Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges.

PubMed

Revsbech, Inge G; Tufts, Danielle M; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E; Storz, Jay F; Fago, Angela

2013-11-15

Semi-fossorial ground squirrels face challenges to respiratory gas transport associated with the chronic hypoxia and hypercapnia of underground burrows, and such challenges are compounded in species that are native to high altitude. During hibernation, such species must also contend with vicissitudes of blood gas concentrations and plasma pH caused by episodic breathing. Here, we report an analysis of hemoglobin (Hb) function in six species of marmotine ground squirrels with different altitudinal distributions. Regardless of their native altitude, all species have high Hb-O2 affinities, mainly due to suppressed sensitivities to allosteric effectors [2,3-diphosphoglycerate (DPG) and chloride ions]. This suppressed anion sensitivity is surprising given that all canonical anion-binding sites are conserved. Two sciurid species, the golden-mantled and thirteen-lined ground squirrel, have Hb-O2 affinities that are characterized by high pH sensitivity and low thermal sensitivity relative to the Hbs of humans and other mammals. The pronounced Bohr effect is surprising in light of highly unusual amino acid substitutions at the C-termini that are known to abolish the Bohr effect in human HbA. Taken together, the high O2 affinity of sciurid Hbs suggests an enhanced capacity for pulmonary O2 loading under hypoxic and hypercapnic conditions, while the large Bohr effect should help to ensure efficient O2 unloading in tissue capillaries. In spite of the relatively low thermal sensitivities of the sciurid Hbs, our results indicate that the effect of hypothermia on Hb oxygenation is the main factor contributing to the increased blood-O2 affinity in hibernating ground squirrels.

Decrease in the red cell cofactor 2,3-diphosphoglycerate increases hemoglobin oxygen affinity in the hibernating brown bear Ursus arctos.

PubMed

Revsbech, Inge G; Malte, Hans; Fröbert, Ole; Evans, Alina; Blanc, Stéphane; Josefsson, Johan; Fago, Angela

2013-01-01

During winter hibernation, brown bears (Ursus arctos) reduce basal O(2) consumption rate to ∼25% compared with the active state, while body temperature decreases moderately (to ∼30°C), suggesting a temperature-independent component in their metabolic depression. To establish whether changes in O(2) consumption during hibernation correlate with changes in blood O(2) affinity, we took blood samples from the same six individuals of hibernating and nonhibernating free-ranging brown bears during winter and summer, respectively. A single hemoglobin (Hb) component was detected in all samples, indicating no switch in Hb synthesis. O(2) binding curves measured on red blood cell lysates at 30°C and 37°C showed a less temperature-sensitive O(2) affinity than in other vertebrates. Furthermore, hemolysates from hibernating bears consistently showed lower cooperativity and higher O(2) affinity than their summer counterparts, regardless of the temperature. We found that this increase in O(2) affinity was associated with a significant decrease in the red cell Hb-cofactor 2,3-diphosphoglycerate (DPG) during hibernation to approximately half of the summer value. Experiments performed on purified Hb, to which DPG had been added to match summer and winter levels, confirmed that the low DPG content was the cause of the left shift in the Hb-O(2) equilibrium curve during hibernation. Levels of plasma lactate indicated that glycolysis is not upregulated during hibernation and that metabolism is essentially aerobic. Calculations show that the increase in Hb-O(2) affinity and decrease in cooperativity resulting from decreased red cell DPG may be crucial in maintaining a fairly constant tissue oxygen tension during hibernation in vivo.

Pharmacological and gene regulation properties point to the SlHAK5 K+ transporter as a system for high-affinity Cs+ uptake in tomato plants.

PubMed

Ródenas, Reyes; Nieves-Cordones, Manuel; Rivero, Rosa M; Martinez, Vicente; Rubio, Francisco

2018-04-01

Potassium (K + ) and cesium (Cs + ) are chemically similar but while K + is an essential nutrient, Cs + can be toxic for living organisms, plants included. Two different situations could lead to problems derived from the presence of Cs + in agricultural systems: (1) presence of Cs + at high concentrations that could produce toxic effects on plants, (2) presence of micromolar concentrations of radiocesium, which can be accumulated in the plant and affect animal and human health through the food chain. While K + uptake has been well described in tomato plants, information on molecular mechanisms involved in Cs + accumulation in this species is absent. Here, we show that in tomato plants, high concentrations of Cs + produce deficiency of K + but do not induce high-affinity K + uptake or the gene encoding the high-affinity K + transporter SlHAK5. At these concentrations, Cs + uptake takes place through a Ca 2+ -sensitive pathway, probably a non-selective cation channel. At micromolar concentrations, Cs + is accumulated by a high-affinity uptake system upregulated in K + -starved plants. This high-affinity Cs + uptake shares features with high-affinity K + uptake. It is sensitive to NH 4 + and insensitive to Ba 2+ and Ca 2+ and its presence parallels the pattern of SlHAK5 expression. Moreover, blockers of reactive oxygen species and ethylene action repress SlHAK5 and negatively regulate both high-affinity K + and Cs + uptake. Thus, we propose that SlHAK5 contributes to Cs + uptake from micromolar concentrations in tomato plants and can constitute a pathway for radiocesium transfer from contaminated areas to the food chain. © 2017 Scandinavian Plant Physiology Society.

Life on the edge: O2 binding in Atlantic cod red blood cells near their southern distribution limit is not sensitive to temperature or haemoglobin genotype

PubMed Central

Barlow, Samantha L.; Metcalfe, Julian; Righton, David A.

2017-01-01

ABSTRACT Atlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O2 saturation difference, Sa–vO2, another major determinant of circulatory O2 supply rate. The results showed statistically indistinguishable red blood cell O2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O2 binding (Bohr and Root effects). Modelling of Sa–vO2 at physiological pH, temperature and O2 partial pressures revealed a substantial capacity for increases in Sa–vO2 to meet rising tissue O2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa–vO2 with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O2 supply to tissue demand. PMID:28148818

Life on the edge: O2 binding in Atlantic cod red blood cells near their southern distribution limit is not sensitive to temperature or haemoglobin genotype.

PubMed

Barlow, Samantha L; Metcalfe, Julian; Righton, David A; Berenbrink, Michael

2017-02-01

Atlantic cod are a commercially important species believed to be threatened by warming seas near their southern, equatorward upper thermal edge of distribution. Limitations to circulatory O 2 transport, in particular cardiac output, and the geographic distribution of functionally different haemoglobin (Hb) genotypes have separately been suggested to play a role in setting thermal tolerance in this species. The present study assessed the thermal sensitivity of O 2 binding in Atlantic cod red blood cells with different Hb genotypes near their upper thermal distribution limit and modelled its consequences for the arterio-venous O 2 saturation difference, Sa-v O 2 , another major determinant of circulatory O 2 supply rate. The results showed statistically indistinguishable red blood cell O 2 binding between the three HbI genotypes in wild-caught Atlantic cod from the Irish Sea (53° N). Red blood cells had an unusually low O 2 affinity, with reduced or even reversed thermal sensitivity between pH 7.4 and 7.9, and 5.0 and 20.0°C. This was paired with strongly pH-dependent affinity and cooperativity of red blood cell O 2 binding (Bohr and Root effects). Modelling of Sa-v O 2  at physiological pH, temperature and O 2 partial pressures revealed a substantial capacity for increases in Sa-v O 2  to meet rising tissue O 2 demands at 5.0 and 12.5°C, but not at 20°C. Furthermore, there was no evidence for an increase of maximal Sa-v O 2  with temperature. It is suggested that Atlantic cod at such high temperatures may solely depend on increases in cardiac output and blood O 2 capacity, or thermal acclimatisation of metabolic rate, for matching circulatory O 2 supply to tissue demand. © 2017. Published by The Company of Biologists Ltd.

Hemoglobin function and allosteric regulation in semi-fossorial rodents (family Sciuridae) with different altitudinal ranges

PubMed Central

Revsbech, Inge G.; Tufts, Danielle M.; Projecto-Garcia, Joana; Moriyama, Hideaki; Weber, Roy E.; Storz, Jay F.; Fago, Angela

2013-01-01

SUMMARY Semi-fossorial ground squirrels face challenges to respiratory gas transport associated with the chronic hypoxia and hypercapnia of underground burrows, and such challenges are compounded in species that are native to high altitude. During hibernation, such species must also contend with vicissitudes of blood gas concentrations and plasma pH caused by episodic breathing. Here, we report an analysis of hemoglobin (Hb) function in six species of marmotine ground squirrels with different altitudinal distributions. Regardless of their native altitude, all species have high Hb–O2 affinities, mainly due to suppressed sensitivities to allosteric effectors [2,3-diphosphoglycerate (DPG) and chloride ions]. This suppressed anion sensitivity is surprising given that all canonical anion-binding sites are conserved. Two sciurid species, the golden-mantled and thirteen-lined ground squirrel, have Hb–O2 affinities that are characterized by high pH sensitivity and low thermal sensitivity relative to the Hbs of humans and other mammals. The pronounced Bohr effect is surprising in light of highly unusual amino acid substitutions at the C-termini that are known to abolish the Bohr effect in human HbA. Taken together, the high O2 affinity of sciurid Hbs suggests an enhanced capacity for pulmonary O2 loading under hypoxic and hypercapnic conditions, while the large Bohr effect should help to ensure efficient O2 unloading in tissue capillaries. In spite of the relatively low thermal sensitivities of the sciurid Hbs, our results indicate that the effect of hypothermia on Hb oxygenation is the main factor contributing to the increased blood–O2 affinity in hibernating ground squirrels. PMID:24172889

Detection of ovomucoid-specific low-affinity IgE in infants and its relationship to eczema.

PubMed

Kawamoto, Norio; Kamemura, Norio; Kido, Hiroshi; Fukao, Toshiyuki

2017-06-01

Allergen-specific low-affinity IgE was previously detected in cord blood by a highly sensitive densely carboxylated protein (DCP) chip, but not by ImmunoCAP. Here, we investigated the presence of low-affinity IgE during the early life of infants and observed its relationship with eczema. We conducted a birth cohort study, collecting sera at birth and 6 and 14 months of age (n = 110). We monitored the ovomucoid (OM)- and egg white (EW)-specific IgE (sIgE) by ImmunoCAP or DCP chip and analyzed the antigen affinity of sIgE by binding inhibition assays in the presence or absence of a mild chaotropic agent, diethyl amine (DEA). The low- and high-affinity OM-sIgEs and sensitization risk factors were analyzed by a multivariate logistic analysis. The OM-sIgE measured by DCP chip significantly correlated with that measured by ImmunoCAP, but some samples assessed as OM-sIgE positive by DCP chip were considered OM-sIgE negative by ImmunoCAP. Binding inhibition analysis after DEA treatment was performed for participants judged as OM-sIgE positive by DCP chip at 14 M. The group assessed as negative for OM- and EW-sIgE by ImmunoCAP at 6 and 14 months showed a larger binding inhibition curve shift after DEA treatment than did the group assessed as positive at these times, indicating the presence of low-affinity sIgE antibodies at 14 months. The logistic regression analysis found that persistent eczema from 6 to 14 months is a significant risk factor for developing high-affinity, but not low-affinity, sIgE. Human infant peripheral blood contains allergen-specific low-affinity sIgE. Persistent eczema is related to the development of high-affinity, but not low-affinity, IgE. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hypothyroidism leads to increased dopamine receptor sensitivity and concentration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crocker, A.D.; Overstreet, D.H.; Crocker, J.M.

1986-06-01

Rats treated with iodine-131 were confirmed to be hypothyroid by their reduced baseline core body temperatures, reduced serum thyroxine concentrations and elevated serum thyroid stimulating hormone concentrations. When hypothyroid rats were compared to euthyroid controls they were more sensitive to the effects of apomorphine (1.0 mumol/kg) on stereotypy, operant responding and body temperature and showed a smaller reduction in locomotor activity after injection of haloperidol (0.25 mumol/kg). Receptor binding studies on striatal homogenates indicated that hypothyroid rats had increased concentrations of D2 dopamine receptors but there was no change in the affinity. It is concluded that hypothyroidism increases dopamine receptormore » sensitivity by increasing receptor concentration.« less

Thermal-depth matching in dynamic scene based on affine projection and feature registration

NASA Astrophysics Data System (ADS)

Wang, Hongyu; Jia, Tong; Wu, Chengdong; Li, Yongqiang

2018-03-01

This paper aims to study the construction of 3D temperature distribution reconstruction system based on depth and thermal infrared information. Initially, a traditional calibration method cannot be directly used, because the depth and thermal infrared camera is not sensitive to the color calibration board. Therefore, this paper aims to design a depth and thermal infrared camera calibration board to complete the calibration of the depth and thermal infrared camera. Meanwhile a local feature descriptors in thermal and depth images is proposed. The belief propagation matching algorithm is also investigated based on the space affine transformation matching and local feature matching. The 3D temperature distribution model is built based on the matching of 3D point cloud and 2D thermal infrared information. Experimental results show that the method can accurately construct the 3D temperature distribution model, and has strong robustness.

2D zirconium-based metal-organic framework nanosheets for highly sensitive detection of mucin 1: consistency between electrochemical and surface plasmon resonance methods

NASA Astrophysics Data System (ADS)

He, Linghao; Duan, Fenghe; Song, Yingpan; Guo, Chuanpan; Zhao, Hui; Tian, Jia-Yue; Zhang, Zhihong; Liu, Chun-Sen; Zhang, Xiaojing; Wang, Peiyuan; Du, Miao; Fang, Shao-Ming

2017-06-01

Two-dimensional (2D) zirconium-based metal-organic framework (denoted as 521-MOF) nanosheets with the thickness of 6.0-7.5 nm were prepared with the aid of polyvinyl pyrrolidone (PVP) under the mild conditions and low temperature (50 °C). Since 521-MOF nanosheets displayed good electrochemical activity, high surface area, and strong affinity interaction between the MOF and the oligonucleotides sequences, they can impel the immobilization of large amounts of aptamer strands when applied as a platform of biosensor. As a result, the developed aptasensor exhibited sensitive bio-recognition for the cancer determination marker protein, mucin 1 (MUC1). The combination of electrochemical techniques and surface plasmon resonance spectroscopy (SPR) was performed to probe the kinetic processes of the aptamer immobilization and the MUC1 detection. The consistency between different determination approaches was observed, in which the developed aptasensor based on 521-MOF nanosheets exhibits pretty high sensitivity for detecting MUC1 with a low detect limit of 0.12 and 0.65 pg·ml-1 deduced from electrochemical impedance spectroscopy and SPR, respectively, within the broad concentration range of MUC1 from 0.001 to 0.5 ng·ml-1. Simultaneously, a comparable affinity constant, K a, was derived from EIS and SPR, which also demonstrates that this new biosensing strategy has high selectivity, stability, reproducibility, and good applicability for the MUC1 detection in the human serum. The present finding indicates that the synthesized 521-MOF nanosheets can be employed in the fields of the biosensing or biomedical diagnosis and explored for different kinds of biosensors.

The jumbo squid, Dosidicus gigas (Ommastrephidae), living in oxygen minimum zones II: Blood-oxygen binding

NASA Astrophysics Data System (ADS)

Seibel, Brad A.

2013-10-01

Dosidicus gigas is a large, metabolically active squid that migrates across a strong oxygen and temperature gradient in the Eastern Pacific. Here we analyze the oxygen-binding properties of the squid's respiratory protein (hemocyanin, Hc) that facilitate such activity. A high Hc-oxygen affinity, strong temperature dependence, and pronounced pH sensitivity (P50=0.009T2.03, pH 7.4; Bohr coefficient=ΔlogP50/ΔpH=-1.55+0.034T) of oxygen binding facilitate night-time foraging in the upper water column, and support suppressed oxygen demand in hypoxic waters at greater depths. Expanding hypoxia may act to alter the species habitable depth range. This analysis supports the contention that ocean acidification could limit oxygen carrying capacity in squids at warmer temperature leading to reduced activity levels or altered distribution.

Determination of alkaline phosphatase based on affinity adsorption solid-substrate room temperature phosphorimetry using rhodamine 6G-dibromoluciferin luminescent nanoparticle to label lectin and prediction of diseases.

PubMed

Liu, Jia-Ming; Liu, Zhen-Bo; Hu, Li-Xiang; He, Hang-Xia; Yang, Min-Lan; Zhou, Ping; Chen, Xin-Hua; Zheng, Min-Min; Zeng, Xiao-Yi; Xu, Yue-Long

2006-10-15

In the presence of heavy atom perturber LiAc, the silicon dioxide nanoparticle containing rhodamine 6G (R) and dibromoluciferin (D) (R-D-SiO(2)) can emit strong and stable solid-substrate room temperature phosphorescence signal of R (lambda(ex)/lambda(em)=481/648 nm) and D (lambda(ex)/lambda(em)=457/622 nm) on the surface of acetyl cellulose membrane (ACM). R-D-SiO(2) is used to label triticum vulgare lectin (WGA). Then two types of affinity adsorption reactions, R-D-SiO(2)-WGA- alkaline phosphatase (ALP) (direct method) and WGA-ALP-WGA-R-D-SiO(2) (sandwich method), are carried out on ACM. The conditions and the analytical characteristics for the determination of ALP using affinity adsorption solid-substrate room temperature phosphorimetry (AA-SS-RTP) were studied. For a 0.40-microl drop of sample, results show that the detection limits of the sandwich method are 0.16 ag spot(-1)(457/622 nm) and 0.17 ag spot(-1)(481/648 nm), and the detection limits of the direct method are 0.41 ag spot(-1) (457/622 nm) and 0.44 ag spot(-1) (481/648 nm). The contents of ALP in human serum correlated well with those obtained by enzyme-linked immunoassay. This study shows that AA-SS-RTP whether by the sandwich method or the direct method, can combine very well the characteristics of both high sensitivity of SS-RTP and specificity of the immunoreaction. Simultaneously, whether the phosphorescence excitation/emission wavelength of either R or D in R-D-SiO(2) is chosen to determine ALP, this can promote the agility and widen the adaptability of AA-SS-RTP.

Expression and GTP sensitivity of peptide histidine isoleucine high-affinity-binding sites in rat.

PubMed

Debaigt, Colin; Meunier, Annie-Claire; Goursaud, Stephanie; Montoni, Alicia; Pineau, Nicolas; Couvineau, Alain; Laburthe, Marc; Muller, Jean-Marc; Janet, Thierry

2006-07-01

High-affinity-binding sites for the vasoactive intestinal peptide (VIP) analogs peptide histidine/isoleucine-amide (PHI)/carboxyterminal methionine instead of isoleucine (PHM) are expressed in numerous tissues in the body but the nature of their receptors remains to be elucidated. The data presented indicate that PHI discriminated a high-affinity guanosine 5'-triphosphate (GTP)-insensitive-binding subtype that represented the totality of the PHI-binding sites in newborn rat tissues but was differentially expressed in adult animals. The GTP-insensitive PHI/PHM-binding sites were also observed in CHO cells over expressing the VPAC2 but not the VPAC1 VIP receptor.

Hyperthermophilic archaeal prefoldin shows refolding activity at low temperature.

PubMed

Zako, Tamotsu; Banba, Shinya; Sahlan, Muhamad; Sakono, Masafumi; Terada, Naofumi; Yohda, Masafumi; Maeda, Mizuo

2010-01-01

Prefoldin is a molecular chaperone that captures a protein-folding intermediate and transfers it to a group II chaperonin for correct folding. Previous studies of archaeal prefoldins have shown that prefoldin only possesses holdase activity and is unable to fold unfolded proteins by itself. In this study, we have demonstrated for the first time that a prefoldin from hyperthermophilic archaeon, Pyrococcus horikoshii OT3 (PhPFD), exhibits refolding activity for denatured lysozyme at temperatures relatively lower than physiologically active temperatures. The interaction between PhPFD and denatured lysozyme was investigated by use of a surface plasmon resonance sensor at various temperatures. Although PhPFD showed strong affinity for denatured lysozyme at high temperature, it exhibited relatively weak interactions at lower temperature. The protein-folding seems to occur through binding and release from PhPFD by virtue of the weak affinity. Our results also imply that prefoldin might be able to contribute to the folding of some cellular proteins whose affinity with prefoldin is weak. Copyright 2009 Elsevier Inc. All rights reserved.

Active uptake of substance P carboxy-terminal heptapeptide (5-11) into rat brain and rabbit spinal cord slices.

PubMed

Nakata, Y; Kusaka, Y; Yajima, H; Segawa, T

1981-12-01

We previously reported that nerve terminals and glial cells lack an active uptake system capable of terminating transmitter action of substance P (SP). In the present study, we demonstrated the existence of an active uptake system for SP carboxy-terminal heptapeptide, (5-11)SP. When the slices from either rat brain or rabbit spinal cord were incubated with [3H](5-11)SP, the uptake of (5-11)SP into slices was observed. The uptake system has the properties of an active transport mechanism: it is dependent on temperature and sensitive to hypoosmotic treatment and is inhibited by ouabain and dinitrophenol (DNP). In the brain, (5-11)SP was accumulated by means of a high-affinity and a low-affinity uptake system. The Km and the Vmax values for the high-affinity system were 4.20 x 10(-8) M and 7.59 fmol/10 mg wet weight/min, respectively, whereas these values for the low-affinity system were 1.00 x 10(-6) M and 100 fmol/10 mg wet weight/min, respectively. In the spinal cord, there was only one uptake system, with a Km value of 2.16 x 10(-7) M and Vmax value of 26.2 fmol/10 mg wet weight/min. These results suggest that when SP is released from nerve terminals, it is hydrolysed into (5-11)SP before or after acting as a neurotransmitter, which is in turn accumulated into nerve terminals. Therefore, the uptake system may represent a possible mechanism for the inactivation of SP.

Determination of trace alkaline phosphatase by solid-substrate room-temperature phosphorimetry based on Triticum vulgare lectin labeled with fullerenol.

PubMed

Liu, Jia-Ming; Gao, Fei; Huang, Hong-Hua; Zeng, Li-Qing; Huang, Xiao-Mei; Zhu, Guo-Hui; Li, Zhi-Ming

2008-04-01

Fullerenol (F) shows a strong and stable room-temperature phosphorescence (RTP) signal on the surface of nitrocellulose membrane (NCM) at lambda ex max/ lambda em max =542.0/709.4 nm. When modified by dodecylbenzenesulfonic acid sodium salt (DBS), fullerenol emits a stronger signal. It was also found that quantitative specific affinity-adsorption reaction can be carried out between Triticum vulgare lectin (WGA) labeled with DBS-F and alkaline phosphatase (ALP) on the surface of NCM, and the product obtained (WGA-ALP-WGA-F-DBS) emits a strong and stable RTP signal. Furthermore, the content of ALP was proportional to the DeltaI(p) value. Based on the facts above, a new method for the determination of trace amounts of ALP by affinity-adsorption solid-substrate room-temperature phosphorimetry (AA-SS-RTP) was established, using fullerenol modified with DBS to label WGA. The detection limit was 0.011 fg spot(-1) (corresponding concentration: 2.8x10(-14) g ml(-1), namely 2.8x10(-16) mol l(-1)). This method with high sensitivity, accuracy, and precision has been successfully applied to the determination of the content of ALP in human serum survey and forecast human disease, and the results are tallied with those using alkaline phosphatase kits. The mechanism for the determination of ALP using AA-SS-RTP was also discussed.

Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents.

PubMed

O'Brien, Jeffrey; Shea, Kenneth J

2016-06-21

Nanomaterials, when introduced into a complex, protein-rich environment, rapidly acquire a protein corona. The type and amount of proteins that constitute the corona depend significantly on the synthetic identity of the nanomaterial. For example, hydrogel nanoparticles (NPs) such as poly(N-isopropylacrylamide) (NIPAm) have little affinity for plasma proteins; in contrast, carboxylated poly(styrene) NPs acquire a dense protein corona. This range of protein adsorption suggests that the protein corona might be "tuned" by controlling the chemical composition of the NP. In this Account, we demonstrate that small libraries of synthetic polymer NPs incorporating a diverse pool of functional monomers can be screened for candidates with high affinity and selectivity to targeted biomacromolecules. Through directed synthetic evolution of NP compositions, one can tailor the protein corona to create synthetic organic hydrogel polymer NPs with high affinity and specificity to peptide toxins, enzymes, and other functional proteins, as well as to specific domains of large proteins. In addition, many NIPAm NPs undergo a change in morphology as a function of temperature. This transformation often correlates with a significant change in NP-biomacromolecule affinity, resulting in a temperature-dependent protein corona. This temperature dependence has been used to develop NP hydrogels with autonomous affinity switching for the protection of proteins from thermal stress and as a method of biomacromolecule purification through a selective thermally induced catch and release. In addition to temperature, changes in pH or buffer can also alter a NP protein corona composition, a property that has been exploited for protein purification. Finally, synthetic polymer nanoparticles with low nanomolar affinity for a peptide toxin were shown to capture and neutralize the toxin in the bloodstream of living mice. While the development of synthetic polymer alternatives to protein affinity reagents is in its early stages, these recent successes using only small libraries of functional monomers are most encouraging. It is likely that by expanding the chemical diversity of functional hydrogels and other polymers, a much broader range of NP-biomacromolecule affinity pairs will result. Since these robust, nontoxic polymers are readily synthesized in the chemistry laboratory, we believe the results presented in this Account offer a promising future for the development of low cost alternatives to more traditional protein affinity reagents such as antibodies.

A mix-and-read drop-based in vitro two-hybrid method for screening high-affinity peptide binders

PubMed Central

Cui, Naiwen; Zhang, Huidan; Schneider, Nils; Tao, Ye; Asahara, Haruichi; Sun, Zhiyi; Cai, Yamei; Koehler, Stephan A.; de Greef, Tom F. A.; Abbaspourrad, Alireza; Weitz, David A.; Chong, Shaorong

2016-01-01

Drop-based microfluidics have recently become a novel tool by providing a stable linkage between phenotype and genotype for high throughput screening. However, use of drop-based microfluidics for screening high-affinity peptide binders has not been demonstrated due to the lack of a sensitive functional assay that can detect single DNA molecules in drops. To address this sensitivity issue, we introduced in vitro two-hybrid system (IVT2H) into microfluidic drops and developed a streamlined mix-and-read drop-IVT2H method to screen a random DNA library. Drop-IVT2H was based on the correlation between the binding affinity of two interacting protein domains and transcriptional activation of a fluorescent reporter. A DNA library encoding potential peptide binders was encapsulated with IVT2H such that single DNA molecules were distributed in individual drops. We validated drop-IVT2H by screening a three-random-residue library derived from a high-affinity MDM2 inhibitor PMI. The current drop-IVT2H platform is ideally suited for affinity screening of small-to-medium-sized libraries (103–106). It can obtain hits within a single day while consuming minimal amounts of reagents. Drop-IVT2H simplifies and accelerates the drop-based microfluidics workflow for screening random DNA libraries, and represents a novel alternative method for protein engineering and in vitro directed protein evolution. PMID:26940078

Temperature Dependence of Inorganic Nitrogen Uptake: Reduced Affinity for Nitrate at Suboptimal Temperatures in Both Algae and Bacteria

PubMed Central

Reay, David S.; Nedwell, David B.; Priddle, Julian; Ellis-Evans, J. Cynan

1999-01-01

Nitrate utilization and ammonium utilization were studied by using three algal isolates, six bacterial isolates, and a range of temperatures in chemostat and batch cultures. We quantified affinities for both substrates by determining specific affinities (specific affinity = maximum growth rate/half-saturation constant) based on estimates of kinetic parameters obtained from chemostat experiments. At suboptimal temperatures, the residual concentrations of nitrate in batch cultures and the steady-state concentrations of nitrate in chemostat cultures both increased. The specific affinity for nitrate was strongly dependent on temperature (Q10 ≈ 3, where Q10 is the proportional change with a 10°C temperature increase) and consistently decreased at temperatures below the optimum temperature. In contrast, the steady-state concentrations of ammonium remained relatively constant over the same temperature range, and the specific affinity for ammonium exhibited no clear temperature dependence. This is the first time that a consistent effect of low temperature on affinity for nitrate has been identified for psychrophilic, mesophilic, and thermophilic bacteria and algae. The different responses of nitrate uptake and ammonium uptake to temperature imply that there is increasing dependence on ammonium as an inorganic nitrogen source at low temperatures. PMID:10347046

Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility

PubMed Central

Rjasanow, Alexandra; Leitner, Michael G.; Thallmair, Veronika; Halaszovich, Christian R.; Oliver, Dominik

2015-01-01

The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. PMID:26150791

Nanoprobe-Enhanced, Split Aptamer-Based Electrochemical Sandwich Assay for Ultrasensitive Detection of Small Molecules.

PubMed

Zhao, Tao; Liu, Ran; Ding, Xiaofan; Zhao, Juncai; Yu, Haixiang; Wang, Lei; Xu, Qing; Wang, Xuan; Lou, Xinhui; He, Miao; Xiao, Yi

2015-08-04

It is quite challenging to improve the binding affinity of antismall molecule aptamers. We report that the binding affinity of anticocaine split aptamer pairs improved by up to 66-fold by gold nanoparticles (AuNP)-attached aptamers due to the substantially increased local concentration of aptamers and multiple and simultaneous ligand interactions. The significantly improved binding affinity enables the detection of small molecule targets with unprecedented sensitivity, as demonstrated in nanoprobe-enhanced split aptamer-based electrochemical sandwich assays (NE-SAESA). NE-SAESA replaces the traditional molecular reporter probe with AuNPs conjugated to multiple reporter probes. The increased binding affinity allowed us to use 1,000-fold lower reporter probe concentrations relative to those employed in SAESA. We show that the near-elimination of background in NE-SAESA effectively improves assay sensitivity by ∼1,000-100,000-fold for ATP and cocaine detection, relative to equivalent SAESA. With the ongoing development of new strategies for the selection of aptamers, we anticipate that our sensor platform should offer a generalizable approach for the high-sensitivity detection of diverse targets. More importantly, we believe that NE-SAESA represents a novel strategy to improve the binding affinity between a small molecule and its aptamer and potentially can be extended to other detection platforms.

Directed evolution of PDZ variants to generate high-affinity detection reagents.

PubMed

Ferrer, Marc; Maiolo, Jim; Kratz, Patricia; Jackowski, Jessica L; Murphy, Dennis J; Delagrave, Simon; Inglese, James

2005-04-01

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.

Hemoglobin isoform differentiation and allosteric regulation of oxygen binding in the turtle, Trachemys scripta

PubMed Central

Damsgaard, Christian; Storz, Jay F.; Hoffmann, Federico G.

2013-01-01

When freshwater turtles acclimatize to winter hibernation, there is a gradual transition from aerobic to anaerobic metabolism, which may require adjustments of blood O2 transport before turtles become anoxic. Here, we report the effects of protons, anionic cofactors, and temperature on the O2-binding properties of isolated hemoglobin (Hb) isoforms, HbA and HbD, in the turtle Trachemys scripta. We determined the primary structures of the constituent subunits of the two Hb isoforms, and we related the measured functional properties to differences in O2 affinity between untreated hemolysates from turtles that were acclimated to normoxia and anoxia. Our data show that HbD has a consistently higher O2 affinity compared with HbA, whereas Bohr and temperature effects, as well as thiol reactivity, are similar. Although sequence data show amino acid substitutions at two known β-chain ATP-binding site positions, we find high ATP affinities for both Hb isoforms, suggesting an alternative and stronger binding site for ATP. The high ATP affinities indicate that, although ATP levels decrease in red blood cells of turtles acclimating to anoxia, the O2 affinity would remain largely unchanged, as confirmed by O2-binding measurements of untreated hemolysates from normoxic and anoxic turtles. Thus, the increase in blood-O2 affinity that accompanies winter acclimation is mainly attributable to a decrease in temperature rather than in concentrations of organic phosphates. This is the first extensive study on freshwater turtle Hb isoforms, providing molecular evidence for adaptive changes in O2 transport associated with acclimation to severe hypoxia. PMID:23986362

The fourth dimension in immunological space: how the struggle for nutrients selects high-affinity lymphocytes.

PubMed

Wensveen, Felix M; van Gisbergen, Klaas P J M; Eldering, Eric

2012-09-01

Lymphocyte activation via the antigen receptor is associated with radical shifts in metabolism and changes in requirements for nutrients and cytokines. Concomitantly, drastic changes occur in the expression of pro-and anti-apoptotic proteins that alter the sensitivity of lymphocytes to limiting concentrations of key survival factors. Antigen affinity is a primary determinant for the capacity of activated lymphocytes to access these vital resources. The shift in metabolic needs and the variable access to key survival factors is used by the immune system to eliminate activated low-affinity cells and to generate an optimal high-affinity response. In this review, we focus on the control of apoptosis regulators in activated lymphocytes by nutrients, cytokines, and costimulation. We propose that the struggle among individual clones that leads to the formation of high-affinity effector cell populations is in effect an 'invisible' fourth signal required for effective immune responses. © 2012 John Wiley & Sons A/S.

O2 binding and CO2 sensitivity in haemoglobins of subterranean African mole rats.

PubMed

Weber, Roy E; Jarvis, Jennifer U M; Fago, Angela; Bennett, Nigel C

2017-11-01

Inhabiting deep and sealed subterranean burrows, mole rats exhibit a remarkable suite of specializations, including eusociality (living in colonies with single breeding queens), extraordinary longevity, cancer immunity and poikilothermy, and extreme tolerance of hypoxia and hypercapnia. With little information available on adjustments in haemoglobin (Hb) function that may mitigate the impact of exogenous and endogenous constraints on the uptake and internal transport of O 2 , we measured haematological characteristics, as well as Hb-O 2 binding affinity and sensitivity to pH (Bohr effect), CO 2 , temperature and 2,3-diphosphoglycerate (DPG, the major allosteric modulator of Hb-O 2 affinity in red blood cells) in four social and two solitary species of African mole rats (family Bathyergidae) originating from different biomes and soil types across Central and Southern Africa. We found no consistent patterns in haematocrit (Hct) and blood and red cell DPG and Hb concentrations or in intrinsic Hb-O 2 affinity and its sensitivity to pH and DPG that correlate with burrowing, sociality and soil type. However, the results reveal low specific (pH independent) effects of CO 2 on Hb-O 2 affinity compared with humans that predictably safeguard pulmonary loading under hypoxic and hypercapnic burrow conditions. The O 2 binding characteristics are discussed in relation to available information on the primary structure of Hbs from adult and developmental stages of mammals subjected to hypoxia and hypercapnia and the molecular mechanisms underlying functional variation in rodent Hbs. © 2017. Published by The Company of Biologists Ltd.

Biomagnetic separation of Salmonella Typhimurium with high affine and specific ligand peptides isolated by phage display technique

NASA Astrophysics Data System (ADS)

Steingroewer, Juliane; Bley, Thomas; Bergemann, Christian; Boschke, Elke

2007-04-01

Analyses of food-borne pathogens are of great importance in order to minimize the health risk for customers. Thus, very sensitive and rapid detection methods are required. Current conventional culture techniques are very time consuming. Modern immunoassays and biochemical analysis also require pre-enrichment steps resulting in a turnaround time of at least 24 h. Biomagnetic separation (BMS) is a promising more rapid method. In this study we describe the isolation of high affine and specific peptides from a phage-peptide library, which combined with BMS allows the detection of Salmonella spp. with a similar sensitivity as that of immunomagnetic separation using antibodies.

Oxygen transport in blood at high altitude: role of the hemoglobin-oxygen affinity and impact of the phenomena related to hemoglobin allosterism and red cell function.

PubMed

Samaja, Michele; Crespi, Tiziano; Guazzi, Marco; Vandegriff, Kim D

2003-10-01

Altitude hypoxia is a major challenge to the blood O2 transport system, and adjustments of the blood-O2 affinity might contribute significantly to hypoxia adaptation. In principle, lowering the blood-O2 affinity is advantageous because it lowers the circulatory load required to assure adequate tissue oxygenation up to a threshold corresponding to about 5,000 m altitude, whereas at higher altitudes an increased blood-O2 affinity appears more advantageous. However, the rather contradictory experimental evidence raises the question whether other factors superimpose on the apparent changes of the blood-O2 affinity. The most important of these are as follows: (1) absolute temperature and temperature gradients within the body; (2) the intracapillary Bohr effect; (3) the red cell population heterogeneity in terms of O2 affinity; (4) control of altitude alkalosis; (5) the possible role of hemoglobin as a carrier of the vasodilator nitric oxide; (6) the effect of varied red cell transit times through the capillaries.

High-affinity RNA aptamers to C-reactive protein (CRP): newly developed pre-elution methods for aptamer selection

NASA Astrophysics Data System (ADS)

Orito, N.; Umekage, S.; Sato, K.; Kawauchi, S.; Tanaka, H.; Sakai, E.; Tanaka, T.; Kikuchi, Y.

2012-03-01

We have developed a modified SELEX (systematic evolution of ligands by exponential enrichment) method to obtain RNA aptamers with high affinity to C-reactive protein (CRP). CRP is a clinical biomarker present in plasma, the level of which increases in response to infections and noninfectious inflammation. The CRP level is also an important prognostic indicator in patients with several syndromes. At present, CRP content in blood is measured immunochemically using antibodies. To develop a more sensitive method using RNA aptamers, we have attempted to obtain high-affinity RNA aptamers to CRP. We succeeded in obtaining an RNA aptamer with high affinity to CRP using a CRP-immobilized Sepharose column and pre-elution procedure. Pre-elution is a method that removes the weak binding portion from a selected RNA population by washing for a short time with buffer containing CRP. By surface plasmon-resonance (SPR) analysis, the affinity constant of this aptamer for CRP was calculated to be KD = 2.25×10-9 (M). The secondary structure, contact sites with CRP protein, and application of this aptamer will be described.

Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity range of concentrations.

PubMed

Rubio, Francisco; Nieves-Cordones, Manuel; Alemán, Fernando; Martínez, Vicente

2008-12-01

The relative contribution of the high-affinity K(+) transporter AtHAK5 and the inward rectifier K(+) channel AtAKT1 to K(+) uptake in the high-affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col-0). The results obtained with wild-type lines, with T-DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the NH4+-sensitive and the Ba(2+)-sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high-affinity range of Rb(+) concentrations. Using Rb(+) as a K(+) analogue, it was shown that AtHAK5 mediates absorption at lower Rb(+) concentrations than AtAKT1 and depletes external Rb(+) to values around 1 muM. Factors such as the presence of K(+) or NH4+ during plant growth determine the relative contribution of each system. The presence of NH4+ in the growth solution inhibits the induction of AtHAK5 by K(+) starvation. In K(+)-starved plants grown without NH4+, both systems are operative, but when NH4+ is present in the growth solution, AtAKT1 is probably the only system mediating Rb(+) absorption, and the capacity of the roots to deplete Rb(+) is reduced.

Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA – development of a novel assay for vancomycin

PubMed Central

Chianella, Iva; Guerreiro, Antonio; Moczko, Ewa; Caygill, J. Sarah; Piletska, Elena V.; Perez De Vargas Sansalvador, Isabel M.; Whitcombe, Michael J.; Piletsky, Sergey A.

2016-01-01

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop ELISA type assays is presented here for the first time. NanoMIPs were synthesized by a solid phase approach with immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering and electron microscopy. Immobilization, blocking and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a HRP-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was three orders of magnitude better than a previously described ELISA based on antibodies. In these experiments nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA. PMID:23947402

Characterization of Zebrafish Cardiac and Slow Skeletal Troponin C Paralogs by MD Simulation and ITC.

PubMed

Stevens, Charles M; Rayani, Kaveh; Genge, Christine E; Singh, Gurpreet; Liang, Bo; Roller, Janine M; Li, Cindy; Li, Alison Yueh; Tieleman, D Peter; van Petegem, Filip; Tibbits, Glen F

2016-07-12

Zebrafish, as a model for teleost fish, have two paralogous troponin C (TnC) genes that are expressed in the heart differentially in response to temperature acclimation. Upon Ca(2+) binding, TnC changes conformation and exposes a hydrophobic patch that interacts with troponin I and initiates cardiac muscle contraction. Teleost-specific TnC paralogs have not yet been functionally characterized. In this study we have modeled the structures of the paralogs using molecular dynamics simulations at 18°C and 28°C and calculated the different Ca(2+)-binding properties between the teleost cardiac (cTnC or TnC1a) and slow-skeletal (ssTnC or TnC1b) paralogs through potential-of-mean-force calculations. These values are compared with thermodynamic binding properties obtained through isothermal titration calorimetry (ITC). The modeled structures of each of the paralogs are similar at each temperature, with the exception of helix C, which flanks the Ca(2+) binding site; this region is also home to paralog-specific sequence substitutions that we predict have an influence on protein function. The short timescale of the potential-of-mean-force calculation precludes the inclusion of the conformational change on the ΔG of Ca(2+) interaction, whereas the ITC analysis includes the Ca(2+) binding and conformational change of the TnC molecule. ITC analysis has revealed that ssTnC has higher Ca(2+) affinity than cTnC for Ca(2+) overall, whereas each of the paralogs has increased affinity at 28°C compared to 18°C. Microsecond-timescale simulations have calculated that the cTnC paralog transitions from the closed to the open state more readily than the ssTnC paralog, an unfavorable transition that would decrease the ITC-derived Ca(2+) affinity while simultaneously increasing the Ca(2+) sensitivity of the myofilament. We propose that the preferential expression of cTnC at lower temperatures increases myofilament Ca(2+) sensitivity by this mechanism, despite the lower Ca(2+) affinity that we have measured by ITC. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Evidence of paired M2 muscarinic receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Potter, L.T.; Ballesteros, L.A.; Bichajian, L.H.

Binding assays involving various antagonists, including N-(3H) methylscopolamine, (3H)quinuclidinyl benzilate, AFDX-116, pirenzepine, and propylbenzilylcholine mustard, disclosed only a single population of M2 muscarinic receptors in membranes from the rat brainstem (medulla, pons, and colliculi). However, competition curves between N-(3H)methylscopolamine and various agonists, including oxotremorine, cis-dioxolane, and acetylethylcholine mustard, showed approximately equal numbers of guanine nucleotide-sensitive high affinity (H) sites and guanine nucleotide-insensitive low affinity (L) sites. This 50% H phenomenon persisted in different buffers, at different temperatures, after the number of receptors was halved (and, thus, the remaining receptor to guanine nucleotide-binding protein ratio was doubled), after membrane solubilization withmore » digitonin, and when rabbit cardiac membranes were used instead of rat brainstem membranes. Preferential occupation of H sites with acetylethylcholine mustard, and of L sites with quinuclidinyl benzilate or either mustard, yielded residual free receptor populations showing predominantly L and H sites, respectively. Low concentrations of (3H)-oxotremorine-M labeled only H sites, and the Bmax for these sites was 49% of the Bmax found with (3H)quinuclidinyl benzilate plus guanine nucleotide. These and other results are most consistent with the idea that H and L receptor sites exist on separate but dimeric receptor molecules and with the hypothesis that only the H receptors cycle between high and low affinity, depending upon interactions between this receptor molecule and a guanine nucleotide-binding protein.« less

Flying high: a theoretical analysis of the factors limiting exercise performance in birds at altitude.

PubMed

Scott, Graham R; Milsom, William K

2006-11-01

The ability of some bird species to fly at extreme altitude has fascinated comparative respiratory physiologists for decades, yet there is still no consensus about what adaptations enable high altitude flight. Using a theoretical model of O(2) transport, we performed a sensitivity analysis of the factors that might limit exercise performance in birds. We found that the influence of individual physiological traits on oxygen consumption (Vo2) during exercise differed between sea level, moderate altitude, and extreme altitude. At extreme altitude, haemoglobin (Hb) O(2) affinity, total ventilation, and tissue diffusion capacity for O(2) (D(To2)) had the greatest influences on Vo2; increasing these variables should therefore have the greatest adaptive benefit for high altitude flight. There was a beneficial interaction between D(To2) and the P(50) of Hb, such that increasing D(To2) had a greater influence on Vo2 when P(50) was low. Increases in the temperature effect on P(50) could also be beneficial for high flying birds, provided that cold inspired air at extreme altitude causes a substantial difference in temperature between blood in the lungs and in the tissues. Changes in lung diffusion capacity for O(2), cardiac output, blood Hb concentration, the Bohr coefficient, or the Hill coefficient likely have less adaptive significance at high altitude. Our sensitivity analysis provides theoretical suggestions of the adaptations most likely to promote high altitude flight in birds and provides direction for future in vivo studies.

Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

PubMed

Lai, H; Carino, M A

1992-07-01

Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beaumont, K.; Vaughn, D.A.; Fanestil, D.D.

Thiazides and related diuretics inhibit NaCl reabsorption in the distal tubule through an unknown mechanism. The authors report here that ({sup 3}H)metolazone, a diuretic with a thiazide-like mechanism of action, labels a site in rat kidney membranes that has characteristics of the thiazide-sensitive ion transporter. ({sup 3}H)Metolazone bound with high affinity to a site with a density of 0.717 pmol/mg of protein in kidney membranes. The binding site was localized to the renal cortex, with little or not binding in other kidney regions and 11 other tissues. The affinities of thiazide-type diuretics for this binding site were significantly correlated withmore » their clinical potency. Halide anions specifically inhibited high-affinity binding of ({sup 3}H)metolazone to this site. ({sup 3})Metolazone also bound with lower affinity to sites present in kidney as well as in liver, testis, lung, brain, heart, and other tissues. Calcium antagonists and certain smooth muscle relaxants had K{sub i} values of 0.6-10 {mu}M for these low-affinity sites, which were not inhibited by most of the thiazide diuretics tested. Properties of the high-affinity ({sup 3}H)metolazone binding site are consistent with its identity as the receptor for thiazide-type diuretics.« less

Thermophoresis in nanoliter droplets to quantify aptamer binding.

PubMed

Seidel, Susanne A I; Markwardt, Niklas A; Lanzmich, Simon A; Braun, Dieter

2014-07-21

Biomolecule interactions are central to pharmacology and diagnostics. These interactions can be quantified by thermophoresis, the directed molecule movement along a temperature gradient. It is sensitive to binding induced changes in size, charge, or conformation. Established capillary measurements require at least 0.5 μL per sample. We cut down sample consumption by a factor of 50, using 10 nL droplets produced with acoustic droplet robotics (Labcyte). Droplets were stabilized in an oil-surfactant mix and locally heated with an IR laser. Temperature increase, Marangoni flow, and concentration distribution were analyzed by fluorescence microscopy and numerical simulation. In 10 nL droplets, we quantified AMP-aptamer affinity, cooperativity, and buffer dependence. Miniaturization and the 1536-well plate format make the method high-throughput and automation friendly. This promotes innovative applications for diagnostic assays in human serum or label-free drug discovery screening. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthesis of thermo-responsive bovine hemoglobin imprinted nanoparticles by combining ionic liquid immobilization with aqueous precipitation polymerization.

