Supervised Exercise Therapy for Intermittent Claudication: A Propensity Score Matched Analysis of Retrospective Data on Long Term Cardiovascular Outcomes.

OBJECTIVE: This study aimed to explore the long term outcomes of patients with intermittent claudication (IC) who completed supervised exercise therapy (SET) vs. those who declined or prematurely discontinued SET, focusing on the incidence of chronic limb threatening ischaemia (CLTI), revascularisation, major adverse limb events (MALE), and major adverse cardiovascular events (MACE). METHODS: A retrospective registry analysis of consecutive patients with IC who were referred for SET between March 2015 and August 2016 and followed up for a minimum of five years. Serial univariable analysis and logistic regression were performed to identify the statistically significant clinical variables that were independent predictors of each outcome measure. The resulting statistically significant variables were used to guide 1:1 propensity score matching (PSM) using the nearest neighbour method with a calliper of 0.2. Cox proportional hazards regression was used to estimate the hazard ratio (HR) and 95% confidence interval (CI) for the association between SET and the outcomes of interest. RESULTS: Two hundred and sixty-six patients were referred to SET between March 2015 and August 2016. Of these, 64 patients completed SET and 202 patients did not. After PSM, 49 patients were analysed in each cohort. The Cox proportional hazards analysis revealed a significant association between completion of SET and revascularisation requirement (HR 0.46 95% CI 0.25 - 0.84; p = .011), completion of SET and progression to CLTI (HR 0.091, 95% CI 0.04 - 0.24; p < .001), completion of SET and MACE (HR 0.52; 95% CI 0.28 - 0.99; p = .05) and completion of SET and MALE (HR 0.28, 95% CI 0.13 - 0.65; p = .003). The Harrell's C index for all of these models was greater than 0.75, indicating good predictive accuracy. CONCLUSION: Completion of SET is associated with better outcomes in patients who completed SET compared with patients who declined or discontinued SET with respect to clinically important cardiovascular outcomes over seven years.

Clinical outcomes following mechanochemical ablation of superficial venous incompetence compared with endothermal ablation: meta-analysis.

BACKGROUND: Mechanochemical ablation (MOCA) is an alternative method to endovenous thermal ablation (EVTA) for the treatment of superficial venous incompetence that does not require tumescent anaesthesia. The aim of this study was to compare the outcomes from RCTs of MOCA versus EVTA. METHODS: A search was conducted in MEDLINE, Embase, and the Cochrane Central Register of Controlled Trials (CENTRAL). Meta-analysis inclusion was restricted to RCTs comparing MOCA against EVTA. Outcomes included anatomical occlusion rate, disease-specific quality of life using the Aberdeen Varicose Vein Questionnaire, procedural and postprocedural pain, and rates of venous thromboembolism. RESULTS: Four RCTs were included in the meta-analysis comprising 654 patients. The anatomical occlusion rate at 1 year was lower after MOCA than EVTA (risk ratio 0.85, 95 per cent c.i. 0.78 to 0.91; P < 0.001). No significant differences were detected in procedural pain (mean difference -3.25, -14.25 to 7.74; P = 0.560) or postprocedural pain (mean difference -0.63, -2.15 to 0.89; P = 0.420). There were no significant differences in Aberdeen Varicose Vein Questionnaire score at 1 year (mean difference 0.06, -0.50 to 0.62; P = 0.830) or in incidence of venous thromboembolism (risk ratio 0.72, 95 per cent c.i. 0.14 to 3.61; P = 0.690). CONCLUSION: The rate of successful anatomical occlusion after MOCA is significantly lower than that after EVTA, but there is no difference in procedural and postprocedural pain between the two interventions. Long-term data are required to assess the impact of the reduced vein occlusion rate on clinical outcomes such as quality of life and reintervention.

