Nonlinear Regression Curve Fitting Research Articles

Extracellular calcium chelation and attenuation of calcium entry decrease in vivo cholinergic‐induced eccrine sweating sensitivity in humans

What is the central question of this study? Calcium is an important second messenger in eccrine sweating; however, whether modulation of extracellular Ca(2+) and Ca(2+) entry has the capacity to modulate sweat rate in non-glabrous human skin has not been explored. What is the main finding and its importance? Acetylcholine to sweat rate dose-response relationships identify that local in vivo Ca(2+) chelation and L-type Ca(2+) channel antagonism have the capacity to attenuate the cholinergic sensitivity of eccrine sweat glands. Importantly, these data translate previous glabrous in vitro animal studies into non-glabrous in vivo human skin. Calcium is an important second messenger in eccrine sweating, with both internal and external sources being identified in vitro. It is unclear whether in vivo modulation of extracellular Ca(2+) levels or influx has the capacity to modulate sweat rate in non-glabrous human skin. To test the hypothesis that lowering interstitial Ca(2+) levels would decrease the sensitivity of the ACh to sweat rate (via capacitance hygrometry) dose-response relationship, nine healthy subjects received six ACh doses (1 × 10(-5) to 1 × 10(0) m in 10-fold increments) with and without a Ca(2+) chelator (12.5 mg ml(-1) EDTA) via forearm intradermal microdialysis (protocol 1). To test the hypothesis that attenuating Ca(2+) influx via L-type Ca(2+) channels would also decrease the sensitivity of the ACh to sweat rate dose-response relationship, 10 healthy subjects received similar ACh doses with and without a phenylalkylamine Ca(2+) channel blocker (1 mm verapamil; protocol 2). Non-linear regression curve fitting identified a right-shifted ED50 in EDTA-treated sites compared with ACh alone (-1.0 ± 0.1 and -1.5 ± 0.1 logm, respectively; P < 0.05), but unchanged maximal sweat rate (0.60 ± 0.07 and 0.58 ± 0.11 mg cm(-2) min(-1), respectively; P > 0.05) in protocol 1. Protocol 2 also resulted in a right-shifted ED(50) (verapamil, -0.9 ± 0.1 logm; ACh alone, -1.6 ± 0.2 logm; P < 0.05), with unchanged maximal sweat rate (verapamil, 0.45 ± 0.08 mg cm(-2) min(-1); ACh alone, 0.35 ± 0.06 mg cm(-2) min(-1); P > 0.05). Thus, local in vivo Ca(2+) chelation and L-type Ca(2+) channel antagonism have the capacity to attenuate in vivo cholinergic sensitivity of eccrine sweat glands. These data suggest that interstitial Ca(2+) and its influx via Ca(2+) channels play a functional role in eccrine sweating in intact non-glabrous human skin.

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

Topical anaesthesia does not affect cutaneous vasomotor or sudomotor responses in human skin

(1) The effects of local sensory blockade (topical anaesthesia) on eccrine sweat glands and cutaneous circulation are not well understood. This study aimed to determine whether topical lidocaine/prilocaine alters eccrine sweat gland and cutaneous blood vessel responses. (2) Sweating (capacitance hygrometry) was induced via forearm intradermal microdialysis of five acetylcholine (ACh) doses (1 × 10(-4) to 1 × 10(0) m, 10-fold increments) in control and treated forearm sites in six healthy subjects. Nitric oxide-mediated vasodilatory (sodium nitroprusside) and adrenergic vasoconstrictor (noradrenaline) agonists were iontophoresed in lidocaine/prilocaine-treated and control forearm skin in nine healthy subjects during blood flow assessment (laser Doppler flowmetry, expressed as% from baseline cutaneous vascular conductance; CVC; flux/mean arterial pressure). (3) Non-linear regression curve fitting identified no change in the ED50 of ACh-induced sweating after sensory blockade (-1.42 ± 0.23 logM) compared to control (-1.27 ± 0.23 logM; P > .05) or in Emax (0.43 ± 0.08 with, 0.53 ± 0.16 mg cm(-2) min(-1) without lidocaine/prilocaine; P > .05). Sensory blockade did not alter the vasodilator response to sodium nitroprusside (1280 ± 548% change from baseline CVC with, 1204 ± 247% without lidocaine/prilocaine) or vasoconstrictor response to noradrenaline (-14 ± 4% change from baseline CVC with, -22 ± 14% without lidocaine/prilocaine; P > 0.05). (4) Cutaneous sensory blockade does not appear to alter nitric oxide-mediated vasodilation, adrenergic vasoconstriction, or cholinergic eccrine sweating dose-response sensitivity or responsiveness to maximal dose. Thus, lidocaine/prilocaine treatment should not affect sweat gland function or have blood flow implications for subsequent research protocols or clinical procedures.