PubMed

Wang, Yongmei; Yang, Chongchong; Sun, Yan; Qiu, Fengtao; Xiang, Yang; Fu, Guoqi

2018-02-01

Surface molecular imprinting over functionalized nanoparticles has proved to be an effective approach for construction of artificial nanomaterials for protein recognition. Herein, we report a strategy for synthesis of core-shell protein-imprinted nanoparticles by the functionalization of nano-cores with ionic liquids followed by aqueous precipitation polymerization to build thermo-responsive imprinted polymer nano-shells. The immobilized ionic liquids can form multiple interactions with the protein template. The polymerization process can produce thermo-reversible physical crosslinks, which are advantageous to enhancing imprinting and facilitating template removal. With bovine hemoglobin as a model template, the imprinted nanoparticles showed temperature-sensitivity in both dispersion behaviors and rebinding capacities. Compared with the ionic-liquid-modified core nanoparticles, the imprinted particles exhibited greatly increased selectivity and two orders of magnitude higher binding affinity for the template protein. The imprinted nanoparticles achieved relatively high imprinting factor up to 5.0 and specific rebinding capacity of 67.7 mg/g, respectively. These nanoparticles also demonstrated rapid rebinding kinetics and good reproducibility after five cycles of adsorption-regeneration. Therefore, the presented approach may be viable for the fabrication of high-performance protein-imprinted nanoparticles with temperature sensitivity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases

NASA Astrophysics Data System (ADS)

Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

2010-09-01

The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH 2 of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5 zg spot -1. For sample volume of 0.40 μl spot -1, corresponding concentration was 6.2 × 10 -18 g ml -1), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was ±5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule switch technique, lectin science and SSRTP.

Determination of trace alkaline phosphatase by affinity adsorption solid substrate room temperature phosphorimetry based on wheat germ agglutinin labeled with 8-quinolineboronic acid phosphorescent molecular switch and prediction of diseases.

PubMed

Liu, Jia-Ming; Gao, Hui; Li, Fei-Ming; Shi, Xiu-Mei; Lin, Chang-Qing; Lin, Li-Ping; Wang, Xin-Xing; Li, Zhi-Ming

2010-09-01

The 8-quinolineboronic acid phosphorescent molecular switch (abbreviated as PMS-8-QBA. Thereinto, 8-QBA is 8-quinolineboronic acid, and PMS is phosphorescent molecular switch) was found for the first time. PMS-8-QBA, which was in the "off" state, could only emit weak room temperature phosphorescence (RTP) on the acetyl cellulose membrane (ACM). However, PMS-8-QBA turned "on" automatically for its changed structure, causing that the RTP of 8-QBA in the system increased, after PMS-8-QBA-WGA (WGA is wheat germ agglutinin) was formed by reaction between -OH of PMS-8-QBA and -COOH of WGA. More interesting is that the -NH(2) of PMS-8-QBA-WGA could react with the -COOH of alkaline phosphatase (AP) to form the affinity adsorption (AA) product WGA-AP-WGA-8-QBA-PMS (containing -NH-CO- bond), which caused RTP of the system to greatly increase. Thus, affinity adsorption solid substrate room temperature phosphorimetry using PMS-8-QBA as labelling reagent (PMS-8-QBA-AA-SSRTP) for the determination of trace AP was established. The method had many advantages, such as high sensitivity (the detection limit (LD) was 2.5zgspot(-1). For sample volume of 0.40mulspot(-1), corresponding concentration was 6.2x10(-18)gml(-1)), good selectivity (the allowed concentration of coexisting material was higher, when the relative error was +/-5%), high accuracy (applied to detection of AP content in serum samples, the result was coincided with those obtained by enzyme-linked immunoassay), which was suitable for the detection of trace AP content in serum samples and the forecast of human diseases. Meanwhile, the mechanism of PMS-8-QBA-AASSRTP was discussed. The new field of analytical application and clinic diagnosis technique of molecule switch are exploited, based on the phosphorescence characteristic of PMS-8-QBA, the AA reaction between WGA and AP, as well as the relation between AP content and human diseases. The research results promote the development and interpenetrate among molecule switch technique, lectin science and SSRTP. Copyright 2009 Elsevier B.V. All rights reserved.

Selective labeling of serotonin uptake sites in rat brain by (/sup 3/H)citalopram contrasted to labeling of multiple sites by (/sup 3/H)imipramine

DOE Office of Scientific and Technical Information (OSTI.GOV)

D'Amato, R.J.; Largent, B.L.; Snowman, A.M.

1987-07-01

Citalopram is a potent and selective inhibitor of neuronal serotonin uptake. In rat brain membranes (/sup 3/H)citalopram demonstrates saturable and reversible binding with a KD of 0.8 nM and a maximal number of binding sites (Bmax) of 570 fmol/mg of protein. The drug specificity for (/sup 3/H)citalopram binding and synaptosomal serotonin uptake are closely correlated. Inhibition of (/sup 3/H)citalopram binding by both serotonin and imipramine is consistent with a competitive interaction in both equilibrium and kinetic analyses. The autoradiographic pattern of (/sup 3/H)citalopram binding sites closely resembles the distribution of serotonin. By contrast, detailed equilibrium-saturation analysis of (/sup 3/H)imipramine bindingmore » reveals two binding components, i.e., high affinity (KD = 9 nM, Bmax = 420 fmol/mg of protein) and low affinity (KD = 553 nM, Bmax = 8560 fmol/mg of protein) sites. Specific (/sup 3/H)imipramine binding, defined as the binding inhibited by 100 microM desipramine, is displaced only partially by serotonin. Various studies reveal that the serotonin-sensitive portion of binding corresponds to the high affinity sites of (/sup 3/H)imipramine binding whereas the serotonin-insensitive binding corresponds to the low affinity sites. Lesioning of serotonin neurons with p-chloroamphetamine causes a large decrease in (/sup 3/H)citalopram and serotonin-sensitive (/sup 3/H)imipramine binding with only a small effect on serotonin-insensitive (/sup 3/H)imipramine binding. The dissociation rate of (/sup 3/H)imipramine or (/sup 3/H)citalopram is not altered by citalopram, imipramine or serotonin up to concentrations of 10 microM. The regional distribution of serotonin sensitive (/sup 3/H)imipramine high affinity binding sites closely resembles that of (/sup 3/H)citalopram binding.« less

Low-affinity spermine block mediating outward currents through Kir2.1 and Kir2.2 inward rectifier potassium channels

PubMed Central

Ishihara, Keiko; Yan, Ding-Hong

2007-01-01

The outward component of the strong inward rectifier K+ current (IKir) plays a pivotal role in polarizing the membranes of excitable and non-excitable cells and is regulated by voltage-dependent channel block by internal cations. Using the Kir2.1 channel, we previously showed that a small fraction of the conductance susceptible only to a low-affinity mode of block likely carries a large portion of the outward current. To further examine the relevance of the low-affinity block to outward IKir and to explore its molecular mechanism, we studied the block of the Kir2.1 and Kir2.2 channels by spermine, which is the principal Kir2 channel blocker. Current–voltage relations of outward Kir2.2 currents showed a peak, a plateau and two peaks in the presence of 10, 1 and 0.1 μm spermine, respectively, which was explained by the presence of two conductances that differ in their susceptibility to spermine block. When the current–voltage relations showed one peak, like those of native IKir, outward Kir2.2 currents were mediated mostly by the conductance susceptible to the low-affinity block. They also flowed in a narrower range than the corresponding Kir2.1 currents, because of 3- to 4-fold greater susceptibility to the low-affinity block than in Kir2.1. Reducing external [K+] shifted the voltage dependences of both the high- and low-affinity block of Kir2.1 in parallel with the shift in the reversal potential, confirming the importance of the low-affinity block in mediating outward IKir. When Kir2.1 mutants known to have reduced sensitivity to internal blockers were examined, the D172N mutation in the transmembrane pore region made almost all of the conductance susceptible only to low-affinity block, while the E224G mutation in the cytoplasmic pore region reduced the sensitivity to low-affinity block without markedly altering that to the high-affinity block or the high/low conductance ratio. The effects of these mutations support the hypothesis that Kir2 channels exist in two states having different susceptibilities to internal cationic blockers. PMID:17640933

A systematic study of the effect of low pH acid treatment on anti-drug antibodies specific for a domain antibody therapeutic: Impact on drug tolerance, assay sensitivity and post-validation method assessment of ADA in clinical serum samples.

PubMed

Kavita, Uma; Duo, Jia; Crawford, Sean M; Liu, Rong; Valcin, Joan; Gleason, Carol; Dong, Huijin; Gadkari, Snaehal; Dodge, Robert W; Pillutla, Renuka C; DeSilva, Binodh S

2017-09-01

We developed a homogeneous bridging anti-drug antibody (ADA) assay on an electro chemiluminescent immunoassay (ECLIA) platform to support the immunogenicity evaluation of a dimeric domain antibody (dAb) therapeutic in clinical studies. During method development we evaluated the impact of different types of acid at various pH levels on polyclonal and monoclonal ADA controls of differing affinities and on/off rates. The data shows for the first time that acids of different pH can have a differential effect on ADA of various affinities and this in turn impacts assay sensitivity and drug tolerance as defined by these surrogate controls. Acid treatment led to a reduction in signal of intermediate and low affinity ADA, but not high affinity or polyclonal ADA. We also found that acid pretreatment is a requisite for dissociation of drug bound high affinity ADA, but not for low affinity ADA-drug complexes. Although we were unable to identify an acid that would allow a 100% retrieval of ADA signal post-treatment, use of glycine pH3.0 enabled the detection of low, intermediate and high affinity antibodies (Abs) to various extents. Following optimization, the ADA assay method was validated for clinical sample analysis. Consistencies within various parameters of the clinical data such as dose dependent increases in ADA rates and titers were observed, indicating a reliable ADA method. Pre- and post-treatment ADA negative or positive clinical samples without detectable drug were reanalyzed in the absence of acid treatment or presence of added exogenous drug respectively to further assess the effectiveness of the final acid treatment procedure. The overall ADA results indicate that assay conditions developed and validated based on surrogate controls sufficed to provide a reliable clinical data set. The effect of low pH acid treatment on possible pre-existing ADA or soluble multimeric target in normal human serum was also evaluated, and preliminary data indicate that acid type and pH also affect drug-specific signal differentially in individual samples. The results presented here represent the most extensive analyses to date on acid treatment of a wide range of ADA affinities to explore sensitivity and drug tolerance issues. They have led to a refinement of our current best practices for ADA method development and provide a depth of data to interrogate low pH mediated immune complex dissociation. Copyright © 2017 Elsevier B.V. All rights reserved.

Ligand Binding Analysis and Screening by Chemical Denaturation Shift

PubMed Central

Sch n, Arne; Brown, Richard K.; Hutchins, Burleigh M.; Freire, Ernesto

2013-01-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Towards this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Since ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities and the ligand rank order obtained at denaturation temperatures (60°C or higher) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations in which binding changes the cooperativity of the unfolding transition. In this paper we develop the basic analytical equations and provide several experimental examples. PMID:23994566

Ligand binding analysis and screening by chemical denaturation shift.

PubMed

Schön, Arne; Brown, Richard K; Hutchins, Burleigh M; Freire, Ernesto

2013-12-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples. Copyright © 2013 Elsevier Inc. All rights reserved.

Electrochemical immobilization of Fluorescent labelled probe molecules on a FTO surface for affinity detection based on photo-excited current

NASA Astrophysics Data System (ADS)

Haruyama, Tetsuya; Wakabayashi, Ryo; Cho, Takeshi; Matsuyama, Sho-taro

2011-10-01

Photo-excited current can be generated at a molecular interface between a photo-excited molecules and a semi-conductive material in appropriate condition. The system has been recognized for promoting photo-energy devices such as an organic dye sensitized solar-cell. The photo-current generated reactions are totally dependent on the interfacial energy reactions, which are in a highly fluctuated interfacial environment. The authors investigated the photo-excited current reaction to develop a smart affinity detection method. However, in order to perform both an affinity reaction and a photo-excited current reaction at a molecular interface, ordered fabrications of the functional (affinity, photo-excitation, etc.) molecules layer on a semi-conductive surface is required. In the present research, we would like to present the fabrication and functional performance of photo-excited current-based affinity assay device and its application for detection of endocrine disrupting chemicals. On the FTO surface, fluorescent pigment labelled affinity peptide was immobilized through the EC tag (electrochemical-tag) method. The modified FTO produced a current when it was irradiated with diode laser light. However, the photo current decreased drastically when estrogen (ES) coexisted in the reaction solution. In this case, immobilized affinity probe molecules formed a complex with ES and estrogen receptor (ER). The result strongly suggests that the photo-excited current transduction between probe molecule-labelled cyanine pigment and the FTO surface was partly inhibited by a complex that formed at the affinity oligo-peptide region in a probe molecule on the FTO electrode. The bound bulky complex may act as an impediment to perform smooth transduction of photo-excited current in the molecular interface. The present system is new type of photo-reaction-based analysis. This system can be used to perform simple high-sensitive homogeneous assays.

Adjusted active carbon fibers for solid phase microextraction.

PubMed

Jia, Jinping; Feng, Xue; Fang, Nenghu; Wang, Yalin; Chen, Hongjin; Dan, Wu

2002-01-01

Adjusted active carbon fiber (AACF) was evaluated for Solid Phase Microextraction (SPME), which showed higher sensitivity and stability than traditional coating fibers. The characteristics of AACF result from two different activation methods (chemical and water vapor) and from variable activation conditions (temperature and time). The fiber treated by water vapor appears to have stronger affinity to polar compounds, while that treated by chemical activation appears to have stronger affinity to non-polar compounds. For different target compounds ranged from non-polar to polar, AACF design could be effective with specific selections and sensitivities. As applications in this paper, benzoic acid in soy sauce was extracted onto water-vapor-activated-fiber, then analyzed using gas chromatograph-mass spectrometer (GC-MS). The chemical-activated-fiber SPME was applied in the analysis of benzene series compounds (BTEX) in water matrix. Compared with standard carbon disulfide extraction method, chemical-activated-fiber SPME is more convenient due to its simple process and turns to be of relative low detection limits.

An Escherichia coli strain, PGB01, isolated from feral pigeon Faeces, thermally fit to survive in pigeon, shows high level resistance to trimethoprim.

PubMed

Kumar, Arvind; Tiwary, Bipransh Kumar; Kachhap, Sangita; Nanda, Ashis Kumar; Chakraborty, Ranadhir

2015-01-01

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon's gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr.

Specificity of cell–cell adhesion by classical cadherins: Critical role for low-affinity dimerization through β-strand swapping

PubMed Central

Chen, Chien Peter; Posy, Shoshana; Ben-Shaul, Avinoam; Shapiro, Lawrence; Honig, Barry H.

2005-01-01

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell–cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by “swapping” the N-terminal β-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that “closed” monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell–cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems. PMID:15937105

Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Senogles, S.E.; Caron, M.G.

1986-05-01

The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with /sup 3/H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..Smore » binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D/sub 2/-dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.« less

High-Affinity Binding of Silybin Derivatives to the Nucleotide-Binding Domain of a Leishmania tropica P-Glycoprotein-Like Transporter and Chemosensitization of a Multidrug-Resistant Parasite to Daunomycin

PubMed Central

Pérez-Victoria, José M.; Pérez-Victoria, F. Javier; Conseil, Gwenaëlle; Maitrejean, Mathias; Comte, Gilles; Barron, Denis; Di Pietro, Attilio; Castanys, Santiago; Gamarro, Francisco

2001-01-01

In order to overcome the multidrug resistance mediated by P-glycoprotein-like transporters in Leishmania spp., we have studied the effects produced by derivatives of the flavanolignan silybin and related compounds lacking the monolignol unit on (i) the affinity of binding to a recombinant C-terminal nucleotide-binding domain of the L. tropica P-glycoprotein-like transporter and (ii) the sensitization to daunomycin on promastigote forms of a multidrug-resistant L. tropica line overexpressing the transporter. Oxidation of the flavanonol silybin to the corresponding flavonol dehydrosilybin, the presence of the monolignol unit, and the addition of a hydrophobic substituent such as dimethylallyl, especially at position 8 of ring A, considerably increased the binding affinity. The in vitro binding affinity of these compounds for the recombinant cytosolic domain correlated with their modulation of drug resistance phenotype. In particular, 8-(3,3-dimethylallyl)-dehydrosilybin effectively sensitized multidrug-resistant Leishmania spp. to daunomycin. The cytosolic domains are therefore attractive targets for the rational design of inhibitors against P-glycoprotein-like transporters. PMID:11158738

Lesion-induced plasticity of high affinity choline uptake in the developing rat fascia dentata.

PubMed

Nadler, J V; Shelton, D L; Cotman, C W

1979-03-23

After removal of the perforant path input to the rat fascia dentata at the age of 11 days, cholinergic septohippocampal fibers invade the denervated area. We have examined the effect of this lesion on hemicholinium-sensitive, high affinity choline uptake and its coupling to acetylcholine synthesis, specific properties of the septohippocampal input. Removal of the ipsilateral perforant path fibers increased the velocity of high affinity choline uptake by dentate particulate preparations, usually within 1 day. Studies conducted 5--104 days after operation showed a consistent 50--65% elevation in the molecular (denervated) layer. In contrast, the choline uptake rate in the granular layer eventually decreased slightly. Calculation of choline uptake rates independently of protein (per whole region) revealed that fasciae dentatae from operated and control sides accumulated choline at approximately equal rates, but on the operated side a greater percentage was transported by structures from the molecular layer and a lesser percentage by those from the granular layer. The rate of acetylcholine synthesis from exogenous choline increased to the same extent as high affinity choline uptake from 3 days after operation onwards. The changes in high affinity choline uptake and acetylcholine synthesis coincided spatially and temporally with the reactive growth of septohippocampal fibers. Our results support the view that a perforant path lesion during development permanently alters the distribution of functional septohippocampal boutons in the fascia dentata. Acetylcholine synthesis is regulated to the same extent by high affinity choline uptake in the anomalous boutons as in normally located boutons.

Novel cellulose-based halochromic test strips for naked-eye detection of alkaline vapors and analytes.

PubMed

Abou-Yousef, Hussein; Khattab, Tawfik A; Youssef, Yehia A; Al-Balakocy, Naser; Kamel, Samir

2017-08-01

A simple, portable and highly sensitive naked-eye test strip is successfully prepared for optical detection of gaseous and aqueous alkaline analytes. Novel pH-sensory tricyanofuran-hydrazone (TCFH) disperse colorant containing a hydrazone recognition functional moiety is successfully synthesized via azo-coupling reaction between active methyl-containing tricyanofuran (TCF) heterocycle and diazonium salt of 4-aminobenzaldehyde followed by Knoevenagel condensation with malononitrile. UV-vis absorption spectra display solvatochromism and reversible color changes of the TCFH solution in dimethyl sulfoxide in response to pH variations. We investigate the preparation of hydrophobic cellulose/polyethylene terephthalate composites characterized by their high affinity for disperse dyes. Composite films made from CA, Cell/CA, PET/CA, and Cell/PET-CA are produced via solvent-casting procedure using 10-30% modified cellulose or modified polyethylene terephthalate. The mechanical properties and morphologies of these composite films are investigated. The prepared pH-sensory hydrazone-based disperse dye is then applied to dye the produced cellulose-based composite films employing the high temperature pressure dyeing procedure. The produced halochromic PET-CA-TCFH test strip provide an instant visible signal from orange to purple upon exposure to alkaline conditions as proved by the coloration measurements. The sensor strip exhibits high sensitivity and quick detection toward ammonia in both of aqueous and vapor phases by naked-eye observations at room temperature and atmospheric pressure. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetically based low oxygen affinities of felid hemoglobins: lack of biochemical adaptation to high-altitude hypoxia in the snow leopard

PubMed Central

Janecka, Jan E.; Nielsen, Simone S. E.; Andersen, Sidsel D.; Hoffmann, Federico G.; Weber, Roy E.; Anderson, Trevor; Storz, Jay F.; Fago, Angela

2015-01-01

ABSTRACT Genetically based modifications of hemoglobin (Hb) function that increase blood–O2 affinity are hallmarks of hypoxia adaptation in vertebrates. Among mammals, felid Hbs are unusual in that they have low intrinsic O2 affinities and reduced sensitivities to the allosteric cofactor 2,3-diphosphoglycerate (DPG). This combination of features compromises the acclimatization capacity of blood–O2 affinity and has led to the hypothesis that felids have a restricted physiological niche breadth relative to other mammals. In seeming defiance of this conjecture, the snow leopard (Panthera uncia) has an extraordinarily broad elevational distribution and occurs at elevations above 6000 m in the Himalayas. Here, we characterized structural and functional variation of big cat Hbs and investigated molecular mechanisms of Hb adaptation and allosteric regulation that may contribute to the extreme hypoxia tolerance of the snow leopard. Experiments revealed that purified Hbs from snow leopard and African lion exhibited equally low O2 affinities and DPG sensitivities. Both properties are primarily attributable to a single amino acid substitution, β2His→Phe, which occurred in the common ancestor of Felidae. Given the low O2 affinity and reduced regulatory capacity of feline Hbs, the extreme hypoxia tolerance of snow leopards must be attributable to compensatory modifications of other steps in the O2-transport pathway. PMID:26246610

Genetically based low oxygen affinities of felid hemoglobins: lack of biochemical adaptation to high-altitude hypoxia in the snow leopard.

PubMed

Janecka, Jan E; Nielsen, Simone S E; Andersen, Sidsel D; Hoffmann, Federico G; Weber, Roy E; Anderson, Trevor; Storz, Jay F; Fago, Angela

2015-08-01

Genetically based modifications of hemoglobin (Hb) function that increase blood-O2 affinity are hallmarks of hypoxia adaptation in vertebrates. Among mammals, felid Hbs are unusual in that they have low intrinsic O2 affinities and reduced sensitivities to the allosteric cofactor 2,3-diphosphoglycerate (DPG). This combination of features compromises the acclimatization capacity of blood-O2 affinity and has led to the hypothesis that felids have a restricted physiological niche breadth relative to other mammals. In seeming defiance of this conjecture, the snow leopard (Panthera uncia) has an extraordinarily broad elevational distribution and occurs at elevations above 6000 m in the Himalayas. Here, we characterized structural and functional variation of big cat Hbs and investigated molecular mechanisms of Hb adaptation and allosteric regulation that may contribute to the extreme hypoxia tolerance of the snow leopard. Experiments revealed that purified Hbs from snow leopard and African lion exhibited equally low O2 affinities and DPG sensitivities. Both properties are primarily attributable to a single amino acid substitution, β2His→Phe, which occurred in the common ancestor of Felidae. Given the low O2 affinity and reduced regulatory capacity of feline Hbs, the extreme hypoxia tolerance of snow leopards must be attributable to compensatory modifications of other steps in the O2-transport pathway. © 2015. Published by The Company of Biologists Ltd.

Fullerene Cyanation Does Not Always Increase Electron Affinity: Experimental and Theoretical Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clikeman, Tyler T.; Deng, Shihu; Popov, Alexey A.

2015-01-01

The electron affinities of C70 derivatives with trifluoromethyl, methyl and cyano groups were studied experimentally and theoretically using low-temperature photoelectron spectroscopy (LT PES) and density functional theory (DFT). The electronic effects of these functional groups were determined and found to be highly dependent on the addition patterns. Substitution of CF3 for CN for the same addition pattern increases the experimental electron affinity by 70 meV per substitution. The synthesis of a new fullerene derivative, C70(CF3)10(CN)2, is reported for the first time

High sensitivity and high Q-factor nanoslotted parallel quadrabeam photonic crystal cavity for real-time and label-free sensing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Daquan; State Key Laboratory of Information Photonics and Optical Communications, School of Information and Communication Engineering, Beijing University of Posts and Telecommunications, Beijing 100876; School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138

We experimentally demonstrate a label-free sensor based on nanoslotted parallel quadrabeam photonic crystal cavity (NPQC). The NPQC possesses both high sensitivity and high Q-factor. We achieved sensitivity (S) of 451 nm/refractive index unit and Q-factor >7000 in water at telecom wavelength range, featuring a sensor figure of merit >2000, an order of magnitude improvement over the previous photonic crystal sensors. In addition, we measured the streptavidin-biotin binding affinity and detected 10 ag/mL concentrated streptavidin in the phosphate buffered saline solution.

Direct replacement of antibodies with molecularly imprinted polymer nanoparticles in ELISA--development of a novel assay for vancomycin.

PubMed

Chianella, Iva; Guerreiro, Antonio; Moczko, Ewa; Caygill, J Sarah; Piletska, Elena V; De Vargas Sansalvador, Isabel M Perez; Whitcombe, Michael J; Piletsky, Sergey A

2013-09-03

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent assay (ELISA) is presented here for the first time. NanoMIPs were synthesized by a solid-phase approach with an immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering, and electron microscopy. Immobilization, blocking, and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a horseradish peroxidase-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range of 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was 3 orders of magnitude better than a previously described ELISA based on antibodies. In these experiments, nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.

Rapid, Sensitive Detection of Botulinum Toxin on a Flexible Microfluidics Platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Dockendorff, Brian P.; Feldhaus, Michael J.

2004-10-30

In this paper we will describe how high affinity reagents and a sensor configuration enabling rapid mass transport can be combined for rapid, sensitive biodetection. The system that we have developed includes a renewable surface immunoassay, which involves on-column detection of a fluorescently labeled secondary antibody in a sandwich immunoassay. Yeast display and directed molecular evolution were used to create high affinity antibodies to the botulinum toxin heavy chain receptor binding domain, AR1 and 3D12. A rotating rod renewable surface microcolumn was used to form a microliter-sized column containing beads functionalized with the capture antibody (AR1). The column was perfusedmore » with sample, wash solutions, and a fluorescently labeled secondary antibody (3D12) while the on-column fluorescence was monitored. Detection was accomplished in less than 5 minutes, with a total processing time of about 10 minutes. On-column detection of botulinum toxin was more sensitive and much faster than flow cytometry analysis on microbeads using the same reagents.« less

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

2000-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1998-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Surface sensitization mechanism on negative electron affinity p-GaN nanowires

NASA Astrophysics Data System (ADS)

Diao, Yu; Liu, Lei; Xia, Sihao; Feng, Shu; Lu, Feifei

2018-03-01

The surface sensitization is the key to prepare negative electron affinity photocathode. The thesis emphasizes on the study of surface sensitization mechanism of p-type doping GaN nanowires utilizing first principles based on density function theory. The adsorption energy, work function, dipole moment, geometry structure, electronic structure and optical properties of Mg-doped GaN nanowires surfaces with various coverages of Cs atoms are investigated. The GaN nanowire with Mg doped in core position is taken as the sensitization base. At the initial stage of sensitization, the best adsorption site for Cs atom on GaN nanowire surface is BN, the bridge site of two adjacent N atoms. Surface sensitization generates a p-type internal surface with an n-type surface state, introducing a band bending region which can help reduce surface barrier and work function. With increasing Cs coverage, work functions decrease monotonously and the "Cs-kill" phenomenon disappears. For Cs coverage of 0.75 ML and 1 ML, the corresponding sensitization systems reach negative electron affinity state. Through surface sensitization, the absorption curves are red shifted and the absorption coefficient is cut down. All theoretical calculations can guide the design of negative electron affinity Mg doped GaN nanowires photocathode.

A simple highly sensitive and selective aptamer-based colorimetric sensor for environmental toxins microcystin-LR in water samples.

PubMed

Li, Xiuyan; Cheng, Ruojie; Shi, Huijie; Tang, Bo; Xiao, Hanshuang; Zhao, Guohua

2016-03-05

A simple and highly sensitive aptamer-based colorimetric sensor was developed for selective detection of Microcystin-LR (MC-LR). The aptamer (ABA) was employed as recognition element which could bind MC-LR with high-affinity, while gold nanoparticles (AuNPs) worked as sensing materials whose plasma resonance absorption peaks red shifted upon binding of the targets at a high concentration of sodium chloride. With the addition of MC-LR, the random coil aptamer adsorbed on Au NPs altered into regulated structure to form MC-LR-aptamer complexes and broke away from the surface of Au NPs, leading to the aggregation of AuNPs, and the color converted from red to blue due to the interparticle plasmon coupling. Results showed that our aptamer-based colorimetric sensor exhibited rapid and sensitive detection performance for MC-LR with linear range from 0.5 nM to 7.5 μM and the detection limit reached 0.37 nM. Meanwhile, the pollutants usually coexisting with MC-LR in pollutant water samples had not demonstrated disturbance for detecting of MC-LR. The mechanism was also proposed suggesting that high affinity interaction between aptamer and MC-LR significantly enhanced the sensitivity and selectivity for MC-LR detection. Besides, the established method was utilized in analyzing real water samples and splendid sensitivity and selectivity were obtained as well. Copyright © 2015 Elsevier B.V. All rights reserved.

High-resolution mapping of transcription factor binding sites on native chromatin

PubMed Central

Kasinathan, Sivakanthan; Orsi, Guillermo A.; Zentner, Gabriel E.; Ahmad, Kami; Henikoff, Steven

2014-01-01

Sequence-specific DNA-binding proteins including transcription factors (TFs) are key determinants of gene regulation and chromatin architecture. Formaldehyde cross-linking and sonication followed by Chromatin ImmunoPrecipitation (X-ChIP) is widely used for profiling of TF binding, but is limited by low resolution and poor specificity and sensitivity. We present a simple protocol that starts with micrococcal nuclease-digested uncross-linked chromatin and is followed by affinity purification of TFs and paired-end sequencing. The resulting ORGANIC (Occupied Regions of Genomes from Affinity-purified Naturally Isolated Chromatin) profiles of Saccharomyces cerevisiae Abf1 and Reb1 provide highly accurate base-pair resolution maps that are not biased toward accessible chromatin, and do not require input normalization. We also demonstrate the high specificity of our method when applied to larger genomes by profiling Drosophila melanogaster GAGA Factor and Pipsqueak. Our results suggest that ORGANIC profiling is a widely applicable high-resolution method for sensitive and specific profiling of direct protein-DNA interactions. PMID:24336359

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2015-02-24

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and ~90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. Finally, this combined approach offers a way to study the role of trace elements in their structural context.« less

Simultaneous cryo X-ray ptychographic and fluorescence microscopy of green algae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deng, Junjing; Vine, David J.; Chen, Si

Trace metals play important roles in normal and in disease-causing biological functions. X-ray fluorescence microscopy reveals trace elements with no dependence on binding affinities (unlike with visible light fluorophores) and with improved sensitivity relative to electron probes. However, X-ray fluorescence is not very sensitive for showing the light elements that comprise the majority of cellular material. Here we show that X-ray ptychography can be combined with fluorescence to image both cellular structure and trace element distribution in frozen-hydrated cells at cryogenic temperatures, with high structural and chemical fidelity. Ptychographic reconstruction algorithms deliver phase and absorption contrast images at a resolutionmore » beyond that of the illuminating lens or beam size. Using 5.2-keV X-rays, we have obtained sub-30-nm resolution structural images and similar to 90-nm-resolution fluorescence images of several elements in frozen-hydrated green algae. This combined approach offers a way to study the role of trace elements in their structural context.« less

Quantitative Detection of Trace Explosive Vapors by Programmed Temperature Desorption Gas Chromatography-Electron Capture Detector

PubMed Central

Field, Christopher R.; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C.; Rose-Pehrsson, Susan L.

2014-01-01

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples. PMID:25145416

Quantitative detection of trace explosive vapors by programmed temperature desorption gas chromatography-electron capture detector.

PubMed

Field, Christopher R; Lubrano, Adam; Woytowitz, Morgan; Giordano, Braden C; Rose-Pehrsson, Susan L

2014-07-25

The direct liquid deposition of solution standards onto sorbent-filled thermal desorption tubes is used for the quantitative analysis of trace explosive vapor samples. The direct liquid deposition method yields a higher fidelity between the analysis of vapor samples and the analysis of solution standards than using separate injection methods for vapors and solutions, i.e., samples collected on vapor collection tubes and standards prepared in solution vials. Additionally, the method can account for instrumentation losses, which makes it ideal for minimizing variability and quantitative trace chemical detection. Gas chromatography with an electron capture detector is an instrumentation configuration sensitive to nitro-energetics, such as TNT and RDX, due to their relatively high electron affinity. However, vapor quantitation of these compounds is difficult without viable vapor standards. Thus, we eliminate the requirement for vapor standards by combining the sensitivity of the instrumentation with a direct liquid deposition protocol to analyze trace explosive vapor samples.

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

PubMed

Lips, Katrin S; Pfeil, Uwe; Reiners, Katja; Rimasch, Christoph; Kuchelmeister, Klaus; Braun-Dullaeus, Ruediger C; Haberberger, Rainer V; Schmidt, Rupert; Kummer, Wolfgang

2003-12-01

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, A.N.; Benson, S.C.

1998-07-21

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a cationic chain. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye. 10 figs.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zukin, R.S.; Eghbali, M.; Olive, D.

{kappa} opioid receptors ({kappa} receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. {kappa} receptors were labeled by using the tritiated prototypic {kappa} opioid ethylketocyclazocine under conditions in which {mu} and {delta} opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity {kappa} opioid receptor sites was observed. In contrast, in the case of rat brain, two populations of {kappa} sites were observed. To test the hypothesis that the high- and low-affinity {kappa} sites represent two distinct {kappa} receptor subtypes, a series of opioids weremore » tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity {kappa} site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other {kappa} opioid drugs competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of {kappa} receptors in rat and guinea pig brain. The distribution patterns of the two {kappa} receptor subtypes of rat brain were clearly different. Collectively, these data provide direct evidence for the presence of two {kappa} receptor subtypes; the U-69,593-sensitive, high-affinity {kappa}{sub 1} site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity {kappa}{sub 2} site predominates in rat brain.« less

Basis for changes in the auxin-sensitivity of Avena sativa (oat) leaf-sheath pulvini during the gravitropic response

NASA Technical Reports Server (NTRS)

Kim, D.; Kaufman, P. B.

1995-01-01

During the gravitropic response, auxin-sensitivity of the lower flanks of leaf-sheath pulvini of Avena sativa (oat) is at least 1000-fold higher than those of the upper flanks and non-gravistimulated pulvini. When the pulvini are treated with 1 mM Ca2+, a 10-fold increase in auxin-sensitivity of the pulvini is observed. Related to this difference in auxin-sensitivity, in vitro activation of the vanadate-sensitive H(-)-ATPase by IAA was observed. Results show that the activation of the H(+)-ATPase by IAA is probably mediated by soluble protein factors and that the H(+)-ATPase prepared from the lower flanks is activated by IAA with a 1000-fold higher auxin-sensitivity as compared with that from the upper flanks of the graviresponding pulvini. Ammonium sulfate fractionation experiments show that these soluble protein factors are in the 30 to 60% fraction. Auxin-binding assays reveal that lower flanks contain more high-affinity soluble auxin-binding sites (kD; on the order of 10(-9) M) and less low-affinity soluble auxin-binding sites (kD; on the order of 10(-6) M) than upper flanks. It is concluded that differential auxin-sensitivity of graviresponding oat-shoot pulvini is achieved by the modulation of affinities of auxin-binding sites in upper and lower flanks of the pulvini, that Ca2+ is involved in such modulation, and that one of the probable cellular functions of these auxin binding sites is the activation of the proton pump on the plasma membranes.

Sensitivity and specificity: twin goals of proteomics assays. Can they be combined?

PubMed

Wilson, Robert

2013-04-01

A major ambition of proteomics is the provision of assays that can diagnose disease and monitor therapies. These assays are required to be sensitive and specific for individual proteins, and in most cases to quantify more than one protein in the same sample. The two main technologies currently used for proteomics assays are based on mass spectrometry and panels of affinity molecules such as antibodies. In the first part of this review the most sensitive existing assays based on these technologies are described and compared with the gold standard of ELISA. Analytical sensitivity is defined and related to the limit of detection, and analytical specificity is defined and shown to depend on molecular proofreading steps, similar to those applied in living systems whenever there is a need for high fidelity. It is shown that at present neither mass spectrometry nor panels of affinity molecules offer the necessary combination of sensitivity and specificity required for multiplexed assays. In the second part of this review the growing numbers of assays that use additional proofreading steps to combine sensitivity with specificity are described. These include assays based on proximity ligation and slow off-rate modified aptamers. Finally the review considers what improvements might be possible in the near future, and concludes that further development of proteomics assays incorporating advanced proofreading steps are most likely to provide the necessary combination of sensitivity and specificity, without incurring high development costs.

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

PubMed

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Evaluation of back scatter interferometry, a method for detecting protein binding in solution.

PubMed

Jepsen, S T; Jørgensen, T M; Zong, W; Trydal, T; Kristensen, S R; Sørensen, H S

2015-02-07

Back Scatter Interferometry (BSI) has been proposed to be a highly sensitive and versatile refractive index sensor usable for analytical detection of biomarker and protein interactions in solution. However the existing literature on BSI lacks a physical explanation of why protein interactions in general should contribute to the BSI signal. We have established a BSI system to investigate this subject in further detail. We contribute with a thorough analysis of the robustness of the sensor including unwanted contributions to the interferometric signal caused by temperature variation and dissolved gasses. We report a limit of the effective minimum detectability of refractive index at the 10(-7) level. Long term stability was examined by simultaneously monitoring the temperature inside the capillary revealing an average drift of 2.0 × 10(-7) per hour. Finally we show that measurements on protein A incubated with immunoglobulin G do not result in a signal that can be attributed to binding affinities as otherwise claimed in literature.

Mechanism of Dynamic Strain Aging in a Niobium-Stabilized Austenitic Stainless Steel

NASA Astrophysics Data System (ADS)

Zhou, Hongwei; Bai, Fengmei; Yang, Lei; Wei, Hailian; Chen, Yan; Peng, Guosheng; He, Yizhu

2018-04-01

Dynamic strain aging (DSA) behavior of a niobium (Nb)-stabilized austenitic stainless steel (TP347H) was studied from room temperature (RT) to 973 K via tensile testing, transmission electron microscopy (TEM), and internal friction (IF) measurements. The DSA effect is nearly negligible from 573 K to 673 K, and it becomes significant at temperatures between 773 K and 873 K with strain rates of 3 × 10-3 s-1, 8 × 10-4 s-1, and 8 × 10-5 s-1, respectively. The results indicate that a dislocation planar slip is dominant in the strong DSA regime. The Snoek-like peak located at 625 K is highly sensitive to the diffusion of free carbon (C) atoms in solid solution. C-Nb octahedrons are formed by C chemical affinity to substitutional Nb solute atoms. Octahedron structure is very stable and captures most free C atoms and inhibits DSA at low tensile test temperatures of 573 K to 673 K. At high test temperatures in the range from 773 K to 873 K, C-Nb octahedrons break up and release free C and Nb atoms, resulting in the stronger Snoek-like peak. The interaction between C atoms and dislocations is responsible for DSA at low temperatures ranging from 573 K to 673 K. At higher temperature of 773 K to 873 K, the Cr and Nb atoms lock the dislocations, and this formation contributes to DSA.

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

PubMed

Sueck, Franziska; Poór, Miklós; Faisal, Zelma; Gertzen, Christoph G W; Cramer, Benedikt; Lemli, Beáta; Kunsági-Máté, Sándor; Gohlke, Holger; Humpf, Hans-Ulrich

2018-06-22

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus . 2′ R -Ochratoxin A (2′ R -OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′ R -OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′ R -OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′ R -OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′ R -OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a log K of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′ R -OTA, a lower, but still high, log K of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′ R -OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′ R -OTA found in human blood samples.

Generation and characterization of a human-mouse chimeric high-affinity antibody that detects the DYKDDDDK FLAG peptide.

PubMed

Ikeda, Koki; Koga, Tomoaki; Sasaki, Fumiyuki; Ueno, Ayumi; Saeki, Kazuko; Okuno, Toshiaki; Yokomizo, Takehiko

2017-05-13

DYKDDDDK peptide (FLAG) is a useful tool for investigating the function and localization of proteins whose antibodies (Abs) are not available. We recently established a high-affinity monoclonal antibody (mAb) for FLAG (clone 2H8). The 2H8 Ab is highly sensitive for detecting FLAG-tagged proteins by flowcytometry and immunoprecipitation, but it can yield nonspecific signals in immunohistochemistry of mouse tissues because it is of mouse origin. In this study, we reduced nonspecific signals by generating a chimeric 2H8 Ab with Fc fragments derived from human immunoglobulin. We fused a 5' terminal cDNA fragments for the Fab region of 2H8 mAb with 3' terminal cDNA fragments for Fc region of human IgG1. We transfected both chimeric plasmids and purified the resulting human-mouse chimeric 2H8. The chimeric 2H8 Ab successfully detected FLAG-tagged proteins in flowcytometry with anti-human IgG secondary Ab with comparable sensitivity to 2H8 mAb. Importantly, chimeric 2H8 detected specific FLAG peptide signals without nonspecific signals in immunohistochemical analysis with mouse tissues. This human-mouse chimeric high-affinity anti-FLAG Ab will prove useful for future immunohistochemical analysis of mouse tissues. Copyright © 2017 Elsevier Inc. All rights reserved.

Reversibly extracellular pH controlled cellular uptake and photothermal therapy by PEGylated mixed-charge gold nanostars.

PubMed

Wang, Shouju; Teng, Zhaogang; Huang, Peng; Liu, Dingbin; Liu, Ying; Tian, Ying; Sun, Jing; Li, Yanjun; Ju, Huangxian; Chen, Xiaoyuan; Lu, Guangming

2015-04-17

Shielding nanoparticles from nonspecific interactions with normal cells/tissues before they reach and after they leave tumors is crucial for the selective delivery of NPs into tumor cells. By utilizing the reversible protonation of weak electrolytic groups to pH changes, long-chain amine/carboxyl-terminated polyethylene glycol (PEG) decorated gold nanostars (GNSs) are designed, exhibiting reversible, significant, and sensitive response in cell affinity and therapeutic efficacy to the extracellular pH (pHe) gradient between normal tissues and tumors. This smart nanosystem shows good dispersity and unimpaired photothermal efficacy in complex bioenvironment at pH 6.4 and 7.4 even when their surface charge is neutral. One PEGylated mixed-charge GNSs with certain surface composition, GNS-N/C 4, exhibits high cell affinity and therapeutic efficacy at pH 6.4, and low affinity and almost "zero" damage to cells at pH 7.4. Remarkably, this significant and sensitive response in cell affinity and therapeutic efficacy is reversible as local pH alternated. In vivo, GNS-N/C 4 shows higher accumulation in tumors and improved photothermal therapeutic efficacy than pH-insensitive GNSs. This newly developed smart nanosystem, whose cell affinity reversibly transforms in response to pHe gradient with unimpaired biostability, provides a novel effective means of tumor-selective therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

PubMed

Haraya, Shiomi; Karasawa, Koji; Sano, Yoshihiro; Ozawa, Kimiko; Kato, Nobumasa; Arakawa, Hidetoshi

2017-01-01

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

AFFINE-CORRECTED PARADISE: FREE-BREATHING PATIENT-ADAPTIVE CARDIAC MRI WITH SENSITIVITY ENCODING

PubMed Central

Sharif, Behzad; Bresler, Yoram

2013-01-01

We propose a real-time cardiac imaging method with parallel MRI that allows for free breathing during imaging and does not require cardiac or respiratory gating. The method is based on the recently proposed PARADISE (Patient-Adaptive Reconstruction and Acquisition Dynamic Imaging with Sensitivity Encoding) scheme. The new acquisition method adapts the PARADISE k-t space sampling pattern according to an affine model of the respiratory motion. The reconstruction scheme involves multi-channel time-sequential imaging with time-varying channels. All model parameters are adapted to the imaged patient as part of the experiment and drive both data acquisition and cine reconstruction. Simulated cardiac MRI experiments using the realistic NCAT phantom show high quality cine reconstructions and robustness to modeling inaccuracies. PMID:24390159

Interrelations of secondary structure stability and DNA-binding affinity in the bacteriophage SPO1-encoded type II DNA-binding protein TF1.

PubMed

Andera, L; Spangler, C J; Galeone, A; Mayol, L; Geiduschek, E P

1994-02-11

TF1, a homodimeric DNA-binding and -bending protein with a preference for hydroxymethyluracil-containing DNA is the Bacillus subtilis-encoded homolog of the bacterial HU proteins and of the E. coli integration host factor. A temperature-sensitive mutation at amino acid 25 of TF1 (L25-->A) and two intragenic second site revertants at amino acids 15 (E15-->G) and 32 (L32-->I) were previously identified and their effects on virus development were examined. The DNA-binding properties of these proteins and the thermal stability of their secondary structures have now been analyzed. Amino acids 15 and 32 are far removed from the putative DNA-binding domains of TF1 but changes there exert striking effects on DNA affinity that correlate with effects on structure. The double mutant protein TF1-G15I32 binds to a preferred site in hydroxymethyluracil-containing DNA 40 times more tightly, denatures at higher temperature (delta tm = 21 degrees C), and also exchanges subunits much more slowly than does the wild-type protein. The L25-->A mutation makes TF1 secondary structure and DNA-binding highly salt concentration-dependent. The E15-->G mutation partly suppresses this effect: secondary structure of TF1-A25G15 is restored at 21 degrees C by 1 M NaCl or, at low NaCl concentration, by binding to DNA.

Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors

PubMed Central

2018-01-01

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity. PMID:29768011

Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors.

PubMed

Gao, Zhaoli; Xia, Han; Zauberman, Jonathan; Tomaiuolo, Maurizio; Ping, Jinglei; Zhang, Qicheng; Ducos, Pedro; Ye, Huacheng; Wang, Sheng; Yang, Xinping; Lubna, Fahmida; Luo, Zhengtang; Ren, Li; Johnson, Alan T Charlie

2018-06-13

All-electronic DNA biosensors based on graphene field-effect transistors (GFETs) offer the prospect of simple and cost-effective diagnostics. For GFET sensors based on complementary probe DNA, the sensitivity is limited by the binding affinity of the target oligonucleotide, in the nM range for 20 mer targets. We report a ∼20 000× improvement in sensitivity through the use of engineered hairpin probe DNA that allows for target recycling and hybridization chain reaction. This enables detection of 21 mer target DNA at sub-fM concentration and provides superior specificity against single-base mismatched oligomers. The work is based on a scalable fabrication process for biosensor arrays that is suitable for multiplexed detection. This approach overcomes the binding-affinity-dependent sensitivity of nucleic acid biosensors and offers a pathway toward multiplexed and label-free nucleic acid testing with high accuracy and selectivity.

IFN-γ regulates CD8+ memory T cell differentiation and survival in response to weak, but not strong, TCR signals.