The current first-line treatment for varicose veins uses heat to block the diseased veins and is called endovenous thermal ablation (EVTA). Mechanochemical ablation (MOCA) is an alternative method of treatment using a chemical and a fast-spinning wire to block the veins instead. The potential benefits of MOCA include less pain and fewer complications. The aim of this study was to identify high-quality clinical trials comparing MOCA with EVTA, and to assess any differences in the results of treatment. The results showed that MOCA was less successful in blocking the diseased veins than EVTA. There were no differences in the amount of pain or discomfort during or after the procedures (which was low). At 1 year, those treated with both techniques reported the same quality of life. Both techniques were effective over 1 year in terms of improving quality of life; however, the potential benefits of MOCA were not clearly proven in the trials, and the poorer rates of successfully blocking the veins may cause the varicose veins to come back sooner, or the quality-of-life improvement to be shorter lived. There was no evidence to support MOCA replacing EVTA as the first-line treatment in the majority of patients, but it is a viable treatment for selected people.

High proportions of asymptomatic and submicroscopic Plasmodium vivax infections in a peri-urban area of low transmission in the Brazilian Amazon.

BACKGROUND: Population-based studies conducted in Latin America have shown a high proportion of asymptomatic and submicroscopic malarial infections. Considering efforts aiming at regional elimination, it is important to investigate the role of this asymptomatic reservoir in malaria transmission in peri-urban areas. This study aimed to estimate the prevalence of Plasmodium spp. and gametocyte burden on symptomatic and asymptomatic infections in the Brazilian Amazon. RESULTS: Two cross-sectional household surveys (CS) were conducted including all inhabitants in a peri-urban area of Manaus, western Amazonas State, Brazil. Malaria parasites were detected by light microscopy (LM) and qPCR. Sexual stages of Plasmodium spp. were detected by LM and RT-qPCR. A total of 4083 participants were enrolled during the two surveys. In CS1, the prevalence of Plasmodium vivax infections was 4.3% (86/2010) by qPCR and 1.6% (32/2010) by LM. Fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In CS2, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive. The P. vivax parasite density was associated with gametocyte carriage (P < 0.001). Sixty-seven percent of P. vivax infected individuals and 53.4% of P. vivax gametocyte carriers were asymptomatic. CONCLUSIONS: This study confirms a substantial proportion of asymptomatic and submicroscopic P. vivax infections in the study area. Most asymptomatic individuals carried gametocytes and presented low asexual parasitemia. This reservoir actively contributes to malaria transmission in the Brazilian Amazon, underscoring a need to implement more efficient control and elimination strategies.

Intradural calcifying pseudoneoplasm of the neuraxis presenting as a cauda equina syndrome.

BACKGROUND: Calcifying nonneoplastic pseudoneoplasms of the neuraxis (CAPNON) have been reported in 59 cases in literature, however, they rarely involve the spinal cord. Owing to the advances in immunohistochemical markers, their structure and origin are better understood now. CASE REPORT: We present the case of a 72-year-old female who had longstanding history of low back pain that exacerbated 20 days prior to the presentation to the emergency room with a frank cauda equina syndrome. The lumbar computed tomography scan showed a hyperdense lesion, suggestive of calcified tumor, whereas the magnetic resonance imaging revealed a hypointense lesion on theT1 and T2-weighted images, without contrast enhancement or edema on fluid-attenuated inversion recovery. She underwent an emergent L2-L4 laminectomy and L3-L4 discectomy with resection of L2 intradural tumor, following which she regained normal function. CONCLUSION: A 72-year-old female presented with a cauda equina syndrome attributed to an L2 intradural CAPNON. Following gross total resection, the patient was neurologically intact.

Use of surface plasmon-coupled emission for enhancing light transmission through Top-Emitting Organic Light Emitting Diodes.

In this paper, we perform surface plasmon-coupled emission studies on Rhodamine 6G molecules embedded in a corrugated structure of a thin film composed of fluorinated silica particles, and a binding medium. Our results show enhancements of photoluminescence due to surface corrugation. By varying the size of the fluorinated silica nanoparticles we were able to control the surface correlation length scale of the corrugated surface structure. It was found that the coupling efficiency of the directional light emission is strongly correlated to the surface morphology, particularly the surface correlation length, of the corrugated dielectric structure. This substantial enhancement of signal could potentially be utilized in Organic Light Emitting Diode devices to enhance the light emission and transmission through a thin silver layer which can also serve as the cathode in Top-Emitting Organic Light Emitting Diodes.

High-grade dysplasia in Barrett's esophagus is associated with increased expression of calgranulin A and B.