Abstract 2122: The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian cancer cells.

Abstract Introduction: Cell stiffness (Young's E modulus), as measured via Atomic Force Microscopy (AFM) is a newly recognized characteristic of cancer cells. We previously demonstrated that cisplatin-resistant ovarian cancer cells have a stiffer cell membrane as compared to their syngeneic cisplatin-sensitive counterpart, and that cisplatin increases cell stiffness in cisplatin-sensitive cells. Here we examine the role of Rho GTPase in cell stiffness and cisplatin resistance. Experimental Procedures: Prior to AFM analysis, cells were stained with Annexin V-FLUOS and only non-apoptotic cells were chosen for measurements. Cell stiffness was measured via AFM on live cells in physiological buffer. The actin cytoskeleton was visualized using phalliodin-ATTO 647N. Cells were imaged with a Leica TCS SP5 STED confocal system or a Nikon Eclispe 90i. Cisplatin sensitivity was determined via the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt] cell viability assay. A nonlinear regression curve fit (one phase exponential decay) was used to analyze cisplatin dose response experiments and determine the IC50 (GraphPad Prism). Rho inhibition and activation were accomplished using Rho Inhibitor I and Rho Activator II, respectively (Cytoskeleton, Inc.). Results: The cisplatin IC50 of cisplatin-sensitive (A2780, OVCAR5, SKOV3) and cisplatin-resistant (CP70, OVCAR5-CisR, SKOV3-CisR, RMG-1) cells correlated with Young's E modulus, with increasing IC50 corresponding to a stiffer cell phenotype. Cells that are resistant to cisplatin displayed an actin cytoskeleton that consisted of long stress fibers that spanned the width of the cell. Rho inhibition in CP70 and OVCAR5-CisR cells abolished this network of actin stress fibers, decreased cell stiffness, and increased cisplatin sensitivity. In contrast, Rho activation in A2780 and OVCAR5 cells induced the reorganization of the actin cytoskeleton into long stress fibers, increased cell stiffness, and decreased cisplatin sensitivity. We demonstrated previously that cisplatin increases cell stiffness in cisplatin-sensitive OVCAR5 cells, and here we show that cisplatin induces actin stress fiber formation in these cells. Conclusions: Rho-mediated actin stress fiber formation increases cell stiffness and mediates cisplatin resistance in ovarian cancer cells. Citation Format: Chintda Santiskulvong, Shivani Sharma, Laurent Bentolila, Jian Y. Rao, James Gimzewski, Oliver Dorigo. The role of Rho GTPase in cell stiffness and cisplatin resistance in ovarian cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2122. doi:10.1158/1538-7445.AM2013-2122

Exploiting Synergistic and Positive Interactions Between Plant Extracts Towards Rational Antimalarial Phytotherapeutic and Evidenced-Based Drug Discovery