PubMed

Stoycheva, Diana; Deiser, Katrin; Stärck, Lilian; Nishanth, Gopala; Schlüter, Dirk; Uckert, Wolfgang; Schüler, Thomas

2015-01-15

In response to primary Ag contact, naive mouse CD8(+) T cells undergo clonal expansion and differentiate into effector T cells. After pathogen clearance, most effector T cells die, and only a small number of memory T cell precursors (TMPs) survive to form a pool of long-lived memory T cells (TMs). Although high- and low-affinity CD8(+) T cell clones are recruited into the primary response, the TM pool consists mainly of high-affinity clones. It remains unclear whether the more efficient expansion of high-affinity clones and/or cell-intrinsic processes exclude low-affinity T cells from the TM pool. In this article, we show that the lack of IFN-γR signaling in CD8(+) T cells promotes TM formation in response to weak, but not strong, TCR agonists. The IFN-γ-sensitive accumulation of TMs correlates with reduced mammalian target of rapamycin activation and the accumulation of long-lived CD62L(hi)Bcl-2(hi)Eomes(hi) TMPs. Reconstitution of mammalian target of rapamycin or IFN-γR signaling is sufficient to block this process. Hence, our data suggest that IFN-γR signaling actively blocks the formation of TMPs responding to weak TCR agonists, thereby promoting the accumulation of high-affinity T cells finally dominating the TM pool. Copyright © 2015 by The American Association of Immunologists, Inc.

Dyes designed for high sensitivity detection of double-stranded DNA

DOEpatents

Glazer, Alexander N.; Benson, Scott C.

1994-01-01

Novel fluorescent dyes are provided, characterized by having a fluorophore joined to a polycationic chain of at least two positive charges. The dyes are found to provide for high enhancement upon binding to nucleic acid and have strong binding affinities to the nucleic acid, as compared to the fluorophore without the polycationic chain. The dyes find use in detection of dsDNA in gel electrophoresis and solution at substantially higher sensitivities using substantially less dye.

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity.

PubMed

Sharma, P; Postel, S; Sundberg, E J; Kranz, D M

2013-12-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection.

Characterization of the Staphylococcal enterotoxin A: Vβ receptor interaction using human receptor fragments engineered for high affinity

PubMed Central

Sharma, P.; Postel, S.; Sundberg, E.J.; Kranz, D.M.

2013-01-01

Staphylococcal food poisoning is a gastrointestinal disorder caused by the consumption of food containing Staphylococcal enterotoxins. Staphylococcal enterotoxin A (SEA) is the most common enterotoxin recovered from food poisoning outbreaks in the USA. In addition to its enteric activity, SEA also acts as a potent superantigen through stimulation of T cells, although less is known about its interactions than the superantigens SEB, SEC and toxic shock syndrome toxin-1. To understand more about SEA:receptor interactions, and to develop toxin-detection systems for use in food testing, we engineered various SEA-binding receptor mutants. The extracellular domain of the receptor, a variable region of the beta chain (Vβ22) of the T-cell receptor, was engineered for stability as a soluble protein and for high affinity, using yeast-display technology. The highest affinity mutant was shown to bind SEA with a Kd value of 4 nM. This was a 25 000-fold improvement in affinity compared with the wild-type receptor, which bound to SEA with low affinity (Kd value of 100 µM), similar to other superantigen:Vβ interactions. The SEA:Vβ interface was centered around residues within the complementarity determining region 2 loop. The engineered receptor was specific for SEA, in that it did not bind to two other closely related enterotoxins SEE or SED, providing information on the SEA residues possibly involved in the interaction. The specificity and affinity of these high-affinity Vβ proteins also provide useful agents for the design of more sensitive and specific systems for SEA detection. PMID:24167300

An Escherichia coli Strain, PGB01, Isolated from Feral Pigeon Faeces, Thermally Fit to Survive in Pigeon, Shows High Level Resistance to Trimethoprim

PubMed Central

Kachhap, Sangita; Nanda, Ashis Kumar; Chakraborty, Ranadhir

2015-01-01

In this study, of the hundred Escherichia coli strains isolated from feral Pigeon faeces, eighty five strains were resistant to one or more antibiotics and fifteen sensitive to all the antibiotics tested. The only strain (among all antibiotic-resistant E. coli isolates) that possessed class 1 integron was PGB01. The dihydrofolate reductase gene of the said integron was cloned, sequenced and expressed in E. coli JM109. Since PGB01 was native to pigeon’s gut, we have compared the growth of PGB01 at two different temperatures, 42°C (normal body temperature of pigeon) and 37°C (optimal growth temperature of E. coli; also the human body temperature), with E. coli K12. It was found that PGB01 grew better than the laboratory strain E. coli K12 at 37°C as well as at 42°C. In the thermal fitness assay, it was observed that the cells of PGB01 were better adapted to 42°C, resembling the average body temperature of pigeon. The strain PGB01 also sustained more microwave mediated thermal stress than E. coli K12 cells. The NMR spectra of the whole cells of PGB01 varied from E. coli K12 in several spectral peaks relating some metabolic adaptation to thermotolerance. On elevating the growth temperature from 37°C to 42°C, susceptibility to kanamycin (both strains were sensitive to it) of E. coli K12 was increased, but in case of PGB01 no change in susceptibility took place. We have also attempted to reveal the basis of trimethoprim resistance phenotype conferred by the dfrA7 gene homologue of PGB01. Molecular Dynamics (MD) simulation study of docked complexes, PGB01-DfrA7 and E. coli TMP-sensitive-Dfr with trimethoprim (TMP) showed loss of some of the hydrogen and hydrophobic interaction between TMP and mutated residues in PGB01-DfrA7-TMP complex compared to TMP-sensitive-Dfr-TMP complex. This loss of interaction entails decrease in affinity of TMP for PGB01-DfrA7 compared to TMP-sensitive-Dfr. PMID:25750990

Pushing antibody-based labeling systems to higher sensitivity by linker-assisted affinity enhancement.

PubMed

Gorris, Hans H; Bade, Steffen; Röckendorf, Niels; Fránek, Milan; Frey, Andreas

2011-08-17

The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.

Analytical Glycobiology at High Sensitivity: Current Approaches and Directions

PubMed Central

Novotny, Milos V.; Alley, William R.; Mann, Benjamin F.

2013-01-01

This review summarizes the analytical advances made during the last several years in the structural and quantitative determinations of glycoproteins in complex biological mixtures. The main analytical techniques used in the fields of glycomics and glycoproteomics involve different modes of mass spectrometry and their combinations with capillary separation methods such as microcolumn liquid chromatography and capillary electrophoresis. The needs for high-sensitivity measurements have been emphasized in the oligosaccharide profiling used in the field of biomarker discovery through MALDI mass spectrometry. High-sensitivity profiling of both glycans and glycopeptides from biological fluids and tissue extracts has been aided significantly through lectin preconcentration and the uses of affinity chromatography. PMID:22945852

Glycan-functionalized graphene-FETs toward selective detection of human-infectious avian influenza virus

NASA Astrophysics Data System (ADS)

Ono, Takao; Oe, Takeshi; Kanai, Yasushi; Ikuta, Takashi; Ohno, Yasuhide; Maehashi, Kenzo; Inoue, Koichi; Watanabe, Yohei; Nakakita, Shin-ichi; Suzuki, Yasuo; Kawahara, Toshio; Matsumoto, Kazuhiko

2017-03-01

There are global concerns about threat of pandemic caused by the human-infectious avian influenza virus. To prevent the oncoming pandemic, it is crucial to analyze the viral affinity to human-type or avian-type sialoglycans with high sensitivity at high speed. Graphene-FET (G-FET) realizes such high-sensitive electrical detection of the targets, owing to graphene’s high carrier mobility. In the present study, G-FET was functionalized using sialoglycans and employed for the selective detection of lectins from Sambucus sieboldiana and Maackia amurensis as alternatives of the human and avian influenza viruses. Glycan-functionalized G-FET selectively monitored the sialoglycan-specific binding reactions at subnanomolar sensitivity.

Fullerol-fluorescein isothiocyanate-concanavalin agglutinin phosphorescent sensor for the detection of alpha-fetoprotein and forecast of human diseases

NASA Astrophysics Data System (ADS)

Liu, Jia-ming; Lin, Li-ping; Jiang, Shu-Lian; Cui, Ma Lin; Jiao, Li; Zhang, Xiao Yang; Zhang, Li-hong; Zheng, Zhi Yong; Lin, Xuan; Lin, Shao-qin

2013-11-01

Based on the reaction of the active -OH group in fullerol (F) with the dissociated -COOH group in fluorescein isothiocyanate (FITC) to form an F-FITC and the enhanced effect of N, N-dimethylaniline (DMA) on phosphorescence signal of F-FITC, a new phosphorescent labeling reagent (DMA-F-FITC) was developed. What's more, a phosphorescent sensor for the determination of alpha-fetoprotein variant (AFP-V) has been designed via the coupling technique of the high sensitivity for affinity adsorption-solid substrate-room temperature phosphorimetry (AA-SS-RTP) with the strong specificity reaction between DMA-F-FITC-Con A and AFP-V. The DMA-F-FITC increased the number of luminescent molecules in the biological target which improved the sensitivity of phosphorescent sensor. The proposed sensor was responsive, simple, selective and sensitive, and it has been applied to the determination of trace AFP-V in human serum and the forecast of human diseases using phosphorescence emission wavelength of F or FITC, with the results agreed well with those obtained by enzyme-linked immunoassay (ELISA). Meanwhile, the mechanisms for the labeling reaction and the sensing detection of AFP-V were discussed.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva.

PubMed

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui; Xiao, Yi

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples.

High refractive index and temperature sensitivity LPGs for high temperature operation

NASA Astrophysics Data System (ADS)

Nascimento, I. M.; Gouveia, C.; Jana, Surnimal; Bera, Susanta; Baptista, J. M.; Moreira, Paulo; Biwas, Palas; Bandyopadhyay, Somnath; Jorge, Pedro A. S.

2013-11-01

A fiber optic sensor for high sensitivity refractive index and temperature measurement able to withstand temperature up to 450 °C is reported. Two identical LPG gratings were fabricated, whereas one was coated with a high refractive index (~1.78) sol-gel thin film in order to increase its sensitivity to the external refractive index. The two sensors were characterized and compared in refractive index and temperature. Sensitivities of 1063 nm/RIU (1.338 - 1.348) and 260 pm/°C were achieved for refractive index and temperature, respectively.

Spontaneous temperature-sensitive Pluronic(®) based niosomes: Triggered drug release using mild hyperthermia.

PubMed

Tavano, Lorena; Oliviero Rossi, Cesare; Picci, Nevio; Muzzalupo, Rita

2016-09-25

Inclusion of lipids or polymers with a transition temperature closer to physiological body temperature (40-42°C) is a strategy used in tumor therapy for more than 30 years, because it allows induction of drug release from delivery systems by mild hyperthermia. Unfortunately, most of these thermo-sensitive carriers are removed from circulation before completion of their function. Thus, novel multi-functional niosomes possessing spontaneous stealth and thermo-sensitive properties were developed from L64 Pluronic(®) and L64ox as its derivative, in presence or absence of cholesterol. The use of L64 both as amphiphilic constituent and thermo-sensitive molecule, gave the possibility to bypass the use of additional excipients and increased the system biocompatibility. Niosomes diameter ranged from 400 to 750nm and were long term stable. Calcein and 5-FU possess great affinity to niosomal matrices rich in PEO groups. Negative Z-potential values were attributed to the negative charges onto the niosomes surface and generally change according to the temperature. The in vitro drugs release studies were performed at 25°C, 37°C and 42°C, that are representative of certain conditions (storage, physiological condition and mild hyperthermia, respectively). Results showed that L64-based niosomes possess spontaneous thermo-sensitive properties: drugs releases were found to be more pronounced at 42°C. These early results are a promising first step for the development of multi-functional devices that combine several advantages such as stealth properties and temperature controllability at the desired location and time, for a more specific and efficient pharmacological therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Facile Synthesis of Molecularly Imprinted Graphene Quantum Dots for the Determination of Dopamine with Affinity-Adjustable.

PubMed

Zhou, Xi; Wang, Anqi; Yu, Chenfei; Wu, Shishan; Shen, Jian

2015-06-10

A facilely prepared fluorescence sensor was developed for dopamine (DA) determination based on polyindole/graphene quantum dots molecularly imprinted polymers (PIn/GQDs@MIPs). The proposed sensor exhibits a high sensitivity with a linear range of 5 × 10(-10) to 1.2 × 10(-6) M and the limit of detection as low as 1 × 10(-10) M in the determination of DA, which is probably due to the tailor-made imprinted cavities for binding DA thought hydrogen bonds between amine groups of DA and oxygen-containing groups of the novel composite. Furthermore, the prepared sensor can rebind DA in dual-type: a low affinity type (noncovalent interaction is off) and a high affinity type (noncovalent interaction is on), and the rebinding interaction can be adjusted by tuning the pH, which shows a unique potential for adjusting the binding interaction while keeping the specificity, allowing for wider applications.

Neurochemical correlates of. gamma. -aminobutyrate (GABA) inhibition in cat visual cortex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balcar, V.J.; Dreher, B.

1990-01-01

High affinity binding of ({sup 3}H){gamma}-aminobutyric acid (GABA) to neuronal membranes from different parts of cat visual cortex was tested for sensitivity to GABA{sub A} agonists isoguvacine and THIP, GABA{sub A} antagonist SR95531 and GABA{sub B} agonist baclofen. Some of the GABA{sub A}-binding sites were found to have a very low affinity for THIP, suggesting the presence and, possibly, uneven distribution of non-synaptic GABA{sub A} receptors in cat visual cortex. There were no differences in K{sub m} and V{sub max} values of high affinity uptake of GABA and in the potency of K{sup +}-stimulated release of GABA, between primary andmore » association cortices. Consequently, the present results indicate that despite the anatomical and physiological differences between the primary and association feline visual cortices the neurochemical characteristics of GABAergic inhibition are very similar in the two regions.« less

Detachment of affinity-captured bioparticles by elastic deformation of a macroporous hydrogel

PubMed Central

Dainiak, Maria B.; Kumar, Ashok; Galaev, Igor Yu.; Mattiasson, Bo

2006-01-01

Adsorption of bioparticles to affinity surfaces involves polyvalent interactions, complicating greatly the recovery of the adsorbed material. A unique system for the efficient binding and release of different cells and particles is described. Affinity-bound bioparticles and synthetic particles are detached from the macroporous hydrogel matrix, a so-called cryogel, when the cryogel undergoes elastic deformation. The particle detachment upon elastic deformation is believed to be due to breaking of many of the multipoint attachments between the particles and the affinity matrix and the change in the distance between affinity ligands when the matrix is deformed. However, no release of affinity-bound protein occurred upon elastic deformation. The phenomenon of particle detachment upon elastic deformation is believed to be of a generic nature, because it was demonstrated for a variety of bioparticles of different sizes and for synthetic particles, for different ligand–receptor pairs (IgG–protein A, sugar–ConA, metal ion–chelating ligand), and when the deformation was caused by either external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive, macroporous hydrogel upon an increase in temperature). The elasticity of cryogel monoliths ensures high recovery of captured cells under mild conditions, with highly retained viability. This property, along with their continuous porous structure makes cryogel monoliths very attractive for applications in affinity cell separation. PMID:16418282

Aptamer-mediated 'turn-off/turn-on' nanozyme activity of gold nanoparticles for kanamycin detection.

PubMed

Sharma, Tarun Kumar; Ramanathan, Rajesh; Weerathunge, Pabudi; Mohammadtaheri, Mahsa; Daima, Hemant Kumar; Shukla, Ravi; Bansal, Vipul

2014-12-28

A new ultrafast and highly sensitive 'turn-off/turn-on' biosensing approach that combines the intrinsic peroxidase-like activity of gold nanoparticles (GNPs) with the high affinity and specificity of a ssDNA aptamer is presented for the efficient detection of a model small molecule kanamycin.

Alternating carrier models of asymmetric glucose transport violate the energy conservation laws.

PubMed

Naftalin, Richard J

2008-11-01

Alternating access transporters with high-affinity externally facing sites and low-affinity internal sites relate substrate transit directly to the unliganded asymmetric "carrier" (Ci) distribution. When both bathing solutions contain equimolar concentrations of ligand, zero net flow of the substrate-carrier complex requires a higher proportion of unliganded low-affinity inside sites (proportional, variant 1/KD(in)) and slower unliganded "free" carrier transit from inside to outside than in the reverse direction. However, asymmetric rates of unliganded carrier movement, kij, imply that an energy source, DeltaGcarrier = RT ln (koi/kio) = RT ln (Cin/Cout) = RT ln (KD(in)/KD(out)), where R is the universal gas constant (8.314 Joules/M/K degrees), and T is the temperature, assumed here to be 300 K degrees , sustains the asymmetry. Without this invalid assumption, the constraints of carrier path cyclicity, combined with asymmetric ligand affinities and equimolarity at equilibrium, are irreconcilable, and any passive asymmetric uniporter or cotransporter model system, e.g., Na-glucose cotransporters, espousing this fundamental error is untenable. With glucose transport via GLUT1, the higher maximal rate and Km of net ligand exit compared to net ligand entry is only properly simulated if ligand transit occurs by serial dissociation-association reactions between external high-affinity and internal low-affinity immobile sites. Faster intersite transit rates occur from lower-affinity sites than from higher-affinity sites and require no other energy source to maintain equilibrium. Similar constraints must apply to cotransport.

Mass-transport limitations in spot-based microarrays.

PubMed

Zhao, Ming; Wang, Xuefeng; Nolte, David

2010-09-20

Mass transport of analyte to surface-immobilized affinity reagents is the fundamental bottleneck for sensitive detection in solid-support microarrays and biosensors. Analyte depletion in the volume adjacent to the sensor causes deviation from ideal association, significantly slows down reaction kinetics, and causes inhomogeneous binding across the sensor surface. In this paper we use high-resolution molecular interferometric imaging (MI2), a label-free optical interferometry technique for direct detection of molecular films, to study the inhomogeneous distribution of intra-spot binding across 100 micron-diameter protein spots. By measuring intra-spot binding inhomogeneity, reaction kinetics can be determined accurately when combined with a numerical three-dimensional finite element model. To ensure homogeneous binding across a spot, a critical flow rate is identified in terms of the association rate k(a) and the spot diameter. The binding inhomogeneity across a spot can be used to distinguish high-affinity low-concentration specific reactions from low-affinity high-concentration non-specific binding of background proteins.

Simple and Efficient Purification of Recombinant Proteins Using the Heparin-Binding Affinity Tag.

PubMed

Jayanthi, Srinivas; Gundampati, Ravi Kumar; Kumar, Thallapuranam Krishnaswamy Suresh

2017-11-01

Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest. HBP-tagged fusion proteins can be purified by heparin Sepharose affinity chromatography using a simple sodium chloride gradient to elute the bound fusion protein. In addition, owing to the high density of positive charges on the HBP tag, recombinant target proteins are preferably expressed in their soluble forms. The purification of HBP-fusion proteins can also be achieved in the presence of chemical denaturants, including urea. Additionally, polyclonal antibodies raised against the affinity tag can be used to detect HBP-fused target proteins with high sensitivity. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

A multiplex microplatform for the detection of multiple DNA methylation events using gold-DNA affinity.

PubMed

Sina, Abu Ali Ibn; Foster, Matthew Thomas; Korbie, Darren; Carrascosa, Laura G; Shiddiky, Muhammad J A; Gao, Jing; Dey, Shuvashis; Trau, Matt

2017-10-07

We report a new multiplexed strategy for the electrochemical detection of regional DNA methylation across multiple regions. Using the sequence dependent affinity of bisulfite treated DNA towards gold surfaces, the method integrates the high sensitivity of a micro-fabricated multiplex device comprising a microarray of gold electrodes, with the powerful multiplexing capability of multiplex-PCR. The synergy of this combination enables the monitoring of the methylation changes across several genomic regions simultaneously from as low as 500 pg μl -1 of DNA with no sequencing requirement.

Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

PubMed

Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

2014-11-18

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

PubMed

Kane, M A; Portanova, L B; Kelley, K; Holley, M; Ross, S E; Boose, D; Escobedo-Morse, A; Alvarado, B

1994-01-01

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.

Insight into the Meligethes aeneus voltage-sensitive sodium channel structure and an attempt to select the best pyrethroid ligands.

PubMed

Obrępalska-Stęplowska, Aleksandra; Czerwoniec, Anna; Wieczorek, Przemysław; Wrzesińska, Barbara

2016-01-01

The voltage-sensitive sodium channel (VSSC) is a target for the pharmacological action of pyrethroids which are used in controlling pests, including those of agricultural importance. Among these is the pollen beetle (Meligethes aeneus F.) - the most serious pest of Brassica napus. Owing to the heavy use of pyrethroids, a widespread build-up of resistance has occurred. The main cause of pyrethroid insensitivity in M. aeneus is considered to be an increased oxidative metabolism; however, the additional mechanism of resistance associated with mutations in the VSSC might contribute to this phenomenon. We generated a VSSC 3D model to study the docking affinities of pyrethroids to their target site within the channel. Our goal was to identify the pyrethroids for which docking affinity scores were high and not affected by potential mutations in the VSSC. We found that the docking scores of cypermethrin are hardly influenced by the appearance of point mutations. Additionally, tau-fluvalinate, deltamethrin and bifenthrin are VSSC ligands with high affinity scores. Our docking models suggest that point mutations in the VSSC binding pocket might affect the stability of ligand interactions and change the pattern of ligand docking locations, which might have a potential effect on VSSC gating properties. © 2015 Society of Chemical Industry.

Evaluation of an affinity-amplified immunoassay of graphene oxide using surface plasmon resonance biosensors

NASA Astrophysics Data System (ADS)

Chiu, Nan-Fu; Huang, Teng-Yi; Kuo, Chun-Chuan

2015-05-01

We describe a fundamental study on the plasmonic properties and advanced biosensing mechanisms of functionalized graphene. We discuss a specific design using modified carboxyl groups, which can modulate surface plasmon (SP) coupling and provide an advantage for their binding to the sensing layer with high-performance affinity in an immunological reaction. The functionalized graphene-based surface plasmon resonance (SPR) biosensors have three advantages: high performance, high sensitivity, and excellent molecular kinetic response. In the future, functionalized graphene sheets will make a unique contribution to photonic and SPR diagnosis devices. We wish to highlight the essential characteristics of functionalized graphene-based SPR biosensors to assist researchers in developing and advancing suitable biosensors for unique applications.

Distinct functional characteristics of levocabastine sensitive rat neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

PubMed

Yamada, M; Yamada, M; Lombet, A; Forgez, P; Rostène, W

1998-01-01

Neurotensin has been shown to produce pharmacological effects both in brain and periphery. Several of these effects are mediated by a high-affinity neurotensin NT1 receptor. On the other hand, a low-affinity levocabastine-sensitive neurotensin NT2 receptor was molecularly cloned from rodent brain recently. In this study, in contrast to NT1 receptor, levocabastine (a histamine H1 receptor antagonist) and SR48692 (an antagonist for NT1 receptor) strongly stimulated intracellular Ca2+ mobilization in transfected Chinese hamster ovary cells expressing rat NT2 receptor, thus acting as potent NT2 receptor. Furthermore, despite of their affinities for NT2 receptor, the Ca2+ responses to potent NT1 agonists, neurotensin or JMV449 ([Lys8-(CH2NH)-Lys9]Pro-Tyr-Ile-Leu, a peptidase resistant analogue of neurotensin) were much smaller than that observed with SR48692. These findings suggest that NT1 and NT2 receptors present distinct functional characteristics and that SR48692 may act as a potent agonist for NT2 receptor.

Sensitivity enhanced strain and temperature measurements based on FBG and frequency chirp magnification.

PubMed

Du, Jiangbing; He, Zuyuan

2013-11-04

In this work, highly sensitive measurements of strain and temperature have been demonstrated using a fiber Bragg grating (FBG) sensor with significantly enhance sensitivity by all-optical signal processing. The sensitivity enhancement is achieved by degenerated Four Wave Mixing (FWM) for frequency chirp magnification (FCM), which can be used for magnifying the wavelength drift of the FBG sensor induced by strain and temperature change. Highly sensitive measurements of static strain and temperature have been experimentally demonstrated with strain sensitivity of 5.36 pm/με and temperature sensitivity of 54.09 pm/°C. The sensitivity has been enhanced by a factor of five based on a 4-order FWM in a highly nonlinear fiber (HNLF).

Development of a bone-targeted pH-sensitive liposomal formulation containing doxorubicin: physicochemical characterization, cytotoxicity, and biodistribution evaluation in a mouse model of bone metastasis.

PubMed

Ferreira, Diêgo Dos Santos; Faria, Samilla Dornelas; Lopes, Sávia Caldeira de Araújo; Teixeira, Cláudia Salviano; Malachias, Angelo; Magalhães-Paniago, Rogério; de Souza Filho, José Dias; Oliveira, Bruno Luis de Jesus Pinto; Guimarães, Alexander Ramos; Caravan, Peter; Ferreira, Lucas Antônio Miranda; Alves, Ricardo José; Oliveira, Mônica Cristina

2016-01-01

Despite recent advances in cancer therapy, the treatment of bone tumors remains a major challenge. A possible underlying hypothesis, limitation, and unmet need may be the inability of therapeutics to penetrate into dense bone mineral, which can lead to poor efficacy and high toxicity, due to drug uptake in healthy organs. The development of nanostructured formulations with high affinity for bone could be an interesting approach to overcome these challenges. To develop a liposomal formulation with high affinity for hydroxyapatite and the ability to release doxorubicin (DOX) in an acidic environment for future application as a tool for treatment of bone metastases. Liposomes were prepared by thin-film lipid hydration, followed by extrusion and the sulfate gradient-encapsulation method. Liposomes were characterized by average diameter, ζ-potential, encapsulation percentage, X-ray diffraction, and differential scanning calorimetry. Release studies in buffer (pH 7.4 or 5), plasma, and serum, as well as hydroxyapatite-affinity in vitro analysis were performed. Cytotoxicity was evaluated by MTT assay against the MDA-MB-231 cell line, and biodistribution was assessed in bone metastasis-bearing animals. Liposomes presented suitable diameter (~170 nm), DOX encapsulation (~2 mg/mL), controlled release, and good plasma and serum stability. The existence of interactions between DOX and the lipid bilayer was proved through differential scanning calorimetry and small-angle X-ray scattering. DOX release was faster when the pH was in the range of a tumor than at physiological pH. The bone-targeted formulation showed a strong affinity for hydroxyapatite. The encapsulation of DOX did not interfere in its intrinsic cytotoxicity against the MDA-MB-231 cell line. Biodistribution studies demonstrated high affinity of this formulation for tumors and reduction of uptake in the heart. These results suggest that bone-targeted pH-sensitive liposomes containing DOX can be an interesting strategy for selectively delivering this drug into bone-tumor sites, increasing its activity, and reducing DOX-related toxicity.

A high-sensitivity temperature sensor based on Sagnac interferometer employing photonic crystal fiber fully filled with ethanol

NASA Astrophysics Data System (ADS)

Shi, Min; Li, Shuguang; Chen, Hailiang

2018-06-01

A high-sensitivity temperature sensor based on photonic crystal fiber Sagnac interferometer is proposed and studied. All holes of the PCF are filled with ethanol with capillarity. The cladding air holes are uniform arrangements. The two air holes around the core are removed to form new core modes with high birefringence. The sensitivities of the temperature can be up to -8.7657 and 16.8142 nm/°C when temperature rises from 45 to 75 °C and the fiber length is 5.05 cm. And when temperature rises from 10 to 45 °C, the sensitivity can reach -7.848 and 16.655 nm/°C with fiber length 2.11 cm. The performance of the selective-filled and the fully-filled PCF with temperature from 45 to 75 °C and fiber length 5.05 cm are analyzed and compared. The fully filling can better achieve PCF's sensing performance. The simple structure and high sensitivities make the temperature sensor easy to achieve. The temperature sensor with high sensitivities and good linearity has great application value for environmental temperature detecting.

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

PubMed Central

Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

2016-01-01

Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

Evaluation of Disulfide Bond Position to Enhance the Thermal Stability of a Highly Stable Single Domain Antibody

PubMed Central

Zabetakis, Dan; Olson, Mark A.; Anderson, George P.; Legler, Patricia M.; Goldman, Ellen R.

2014-01-01

Single domain antibodies are the small recombinant variable domains derived from camelid heavy-chain-only antibodies. They are renowned for their stability, in large part due to their ability to refold following thermal or chemical denaturation. In addition to refolding after heat denaturation, A3, a high affinity anti-Staphylococcal Enterotoxin B single domain antibody, possesses a melting temperature of ∼84°C, among the highest reported for a single domain antibody. In this work we utilized the recently described crystal structure of A3 to select locations for the insertion of a second disulfide bond and evaluated the impact that the addition of this second bond had on the melting temperature. Four double-disulfide versions of A3 were constructed and each was found to improve the melting temperature relative to the native structure without reducing affinity. Placement of the disulfide bond at a previously published position between framework regions 2 and 3 yielded the largest improvement (>6°C), suggesting this location is optimal, and seemingly provides a universal route to raise the melting temperature of single domain antibodies. This study further demonstrates that even single domain antibodies with extremely high melting points can be further stabilized by addition of disulfide bonds. PMID:25526640

Towards vaporized molecular discrimination: a quartz crystal microbalance (QCM) sensor system using cobalt-containing mesoporous graphitic carbon.

PubMed

Tang, Jing; Torad, Nagy L; Salunkhe, Rahul R; Yoon, Jang-Hee; Al Hossain, Md Shahriar; Dou, Shi Xue; Kim, Jung Ho; Kimura, Tatsuo; Yamauchi, Yusuke

2014-11-01

A recent study on nanoporous carbon based materials (J. Am. Chem. Soc. 2012, 134, 2864) showed that the presence of abundant graphitized sp(2) carbon species in the frameworks led to higher affinity for aromatic hydrocarbons than their aliphatic analogues. Herein, improved understanding of the sensitive and selective detection of aromatic substances by using mesoporous carbon (MPC)-based materials, combined with a quartz crystal microbalance (QCM) sensor system, was obtained. MPCs were synthesized by direct carbonization of mesoporous polymers prepared from resol through a soft templating approach with Pluronic F127. The carbon-based frameworks can be graphitized through the addition of a cobalt source to the precursor solution, according to the catalytic activity of the cobalt nanoparticles formed during the carbonization process. From the Raman data, the degree of the graphitization was clearly increased by increasing the cobalt content and elevating the carbonization temperature. From a QCM study, it was proved that the highly graphitized MPCs exhibited a higher affinity for aromatic hydrocarbons than their aliphatic analogues. By increasing the degree of graphitization in the carbon-based pore walls, the MPCs showed both larger adsorption uptake and faster sensor response towards toxic benzene and toluene vapors. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Recent Advances in Bacteria Identification by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using Nanomaterials as Affinity Probes

PubMed Central

Chiu, Tai-Chia

2014-01-01

Identifying trace amounts of bacteria rapidly, accurately, selectively, and with high sensitivity is important to ensuring the safety of food and diagnosing infectious bacterial diseases. Microbial diseases constitute the major cause of death in many developing and developed countries of the world. The early detection of pathogenic bacteria is crucial in preventing, treating, and containing the spread of infections, and there is an urgent requirement for sensitive, specific, and accurate diagnostic tests. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an extremely selective and sensitive analytical tool that can be used to characterize different species of pathogenic bacteria. Various functionalized or unmodified nanomaterials can be used as affinity probes to capture and concentrate microorganisms. Recent developments in bacterial detection using nanomaterials-assisted MALDI-MS approaches are highlighted in this article. A comprehensive table listing MALDI-MS approaches for identifying pathogenic bacteria, categorized by the nanomaterials used, is provided. PMID:24786089

Recent advances in bacteria identification by matrix-assisted laser desorption/ionization mass spectrometry using nanomaterials as affinity probes.

PubMed

Chiu, Tai-Chia

2014-04-28

Identifying trace amounts of bacteria rapidly, accurately, selectively, and with high sensitivity is important to ensuring the safety of food and diagnosing infectious bacterial diseases. Microbial diseases constitute the major cause of death in many developing and developed countries of the world. The early detection of pathogenic bacteria is crucial in preventing, treating, and containing the spread of infections, and there is an urgent requirement for sensitive, specific, and accurate diagnostic tests. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an extremely selective and sensitive analytical tool that can be used to characterize different species of pathogenic bacteria. Various functionalized or unmodified nanomaterials can be used as affinity probes to capture and concentrate microorganisms. Recent developments in bacterial detection using nanomaterials-assisted MALDI-MS approaches are highlighted in this article. A comprehensive table listing MALDI-MS approaches for identifying pathogenic bacteria, categorized by the nanomaterials used, is provided.

A new real-time method for investigation of affinity properties and binding kinetics of magnetic nanoparticles

NASA Astrophysics Data System (ADS)

Orlov, Alexey V.; Nikitin, Maxim P.; Bragina, Vera A.; Znoyko, Sergey L.; Zaikina, Marina N.; Ksenevich, Tatiana I.; Gorshkov, Boris G.; Nikitin, Petr I.

2015-04-01

A method for quantitative investigation of affinity constants of receptors immobilized on magnetic nanoparticles (MP) is developed based on spectral correlation interferometry (SCI). The SCI records with a picometer resolution the thickness changes of a layer of molecules or nanoparticles due to a biochemical reaction on a cover slip, averaged over the sensing area. The method is compatible with other types of sensing surfaces employed in biosensing. The measured values of kinetic association constants of magnetic nanoparticles are 4 orders of magnitude higher than those of molecular antibody association with antigen. The developed method also suggests highly sensitive detection of antigens in a wide dynamic range. The limit of detection of 92 pg/ml has been demonstrated for prostate-specific antigen (PSA) with 50-nm MP employed as labels, which produce 3-order amplification of the SCI signals. The calibration curve features high sensitivity (slope) of 3-fold signal raise per 10-fold increase of PSA concentration within 4-order dynamic range, which is an attractive compromise for precise quantitative and highly sensitive immunoassay. The proposed biosensing technique offers inexpensive disposable sensor chips of cover slips and represents an economically sound alternative to traditional immunoassays for disease diagnostics, detection of pathogens in food and environmental monitoring.

Temperatures and cooling rates recorded in REE in coexisting pyroxenes in ophiolitic and abyssal peridotites

NASA Astrophysics Data System (ADS)

Dygert, Nick; Liang, Yan

2015-06-01

Mantle peridotites from ophiolites are commonly interpreted as having mid-ocean ridge (MOR) or supra-subduction zone (SSZ) affinity. Recently, an REE-in-two-pyroxene thermometer was developed (Liang et al., 2013) that has higher closure temperatures (designated as TREE) than major element based two-pyroxene thermometers for mafic and ultramafic rocks that experienced cooling. The REE-in-two-pyroxene thermometer has the potential to extract meaningful cooling rates from ophiolitic peridotites and thus shed new light on the thermal history of the different tectonic regimes. We calculated TREE for available literature data from abyssal peridotites, subcontinental (SC) peridotites, and ophiolites around the world (Alps, Coast Range, Corsica, New Caledonia, Oman, Othris, Puerto Rico, Russia, and Turkey), and augmented the data with new measurements for peridotites from the Trinity and Josephine ophiolites and the Mariana trench. TREE are compared to major element based thermometers, including the two-pyroxene thermometer of Brey and Köhler (1990) (TBKN). Samples with SC affinity have TREE and TBKN in good agreement. Samples with MOR and SSZ affinity have near-solidus TREE but TBKN hundreds of degrees lower. Closure temperatures for REE and Fe-Mg in pyroxenes were calculated to compare cooling rates among abyssal peridotites, MOR ophiolites, and SSZ ophiolites. Abyssal peridotites appear to cool more rapidly than peridotites from most ophiolites. On average, SSZ ophiolites have lower closure temperatures than abyssal peridotites and many ophiolites with MOR affinity. We propose that these lower temperatures can be attributed to the residence time in the cooling oceanic lithosphere prior to obduction. MOR ophiolites define a continuum spanning cooling rates from SSZ ophiolites to abyssal peridotites. Consistent high closure temperatures for abyssal peridotites and the Oman and Corsica ophiolites suggests hydrothermal circulation and/or rapid cooling events (e.g., normal faulting, unroofing) control the late thermal histories of peridotites from transform faults and slow and fast spreading centers with or without a crustal section.

Highly sensitive long-period fiber-grating strain sensor with low temperature sensitivity

NASA Astrophysics Data System (ADS)

Wang, Yi-Ping; Xiao, Limin; Wang, D. N.; Jin, Wei

2006-12-01

A long-period fiber-grating sensor with a high strain sensitivity of -7.6 pm/μɛ and a low temperature sensitivity of 3.91 pm/°C is fabricated by use of focused CO2 laser beam to carve periodic grooves on a large- mode-area photonic crystal fiber. Such a strain sensor can effectively reduce the cross-sensitivity between strain and temperature, and the temperature-induced strain error obtained is only 0.5 μɛ/°C without using temperature compensation.

PolyCheck: Dynamic Verification of Iteration Space Transformations on Affine Programs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bao, Wenlei; Krishnamoorthy, Sriram; Pouchet, Louis-noel

2016-01-11

High-level compiler transformations, especially loop transformations, are widely recognized as critical optimizations to restructure programs to improve data locality and expose parallelism. Guaranteeing the correctness of program transformations is essential, and to date three main approaches have been developed: proof of equivalence of affine programs, matching the execution traces of programs, and checking bit-by-bit equivalence of the outputs of the programs. Each technique suffers from limitations in either the kind of transformations supported, space complexity, or the sensitivity to the testing dataset. In this paper, we take a novel approach addressing all three limitations to provide an automatic bug checkermore » to verify any iteration reordering transformations on affine programs, including non-affine transformations, with space consumption proportional to the original program data, and robust to arbitrary datasets of a given size. We achieve this by exploiting the structure of affine program control- and data-flow to generate at compile-time lightweight checker code to be executed within the transformed program. Experimental results assess the correctness and effectiveness of our method, and its increased coverage over previous approaches.« less

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.

Comprehensive analysis of an Antarctic bacterial community with the adaptability of growth at higher temperatures than those in Antarctica.

PubMed

Hosoi-Tanabe, Shoko; Zhang, Hongyan; Zhu, Daochen; Nagata, Shinichi; Ban, Syuhei; Imura, Satoshi

2010-06-01

To investigate the adaptability to higher temperatures of Antarctic microorganisms persisting in low temperature conditions for a long time, Antarctic lake samples were incubated in several selection media at 25 degrees C and 30 degrees C. The microorganisms did not grow at 30 degrees C; however, some of them grew at 25 degrees C, indicating that the bacteria in Antarctic have the ability to grow at a wide range of temperatures. Total DNA was extracted from these microorganisms and amplified using the bacteria-universal primers. The amplified fragments were cloned, and randomly selected 48 clones were sequenced. The sequenced clones showed high similarity to the alpha-subdivision of the Proteobacteria with specific affinity to the genus Agrobacterium, Caulobacter and Brevundimonas, the ss-subdivision of Proteobacteria with specific affinity to the genus Cupriavidus, and Bacillus of the phylum Firmicutes. These results showed the presence of universal genera, suggesting that the bacteria in the Antarctic lake were not specific to this environment.

Single particle electrochemical sensors and methods of utilization

DOEpatents

Schoeniger, Joseph [Oakland, CA; Flounders, Albert W [Berkeley, CA; Hughes, Robert C [Albuquerque, NM; Ricco, Antonio J [Los Gatos, CA; Wally, Karl [Lafayette, CA; Kravitz, Stanley H [Placitas, NM; Janek, Richard P [Oakland, CA

2006-04-04

The present invention discloses an electrochemical device for detecting single particles, and methods for using such a device to achieve high sensitivity for detecting particles such as bacteria, viruses, aggregates, immuno-complexes, molecules, or ionic species. The device provides for affinity-based electrochemical detection of particles with single-particle sensitivity. The disclosed device and methods are based on microelectrodes with surface-attached, affinity ligands (e.g., antibodies, combinatorial peptides, glycolipids) that bind selectively to some target particle species. The electrodes electrolyze chemical species present in the particle-containing solution, and particle interaction with a sensor element modulates its electrolytic activity. The devices may be used individually, employed as sensors, used in arrays for a single specific type of particle or for a range of particle types, or configured into arrays of sensors having both these attributes.

Detection of Volatile Organic Compounds by Self-assembled Monolayer Coated Sensor Array with Concentration-independent Fingerprints

PubMed Central

Chang, Ye; Tang, Ning; Qu, Hemi; Liu, Jing; Zhang, Daihua; Zhang, Hao; Pang, Wei; Duan, Xuexin

2016-01-01

In this paper, we have modeled and analyzed affinities and kinetics of volatile organic compounds (VOCs) adsorption (and desorption) on various surface chemical groups using multiple self-assembled monolayers (SAMs) functionalized film bulk acoustic resonator (FBAR) array. The high-frequency and micro-scale resonator provides improved sensitivity in the detections of VOCs at trace levels. With the study of affinities and kinetics, three concentration-independent intrinsic parameters (monolayer adsorption capacity, adsorption energy constant and desorption rate) of gas-surface interactions are obtained to contribute to a multi-parameter fingerprint library of VOC analytes. Effects of functional group’s properties on gas-surface interactions are also discussed. The proposed sensor array with concentration-independent fingerprint library shows potential as a portable electronic nose (e-nose) system for VOCs discrimination and gas-sensitive materials selections. PMID:27045012

Functional identification of activity-regulated, high-affinity glutamine transport in hippocampal neurons inhibited by riluzole.

PubMed

Erickson, Jeffrey D

2017-07-01

Glutamine (Gln) is considered the preferred precursor for the neurotransmitter pool of glutamate (Glu), the major excitatory transmitter in the mammalian CNS. Here, an activity-regulated, high-affinity Gln transport system is described in developing and mature neuron-enriched hippocampal cultures that is potently inhibited by riluzole (IC 50 1.3 ± 0.5 μM), an anti-glutamatergic drug, and is blocked by low concentrations of 2-(methylamino)isobutyrate (MeAIB), a system A transport inhibitor. K + -stimulated MeAIB transport displays an affinity (K m ) for MeAIB of 37 ± 1.2 μM, saturates at ~ 200 μM, is dependent on extracellular Ca 2+ , and is blocked by inhibition of voltage-gated Ca 2+ channels. Spontaneous MeAIB transport is also dependent on extracellullar Ca 2+ and voltage-gated calcium channels, but is also blocked by the Na + channel blocker tetrodotoxin, by Glu receptor antagonists, and by GABA indicating its dependence on intact neural circuits driven by endogenous glutamatergic activity. The transport of MeAIB itself does not rely on Ca 2+ , but on Na + ions, and is pH sensitive. Activity-regulated, riluzole-sensitive spontaneous and K + -stimulated transport is minimal at 7-8 days in vitro, coordinately induced during the next 2 weeks and is maximally expressed by days in vitro > 20; the known period for maturation of the Glu/Gln cycle and regulated pre-synaptic Glu release. Competition analyses with various amino acids indicate that Gln is the most likely physiological substrate. Activity-regulated Gln/MeAIB transport is not observed in astrocytes. The functional identification of activity-regulated, high-affinity, riluzole-sensitive Gln/MeAIB transport in hippocampal neurons may have important ramifications in the neurobiology of activity-stimulated pre-synaptic Glu release, the Glu/Gln cycle between astrocytes and neurons, and neuronal Glu-induced excitotoxicity. Cover Image for this issue: doi: 10.1111/jnc.13805. © 2017 International Society for Neurochemistry.

A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1

NASA Astrophysics Data System (ADS)

Yang, Shilong; Yang, Fan; Wei, Ningning; Hong, Jing; Li, Bowen; Luo, Lei; Rong, Mingqiang; Yarov-Yarovoy, Vladimir; Zheng, Jie; Wang, Kewei; Lai, Ren

2015-09-01

The capsaicin receptor TRPV1 ion channel is a polymodal nociceptor that responds to heat with exquisite sensitivity through an unknown mechanism. Here we report the identification of a novel toxin, RhTx, from the venom of the Chinese red-headed centipede that potently activates TRPV1 to produce excruciating pain. RhTx is a 27-amino-acid small peptide that forms a compact polarized molecule with very rapid binding kinetics and high affinity for TRPV1. We show that RhTx targets the channel's heat activation machinery to cause powerful heat activation at body temperature. The RhTx-TRPV1 interaction is mediated by the toxin's highly charged C terminus, which associates tightly to the charge-rich outer pore region of the channel where it can directly interact with the pore helix and turret. These findings demonstrate that RhTx binding to the outer pore can induce TRPV1 heat activation, therefore providing crucial new structural information on the heat activation machinery.

Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides

NASA Astrophysics Data System (ADS)

Chen, Yajing; Xiong, Zhichao; Zhang, Lingyi; Zhao, Jiaying; Zhang, Quanqing; Peng, Li; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

2015-02-01

Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g-1), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g-1), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05955g

A Protein Microarray ELISA for the Detection of Botulinum neurotoxin A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varnum, Susan M.

An enzyme-linked immunosorbent assay (ELISA) microarray was developed for the specific and sensitive detection of botulinum neurotoxin A (BoNT/A), using high-affinity recombinant monoclonal antibodies against the receptor binding domain of the heavy chain of BoNT/A. The ELISA microarray assay, because of its sensitivity, offers a screening test with detection limits comparable to the mouse bioassay, with results available in hours instead of days.

Affinity interactions between natural pigments and human whole saliva.

PubMed

Yao, Jiang-Wu; Lin, Feng; Tao, Tao; Lin, Chang-Jian

2011-03-01

The aim of the present study was to assess the null hypothesis that there are no differences of affinity between pigments and human whole saliva (WS), and the affinity is not influenced by the functional groups of pigments, temperatures, pH values, and salt concentrations. The affinity constants of interactions between WS and theaflavin (TF)/curcumin (Cur)/cyanidin (Cy) were determined by surface plasmon resonance (SPR) and fluorescence quenching. Mass-uptake at various temperatures, pH values, and salt concentrations was also carried out. The order of affinity of the pigments binding to WS is TF>Cur>Cy. A large number of complexes and precipitations of pigments/proteins were formed through a quick, strong, and almost irreversible binding process. The mass-uptake of pigments was affected not only by the functional groups, but also by molecular weight of pigments, temperatures, pH values, and salt concentrations. The complex of pigments may easily and rapidly deposit onto the WS film, and are difficult to remove from the WS surface. However, the complex of pigments can be reduced by properly regulating the physicochemical conditions, such as temperatures, pH values, and salt concentrations. Copyright © 2010 Elsevier Ltd. All rights reserved.

Current advances in screening for bioactive components from medicinal plants by affinity ultrafiltration mass spectrometry.

PubMed

Chen, Guilin; Huang, Bill X; Guo, Mingquan

2018-05-21

Medicinal plants have played an important role in maintaining human health for thousands of years. However, the interactions between the active components in medicinal plants and some certain biological targets during a disease are still unclear in most cases. To conduct the high-throughput screening for small active molecules that can interact with biological targets, which is of great theoretical significance and practical value. The ultrafiltration mass spectrometry (UF-LC/MS) is a powerful bio-analytical method by combining affinity ultrafiltration and liquid chromatography-mass spectrometry (LC/MS), which could rapidly screen and identify small active molecules that bind to biological targets of interest at the same time. Compared with other analytical methods, affinity UF-LC/MS has the characteristics of fast, sensitive and high throughput, and is especially suitable for the complicated extracts of medicinal plants. In this review, the basic principle, characteristics and some most recent challenges in UF-LC/MS have been demonstrated. Meanwhile, the progress and applications of affinity UF-LC/MS in the discovery of the active components from natural medicinal plants and the interactions between small molecules and biological target proteins are also briefly summarised. In addition, the future directions for UF-LC/MS are also prospected. Affinity UF-LC/MS is a powerful tool in studies on the interactions between small active molecules and biological protein targets, especially in the high-throughput screening of active components from the natural medicinal plants. Copyright © 2018 John Wiley & Sons, Ltd.