OBJECTIVE: Patients with Barrett's esophagus (BE) are at risk of developing esophageal adenocarcinoma, which is usually preceded by dysplastic changes of the metaplastic mucosa. The aim of this study was to increase the understanding of the development of dysplastic lesions in BE through the identification of genes that are differentially transcribed in these tissue types. MATERIAL AND METHODS: Paired biopsy samples from non-dysplastic BE, and high-grade dysplasia from a single patient were used for histological evaluation and gene expression profile analysis. In addition, relative mRNA levels of differentially expressed genes were tested to validate the association with the presence or absence of dysplasia by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) (58 biopsy samples containing squamous epithelium, non-dysplastic BE, high-grade dysplasia, or adenocarcinoma from 23 unrelated patients) and immunohistochemistry (9 sets of paired non-dysplastic/high-grade dysplasiac samples from 9 unrelated patients). RESULTS: Microarray results from high-grade dysplasia showed 866 genes with a>2-fold difference in mRNA levels compared with non-dysplastic BE. Subsequent comparison of mRNA levels of the 22 genes with a>10-fold difference in 76 unrelated biopsies showed that only two of these genes, i.e. calgranulin A (S100A8; p=0.017) and calgranulin B (S100A9; p=0.022), were consistently up-regulated in high-grade dysplasia, as were protein levels for calgranulin A and B. CONCLUSIONS: This is the first report of an association between the calprotectin complex, which is involved in chemotaxis of neutrophils, and the progression towards high-grade dysplasia in BE. It remains to be established whether differentially expressed proteins in biopsies form BE can be used to facilitate the diagnosis of advanced dysplasia in BE.

Evaluation of the in situ, time-integrated DGT technique by monitoring changes in heavy metal concentrations in estuarine waters.

Various natural and anthropogenic processes influence heavy metal concentrations within estuaries. In situ, time-integrated DGT measurements made over concurrent tidal phases found significantly higher concentrations of Cu (probability p=0.017), Zn (p=0.003) and Ni (p=0.003) during the flood phase, because the incoming tide passes several point sources. DGT-reactive Cu concentrations significantly decreased with increased tidal-flushing and vice versa within a marina (correlation r=-0.788, p=0.02). DGT measurements also recorded significant increases in Cu (4 out of 4 sites, p<0.001) and Zn (3 out of 4 sites, p< or =0.015) after a 24 mm rainfall event. Finally, DGT-reactive Cu increased significantly (p<0.001) during peak boating times, due to increased numbers of Cu-antifouled boats. This study demonstrates that, with judicious selection of deployment times, DGT measurements enable changes in heavy metal concentrations to be related to various cycles and events within estuaries.

Analysis of restriction fragment length polymorphism in the kappa-casein gene related to weight expected progeny difference in Nellore cattle

Restriction fragment length polymorphism (RFLP) has been detected at the bovine kappa-casein locus. The polymorphism has been analyzed for its effects in cattle production, mostly for milk traits and even for maternal effect on pre-weaning weights. We used polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) to genotype 408 Nellore animals for the non-silent mutation (Thr/Ile136 and Asp/Ala148) that characterizes the A and B variants of the polymorphism and compared expected progeny difference (EPD) for a maternal effect on 120 and 210 days weights and direct EPD for 120, 210, 450 and 550 day weight between AA and AB animals. The EPD values were obtained from the University of São Paulo (Brazil) Nellore Cattle Breeding Program, which evaluated 266,272 animals in 2001. Analysis of Variance was used to compare weight expected progeny differences (EPDs) between animals genotyped as AA and AB. The A allele frequency was 0.911. Although the AA animals had higher weight EPDs than AB animals the differences were not statistically significant (p > 0.05).

The calcium-sensing receptor regulates calcium absorption in MDCK cells by inhibition of PMCA.

Calcium transport across a monolayer of Madin-Darby canine kidney (MDCK) cells was measured in response to stimulation of the basal surface with calcium-sensing receptor (CaR) agonists. Stimulation of the CaR resulted in a time- and concentration-dependent inhibition of calcium transport but did not change transepithelial voltage or resistance. Inhibition of transport was not altered by pretreatment of cells with pertussis toxin but was blocked by the phospholipase C (PLC) inhibitor U-73122. To determine a potential mechanism by which the CaR could inhibit calcium transport, we measured activity of the plasma membrane calcium ATPase (PMCA). Stimulation of the CaR on the basal surface resulted in an inhibition of the PMCA in a concentration- and PLC-dependent manner. Thus stimulation of the CaR inhibits both calcium transport and PMCA activity through a PLC-dependent pathway. These studies provide the first direct evidence that calcium can inhibit its own transcellular absorption in a model of the distal tubule. In addition, they provide a potential mechanism for the CaR to inhibit calcium transport, inhibition of PMCA.