Ethnopharmacological evidence of the folk use of Nefang, a polyherbal product composed of Mangifera indica (bark and leaf), Psidium guajava, Carica papaya, Cymbopogon citratus, Citrus sinensis, Ocimum gratissimum (leaves), for the treatment of malaria has been reported previously [1]. In vitro antiplasmodial activities of the constituent plant extracts on both 3D7 (CQ-sensitive) and Dd2 (multi-drug resistant) strains of P. falciparum were analyzed using the SYBR Green 1-based fluorescence assay [2, 3], and the interactions between various extract pairs analyzed using a fixed-ratio drug combination approach [4]. Effective concentration 50 (EC50) values were determined by nonlinear regression curve-fitting using GraphPad prism 6.0, and used to calculate the Fractional Inhibitory Concentration 50 (FICA= EC50A in combination/EC50A) and combination indices (CI) (CIA/B= FICA+FICB) for each pair. The derived EC50 values were as follows (3D7/Dd2): MiB-65.33/34.58, MiL-82.56/40.04, Pg-47.02/25.79, Cp-1188/317.5, Cc-723.3/141, Cs-184.4/105.1, Og-778.5/118.9, Nefang-96.96/55.08 (µg/mL), and the best extract pairs gave EC50 ratios (and CI) of: MiB/Pg-6.07/4.53 (CI = 0.351), MiL/Pg-7.18/4.62 (0.358), MiB/Cs-6.34/19.25 (0.366), MiL/Cs-9.63/25.27 (0.482), Pg/Cs-6.22/25.36 (0.483), Cs/Og-21.78/24.64 (0.414) when combined at equipotency ratios, indicating apparent synergism and/or additive interactions between the different extracts[5]. Taken together, this study confirms the antimalarial activities of Nefang and has identified extract pairs with possible positive interactions for exploitation towards a rational, evidence-based and phytotherapeutic drug discovery in malaria. Acknowledgements: Thanks go to Institut Pasteur, Korea for the internship/training course in Biosafety and Drug Discovery.

Combining simulation to nonlinear fitting for the determination of rate constants of elementary reactions by using Excel spreadsheets

A common problem in chemistry is to determine parameters (constants) in an equation used to represent experimental data. Examples are fitting a set of data to a model equation (straight line or curve) to obtain unknown parameters. In chemical kinetics, a set of data is usually a number of concentrations versus time, but the model equation is not well defined! Instead of a well defined model equation we have a set of coupled ODE’s (ordinary differential equations) which represent rate equations for reactants and products. The analytical integration of these ODE’s is rarely possible. The numerical integration is the alternative. In this work are combined the simulation of chemical reactions, by using numerical integration, and nonlinear regression (curve fitting) by using “Solver add-in” of Microsoft Excel to find rate constants of elementary reactions from experimental data. This method is illustrated on three complex mechanisms. The simulation of chemical reactions in Excel spreadsheets is illustrated with/without VBA programming. The automation (automatic obtaining of rate equations from mechanism: no need of chemical kinetics knowledge from the end user!) of mechanism simulation is demonstrated on many example.

English

The use of plants for the treatment of human and animal diseases continues to rise although there are few studies providing proof of these effects. Among them is theAnacardium occidentale&nbsp;L., popularly known as cashew.&nbsp;In vitro&nbsp;egg hatch and larval development and viability assays was conducted to determine possible direct anthelmintic effect of acetone extract and fractions of&nbsp;A. occidentale&nbsp;against nematode of sheep, predominantly,&nbsp;Haemonchus contortus. The effect of the extracts on hatching of eggs and development and survival of infective larvae (L3) was assessed. The best-fit LC50&nbsp;values were computed by global model of non-linear regression curve fitting (95% confidence interval). The presence of&nbsp;A. occidentale&nbsp;extracts in the cultures decreased the hatchability of eggs and survival of L3&nbsp;larvae in a concentration dependent manner. The LC50&nbsp;values of acetone extract was 0.311 and 1.72 mg/ml for egg hatch and larval viability test, respectively. The fractions of&nbsp;A. occidentale&nbsp;were more active, demonstrating a lower LC50&nbsp;compare with the acetone extract. The activities of the fractions were not significantly different against the eggs and larvae of&nbsp;H. contortus&nbsp;(p &gt; 0.05). Further studies are required to identify the compound(s) responsible for activity and more clearly comprehend the anthelmintic mechanism detected in this study. &nbsp; Key words:&nbsp;Anacardium occidentale, anthelmintic,&nbsp;Haemonchus contortus,&nbsp;in&nbsp;vitrodetection, larvae, ova.