Dynein and dynactin leverage their bivalent character to form a high-affinity interaction.

PubMed

Siglin, Amanda E; Sun, Shangjin; Moore, Jeffrey K; Tan, Sarah; Poenie, Martin; Lear, James D; Polenova, Tatyana; Cooper, John A; Williams, John C

2013-01-01

Cytoplasmic dynein and dynactin participate in retrograde transport of organelles, checkpoint signaling and cell division. The principal subunits that mediate this interaction are the dynein intermediate chain (IC) and the dynactin p150(Glued); however, the interface and mechanism that regulates this interaction remains poorly defined. Herein, we use multiple methods to show the N-terminus of mammalian dynein IC, residues 10-44, is sufficient for binding p150(Glued). Consistent with this mapping, monoclonal antibodies that antagonize the dynein-dynactin interaction also bind to this region of the IC. Furthermore, double and triple alanine point mutations spanning residues 6 to 19 in the yeast IC homolog, Pac11, produce significant defects in spindle positioning. Using the same methods we show residues 381 to 530 of p150(Glued) form a minimal fragment that binds to the dynein IC. Sedimentation equilibrium experiments indicate that these individual fragments are predominantly monomeric, but admixtures of the IC and p150(Glued) fragments produce a 2:2 complex. This tetrameric complex is sensitive to salt, temperature and pH, suggesting that the binding is dominated by electrostatic interactions. Finally, circular dichroism (CD) experiments indicate that the N-terminus of the IC is disordered and becomes ordered upon binding p150(Glued). Taken together, the data indicate that the dynein-dynactin interaction proceeds through a disorder-to-order transition, leveraging its bivalent-bivalent character to form a high affinity, but readily reversible interaction.

Ebolavirus Nucleoprotein C-Termini Potently Attract Single Domain Antibodies Enabling Monoclonal Affinity Reagent Sandwich Assay (MARSA) Formulation

PubMed Central

Sherwood, Laura J.; Hayhurst, Andrew

2013-01-01

Background Antigen detection assays can play an important part in environmental surveillance and diagnostics for emerging threats. We are interested in accelerating assay formulation; targeting the agents themselves to bypass requirements for a priori genome information or surrogates. Previously, using in vitro affinity reagent selection on Marburg virus we rapidly established monoclonal affinity reagent sandwich assay (MARSA) where one recombinant antibody clone was both captor and tracer for polyvalent nucleoprotein (NP). Hypothesizing that the closely related Ebolavirus genus may share the same Achilles' heel, we redirected the scheme to see whether similar assays could be delivered and began to explore their mechanism. Methods and Findings In parallel we selected panels of llama single domain antibodies (sdAb) from a semi-synthetic library against Zaire, Sudan, Ivory Coast, and Reston Ebola viruses. Each could perform as both captor and tracer in the same antigen sandwich capture assay thereby forming MARSAs. All sdAb were specific for NP and those tested required the C-terminal domain for recognition. Several clones were cross-reactive, indicating epitope conservation across the Ebolavirus genus. Analysis of two immune shark sdAb revealed they also targeted the C-terminal domain, and could be similarly employed, yet were less sensitive than a comparable llama sdAb despite stemming from immune selections. Conclusions The C-terminal domain of Ebolavirus NP is a strong attractant for antibodies and enables sensitive sandwich immunoassays to be rapidly generated using a single antibody clone. The polyvalent nature of nucleocapsid borne NP and display of the C-terminal region likely serves as a bountiful affinity sink during selections, and a highly avid target for subsequent immunoassay capture. Combined with the high degree of amino acid conservation through 37 years and across wide geographies, this domain makes an ideal handle for monoclonal affinity reagent driven antigen sandwich assays for the Ebolavirus genus. PMID:23577211

Tuning sensitivity of CAR to EGFR density limits recognition of normal tissue while maintaining potent anti-tumor activity

PubMed Central

Caruso, Hillary G.; Hurton, Lenka V.; Najjar, Amer; Rushworth, David; Ang, Sonny; Olivares, Simon; Mi, Tiejuan; Switzer, Kirsten; Singh, Harjeet; Huls, Helen; Lee, Dean A.; Heimberger, Amy B.; Champlin, Richard E.; Cooper, Laurence J. N.

2015-01-01

Many tumors over express tumor-associated antigens relative to normal tissue, such as epidermal growth factor receptor (EGFR). This limits targeting by human T cells modified to express chimeric antigen receptors (CARs) due to potential for deleterious recognition of normal cells. We sought to generate CAR+ T cells capable of distinguishing malignant from normal cells based on the disparate density of EGFR expression by generating two CARs from monoclonal antibodies which differ in affinity. T cells with low affinity Nimo-CAR selectively targeted cells over-expressing EGFR, but exhibited diminished effector function as the density of EGFR decreased. In contrast, the activation of T cells bearing high affinity Cetux-CAR was not impacted by the density of EGFR. In summary, we describe the generation of CARs able to tune T-cell activity to the level of EGFR expression in which a CAR with reduced affinity enabled T cells to distinguish malignant from non-malignant cells. PMID:26330164

Fundamental molecular design for precise control of thermoresponsiveness of organic polymers by using ternary systems.

PubMed

Amemori, Shogo; Kokado, Kenta; Sada, Kazuki

2012-05-23

The de novo design of thermosensitive polymers in solution has been achieved by using the addition of small organic molecules (or "effectors"). Hydrogen bonding as an attractive polymer-polymer or polymer-effector interaction substantially dominates the responsivity, causing facile switching between LCST-type and UCST-type phase transitions, control of the transition temperature, and further coincidence of the two transitions. Small molecules having a high affinity for the polymer induce UCST-type phase behavior, whereas those having a low affinity for the polymer showed LCST-type phase behavior.

Label-Free Bioanalyte Detection from Nanometer to Micrometer Dimensions-Molecular Imprinting and QCMs †.

PubMed

Mujahid, Adnan; Mustafa, Ghulam; Dickert, Franz L

2018-06-01

Modern diagnostic tools and immunoassay protocols urges direct analyte recognition based on its intrinsic behavior without using any labeling indicator. This not only improves the detection reliability, but also reduces sample preparation time and complexity involved during labeling step. Label-free biosensor devices are capable of monitoring analyte physiochemical properties such as binding sensitivity and selectivity, affinity constants and other dynamics of molecular recognition. The interface of a typical biosensor could range from natural antibodies to synthetic receptors for example molecular imprinted polymers (MIPs). The foremost advantages of using MIPs are their high binding selectivity comparable to natural antibodies, straightforward synthesis in short time, high thermal/chemical stability and compatibility with different transducers. Quartz crystal microbalance (QCM) resonators are leading acoustic devices that are extensively used for mass-sensitive measurements. Highlight features of QCM devices include low cost fabrication, room temperature operation, and most importantly ability to monitor extremely low mass shifts, thus potentially a universal transducer. The combination of MIPs with quartz QCM has turned out as a prominent sensing system for label-free recognition of diverse bioanalytes. In this article, we shall encompass the potential applications of MIP-QCM sensors exclusively label-free recognition of bacteria and virus species as representative micro and nanosized bioanalytes.

The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells.

PubMed

Larionova, Marina D; Markova, Svetlana V; Vysotski, Eugene S

2017-01-29

The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazine-dependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M. longa encoding extremely psychrophilic isoforms of MLuc (MLuc2-type). The novel isoforms share only ∼54-64% of protein sequence identity with the previously cloned isoforms and, consequently, are the product of a separate group of paralogous genes. The MLuc2 isoform with consensus sequence was produced as a natively folded protein using baculovirus/insect cell expression system, purified, and characterized. The MLuc2 displays a very high bioluminescent activity and high thermostability similar to those of the previously characterized M. longa luciferase isoform MLuc7. However, in contrast to MLuc7 revealing the highest activity at 12-17 °C and 0.5 M NaCl, the bioluminescence optima of MLuc2 isoforms are at ∼5 °C and 1 M NaCl. The MLuc2 adaptation to cold is also accompanied by decrease of melting temperature and affinity to substrate suggesting a more conformational flexibility of a protein structure. The luciferase isoforms with different temperature optima may provide adaptability of the M. longa bioluminescence to the changes of water temperature during diurnal vertical migrations. Copyright © 2016 Elsevier Inc. All rights reserved.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.

PubMed

Schröter, Christian; Günther, Ralf; Rhiel, Laura; Becker, Stefan; Toleikis, Lars; Doerner, Achim; Becker, Janine; Schönemann, Andreas; Nasu, Daichi; Neuteboom, Berend; Kolmar, Harald; Hock, Björn

2015-01-01

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.

Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds.

PubMed

Troncoso-Ponce, M A; Rivoal, J; Venegas-Calerón, M; Dorion, S; Sánchez, R; Cejudo, F J; Garcés, R; Martínez-Force, E

2012-07-01

Three cDNAs encoding different phosphoglycerate kinase (PGK, EC 2.7.2.3) isoforms, two cytosolic (HacPGK1 and HacPGK2) and one plastidic (HapPGK), were cloned and characterized from developing sunflower (Helianthus annuus L.) seeds. The expression profiles of these genes showed differences in heterotrophic tissues, such as developing seeds and roots, where HacPGK1 was predominant, while HapPGK was highly expressed in photosynthetic tissues. The cDNAs were expressed in Escherichia coli, and the corresponding proteins purified to electrophoretic homogeneity, using immobilized metal ion affinity chromatography, and biochemically characterized. Despite the high level of identity between sequences, the HacPGK1 isoform showed strong differences in terms of specific activity, temperature stability and pH sensitivity in comparison to HacPGK2 and HapPGK. A polyclonal immune serum was raised against the purified HacPGK1 isoform, which showed cross-immunoreactivity with the other PGK isoforms. This serum allowed the localization of high expression levels of PGK isozymes in embryo tissues. Copyright © 2012 Elsevier Ltd. All rights reserved.

Paper-based immune-affinity arrays for detection of multiple mycotoxins in cereals.

PubMed

Li, Li; Chen, Hongpu; Lv, Xiaolan; Wang, Min; Jiang, Xizhi; Jiang, Yifei; Wang, Heye; Zhao, Yongfu; Xia, Liru

2018-03-01

Mycotoxins produced by different species of fungi may coexist in cereals and feedstuffs, and could be highly toxic for humans and animals. For quantification of multiple mycotoxins in cereals, we developed a paper-based mycotoxin immune-affinity array. First, paper-based microzone arrays were fabricated by photolithography. Then, monoclonal mycotoxin antibodies were added in a copolymerization reaction with a cross-linker to form an immune-affinity monolith on the paper-based microzone array. With use of a competitive immune-response format, paper-based mycotoxin immune-affinity arrays were successfully applied to detect mycotoxins in samples. The detection limits for deoxynivalenol, zearalenone, T-2 toxin, and HT-2 toxin were 62.7, 10.8, 0.36, and 0.23 μg·kg -1 , respectively, which meet relevant requirements for these compounds in food. The recovery rates were 81-86% for deoxynivalenol, 89-117% for zearalenone, 79-86% for T-2 toxin, and 78-83% for HT-2 toxin, and showed the paper-based immune-affinity arrays had good reproducibility. In summary, the paper-based mycotoxin immune-affinity array provides a sensitive, rapid, accurate, stable, and convenient platform for detection of multiple mycotoxins in agro-foods. Graphical abstract Paper-based immune-affinity monolithic array. DON deoxynivalenol, HT-2 HT-2 toxin, T-2 T-2 toxin, PEGDA polyethylene glycol diacrylate, ZEN zearalenone.

Computational design of a pH-sensitive IgG binding protein.

PubMed

Strauch, Eva-Maria; Fleishman, Sarel J; Baker, David

2014-01-14

Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ∼ 4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.

Comparison of an antibody and its recombinant derivative for the detection of the small molecule explosive 2,4,6-trinitrotoluene.

PubMed

Liu, Jinny L; Zabetakis, Dan; Acevedo-Vélez, Glendalys; Goldman, Ellen R; Anderson, George P

2013-01-08

Antibodies are commonly used as recognition elements in immunoassays because of their high specificity and affinity, and have seen extensive use in competitive assays for the detection of small molecules. However, these complex molecules require production either in animals or by mammalian cell cultures, and are not easily tailored through genetic manipulation. Single chain antibodies (scFv), recombinantly expressed molecules consisting of only the antibody's binding region joined via a linking peptide, can provide an alternative to intact antibodies. We describe the characterization of a new monoclonal antibody (mAb), 2G5B5, able to detect the small molecule explosive 2,4,6-trinitrotoluene (TNT) and the scFv derived from its variable regions. The mAb and scFv were tested by surface plasmon resonance to determine their affinity for an immobilized TNT surrogate; dissociation constants were determined to be 1.5×10(-13) M and 4.8×10(-10) M respectively. Circular dichroism was used to determine their melting temperatures. The mAb is more stable melting at ∼75°C while the scFv melts at ∼65°C. The recognition elements were incorporated into a competitive assay format using a bead-based multiplexing platform to examine their sensitivity and specificity. The scFv was able to detect TNT ∼10-fold more sensitively than the mAb in this assay format, allowing detection of TNT concentrations down to at least 1 μg L(-1). The 2G5B gave similar detection limits to a commercial anti-TNT mAb, but was less specific, recognizing 1,3,5-trinitrobenzene (TNB) equally well as TNT. Published by Elsevier B.V.

A comprehensive review on nano-molybdenum disulfide/DNA interfaces as emerging biosensing platforms.

PubMed

Kukkar, Manil; Mohanta, Girish C; Tuteja, Satish K; Kumar, Parveen; Bhadwal, Akhshay Singh; Samaddar, Pallabi; Kim, Ki-Hyun; Deep, Akash

2018-06-01

The development of nucleic acid-based portable platforms for the real-time analysis of diseases has attracted considerable scientific and commercial interest. Recently, 2D layered molybdenum sulfide (2D MoS 2 from here on) nanosheets have shown great potential for the development of next-generation platforms for efficient signal transduction. Through combination with DNA as a biorecognition medium, MoS 2 nanostructures have opened new opportunities to design and construct highly sensitive, specific, and commercially viable sensing devices. The use of specific short ssDNA sequences like aptamers has been proven to bind well with the unique transduction properties of 2D MoS 2 nanosheets to realize aptasensing devices. Such sensors can be operated on the principles of fluorescence, electro-cheumuluminescence, and electrochemistry with many advantageous features (e.g., robust biointerfacing through various conjugation chemistries, facile sensor assembly, high stability with regard to temperature/pH, and high affinity to target). This review encompasses the state of the art information on various design tactics and working principles of MoS 2 /DNA sensor technology which is emerging as one of the most sought-after and valuable fields with the advent of nucleic acid inspired devices. To help achieve a new milestone in biosensing applications, great potential of this emerging technique is described further with regard to sensitivity, specificity, operational convenience, and versatility. Copyright © 2018 Elsevier B.V. All rights reserved.

A Micro-Preconcentrator Combined Olfactory Sensing System with a Micromechanical Cantilever Sensor for Detecting 2,4-Dinitrotoluene Gas Vapor

PubMed Central

Chae, Myung-Sic; Kim, Jinsik; Yoo, Yong Kyoung; Kang, Ji Yoon; Lee, Jeong Hoon; Hwang, Kyo Seon

2015-01-01

Preventing unexpected explosive attacks and tracing explosion-related molecules require the development of highly sensitive gas-vapor detection systems. For that purpose, a micromechanical cantilever-based olfactory sensing system including a sample preconcentrator was developed to detect 2,4-dinitrotoluene (2,4-DNT), which is a well-known by-product of the explosive molecule trinitrotoluene (TNT) and exists in concentrations on the order of parts per billion in the atmosphere at room temperature. A peptide receptor (His-Pro-Asn-Phe-Ser-Lys-Tyr-Ile-Leu-His-Gln-Arg) that has high binding affinity for 2,4-DNT was immobilized on the surface of the cantilever sensors to detect 2,4-DNT vapor for highly selective detection. A micro-preconcentrator (µPC) was developed using Tenax-TA adsorbent to produce higher concentrations of 2,4-DNT molecules. The preconcentration was achieved via adsorption and thermal desorption phenomena occurring between target molecules and the adsorbent. The µPC directly integrated with a cantilever sensor and enhanced the sensitivity of the cantilever sensor as a pretreatment tool for the target vapor. The response was rapidly saturated within 5 min and sustained for more than 10 min when the concentrated vapor was introduced. By calculating preconcentration factor values, we verified that the cantilever sensor provides up to an eightfold improvement in sensing performance. PMID:26213944

The lethal interaction of x ray and penicillin induced lesions following x-irradiation of Escherichia coli B/r in the presence of hypoxic cell sensitizers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gillies, N.E.; Obioha, F.I.

When Escherichia coli B/r were x-irradiated under anoxia in the presence of different electron-affinic sensitizers and then incubated in broth containing penicillin (at a concentration that did not kill unirradiated cells) additional killing of the bacteria occurred provided the sensitizers were of relatively high lipophilicity. The overall effect was to increase the efficiency of these sensitizers. It is concluded that sensitizer-dependent latent radiation lesions(s) are produced in membrane components of the cell envelope that interact with damage caused by penicillin in the peptidoglycan layer and this causes the additional lethality.

Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

PubMed

Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

2016-10-01

Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

Antibody-free PRISM-SRM for multiplexed protein quantification: Is this the new competition for immunoassays in bioanalysis?

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Qian, Weijun

2013-02-01

Highly sensitive technologies for multiplexed quantification of a large number of candidate proteins will play an increasingly important role in clinical biomarker discovery, systems biology, and general biomedical research. Herein we introduce the new PRISM-SRM technology, which represents a highly sensitive multiplexed quantification technology capable of simultaneous quantification of many low-abundance proteins without the need of affinity reagents. The versatility of antibody-free PRISM-SRM for quantifying various types of targets including protein isoforms, protein modifications, metabolites, and others, thus offering new competition with immunoassays.

Development of a high specific activity radioligand, /sup 125/I-LSD, and its application to the study of serotonin receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kadan, M.J.

/sup 125/I-Labeled receptor ligands can be synthesized with specific activities exceeding 2000 Ci/mmol, making them nearly 70-fold more sensitive in receptor site assays than (mono) tritiated ligands. We have synthesized and characterized /sup 125/I-lysergic acid diethylamide (/sup 125/I-LSD), the first radioiodinated ligand for serotonin receptor studies. The introduction of /sup 125/I at the 2 position of LSD increased both the affinity and selectivity of this compound for serotonin 5-HT/sub 2/ receptors in rat cortex. The high specific activity of /sup 125/I-LSD and its high ratio of specific to nonspecific binding make this ligand especially useful for autoradiographic studies of serotoninmore » receptor distribution. We have found that /sup 125/I-LSD binds with high affinity to a class of serotonin receptors in the CNS of the marine mollusk Aplysia californica.« less

Compound immobilization and drug-affinity chromatography.

PubMed

Rix, Uwe; Gridling, Manuela; Superti-Furga, Giulio

2012-01-01

Bioactive small molecules act through modulating a yet unpredictable number of targets. It is therefore of critical importance to define the cellular target proteins of a compound as an entry point to understanding its mechanism of action. Often, this can be achieved in a direct fashion by chemical proteomics. As with any affinity chromatography, immobilization of the bait to a solid support is one of the earliest and most crucial steps in the process. Interfering with structural features that are important for identification of a target protein will be detrimental to binding affinity. Also, many molecules are sensitive to heat or to certain chemicals, such as acid or base, and might be destroyed during the process of immobilization, which therefore needs to be not only efficient, but also mild. The subsequent affinity chromatography step needs to preserve molecular and conformational integrity of both bait compound and proteins in order to result in the desired specific enrichment while ensuring a high level of compatibility with downstream analysis by mass spectrometry. Thus, the right choice of detergent, buffer, and protease inhibitors is also essential. This chapter describes a widely applicable procedure for the immobilization of small molecule drugs and for drug-affinity chromatography with subsequent protein identification by mass spectrometry.

Kinetics of N-Utilization By Natural Phytoplankton Assemblages During Upwelling Events At The NW Iberian Shelf

NASA Astrophysics Data System (ADS)

Brion, N.; Elskens, M.; Dehairs, F.; Baeyens, W.

2003-04-01

The concentration-dependent uptakes of nitrate, ammonium and the effect of ammo-nium on the f-ratio were surveyed in surface waters of the NW Iberian shelf during June 1997, July 1998 and September 1999. Because relationships between rates and substrate concentrations were quite variable, ranging from linear to convex shaped curves, they were fitted to rational functions. Stepwize regression analysis yielded subsequent model equations with reasonable statistical properties which allowed describing all but all a few cases. Differentiating these equations with respect to the concentration gave the slope of the tangent to the curve, i.e., the variation in rate expected for a given perturbation of the ambient substrate concentration. The initial slope value was then used as an index to gauge the "affinity" of the plankton community for the nitrogen substrate utilization. In June 1997, the situation at the Iberian shelf showed no upwelling except near Cape Finistère. Overall, the phytoplankton community displayed a high "affinity" for both nitrate and ammonium and low f-ratio values, which is indicative of a re-generated production regime. High ammonium regeneration rates supported furthermore these observations. It was also demonstrated that the new production rates is only marginally sensitive to changes of the ambient nitrate and/or ammonium concentrations. This indicates that the production regime is rather stable throughout. Only at Cape Finistère, nitrate concentrations were high reflecting the onset of an upwelling event. In this zone, the phytoplankton community, taking advantage of its high affinity for nitrate enhanced both total N-uptake rate and f-ratio. In July 1998, the situation evolved towards an extension to the south of the upwelling event starting at Cape Finistère. In this southern zone of the upwelling the phytoplankton community displayed generally a lower affinity for nitrate (but not for ammonium) than in 1997. In spite of this lower affinity, nitrate uptake rate was dominant resulting in f-ratio values greater than 0.5, a characteristic of a new production regime. The new production rate is only marginally sensitive to increases of the ambient nitrate, but is drastically inhibited by small increases of the ambient ammonium. The situation of September 1999 was very close to that observed in July 1998, with higher nitrate concentrations in the coastal northern part of the sampling area dominated by upwelling.

High temperature sensitivity is intrinsic to voltage-gated potassium channels

PubMed Central

Yang, Fan; Zheng, Jie

2014-01-01

Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors. DOI: http://dx.doi.org/10.7554/eLife.03255.001 PMID:25030910

Calculating the sensitivity and robustness of binding free energy calculations to force field parameters

PubMed Central

Rocklin, Gabriel J.; Mobley, David L.; Dill, Ken A.

2013-01-01

Binding free energy calculations offer a thermodynamically rigorous method to compute protein-ligand binding, and they depend on empirical force fields with hundreds of parameters. We examined the sensitivity of computed binding free energies to the ligand’s electrostatic and van der Waals parameters. Dielectric screening and cancellation of effects between ligand-protein and ligand-solvent interactions reduce the parameter sensitivity of binding affinity by 65%, compared with interaction strengths computed in the gas-phase. However, multiple changes to parameters combine additively on average, which can lead to large changes in overall affinity from many small changes to parameters. Using these results, we estimate that random, uncorrelated errors in force field nonbonded parameters must be smaller than 0.02 e per charge, 0.06 Å per radius, and 0.01 kcal/mol per well depth in order to obtain 68% (one standard deviation) confidence that a computed affinity for a moderately-sized lead compound will fall within 1 kcal/mol of the true affinity, if these are the only sources of error considered. PMID:24015114

GZ-793A, a lobelane analog, interacts with the vesicular monoamine transporter-2 to inhibit the effect of methamphetamine

PubMed Central

Horton, David B.; Nickell, Justin R.; Zheng, Guangrong; Crooks, Peter A.; Dwoskin, Linda P.

2013-01-01

GZ-793A inhibits methamphetamine-evoked dopamine release from striatal slices and methamphetamine self-administration in rats. GZ-793A potently and selectively inhibits dopamine uptake at the vesicular monoamine transporter-2 (VMAT2). The present study determined GZ-793A’s ability to evoke [3H]dopamine release and inhibit methamphetamine-evoked [3H]dopamine release from isolated striatal synaptic vesicles. Results show GZ-793A concentration-dependent [3H]dopamine release; nonlinear regression revealed a two-site model of interaction with VMAT2 (High- and Low-EC50 = 15.5 nM and 29.3 µM, respectively). Tetrabenazine and reserpine completely inhibited the GZ-793A-evoked [3H]dopamine release, however, only at the High-affinity site. Low concentrations of GZ-793A that interact with the extravesicular dopamine uptake site and the High-affinity intravesicular DA release site also inhibited methamphetamine-evoked [3H]dopamine release from synaptic vesicles. A rightward shift in the methamphetamine concentration-response was evident with increasing concentrations of GZ-793A, and the Schild regression slope was 0.49±0.08, consistent with surmountable allosteric inhibition. These results support a hypothetical model of GZ-793A interaction at more than one site on VMAT2 protein, which explains its potent inhibition of dopamine uptake, dopamine release via a High-affinity tetrabenazine- and reserpine-sensitive site, dopamine release via a Low-affinity tetrabenazine- and reserpine-insensitive site, and low-affinity interaction with the dihydrotetrabenazine binding site on VMAT2. GZ-793A-inhibition of the effects of methamphetamine supports its potential as a therapeutic agent for the treatment of methamphetamine abuse. PMID:23875622

Two-phase positive inotropic effects of ouabain and the presence of multiple classes of ouabain binding sites in the ferret heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ng, Y.C.; Akera, T.

1986-03-05

Characteristics of more than one class of ouabain receptors which appear to exist in ferret heart were examined. In isolated papillary muscle, 1 to 30 nM ouabain produced a positive inotropic effect in the presence of 5 ..mu..M propranolol and 2 ..mu..M phentolamine. Higher concentrations of ouabain (0.1 to 10 ..mu..M) produced an additional and prominent inotropic effect. In partially purified Na, K-ATPase, ouabain caused a monophasic inhibition; however, the concentration-inhibition curve spanned over 5 log units, indicating that ouabain is interacting with more than a single class of the enzyme. Scatchard analysis of specific /sup 3/H-ouabain binding revealed approximatelymore » equal abundance of high and low affinity binding sites. The K/sub D/ value for high affinity sites was approximately 20 nM whereas that for low affinity sites was about 45 times higher. When phosphoenzyme was formed in the presence of (..gamma..-/sup 32/P)-ATP, Mg/sup 2 +/ and Na/sup +/ and subjected to SDS gel electrophoresis, two distinct K/sup +/-sensitive bands with about 100,000 dalton molecular weight were detected. Molecular weight difference between these two bands was approximately 2500 dalton. Phosphorylation of either band was abolished by 1 ..mu..M ouabain suggesting that both bands may correspond to the high-affinity binding sites. These results indicate that high and low affinity ouabain binding sites exists in approximately equal abundance in the ferret heart, and that binding of ouabain to these sites cases Na,K-ATPase inhibition and the positive inotropic effect.« less

AtCHX13 is a plasma membrane K+ transporter.

PubMed

Zhao, Jian; Cheng, Ning-Hui; Motes, Christy M; Blancaflor, Elison B; Moore, Miranda; Gonzales, Naomi; Padmanaban, Senthilkumar; Sze, Heven; Ward, John M; Hirschi, Kendal D

2008-10-01

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using (86)Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a K(m) of 136 and 196 microm, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment.

Chromatographic removal combined with heat, acid and chaotropic inactivation of four model viruses.

PubMed

Valdés, R; Ibarra, Neysi; Ruibal, I; Beldarraín, A; Noa, E; Herrera, N; Alemán, R; Padilla, S; Garcia, J; Pérez, M; Morales, R; Chong, E; Reyes, B; Quiñones, Y; Agraz, A; Herrera, L

2002-07-03

The virus removal of protein A affinity chromatography, inactivation capacity, acid pH and a combination of high temperature with a chaotropic agent was determined in this work. The model viruses studied were sendaivirus, human immunodeficency virus (HIV-IIIb), human poliovirus type-II, human herpesvirus I and canine parvovirus. The protein A affinity chromatography showed a maximum reduction factor of 8 logs in the case of viruses larger than 120 nm size, while for small viruses (18-30 nm) the maximum reduction factor was about 5 logs. Non viral inactivation was observed during the monoclonal antibody elution step. Low pH treatment showed a maximum inactivation factor of 7.1 logs for enveloped viruses. However, a weak inactivation factor (3.4 logs) was obtained for DNA nonenveloped viruses. The combination of high temperature with 3 M KSCN showed a high inactivation factor for all of the viruses studied. The total clearance factor was 23.1, 15.1, 13.6, 20.0 and 16.0 logs for sendaivirus, HIV-IIIb, human poliovirus type-II, human herpesvirus I and canine parvovirus, respectively.

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

PubMed

Ciossek, T; Ullrich, A

1997-01-09

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hao, Z.; Chen, S.; Wilson, D.B.

1999-11-01

An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake systemmore » that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.« less

Noninvasive imaging of multiple myeloma using near infrared fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Hathi, Deep; Zhou, Haiying; Bollerman-Nowlis, Alex; Shokeen, Monica; Akers, Walter J.

2016-03-01

Multiple myeloma is a plasma cell malignancy characterized by monoclonal gammopathy and osteolytic bone lesions. Multiple myeloma is most commonly diagnosed in late disease stages, presenting with pathologic fracture. Early diagnosis and monitoring of disease status may improve quality of life and long-term survival for multiple myeloma patients from what is now a devastating and fatal disease. We have developed a near-infrared targeted fluorescent molecular probe with high affinity to the α4β1 integrin receptor (VLA-4)overexpressed by a majority of multiple myeloma cells as a non-radioactive analog to PET/CT tracer currently being developed for human diagnostics. A near-infrared dye that emits about 700 nm was conjugated to a high affinity peptidomimmetic. Binding affinity and specificity for multiple myeloma cells was investigated in vitro by tissue staining and flow cytometry. After demonstration of sensitivity and specificity, preclinical optical imaging studies were performed to evaluate tumor specificity in murine subcutaneous and metastatic multiple myeloma models. The VLA-4-targeted molecular probe showed high affinity for subcutaneous MM tumor xenografts. Importantly, tumor cells specific accumulation in the bone marrow of metastatic multiple myeloma correlated with GFP signal from transfected cells. Ex vivo flow cytometry of tumor tissue and bone marrow further corroborated in vivo imaging data, demonstrating the specificity of the novel agent and potential for quantitative imaging of multiple myeloma burden in these models.

Thermochemistry of the specific binding of C12 surfactants to bovine serum albumin.

PubMed

Nielsen, A D; Borch, K; Westh, P

2000-06-15

The specific binding to bovine serum albumin (BSA) of anionic and non-ionic surfactants with C12 acyl chains has been studied by high sensitivity isothermal titration calorimetry. This method proved particularly effective in resolving the binding of anionic surfactants into separate classes of sites with different affinity. For sodium dodecylsulfate (SDS) the measured binding curves could be rationalized as association to two classes (high affinity/low affinity) of sites comprising, respectively, three and six similar (i.e. thermodynamically equivalent), independent sites. Changes in the thermodynamic functions enthalpy, standard free energy, standard entropy and heat capacity could be discerned for each class of binding site, as well as for micelle formation. These data suggest that binding to low affinity sites (in analogy with micelle formation) exhibits energetic parameters; in particular, a large negative change in heat capacity, which is characteristic of hydrophobic interactions. The thermodynamics of high affinity binding, on the other hand, is indicative of other dominant forces; most likely electrostatic interactions. Other anionic ligands investigated (laurate and dodecyl benzylsulfonate) showed a behavior similar to SDS, the most significant difference being the high affinity binding of the alkylbenzyl sulfonate. For this ligand, the thermodynamic data is indicative of a more loosely associated complex than for SDS and laurate. BSA was found to bind one or two of the non-ionic surfactants (NIS) hepta- or penta(ethylene glycol) monododecyl ether (C12EO7 and C12EO5) with binding constants about three orders of magnitude lower than for SDS. Hence, the free energy of the surfactant in the weakly bound BSA-NIS complex is only slightly favored over the micellar state. The binding process is characterized by very large exothermic enthalpy changes (larger than for the charged surfactants) and a large, positive increment in heat capacity. These observations cannot be reconciled with a molecular picture based on simple hydrophobic condensation onto non-polar patches on the protein surface.

The roles of two O-donor ligands in the Fe2+-binding and H2O2-sensing by the Fe2+-dependent H2O2 sensor PerR.

PubMed

Ji, Chang-Jun; Yang, Yoon-Mo; Kim, Jung-Hoon; Ryu, Su-Hyun; Youn, Hwan; Lee, Jin-Won

2018-05-10

PerR is a metal-dependent peroxide sensing transcription factor which controls the expression of genes involved in peroxide resistance. The function of Bacillus subtilis PerR is mainly dictated by the regulatory metal ion (Fe 2+ or Mn 2+ ) coordinated by three N-donor ligands (His37, His91, and His93) and two O-donor ligands (Asp85 and Asp104). While H 2 O 2 sensing by PerR is mediated by Fe 2+ -dependent oxidation of N-donor ligand (either His37 or His91), one of the O-donor ligands (Asp104), but not Asp85, has been proposed as the key residue that regulates the sensitivity of PerR to H 2 O 2 . Here we systematically investigated the relative roles of two O-donor ligands of PerR in metal-binding affinity and H 2 O 2 sensitivity in vivo and in vitro. Consistent with the previous report, in vitro the D104E-PerR could not sense low levels of H 2 O 2 in the presence of excess Fe 2+ sufficient for the formation of the Fe 2+ -bound D104E-PerR. However, the expression of PerR-regulated reporter fusion was not repressed by D104E-PerR in the presence of Fe 2+ , suggesting that Fe 2+ is not an effective corepressor for this mutant protein in vivo. Furthermore, in vitro metal titration assays indicate that D104E-PerR has a significantly reduced affinity for Fe 2+ , but not for Mn 2+ , when compared to wild type PerR. These data indicate that the type of O-donor ligand (Asp vs. Glu) at position 104 is an important determinant in providing high Fe 2+ -binding affinity required for the sensing of the physiologically relevant Fe 2+ -levels, in addition to its role in rendering PerR highly sensitive to physiological levels of H 2 O 2 . In comparison, the D85E-PerR did not show a perturbed change in Fe 2+ -binding affinity, however, it displayed a slightly decreased sensitivity to H 2 O 2 both in vivo and in vitro, suggesting that the type of O-donor ligand (Asp vs. Glu) at position 85 may be important for the fine-tuning of H 2 O 2 sensitivity. Copyright © 2018 Elsevier Inc. All rights reserved.

Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf.

PubMed

Tao, Yi; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

2013-06-27

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography-mass spectrometry. Three flavonoids including quercetin-3-O-β-D-arabinopyranosyl-(1→2)-β-D-galactopyranoside, quercetin-3-O-β-D-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources. Copyright © 2013 Elsevier B.V. All rights reserved.

Oxytocin and vasopressin: distinct receptors in myometrium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guillon, G.; Balestre, M.N.; Roberts, J.M.

1987-06-01

The binding characteristics of (/sup 3/H)oxytocin (( /sup 3/H)OT) and (/sup 3/H)lysine vasopressin (( /sup 3/H)LVP) to nonpregnant human myometrium were investigated. Binding of both radioligands was saturable, time dependent, and reversible. Whereas (/sup 3/H)OT was found to bind to a single class of sites with high affinity (Kd, 1.5 +/- 0.4 (+/- SEM) nM) and low capacity (maximum binding (Bmax), 34 +/- 6 fmol/mg protein), (/sup 3/H)LVP bound to two classes of sites, one with high affinity (Kd, 2.2 +/- 0.1 nM) and low capacity (Bmax, 198 +/- 7 fmol/mg protein) and another with low affinity (Kd, 655 +/-more » 209 nM) and high capacity (Bmax, 5794 +/- 1616 fmol/mg protein). The binding of the labeled peptides also displayed a marked difference in sensitivity to Mg2+ and guanine nucleotides. These differences in binding characteristics as well as the differences in potency of analogs in competing for (/sup 3/H)OT and (/sup 3/H)LVP binding indicate the presence of distinct receptors for OT and vasopressin in human myometrium. Pharmacological characterization of the high affinity binding sites for (/sup 3/H)LVP indicated that these are of the V1 subtype. Although, as suggested by others, vasopressin and OT can bind to the same sites, the presence of distinct receptors for both peptides provides an explanation for the previously reported difference in myometrial responsiveness to OT and vasopressin.« less

Screening the sequence selectivity of DNA-binding molecules using a gold nanoparticle-based colorimetric approach.

PubMed

Hurst, Sarah J; Han, Min Su; Lytton-Jean, Abigail K R; Mirkin, Chad A

2007-09-15

We have developed a novel competition assay that uses a gold nanoparticle (Au NP)-based, high-throughput colorimetric approach to screen the sequence selectivity of DNA-binding molecules. This assay hinges on the observation that the melting behavior of DNA-functionalized Au NP aggregates is sensitive to the concentration of the DNA-binding molecule in solution. When short, oligomeric hairpin DNA sequences were added to a reaction solution consisting of DNA-functionalized Au NP aggregates and DNA-binding molecules, these molecules may either bind to the Au NP aggregate interconnects or the hairpin stems based on their relative affinity for each. This relative affinity can be measured as a change in the melting temperature (Tm) of the DNA-modified Au NP aggregates in solution. As a proof of concept, we evaluated the selectivity of 4',6-diamidino-2-phenylindone (an AT-specific binder), ethidium bromide (a nonspecific binder), and chromomycin A (a GC-specific binder) for six sequences of hairpin DNA having different numbers of AT pairs in a five-base pair variable stem region. Our assay accurately and easily confirmed the known trends in selectivity for the DNA binders in question without the use of complicated instrumentation. This novel assay will be useful in assessing large libraries of potential drug candidates that work by binding DNA to form a drug/DNA complex.

2017 Guralp Affinity Digitizer Evaluation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Merchant, Bion J.

Sandia National Laboratories has tested and evaluated two Guralp Affinity digitizers. The Affinity digitizers are intended to record sensor output for seismic and infrasound monitoring applications. The purpose of this digitizer evaluation is to measure the performance characteristics in such areas as power consumption, input impedance, sensitivity, full scale, self- noise, dynamic range, system noise, response, passband, and timing. The Affinity digitizers are being evaluated for potential use in the International Monitoring System (IMS) of the Comprehensive Nuclear Test-Ban-Treaty Organization (CTBTO).

Method development of enantiomer separations by affinity capillary electrophoresis, cyclodextrin electrokinetic chromatography and capillary electrophoresis-mass spectrometry.

PubMed

Tanaka, Yoshihide

2002-07-01

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 mumol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE-mass spectrometry (CE-MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE-MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE-MS.

Okadaic acid mediates p53 hyperphosphorylation and growth arrest in cells with wild-type p53 but increases aberrant mitoses in cells with non-functional p53.

PubMed

Milczarek, G J; Chen, W; Gupta, A; Martinez, J D; Bowden, G T

1999-06-01

The protein phosphatase inhibitor and tumor promoting agent okadaic acid (OA), has been shown previously to induce hyperphosphorylation of p53 protein, which in turn correlated with increased transactivation or apoptotic function. However, how the tumor promotion effects of OA relate to p53 tumor supressor function (or dysfunction) remain unclear. Rat embryonic fibroblasts harboring a temperature-sensitive mouse p53 transgene were treated with 50 nM doses of OA. At the wild-type permissive temperature this treatment resulted in: (i) the hyperphosphorylation of sites within tryptic peptides of the transactivation domain of p53; (ii) an increase in p53 affinity for a p21(waf1) promotor oligonucleotide; (iii) an increase in cellular steady state levels of p21(waf1) message; (iv) a G2/M cell cycle blockage in addition to the G1/S arrest previously associated with p53; and (v) no increased incidence of apoptosis. On the other hand, OA treatment at the mutated p53 permissive temperature resulted in a relatively high incidence of aberrant mitosis with no upregulation of p21(waf1) message. These results suggest that while wild-type p53 blocks the proliferative effects of OA through p21(waf1)-mediated growth arrest, cells with non-functional p53 cannot arrest and suffer relatively high levels of OA-mediated aberrant mitoses.

Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides.

PubMed

Chen, Yajing; Xiong, Zhichao; Zhang, Lingyi; Zhao, Jiaying; Zhang, Quanqing; Peng, Li; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

2015-02-21

Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g(-1)), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.

Quantitation of bovine immunoglobulin isotypes and allotypes using monoclonal antibodies.

PubMed

Williams, D J; Newson, J; Naessens, J

1990-03-01

A panel of 10 monoclonal antibodies specific for bovine immunoglobulins M, A, G1, G2 and light chains were produced and enzyme-linked immunosorbent assays developed to measure Ig levels in body fluids and culture supernatants using this panel of MAbs. An inhibition ELISA was accurate and sensitive for MAbs of high affinity, detecting levels as low as 10 ng ml-1 of IgM using a high-affinity MAb, IL-A50 (dissociation constant = 1.3 X 10(-11) M). For MAbs of lower affinity (KD of less than 0.25 X 10(-9) M) a sandwich ELISA was more sensitive, detecting 0.1-1.0 microgram ml-1 Ig, provided a conjugate of an anti-light chain MAb was used. Using these ELISA techniques, four pairs of MAbs specific for bovine IgM, IgA, IgG1 and IgG2 respectively, were screened on sera from over 100 cattle of different breeds to determine whether any detected a polymorphic epitope. MAbs IL-A30, IL-A60, IL-A66, IL-A71, IL-A72, IL-A73 and IL-A74 were shown to recognise monomorphic determinants on their respective heavy chains. In contrast, the epitope recognised on the mu-heavy chain by MAb IL-A50, which had previously been shown to be polymorphic, was found to be allelic and inherited under the control of a single gene, probably Cu.

Magneto-nanosensor platform for probing low-affinity protein–protein interactions and identification of a low-affinity PD-L1/PD-L2 interaction

PubMed Central

Lee, Jung-Rok; Bechstein, Daniel J. B.; Ooi, Chin Chun; Patel, Ashka; Gaster, Richard S.; Ng, Elaine; Gonzalez, Lino C.; Wang, Shan X.

2016-01-01

Substantial efforts have been made to understand the interactions between immune checkpoint receptors and their ligands targeted in immunotherapies against cancer. To carefully characterize the complete network of interactions involved and the binding affinities between their extracellular domains, an improved kinetic assay is needed to overcome limitations with surface plasmon resonance (SPR). Here, we present a magneto-nanosensor platform integrated with a microfluidic chip that allows measurement of dissociation constants in the micromolar-range. High-density conjugation of magnetic nanoparticles with prey proteins allows multivalent receptor interactions with sensor-immobilized bait proteins, more closely mimicking natural-receptor clustering on cells. The platform has advantages over traditional SPR in terms of insensitivity of signal responses to pH and salinity, less consumption of proteins and better sensitivities. Using this platform, we characterized the binding affinities of the PD-1—PD-L1/PD-L2 co-inhibitory receptor system, and discovered an unexpected interaction between the two known PD-1 ligands, PD-L1 and PD-L2. PMID:27447090

Alleviation of rapid, futile ammonium cycling at the plasma membrane by potassium reveals K+-sensitive and -insensitive components of NH4+ transport.

PubMed

Szczerba, Mark W; Britto, Dev T; Balkos, Konstantine D; Kronzucker, Herbert J

2008-01-01

Futile plasma membrane cycling of ammonium (NH4+) is characteristic of low-affinity NH4+ transport, and has been proposed to be a critical factor in NH4+ toxicity. Using unidirectional flux analysis with the positron-emitting tracer 13N in intact seedlings of barley (Hordeum vulgare L.), it is shown that rapid, futile NH4+ cycling is alleviated by elevated K+ supply, and that low-affinity NH4+ transport is mediated by a K+-sensitive component, and by a second component that is independent of K+. At low external [K+] (0.1 mM), NH4+ influx (at an external [NH4+] of 10 mM) of 92 micromol g(-1) h(-1) was observed, with an efflux:influx ratio of 0.75, indicative of rapid, futile NH4+ cycling. Elevating K+ supply into the low-affinity K+ transport range (1.5-40 mM) reduced both influx and efflux of NH4+ by as much as 75%, and substantially reduced the efflux:influx ratio. The reduction of NH4+ fluxes was achieved rapidly upon exposure to elevated K+, within 1 min for influx and within 5 min for efflux. The channel inhibitor La3+ decreased high-capacity NH4+ influx only at low K+ concentrations, suggesting that the K+-sensitive component of NH4+ influx may be mediated by non-selective cation channels. Using respiratory measurements and current models of ion flux energetics, the energy cost of concomitant NH4+ and K+ transport at the root plasma membrane, and its consequences for plant growth are discussed. The study presents the first demonstration of the parallel operation of K+-sensitive and -insensitive NH4+ flux mechanisms in plants.

Anion exchange pathways for Cl sup minus transport in rabbit renal microvillus membranes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Karniski, L.P.; Aronson, P.S.