The calcium-sensing receptor stimulates JNK in MDCK cells.

The calcium-sensing receptor (CaR) stimulates ERK1 in rat fibroblasts, but its effect on other MAP kinases is not known. We used a model of renal distal tubule, the MDCK cell, to determine the effects of CaR stimulation on Jun kinase (JNK) activity. Stimulation of the CaR with 5 mM Ca(2+) resulted in a time-dependent increase in JNK activity. Activation of JNK occurred preferentially with stimulation on the basal surface relative to the apical surface. Basal administration of the CaR agonist gadolinium (30 microm) also stimulated JNK activity. Pertussis toxin blocked the ability of both CaR agonists to stimulate JNK, indicating that the effect was mediated through G(ialpha) class G proteins. Finally, we used confocal microscopy to determine that the CaR was located predominantly on the basal surface. These studies demonstrate for the first time that the CaR stimulates JNK activity.

Interaction of cyclosporine and FK506 with diuretics in transplant patients.

BACKGROUND: The calcineurin inhibitors cyclosporine and FK506 are widely used for immunosuppression in solid organ transplantation. One of the side effects of these agents is renal magnesium wasting. The site of action and molecular mechanism of this effect are not known. We hypothesized that agents such as diuretics that cause renal magnesium wasting through a similar action would not have an additive effect on magnesium deficiency with calcineurin inhibitors. METHODS: The records of 50 heart transplant patients on calcineurin inhibitors were reviewed to determine levels of serum magnesium and required replacement dose of magnesium, diuretic usage, and other laboratory values. RESULTS: Loop diuretics did not change either the magnesium level or magnesium replacement requirements in patients on calcineurin inhibitors. In contrast, the thiazide diuretic resulted in an increase in serum magnesium and a decrease in magnesium replacement. Results were similar when the cyclosporine or FK506 groups were evaluated alone. Patients taking FK506 had lower serum magnesium values and higher requirements for magnesium replacement compared with patients taking cyclosporine. CONCLUSION: We conclude that calcineurin inhibitors and loop diuretics have a similar site of action.

Deficient homologous desensitization of formyl peptide receptors stably expressed in undifferentiated HL-60 cells.

The ability of formyl peptide receptors (FPRs) stably expressed in undifferentiated HL-60 cells to undergo ligand-induced desensitization was compared with their ability in normal and vector-transfected HL-60 cells following granulocyte differentiation with DMSO. fMet-Leu-Phe failed to induce uncoupling of FPRs from G-proteins in FPR-transfected cells, whereas uncoupling was induced in differentiated HL-60 cells and differentiated vector-transfected HL-60 cells, as determined by ligand-stimulated guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) binding and GTPgammaS inhibition of fMet-Leu-Phe binding to isolated membranes. Immunoprecipitation of Galpha(i2) from solubilized, azidoanalide (AA-gammaGTP) photolabeled membranes showed that receptors in desensitized FPR-transfected HL-60 cells remained coupled to Galpha(i2), whereas desensitized receptors in differentiated HL-60 cell membranes were uncoupled from Galpha(i2). As determined by immunoblotting, Galpha(i2) expression was similar in undifferentiated and differentiated HL-60 cells and FPR-transfected cells. Ligand-stimulated receptor internalization and desensitization of calcium redistribution were similar in all three groups of cells. Immunoblotting also indicated that G-protein-coupled receptor kinases (GRKs) 2 and 4 were present in undifferentiated FPR-transfected HL-60 cells at 50% of the level seen in differentiated HL-60 cells. However, differentiation did not increase GRK2 or GRK4 expression, indicating that differences in GRK expression do not explain deficient desensitization. The data indicated that undifferentiated HL-60 cells are unable to induce homologous desensitization of FPRs.

The MDCK cell line is made up of populations of cells with diverse resistive and transport properties.