Development and Characterization of High Affinity Leptins and Leptin Antagonists

Leptin is a pleiotropic hormone acting both centrally and peripherally. It participates in a variety of biological processes, including energy metabolism, reproduction, and modulation of the immune response. So far, structural elements affecting leptin binding to its receptor remain unknown. We employed random mutagenesis of leptin, followed by selection of high affinity mutants by yeast surface display and discovered that replacing residue Asp-23 with a non-negatively charged amino acid leads to dramatically enhanced affinity of leptin for its soluble receptor. Rational mutagenesis of Asp-23 revealed the D23L substitution to be most effective. Coupling the Asp-23 mutation with alanine mutagenesis of three amino acids (L39A/D40A/F41A) previously reported to convert leptin into antagonist resulted in potent antagonistic activity. These novel superactive mouse and human leptin antagonists (D23L/L39A/D40A/F41A), termed SMLA and SHLA, respectively, exhibited over 60-fold increased binding to leptin receptor and 14-fold higher antagonistic activity in vitro relative to the L39A/D40A/F41A mutants. To prolong and enhance in vivo activity, SMLA and SHLA were monopegylated mainly at the N terminus. Administration of the pegylated SMLA to mice resulted in a remarkably rapid, significant, and reversible 27-fold more potent increase in body weight (as compared with pegylated mouse leptin antagonist), because of increased food consumption. Thus, recognition and mutagenesis of Asp-23 enabled construction of novel compounds that induce potent and reversible central and peripheral leptin deficiency. In addition to enhancing our understanding of leptin interactions with its receptor, these antagonists enable in vivo study of the role of leptin in metabolic and immune processes and hold potential for future therapeutic use in disease pathologies involving leptin.

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H(2)O(2)-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.

Enhancement of on chip chemiluminescence signal intensity of tris(1,10-phenanthroline)-ruthenium(II) peroxydisulphate system for analysis of chlorpheniramine maleate in pharmaceutical formulations

The effect of detection chip geometry on chemiluminescence (CL) signal intensity of tris(1,10-phenanthroline)-ruthenium(II) peroxydisulphate system for analysis of chlorpheniramine maleate (CPM) in pharmaceutical formulations was investigated. It was observed that the design of the detection chip is very crucial and can play an important role in enhancing the CL signal intensity in this system. The CL signal intensity was enhanced 250% when a teardrop micromixer chip was used, compared to the commonly used serpentine chip geometry. The study was conducted using a multi-chip device. In this device, chip 1 was used to prepare and pump the reagent mixture, whereas chip 3 was used for pumping the sample. The two chips were connected to the teardrop chip (2) via silica capillary where detection took place. Non-linear regression curve fitting of the calibration data revealed that the calibration curves are best described by third order polynomial equation with excellent correlation coefficients ( R 2 = 0.9998) for the concentration range 7.69 × 10 −8 to 5.12 × 10 −5 mol L −1. A linear response is also observed over the range 7.69 × 10 −8 to 1.28 × 10 −5 mol L −1 ( R 2 = 0.9996) and the detection limit was found to be 5.49 × 10 −8 mol L −1. The device was successfully used for the analysis of CPM in tablets and a multi-component cough syrup. Results were reproducible with relative standard deviation (RSD) of 0.6–1.1%.

In vitro anthelmintic activity of Combretum molle (R. Br. ex G. Don) (Combretaceae) against Haemonchus contortus ova and larvae

Parasitic nematodes, especially Haemonchus contortus (Rudolphi), are among the most common and economically important causes of disease in sheep and goats owned by pastoralists and small holder farmers in Africa. The control of these infections relies mainly on the use of anthelmintic drugs. However, herbal preparations are widely used by pastoralists and small holder farmers for the treatment of their livestock against helminth parasites. The anthelmintic effect of acetone leaf extract and fractions of Combretum molle was investigated to determine the relative efficacy of the components against gastrointestinal sheep nematodes. The fractions were obtained by solvent:solvent extraction from the acetone extract. These were evaluated for nematocidal activity by means of an egg hatch (EHA) and larval a development and viability assay (LDVA) in vitro. The effect of the test extracts on the hatchability of eggs and development of first to third stage larvae and the survival rate of the third stage larvae. H. contortus, were used to determine the relative bioactivities. Best-fit LC 50 values were computed using global model of nonlinear regression curve-fitting. The extracts inhibited egg hatching and development of the larvae of H. contortus in a concentration-dependent manner. Best-fit LC 50 values for the egg hatch test were 0.866, 0.333, 0.833, 0.747, and 0.065 mg/mL for acetone extract, n-butanol, hexane, chloroform, and 35% water in methanol fractions, respectively. The best-fit LC 50 values for the LDVA were 0.604, 0.362, 1.077, 0.131 and 0.318 mg/mL for the acetone extract, butanol, hexane, chloroform, and 35% water in methanol fractions, respectively. In the EHA the 35% water in methanol fraction was significantly more active than all the other fractions ( p < 0.05); however the activity was not significantly different with the LDVA. C. molle leaf could find application in anthelmintic therapy in veterinary practice.