1987-09-01

The authors evaluated the mechanisms of chloride transport in microvillus membrane vesicles isolated from the rabbit renal cortex. The presence of Cl-formate exchange was confirmed. Outward gradients of oxaloacetate, HCO{sub 3}, acetate, lactate, succinate, sulfate, and p-aminohippurate (PAH) stimulated the rate of Cl uptake minimally or not at all. However, an outward gradient of oxalate stimulated Cl uptake by 70%, and an outward Cl gradient induced uphill oxalate uptake, indicting Cl-oxalate exchange. Moreover, an outward formate gradient induced uphill oxalate uptake, indicating formate-oxalate exchange. Studies of inhibitor and substrate specificity indicated the probably operation of at least two separate anionmore » exchangers in mediating Cl transport. The Cl-formate exchanger accepted Cl and formate as substrates, had little or no affinity for oxalate, was sensitive to inhibition by furosemide, and was less sensitive to inhibition by 4,4{prime}-diisothiocyanostilbene-2,2{prime}-disulfonic acid (DIDS). The Cl (formate)-oxalate exchanger also accepted Cl and formate as substrates but had high affinity for oxalate, was highly sensitive to inhibition by DIDS, and was less sensitive to inhibition by furosemide. The Cl-formate exchanger was electroneutral, whereas the Cl (formate)-oxalate exchanger was electrogenic. They conclude that at least separate anion exchangers mediating Cl transport are present on the luminal membrane of the rabbit proximal tubule cell. These exchangers may play important roles in mediating transtubular Cl and oxalate transport in this nephron segment.« less

Design and Synthesis of High Affinity Inhibitors of Plasmodium falciparum and Plasmodium vivax N-Myristoyltransferases Directed by Ligand Efficiency Dependent Lipophilicity (LELP)

PubMed Central

2014-01-01

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization. PMID:24641010

Effect of processing conditions on microstructural features in Mn–Si sintered steels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oro, Raquel, E-mail: raqueld@chalmers.se; Hryha, Eduard, E-mail: hryha@chalmers.se; Campos, Mónica, E-mail: campos@ing.uc3m.es

2014-09-15

Sintering of steels containing oxidation sensitive elements is possible if such elements are alloyed with others which present lower affinity for oxygen. In this work, a master alloy powder containing Fe–Mn–Si–C, specifically designed to create a liquid phase during sintering, has been used for such purpose. The effect of processing conditions such as sintering temperature and atmosphere was studied with the aim of describing the microstructural evolution as well as the morphology and distribution of oxides in the sintered material, evaluating the potential detrimental effect of such oxides on mechanical properties. Chemical analyses, metallography and fractography studies combined with X-raymore » photoelectron spectroscopy analyses on the fracture surfaces were used to reveal the main mechanism of fracture and their correlation with the chemical composition of the different fracture surfaces. The results indicate that the main mechanism of failure in these steels is brittle fracture in the surrounding of the original master alloy particles due to degradation of grain boundaries by the presence of oxide inclusions. Mn–Si oxide inclusions were observed on intergranular decohesive facets. The use of reducing atmospheres and high sintering temperatures reduces the amount and size of such oxide inclusions. Besides, high heating and cooling rates reduce significantly the final oxygen content in the sintered material. A model for microstructure development and oxide evolution during different stages of sintering is proposed, considering the fact that when the master alloy melts, the liquid formed can dissolve some of the oxides as well as the surface of the surrounding iron base particles. - Highlights: • Oxide distribution in steels containing oxidation-sensitive elements • Mn, Si introduced in a master alloy powder, mixed with a base iron powder • Selective oxidation of Mn and Si on iron grain boundaries • Decohesive fracture caused by degradation of grain boundaries by oxide inclusions • Reducing agents efficient at low temperatures critical for avoiding oxide inclusions.« less

Substrate-specific modifications on magnetic iron oxide nanoparticles as an artificial peroxidase for improving sensitivity in glucose detection.

PubMed

Liu, Yanping; Yu, Faquan

2011-04-08

Magnetic iron oxide nanoparticles (MION) were recently found to act as a peroxidase with intrinsic advantages over natural counterparts. Their limited affinity toward catalysis substrates, however, dramatically reduces their utility. In this paper, some effective groups were screened out and conjugated on MION as substrate-specific modifications for improving MION's affinity to substrates and hence utility. Nanoparticles of four different superficial structures were synthesized and characterized by TEM, size, zeta potential and SQUID, and assayed for peroxidase activity. Glucose detection was selected as an application model system to evaluate the bonus thereof. Catalysis was found to follow Michaelis-Menten kinetics. Sulfhydryl groups incorporated on MION (SH-MION) notably improve the affinity toward a substrate (hydrogen peroxide) and so do amino groups (NH₂-MION) toward another substrate, proved by variation in the determined kinetic parameters. A synergistically positive effect was observed and an apparently elevated detection sensitivity and a significantly lowered detection limit of glucose were achieved when integrated with both sulfhydryl and amino groups (SH-NH₂-MION). Our findings suggest that substrate-specific surface modifications are a straightforward and robust strategy to improve MION peroxidase-like activity. The high activity extends magnetic nanoparticles to wide applications other than glucose detection.

Highly sensitive evanescent wave combination tapered fiber optic fluorosensor for protein detection

NASA Astrophysics Data System (ADS)

Nardone, Vincent; Kapoor, Rakesh

2008-02-01

In this paper we are reporting the development of a highly sensitive evanescent wave combination tapered fiber optic fluorosensor. We have demonstrated detection of 5 pM Bovine Serum Albumin (BSA) protein using these fiber optic sensors. The sensor can be easily adopted for detection of other proteins. Six identical probes were prepared and affinity pure Goat anti-BSA antibodies were immobilized on the probe surface. We could detect signal from all the probes kept in 5 pM to 1 nM BSA solution while no signal was detected from the probes kept in 20 nM labeled ESA solution.

Fabrication of High-Sensitivity Skin-Attachable Temperature Sensors with Bioinspired Microstructured Adhesive.

PubMed

Oh, Ju Hyun; Hong, Soo Yeong; Park, Heun; Jin, Sang Woo; Jeong, Yu Ra; Oh, Seung Yun; Yun, Junyeong; Lee, Hanchan; Kim, Jung Wook; Ha, Jeong Sook

2018-02-28

In this study, we demonstrate the fabrication of a highly sensitive flexible temperature sensor with a bioinspired octopus-mimicking adhesive. A resistor-type temperature sensor consisting of a composite of poly(N-isopropylacrylamide) (pNIPAM)-temperature sensitive hydrogel, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate, and carbon nanotubes exhibits a very high thermal sensitivity of 2.6%·°C -1 between 25 and 40 °C so that the change in skin temperature of 0.5 °C can be accurately detected. At the same time, the polydimethylsiloxane adhesive layer of octopus-mimicking rim structure coated with pNIPAM is fabricated through the formation of a single mold by utilizing undercut phenomenon in photolithography. The fabricated sensor shows stable and reproducible detection of skin temperature under repeated attachment/detachment cycles onto skin without any skin irritation for a long time. This work suggests a high potential application of our skin-attachable temperature sensor to wearable devices for medical and health-care monitoring.

Kinetic sensitivity of a receptor-binding radiopharmaceutical: Technetium-99m galactosyl-neoglycoalbumin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vera, D.R.; Woodle, E.S.; Stadalnik, R.C.

1989-09-01

Kinetic sensitivity is the ability of a physiochemical parameter to alter the time-activity curve of a radiotracer. The kinetic sensitivity of liver and blood time-activity data resulting from a single bolus injection of ({sup 99m}Tc)galactosyl-neoglycoalbumin (( Tc)NGA) into healthy pigs was examined. Three parameters, hepatic plasma flow scaled as flow per plasma volume, ligand-receptor affinity, and total receptor concentration, were tested using (Tc)NGA injections of various molar doses and affinities. Simultaneous measurements of plasma volume (iodine-125 human serum albumin dilution), and hepatic plasma flow (indocyanine green extraction) were performed during 12 (Tc)NGA studies. Paired data sets demonstrated differences (P(chi v2)more » less than 0.01) in liver and blood time-activity curves in response to changes in each of the tested parameters. We conclude that the (Tc)NGA radiopharmacokinetic system is therefore sensitive to hepatic plasma flow, ligand-receptor affinity, and receptor concentration. In vivo demonstration of kinetic sensitivity permits delineation of the physiologic parameters that determine the biodistribution of a radiopharmaceutical. This delineation is a prerequisite to a valid analytic assessment of receptor biochemistry via kinetic modeling.« less

A rapid electrochemical monitoring platform for sensitive determination of thiamethoxam based on β-cyclodextrin-graphene composite.

PubMed

Zhai, XingChen; Zhang, Hua; Zhang, Min; Yang, Xin; Gu, Cheng; Zhou, GuoPeng; Zhao, HaiTian; Wang, ZhenYu; Dong, AiJun; Wang, Jing

2017-08-01

A rapid monitoring platform for sensitive voltammetric detection of thiamethoxam residues is reported in the present study. A β-cyclodextrin-reduced graphene oxide composite was used as a reinforcing material in electrochemical determination of thiamethoxam. Compared with bare glassy carbon electrodes, the reduction peak currents of thiamethoxam at reduced graphene oxide/glassy carbon electrode and β-cyclodextrin-reduced graphene oxide/glassy carbon electrode were increased by 70- and 124-fold, respectively. The experimental conditions influencing voltammetric determination of thiamethoxam, such as the amount of β-cyclodextrin-reduced graphene oxide, solution pH, temperature, and accumulation time, were optimized. The reduction mechanism and binding affinity of this material is also discussed. Under optimal conditions, the reduction peak currents increased linearly between 0.5 µM and 16 µM concentration of thiamethoxam. The limit of detection was 0.27 µM on the basis of a signal-to-noise ratio of 3. When the proposed method was applied to brown rice in a recovery test, the recoveries were between 92.20% and 113.75%. The results were in good concordance with the high-performance liquid chromatography method. The proposed method therefore provides a promising and effective platform for sensitive and rapid determination of thiamethoxam. Environ Toxicol Chem 2017;36:1991-1997. © 2017 SETAC. © 2017 SETAC.

Spectral and Concentration Sensitivity of Multijunction Solar Cells at High Temperature: Preprint

DOE Office of Scientific and Technical Information (OSTI.GOV)

Friedman, Daniel J.; Steiner, Myles A.; Perl, Emmett E.

2017-06-14

We model the performance of two-junction solar cells at very high temperatures of ~400 degrees C and beyond for applications such as hybrid PV/solar-thermal power production, and identify areas in which the design and performance characteristics behave significantly differently than at more conventional near-room-temperature operating conditions. We show that high-temperature operation reduces the sensitivity of the cell efficiency to spectral content, but increases the sensitivity to concentration, both of which have implications for energy yield in terrestrial PV applications. For other high-temperature applications such as near-sun space missions, our findings indicate that concentration may be a useful tool to enhancemore » cell efficiency.« less

A novel method for detection of H9N2 influenza viruses by an aptamer-real time-PCR.

PubMed

Hmila, Issam; Wongphatcharachai, Manoosak; Laamiri, Nacira; Aouini, Rim; Marnissi, Boutheina; Arbi, Marwa; Sreevatsan, Srinand; Ghram, Abdeljelil

2017-05-01

H9N2 Influenza subtype has emerged in Tunisia causing epidemics in poultry and resulting in major economic losses. New mutations in their hemagglutinin and neuraminidase proteins were acquired, suggesting their potential to directly infect humans. Effective surveillance tools should be implemented to help prevent potential spillover of the virus across species. We have developed a highly sensitive real time immuno-polymerase chain reaction (RT-I-PCR) method for detecting H9N2 virus. The assay applies aptamers as ligands to capture and detect the virus. First, a panel of specific ssDNA aptamers was selected via a one step high stringency protocol. Next, the panel of selected aptamers was characterized for their affinities and their specificity to H9N2 virus. The aptamer showing the highest binding affinity to the virus was used as ligand to develop a highly sensitive sandwich Aptamer I-PCR. A 3-log increase in analytical sensitivity was achieved as compared to a routinely used ELISA antigen test, highlighting the potential of this approach to detect very low levels of virus particles. The test was validated using clinical samples and constitutes a rapid and a label-free platform, opening a new venue for the development of aptamer -based viability sensing for a variety of microorganisms of economic importance in Tunisia and surrounding regions. Copyright © 2017 Elsevier B.V. All rights reserved.

Denervation does not alter the number of neuronal bungarotoxin binding sites on autonomic neurons in the frog cardiac ganglion.

PubMed

Sargent, P B; Bryan, G K; Streichert, L C; Garrett, E N

1991-11-01

The binding of neuronal bungarotoxin (n-BuTX; also known as bungarotoxin 3.1, kappa-bungarotoxin, and toxin F) was analyzed in normal and denervated parasympathetic cardiac ganglia of the frog Rana pipiens, n-BuTX blocks both EPSPs and ACh potentials at 5-20 nM, as determined by intracellular recording techniques. Scatchard analysis on homogenates indicates that cardiac ganglia have two classes of binding sites for 125I-n-BuTX: a high-affinity site with an apparent dissociation constant (Kd,app) of 1.7 nM and a Bmax (number of binding sites) of 3.8 fmol/ganglion and a low-affinity site with a Kd,app of 12 microM and a Bmax of 14 pmol/ganglion. alpha-Bungarotoxin does not appear to interfere with the binding of 125I-n-BuTX to either site. The high-affinity binding site is likely to be the functional nicotinic ACh receptor (AChR), given the similarity between its affinity for 125I-n-BuTX and the concentration of n-BuTX required to block AChR function. Light microscopic autoradiographic analysis of 125I-n-BuTX binding to the ganglion cell surface reveals that toxin binding is concentrated at synaptic sites, which were identified using a synaptic vesicle-specific antibody. Scatchard analysis of autoradiographic data reveals that 125I-n-BuTX binding to the neuronal surface is saturable and has a Kd,app similar to that of the high-affinity binding site characterized in homogenates. Surface binding of 125I-n-BuTX is blocked by nicotine, carbachol, and d-tubocurarine (IC50 less than 20 microM), but not by atropine (IC50 greater than 10 mM). Denervation of the heart increases the ACh sensitivity of cardiac ganglion cells but has no effect upon the number of high-affinity binding sites for 125I-n-BuTX in tissue homogenates. Moreover, autoradiographic analysis indicates that denervation does not alter the number of 125I-n-BuTX binding sites on the ganglion cell surface. n-BuTX is as effective in reducing ganglion cell responses to ACh in denervated ganglia as it is in normally innervated ganglia. These results suggest that denervation alters neither the total number of nicotinic AChRs in the cardiac ganglion nor the number found on the surface of ganglion cells. These autonomic neurons thus respond differently to denervation than do skeletal myofibers. The increase in ACh sensitivity displayed by cardiac ganglion cells upon denervation cannot be explained by changes in AChR number.

Affinity immunoblotting - High resolution isoelectric focusing analysis of antibody clonotype distribution

NASA Technical Reports Server (NTRS)

Knisley, Keith A.; Rodkey, L. Scott

1986-01-01

A sensitive and specific method is proposed for the analysis of specific antibody clonotype changes occurring during an immune response and for comparing multiple sera for antibody clonotype similarities. Polyclonal serum antibodies separated by isoelectric focusing (IEF) were analyzed by an affinity immunoblotting method using antigen-coated nitrocellulose membranes. Antibodies present on the surface of the acrylamide gels following IEF bind the antigen on the nitrocellulose when the coated nitrocellulose is laid over the gels. The technique has been used to analyze Ig clonotypes specific for five protein antigens and two carbohydrate antigens. Optimal antigen concentrations for coating the nitrocellulose membranes were found to range from 10-100 microgram/ml.

Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry.

PubMed

Sweadner, K J

1985-09-25

There are two isozymes of the Na,K-ATPase, which can be purified separately from rat renal medulla and brainstem axolemma. Here the basic kinetic properties of the two Na,K-ATPases have been compared in conditions permitting enzyme turnover. The two isozymes are half-maximally activated at different concentrations of ATP, the axolemma Na,K-ATPase having the higher affinity. They are half-maximally activated by Na+ and K+ at very similar concentrations but show differences in cooperativity toward Na+. The affinities of both isozymes for ATP and Na+ are affected in a qualitatively similar way by variations in the concentration of K+. Both isozymes transport 22Na+ and 42K+ in a ratio close to 3:2 in artificial lipid vesicles. The two isozymes differ most strikingly in the inhibition of ATPase activity by ouabain. The axolemma Na,K-ATPase has a high affinity for ouabain with positive cooperativity, while the renal medulla Na,K-ATPase has a lower affinity with negative cooperativity. It is likely that the cooperativity differences are due to kinetic effects, reflecting different rates of conformation transitions during enzyme turnover. The functional result of the contrasting cooperativities is that the difference in sensitivity to ouabain is amplified.

Variable sensitivity of US maize yield to high temperatures across developmental stages

NASA Astrophysics Data System (ADS)

Butler, E. E.; Huybers, P. J.

2013-12-01

The sensitivity of maize to high temperatures has been widely demonstrated. Furthermore, field work has indicated that reproductive development stages are particularly sensitive to stress, but this relationship has not been quantified across a wide geographic region. Here, the relationship between maize yield and temperature variations is examined as a function of developmental stage. US state-level data from the National Agriculture Statistics Service provide dates for six growing stages: planting, silking, doughing, dented, mature, and harvested. Temperatures that correspond to each developmental stage are then inferred from a network of weather station observations interpolated to the county level, and a multiple linear regression technique is employed to estimate the sensitivity of county yield outcomes to variations in growing-degree days and an analogous measure of high temperatures referred to as killing-degree days. Uncertainties in the transition times between county-level growth stages are accounted for. Results indicate that the silking and dented stages are generally the most sensitive to killing degree days, with silking the most sensitive stage in the US South and dented the most sensitive in the US North. These variable patterns of sensitivity aid in interpreting which weather events are of greatest significance to maize yields and provide some insight into how shifts in planting time or changes in developmental timing would influence the risks associated with exposure to high temperatures.

Anther response to high-temperature stress during development and pollen thermotolerance heterosis as revealed by pollen tube growth and in vitro pollen vigor analysis in upland cotton.

PubMed

Song, Guicheng; Wang, Miaomiao; Zeng, Bin; Zhang, Jing; Jiang, Chenliang; Hu, Qirui; Geng, Guangtao; Tang, Canming

2015-05-01

Pollen tube growth in styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the adverse effect of high temperatures during square development. High-temperature stress during flowering influences the square development of upland cotton (Gossypium hirsutum L.) and cotton yield. Although it is well known that square development is sensitive to high temperature, high-temperature sensitive stages of square development and the effects of high temperature on pollen tube growth in the styles are unknown. The effect of high temperature on anther development corresponding to pollen vigor is unknown during anther development. The objectives of this study were to identify the stages of square development that are sensitive to high temperatures (37/30 and 40/34 °C), to determine whether the abnormal development of squares influenced by high temperature is responsible for the variation in the in vitro germination percent of pollen grains at anthesis, to identify the effect of high temperature on pollen germination in the styles, and to determine pollen thermotolerance heterosis. Our results show that the stages from the sporogenous cell to tetrad stage (square length <6.0 mm) were the most sensitive to high temperature, and the corresponding pollen viability at anthesis was consistent with the changes in the square development stage. Pollen tube growth in the styles was strongly inhibited by temperature above 35 °C, and the yield of cotton decreased because of the effect of high temperature during square development. The thermotolerance of hybrid F1 pollen showed heterosis, and pollen viability could be used as a criterion for screening for high-temperature tolerance cultivars. These results can be used in breeding to develop new cotton cultivars that can withstand high-temperature conditions, particularly in a future warmer climate.

Comparative investigation of surface transfer doping of hydrogen terminated diamond by high electron affinity insulators

DOE Office of Scientific and Technical Information (OSTI.GOV)

Verona, C.; Marinelli, Marco; Verona-Rinati, G.

We report on a comparative study of transfer doping of hydrogenated single crystal diamond surface by insulators featured by high electron affinity, such as Nb{sub 2}O{sub 5}, WO{sub 3}, V{sub 2}O{sub 5}, and MoO{sub 3}. The low electron affinity Al{sub 2}O{sub 3} was also investigated for comparison. Hole transport properties were evaluated in the passivated hydrogenated diamond films by Hall effect measurements, and were compared to un-passivated diamond films (air-induced doping). A drastic improvement was observed in passivated samples in terms of conductivity, stability with time, and resistance to high temperatures. The efficiency of the investigated insulators, as electron acceptingmore » materials in hydrogenated diamond surface, is consistent with their electronic structure. These surface acceptor materials generate a higher hole sheet concentration, up to 6.5 × 10{sup 13} cm{sup −2}, and a lower sheet resistance, down to 2.6 kΩ/sq, in comparison to the atmosphere-induced values of about 1 × 10{sup 13} cm{sup −2} and 10 kΩ/sq, respectively. On the other hand, hole mobilities were reduced by using high electron affinity insulator dopants. Hole mobility as a function of hole concentration in a hydrogenated diamond layer was also investigated, showing a well-defined monotonically decreasing trend.« less

Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

NASA Astrophysics Data System (ADS)

Taft, William C.; Delorenzo, Robert J.

1984-05-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

PubMed Central

Taft, W C; DeLorenzo, R J

1984-01-01

Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance. PMID:6328498

Selection of specific aptamer against enrofloxacin and fabrication of graphene oxide based label-free fluorescent assay.

PubMed

Dolati, Somayeh; Ramezani, Mohammad; Nabavinia, Maryam Sadat; Soheili, Vahid; Abnous, Khalil; Taghdisi, Seyed Mohammad

2018-05-15

Specific ssDNA aptamers for the antibiotic enrofloxacin (ENR) were isolated from an enriched nucleotide library by SELEX (Systematic Evolution of Ligands by EXponential enrichment) method with high binding affinity. After seven rounds, five aptamers were selected and identified. Apt58 with highest affinity and sensitivity (K d  = 14.19 nM) was employed to develop a label-free fluorescent biosensing approach based on aptamer, graphene oxide (GO) and native fluorescence of ENR for determination of ENR residue in raw milk samples. Under optimized experimental conditions, the linear range was from 5 nM to 250 nM and LOD was calculated to be 3.7 nM, and the recovery rate was between 94.1% and 108.5%. The integration of aptamer and GO in this bioassay provides a promising way for rapid, sensitive and cost-effective detection of ENR in real samples like raw milk. Copyright © 2018 Elsevier Inc. All rights reserved.

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

PubMed Central

Schotte, Lise; Rombaut, Bart; Thys, Bert

2012-01-01

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads. PMID:22688388

Fragment screening of cyclin G-associated kinase by weak affinity chromatography.

PubMed

Meiby, Elinor; Knapp, Stefan; Elkins, Jonathan M; Ohlson, Sten

2012-11-01

Fragment-based drug discovery (FBDD) has become a new strategy for drug discovery where lead compounds are evolved from small molecules. These fragments form low affinity interactions (dissociation constant (K(D)) = mM - μM) with protein targets, which require fragment screening methods of sufficient sensitivity. Weak affinity chromatography (WAC) is a promising new technology for fragment screening based on selective retention of fragments by a drug target. Kinases are a major pharmaceutical target, and FBDD has been successfully applied to several of these targets. In this work, we have demonstrated the potential to use WAC in combination with mass spectrometry (MS) detection for fragment screening of a kinase target-cyclin G-associated kinase (GAK). One hundred seventy fragments were selected for WAC screening by virtual screening of a commercial fragment library against the ATP-binding site of five different proteins. GAK protein was immobilized on a capillary HPLC column, and compound binding was characterized by frontal affinity chromatography. Compounds were screened in sets of 13 or 14, in combination with MS detection for enhanced throughput. Seventy-eight fragments (46 %) with K(D) < 200 μM were detected, including a few highly efficient GAK binders (K(D) of 2 μM; ligand efficiency = 0.51). Of special interest is that chiral screening by WAC may be possible, as two stereoisomeric fragments, which both contained one chiral center, demonstrated twin peaks. This ability, in combination with the robustness, sensitivity, and simplicity of WAC makes it a new method for fragment screening of considerable potential.

Fast probing of glucose and fructose in plant tissues via plasmonic affinity sandwich assay with molecularly-imprinted extraction microprobes.

PubMed

Muhammad, Pir; Liu, Jia; Xing, Rongrong; Wen, Yanrong; Wang, Yijia; Liu, Zhen

2017-12-01

Determination of specific target compounds in agriculture food and natural plant products is essential for many purposes; however, it is often challenging due to the complexity of the sample matrices. Herein we present a new approach called plasmonic affinity sandwich assay for the facile and rapid probing of glucose and fructose in plant tissues. The approach mainly relies on molecularly imprinted plasmonic extraction microprobes, which were prepared on gold-coated acupuncture needles via boronate affinity controllable oriented surface imprinting with the target monosaccharide as the template molecules. An extraction microprobe was inserted into plant tissues under investigation, which allowed for the specific extraction of glucose or fructose from the tissues. The glucose or fructose molecules extracted on the microprobe were labeled with boronic acid-functionalized Raman-active silver nanoparticles, and thus affinity sandwich complexes were formed on the microprobes. After excess Raman nanotags were washed away, the microprobe was subjected to Raman detection. Upon being irradiated with a laser beam, surface plasmon on the gold-coated microprobes was generated, which further produced plasmon-enhanced Raman scattering of the silver-based nanotags and thereby provided sensitive detection. Apple fruits, which contain abundant glucose and fructose, were used as a model of plant tissues. The approach exhibited high specificity, good sensitivity (limit of detection, 1 μg mL -1 ), and fast speed (the whole procedure required only 20 min). The spatial distribution profiles of glucose and fructose within an apple were investigated by the developed approach. Copyright © 2017 Elsevier B.V. All rights reserved.

The size of the hydroxyl group and its contribution to the affinity of atropine for muscarine-sensitive acetylcholine receptors.

PubMed Central

Barlow, R. B.; Ramtoola, S.

1980-01-01

1 From measurements of the affinity constants of hydratropyltropine and its methiodide for muscarine-sensitive acetylcholine receptors in the guinea-pig ileum, the increment in log K for the hydroxyl group in atropine is 2.06 and in the methiodide it is 2.16. These effects are slightly bigger than any so far recorded with these receptors. 2 The estimate of the increment in apparent molal volume for the hydroxyl group is 1.1 cm3/mol in atropine and 1.0 cm3/mol in the methobromide. 3 The large effect of the group on affinity may be linked to its small apparent size in water as suggested in the previous paper. PMID:7470742

AtCHX13 Is a Plasma Membrane K+ Transporter1[C][W][OA

PubMed Central

Zhao, Jian; Cheng, Ning-Hui; Motes, Christy M.; Blancaflor, Elison B.; Moore, Miranda; Gonzales, Naomi; Padmanaban, Senthilkumar; Sze, Heven; Ward, John M.; Hirschi, Kendal D.

2008-01-01

Potassium (K+) homeostasis is essential for diverse cellular processes, although how various cation transporters collaborate to maintain a suitable K+ required for growth and development is poorly understood. The Arabidopsis (Arabidopsis thaliana) genome contains numerous cation:proton antiporters (CHX), which may mediate K+ transport; however, the vast majority of these transporters remain uncharacterized. Here, we show that AtCHX13 (At2g30240) has a role in K+ acquisition. AtCHX13 suppressed the sensitivity of yeast (Saccharomyces cerevisiae) mutant cells defective in K+ uptake. Uptake experiments using 86Rb+ as a tracer for K+ demonstrated that AtCHX13 mediated high-affinity K+ uptake in yeast and in plant cells with a Km of 136 and 196 μm, respectively. Functional green fluorescent protein-tagged versions localized to the plasma membrane of both yeast and plant. Seedlings of null chx13 mutants were sensitive to K+ deficiency conditions, whereas overexpression of AtCHX13 reduced the sensitivity to K+ deficiency. Collectively, these results suggest that AtCHX13 mediates relatively high-affinity K+ uptake, although the mode of transport is unclear at present. AtCHX13 expression is induced in roots during K+-deficient conditions. These results indicate that one role of AtCHX13 is to promote K+ uptake into plants when K+ is limiting in the environment. PMID:18676662

Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles

NASA Astrophysics Data System (ADS)

Mahmoudi, Morteza; Shokrgozar, Mohammad A.; Behzadi, Shahed

2013-03-01

It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr32551b

Predicting the toxicity of metal mixtures

USGS Publications Warehouse

Balistrieri, Laurie S.; Mebane, Christopher A.

2013-01-01

The toxicity of single and multiple metal (Cd, Cu, Pb, and Zn) solutions to trout is predicted using an approach that combines calculations of: (1) solution speciation; (2) competition and accumulation of cations (H, Ca, Mg, Na, Cd, Cu, Pb, and Zn) on low abundance, high affinity and high abundance, low affinity biotic ligand sites; (3) a toxicity function that accounts for accumulation and potency of individual toxicants; and (4) biological response. The approach is evaluated by examining water composition from single metal toxicity tests of trout at 50% mortality, results of theoretical calculations of metal accumulation on fish gills and associated mortality for single, binary, ternary, and quaternary metal solutions, and predictions for a field site impacted by acid rock drainage. These evaluations indicate that toxicity of metal mixtures depends on the relative affinity and potency of toxicants for a given aquatic organism, suites of metals in the mixture, dissolved metal concentrations and ratios, and background solution composition (temperature, pH, and concentrations of major ions and dissolved organic carbon). A composite function that incorporates solution composition, affinity and competition of cations for two types of biotic ligand sites, and potencies of hydrogen and individual metals is proposed as a tool to evaluate potential toxicity of environmental solutions to trout.

Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.

PubMed

Wang, Yuling; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Schlücker, Sebastian; Trau, Matt

2014-10-07

Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

Graphene Field-Effect Transistors for the Sensitive and Selective Detection of Escherichia coli Using Pyrene-Tagged DNA Aptamer.

PubMed

Wu, Guangfu; Dai, Ziwen; Tang, Xin; Lin, Zihong; Lo, Pik Kwan; Meyyappan, M; Lai, King Wai Chiu

2017-10-01

This study reports biosensing using graphene field-effect transistors with the aid of pyrene-tagged DNA aptamers, which exhibit excellent selectivity, affinity, and stability for Escherichia coli (E. coli) detection. The aptamer is employed as the sensing probe due to its advantages such as high stability and high affinity toward small molecules and even whole cells. The change of the carrier density in the probe-modified graphene due to the attachment of E. coli is discussed theoretically for the first time and also verified experimentally. The conformational change of the aptamer due to the binding of E. coli brings the negatively charged E. coli close to the graphene surface, increasing the hole carrier density efficiently in graphene and achieving electrical detection. The binding of negatively charged E. coli induces holes in graphene, which are pumped into the graphene channel from the contact electrodes. The carrier mobility, which correlates the gate voltage to the electrical signal of the APG-FETs, is analyzed and optimized here. The excellent sensing performance such as low detection limit, high sensitivity, outstanding selectivity and stability of the graphene biosensor for E. coli detection paves the way to develop graphene biosensors for bacterial detection. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATP regulation of the ligand-binding properties in temperate and cold-adapted haemoglobins. X-ray structure and ligand-binding kinetics in the sub-Antarctic fish Eleginops maclovinus.

PubMed

Coppola, Daniela; Abbruzzetti, Stefania; Nicoletti, Francesco; Merlino, Antonello; Gambacurta, Alessandra; Giordano, Daniela; Howes, Barry D; De Sanctis, Giampiero; Vitagliano, Luigi; Bruno, Stefano; di Prisco, Guido; Mazzarella, Lelio; Smulevich, Giulietta; Coletta, Massimo; Viappiani, Cristiano; Vergara, Alessandro; Verde, Cinzia

2012-10-30

The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.

Identification and measurement of chlorinated organic pesticides in water by electron-capture gas chromatography

USGS Publications Warehouse

Lamar, William L.; Goerlitz, Donald F.; Law, LeRoy M.

1965-01-01

Pesticides, in minute quantities, may affect the regimen of streams, and because they may concentrate in sediments, aquatic organisms, and edible aquatic foods, their detection and their measurement in the parts-per-trillion range are considered essential. In 1964 the U.S. Geological Survey at Menlo Park, Calif., began research on methods for monitoring pesticides in water. Two systems were selected--electron-capture gas chromatography and microcoulometric-titration gas chromatography. Studies on these systems are now in progress. This report provides current information on the development and application of an electron-capture gas chromatographic procedure. This method is a convenient and extremely sensitive procedure for the detection and measurement of organic pesticides having high electron affinities, notably the chlorinated organic pesticides. The electron-affinity detector is extremely sensitive to these substances but it is not as sensitive to many other compounds. By this method, the chlorinated organic pesticide may be determined on a sample of convenient size in concentrations as low as the parts-per-trillion range. To insure greater accuracy in the identifications, the pesticides reported were separated and identified by their retention times on two different types of gas chromatographic columns.

Inter- and intra-variability of seed germination traits of Carpobrotus edulis N.E.Br. and its hybrid C. affine acinaciformis.

PubMed

Podda, Lina; Santo, Andrea; Mattana, Efisio; Mayoral, Olga; Bacchetta, Gianluigi

2018-06-22

Invasions by alien Carpobrotus spp. have been recognized as one of the most severe threats to Mediterranean-climate coastal ecosystems and Carpobrotus is considered one of the most widespread invasive alien genera in the Mediterranean Basin. The aims of this study were to characterize seed germination of both C. edulis and its hybrid C. affine acinaciformis, in terms of photoperiod, temperature and salinity. Inter- and intra-specific variability in the responses to photoperiod (12/12 h light and total darkness), constant temperatures (5, 10, 15, 20, 25°C) and an alternating temperature regime (25/10°C), salt stress (0, 125, 250, 500 mM NaCl) and the recovery of seed germination were evaluated for two seed lots of C. edulis and two of its hybrid C. affine acinaciformis. All the tested seed lots achieved higher germination percentages in the light, respect to total darkness. In relation to temperature, the two C. edulis seed lots did not show a preference, while the two C. affine acinaciformis seed lots differed in their germination response, one germinating more at the lowest temperatures (5 and 10°C) and one at the highest (20 and 25°C). For all the seed lots, highest germination occurred without NaCl (0 mM) and germination decreased with increasing salinity. Different germination requirements in saline substrate were not detected for C. edulis, while were observed for C. affine acinaciformis. Marked differences were detected in recovery responses between the two taxa. C. edulis demonstrated to have the ability to germinate in a wide time window during the year. This study identified significant differences in seed production, seed mass, germination requirements (temperature) and salinity tolerance for both C. edulis and C. affine acinaciformis. Our results indicated the extreme versatility of the hybrid forms to germinate in a wide range of natural conditions and habitats. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Affinity Biosensors for Detection of Mycotoxins in Food.

PubMed

Evtugyn, Gennady; Subjakova, Veronika; Melikishvili, Sopio; Hianik, Tibor

2018-01-01

This chapter reviews recent achievements in methods of detection of mycotoxins in food. Special focus is on the biosensor technology that utilizes antibodies and nucleic acid aptamers as receptors. Development of biosensors is based on the immobilization of antibodies or aptamers onto various conventional supports like gold layer, but also on nanomaterials such as graphene oxide, carbon nanotubes, and quantum dots that provide an effective platform for achieving high sensitivity of detection using various physical methods, including electrochemical, mass sensitive, and optical. The biosensors developed so far demonstrate high sensitivity typically in subnanomolar limit of detection. Several biosensors have been validated in real samples. The sensitivity of biosensors is similar and, in some cases, even better than traditional analytical methods such as ELISA or chromatography. We believe that future trends will be focused on improving biosensor properties toward practical application in food industry. © 2018 Elsevier Inc. All rights reserved.

The Functions of BRCA2 in Homologous Recombinational Repair

DTIC Science & Technology

2004-07-01

chromatography with hydroxyapatite , Q-Sepharose, heparin affinity and MonoQ column (Fig. 4.). We have been able to obtain about 10 mg of the purified Rad51...and DNA- PKcs (the XR -1, xrs5/6, and V3 cell lines, respectively) are highly sensitive to IR in G1 and early S phases, compared to the wild-type, but

Microfluidics-assisted generation of stimuli-responsive hydrogels based on alginates incorporated with thermo-responsive and amphiphilic polymers as novel biomaterials.

PubMed

Karakasyan, C; Mathos, J; Lack, S; Davy, J; Marquis, M; Renard, D

2015-11-01

We used a droplet-based microfluidics technique to produce monodisperse responsive alginate-block-polyetheramine copolymer microgels. The polyetheramine group (PEA), corresponding to a propylene oxide /ethylene oxide ratio (PO/EO) of 29/6 (Jeffamine(®) M2005), was condensed, via the amine link, to alginates with various mannuronic/guluronic acids ratios and using two alginate:jeffamine mass ratios. The size of the grafted-alginate microgels varied from 60 to 80 μm depending on the type of alginate used and the degree of substitution. The droplet-based microfluidics technique offered exquisite control of both the dimension and physical chemical properties of the grafted-alginate microgels. These microgels were therefore comparable to isolated grafted-alginate chains in retaining both their amphiphilic and thermo-sensitive properties. Amphiphilicity was demonstrated at the oil-water interface where grafted-alginate microgels were found to decrease interfacial tension by ∼ 50%. The thermo-sensitivity of microgels was clearly demonstrated and a 10 to 20% reduction in size between was evidenced on increasing the temperature above the lower critical solution temperature (TLCST) of Jeffamine. In addition, the reversibility of thermo-sensitivity was demonstrated by studying the oil-water affinity of microgels with temperature after Congo red labeling. Finally, droplet-based microfluidics was found to be a good and promising tool for generating responsive biobased hydrogels for drug delivery applications and potential new colloidal stabilizers for dispersed systems such as Pickering emulsions. Copyright © 2015 Elsevier B.V. All rights reserved.

Ropizine concurrently enhances and inhibits ( sup 3 H) dextromethorpan binding to different structures of the guinea pig brain: Autoradiographic evidence for multiple binding sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canoll, P.D.; Smith, P.R.; and Musacchio, J.M.

1990-01-01

Ropizine produces a simultaneous enhancement and inhibition of ({sup 3}H) dextromethorphan (DM) high-affinity binding to different areas of the guinea pig brain. These results imply that there are two distinct types of high-affinity ({sup 3}H)DM binding sites, which are present in variable proportions in different brain structures. The ropizine-enhances ({sup 3}H)DM binding type was preferentially inhibited by (+)-pentazocine. This is consistent with the presumption that the (+)-pentazocine-sensitive site is identical with the common site for DM and 3-(-3-Hydroxphenyl)-N-(1-propyl)piperidine ((+)-3-PPP). The second binding type, which is inhibited by ropizine and is not so sensitive to (+){minus} pentazocine, has not been fullymore » characterized. This study demonstrates that the biphasic effects to ropizine are due, at least in part, to the effects of ropizine on two different types of ({sup 3}H)DM binding sites. However, this study does not rule out that the common DM/(+)-3-PPP site also might be inhibited by higher concentrations of ropizine.« less

Canceling effect leads temperature insensitivity of hydrolytic enzymes in soil

NASA Astrophysics Data System (ADS)

Razavi, Bahar S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

2015-04-01

Extracellular enzymes are important for decomposition of many macromolecules abundant in soil such as cellulose, hemicelluloses and proteins (Allison et al., 2010; Chen et al., 2012). The temperature sensitivity of enzymes responsible for organic matter decomposition is the most crucial parameter for prediction of the effects of global warming on carbon cycle. Temperature responses of biological systems are often expressed as a Q10 functions; The Q10 describes how the rate of a chemical reaction changes with a temperature increase for 10 °C The aim of this study was to test how the canceling effect will change with variation in temperature interval, during short-term incubation. We additionally investigated, whether canceling effect occurs in a broad range of concentrations (low to high) and whether it is similar for the set of hydrolytic enzymes within broad range of temperatures. To this end, we performed soil incubation over a temperature range of 0-40°C (with 5°C steps). We determined the activities of three enzymes involved in plant residue decomposition: β-glucosidase and cellobiohydrolase, which are commonly measured as enzymes responsible for degrading cellulose (Chen et al., 2012), and xylanase, which degrades xylooligosaccharides (short xylene chain) in to xylose, thus being responsible for breaking down hemicelluloses (German et al., 2011). Michaelis-Menten kinetics measured at each temperature allowed to calculate Q10 values not only for the whole reaction rates, but specifically for maximal reaction rate (Vmax) and substrate affinity (Km). Subsequently, the canceling effect - simultaneous increase of Vmax and Km with temperature was analyzed within 10 and 5 degree of temperature increase. Three temperature ranges (below 10, between 15 and 25, and above 30 °C) clearly showed non-linear but stepwise increase of temperature sensitivity of all three enzymes and allowed to conclude for predominance of psychrophilic, mesophilic and thermophilic microorganisms in soil at these temperature ranges. We conclude that the temperature sensitivity (Q10) of enzyme activity declines at higher temperature and lower concentration of substrates in soil. Overall, our results suggest that the fine-scale (five degree) temperature resolution level needs to be considered in global earth system models especially at temperature thresholds for physiological groups of soil microorganisms. Refcences: Allison, S.D., Wallenstein, M.D., Bradford, M.A., 2010. Soil-carbon response to warming dependent on microbial physiology. Nat. Geosci. 3, 336-340. doi:10.1038/ngeo846 Chen, R., Blagodatskaya, E., Senbayram, M., Blagodatsky, S., Myachina, O., Dittert, K., Kuzyakov, Y., 2012. Decomposition of biogas residues in soil and their effects on microbial growth kinetics and enzyme activities. Biomass Bioenergy 45, 221-229. doi:10.1016/j.biombioe.2012.06.014 German, D.P., Weintraub, M.N., Grandy, A.S., Lauber, C.L., Rinkes, Z.L., Allison, S.D., 2011. Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biol. Biochem. 43, 1387-1397. doi:10.1016/j.soilbio.2011.03.017

The effects of magnesium on potassium transport in ferret red cells.

PubMed Central

Flatman, P W

1988-01-01

1. The magnesium dependence of net and isotopic (using 86Rb as tracer) potassium transport was measured in fed ferret red cells. Bumetanide (0.1 mM) was used to dissect total flux into two components: bumetanide sensitive and bumetanide resistant. 2. Increasing the external magnesium concentration from zero (added) to 2 mM stimulated bumetanide-sensitive uptake by 16% but inhibited the bumetanide-resistant component by about 20%. 3. Ionophore A23187 was used to control internal magnesium concentration. A23187 was usually present in the cells during measurement of isotopic fluxes but was washed away before measurement of net fluxes. The magnesium-buffering characteristics of fed ferret red cells were assessed during these experiments. The cytoplasm acts as a high-capacity, low-affinity magnesium buffer over most of the range. Some high-affinity binding was seen in the presence of A23187 and 2 mM-EDTA. 4. A23187 itself slightly inhibits bumetanide-sensitive potassium transport. 5. Bumetanide-sensitive potassium transport is strongly dependent on the concentration of internal ionized magnesium. Transport is 35% maximal at 10(-7) M and increases up to the maximal rate at 1.3 mM. Further increase in ionized magnesium concentration to 3.5 mM has no additional effect. The curve relating activity to magnesium concentration is steepest at the physiological magnesium concentration. The effects of changing magnesium concentration are fully reversible. 6. Reduction of internal ionized magnesium concentration to 10(-7) M with A23187 and EDTA approximately doubles bumetanide-resistant potassium transport. 7. Bumetanide-sensitive fluxes occur via the sodium-potassium-chloride co-transport system under the conditions used. Results described in this paper thus suggest that internal magnesium may be an important physiological controller of sodium-potassium-chloride co-transport activity. PMID:3137332

Effect of atomic layer deposition temperature on current conduction in Al2O3 films formed using H2O oxidant

NASA Astrophysics Data System (ADS)

Hiraiwa, Atsushi; Matsumura, Daisuke; Kawarada, Hiroshi

2016-08-01

To develop high-performance, high-reliability gate insulation and surface passivation technologies for wide-bandgap semiconductor devices, the effect of atomic layer deposition (ALD) temperature on current conduction in Al2O3 films is investigated based on the recently proposed space-charge-controlled field emission model. Leakage current measurement shows that Al2O3 metal-insulator-semiconductor capacitors formed on the Si substrates underperform thermally grown SiO2 capacitors at the same average field. However, using equivalent oxide field as a more practical measure, the Al2O3 capacitors are found to outperform the SiO2 capacitors in the cases where the capacitors are negatively biased and the gate material is adequately selected to reduce virtual dipoles at the gate/Al2O3 interface. The Al2O3 electron affinity increases with the increasing ALD temperature, but the gate-side virtual dipoles are not affected. Therefore, the leakage current of negatively biased Al2O3 capacitors is approximately independent of the ALD temperature because of the compensation of the opposite effects of increased electron affinity and permittivity in Al2O3. By contrast, the substrate-side sheet of charge increases with increasing ALD temperature above 210 °C and hence enhances the current of positively biased Al2O3 capacitors more significantly at high temperatures. Additionally, an anomalous oscillatory shift of the current-voltage characteristics with ALD temperature was observed in positively biased capacitors formed by low-temperature (≤210 °C) ALD. This shift is caused by dipoles at the Al2O3/underlying SiO2 interface. Although they have a minimal positive-bias leakage current, the low-temperature-grown Al2O3 films cause the so-called blisters problem when heated above 400 °C. Therefore, because of the absence of blistering, a 450 °C ALD process is presently the most promising technology for growing high-reliability Al2O3 films.

Two-dimensional turbulent flow chromatography coupled on-line to liquid chromatography-mass spectrometry for solution-based ligand screening against multiple proteins.

PubMed

Zhou, Jian-Liang; An, Jing-Jing; Li, Ping; Li, Hui-Jun; Jiang, Yan; Cheng, Jie-Fei

2009-03-20

We present herein a novel bioseparation/chemical analysis strategy for protein-ligand screening and affinity ranking in compound mixtures, designed to increase screening rates and improve sensitivity and ruggedness in performance. The strategy is carried out by combining on-line two-dimensional turbulent flow chromatography (2D-TFC) with liquid chromatography-mass spectrometry (LC-MS), and accomplished through the following steps: (1) a reversed-phase TFC stage to separate the protein/ligand complex from the unbound free molecules, (2) an on-line dissociation process to release the bound ligands from the complexes, and (3) a second mixed-mode cation-exchange/reversed-phase TFC stage to trap the bound ligands and to remove the proteins and salts, followed by LC-MS analysis for identification and determination of the binding affinities. The technique can implement an ultra-fast isolation of protein/ligand complex with the retention time of a complex peak in about 5s, and on-line prepare the "clean" sample to be directly compatible with the LC-MS analysis. The improvement in performance of this 2D-TFC/LC-MS approach over the conventional approach has been demonstrated by determining affinity-selected ligands of the target proteins acetylcholinesterase and butyrylcholinesterase from a small library with known binding affinities and a steroidal alkaloid library composed of structurally similar compounds. Our results show that 2D-TFC/LC-MS is a generic and efficient tool for high-throughput screening of ligands with low-to-high binding affinities, and structure-activity relationship evaluation.