The MDCK cell line has been used as a model for the renal distal tubule. When grown on permeable supports, the cells form tight junctions and transport sodium and calcium. Widely different values have, however, been obtained for resistance and transports by different investigators. Since the MDCK cell line is genetically heterogeneous, one potential reason for observed differences is the use of different populations of cells. Dilutional techniques have been used to obtain eleven MDCK cell clones. The clones exhibit a range of values for resistance (300-4000 ohms cm2), net Ca2+ transport (3.2-81 pmol/cm2/min) and net Na+ transport (0.4-6.0 nmol/cm2/min). To determine if the characteristics of clonal cell lines changed with increasing passage number, resistance was measured at relatively early and late passage number. Resistance increased with increasing passage number in two of six lines and decreased in one. These studies demonstrate that differences between previous studies may be due to differences in cell population, length of time since passage, and passage number. Clonal cell lines should provide a useful tool to investigate ion transport and mechanisms of regulation of transport in this model of renal distal tubule.

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3).

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.

Agonist-induced translocation of Gq/11alpha immunoreactivity directly from plasma membrane in MDCK cells.

Both Gsalpha and Gqalpha are palmitoylated and both can move from a crude membrane fraction to a soluble fraction in response to stimulation with agonists. This response may be mediated through depalmitoylation. Previous studies have not demonstrated that endogenous guanine nucleotide-binding regulatory protein (G protein) alpha-subunits are released directly from the plasma membrane. We have examined the effect of agonist stimulation on the location of Gq/11alpha immunoreactivity in Madin-Darby canine kidney (MDCK) cells. Bradykinin (BK; 0.1 microM) caused Gq/11alpha, but not Gialpha, to rapidly translocate from purified plasma membranes to the supernatant. AlF and GTP also caused translocation of Gq/11alpha immunoreactivity from purified plasma membranes. BK caused translocation of Gq/11alpha immunoreactivity in intact cells from the basal and lateral plasma membranes to an intracellular compartment as assessed by confocal microscopy. Thus Gq/11alpha is released directly from the plasma membrane to an intracellular location in response to activation by an agonist and direct activation of G proteins. G protein translocation may be a mechanism for desensitization or for signaling specificity.

Activation of mitogen-activated protein kinases by formyl peptide receptors is regulated by the cytoplasmic tail.

Wild type formyl peptide receptors (FPRwt) and receptors deleted of the carboxyl-terminal 45 amino acids (FPRdel) were stably expressed in undifferentiated HL-60 promyelocytes. Expression of FPRwt reconstituted N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated extracellular signal-regulated kinase (ERK) and p38 kinase activity. Expression of FPRdel resulted in a 2-5-fold increase in basal ERK and p38 kinase activity, whereas FMLP failed to stimulate either mitogen-activated protein kinase (MAPK). Pertussis toxin abolished FMLP stimulation of both MAPKs in FPRwt cells but had no effect on either basal or FMLP-stimulated MAPK activity in FPRdel cells. FMLP stimulated a concentration-dependent increase in guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding in membranes from FPRwt but not FPRdel cells. GTPgammaS inhibited FMLP binding to FPRwt but not FPRdel membranes. Photoaffinity labeling with azidoanilide-[gamma-32P]GTP in the presence or absence of FMLP showed increased labeling only in FPRwt membranes. Immunoprecipitation of alphai2 and alphaq/11 from solubilized, photolabeled membranes showed that FPRwt were coupled to alphai2 but not to alphaq/11. FPRwt cells demonstrated calcium mobilization following stimulation with FMLP, whereas FPRdel cells showed no increase in intracellular calcium. We conclude that the carboxyl-terminal tail of FPRs is necessary for ligand-mediated activation of Gi proteins and MAPK cascades. Deletion of the carboxyl-terminal tail results in constitutive activation of ERK and p38 kinase through a Gi2-independent pathway.

Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase.