Characterization of the Mycobacterial AdnAB DNA Motor Provides Insights into the Evolution of Bacterial Motor-Nuclease Machines

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

Molecular Insight into the Synergism between the Minor Allele of Human Liver Peroxisomal Alanine:Glyoxylate Aminotransferase and the F152I Mutation

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

JNK-mediated Phosphorylation of Paxillin in Adhesion Assembly and Tension-induced Cell Death by the Adenovirus Death Factor E4orf4

The adenovirus type 2 Early Region 4 ORF4 (E4orf4) protein induces a caspase-independent death program in tumor cells involving changes in actin dynamics that are functionally linked to cell killing. Because an increase in myosin II-based contractility is needed for the death of E4orf4-expressing cells, we have proposed that alteration of cytoskeletal tension is part of the signals engaging the death pathway. Yet the mechanisms involved are poorly defined. Herein, we show that the Jun N-terminal kinase JNK is activated in part through a pathway involving Src, Rho, and ROCK (Rho kinase) and contributes to dysregulate adhesion dynamics and to kill cells in response to E4orf4. JNK supports the formation of atypically robust focal adhesions, which are bound to the assembly of the peculiar actomyosin network typifying E4orf4-induced cell death and which are required for driving nuclear condensation. Remarkably, the dramatic enlargement of focal adhesions, actin remodeling, and cell death all rely on paxillin phosphorylation at Ser-178, which is induced by E4orf4 in a JNK-dependent way. Furthermore, we found that Ser-178-paxillin phosphorylation is necessary to decrease adhesion turnover and to enhance the time residency of paxillin at focal adhesions, promoting its recruitment from an internal pool. Our results indicate that perturbation of tensional homeostasis by E4orf4 involves JNK-regulated changes in paxillin adhesion dynamics that are required to engage the death pathway. Moreover, our findings support a role for JNK-mediated paxillin phosphorylation in adhesion growth and stabilization during tension signaling.

Intracellular Interaction of Interleukin-15 with Its Receptor α during Production Leads to Mutual Stabilization and Increased Bioactivity

We show that co-expression of interleukin 15 (IL-15) and IL-15 receptor alpha (IL-15Ralpha) in the same cell allows for the intracellular interaction of the two proteins early after translation, resulting in increased stability and secretion of both molecules as a complex. In the absence of co-expressed IL-15Ralpha, a large portion of the produced IL-15 is rapidly degraded immediately after synthesis. Co-injection into mice of IL-15 and IL-15Ralpha expression plasmids led to significantly increased levels of the cytokine in serum as well as increased biological activity of IL-15. Examination of natural killer cells and T lymphocytes in mouse organs showed a great expansion of both cell types in the lung, liver, and spleen. The presence of IL-15Ralpha also increased the number of CD44(high) memory cells with effector phenotype (CD44(high)CD62L-). Thus, mutual stabilization of IL-15 and IL-15Ralpha leads to remarkable increases in production, stability, and tissue availability of bioactive IL-15 in vivo. The in vivo data show that the most potent form of IL-15 is as part of a complex with its receptor alpha either on the surface of the producing cells or as a soluble extracellular complex. These results explain the reason for coordinate expression of IL-15 and IL-15Ralpha in the same cell and suggest that the IL-15Ralpha is part of the active IL-15 cytokine rather than part of the receptor.

