vitamin B12 concentrations positively and with tHcy concentration negatively. By categories of beer intake, subjects with intake of 1 l daily or more had significantly lower tHcy and higher folate concentrations than those reporting lower daily beer intake. CONCLUSION: Moderate beer consumption may help to maintain the tHcy levels in the normal range due to high folate content. Folate from beer may thus contribute to the protective effect of alcohol consumption on cardiovascular disease in population with generally low folate intake from other nutrients. SPONSORSHIP: Supported by grant 301/00/P089 Grant Agency of Czech Republic and Internal Grant Agency of the Ministry of Health.
The electron-transport chain that catalyzes nitrite respiration with formate in Wolinella succinogenes consists of formate dehydrogenase, menaquinone and the nitrite reductase complex. The latter catalyzes nitrite reduction by menaquinol and is made up of NrfA and NrfH, two c-type cytochromes. NrfA is the catalytic subunit; its crystal structure is known. NrfH belongs to the NapC/NirT family of membrane-bound c-type cytochromes and mediates electron transport between menaquinol and NrfA. It is demonstrated here by MALDI MS that four heme groups are attached to NrfH. A Delta nrfH deletion mutant of W. succinogenes was constructed by replacing the nrfH gene with a kanamycin-resistance gene cartridge. This mutant did not form the NrfA protein, probably because of a polar effect of the mutation on nrfA expression. The nrfHAIJ gene cluster was restored by integration of an nrfH-containing plasmid into the genome of the Delta nrfH mutant. The resulting strain had wild-type properties with respect to growth by nitrite respiration and nitrite reductase activity. A mutant (stopH) that contained the nrfHAIJ locus with nrfH modified by two artificial stop codons near its 5' end produced wild-type amounts of NrfA in the absence of the NrfH protein. NrfA was located exclusively in the soluble cell fraction of the stopH mutant, indicating that NrfH acts as the membrane anchor of the NrfHA complex in wild-type bacteria. The stopH mutant did not grow by nitrite respiration and did not catalyze nitrite reduction by formate, indicating that the electron transport is strictly dependent on NrfH. The NrfH protein seems to be an unusual member of the NapC/NirT family as it forms a stable complex with its redox partner protein NrfA.
We present two interesting cases of a 24-year-old man and a 14-year-old boy, uncle and nephew, with lower urinary tract symptoms, café au lait patches and subcutaneous nodules. Ultrasonography and computed tomography scans showed a large, irregular lobulated soft tissue mass between the bladder and sacrum. Cystoscopy, laparotomy and biopsies revealed neurofibromatosis involving the urinary bladder. No enlargement of the tumor or upper urinary tract obstruction has occurred during the long-term follow up. We recommend meticulous follow up of patients with giant intrapelvic neurofibromatosis.