Formyl peptide receptor activation of three mitogen-activated protein kinase (MAPK) cascades, extracellular signal-regulated kinases (ERKs), N-terminal kinases (JNKs), and p38 MAPK was examined in differentiated HL-60 granulocytes. FMLP stimulated a concentration- and time-dependent increase in ERK, JNK, and p38 MAPK activities, all of which were dependent on a pertussis toxin-sensitive G protein. Pharmacologic inhibitors were used to examine the roles of tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C. FMLP-stimulated ERK activity was dependent on tyrosine kinases, phosphatidylinositol 3-kinase, protein kinase C, and phospholipase C; p38 MAPK activation was dependent on phosphatidylinositol 3-kinase and phospholipase C; while JNK activation was independent of all of these signaling components. The mitogen-activated protein kinase/ERK kinase inhibitor PD098059 reduced ERK activation by 90%, while an inhibitor of p38 MAPK, SB203580, inhibited p38 MAPK activation by 80%. Both PD098059 and SB203580 inhibited FMLP-stimulated superoxide release, as did inhibitors directed against protein kinase C, tyrosine kinases, and phosphatidylinositol 3-kinase. We conclude that formyl peptide receptors are coupled to three MAPK cascades by Gi proteins. ERKs, p38 MAPK, and JNKs are each activated by distinct proximal signal transduction pathways. Activation of p38 MAPK is necessary for FMLP stimulation of respiratory burst activity; however, a second signal that may involve ERK is also required for this activity.

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells.

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

Configurational Entropy Approach to the Kinetics of Glasses.

A kinetic theory of glasses is developed using equilibrium theory as a foundation. After establishing basic criteria for glass formation and the capability of the equilibrium entropy theory to describe the equilibrium aspects of glass formation, a minimal model for the glass kinetics is proposed. Our kinetic model is based on a trapping description of particle motion in which escapes from deep wells provide the rate-determining steps for motion. The formula derived for the zero frequency viscosity η (0,T) is log η (0,T) = B - AF(T)kT where F is the free energy and T the temperature. Contrast this to the Vogel-Fulcher law log η (0,T) = B + A/(T - Tc). A notable feature of our description is that even though the location of the equilibrium second-order transition in temperature-pressure space is given by the break in the entropy or volume curves the viscosity and its derivative are continuous through the transition. The new expression for η (0,T) has no singularity at a critical temperature Tc as in the Vogel-Fulcher law and the behavior reduces to the Arrhenius form in the glass region. Our formula for η (0,T) is discussed in the context of the concepts of strong and fragile glasses, and the experimentally observed connection of specific heat to relaxation response in a homologous series of polydimethylsiloxane is explained. The frequency and temperature dependencies of the complex viscosity η (ω< T), the diffusion coefficient D(ω< T), and the dielectric response ε (ω< T) are also obtained for our kinetic model and found to be consistent with stretched exponential behavior.

5-Hydroxytryptamine2A receptors expressed in rat renal mesangial cells inhibit cyclic AMP accumulation.

Second messenger coupling of the 5-hydroxytryptamine (5-HT)2A receptor endogenous to cultured rat glomerular mesangial cells was studied. 5-HT induced an increase in total inositol phosphate levels (EC50 = 265 +/- 55 nM, maximum stimulation = 150 +/- 23%). That effect was sensitive to antagonists of the 5-HT2A receptor and was insensitive to pertussis toxin at doses that eliminated detectable pertussis toxin substrate, as determined by membrane ADP-ribosylation. Surprisingly, 5-HT also induced an inhibition of forskolin-stimulated cAMP accumulation (55 +/- 6%, IC50 = 5 +/- 3 nM). This effect was competitively antagonized by the 5-HT2A receptor antagonists ketanserin, ritanserin, and spiperone and could be produced by the 5-HT2 receptor agonists alpha-methyl-5-HT (66 +/- 13%, IC50 = 23 +/- 14 nM) and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (65 +/- 4%, IC50 = 14 +/- 7 nM). The inhibition of cAMP accumulation occurred in the presence of a number of agents that either stimulate or inhibit protein kinase C activity, arachidonic acid metabolism, or Ca2+ mobilization. In isolated membranes, 5-HT induced a 36 +/- 5% inhibition of adenylyl cyclase activity (IC50 = 8 +/- 4 nM). Inhibition of cAMP accumulation in intact cells and of adenylyl cyclase activity in washed membranes was (> 50%) sensitive to pertussis toxin, implicating Gi alpha or Go alpha subunits in the inhibitory signal. These data suggest that the 5-HT2A receptor can be permissive in its coupling to G proteins and second messengers.