Structure and Function of the Smallest Vertebrate Telomerase RNA from Teleost Fish

Telomerase extends chromosome ends by copying a short template sequence within its intrinsic RNA component. Telomerase RNA (TR) from different groups of species varies dramatically in sequence and size. We report here the bioinformatic identification, secondary structure comparison, and functional analysis of the smallest known vertebrate TRs from five teleost fishes. The teleost TRs (312-348 nucleotides) are significantly smaller than the cartilaginous fish TRs (478-559 nucleotides) and tetrapod TRs. This remarkable length reduction of teleost fish TRs correlates positively with the genome size, reflecting an unusual structural plasticity of TR during evolution. The teleost TR consists of a compact three-domain structure, lacking most of the sequences in regions that are variable in other vertebrate TR structures. The medaka and fugu TRs, when assembled with their telomerase reverse transcriptase (TERT) protein counterparts, reconstituted active and processive telomerase enzymes. Titration analysis of individual RNA domains suggests that the efficient assembly of the telomerase complex is influenced more by the telomerase reverse transcriptase (TERT) binding of the CR4-CR5 domain than the pseudoknot domain of TR. The remarkably small teleost fish TR further expands our understanding about the evolutionary divergence of vertebrate TR.

Cutaneous Endothelial Dependent and Independent Vascular Responsiveness after Prolonged Head-Down Bed Rest

Spaceflight, or its ground based analog head-down bed rest (HDBR), reduces the elevation in skin blood flow to whole-body and local heating. The mechanisms that underlie these microgravity-related changes are unclear and may be related to altered cutaneous post-synaptic function (i.e., decreased sensitivity to vasodilator neurotransmitters and co-factors). PURPOSE: Test the hypothesis that HDBR alters post-synaptic cutaneous vascular responsiveness to endothelial-dependent and independent vasodilators. METHODS: Intradermal microdialysis membranes were placed in forearm skin of seven healthy subjects (5 males, 2 females) before and, in a randomised fashion, on day 16 or 18 of 6° HDBR, thereby allowing local delivery of the endothelial-dependent vasodilator, acetylcholine (ACh; 1x10-7 to 1x10-1 M at 10-fold increments) and the endothelial-independent nitric oxide donor, sodium nitroprusside (SNP; 5x10-8 to 5x10-2 M at 10-fold increments). Local skin temperature was clamped at 34 °C throughout the protocols. Cutaneous vascular conductance (CVC) was calculated from the ratio of laser-Doppler derived skin blood flux (assessed directly over the membranes) to mean arterial blood pressure. CVC dose-response curves to ACh and SNP were modeled via nonlinear regression curve fitting to identify the dose of ACh and SNP causing 50% of the maximal vasodilator response (EC50), used as an index of cutaneous vascular responsiveness. RESULTS: The EC50 of the ACh dose response curve was unchanged after HDBR (pre-HDBR: −3.71 ± 0.35 vs post HDBR: −3.82 ± 0.64 log M, P = 0.88) as was the EC50 of the SNP dose response curve (pre-HDBR: −3.98 ± 0.25 vs post HDBR: −4.37 ± 0.33 log M, P = 0.30). CONCLUSIONS: HDBR does not affect post-synaptic cutaneous endothelial dependent and independent responsiveness, as indexed by the EC50 of the assessed dose-response curves. Given these findings, reduced increases in skin blood flow during whole-body and local heating after HDBR are unlikely to be due to impaired endothelial dependent and independent vasodilator responsiveness. This research project was funded by grants from the National Heart, Lung and Blood Institute (HL-61388, HL-67422, HL-84072, GM-68865).