Low affinity PEGylated hemoglobin from Trematomus bernacchii, a model for hemoglobin-based blood substitutes

PubMed Central

2011-01-01

Background Conjugation of human and animal hemoglobins with polyethylene glycol has been widely explored as a means to develop blood substitutes, a novel pharmaceutical class to be used in surgery or emergency medicine. However, PEGylation of human hemoglobin led to products with significantly different oxygen binding properties with respect to the unmodified tetramer and high NO dioxygenase reactivity, known causes of toxicity. These recent findings call for the biotechnological development of stable, low-affinity PEGylated hemoglobins with low NO dioxygenase reactivity. Results To investigate the effects of PEGylation on protein structure and function, we compared the PEGylation products of human hemoglobin and Trematomus bernacchii hemoglobin, a natural variant endowed with a remarkably low oxygen affinity and high tetramer stability. We show that extension arm facilitated PEGylation chemistry based on the reaction of T. bernacchii hemoglobin with 2-iminothiolane and maleimido-functionalyzed polyethylene glycol (MW 5000 Da) leads to a tetraPEGylated product, more homogeneous than the corresponding derivative of human hemoglobin. PEGylated T. bernacchii hemoglobin largely retains the low affinity of the unmodified tetramer, with a p50 50 times higher than PEGylated human hemoglobin. Moreover, it is still sensitive to protons and the allosteric effector ATP, indicating the retention of allosteric regulation. It is also 10-fold less reactive towards nitrogen monoxide than PEGylated human hemoglobin. Conclusions These results indicate that PEGylated hemoglobins, provided that a suitable starting hemoglobin variant is chosen, can cover a wide range of oxygen-binding properties, potentially meeting the functional requirements of blood substitutes in terms of oxygen affinity, tetramer stability and NO dioxygenase reactivity. PMID:22185675

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α2β2Pro100Leu)

PubMed Central

Mollan, Todd L; Abraham, Bindu; Strader, Michael Brad; Jia, Yiping; Lozier, Jay N; Olson, John S; Alayash, Abdu I

2012-01-01

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O2 affinity compared with normal cells (P50 = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O2 affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO2, decreasing the rate approximately twofold. These kinetic data help explain the high O2 affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure. PMID:22821886

Role of β/δ101Gln in Regulating the Effect of Temperature and Allosteric Effectors on Oxygen Affinity in Woolly Mammoth Hemoglobin†

PubMed Central

Yuan, Yue; Byrd, Catherine; Shen, Tong-Jian; Simplaceanu, Virgil; Tam, Tsuey Chyi S.; Ho, Chien

2013-01-01

The oxygen affinity of woolly mammoth hemoglobin (rHb WM) is less affected by temperature change than that of Asian elephant hemoglobin (rHb AE) or human adult hemoglobin (Hb A). We report here a biochemical-biophysical study of Hb A, rHb AE, rHb WM and three rHb WM mutants with amino acid substitutions at β/δ101 (β/δ101Gln→Glu, Lys, or Asp) plus a double and a triple mutant, designed to clarify the role of the β/δ101 residue. The β/δ101Gln residue is important for responding to allosteric effectors, such as phosphate, inositol hexaphosphate (IHP), and chloride. The rHb WM mutants studied generally have higher affinity for oxygen under various conditions of pH, temperature, and salt concentration, and in the presence or absence of organic phosphate, than do rHb WM, rHb AE and Hb A. Titrations for the O2 affinity of these mutant rHbs as a function of chloride concentration indicate a lower heterotopic effect of this anion due to the replacement of β/δ101Gln in rHb WM. The alkaline Bohr effect of rHb WM and its mutants is reduced by 20–50% compared to that of Hb A and is independent of changes in temperature, in contrast to what has been observed in the hemoglobins of most mammalian species, including human. The results of our study on the temperature dependence of the O2 affinity of rHb WM and its mutant rHbs illustrate the important role of β/δ101Gln in regulating the functional properties of these hemoglobins. PMID:24228693

Fundamentals of affinity cell separations.

PubMed

Zhang, Ye; Lyons, Veronica; Pappas, Dimitri

2018-03-01

Cell separations using affinity methods continue to be an enabling science for a wide variety of applications. In this review, we discuss the fundamental aspects of affinity separation, including the competing forces for cell capture and elution, cell-surface interactions, and models for cell adhesion. Factors affecting separation performance such as bond affinity, contact area, and temperature are presented. We also discuss and demonstrate the effects of nonspecific binding on separation performance. Metrics for evaluating cell separations are presented, along with methods of comparing separation techniques for cell isolation using affinity capture. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The M sub 1 muscarinic receptor and its second messenger coupling in human neuroblastoma cells and transfected murine fibroblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mei, Lin.

1989-01-01

The data of this study indicate that pirenzepine (PZ)-high affinity muscarinic receptors (mAChRs) are coupled to the hydrolysis of inositol lipids and not to the adenylate cyclase system in human neuroblastoma SH-SY5Y cells. The maximal carbachol(CCh)-stimulated ({sup 3}H)IP{sub 1} accumulation in the SH-SY5Y cells was decreased in the presence of 1{mu}g/ml pertussis toxin, suggesting that a pertussis toxin sensitive G-protein may be involved in the coupling. Several cell clones which express only M{sub 1} mAChR were generated by transfecting the murine fibroblast B82 cells with the cloned rat genomic m{sub 1} gene. The transfected B82 cells (cTB10) showed specific ({supmore » 3}H)(-)QNB binding activity. The mAChRs in these cells are of the M{sub 1} type defined by their high affinity for PZ and low affinity for AF-DX 116 and coupled to hydrolysis of inositol lipids, possibly via a pertussis toxin sensitive G protein. The relationship between the M{sub 1} mAChR density and the receptor-mediated hydrolysis of inositol lipids was studied in 7 clones. The M{sub 1} mAChR densities in these cells characterized by ({sup 3}H)(-)MQNB binding ranged from 12 fmol/10{sup 6} cells in LK3-1 cells to 260 fmol/10{sup 6} cells in the LK3-8 cells.« less

Complex interactions between climate change and toxicants: evidence that temperature variability increases sensitivity to cadmium.

PubMed

Kimberly, David A; Salice, Christopher J

2014-07-01

The Intergovernmental Panel on Climate Change projects that global climate change will have significant impacts on environmental conditions including potential effects on sensitivity of organisms to environmental contaminants. The objective of this study was to test the climate-induced toxicant sensitivity (CITS) hypothesis in which acclimation to altered climate parameters increases toxicant sensitivity. Adult Physa pomilia snails were acclimated to a near optimal 22 °C or a high-normal 28 °C for 28 days. After 28 days, snails from each temperature group were challenged with either low (150 μg/L) or high (300 μg/L) cadmium at each temperature (28 or 22 °C). In contrast to the CITS hypothesis, we found that acclimation temperature did not have a strong influence on cadmium sensitivity except at the high cadmium test concentration where snails acclimated to 28 °C were more cadmium tolerant. However, snails that experienced a switch in temperature for the cadmium challenge, regardless of the switch direction, were the most sensitive to cadmium. Within the snails that were switched between temperatures, snails acclimated at 28 °C and then exposed to high cadmium at 22 °C exhibited significantly greater mortality than those snails acclimated to 22 °C and then exposed to cadmium at 28 °C. Our results point to the importance of temperature variability in increasing toxicant sensitivity but also suggest a potentially complex cost of temperature acclimation. Broadly, the type of temporal stressor exposures we simulated may reduce overall plasticity in responses to stress ultimately rendering populations more vulnerable to adverse effects.

Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities

PubMed Central

Petrič, Andrej; Johnson, Scott A.; Pham, Hung V.; Li, Ying; Čeh, Simon; Golobič, Amalija; Agdeppa, Eric D.; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N.; Barrio, Jorge R.

2012-01-01

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. PMID:23012452

Enhanced production of a single domain antibody with an engineered stabilizing extra disulfide bond.

PubMed

Liu, Jinny L; Goldman, Ellen R; Zabetakis, Dan; Walper, Scott A; Turner, Kendrick B; Shriver-Lake, Lisa C; Anderson, George P

2015-10-09

Single domain antibodies derived from the variable region of the unique heavy chain antibodies found in camelids yield high affinity and regenerable recognition elements. Adding an additional disulfide bond that bridges framework regions is a proven method to increase their melting temperature, however often at the expense of protein production. To fulfill their full potential it is essential to achieve robust protein production of these stable binding elements. In this work, we tested the hypothesis that decreasing the isoelectric point of single domain antibody extra disulfide bond mutants whose production fell due to the incorporation of the extra disulfide bond would lead to recovery of the protein yield, while maintaining the favorable melting temperature and affinity. Introduction of negative charges into a disulfide bond mutant of a single domain antibody specific for the L1 antigen of the vaccinia virus led to approximately 3.5-fold increase of protein production to 14 mg/L, while affinity and melting temperature was maintained. In addition, refolding following heat denaturation improved from 15 to 70 %. It also maintained nearly 100 % of its binding function after heating to 85 °C for an hour at 1 mg/mL. Disappointingly, the replacement of neutral or positively charged amino acids with negatively charged ones to lower the isoelectric point of two anti-toxin single domain antibodies stabilized with a second disulfide bond yielded only slight increases in protein production. Nonetheless, for one of these binders the charge change itself stabilized the structure equivalent to disulfide bond addition, thus providing an alternative route to stabilization which is not accompanied by loss in production. The ability to produce high affinity, stable single domain antibodies is critical for their utility. While the addition of a second disulfide bond is a proven method for enhancing stability of single domain antibodies, it frequently comes at the cost of reduced yields. While decreasing the isoelectric point of double disulfide mutants of single domain antibodies may improve protein production, charge addition appears to consistently improve refolding and some charge changes can also improve thermal stability, thus providing a number of benefits making the examination of such mutations worth consideration.

Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang Yongmin; IgE Therapeutics, Inc., San Diego, CA 92121-2233; Barankiewicz, Teresa J.

2007-07-27

Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes onmore » either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.« less

Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

PubMed Central

Camps, M; Gumà, A; Viñals, F; Testar, X; Palacín, M; Zorzano, A

1992-01-01

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle. PMID:1323279

Evidence for the lack of spare high-affinity insulin receptors in skeletal muscle.

PubMed

Camps, M; Gumà, A; Viñals, F; Testar, X; Palacín, M; Zorzano, A

1992-08-01

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle.

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes.

PubMed

Ribeiro, Mariana Borsoi; Vijayalakshmi, Mookambesvaran; Todorova-Balvay, Daniele; Bueno, Sonia Maria Alves

2008-01-01

The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.

Modular, Antibody-free Time-Resolved LRET Kinase Assay Enabled by Quantum Dots and Tb3+-sensitizing Peptides

NASA Astrophysics Data System (ADS)

Cui, Wei; Parker, Laurie L.

2016-07-01

Fluorescent drug screening assays are essential for tyrosine kinase inhibitor discovery. Here we demonstrate a flexible, antibody-free TR-LRET kinase assay strategy that is enabled by the combination of streptavidin-coated quantum dot (QD) acceptors and biotinylated, Tb3+ sensitizing peptide donors. By exploiting the spectral features of Tb3+ and QD, and the high binding affinity of the streptavidin-biotin interaction, we achieved multiplexed detection of kinase activity in a modular fashion without requiring additional covalent labeling of each peptide substrate. This strategy is compatible with high-throughput screening, and should be adaptable to the rapidly changing workflows and targets involved in kinase inhibitor discovery.

Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes.

PubMed

Anbazhagan, V; Sankhala, Rajeshwer S; Singh, Bhanu Pratap; Swamy, Musti J

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.

Isothermal Titration Calorimetric Studies on the Interaction of the Major Bovine Seminal Plasma Protein, PDC-109 with Phospholipid Membranes

PubMed Central

Anbazhagan, V.; Sankhala, Rajeshwer S.; Singh, Bhanu Pratap; Swamy, Musti J.

2011-01-01

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process. PMID:22022488

Sodium channel from rat brain. Reconstitution of voltage-dependent scorpion toxin binding in vesicles of defined lipid composition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feller, D.J.; Talvenheimo, J.A.; Catterall, W.A.

1985-09-25

Purified sodium channels incorporated into phosphatidylcholine (PC) vesicles mediate neurotoxin-activated SSNa influx but do not bind the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx) with high affinity. Addition of phosphatidylethanolamine (PE) or phosphatidylserine to the reconstitution mixture restores high affinity LqTx binding with KD = 1.9 nM for PC/PE vesicles at -90 mV and 36 degrees C in sucrose-substituted medium. Other lipids tested were markedly less effective. The binding of LqTx in vesicles of PC/PE (65:35) is sensitive to both the membrane potential formed by sodium gradients across the reconstituted vesicle membrane and the cation concentration in the extravesicular medium. Bindingmore » of LqTx is reduced 3- to 4-fold upon depolarization to 0 mV from -50 to -60 mV in experiments in which (Na+)out/(Na+)in is varied by changing (Na+)in or (Na+)out at constant extravesicular ionic strength. It is concluded that the purified sodium channel contains the receptor site for LqTx in functional form and that restoration of high affinity, voltage-dependent binding of LqTx by the purified sodium channel requires an appropriate ratio of PC to PE and/or phosphatidylserine in the vesicle membrane.« less

A sensitive and selective resonance Rayleigh scattering method for quick detection of avidin using affinity labeling Au nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Qi; Huang, Xi; Fu, Xuan; Deng, Huan; Ma, Meihu; Cai, Zhaoxia

2016-06-01

Avidin is a glycoprotein with antinutritional property, which should be limited in daily food. We developed an affinity biosensor system based on resonance Rayleigh scattering (RRS) and using affinity biotin labeling Au nanoparticles (AuNPs). This method was selective and sensitive for quick avidin detection due to the avidin-biotin affinitive interaction. Under optimal conditions, RRS intensity of biotin-AuNPs increase linearly with an increasing concentration of avidin from 5 to 160 ng/mL. The lower limit of detection was 0.59 ng/mL. This rapid and selective avidin detection method was used in synthetic samples and egg products with recoveries of between 102.97 and 107.92%, thereby demonstrating the feasible and practical application of this assay.

Optimizing pH response of affinity between protein G and IgG Fc: how electrostatic modulations affect protein-protein interactions.

PubMed

Watanabe, Hideki; Matsumaru, Hiroyuki; Ooishi, Ayako; Feng, Yanwen; Odahara, Takayuki; Suto, Kyoko; Honda, Shinya

2009-05-01

Protein-protein interaction in response to environmental conditions enables sophisticated biological and biotechnological processes. Aiming toward the rational design of a pH-sensitive protein-protein interaction, we engineered pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG constant region. We systematically introduced histidine residues into the binding interface to cause electrostatic repulsion on the basis of a rigid body model. Exquisite pH sensitivity of this interaction was confirmed by surface plasmon resonance and affinity chromatography employing a clinically used human IgG. The pH-sensitive mechanism of the interaction was analyzed and evaluated from kinetic, thermodynamic, and structural viewpoints. Histidine-mediated electrostatic repulsion resulted in significant loss of exothermic heat of the binding that decreased the affinity only at acidic conditions, thereby improving the pH sensitivity. The reduced binding energy was partly recovered by "enthalpy-entropy compensation." Crystal structures of the designed mutants confirmed the validity of the rigid body model on which the effective electrostatic repulsion was based. Moreover, our data suggested that the entropy gain involved exclusion of water molecules solvated in a space formed by the introduced histidine and adjacent tryptophan residue. Our findings concerning the mechanism of histidine-introduced interactions will provide a guideline for the rational design of pH-sensitive protein-protein recognition.

Introduction of structural affinity handles as a tool in selective nucleic acid separations

NASA Technical Reports Server (NTRS)

Willson, III, Richard Coale (Inventor); Cano, Luis Antonio (Inventor)

2011-01-01

The method is used for separating nucleic acids and other similar constructs. It involves selective introduction, enhancement, or stabilization of affinity handles such as single-strandedness in the undesired (or desired) nucleic acids as compared to the usual structure (e.g., double-strandedness) of the desired (or undesired) nucleic acids. The undesired (or desired) nucleic acids are separated from the desired (or undesired) nucleic acids due to capture by methods including but not limited to immobilized metal affinity chromatography, immobilized single-stranded DNA binding (SSB) protein, and immobilized oligonucleotides. The invention is useful to: remove contaminating genomic DNA from plasmid DNA; remove genomic DNA from plasmids, BACs, and similar constructs; selectively separate oligonucleotides and similar DNA fragments from their partner strands; purification of aptamers, (deoxy)-ribozymes and other highly structured nucleic acids; Separation of restriction fragments without using agarose gels; manufacture recombinant Taq polymerase or similar products that are sensitive to host genomic DNA contamination; and other applications.

Preparation and characterization of monoclonal antibody against digoxin.

PubMed

Kashanian, S; Rasaee, M J; Paknejad, M; Omidfar, K; Pour-Amir, M; Rajabi, Bazl M

2002-10-01

Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.

A cooperative-binding split aptamer assay for rapid, specific and ultra-sensitive fluorescence detection of cocaine in saliva† †Electronic supplementary information (ESI) available: Optimization of Mg2+ and ATMND concentrations for our CBSA-based ATMND-binding assay; ATMND-reported calibration curve for CBSA-5325 at various cocaine concentrations; ATMND binding affinity for the cocaine-assembled CBSA-5325; K D of 38-GC and different 38-GC mutants for cocaine as characterized by ITC; stem length effects on cocaine-induced CBSA assembly; spectra of CBSA-5335-based fluorescence detection of cocaine in 1× binding buffer; characterization of cocaine binding affinity of CBSA-5335 and PSA using ITC; fluorescence detection of cocaine in saliva with our fluorophore/quencher modified CBSA-5335; calibration curve of our CBSA-5335-based fluorophore/quencher assay in 1× binding buffer and 10% saliva at cocaine concentrations ranging from 0 to 10 μM; bias and precision of the CBSA-5335-based fluorophore/quencher assay; comparison of amplification-free split-aptamer assays for cocaine detection; sequence ID and DNA sequences used in this work. See DOI: 10.1039/c6sc01833e Click here for additional data file.

PubMed Central

Yu, Haixiang; Canoura, Juan; Guntupalli, Bhargav; Lou, Xinhui

2017-01-01

Sensors employing split aptamers that reassemble in the presence of a target can achieve excellent specificity, but the accompanying reduction of target affinity mitigates any overall gains in sensitivity. We for the first time have developed a split aptamer that achieves enhanced target-binding affinity through cooperative binding. We have generated a split cocaine-binding aptamer that incorporates two binding domains, such that target binding at one domain greatly increases the affinity of the second domain. We experimentally demonstrate that the resulting cooperative-binding split aptamer (CBSA) exhibits higher target binding affinity and is far more responsive in terms of target-induced aptamer assembly compared to the single-domain parent split aptamer (PSA) from which it was derived. We further confirm that the target-binding affinity of our CBSA can be affected by the cooperativity of its binding domains and the intrinsic affinity of its PSA. To the best of our knowledge, CBSA-5335 has the highest cocaine affinity of any split aptamer described to date. The CBSA-based assay also demonstrates excellent performance in target detection in complex samples. Using this CBSA, we achieved specific, ultra-sensitive, one-step fluorescence detection of cocaine within fifteen minutes at concentrations as low as 50 nM in 10% saliva without signal amplification. This limit of detection meets the standards recommended by the European Union's Driving under the Influence of Drugs, Alcohol and Medicines program. Our assay also demonstrates excellent reproducibility of results, confirming that this CBSA-platform represents a robust and sensitive means for cocaine detection in actual clinical samples. PMID:28451157

Electrical Detection of C-Reactive Protein Using a Single Free-Standing, Thermally Controlled Piezoresistive Microcantilever for Highly Reproducible and Accurate Measurements

PubMed Central

Yen, Yi-Kuang; Lai, Yu-Cheng; Hong, Wei-Ting; Pheanpanitporn, Yotsapoom; Chen, Chuin-Shan; Huang, Long-Sun

2013-01-01

This study demonstrates a novel method for electrical detection of C-reactive protein (CRP) as a means of identifying an infection in the body, or as a cardiovascular disease risk assay. The method uses a single free-standing, thermally controlled piezoresistive microcantilever biosensor. In a commonly used sensing arrangement of conventional dual cantilevers in the Wheatstone bridge circuit, reference and gold-coated sensing cantilevers that inherently have heterogeneous surface materials and different multilayer structures may yield independent responses to the liquid environmental changes of chemical substances, flow field and temperature, leading to unwanted signal disturbance for biosensing targets. In this study, the single free-standing microcantilever for biosensing applications is employed to resolve the dual-beam problem of individual responses in chemical solutions and, in a thermally controlled system, to maintain its sensor performance due to the sensitive temperature effect. With this type of single temperature-controlled microcantilever sensor, the electrical detection of various CRP concentrations from 1 μg/mL to 200 μg/mL was performed, which covers the clinically relevant range. Induced surface stresses were measured at between 0.25 N/m and 3.4 N/m with high reproducibility. Moreover, the binding affinity (KD) of CRP and anti-CRP interaction was found to be 18.83 ± 2.99 μg/mL, which agreed with results in previous reported studies. This biosensing technique thus proves valuable in detecting inflammation, and in cardiovascular disease risk assays. PMID:23899933

Graphene-Templated Synthesis of Magnetic Metal Organic Framework Nanocomposites for Selective Enrichment of Biomolecules.

PubMed

Cheng, Gong; Wang, Zhi-Gang; Denagamage, Sachira; Zheng, Si-Yang

2016-04-27

Successful control of homogeneous and complete coating of graphene or graphene-based composites with well-defined metal organic framework (MOF) layers is a great challenge. Herein, novel magnetic graphene MOF composites were constructed via a simple strategy for self-assembly of well-distributed, dense, and highly porous MOFs on both sides of graphene nanosheets. Graphene functionalized with magnetic nanoparticles and carboxylic groups on both sides was explored as the backbone and template to direct the controllable self-assembly of MOFs. The prepared composite materials have a relatively high specific surface area (345.4 m(2) g(-1)), and their average pore size is measured to be 3.2 nm. Their relatively high saturation magnetization (23.8 emu g(-1)) indicates their strong magnetism at room temperature. Moreover, the multifunctional composite was demonstrated to be a highly effective affinity material in selective extraction and separation of low-concentration biomolecules from biological samples, in virtue of the size-selection property of the unique porous structure and the excellent affinity of the composite materials. Besides providing a solution for the construction of well-defined functional graphene-based MOFs, this work could also contribute to selective extraction of biomolecules, in virtue of the universal affinity between immobilized metal ions and biomolecules.

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

PubMed Central

Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

2016-01-01

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

High-temperature microphone system. [for measuring pressure fluctuations in gases at high temperature

NASA Technical Reports Server (NTRS)

Zuckerwar, A. J. (Inventor)

1979-01-01

Pressure fluctuations in air or other gases in an area of elevated temperature are measured using a condenser microphone located in the area of elevated temperature and electronics for processing changes in the microphone capacitance located outside the area the area and connected to the microphone by means of high-temperature cable assembly. The microphone includes apparatus for decreasing the undesirable change in microphone sensitivity at high temperatures. The high temperature cable assembly operates as a half-wavelength transmission line in an AM carrier system and maintains a large temperature gradient between the two ends of the cable assembly. The processing electronics utilizes a voltage controlled oscillator for automatic tuning thereby increasing the sensitivity of the measuring apparatus.

Phase Interrogation Used for a Wireless Passive Pressure Sensor in an 800 °C High-Temperature Environment

PubMed Central

Zhang, Huixin; Hong, Yingping; Liang, Ting; Zhang, Hairui; Tan, Qiulin; Xue, Chenyang; Liu, Jun; Zhang, Wendong; Xiong, Jijun

2015-01-01

A wireless passive pressure measurement system for an 800 °C high-temperature environment is proposed and the impedance variation caused by the mutual coupling between a read antenna and a LC resonant sensor is analyzed. The system consists of a ceramic-based LC resonant sensor, a readout device for impedance phase interrogation, heat insulating material, and a composite temperature-pressure test platform. Performances of the pressure sensor are measured by the measurement system sufficiently, including pressure sensitivity at room temperature, zero drift from room temperature to 800 °C, and the pressure sensitivity under the 800 °C high temperature environment. The results show that the linearity of sensor is 0.93%, the repeatability is 6.6%, the hysteretic error is 1.67%, and the sensor sensitivity is 374 KHz/bar. The proposed measurement system, with high engineering value, demonstrates good pressure sensing performance in a high temperature environment. PMID:25690546

Energetic basis on interactions between ferredoxin and ferredoxin NADP{sup +} reductase at varying physiological conditions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kinoshita, Misaki; Kim, Ju Yaen; Kume, Satoshi

In spite of a number of studies to characterize ferredoxin (Fd):ferredoxin NADP{sup +} reductase (FNR) interactions at limited conditions, detailed energetic investigation on how these proteins interact under near physiological conditions and its linkage to FNR activity are still lacking. We herein performed systematic Fd:FNR binding thermodynamics using isothermal titration calorimetry (ITC) at distinct pH (6.0 and 8.0), NaCl concentrations (0–200 mM), and temperatures (19–28 °C) for mimicking physiological conditions in chloroplasts. Energetically unfavorable endothermic enthalpy changes were accompanied by Fd:FNR complexation at all conditions. This energetic cost was compensated by favorable entropy changes, balanced by conformational and hydrational entropy. Increases inmore » the NaCl concentration and pH weakened interprotein affinity due to the less contribution of favorable entropy change regardless of energetic gains from enthalpy changes, suggesting that entropy drove complexation and modulated affinity. Effects of temperature on binding thermodynamics were much smaller than those of pH and NaCl. NaCl concentration and pH-dependent enthalpy and heat capacity changes provided clues for distinct binding modes. Moreover, decreases in the enthalpy level in the Hammond's postulate-based energy landscape implicated kinetic advantages for FNR activity. All these energetic interplays were comprehensively demonstrated by the driving force plot with the enthalpy-entropy compensation which may serve as an energetic buffer against outer stresses. We propose that high affinity at pH 6.0 may be beneficial for protection from proteolysis of Fd and FNR in rest states, and moderate affinity at pH 8.0 and proper NaCl concentrations with smaller endothermic enthalpy changes may contribute to increase FNR activity. - Highlights: • Energetics of Fd:FNR binding were examined by considering physiological conditions. • NaCl and pH affect energetically Fd:FNR binding with minimal effects of temperature. • Enthalpy and heat capacity may modulate binding kinetics and modes for FNR activity. • Entropy drives complexation by overcoming unfavorable enthalpy and tunes affinity. • Driving force plot reveals condition-dependent energetic interplays for complexation.« less

Single-residue molecular switch for high-temperature dependence of vanilloid receptor TRPV3

PubMed Central

Liu, Beiying; Qin, Feng

2017-01-01

Thermal transient receptor potential (TRP) channels, a group of ion channels from the transient receptor potential family, play important functions in pain and thermal sensation. These channels are directly activated by temperature and possess strong temperature dependence. Furthermore, their temperature sensitivity can be highly dynamic and use-dependent. For example, the vanilloid receptor transient receptor potential 3 (TRPV3), which has been implicated as a warmth detector, becomes responsive to warm temperatures only after intensive stimulation. Upon initial activation, the channel exhibits a high-temperature threshold in the noxious temperature range above 50 °C. This use dependence of heat sensitivity thus provides a mechanism for sensitization of thermal channels. However, how the channels acquire the use dependence remains unknown. Here, by comparative studies of chimeric channels between use-dependent and use-independent homologs, we have determined the molecular basis that underlies the use dependence of temperature sensitivity of TRPV3. Remarkably, the restoration of a single residue that is apparently missing in the use-dependent homologs could largely eliminate the use dependence of heat sensitivity of TRPV3. The location of the region suggests a mechanism of temperature-dependent gating of thermal TRP channels involving an intracellular region assembled around the TRP domain. PMID:28154143

Normalized Synergy Predicts That CD8 Co-Receptor Contribution to T Cell Receptor (TCR) and pMHC Binding Decreases As TCR Affinity Increases in Human Viral-Specific T Cells

PubMed Central

Williams, Chad M.; Schonnesen, Alexandra A.; Zhang, Shu-Qi; Ma, Ke-Yue; He, Chenfeng; Yamamoto, Tori; Eckhardt, S. Gail; Klebanoff, Christopher A.; Jiang, Ning

2017-01-01

The discovery of naturally occurring T cell receptors (TCRs) that confer specific, high-affinity recognition of pathogen and cancer-associated antigens remains a major goal in cellular immunotherapies. The contribution of the CD8 co-receptor to the interaction between the TCR and peptide-bound major histocompatibility complex (pMHC) has previously been correlated with the activation and responsiveness of CD8+ T cells. However, these studies have been limited to model systems of genetically engineered hybridoma TCRs or transgenic mouse TCRs against either a single epitope or an array of altered peptide ligands. CD8 contribution in a native human antigen-specific T cell response remains elusive. Here, using Hepatitis C Virus-specific precursor CTLs spanning a large range of TCR affinities, we discovered that the functional responsiveness of any given TCR correlated with the contribution of CD8 to TCR/pMHC binding. Furthermore, we found that CD8 contribution to TCR/pMHC binding in the two-dimensional (2D) system was more accurately reflected by normalized synergy (CD8 cooperation normalized by total TCR/pMHC bonds) rather than synergy (total CD8 cooperation) alone. While synergy showed an increasing trend with TCR affinity, normalized synergy was demonstrated to decrease with the increase of TCR affinity. Critically, normalized synergy was shown to correlate with CTL functionality and peptide sensitivity, corroborating three-dimensional (3D) analysis of CD8 contribution with respect to TCR affinity. In addition, we identified TCRs that were independent of CD8 for TCR/pMHC binding. Our results resolve the current discrepancy between 2D and 3D analysis on CD8 contribution to TCR/pMHC binding, and demonstrate that naturally occurring high-affinity TCRs are more capable of CD8-independent interactions that yield greater functional responsiveness even with CD8 blocking. Taken together, our data suggest that addition of the normalized synergy parameter to our previously established TCR discovery platform using 2D TCR affinity and sequence test would allow for selection of TCRs specific to any given antigen with the desirable attributes of high TCR affinity, CD8 co-receptor independence and functional superiority. Utilizing TCRs with less CD8 contribution could be beneficial for adoptive cell transfer immunotherapies using naturally occurring or genetically engineered T cells against viral or cancer-associated antigens. PMID:28804489

Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

PubMed Central

Alexander, Crispin G.; Wanner, Randy; Johnson, Christopher M.; Breitsprecher, Dennis; Winter, Gerhard; Duhr, Stefan; Baaske, Philipp; Ferguson, Neil

2014-01-01

Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein–ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide–PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes. PMID:25262836

Surface Effects and Challenges for Application of Piezoelectric Langasite Substrates in Surface Acoustic Wave Devices Caused by High Temperature Annealing under High Vacuum.

PubMed

Seifert, Marietta; Rane, Gayatri K; Kirbus, Benjamin; Menzel, Siegfried B; Gemming, Thomas

2015-12-19

Substrate materials that are high-temperature stable are essential for sensor devices which are applied at high temperatures. Although langasite is suggested as such a material, severe O and Ga diffusion into an O-affine deposited film was observed during annealing at high temperatures under vacuum conditions, leading to a damage of the metallization as well as a change of the properties of the substrate and finally to a failure of the device. Therefore, annealing of bare LGS (La 3 Ga 5 SiO 14 ) substrates at 800 ∘ C under high vacuum conditions is performed to analyze whether this pretreatment improves the suitability and stability of this material for high temperature applications in vacuum. To reveal the influence of the pretreatment on the subsequently deposited metallization, RuAl thin films are used as they are known to oxidize on LGS at high temperatures. A local study of the pretreated and metallized substrates using transmission electron microscopy reveals strong modification of the substrate surface. Micro cracks are visible. The composition of the substrate is strongly altered at those regions. Severe challenges for the application of LGS substrates under high-temperature vacuum conditions arise from these substrate damages, revealing that the pretreatment does not improve the applicability.

Fluorescent vancomycin and terephthalate comodified europium-doped layered double hydroxides nanoparticles: synthesis and application for bacteria labelling

NASA Astrophysics Data System (ADS)

Sun, Jianchao; Fan, Hai; Wang, Nan; Ai, Shiyun

2014-09-01

Vancomycin (Van)- and terephthalate (TA)-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles were successfully prepared by a two-step method, in which, TA acted as a sensitizer to enhance the fluorescent property and Van was modified on the surface of LDH to act as an affinity reagent to bacteria. The obtained products were characterized by X-ray diffraction, transmission electron microscope and fluorescent spectroscopy. The results demonstrated that the prepared Van- and TA-comodified europium-doped layered double hydroxides (Van-TA-Eu-LDHs) nanoparticles with diameter of 50 nm in size showed highly efficient fluorescent property. Furthermore, due to the high affinity of Van to bacteria, the prepared Van-TA-Eu-LDHs nanoparticles showed efficient bacteria labelling by fluorescent property. The prepared nanoparticles may have wide applications in the biological fields, such as biomolecular labelling and cell imaging.

Volatile anesthetics interfere with muscarinic receptor-g protein interactions in rat heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Anthony, B.L.

The influence of halothane and enflurane (0.5-8%) on muscarinic receptor binding in rat atrium was studied using (/sup 3/H) methylscopolamine ((/sup 3/H)MS). Anesthetic-gas mixtures were blown over membrane suspensions for 20 min before and during the binding assays. Halothane and enflurane increased the affinity of cardiac muscarinic receptors for (/sup 3/H)MS by slowing the rate of dissociation. These anesthetics did not affect the affinity of the receptor for carbamylcholine, but significantly reduced the sensitivity of agonist binding to regulation by guanine nucleotides. For example, the fraction of receptors displaying high affinity agonist binding was decreased by a GTP analog frommore » 0.64 to 0.43 in the absence, but only to 0.52 in the presence of 2% halothane. The binding of a radiolabeled agonist, (/sup 3/H)oxotremorine-M, was reduced by 50% by halothane, while its sensitivity to guanine nucleotides was reduced by at least 100 fold. The diminution of the guanine nucleotide effect may reflect a stabilization of the receptor-G proteincomplex due to either a direct action on the receptor complex or to an alteration of the physical state of the membrane. It is also possible that the ability of the G protein to bind guanine nucleotides is adversely affected by anesthetic agents.« less

Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

PubMed

Rojo, Liliana; García-Carreño, Fernando; de Los Angeles Navarrete del Toro, Maria

2013-02-01

Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

Characterization of the three different states of the cholecystokinin (CCK) receptor in pancreatic acini.

PubMed

Talkad, V D; Patto, R J; Metz, D C; Turner, R J; Fortune, K P; Bhat, S T; Gardner, J D

1994-10-20

By measuring binding of [125I]CCK-8 and [3H]L-364,718 to rat pancreatic acini we demonstrated directly that the pancreatic CCK receptor can exist in three different affinity states with respect to CCK--high affinity, low affinity and very low affinity. Binding of [125I]CCK-8 reflects interaction of the tracer with the high and low affinity states, whereas binding of [3H]L-364,718 reflects interaction of the tracer with the low and very low affinity states. Treating acini with carbachol abolished the high affinity state of the CCK receptor and converted approximately 25% of the low affinity receptors to the very low affinity state. Carbachol treatment was particularly useful in establishing the values of Kd for the high and low affinity states for different CCK receptor agonists and antagonists. Of the various CCK receptor agonists tested, CCK-8 had the highest affinity for the high affinity state (Kd approximately 1 nM), whereas CCK-JMV-180 had the highest affinity for the low (Kd 7 nM) and very low affinity (Kd 200 nM) states. Gastrin and de(SO4)CCK-8 had affinities for the high and low affinity states of the receptor that were 100- to 400-fold less than those of CCK-8 but had affinities for the very low affinity state that were only 3- to 10-fold less than that of CCK-8. CCK receptor antagonists showed several patterns in interacting with the different states of the CCK receptor. L-364,718 had the same affinity for each state of the CCK receptor. CR1409 and Bt2cGMP each had similar affinities for the high and low affinity states and lower affinity for the very low affinity state. L-365,260 and CCK-JMV-179 had the highest affinity for the low affinity state and lower affinities for the high and very low affinity states. Different CCK receptor agonists caused the same maximal stimulation of amylase secretion but showed different degrees of amplification in terms of the relationship between their abilities to stimulate amylase secretion and their abilities to occupy the low affinity state of the CCK receptor. When amplification was expressed quantitatively as the value of Kd for the low affinity state divided by the corresponding EC50 for stimulating amylase secretion the values were CCK-8 (1000), de(SO)CCK-8 (1500), gastrin (100) and CCK-JMV-180 (Menozzi, D., Vinayek, R., Jensen, R.T. and Gardner, J.D. (1991) J. Biol. Chem. 266, 10385-1091).(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrahigh-sensitive sensing platform based on p-type dumbbell-like Co3O4 network

NASA Astrophysics Data System (ADS)

Zhou, Tingting; Zhang, Tong; Zhang, Rui; Lou, Zheng; Deng, Jianan; Wang, Lili

2017-12-01

Development of high performance room temperature sensors remains a grand challenge for high demand of practical application. Metal oxide semiconductors (MOSs) have many advantages over others due to their easy functionalization, high surface area, and low cost. However, they typically need a high work temperature during sensing process. Here, p-type sensing layer is reported, consisting of pore-rich dumbbell-like Co3O4 particles (DP-Co3O4) with intrinsic high catalytic activity. The gas sensor (GS) based DP-Co3O4 catalyst exhibits ultrahigh NH3 sensing activity along with excellent stability over other structure based NH3 GSs in room temperature work environment. In addition, the unique structure of DP-Co3O4 with pore-rich and high catalytic activity endows fast gas diffusion rate and high sensitivity at room temperature. Taken together, the findings in this work highlight the merit of integrating highly active materials in p-type materials, offering a framework to develop high-sensitivity room temperature sensing platforms.

Thermodynamic analysis of the interaction of factor VIII with von Willebrand factor.

PubMed

Dimitrov, Jordan D; Christophe, Olivier D; Kang, Jonghoon; Repessé, Yohann; Delignat, Sandrine; Kaveri, Srinivas V; Lacroix-Desmazes, Sébastien

2012-05-22

Factor VIII (FVIII) is a glycoprotein that plays an important role in the intrinsic pathway of coagulation. In circulation, FVIII is protected upon binding to von Willebrand factor (VWF), a chaperone molecule that regulates its half-life, distribution, and activity. Despite the biological significance of this interaction, its molecular mechanisms are not fully characterized. We determined the equilibrium and activation thermodynamics of the interaction between FVIII and VWF. The equilibrium affinity determined by surface plasmon resonance was temperature-dependent with a value of 0.8 nM at 35 °C. The FVIII-VWF interaction was characterized by very fast association (8.56 × 10(6) M(-1) s(-1)) and fast dissociation (6.89 × 10(-3) s(-1)) rates. Both the equilibrium association and association rate constants, but not the dissociation rate constant, were dependent on temperature. Binding of FVIII to VWF was characterized by favorable changes in the equilibrium and activation entropy (TΔS° = 89.4 kJ/mol, and -TΔS(++) = -8.9 kJ/mol) and unfavorable changes in the equilibrium and activation enthalpy (ΔH° = 39.1 kJ/mol, and ΔH(++) = 44.1 kJ/mol), yielding a negative change in the equilibrium Gibbs energy. Binding of FVIII to VWF in solid-phase assays demonstrated a high sensitivity to acidic pH and a sensitivity to ionic strength. Our data indicate that the interaction between FVIII and VWF is mediated mainly by electrostatic forces, and that it is not accompanied by entropic constraints, suggesting the absence of conformational adaptation but the presence of rigid "pre-optimized" binding surfaces.

Sensitive Targeted Quantification of ERK Phosphorylation Dynamics and Stoichiometry in Human Cells without Affinity Enrichment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J.

2014-12-17

Mass spectrometry-based targeted quantification is a promising technology for site-specific quantification of posttranslational modifications (PTMs). However, a major constraint of most targeted MS approaches is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents to enrich specific PTMs. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometries using a highly sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection and multiplexing (PRISM). PRISM provides effective enrichment of target peptides within a given fraction from complex biological matrix with minimal sample losses, followed by selected reactionmore » monitoring (SRM) quantification. The PRISM-SRM approach enabled direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) from as little as 25 µg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided >10-fold improvement in signal intensities, presumably due to the better peptide recovery of PRISM for handling small size samples. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of EGF at both the peak activation (10 min) and steady state (2 h). At 10 min, the maximal ERK activation was observed with 0.3 ng/mL dose, whereas the maximal steady state level of ERK activation at 2 h was at 3 ng/ml dose, corresponding to 1200 and 9000 occupied receptors, respectively. At 10 min, the maximally activated pTpY isoform represented ~40% of total ERK, falling to less than 10% at 2 h. The time course and dose-response profiles of individual phosphorylated ERK isoforms indicated that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed, model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.« less

Coordination Environment of a Site-Bound Metal Ion in the Hammerhead Ribozyme Determined by 15N and 2H ESEEM Spectroscopy

PubMed Central

Vogt, Matthew; Lahiri, Simanti; Hoogstraten, Charles G.; Britt, R. David; DeRose, Victoria J.

2010-01-01

Although site-bound Mg2+ ions have been proposed to influence RNA structure and function, establishing the molecular properties of such sites has been challenging due largely to the unique electrostatic properties of the RNA biopolymer. We have previously determined that, in solution, the hammerhead ribozyme (a self-cleaving RNA) has a high-affinity metal ion binding site characterized by a Kd,app < 10 µM for Mn2+ in 1 M NaCl and speculated that this site has functional importance in the ribozyme cleavage reaction. Here we determine both the precise location and the hydration level of Mn2+ in this site using ESEEM (electron spin–echo envelope modulation) spectroscopy. Definitive assignment of the high-affinity site to the activity-sensitive A9/G10.1 region is achieved by site-specific labeling of G10.1 with 15N guanine. The coordinated metal ion retains four water ligands as measured by 2H ESEEM spectroscopy. The results presented here show that a functionally important, specific metal binding site is uniquely populated in the hammerhead ribozyme even in a background of high ionic strength. Although it has a relatively high thermodynamic affinity, this ion remains partially hydrated and is chelated to the RNA by just two ligands. PMID:17177426

Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries.

PubMed

Robinson, P A; Anderton, B H; Loviny, T L

1988-04-06

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.

Electrochemical affinity biosensors for detection of mycotoxins: A review.

PubMed

Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

2013-11-15

This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago. Copyright © 2013 Elsevier B.V. All rights reserved.

Synthesis and Biological Evaluation of Cyclic [99mTc]-HYNIC-CGPRPPC as a Fibrin-Binding Peptide for Molecular Imaging of Thrombosis and Its Comparison with [99mTc]-HYNIC-GPRPP.

PubMed

Rezaeianpour, Sedigheh; Bozorgi, Atefeh Hajiagha; Moghimi, Abolghasem; Almasi, Ameneh; Balalaie, Saeed; Ramezanpour, Sorour; Nasoohi, Sanaz; Mazidi, Seyed Mohammad; Geramifar, Parham; Bitarafan-Rajabi, Ahmad; Shahhosseini, Soraya

2017-04-01

Many patients worldwide suffer from cardiovascular diseases for which an underlying factor is thrombosis. Devising a molecular imaging technique for early detection of thrombosis in a clinical setting is highly recommended. Because fibrin is a major constituent of clots and is present in all types of thrombi but absent in circulation, it is a highly specific and sensitive target for molecular imaging of thrombi. It is assumed that cyclization of peptides will improve the receptor binding affinity and stability of the peptide. In the present study, we have developed linear and cyclic fibrin-binding peptides for thrombus imaging and compared their biological properties. Linear HYNIC-GPRPP and cyclic HYNIC-CGPRPPC peptides were synthesized using a standard Fmoc strategy and radiolabeled with Tc-99m. The stability of the radiolabeled peptides in human plasma and their affinity for fibrin and blood clots were determined. Blood clearance and biodistribution were evaluated in rats and mice, respectively. The peptide with the highest affinity was injected to a live rabbit femoral thrombosis model, and scintigraphic images were obtained. In vitro studies show that peptides are stable in human plasma and have a high affinity for human fibrin. They also demonstrated fast blood clearance in rats and high thrombus uptake in the Balb/c mice femoral thrombosis model. Femoral thrombosis was visualized 30 min postinjection of cyclic peptide in a live rabbit model using single photon emission computed tomography (SPECT)/X-ray computed tomography. The results indicate that the cyclic peptide is a promising agent for molecular imaging of fibrin using SPECT.

ODE/IM correspondence for modified B2(1) affine Toda field equation

NASA Astrophysics Data System (ADS)

Ito, Katsushi; Shu, Hongfei

2017-03-01

We study the massive ODE/IM correspondence for modified B2(1) affine Toda field equation. Based on the ψ-system for the solutions of the associated linear problem, we obtain the Bethe ansatz equations. We also discuss the T-Q relations, the T-system and the Y-system, which are shown to be related to those of the A3 /Z2 integrable system. We consider the case that the solution of the linear problem has a monodromy around the origin, which imposes nontrivial boundary conditions for the T-/Y-system. The high-temperature limit of the T- and Y-system and their monodromy dependence are studied numerically.

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

PubMed Central

Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Körschen, Heinz G.; Collienne, Ursel; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth

2014-01-01

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

A versatile Escherichia coli strain for identification of biotin transporters and for biotin quantification

PubMed Central

Finkenwirth, Friedrich; Kirsch, Franziska; Eitinger, Thomas

2014-01-01

Biotin is an essential cofactor of carboxylase enzymes in all kingdoms of life. The vitamin is produced by many prokaryotes, certain fungi, and plants. Animals depend on biotin uptake from their diet and in humans lack of the vitamin is associated with serious disorders. Many aspects of biotin metabolism, uptake, and intracellular transport remain to be elucidated. In order to characterize the activity of novel biotin transporters by a sensitive assay, an Escherichia coli strain lacking both biotin synthesis and its endogenous high-affinity biotin importer was constructed. This strain requires artificially high biotin concentrations for growth. When only trace levels of biotin are available, it is viable only if equipped with a heterologous high-affinity biotin transporter. This feature was used to ascribe transport activity to members of the BioY protein family in previous work. Here we show that this strain together with its parent is also useful as a diagnostic tool for wide-concentration-range bioassays. PMID:24256712

Structure/activity relationships for the enhancement by electron-affinic drugs of the anti-tumour effect of CCNU.

PubMed Central

Workman, P.; Twentyman, P. R.

1982-01-01

Using a regrowth-delay assay, we investigated structure/activity relationships for the enhancement by electron-affinic agents of the anti-tumour effect of the nitrosourea CCNU against the KHT sarcoma in C3H mice. A series of neutral 2-nitroimidazoles similar in electron affinity but varying in octanol/water partition coefficient (PC) over 4 orders of magnitude (0.016- greater than 200, Misonidazole = 0.43) were examined at a fixed dose of 2.5 mmol/kg. A parabolic (quadratic) dependence of activity on log PC was observed. Analogues more hydrophilic than misonidazole (MISO) were inactive as were those with very high PCs (greater than 20). Those with PC 0.43--20 were usually more active than MISO, some considerably so. The fairly lipophilic 5-nitroimidazoles nimorazole and metronidazole (METRO) had similar activity to MISO, despite their reduced electron affinity. Two basic 2-nitroimidazoles more efficient as radiosensitizers in vitro likewise showed activity comparable to MISO. We also investigated several agents more electron-affinic than MISO, including some non-nitro compounds. Most were inactive at maximum tolerated doses, but nitrofurazone showed reasonable activity. Sensitizer dose-response curves were obtained for MISO, METRO and two of the most effective agents, benznidazole (Ro 07-1051) and Ro 07-1902. The two latter agents were both considerably more active than MISO at low doses (0.1--0.9 mmol/kg). These studies indicate that the structural features of electron-affinic agents responsible for the enhancement of KHT tumour response to CCNU, are quite different from those affecting radiosensitization, lipophilicity being particularly important. The microsomal enzyme-inhibitor SKF 525A increased the anti-tumour effect of CCNU, suggesting inhibition of CCNU metabolism as one possible mechanism contributing to chemosensitization by lipophilic electron-affinic agents in mice. PMID:7150475

The design of high precision temperature control system for InGaAs short-wave infrared detector

NASA Astrophysics Data System (ADS)

Wang, Zheng-yun; Hu, Yadong; Ni, Chen; Huang, Lin; Zhang, Aiwen; Sun, Xiao-bing; Hong, Jin

2018-02-01

The InGaAs Short-wave infrared detector is a temperature-sensitive device. Accurate temperature control can effectively reduce the background signal and improve detection accuracy, detection sensitivity, and the SNR of the detection system. Firstly, the relationship between temperature and detection background, NEP is analyzed, the principle of TEC and formula between cooling power, cooling current and hot-cold interface temperature difference are introduced. Then, the high precision constant current drive circuit based on triode voltage control current, and an incremental algorithm model based on deviation tracking compensation and PID control are proposed, which effectively suppresses the temperature overshoot, overcomes the temperature inertia, and has strong robustness. Finally, the detector and temperature control system are tested. Results show that: the lower of detector temperature, the smaller the temperature fluctuation, the higher the detection accuracy and the detection sensitivity. The temperature control system achieves the high temperature control with the temperature control rate is 7 8°C/min and the temperature fluctuation is better than +/-0. 04°C.