Skin Grafting Impairs Postsynaptic Cutaneous Vasodilator and Sweating Responses

This study tested the hypothesis that postsynaptic cutaneous vascular responses to endothelial-dependent and -independent vasodilators, as well as sweat gland function, are impaired in split-thickness grafted skin 5 to 9 months after surgery. Intradermal microdialysis membranes were placed in grafted and adjacent control skin, thereby allowing local delivery of the endothelial-dependent vasodilator, acetylcholine (ACh; 1 x 10(-7) to 1 x 10(-1) M at 10-fold increments) and the endothelial-independent nitric oxide donor, sodium nitroprusside (SNP; 5 x 10(-8) to 5 x 10(-2) M at 10-fold increments). Skin blood flow and sweat rate were simultaneously assessed over the semipermeable portion of the membrane. Cutaneous vascular conductance (CVC) was calculated from the ratio of laser Doppler-derived skin blood flow to mean arterial blood pressure. deltaCVC responses from baseline to these drugs were modeled via nonlinear regression curve fitting to identify the dose of ACh and SNP causing 50% of the maximal vasodilator response (EC50). A rightward shift in the CVC dose response curve for ACh was observed in grafted (EC50 = -2.61 +/- 0.44 log M) compared to adjacent control skin (EC50 = -3.34 +/- 0.46 log M; P = .003), whereas the mean EC50 for SNP was similar between grafted (EC50 = -4.21 +/- 0.94 log M) and adjacent control skin (EC50 = -3.87 +/- 0.65 log M; P = 0.332). Only minimal sweating to exogenous ACh was observed in grafted skin whereas normal sweating was observed in control skin. Increased EC50 and decreased maximal CVC responses to the exogenous administration of ACh suggest impairment of endothelial-dependent cutaneous vasodilator responses in grafted skin 5 to 9 months after surgery. Greatly attenuated sweating responses to ACh suggests either abnormal or an absence of functional sweat glands in the grafted skin.

GABAB Receptor Association with the PDZ Scaffold Mupp1 Alters Receptor Stability and Function

gamma-Aminobutyric acid, type B (GABA(B)) receptors are heterodimeric G protein-coupled receptors that mediate slow inhibitory synaptic transmission in the central nervous system. To identify novel interacting partners that might regulate GABA(B) receptor (GABA(B)R) functionality, we screened the GABA(B)R2 carboxyl terminus against a recently created proteomic array of 96 distinct PDZ (PSD-95/Dlg/ZO-1 homology) domains. The screen identified three specific PDZ domains that exhibit interactions with GABA(B)R2: Mupp1 PDZ13, PAPIN PDZ1, and Erbin PDZ. Biochemical analysis confirmed that full-length Mupp1 and PAPIN interact with GABA(B)R2 in cells. Disruption of the GABA(B)R2 interaction with PDZ scaffolds by a point mutation to the carboxyl terminus of the receptor dramatically decreased receptor stability and attenuated the duration of GABA(B) receptor signaling. The effects of mutating the GABA(B)R2 carboxyl terminus on receptor stability and signaling were mimicked by small interference RNA knockdown of endogenous Mupp1. These findings reveal that GABA(B) receptor stability and signaling can be modulated via GABA(B)R2 interactions with the PDZ scaffold protein Mupp1, which may contribute to cell-specific regulation of GABA(B) receptors in the central nervous system.

Anthelmintic activity of Spigelia anthelmia extract against gastrointestinal nematodes of sheep

In vitro (larval development assay) and in vivo studies were conducted to determine possible direct anthelmintic effect of ethanolic and aqueous extracts of Spigelia anthelmia towards different ovine gastrointestinal nematodes. The effect of extracts on development and survival of infective larvae stage (L(3)) was assessed. Best-fit LC(50) values were computed by global model of non-linear regression curve fitting (95% confidence interval). Therapeutic efficacy of the ethanolic extracts administered orally at a dose rate of 125, 250, and 500 mg/kg, relative to a non-medicated control group of sheep harbouring naturally acquired infection of gastrointestinal nematodes, was evaluated in vivo.The presence of S. anthelmia extracts in the cultures decreased the survival of L(3) larvae. The LC(50) of aqueous extract (0.714 mg/ml) differ significantly from the LC(50) of the ethanolic extract (0.628 mg/ml) against the strongyles (p < 0.05, paired t-test). Faecal egg counts on day 12 after treatment showed that the extract is effective, relative to control (one-way analysis of variance [ANOVA], Dunnett's multiple comparison test) at 500 mg/kg against Strongyloides spp. (p < 0.01), 250 mg/kg against Oesophagostomum spp., Trichuris spp. (p < 0.05), and 125 mg/kg against Haemonchus spp. and Trichostrongylus spp. (p < 0.01). The effect of the doses is significant in all cases, the day after treatment is also extremely significant in most cases, whereas interaction between dose and day after treatment is significant (two-way ANOVA). S. anthelmia extract could, therefore, find application in the control of helminth in livestock, by the ethnoveterinary medicine approach.