A minimalist biosensor: Quantitation of cyclic di-GMP using the conformational change of a riboswitch aptamer.

PubMed

Kellenberger, Colleen A; Sales-Lee, Jade; Pan, Yuchen; Gassaway, Madalee M; Herr, Amy E; Hammond, Ming C

2015-01-01

Cyclic di-GMP (c-di-GMP) is a second messenger that is important in regulating bacterial physiology and behavior, including motility and virulence. Many questions remain about the role and regulation of this signaling molecule, but current methods of detection are limited by either modest sensitivity or requirements for extensive sample purification. We have taken advantage of a natural, high affinity receptor of c-di-GMP, the Vc2 riboswitch aptamer, to develop a sensitive and rapid electrophoretic mobility shift assay (EMSA) for c-di-GMP quantitation that required minimal engineering of the RNA.

High sensitivity long-period grating-based temperature monitoring using a wide wavelength range to 2.2 μm

NASA Astrophysics Data System (ADS)

Venugopalan, Thillainathan; Yeo, Teck L.; Sun, Tong; Grattan, Kenneth T. V.

2006-12-01

Temperature effects on the various cladding modes of a long-period grating (LPG) fabricated in B-Ge co-doped fibre have been investigated to create a high sensitivity measurement device. The temperature sensitivities of the attenuation bands of the LPG over the wavelength region 1.2-2.2 μm, for a grating with a 330 μm period, were obtained by monitoring the wavelength shift of each attenuation band, with a temperature increment of 20 °C, over the range from 23 °C to 140 °C. The attenuation band appearing over the 1.8-2.0 μm wavelength range has shown a nearly five times higher temperature sensitivity than that of lower order modes, and thus it shows significant promise for fibre optic temperature sensor applications.

absorption sensor for sensitive temperature and species measurements in high-temperature gases

NASA Astrophysics Data System (ADS)

Spearrin, R. M.; Ren, W.; Jeffries, J. B.; Hanson, R. K.

2014-09-01

A continuous-wave laser absorption diagnostic, based on the infrared CO2 bands near 4.2 and 2.7 μm, was developed for sensitive temperature and concentration measurements in high-temperature gas systems using fixed-wavelength methods. Transitions in the respective R-branches of both the fundamental υ 3 band (~2,350 cm-1) and combination υ 1 + υ 3 band (~3,610 cm-1) were chosen based on absorption line-strength, spectral isolation, and temperature sensitivity. The R(76) line near 2,390.52 cm-1 was selected for sensitive CO2 concentration measurements, and a detection limit of <5 ppm was achieved in shock tube kinetics experiments (~1,300 K). A cross-band, two-line thermometry technique was also established utilizing the R(96) line near 2,395.14 cm-1, paired with the R(28) line near 3,633.08 cm-1. This combination yields high temperature sensitivity (ΔE" = 3,305 cm-1) and expanded range compared with previous intra-band CO2 sensors. Thermometry performance was validated in a shock tube over a range of temperatures (600-1,800 K) important for combustion. Measured temperature accuracy was demonstrated to be better than 1 % over the entire range of conditions, with a standard error of ~0.5 % and µs temporal resolution.

Impact of high temperature stress on floret fertility and individual grain weight of grain sorghum: sensitive stages and thresholds for temperature and duration

PubMed Central

Prasad, P. V. V.; Djanaguiraman, Maduraimuthu; Perumal, Ramasamy; Ciampitti, Ignacio A.

2015-01-01

Sorghum [Sorghum bicolor (L.) Moench] yield formation is severely affected by high temperature stress during reproductive stages. This study pursues to (i) identify the growth stage(s) most sensitive to high temperature stress during reproductive development, (ii) determine threshold temperature and duration of high temperature stress that decreases floret fertility and individual grain weight, and (iii) quantify impact of high daytime temperature during floret development, flowering and grain filling on reproductive traits and grain yield under field conditions. Periods between 10 and 5 d before anthesis; and between 5 d before- and 5 d after-anthesis were most sensitive to high temperatures causing maximum decreases in floret fertility. Mean daily temperatures >25°C quadratically decreased floret fertility (reaching 0% at 37°C) when imposed at the start of panicle emergence. Temperatures ranging from 25 to 37°C quadratically decreased individual grain weight when imposed at the start of grain filling. Both floret fertility and individual grain weights decreased quadratically with increasing duration (0–35 d or 49 d during floret development or grain filling stage, respectively) of high temperature stress. In field conditions, imposition of temperature stress (using heat tents) during floret development or grain filling stage also decreased floret fertility, individual grain weight, and grain weight per panicle. PMID:26500664

Structural implications of hERG K+ channel block by a high-affinity minimally structured blocker

PubMed Central

Helliwell, Matthew V.; Zhang, Yihong; El Harchi, Aziza; Du, Chunyun; Hancox, Jules C.; Dempsey, Christopher E.

2018-01-01

Cardiac potassium channels encoded by human ether-à-go-go–related gene (hERG) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK293 cells was inhibited with an IC50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block. PMID:29545312

Evaluation of 111In-labeled EPep and FibPep as tracers for fibrin SPECT imaging.

PubMed

Starmans, Lucas W E; van Duijnhoven, Sander M J; Rossin, Raffaella; Berben, Monique; Aime, Silvio; Daemen, Mat J A P; Nicolay, Klaas; Grüll, Holger

2013-11-04

Fibrin targeting is an attractive strategy for nuclear imaging of thrombosis, atherosclerosis and cancer. Recently, FibPep, an (111)In-labeled fibrin-binding peptide, was established as a tracer for fibrin SPECT imaging and was reported to allow sensitive detection of minute thrombi in mice using SPECT. In this study, we developed EPep, a novel (111)In-labeled fibrin-binding peptide containing the fibrin-binding domain of the clinically verified EP-2104R peptide, and sought to compare the potential of EPep and FibPep as tracers for fibrin SPECT imaging. In vitro, both EPep and FibPep showed high stability in serum, but were less stable in liver and kidney homogenate assays. Both peptide probes displayed comparable affinities toward human and mouse derived fibrin (Kd ≈ 1 μM), and similarly to FibPep, EPep showed fast blood clearance, low nontarget uptake and high thrombus uptake (6.8 ± 1.2% ID g(-1)) in a mouse carotid artery thrombosis model. Furthermore, EPep showed a similar affinity toward rat derived fibrin (Kd ≈ 1 μM), displayed high thrombus uptake in a rat carotid artery thrombosis model (0.74 ± 0.39% ID g(-1)), and allowed sensitive detection of thrombosis in rats using SPECT. In contrast, FibPep displayed a significantly lower affinity toward rat derived fibrin (Kd ≈ 14 μM) and low uptake in rat thrombi (0.06 ± 0.02% ID g(-1)) and did not allow clear visualization of carotid artery thrombosis in rats using SPECT. These results were confirmed ex vivo by autoradiography, which showed a 7-fold higher ratio of activity in the thrombus over the contralateral carotid artery for EPep in comparison to FibPep. These findings suggest that the FibPep binding fibrin epitope is not fully homologous between humans and rats, and that preclinical rat models of disease should not be employed to gauge the clinical potential of FibPep. In conclusion, both peptides showed approximately similar metabolic stability and affinity toward human and mouse derived fibrin, and displayed high thrombus uptake in a mouse carotid artery thrombosis model. Therefore, both EPep and FibPep are promising fibrin targeted tracers for translation into clinical settings to serve as novel tools for molecular imaging of fibrin.

Highly sensitive and selective detection of Al(III) ions in aqueous buffered solution with fluorescent peptide-based sensor.

PubMed

In, Byunggyu; Hwang, Gi Won; Lee, Keun-Hyeung

2016-09-15

A fluorescent sensor based on a tripeptide (SerGluGlu) with a dansyl fluorophore detected selectively Al(III) among 16 metal ions in aqueous buffered solutions without any organic cosolvent. The peptide-based sensor showed a highly sensitive turn on response to aluminium ion with high binding affinity (1.84×10(4)M(-1)) in aqueous buffered solutions. The detection limit (230nM, 5.98ppb) of the peptide-based sensor was much lower than the maximum allowable level (7.41μM) of aluminium ions in drinking water demanded by EPA. The binding mode of the peptide sensor with aluminium ions was characterized using ESI mass spectrometry, NMR titration, and pH titration experiments. Copyright © 2016 Elsevier Ltd. All rights reserved.

Lysine Decarboxylase with an Enhanced Affinity for Pyridoxal 5-Phosphate by Disulfide Bond-Mediated Spatial Reconstitution.

PubMed

Sagong, Hye-Young; Kim, Kyung-Jin

2017-01-01

Lysine decarboxylase (LDC) catalyzes the decarboxylation of l-lysine to produce cadaverine, an important industrial platform chemical for bio-based polyamides. However, due to high flexibility at the pyridoxal 5-phosphate (PLP) binding site, use of the enzyme for cadaverine production requires continuous supplement of large amounts of PLP. In order to develop an LDC enzyme from Selenomonas ruminantium (SrLDC) with an enhanced affinity for PLP, we introduced an internal disulfide bond between Ala225 and Thr302 residues with a desire to retain the PLP binding site in a closed conformation. The SrLDCA225C/T302C mutant showed a yellow color and the characteristic UV/Vis absorption peaks for enzymes with bound PLP, and exhibited three-fold enhanced PLP affinity compared with the wild-type SrLDC. The mutant also exhibited a dramatically enhanced LDC activity and cadaverine conversion particularly under no or low PLP concentrations. Moreover, introduction of the disulfide bond rendered SrLDC more resistant to high pH and temperature. The formation of the introduced disulfide bond and the maintenance of the PLP binding site in the closed conformation were confirmed by determination of the crystal structure of the mutant. This study shows that disulfide bond-mediated spatial reconstitution can be a platform technology for development of enzymes with enhanced PLP affinity.

TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+.

PubMed

Pitt, Samantha J; Funnell, Tim M; Sitsapesan, Mano; Venturi, Elisa; Rietdorf, Katja; Ruas, Margarida; Ganesan, A; Gosain, Rajendra; Churchill, Grant C; Zhu, Michael X; Parrington, John; Galione, Antony; Sitsapesan, Rebecca

2010-11-05

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca(2+) required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca(2+) from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca(2+) release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca(2+) that will enable it to act as a Ca(2+) release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca(2+)] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca(2+) release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca(2+) release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μM but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

Temperature sensitive surfaces and methods of making same

DOEpatents

Liang, Liang [Richland, WA; Rieke, Peter C [Pasco, WA; Alford, Kentin L [Pasco, WA

2002-09-10

Poly-n-isopropylacrylamide surface coatings demonstrate the useful property of being able to switch charateristics depending upon temperature. More specifically, these coatings switch from being hydrophilic at low temperature to hydrophobic at high temperature. Research has been conducted for many years to better characterize and control the properties of temperature sensitive coatings. The present invention provides novel temperature sensitive coatings on articles and novel methods of making temperature sensitive coatings that are disposed on the surfaces of various articles. These novel coatings contain the reaction products of n-isopropylacrylamide and are characterized by their properties such as advancing contact angles. Numerous other characteristics such as coating thickness, surface roughness, and hydrophilic-to-hydrophobic transition temperatures are also described. The present invention includes articles having temperature-sensitve coatings with improved properties as well as improved methods for forming temperature sensitive coatings.

The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal

PubMed Central

Vergis, James M.; Wiener, Michael C.

2011-01-01

Recombinant proteins typically include one or more affinity tags to facilitate purification and/or detection. Expression constructs with affinity tags often include an engineered protease site for tag removal. Like other enzymes, the activities of proteases can be affected by buffer conditions. The buffers used for integral membrane proteins contain detergents, which are required to maintain protein solubility. We examined the detergent sensitivity of six commonly-used proteases (Enterokinase, Factor Xa, Human Rhinovirus 3C Protease, SUMOstar, Tobacco Etch Virus Protease, and Thrombin) by use of a panel of ninety-four individual detergents. Thrombin activity was insensitive to the entire panel of detergents, thus suggesting it as the optimal choice for use with membrane proteins. Enterokinase and Factor Xa were only affected by a small number of detergents, making them good choices as well. PMID:21539919

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Approaching the evolutionary advantage of ancillary types of haemoglobin in Daphnia magna by simulation of oxygen supply.

PubMed

Moenickes, S; Richter, O; Pirow, R

2010-02-01

The planktonic crustacean Daphnia magna synthesizes haemoglobin (Hb) macromolecules of variant subunit composition and oxygen affinity. This is one of the strategies by which the animals cope with variations in environmental conditions such as ambient oxygen tension. The enrichment of high-affinity Hb molecules in the haemolymph of hypoxia-exposed animals is thought to reduce Hb synthesis costs due to an enhanced transport efficiency of these molecules in comparison to the low-affinity Hb molecules. How great this economic advantage is, and under which conditions this benefit disappears, is still not fully understood. Here we implemented a rigorously simplified model of the daphnid body and described the transport of oxygen from the environment via the haemolymph to the tissues in terms of the convection-diffusion-reaction equation. The model was validated by comparing various model predictions with experimental data. A sensitivity analysis was used to evaluate the influence of parameter uncertainties on the model predictions. Cost-benefit analysis revealed in which way at the system's level the increase in Hb oxygen affinity improves the oxygen loading at the respiratory surfaces and impairs the release of oxygen to the tissues. The benefit arising from the improved oxygen loading exceeds the disadvantage of impaired unloading only under conditions where the ambient oxygen tension is critically low and the Hb concentration is high. The low-affinity Hb, on the other hand, provides an advantage given that the Hb concentration is low and the ambient oxygen tension is well above the critical level. Computer-aided modelling and simulation therefore provide valuable mechanistic insights into the driving forces that could have shaped the evolution of globin genes in daphnids.

Low-Temperature Photochemically Activated Amorphous Indium-Gallium-Zinc Oxide for Highly Stable Room-Temperature Gas Sensors.

PubMed

Jaisutti, Rawat; Kim, Jaeyoung; Park, Sung Kyu; Kim, Yong-Hoon

2016-08-10

We report on highly stable amorphous indium-gallium-zinc oxide (IGZO) gas sensors for ultraviolet (UV)-activated room-temperature detection of volatile organic compounds (VOCs). The IGZO sensors fabricated by a low-temperature photochemical activation process and exhibiting two orders higher photocurrent compared to conventional zinc oxide sensors, allowed high gas sensitivity against various VOCs even at room temperature. From a systematic analysis, it was found that by increasing the UV intensity, the gas sensitivity, response time, and recovery behavior of an IGZO sensor were strongly enhanced. In particular, under an UV intensity of 30 mW cm(-2), the IGZO sensor exhibited gas sensitivity, response time and recovery time of 37%, 37 and 53 s, respectively, against 750 ppm concentration of acetone gas. Moreover, the IGZO gas sensor had an excellent long-term stability showing around 6% variation in gas sensitivity over 70 days. These results strongly support a conclusion that a low-temperature solution-processed amorphous IGZO film can serve as a good candidate for room-temperature VOCs sensors for emerging wearable electronics.

High-sensitivity temperature sensor based on highly-birefringent microfiber

NASA Astrophysics Data System (ADS)

Sun, Li-Peng; Li, Jie; Jin, Long; Gao, Shuai; Tian, Zhuang; Ran, Yang; Guan, Bai-Ou

2013-09-01

We demonstrate an ultrasensitive temperature sensor by sealing a highly-birefringent microfiber into an alcoholinfiltrated copper capillary. With a Sagnac loop configuration, the interferometric spectrum is strongly dependent on the external refractive index (RI) with sensitivity of 36800nm/RIU around RI=1.356. As mainly derived from the ultrahigh RI sensitivity, the temperature response can reach as high as -14.72 nm/°C in the range of 30.9-36.9 °C. The measured response time is ~8s, as determined by the heat-conducting characteristic of the device and the diameter of the copper capillary. Our sensor is featured with low cost, easy fabrication and robustness.

Advances in the Breeding and Genetics of Heat Tolerance to Alleviate the Effects of Climate Change, with a Focus on Common Bean

USDA-ARS?s Scientific Manuscript database

Crop plants are broadly sensitive to high ambient temperatures during reproductive development while breeding efforts are helping to alleviate the impact of heat stress. Common bean, Phaseolus vulgaris L., is sensitive to moderately high ambient temperature, where temperatures greater than 25C have ...

Hollow glass microsphere-structured Fabry-Perot interferometric sensor for highly sensitive temperature measurement

NASA Astrophysics Data System (ADS)

Cheng, Junna; Zhou, Ciming; Fan, Dian; Ou, Yiwen

2017-04-01

We propose and demonstrate a miniature Fabry-Perot (F-P) interferometric sensor based on a hollow glass microsphere (HGM) for highly sensitive temperature measurement. The sensor head is fabricated by sticking a HGM on the end face of a single-mode fiber, and it consists of a short air F-P cavity between the front and the rear surfaces of the HGM. A sensor with 135.7280-μm cavity length was tested for temperature measurement from -5 °C to 50 °C. The obtained sensitivity reached up to 24.5 pm/°C and the variation rate of the HGM- F-P's cavity length was2.1 nm/°C. The advantages of compact size, easy fabrication and low cost make the sensor suitable for highly sensitive temperature sensing.

Fiber specklegram sensors sensitivities at high temperatures

NASA Astrophysics Data System (ADS)

Rodriguez-Cobo, L.; Lomer, M.; Lopez-Higuera, J. M.

2015-09-01

In this work, the sensitivity of Fiber Specklegram Sensors to high temperatures (up to 800ºC) have been studied. Two multimode silica fibers have been introduced into a tubular furnace while a HeNe laser source was launched into a fiber edge, projecting speckle patterns to a commercial webcam. A computer generated different heating and cooling sweeps while the specklegram evolution was recorded. The achieved results exhibit a remarkably linearity in FSS's sensitivity for temperatures under 800ºC, following the thermal expansion of fused silica.

Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle.

PubMed Central

Godt, R E; Nosek, T M

1989-01-01

1. Maximal calcium-activated force (Fmax) and calcium sensitivity were markedly decreased in detergent-skinned fibres from skeletal and cardiac muscle by solutions that mimicked the total milieu changes associated with fatigue and hypoxia. Further experiments determined the relative contribution of each of the individual changes in milieu. 2. Both Ca2+ sensitivity and Fmax of skeletal and cardiac fibres were decreased with increased [H+] or inorganic phosphate (Pi). These effects were greater in cardiac muscle. 3. Decreasing MgATP over the range observed with fatigue and hypoxia (6.8-4.7 mM) had no effect on Fmax or Ca2+ sensitivity of either muscle type. 4. Decreasing phosphocreatine (PCr: 15-1 mM) increased Fmax but had little effect on Ca2+ sensitivity in both muscle types. In cardiac fibres, the effect on Fmax could be mimicked by inhibition of endogenous creatine kinase. 5. ADP (0.7 mM) increased Fmax and Ca2+ sensitivity, while AMP (0.06 mM) slightly increased Fmax but had no effect on Ca2+ sensitivity of either skeletal or cardiac fibres. 6. Creatine (25 mM) had no significant effect on either Ca2+ sensitivity or Fmax of skeletal and cardiac muscle fibres. At higher levels (50 mM), however, creatine depressed Fmax and slightly altered Ca2+ sensitivity. 7. Thiophosphorylation of myosin P light chains (phosphorylatable light chains of myosin) in rabbit psoas fibres had no effect on Ca2+ sensitivity, yet slightly but significantly increased Fmax under fatigue conditions. 8. Reducing the affinity for ATP hydrolysis (by adding ADP, AMP and creatine) over the range calculated for fatigue/hypoxia (60-45 kJ/mol) produced the enhancement in Fmax expected from added ADP and AMP in cardiac but not skeletal muscle, indicating that changes in affinity influence Fmax of skeletal muscle. Reducing affinity produced little change in Ca2+ sensitivity of skeletal muscle. In contrast, the change produced in cardiac muscle was greater than that expected from addition of ADP and AMP; i.e. decreasing affinity increases calcium sensitivity of the heart. 9. Simple summation of all significant changes expected from each constituent altered by fatigue/hypoxia adequately predicted the observed changes in Fmax and Ca2+ sensitivity in both cardiac and skeletal muscle fibres with but one exception (the change in Ca2+ sensitivity of skeletal muscle at pH 7 was slightly overestimated). PMID:2600830

The fast release of sticky protons: Kinetics of substrate binding and proton release in a multidrug transporter

PubMed Central

Adam, Yoav; Tayer, Naama; Rotem, Dvir; Schreiber, Gideon; Schuldiner, Shimon

2007-01-01

EmrE is an Escherichia coli H+-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP+, a high-affinity substrate, is 2 × 107 M−1·s−1, a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the koff of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs. PMID:17984053

Titration calorimetry of anesthetic-protein interaction: negative enthalpy of binding and anesthetic potency.

PubMed

Ueda, I; Yamanaka, M

1997-04-01

Anesthetic potency increases at lower temperatures. In contrast, the transfer enthalpy of volatile anesthetics from water to macromolecules is usually positive. The transfer decreases at lower temperature. It was proposed that a few selective proteins bind volatile anesthetics with negative delta H, and these proteins are involved in signal transduction. There has been no report on direct estimation of binding delta H of anesthetics to proteins. This study used isothermal titration calorimetry to analyze chloroform binding to bovine serum albumin. The calorimetrically measured delta H cal was -10.37 kJ.mol-1. Thus the negative delta H of anesthetic binding is not limited to signal transduction proteins. The binding was saturable following Fermi-Dirac statistics and is characterized by the Langmuir adsorption isotherms, which is interfacial. The high-affinity association constant, K, was 2150 +/- 132 M-1 (KD = 0.47 mM) with the maximum binding number, Bmax = 3.7 +/- 0.2. The low-affinity K was 189 +/- 3.8 M-1 (KD = 5.29 mM), with a Bmax of 13.2 +/- 0.3. Anesthetic potency is a function of the activity of anesthetic molecules, not the concentration. Because the sign of delta H determines the temperature dependence of distribution of anesthetic molecules, it is irrelevant to the temperature dependence of anesthetic potency.

Titration calorimetry of anesthetic-protein interaction: negative enthalpy of binding and anesthetic potency.

PubMed Central

Ueda, I; Yamanaka, M

1997-01-01

Anesthetic potency increases at lower temperatures. In contrast, the transfer enthalpy of volatile anesthetics from water to macromolecules is usually positive. The transfer decreases at lower temperature. It was proposed that a few selective proteins bind volatile anesthetics with negative delta H, and these proteins are involved in signal transduction. There has been no report on direct estimation of binding delta H of anesthetics to proteins. This study used isothermal titration calorimetry to analyze chloroform binding to bovine serum albumin. The calorimetrically measured delta H cal was -10.37 kJ.mol-1. Thus the negative delta H of anesthetic binding is not limited to signal transduction proteins. The binding was saturable following Fermi-Dirac statistics and is characterized by the Langmuir adsorption isotherms, which is interfacial. The high-affinity association constant, K, was 2150 +/- 132 M-1 (KD = 0.47 mM) with the maximum binding number, Bmax = 3.7 +/- 0.2. The low-affinity K was 189 +/- 3.8 M-1 (KD = 5.29 mM), with a Bmax of 13.2 +/- 0.3. Anesthetic potency is a function of the activity of anesthetic molecules, not the concentration. Because the sign of delta H determines the temperature dependence of distribution of anesthetic molecules, it is irrelevant to the temperature dependence of anesthetic potency. PMID:9083685

Diagnostics Strategies with Electrochemical Affinity Biosensors Using Carbon Nanomaterials as Electrode Modifiers

PubMed Central

Campuzano, Susana; Yáñez-Sedeño, Paloma; Pingarrón, José M.

2016-01-01

Early diagnosis is often the key to successful patient treatment and survival. The identification of various disease signaling biomarkers which reliably reflect normal and disease states in humans in biological fluids explain the burgeoning research field in developing new methodologies able to determine the target biomarkers in complex biological samples with the required sensitivity and selectivity and in a simple and rapid way. The unique advantages offered by electrochemical sensors together with the availability of high affinity and specific bioreceptors and their great capabilities in terms of sensitivity and stability imparted by nanostructuring the electrode surface with different carbon nanomaterials have led to the development of new electrochemical biosensing strategies that have flourished as interesting alternatives to conventional methodologies for clinical diagnostics. This paper briefly reviews the advantages of using carbon nanostructures and their hybrid nanocomposites as electrode modifiers to construct efficient electrochemical sensing platforms for diagnosis. The review provides an updated overview of some selected examples involving attractive amplification and biosensing approaches which have been applied to the determination of relevant genetic and protein diagnostics biomarkers. PMID:28035946

Temperature sensitivity of silicon nitride nanocoated long-period gratings working in various surrounding media

NASA Astrophysics Data System (ADS)

Smietana, M.; Bock, W. J.; Mikulic, P.

2011-11-01

This paper presents the temperature sensing properties of a silicon nitride (SiNx) nanocoated long-period grating (LPG). A high-temperature, radio-frequency plasma-enhanced chemical-vapor-deposited SiNx nanocoating was applied to tune the external refractive index (RI) sensitivity of LPGs written with UV and electric arc techniques in boron co-doped and standard germanium doped fibers, respectively. The technique allows for deposition of good quality, hard and wear-resistant nanofilms as are required for optical sensors. Thanks to the high-RI SiNx nanocoating, which is less than 90 nm thick, it is possible to reduce RI sensitivity over a wide range (from nD = 1.333 to 1.479), simultaneously decreasing its cross-sensitivity to temperature. For the presented nanocoated LPGs, the temperature effect on resonance wavelength is linear and slightly dependent on the thermo-optic coefficient of the surrounding liquid. The other advantage of the nanocoating is that it makes the resonance clearly visible in the whole investigated external RI range. To the best of our knowledge, this work presents for the first time a nanocoating able to simultaneously tune the RI sensitivity and enable temperature measurements in high-RI liquids applied to LPGs.

Characterization of high affinity (/sup 3/H)triazolam binding in rat brain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Earle, M.; Concas, A.; Yamamura, H.I.

1986-03-01

The hypnotic Triazolam (TZ), a triazolo (1,4)-benzodiazepine, displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. Specific binding properties of this recently tritiated TZ were characterized. The authors major objectives were the direct measurement of the temperature dependence and the GABA effect on (/sup 3/H)TZ binding. Saturation studies showed a shift to lower affinity at 37/sup 0/C (K/sub d/ = 0.25 +/- 0.01 nM at O/sup 0/C; K/sub d/ = 1.46 +/- 0.03 nM at 37/sup 0/C) while the B/sub max/ values remained unchanged (1003 +/- 37 fmoles/mg prot. atmore » 0/sup 0/C and 1001 +/- 43 fmoles/mg prot. at 37/sup 0/C). Inhibition studies showed that (/sup 3/H)TZ binding displayed no GABA shift at 0/sup 0/C(K/sub i/ 0.37 +/- 0.03 nM/- GABA and K/sub i/ = 0.55 +/- 0.13 nM/+GABA) but a nearly two-fold shift was apparent at 37/sup 0/C (K/sub i/ = 2.92 +/- 0.2 nM/-GABA; K/sub i/ = 1.37 +/- 0.11 mM/+GABA). These results were also confirmed by saturation studies in the presence or absence of GABA showing a shift to higher affinity in the presence of GABA only at 37/sup 0/C. In Ro 15-1788/(/sup 3/H)TZ competition experiments the presence of GABA did not affect the inhibitory potency of Ro 15-1788 on (/sup 3/H)TZ binding at both temperatures. In conclusion (/sup 3/H)TZ binding showed an extremely high affinity for benzodiazepine receptors. In contrast to reported literature, the findings suggest that TZ interacts with benzodiazepine receptors similar to other benzodiazepine agonists.« less

Fluorescence spectroscopy approaches for the development of a real-time organophosphate detection system using an enzymatic sensor.

PubMed

Carullo, Paola; Cetrangolo, Giovanni Paolo; Mandrich, Luigi; Manco, Giuseppe; Febbraio, Ferdinando

2015-02-09

Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

Middle-Down and Chemical Proteomic Approaches to Reveal Histone H4 Modification Dynamics in Cell Cycle: Label-Free Semi-Quantification of Histone Tail Peptide Modifications Including Phosphorylation and Highly Sensitive Capture of Histone PTM Binding Proteins Using Photo-Reactive Crosslinkers

PubMed Central

Yamamoto, Kazuki; Chikaoka, Yoko; Hayashi, Gosuke; Sakamoto, Ryosuke; Yamamoto, Ryuji; Sugiyama, Akira; Kodama, Tatsuhiko; Okamoto, Akimitsu; Kawamura, Takeshi

2015-01-01

Mass spectrometric proteomics is an effective approach for identifying and quantifying histone post-translational modifications (PTMs) and their binding proteins, especially in the cases of methylation and acetylation. However, another vital PTM, phosphorylation, tends to be poorly quantified because it is easily lost and inefficiently ionized. In addition, PTM binding proteins for phosphorylation are sometimes resistant to identification because of their variable binding affinities. Here, we present our efforts to improve the sensitivity of detection of histone H4 tail peptide phosphorylated at serine 1 (H4S1ph) and our successful identification of an H4S1ph binder candidate by means of a chemical proteomics approach. Our nanoLC-MS/MS system permitted semi-quantitative label-free analysis of histone H4 PTM dynamics of cell cycle-synchronized HeLa S3 cells, including phosphorylation, methylation, and acetylation. We show that H4S1ph abundance on nascent histone H4 unmethylated at lysine 20 (H4K20me0) peaks from late S-phase to M-phase. We also attempted to characterize effects of phosphorylation at H4S1 on protein–protein interactions. Specially synthesized photoaffinity bait peptides specifically captured 14-3-3 proteins as novel H4S1ph binding partners, whose interaction was otherwise undetectable by conventional peptide pull-down experiments. This is the first report that analyzes dynamics of PTM pattern on the whole histone H4 tail during cell cycle and enables the identification of PTM binders with low affinities using high-resolution mass spectrometry and photo-affinity bait peptides. PMID:26819910

The influence of adhesive on fiber Bragg grating strain sensor

NASA Astrophysics Data System (ADS)

Chen, Jixuan; Gong, Huaping; Jin, Shangzhong; Li, Shuhua

2009-08-01

A fiber Bragg grating (FBG) sensor was fixed on the uniform strength beam with three adhesives, which were modified acrylate, glass glue and epoxy resin. The influence of adhesive on FBG strain sensor was investigated. The strain of FBG sensor was varied by loading weight to the uniform strength beam. The wavelength shift of the FBG sensor fixed by the three kinds of adhesive were measured with different weight at the temperatures 0°C, 10°C, 20°C, 30°C, 40°C. The linearity, sensitivity and their stability at different temperature of FBG sensor which fixed by every kind of adhesives were analyzed. The results show that, the FBG sensor fixed by the modified acrylate has a high linearity, and the linear correlation coefficient is 0.9996. It also has a high sensitivity which is 0.251nm/kg. The linearity and the sensitivity of the FBG sensor have a high stability at different temperatures. The FBG sensor fixed by the glass glue also has a high linearity, and the linear correlation coefficient is 0.9986, but it has a low sensitivity which is only 0.041nm/kg. The linearity and the sensitivity of the FBG sensor fixed by the glass glue have a high stability at different temperatures. When the FBG sensor is fixed by epoxy resin, the sensitivity and linearity is affected significantly by the temperature. When the temperature changes from 0°C to 40°C, the sensitivity decreases from 0.302nm/kg to 0.058nm/kg, and the linear correlation coefficient decreases from 0.9999 to 0.9961.

Significant relaxation of residual negative carrier in polar Alq3 film directly detected by high-sensitivity photoemission

NASA Astrophysics Data System (ADS)

Kinjo, Hiroumi; Lim, Hyunsoo; Sato, Tomoya; Noguchi, Yutaka; Nakayama, Yasuo; Ishii, Hisao

2016-02-01

Tris(8-hydroxyquinoline)aluminum (Alq3) has been widely applied as a good electron-injecting layer (EIL) in organic light-emitting diodes. High-sensitivity photoemission measurement revealed a clear photoemission by visible light, although its ionization energy is 5.7 eV. This unusual photoemission is ascribed to Alq3 anions captured by positive polarization charges. The observed electron detachment energy of the anion was about 1 eV larger than the electron affinity reported by inverse photoemission. This difference suggests that the injected electron in the Alq3 layer is energetically relaxed, leading to the reduction in injection barrier. This nature is one of the reasons why Alq3 worked well as the EIL.

A compact, high temperature nuclear magnetic resonance probe for use in a narrow-bore superconducting magnet

NASA Astrophysics Data System (ADS)

Adler, Stuart B.; Michaels, James N.; Reimer, Jeffrey A.

1990-11-01

The design of a nuclear magnetic resonance (NMR) probe is reported, that can be used in narrow-bore superconducting solenoids for the observation of nuclear induction at high temperatures. The probe is compact, highly sensitive, and stable in continuous operation at temperatures up to 1050 C. The essential feature of the probe is a water-cooled NMR coil that contains the sample-furnace; this design maximizes sensitivity and circuit stability by maintaining the probe electronics at ambient temperature. The design is demonstrated by showing high temperature O-17 NMR spectra and relaxation measurements in solid barium bismuth oxide and yttria-stabilized zirconia.

High-affinity copper transport and Snq2 export permease of saccharomyces cerevisiae modulate cytotoxicity of PR-10 from Theobroma cacao.

PubMed

Pungartnik, Cristina; da Silva, Aline Clara; de Melo, Sarah Alves; Gramacho, Karina Peres; de Mattos Cascardo, Júlio Cézar; Brendel, Martin; Micheli, Fabienne; da Silva Gesteira, Abelmon

2009-01-01

A pathogenesis-related (PR) protein from Theobroma cacao (TcPR-10) was identified from a cacao-Moniliophthora perniciosa interaction cDNA library. Nucleotide and amino acid sequences showed homology with other PR-10 proteins having P loop motif and Betv1 domain. Recombinant TcPR-10 showed in vitro and in vivo ribonuclease activity, and antifungal activity against the basidiomycete cacao pathogen M. perniciosa and the yeast Saccharomyces cerevisiae. Fluorescein isothiocyanate-labeled TcPR-10 was internalized by M. perniciosa hyphae and S. cerevisiae cells and inhibited growth of both fungi. Energy and temperature-dependent internalization of the TcPR-10 suggested an active importation into the fungal cells. Chronical exposure to TcPR-10 of 29 yeast mutants with single gene defects in DNA repair, general membrane transport, metal transport, and antioxidant defenses was tested. Two yeast mutants were hyperresistant compared with their respective isogenic wild type: ctr3Delta mutant, lacking the high-affinity plasma membrane copper transporter and mac1Delta, the copper-sensing transcription factor involved in regulation of high-affinity copper transport. Acute exposure of exponentially growing yeast cells revealed that TcPR-10 resistance is also enhanced in the Snq2 export permease-lacking mutant which has reduced intracellular presence of TcPR-10.

Familial Hypertrophic Cardiomyopathy Related Cardiac Troponin C L29Q Mutation Alters Length-Dependent Activation and Functional Effects of Phosphomimetic Troponin I*

PubMed Central

Li, Alison Y.; Stevens, Charles M.; Liang, Bo; Rayani, Kaveh; Little, Sean; Davis, Jonathan; Tibbits, Glen F.

2013-01-01

The Ca2+ binding properties of the FHC-associated cardiac troponin C (cTnC) mutation L29Q were examined in isolated cTnC, troponin complexes, reconstituted thin filament preparations, and skinned cardiomyocytes. While higher Ca2+ binding affinity was apparent for the L29Q mutant in isolated cTnC, this phenomenon was not observed in the cTn complex. At the level of the thin filament in the presence of phosphomimetic TnI, L29Q cTnC further reduced the Ca2+ affinity by 27% in the steady-state measurement and increased the Ca2+ dissociation rate by 20% in the kinetic studies. Molecular dynamics simulations suggest that L29Q destabilizes the conformation of cNTnC in the presence of phosphomimetic cTnI and potentially modulates the Ca2+ sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca2+ sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca2+ sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca2+ sensitivity. This might result from its modified interaction with cTnI which altered the feedback effects of cross-bridges on the L29Q cTnC-cTnI-Tm complex. This study demonstrates that the L29Q mutation alters the contractility and the functional effects of the phosphomimetic cTnI in both thin filament and single skinned cardiomyocytes and importantly that this effect is highly sarcomere length dependent. PMID:24260207

Photonic crystal fiber temperature sensor with high sensitivity based on surface plasmon resonance

NASA Astrophysics Data System (ADS)

Wu, Junjun; Li, Shuguang; shi, Min; Feng, Xinxing

2018-07-01

A high sensitivity photonic crystal fiber (PCF) temperature sensor based on surface plasmon resonance is proposed and evaluated using the finite element method. Besides, the coupling phenomenon is studied. The gold layer deposited on the polishing surface of D-shape PCF is used as the metal to stimulate surface plasma, which can improves the sensitivity. Through exquisite design, the birefringence of the fiber is improved, which makes the loss of y-polarization far greater than the loss of x-polarization. The D-shape fiber avoids filling metal and liquid into the air-holes, which can contact with fluid directly to feel temperature. When the phase matching condition is satisfied, the core mode will couple with the surface plasma mode. The resonance position of y-polarization is very sensitive to the temperature change. The simulation shows that the PCF has high sensitivity of 36.86 nm/°C in y-polarization and wide detection that from 10 °C to 85 °C.

The Mechanisms and Biomedical Applications of an NIR BODIPY-Based Switchable Fluorescent Probe

PubMed Central

Cheng, Bingbing; Bandi, Venugopal; Yu, Shuai; D’Souza, Francis; Nguyen, Kytai T.; Hong, Yi; Tang, Liping; Yuan, Baohong

2017-01-01

Highly environment-sensitive fluorophores have been desired for many biomedical applications. Because of the noninvasive operation, high sensitivity, and high specificity to the microenvironment change, they can be used as excellent probes for fluorescence sensing/imaging, cell tracking/imaging, molecular imaging for cancer, and so on (i.e., polarity, viscosity, temperature, or pH measurement). In this work, investigations of the switching mechanism of a recently reported near-infrared environment-sensitive fluorophore, ADP(CA)2, were conducted. Besides, multiple potential biomedical applications of this switchable fluorescent probe have been demonstrated, including wash-free live-cell fluorescence imaging, in vivo tissue fluorescence imaging, temperature sensing, and ultrasound-switchable fluorescence (USF) imaging. The fluorescence of the ADP(CA)2 is extremely sensitive to the microenvironment, especially polarity and viscosity. Our investigations showed that the fluorescence of ADP(CA)2 can be switched on by low polarity, high viscosity, or the presence of protein and surfactants. In wash-free live-cell imaging, the fluorescence of ADP(CA)2 inside cells was found much brighter than the dye-containing medium and was retained for at least two days. In all of the fluorescence imaging applications conducted in this study, high target-to-noise (>5-fold) was achieved. In addition, a high temperature sensitivity (73-fold per Celsius degree) of ADP(CA)2-based temperature probes was found in temperature sensing. PMID:28208666

Picomolar-affinity binding and inhibition of adenylate cyclase activity by melatonin in Syrian hamster hypothalamus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niles, L.P.; Hashemi, F.

1. The effect of melatonin on forskolin-stimulated adenylate cyclase activity was measured in homogenates of Syrian hamster hypothalamus. In addition, the saturation binding characteristics of the melatonin receptor ligand, ({sup 125}I)iodomelatonin, was examined using an incubation temperature (30{degree}C) similar to that used in enzyme assays. 2. At concentrations ranging from 10 pM to 1 nM, melatonin caused a significant decrease in stimulated adenylate cyclase activity with a maximum inhibition of approximately 22%. 3. Binding experiments utilizing ({sup 125}I)iodomelatonin in a range of approximately 5-80 pM indicated a single class of high-affinity sites: Kd = 55 +/- 9 pM, Bmax =more » 1.1 +/- 0.3 fmol/mg protein. 4. The ability of picomolar concentrations of melatonin to inhibit forskolin-stimulated adenylate cyclase activity suggests that this affect is mediated by picomolar-affinity receptor binding sites for this hormone in the hypothalamus.« less

High-Temperature Proton-Conducting Ceramics Developed

NASA Technical Reports Server (NTRS)

Sayir, Ali; Dynys, Frederick W.; Berger, M. H.

2005-01-01

High-temperature protonic conductors (HTPC) are needed for hydrogen separation, hydrogen sensors, fuel cells, and hydrogen production from fossil fuels. The HTPC materials for hydrogen separation at high temperatures are foreseen to be metal oxides with the perovskite structure A(sup 2+)B(sup 4+)C(sup 2-, sub 3) and with the trivalent cation (M(sup 3+)) substitution at the B(sup 4+)-site to introduce oxygen vacancies. The high affinity for hydrogen ions (H(sup +)) is advantageous for protonic transport, but it increases the reactivity toward water (H2O) and carbon dioxide (CO2), which can lead to premature membrane failure. In addition, there are considerable technological challenges related to the processing of HTPC materials. The high melting point and multi-cation chemistry of HTPC materials creates difficulties in in achieving high-density, single-phase membranes by solid-state sintering. The presence of secondary phases and grain-boundary interfaces are detrimental to the protonic conduction and environmental stability of polycrystalline HTPC materials.

Oxygen transport by hemoglobin.

PubMed

Mairbäurl, Heimo; Weber, Roy E

2012-04-01

Hemoglobin (Hb) constitutes a vital link between ambient O2 availability and aerobic metabolism by transporting oxygen (O2) from the respiratory surfaces of the lungs or gills to the O2-consuming tissues. The amount of O2 available to tissues depends on the blood-perfusion rate, as well as the arterio-venous difference in blood O2 contents, which is determined by the respective loading and unloading O2 tensions and Hb-O2-affinity. Short-term adjustments in tissue oxygen delivery in response to decreased O2 supply or increased O2 demand (under exercise, hypoxia at high altitude, cardiovascular disease, and ischemia) are mediated by metabolically induced changes in the red cell levels of allosteric effectors such as protons (H(+)), carbon dioxide (CO2), organic phosphates, and chloride (Cl(-)) that modulate Hb-O2 affinity. The long-term, genetically coded adaptations in oxygen transport encountered in animals that permanently are subjected to low environmental O2 tensions commonly result from changes in the molecular structure of Hb, notably amino acid exchanges that alter Hb's intrinsic O2 affinity or its sensitivity to allosteric effectors. Structure-function studies of animal Hbs and human Hb mutants illustrate the different strategies for adjusting Hb-O2 affinity and optimizing tissue oxygen supply. © 2012 American Physiological Society. Compr Physiol 2:1491-1539, 2012.

Effect of atomic layer deposition temperature on current conduction in Al{sub 2}O{sub 3} films formed using H{sub 2}O oxidant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hiraiwa, Atsushi, E-mail: hiraiwa@aoni.waseda.jp, E-mail: qs4a-hriw@asahi-net.or.jp; Matsumura, Daisuke; Kawarada, Hiroshi, E-mail: kawarada@waseda.jp

To develop high-performance, high-reliability gate insulation and surface passivation technologies for wide-bandgap semiconductor devices, the effect of atomic layer deposition (ALD) temperature on current conduction in Al{sub 2}O{sub 3} films is investigated based on the recently proposed space-charge-controlled field emission model. Leakage current measurement shows that Al{sub 2}O{sub 3} metal-insulator-semiconductor capacitors formed on the Si substrates underperform thermally grown SiO{sub 2} capacitors at the same average field. However, using equivalent oxide field as a more practical measure, the Al{sub 2}O{sub 3} capacitors are found to outperform the SiO{sub 2} capacitors in the cases where the capacitors are negatively biased andmore » the gate material is adequately selected to reduce virtual dipoles at the gate/Al{sub 2}O{sub 3} interface. The Al{sub 2}O{sub 3} electron affinity increases with the increasing ALD temperature, but the gate-side virtual dipoles are not affected. Therefore, the leakage current of negatively biased Al{sub 2}O{sub 3} capacitors is approximately independent of the ALD temperature because of the compensation of the opposite effects of increased electron affinity and permittivity in Al{sub 2}O{sub 3}. By contrast, the substrate-side sheet of charge increases with increasing ALD temperature above 210 °C and hence enhances the current of positively biased Al{sub 2}O{sub 3} capacitors more significantly at high temperatures. Additionally, an anomalous oscillatory shift of the current-voltage characteristics with ALD temperature was observed in positively biased capacitors formed by low-temperature (≤210 °C) ALD. This shift is caused by dipoles at the Al{sub 2}O{sub 3}/underlying SiO{sub 2} interface. Although they have a minimal positive-bias leakage current, the low-temperature-grown Al{sub 2}O{sub 3} films cause the so-called blisters problem when heated above 400 °C. Therefore, because of the absence of blistering, a 450 °C ALD process is presently the most promising technology for growing high-reliability Al{sub 2}O{sub 3} films.« less

Wheat seed weight and quality differ temporally in sensitivity to warm or cool conditions during seed development and maturation

PubMed Central

Nasehzadeh, M

2017-01-01

Abstract Background and aims Short periods of extreme temperature may affect wheat (Triticum aestivum) seed weight, but also quality. Temporal sensitivity to extreme temperature during seed development and maturation was investigated. Methods Plants of ‘Tybalt’ grown at ambient temperature were moved to growth cabinets at 29/20°C or 34/20°C (2010), or 15/10°C or 34/20°C (2011), for successive 7-d periods from 7 DAA (days after anthesis) onwards, and also 7–65 DAA in 2011. Seed samples were harvested serially and moisture content, weight, ability to germinate, subsequent longevity in air-dry storage and bread-making quality were determined. Key Results High temperature (34/20°C) reduced final seed weight, with greatest temporal sensitivity at 7–14 or 14–21 DAA. Several aspects of bread-making quality were also most sensitive to high temperature then, but whereas protein quality decreased protein and sulphur concentrations improved. Early exposure to high temperature provided earlier development of ability to germinate and tolerate desiccation, but had little effect on maximum germination capacity. All treatments at 15/10°C resulted in ability to germinate declining between 58 and 65 DAA. Early exposure to high temperature hastened improvement in seed storage longevity, but the subsequent decline in late maturation preceded that in the control. Long (7–65 DAA) exposure to 15/10°C disrupted the development of seed longevity, with no improvement after seed filling ended. Longevity improved during maturation drying in other treatments. Early (7–14 DAA) exposure to high temperature reduced and low temperature increased subsequent longevity at harvest maturity, whereas late (35 or 42–49 DAA) exposure to high temperature increased and low temperature reduced it. Conclusions Temporal sensitivity to extreme temperature was detected. It varied considerably amongst the contrasting seed variables investigated. Subsequent seed longevity at harvest maturity responded negatively to temperature early in development, but positively later in development and throughout maturation. PMID:28637252

Ccl22/MDC, is a prostaglandin dependent pyrogen, acting in the anterior hypothalamus to induce hyperthermia via activation of brown adipose tissue

PubMed Central

Osborn, Olivia; Sanchez-Alavez, Manuel; Dubins, Jeffrey S.; Gonzalez, Alejandro Sanchez; Morrison, Brad; Hadcock, John R.; Bartfai, Tamas

2011-01-01

CC Chemokine ligand 22 (Ccl22) is a selective, high affinity ligand at the CC chemokine receptor 4 (Ccr4). We have identified cDNAs encoding both ligand and receptor of the Ccl22-Ccr4 pair in cDNA libraries of the anterior hypothalamus/preoptic area (AH/POA) by PCR. The AH/POA is the key brain region where endogenous pyrogens have been shown to act on warm sensitive neurons to affect thermogenesis in brown adipose tissue (BAT) and other thermogenically responsive tissues. We show that functional Ccr4 receptors are present in the AH/POA neurons as injection of Ccl22 into the POA but not to other hypothalamic nuclei induces an increase in core body temperature as measured by radiotelemetry. Indomethacin (5mg/kg s.c) pretreatment markedly reduced the hyperthermia evoked by POA injection of Ccl22 (10 ng/0.5ul) and thus suggests that this hyperthermia is mediated through cyclooxygenase activation and thus likely through the formation and action of the pyrogen prostaglandin E2. The temperature elevation involves a decrease in the respiratory exchange ratio and increased activation of the brown adipose tissue as demonstrated by 18F-FDG –PET imaging. We describe a novel role to the ligand Ccl22 and its receptor Ccr4 in the anterior hypothalamus in temperature regulation that depends on the synthesis of the endogenous pyrogen, prostaglandin E2. PMID:21177120

Effects of Temperature and Supply Voltage on SEU- and SET-Induced Errors in Bulk 40-nm Sequential Circuits

NASA Astrophysics Data System (ADS)

Chen, R. M.; Diggins, Z. J.; Mahatme, N. N.; Wang, L.; Zhang, E. X.; Chen, Y. P.; Zhang, H.; Liu, Y. N.; Narasimham, B.; Witulski, A. F.; Bhuva, B. L.; Fleetwood, D. M.

2017-08-01

The single-event sensitivity of bulk 40-nm sequential circuits is investigated as a function of temperature and supply voltage. An overall increase in SEU cross section versus temperature is observed at relatively high supply voltages. However, at low supply voltages, there is a threshold temperature beyond which the SEU cross section decreases with further increases in temperature. Single-event transient induced errors in flip-flops also increase versus temperature at relatively high supply voltages and are more sensitive to temperature variation than those caused by single-event upsets.

Metal-Chelate Affinity Precipitation with Thermo-Responsive Polymer for Purification of ε-Poly-L-Lysine.

PubMed

Li, Sipeng; Ding, Zhaoyang; Liu, Jifu; Cao, Xuejun

2017-12-01

ε-Poly-L-lysine (ε-PL) is a natural preservative for food processing industry. A thermo-responsive polymer, attached with Cu 2+ or Ni 2+ , was prepared for metal-chelate affinity precipitation for purification of ε-PL. The low critical solution temperatures (LCSTs) of these polymers were close to the room temperature (31.0-35.0 °C). The optimal adsorption conditions were as follows: pH 4.0, 0 mol/L NaCl, ligand density 75.00 μmol/g, and 120 min. The ligand Cu 2+ showed a stronger affinity interaction with ε-PL and the highest adsorption amount reached 251.93 mg/g polymer. The elution recovery of ε-PL could be 98.42% with 0.50 mol/L imidazole (pH = 8.0) as the eluent. The method could purify ε-PL from fermentation broth and the final product was proved as electrophoretic pure by SDS-PAGE. Moreover, these affinity polymers could be recycled after the purification of ε-PL and the recoveries were above 95.00%. Graphical Abstract Scheme for affinity precipitation of ε-PL.

Methacrylate gels with epoxide groups as supports for immobilization of enzymes in pH range 3-12.

PubMed

Turková, J; Bláha, K; Malaníková, M; Vancurová, D; Svec, F; Kálal, J

1978-05-11

Glycidyl methacrylate gels are carriers suitable for attachment of enzymes and for use in affinity chromatography. Experiments on the coupling of glycyl-L-leucine and acetyl-L-leucine to these gels have shown a high pH-dependence of the bond formation between the support and the alpha-amino group (pH optimum 9.7); the coupling reaction between the epoxide group and the carboxyl group is practically pH-independent. Serum albumin and trypsin were attached to a greater extent in acidic than in alkaline media. The effects of time and temperature were also studied. The catalytic action of immobilized trypsin, as well as its use for affinity chromatography of trypsin inhibitor, were studied.

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

PubMed Central

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathy L.; Marks, James D.; Varnum, Susan M.

2012-01-01

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples. PMID:22935296

Modification of a deoxynivalenol-antigen-mimicking nanobody to improve immunoassay sensitivity by site-saturation mutagenesis.

PubMed

Qiu, Yu-Lou; He, Qing-Hua; Xu, Yang; Wang, Wei; Liu, Yuan-Yuan

2016-01-01

A nanobody (N-28) which can act as a deoxynivalenol (DON) antigen has been generated, and its residues Thr102-Ser106 were identified to bind with anti-DON monoclonal antibody by alanine-scanning mutagenesis. Site-saturation mutagenesis was used to analyze the plasticity of five residues and to improve the sensitivity of the N-28-based immunoassay. After mutagenesis, three mutants were selected by phage immunoassay and were sequenced. The half-maximal inhibitory concentrations of the immunoassay based on mutants N-28-T102Y, N-28-V103L, and N-28-Y105F were 24.49 ± 1.0, 51.83 ± 2.5, and 35.65 ± 1.6 ng/mL, respectively, showing the assay was, respectively, 3.2, 1.5, and 2.2 times more sensitive than the wild-type-based assay. The best mutant, N-28-T102Y, was used to develop a competitive phage ELISA to detect DON in cereals with high specificity and accuracy. In addition, the structural properties of N-28-T102Y and N-28 were investigated, revealing that the affinity of N-28-T102Y decreased because of increased steric hindrance with the large side chain. The lower-binding-affinity antigen mimetic may contribute to the improvement of the sensitivity of competitive immunoassays. These results demonstrate that nanobodies would be a favorable tool for engineering. Moreover, our results have laid a solid foundation for site-saturation mutagenesis of antigen-mimicking nanobodies to improve immunoassay sensitivity for small molecules.

Polarization-sensitive and broadband germanium sulfide photodetectors with excellent high-temperature performance.

PubMed

Tan, Dezhi; Zhang, Wenjin; Wang, Xiaofan; Koirala, Sandhaya; Miyauchi, Yuhei; Matsuda, Kazunari

2017-08-31

Layered materials, such as graphene, transition metal dichalcogenides and black phosphorene, have been established rapidly as intriguing building blocks for optoelectronic devices. Here, we introduce highly polarization sensitive, broadband, and high-temperature-operation photodetectors based on multilayer germanium sulfide (GeS). The GeS photodetector shows a high photoresponsivity of about 6.8 × 10 3 A W -1 , an extremely high specific detectivity of 5.6 × 10 14 Jones, and broad spectral response in the wavelength range of 300-800 nm. More importantly, the GeS photodetector has high polarization sensitivity to incident linearly polarized light, which provides another degree of freedom for photodetectors. Tremendously enhanced photoresponsivity is observed with a temperature increase, and high responsivity is achievable at least up to 423 K. The establishment of larger photoinduced reduction of the Schottky barrier height will be significant for the investigation of the photoresponse mechanism of 2D layered material-based photodetectors. These attributes of high photocurrent generation in a wide temperature range, broad spectral response, and polarization sensitivity coupled with environmental stability indicate that the proposed GeS photodetector is very suitable for optoelectronic applications.

Low soil moisture during hot periods drives apparent negative temperature sensitivity of soil respiration in a dryland ecosystem: A multi-model comparison

USGS Publications Warehouse

Tucker, Colin; Reed, Sasha C.

2016-01-01

Arid and semiarid ecosystems (drylands) may dominate the trajectory of biosphere-to-atmosphere carbon (C) flux over the coming century. Accordingly, understanding dryland CO2 efflux controls is important for understanding C cycling at the global-scale: key unknowns regarding how temperature and moisture interact to regulate dryland C cycling remain. Further, the patchiness of dryland vegetation can create ‘islands of fertility’, with spatially heterogeneous rates of soil respiration (Rs). At our study site in southeastern Utah, USA we added or removed litter (0 to 650% of control) in paired plots that were either associated with a shrub or with interspaces between vascular plants. We measured Rs, soil temperature, and water content (θ) on eight sampling dates between October 2013 and November 2014. Rs was highest following monsoon rains in late summer when soil temperature was ~30°C. During mid-summer, Rs was low, associated with high soil temperatures (>40°C), resulting in an apparent negative temperature sensitivity of Rs at high temperatures, and positive temperature sensitivity at low-moderate temperatures. We used Bayesian statistical methods to compare multiple competing models capturing a wide range of hypothesized relationships between temperature, moisture, and Rs. The best fit model indicates apparent negative temperature sensitivity of soil respiration at high temperatures reflects the control of soil moisture – not high temperatures – in limiting Rs. The modeled Q10 ranged from 2.7 at 5°C to 1.4 at 45°C. Litter addition had no effect on temperature sensitivity or reference respiration (Rref = Rs at 20°C and optimum moisture) beneath shrubs, and little effect on Rref in interspaces, yet Rref was 1.5 times higher beneath shrubs than in interspaces. Together, these results suggest reduced Rs often observed at high temperatures in drylands is dominated by the control of moisture, and that variable litter inputs – at least over the short-term – exert minimal control over Rs.

Polymer/silica hybrid waveguide temperature sensor based on asymmetric Mach-Zehnder interferometer

NASA Astrophysics Data System (ADS)

Niu, Donghai; Wang, Xibin; Sun, Shiqi; Jiang, Minghui; Xu, Qiang; Wang, Fei; Wu, Yuanda; Zhang, Daming

2018-04-01

A highly sensitive waveguide temperature sensor based on asymmetric Mach-Zehnder interferometer was designed and experimentally demonstrated. The interferometer is based on the polymer/silica hybrid waveguide structure, and Norland Optical Adhesive 73 (NOA 73) was employed as the waveguide core to enhance the temperature sensitivity. The influence of the different length differences between the two interferometer arms on the sensitivity of the sensor was systemically studied. It is shown that the maximum temperature sensitivity of -431 pm °C-1 can be obtained in the range of 25 °C-75 °C, while the length difference is 92 μm. Moreover, the temperature sensitivity contributions from different core materials were also investigated experimentally. It is shown that the waveguide material and microstructure of the device have significant influences on the sensitivity of the waveguide temperature sensor.

Purification of camel liver catalase by zinc chelate affinity chromatography and pH gradient elution: An enzyme with interesting properties.

PubMed

Chafik, Abdelbasset; Essamadi, Abdelkhalid; Çelik, Safinur Yildirim; Mavi, Ahmet

2017-12-01

Climate change and increasing temperatures are global concerns. Camel (Camelus dromedarius) lives most of its life under high environmental stress in the desert and represent ideal model for studying desert adaptation among mammals. Catalase plays a key role in protecting cells against oxidative stress. For the first time, catalase from camel liver was purified to homogeneity by zinc chelate affinity chromatography using pH gradient elution, a better separation was obtained. A purification fold of 201.81 with 1.17% yield and a high specific activity of 1132539.37U/mg were obtained. The native enzyme had a molecular weight of 268kDa and was composed of four subunits of equal size (65kDa). The enzyme showed optimal activity at a temperature of 45°C and pH 7.2. Thiol reagents, β-Mercaptoethanol and D,L-Dithiothreitol, inhibited the enzyme activity. The enzyme was inhibited by Al 3+ , Cd 2+ and Mg 2+ , whereas Ca 2+ , Co 2+ and Ni 2+ stimulated the catalase activity. Reduced glutathione has no effect on catalase activity. The K m and V max of the enzyme for hydrogen peroxide were 37.31mM and 6185157U/mg, respectively. Sodium azide inhibited the enzyme noncompetitively with K i value of 14.43μM, the IC 50 was found to be 16.71μM. The properties of camel catalase were different comparing to those of mammalian species. Relatively higher molecular weight, higher optimum temperature, protection of reduced glutathione from hydrogen peroxide oxidation and higher affinity for hydrogen peroxide and sodium azide, these could be explained by the fact that camel is able to live in the intense environmental stress in the desert. Copyright © 2017 Elsevier B.V. All rights reserved.

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI.

PubMed

Sabban, Sari; Ye, Hongtu; Helm, Birgit

2014-11-01

The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml(-1) of antigen. This assay was modified from previous assays used to study human and canine allergic responses. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease.

Development of an in vitro model system for studying the interaction of Equus caballus IgE with its high-affinity receptor FcεRI

PubMed Central

Sabban, Sari; Ye, Hongtu; Helm, Birgit

2014-01-01

The interaction of IgE with its high-affinity Fc receptor (FcεRI) followed by an antigenic challenge is the principal pathway in IgE mediated allergic reactions. As a consequence of the high affinity binding between IgE and FcεRI, along with the continuous production of IgE by B cells, allergies usually persist throughout life, with currently no permanent cure available. Horses, especially race horses, which are commonly inbred, are a species of mammals that are very prone to the development of hypersensitivity responses, which can seriously affect their performance. Physiological responses to allergic sensitization in horses mirror that observed in humans and dogs. In this paper we describe the development of an in situ assay system for the quantitative assessment of the release of mediators of the allergic response pertaining to the equine system. To this end, the gene encoding equine FcεRIα was transfected into and expressed onto the surface of parental Rat Basophil Leukemia (RBL-2H3.1) cells. The gene product of the transfected equine α-chain formed a functional receptor complex with the endogenous rat β- and γ-chains 1. The resultant assay system facilitated an assessment of the quantity of mediator secreted from equine FcεRIα transfected RBL-2H3.1 cells following sensitization with equine IgE and antigenic challenge using β-hexosaminidase release as a readout 2, 3. Mediator release peaked at 36.68% ± 4.88% at 100 ng ml-1 of antigen. This assay was modified from previous assays used to study human and canine allergic responses 4, 5. We have also shown that this type of assay system has multiple applications for the development of diagnostic tools and the safety assessment of potential therapeutic intervention strategies in allergic disease 6, 2, 3. PMID:25406512

Highly Sensitive Liquid Core Temperature Sensor Based on Multimode Interference Effects

PubMed Central

Fuentes-Fuentes, Miguel A.; May-Arrioja, Daniel A.; Guzman-Sepulveda, José R.; Torres-Cisneros, Miguel; Sánchez-Mondragón, José J.

2015-01-01

A novel fiber optic temperature sensor based on a liquid-core multimode interference device is demonstrated. The advantage of such structure is that the thermo-optic coefficient (TOC) of the liquid is at least one order of magnitude larger than that of silica and this, combined with the fact that the TOC of silica and the liquid have opposite signs, provides a liquid-core multimode fiber (MMF) highly sensitive to temperature. Since the refractive index of the liquid can be easily modified, this allows us to control the modal properties of the liquid-core MMF at will and the sensor sensitivity can be easily tuned by selecting the refractive index of the liquid in the core of the device. The maximum sensitivity measured in our experiments is 20 nm/°C in the low-temperature regime up to 60 °C. To the best of our knowledge, to date, this is the largest sensitivity reported for fiber-based MMI temperature sensors. PMID:26512664

Optimal use of tandem biotin and V5 tags in ChIP assays

PubMed Central

Kolodziej, Katarzyna E; Pourfarzad, Farzin; de Boer, Ernie; Krpic, Sanja; Grosveld, Frank; Strouboulis, John

2009-01-01

Background Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. PMID:19196479

Determination and experimental verification of high-temperature SAW orientations on langatate.

PubMed

Davulis, Peter M; da Cunha, Mauricio Pereira

2012-02-01

Langatate (LGT) is a member of the langasite family of crystals appropriate for high-temperature frequency control and sensing applications. This paper identifies multiple LGT SAW orientations for use at high temperature, specifically in the 400°C to 900°C range. Orientations with low sensitivity to temperature are desired for frequency control devices and many sensors, conversely large temperature sensitivity is a benefit for temperature sensors. The LGT SAW temperature behavior has been calculated for orientations sweeping the Euler angles (0°, Θ, ψ), (90°, Θ, ψ), and (ψ, 90°, ψ), based on newly identified high-temperature elastic constants and temperature coefficients for this material. The temperature coefficient of delay (TCD) and total frequency change over the temperature range were analyzed from 400°C to 900°C. Multiple SAW orientations were identified with zero-TCD between 400°C and 500°C. Although no orientations that have turn-over temperatures above 500°C were identified, several have low frequency variation with temperature, of the order of -0.8% over the range 400°C to 800°C. Temperature-sensitive orientations with TCD up to 75 ppm/°C at 900°C were identified, with potential for high-temperature sensor applications. The reported predictions are shown to agree with measured behavior of LGT SAW delay lines fabricated along 6 orientations in the (90°, 23°, ψ) plane. In addition, this work demonstrates that concurrently operated LGT SAW devices fabricated on the same wafer provide means of temperature sensing. In particular, the measured frequency difference between delay lines oriented along (90°, 23°, 0°) and (90°, 23°, 48°) has fractional temperature sensitivity that ranges from -172 ppm/°C at 25°C to -205 ppm/°C at 900°C.

Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine.

PubMed

Rahbarizadeh, F; Rasaee, M J; Madani, R; Rahbarizadeh, M H; Omidfar, K

2000-10-01

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivative of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody produced in these experiments. After two successive limiting dilutions, antibodies produced by 5 clones with good affinities ranging from 10(8) to 10(12) M(-1) and less cross-reaction (least for codeine and other structurally related molecules) were selected. These clones were found to be of IgG class with kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antibody was coated onto the wells of microtiter plate. The standard curve was constructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffer and urine. The slope of the standard curve for selected clone in buffer and urine was calculated to be -0.7 and -0.64, respectively.

Pleistocene tropical Pacific temperature sensitivity to radiative greenhouse gas forcing

NASA Astrophysics Data System (ADS)

Dyck, K. A.; Ravelo, A. C.

2011-12-01

How high will Earth's global average surface temperature ultimately rise as greenhouse gas concentrations increase in the future? One way to tackle this question is to compare contemporaneous temperature and greenhouse gas concentration data from paleoclimate records, while considering that other radiative forcing mechanisms (e.g. changes in the amount and distribution of incoming solar radiation associated with changes in the Earth's orbital configuration) also contribute to surface temperature change. Since the sensitivity of surface temperature varies with location and latitude, here we choose a central location representative of the west Pacific warm pool, far from upwelling regions or surface temperature gradients in order to minimize climate feedbacks associated with high-latitude regions or oceanic dynamics. The 'steady-state' or long-term temperature change associated with greenhouse gas radiative forcing is often labeled as equilibrium (or 'Earth system') climate sensitivity to the doubling of atmospheric greenhouse gas concentration. Climate models suggest that Earth system sensitivity does not change dramatically over times when CO2 was lower or higher than the modern atmospheric value. Thus, in our investigation of the changes in tropical SST, from the glacial to interglacial states when greenhouse gas forcing nearly doubled, we use Late Pleistocene paleoclimate records to constrain earth system sensitivity for the tropics. Here we use Mg/Ca-paleothermometry using the foraminifera G. ruber from ODP Site 871 from the past 500 kyr in the western Pacific warm pool to estimate tropical Pacific equilibrium climate sensitivity to a doubling of greenhouse gas concentrations to be ~4°C. This tropical SST sensitivity to greenhouse gas forcing is ~1-2°C higher than that predicted by climate models of past glacial periods or future warming for the tropical Pacific. Equatorial Pacific SST sensitivity may be higher than predicted by models for a number of reasons. First, models may not be adequately representing long-term deep ocean feedbacks. Second, models may incorrectly parameterize tropical cloud (or other short-term) feedback processes. Lastly, either paleo-temperature or radiative forcing may have been incorrectly estimated (e.g. through calibration of paleoclimate evidence for temperature change). Since theory suggests that surface temperature in the high latitudes is more sensitive to radiative forcing changes than surface temperature in the tropics, the results of this study also imply that globally averaged Earth system sensitivity to greenhouse gas concentrations may be higher than most climate models predict.

Affinity Versus Label-Free Isolation of Circulating Tumor Cells: Who Wins?

PubMed

Murlidhar, Vasudha; Rivera-Báez, Lianette; Nagrath, Sunitha

2016-09-01

The study of circulating tumor cells (CTCs) has been made possible by many technological advances in their isolation. Their isolation has seen many fronts, but each technology brings forth a new set of challenges to overcome. Microfluidics has been a key player in the capture of CTCs and their downstream analysis, with the aim of shedding light into their clinical application in cancer and metastasis. Researchers have taken diverging paths to isolate such cells from blood, ranging from affinity-based isolation targeting surface antigens expressed on CTCs, to label-free isolation taking advantage of the size differences between CTCs and other blood cells. For both major groups, many microfluidic technologies have reported high sensitivity and specificity for capturing CTCs. However, the question remains as to the superiority among these two isolation techniques, specifically to identify different CTC populations. This review highlights the key aspects of affinity and label-free microfluidic CTC technologies, and discusses which of these two would be the highest benefactor for the study of CTCs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Affinity fluorescence-labeled peptides for the early detection of cancer in Barrett's esophagus

NASA Astrophysics Data System (ADS)

Li, Meng; Lu, Shaoying; Piraka, Cyrus; Appelman, Henry; Kwon, Rich; Soetikno, Roy; Kaltenbach, Tonya; Wang, Thomas D.

2009-02-01

Fluorescence-labeled peptides that affinity bind to neoplastic mucsosa are promising for use as a specific contrast agent in the detection of pre-malignant tissue in the esophagus. This method is can be used to identify expression of biological markers associated with dysplasia on endoscopic imaging as a guide for biopsy and represents a novel method for the early detection and prevention of cancer. We demonstrate the use of phage display to select affinity peptides and identify the sequence "ASYNYDA" that binds with high target-to-background ratio to dysplastic esophageal mucosa compared to that of intestinal metaplasia. Validation of preferential binding is demonstrated for neoplasia in the setting of Barrett's esophagus. An optimal tradeoff between sensitivity and specificity of 82% and 85% was found at the relative threshold of 0.60 with a target-to-background ratio of 1.81 and an area under the ROC curve of 0.87. Peptides are a novel class of ligand for targeted detection of pre-malignant mucosa for purposes of screening and surveillance.

CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

PubMed Central

Liu, K H; Huang, C Y; Tsay, Y F

1999-01-01

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis. PMID:10330471

Electrochemical high-temperature gas sensors

NASA Astrophysics Data System (ADS)

Saruhan, B.; Stranzenbach, M.; Yüce, A.; Gönüllü, Y.

2012-06-01

Combustion produced common air pollutant, NOx associates with greenhouse effects. Its high temperature detection is essential for protection of nature. Component-integration capable high-temperature sensors enable the control of combustion products. The requirements are quantitative detection of total NOx and high selectivity at temperatures above 500°C. This study reports various approaches to detect NO and NO2 selectively under lean and humid conditions at temperatures from 300°C to 800°C. All tested electrochemical sensors were fabricated in planar design to enable componentintegration. We suggest first an impedance-metric gas sensor for total NOx-detection consisting of NiO- or NiCr2O4-SE and PYSZ-electrolyte. The electrolyte-layer is about 200μm thickness and constructed of quasi-single crystalline columns. The sensing-electrode (SE) is magnetron sputtered thin-layers of NiO or NiCr2O4. Sensor sensitivity for detection of total NOx has been measured by applying impedance analysis. The cross-sensitivity to other emission gases such as CO, CO2, CH4 and oxygen (5 vol.%) has been determined under 0-1000ppm NO. Sensor maintains its high sensitivity at temperatures up to 550°C and 600°C, depending on the sensing-electrode. NiO-SE yields better selectivity to NO in the presence of oxygen and have shorter response times comparing to NiCr2O4-SE. For higher temperature NO2-sensing capability, a resistive DC-sensor having Al-doped TiO2-sensing layers has been employed. Sensor-sensitivity towards NO2 and cross-sensitivity to CO has been determined in the presence of H2O at temperatures 600°C and 800°C. NO2 concentrations varying from 25 to 100ppm and CO concentrations from 25 to 75ppm can be detected. By nano-tubular structuring of TiO2, NO2 sensitivity of the sensor was increased.

Multimode fiber tip Fabry-Perot cavity for highly sensitive pressure measurement.

PubMed

Chen, W P; Wang, D N; Xu, Ben; Zhao, C L; Chen, H F

2017-03-23

We demonstrate an optical Fabry-Perot interferometer fiber tip sensor based on an etched end of multimode fiber filled with ultraviolet adhesive. The fiber device is miniature (with diameter of less than 60 μm), robust and low cost, in a convenient reflection mode of operation, and has a very high gas pressure sensitivity of -40.94 nm/MPa, a large temperature sensitivity of 213 pm/°C within the range from 55 to 85 °C, and a relatively low temperature cross-sensitivity of 5.2 kPa/°C. This device has a high potential in monitoring environment of high pressure.

Identification and analysis of o-acetylated sialoglycoproteins.

PubMed

Mandal, Chandan; Mandal, Chitra

2013-01-01

5-N-acetylneuraminic acid, commonly known as sialic acid (Sia), constitutes a family of N- and O-substituted 9-carbon monosaccharides. Frequent modification of O-acetylations at positions C-7, C-8, or C-9 of Sias generates a family of O-acetylated sialic acid (O-AcSia) and plays crucial roles in many cellular events like cell-cell adhesion, proliferation, migration, etc. Therefore, identification and analysis of O-acetylated sialoglycoproteins (O-AcSGPs) are important. In this chapter, we describe several approaches for successful identification of O-AcSGPs. We broadly divide them into two categories, i.e., invasive and noninvasive methods. Several O-AcSias-binding probes are used for this purpose. Detailed methodologies for step-by-step identification using these probes have been discussed. We have also included a few invasive analytical methods for identification and quantitation of O-AcSias. Several indirect methods are also elaborated for such purpose, in which O-acetyl group from sialic acids is initially removed followed by detection of Sias by several approaches. For molecular identification, we have described methods for affinity purification of O-AcSGPs using an O-AcSias-binding lectin as an affinity matrix followed by sequencing using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF-TOF) mass spectroscopy (MS). In spite of special attention, loss of O-acetyl groups due to its sensitivity towards alkaline pH and high temperature along with migration of labile O-acetyl groups from C7-C8-C9 during sample preparation is difficult to avoid. Therefore there is always a risk for underestimation of O-AcSias.

Harnessing Fluorine–Sulfur Contacts and Multipolar Interactions for the Design of p53 Mutant Y220C Rescue Drugs

PubMed Central

2016-01-01

Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutation-induced surface crevice with a KD = 150 μM, thereby increasing the melting temperature of the protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small molecule ligands can substantially improve binding affinity to their protein targets. We have, therefore, harnessed fluorine–protein interactions to improve the affinity of this ligand. Step-wise introduction of fluorines at the carbazole ethyl anchor, which is deeply buried within the binding site in the Y220C–PhiKan083 complex, led to a 5-fold increase in affinity for a 2,2,2-trifluoroethyl anchor (ligand efficiency of 0.3 kcal mol–1 atom–1). High-resolution crystal structures of the Y220C–ligand complexes combined with quantum chemical calculations revealed favorable interactions of the fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur of Cys220 at the mutation site. Affinity gains were, however, only achieved upon trifluorination, despite favorable interactions of the mono- and difluorinated anchors with the binding pocket, indicating a trade-off between energetically favorable protein–fluorine interactions and increased desolvation penalties. Taken together, the optimized carbazole scaffold provides a promising starting point for the development of high-affinity ligands to reactivate the tumor suppressor function of the p53 mutant Y220C in cancer cells. PMID:27267810

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Advantages and application of label-free detection assays in drug screening.

PubMed

Cunningham, Brian T; Laing, Lance G

2008-08-01

Adoption is accelerating for a new family of label-free optical biosensors incorporated into standard format microplates owing to their ability to enable highly sensitive detection of small molecules, proteins and cells for high-throughput drug discovery applications. Label-free approaches are displacing other detection technologies owing to their ability to provide simple assay procedures for hit finding/validation, accessing difficult target classes, screening the interaction of cells with drugs and analyzing the affinity of small molecule inhibitors to target proteins. This review describes several new drug discovery applications that are under development for microplate-based photonic crystal optical biosensors and the key issues that will drive adoption of the technology. Microplate-based optical biosensors are enabling a variety of cell-based assays, inhibition assays, protein-protein binding assays and protein-small molecule binding assays to be performed with high-throughput and high sensitivity.

A highly sensitive and specific capacitive aptasensor for rapid and label-free trace analysis of Bisphenol A (BPA) in canned foods.

PubMed

Mirzajani, Hadi; Cheng, Cheng; Wu, Jayne; Chen, Jiangang; Eda, Shigotoshi; Najafi Aghdam, Esmaeil; Badri Ghavifekr, Habib

2017-03-15

A rapid, highly sensitive, specific and low-cost capacitive affinity biosensor is presented here for label-free and single step detection of Bisphenol A (BPA). The sensor design allows rapid prototyping at low-cost using printed circuit board material by benchtop equipment. High sensitivity detection is achieved through the use of a BPA-specific aptamer as probe molecule and large electrodes to enhance AC-electroelectrothermal effect for long-range transport of BPA molecules toward electrode surface. Capacitive sensing technique is used to determine the bounded BPA level by measuring the sample/electrode interfacial capacitance of the sensor. The developed biosensor can detect BPA level in 20s and exhibits a large linear range from 1 fM to 10 pM, with a limit of detection (LOD) of 152.93 aM. This biosensor was applied to test BPA in canned food samples and could successfully recover the levels of spiked BPA. This sensor technology is demonstrated to be highly promising and reliable for rapid, sensitive and on-site monitoring of BPA in food samples. Copyright © 2016 Elsevier B.V. All rights reserved.

[Changes in blood gases with temperature: implications for clinical practice].

PubMed

Tremey, B; Vigué, B

2004-05-01

To understand changes in blood gases results with core temperature. Analysis from two case reports. Hypothermia induces a decrease in PaCO(2) with a related increase in pH, thus a physiologic alkalosis. Decrease in PaCO(2) is due to an increase of gas solubility and a decrease of peripheral consumption that can be estimated from comparison between corrected and non-corrected for temperature blood gases. For O(2), variations of temperature induce variations of solubility but also of haemoglobin affinity for O(2). During hyperthermia, haemoglobin affinity for O(2) is decreased with a decreased SvO(2) for a same PvO(2). SvO(2) ischemic or therapeutic thresholds are thus modified with core temperature. Blood gases cannot be understood without patient core temperature. Physiologic variations of PaCO(2) and pH must probably be tolerated. Ischemic threshold should be estimated on PvO(2), not only on PvO(2).

SU-E-T-112: Experimental Characterization of a Novel Thermal Reservoir for Consistent and Accurate Annealing of High-Sensitivity TLDs.

PubMed

Donahue, W; Bongiorni, P; Hearn, R; Rodgers, J; Nath, R; Chen, Z

2012-06-01

To develop and characterize a novel thermal reservoir for consistent and accurate annealing of high-sensitivity thermoluminescence dosimeters (TLD-100H) for dosimetry of brachytherapy sources. The sensitivity of TLD-100H is about 18 times that of TLD-100 which has clear advantages in for interstitial brachytherapy sources. However, the TLD-100H requires a short high temperature annealing cycle (15 min.) and opening and closing the oven door causes significant temperature fluctuations leading to unreliable measurements. A new thermal reservoir made of aluminum alloy was developed to provide stable temperature environment in a standard hot air oven. The thermal reservoir consisted of a 20 cm × 20 cm × 8 cm Al block with a machine-milled chamber in the middle to house the aluminum TLD holding tray. The thermal reservoir was placed inside the oven until it reaches thermal equilibrium with oven chamber. The temperatures of the oven chamber, heat reservoir, and TLD holding tray were monitored by two independent thermo-couples which interfaced digitally to a control computer. A LabView interface was written for monitoring and recording the temperatures in TLD holding tray, the thermal reservoir, and oven chamber. The temperature profiles were measured as a function of oven-door open duration. The settings for oven chamber temperature and oven door open-close duration were optimized to achieve a stable temperature of 240 0C in the TLD holding tray. Complete temperature profiles of the TLD annealing tray over the entire annealing process were obtained. A LabView interface was written for monitoring and recording the temperatures in TLD holding The use of the thermal reservoir has significantly reduced the temperature fluctuations caused by the opening of oven door when inserting the TLD holding tray into the oven chamber. It has enabled consistent annealing of high-sensitivity TLDs. A comprehensive characterization of a custom-built novel thermal reservoir for annealing high-sensitivity TLD has been carried out. It enabled consistent and accurate annealing of high- sensitivity TLDs which could significantly improve the efficiency of brachytherapy source characterizations. Supported in part by NIH grant R01-CA134627. © 2012 American Association of Physicists in Medicine.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand.

PubMed

Ding, Zhaoyang; Cao, Xuejun

2013-12-17

Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris-HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand

PubMed Central

2013-01-01

Background Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. Results A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. Conclusion A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum. PMID:24341315

High-performance, room-temperature, and no-humidity-impact ammonia sensor based on heterogeneous nickel oxide and zinc oxide nanocrystals.

PubMed

Wang, Jian; Yang, Pan; Wei, Xiaowei

2015-02-18

NiO nanocones decorated with ZnO nanothorns on NiO foil substrates are shown to be an ammonia sensor with excellent comprehensive performance, which could, in real-time, detect and monitor NH3 in the surrounding environment. Gas-sensing measurements indicate that assembling nanocones decorated with nanothorns on NiO foil substrate is an effective strategy for simultaneously promoting the stability, reproducibility, and sensitivity of the sensor, because the NiO foil substrate as a whole can quickly and stably transfer electrons between the gas molecules and the sensing materials and the large specific surface area of both nanocones and nanothorns provide good accessibility of the gas molecules to the sensing materials. Moreover, p-type NiO, with majority charge carriers of holes, has higher binding affinity for the electron-donating ammonia, resulting in a significant increase in selectivity toward NH3 over other organic gases. Compared with the NiO nanowires and pure NiO nanocones, the heterogeneous NiO nanocones/ZnO nanothorns exhibit less dependence on the temperature and humidity in response/recovery speed and sensitivity of sensing NH3. Our investigation indicates that two factors are responsible for reducing the dependence on the gas sensing characteristics under various environmental conditions. One is that the n-type ZnO nanothorns growing on the surface of nanocones, with majority charge carriers of electrons, speed up adsorption and desorption of gas molecules. The other is that the abundant cone-shaped and thornlike superstructures on the substrate are favorable for constructing a hydrophobic surface, which prevents the gas sensing material from being wetted.

The oxygen-binding properties of hemocyanin from the mollusk Concholepas concholepas.

PubMed

González, Andrea; Nova, Esteban; Del Campo, Miguel; Manubens, Augusto; De Ioannes, Alfredo; Ferreira, Jorge; Becker, María Inés

2017-12-01

Hemocyanins have highly conserved copper-containing active sites that bind oxygen. However, structural differences among the hemocyanins of various mollusks may affect their physicochemical properties. Here, we studied the oxygen-binding cooperativity and affinity of Concholepas concholepas hemocyanin (CCH) and its two isolated subunits over a wide range of temperatures and pH values. Considering the differences in the quaternary structures of CCH and keyhole limpet hemocyanin (KLH), we hypothesized that the heterodidecameric CCH has different oxygen-binding parameters than the homodidecameric KLH. A novel modification of the polarographic method was applied in which rat liver submitochondrial particles containing cytochrome c oxidase were introduced to totally deplete oxygen of the test solution using ascorbate as the electron donor. This method was both sensitive and reproducible. The results showed that CCH, like other hemocyanins, exhibits cooperativity, showing an inverse relationship between the oxygen-binding parameters and temperature. According to their Hill coefficients, KLH has greater cooperativity than CCH at physiological pH; however, CCH is less sensitive to pH changes than KLH. Appreciable differences in binding behavior were found between the CCH subunits: the cooperativity of CCH-A was not only almost double that of CCH-B, but it was also slightly superior to that of CCH, thus suggesting that the oxygen-binding domains of the CCH subunits are different in their primary structure. Collectively, these data suggest that CCH-A is the main oxygen-binding domain in CCH; CCH-B may play a more structural role, perhaps utilizing its surprising predisposition to form tubular polymers, unlike CCH-A, as demonstrated here using electron microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

Optical fiber extrinsic Fabry-Perot interferometric (EFPI)-based biosensors

NASA Astrophysics Data System (ADS)

Elster, Jennifer L.; Jones, Mark E.; Evans, Mishell K.; Lenahan, Shannon M.; Boyce, Christopher A.; Velander, William H.; VanTassell, Roger

2000-05-01

A novel system incorporating optical fiber extrinsic Fabry- Perot interferometric (EFPI)-based sensors for rapid detection of biological targets is presented. With the appropriate configuration, the EFPI senor is able to measure key environmental parameters by monitoring the interferometric fringes resulting from an optical path differences of reflected signals. The optical fiber EFPI sensor has been demonstrated for strain, pressure, and temperature measurements and can be readily modified for refractive index measurements by allowing solutions to flow into an open cavity. The sensor allows for highly sensitive, real-time, refractive index measurements and by applying affinity coatings containing ligands within this cavity, specific binding of target molecules can be accomplished. As target molecules bind to the coating, there is an increased density within the film, causing a measurable refractive index change that correlates to the concentration of detected target molecules. This sensor platform offers enhanced sensing capabilities for clinical diagnostics, pharmaceutical screening, environmental monitoring, food pathogen detection, biological warfare agent detection, and industrial bioprocessing. Promising applications also exist for process monitoring within the food/beverage, petroleum, and chemical industry.

Diselenolane-mediated cellular uptake.

PubMed

Chuard, Nicolas; Poblador-Bahamonde, Amalia I; Zong, Lili; Bartolami, Eline; Hildebrandt, Jana; Weigand, Wolfgang; Sakai, Naomi; Matile, Stefan

2018-02-21

The emerging power of thiol-mediated uptake with strained disulfides called for a move from sulfur to selenium. We report that according to results with fluorescent model substrates, cellular uptake with 1,2-diselenolanes exceeds uptake with 1,2-dithiolanes and epidithiodiketopiperazines with regard to efficiency as well as intracellular localization. The diselenide analog of lipoic acid performs best. This 1,2-diselenolane delivers fluorophores efficiently to the cytosol of HeLa Kyoto cells, without detectable endosomal capture as with 1,2-dithiolanes or dominant escape into the nucleus as with epidithiodiketopiperazines. Diselenolane-mediated cytosolic delivery is non-toxic (MTT assay), sensitive to temperature but insensitive to inhibitors of endocytosis (chlorpromazine, methyl-β-cyclodextrin, wortmannin, cytochalasin B) and conventional thiol-mediated uptake (Ellman's reagent), and to serum. Selenophilicity, the extreme CSeSeC dihedral angle of 0° and the high but different acidity of primary and secondary selenols might all contribute to uptake. Thiol-exchange affinity chromatography is introduced as operational mimic of thiol-mediated uptake that provides, in combination with rate enhancement of DTT oxidation, direct experimental evidence for existence and nature of the involved selenosulfides.

Differences in the distribution and characteristics of tachykinin NK1 binding sites between human and guinea pig lung.

PubMed Central

Walsh, D A; Salmon, M; Featherstone, R; Wharton, J; Church, M K; Polak, J M

1994-01-01

1. The distribution and characteristics of tachykinin NK1 binding sites have been compared in human and guinea pig lung using quantitative in vitro receptor autoradiography with [125I]-Bolton Hunter-labelled substance P ([125I]-BH-SP). In addition, the effects on these sites of ovalbumin sensitization and challenge have been determined in guinea pig lung. 2. [125I]-BH-SP bound specifically and with high affinity to microvascular endothelium in both human and guinea pig lung, but to bronchial smooth muscle and pulmonary artery media in only guinea pig lung. 3. Specific binding of [125I]-BH-SP to guinea pig bronchial smooth muscle was positively correlated with airway diameter in the range 150-800 microns and was less dense in trachea than in main bronchi. 4. [125I]-BH-SP binding was inhibited by tachykinins with rank orders of affinity of SP > NKA > NKB (human microvessels) and SP > NKA = NKB (guinea pig bronchi and pulmonary arteries). NKA displayed a higher affinity for [125I]-BH-SP binding sites in human microvessels than in guinea pig tissues (P < 0.0001), indicating differences in selectivity for tachykinins between human and guinea pig NK1 receptors. 5. In both human and guinea pig lung, [125I]-BH-SP binding was inhibited by the specific tachykinin receptor antagonists FK888 (NK1 selective antagonist) and FK224 (mixed NK1/NK2 antagonist), with FK888 displaying equal affinity to SP and > 500 times higher affinity than FK224. SP, NKA, NKB and FK888 exhibited similar affinities for [125I]-BH-SP binding sites in both guinea pig arteries and bronchi.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 1 Figure 2 PMID:7534186

Characterization of the interaction between the heavy and light chains of bovine factor Va.

PubMed

Walker, F J

1992-10-05

Bovine factor Va has been previously been shown to consist of heavy (M(r) = 94,000) and light chains (M(r) = 81,000), that interact in a manner dependent upon the presence of either calcium or manganese ions. In an attempt to understand the mechanism of subunit interaction we have studied the effects of temperature and ions on factor Va stability. The rates of formation of factor Va from isolated chains and dissociation were temperature-dependent with an energy of activation of 6.2 and 1.3 kcal mol-1, respectively. The yield of factor Va from isolated chains was inversely related to the amount of time the chains were incubated at 4 degrees C. Incubation of individual chains revealed that the heavy chain is cold-labile, an effect that is reversible. Manganese ion was observed to prevent the conversion to the inactive form. High salt tends to stabilize the two-chain structure of factor Va, but is inhibitory to its formation from isolated chains. High concentrations of either manganese or calcium ions also inhibited reconstitution of activity. The light chain, in particular, was sensitive to the presence of manganese or calcium ion. Heavy chain that had been cleaved by activated protein C had a weakened interaction with the light chain, and the resulting complex had no procoagulant activity. Cooling of the heavy chain to 4 degrees C enhanced its intrinsic fluorescence. Manganese ion prevented some of this enhancement. The heavy chain fluorescence returned to the room temperature value with a half-life of approximately 10 min. In the presence of manganese ion relaxation was accelerated. The intrinsic fluorescence of activated protein C-cleaved heavy chain was not increased when the temperature was decreased. These data suggest that the heavy chain can exist in two forms. Elevated temperature converts it to a form that can bind ions and have a productive interaction with the light chain. However, conditions that prevent the heavy chain from combining with the light chain also stabilize the two subunit structure, suggesting that the high affinity of the complex is due to conformational changes that occur after chain interaction.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

PubMed

Akunne, H C; Demattos, S B; Whetzel, S Z; Wustrow, D J; Davis, D M; Wise, L D; Cody, W L; Pugsley, T A; Heffner, T G

1995-04-18

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Polyploidization mechanisms: temperature environment can induce diploid gamete formation in Rosa sp.

PubMed

Pécrix, Yann; Rallo, Géraldine; Folzer, Hélène; Cigna, Mireille; Gudin, Serge; Le Bris, Manuel

2011-06-01

Polyploidy is an important evolutionary phenomenon but the mechanisms by which polyploidy arises still remain underexplored. There may be an environmental component to polyploidization. This study aimed to clarify how temperature may promote diploid gamete formation considered an essential element for sexual polyploidization. First of all, a detailed cytological analysis of microsporogenesis and microgametogenesis was performed to target precisely the key developmental stages which are the most sensitive to temperature. Then, heat-induced modifications in sporad and pollen characteristics were analysed through an exposition of high temperature gradient. Rosa plants are sensitive to high temperatures with a developmental sensitivity window limited to meiosis. Moreover, the range of efficient temperatures is actually narrow. 36 °C at early meiosis led to a decrease in pollen viability, pollen ectexine defects but especially the appearance of numerous diploid pollen grains. They resulted from dyads or triads mainly formed following heat-induced spindle misorientations in telophase II. A high temperature environment has the potential to increase gamete ploidy level. The high frequencies of diplogametes obtained at some extreme temperatures support the hypothesis that polyploidization events could have occurred in adverse conditions and suggest polyploidization facilitating in a global change context.

Real-time reliable determination of binding kinetics of DNA hybridization using a multi-channel graphene biosensor

NASA Astrophysics Data System (ADS)

Xu, Shicai; Zhan, Jian; Man, Baoyuan; Jiang, Shouzhen; Yue, Weiwei; Gao, Shoubao; Guo, Chengang; Liu, Hanping; Li, Zhenhua; Wang, Jihua; Zhou, Yaoqi

2017-03-01

Reliable determination of binding kinetics and affinity of DNA hybridization and single-base mismatches plays an essential role in systems biology, personalized and precision medicine. The standard tools are optical-based sensors that are difficult to operate in low cost and to miniaturize for high-throughput measurement. Biosensors based on nanowire field-effect transistors have been developed, but reliable and cost-effective fabrication remains a challenge. Here, we demonstrate that a graphene single-crystal domain patterned into multiple channels can measure time- and concentration-dependent DNA hybridization kinetics and affinity reliably and sensitively, with a detection limit of 10 pM for DNA. It can distinguish single-base mutations quantitatively in real time. An analytical model is developed to estimate probe density, efficiency of hybridization and the maximum sensor response. The results suggest a promising future for cost-effective, high-throughput screening of drug candidates, genetic variations and disease biomarkers by using an integrated, miniaturized, all-electrical multiplexed, graphene-based DNA array.

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

PubMed

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Specific binding, internalization, and degradation of human neutrophil activating factor by human polymorphonuclear leukocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Besemer, J.; Hujber, A.; Kuhn, B.

1989-10-15

The interaction of {sup 125}I-labeled recombinant human neutrophil activating factor (NAF) with polymorphonuclear leukocytes (PMN) was studied by means of a radioreceptor assay. The binding was characterized by a rapid transition (t1/2 less than or equal to 1 min) from a pH 3-sensitive state at 4{degree}C to pH 3 resistance at 37{degree}C. This was not caused by internalization of NAF since pH 3-resistant bound iodinated NAF could still be exchanged by an excess of nonlabeled NAF, i.e. was dissociable. Internalized iodinated NAF was processed into trichloroacetic acid-soluble forms. Scatchard transformation of binding isotherms at 4 and 37{degree}C led to nonlinearmore » curves, a finding which is consistent with the expression of two receptor populations, one with high (KD = 11-35 pM) and the other with lower affinity (KD = 640-830 pM) at 4 degrees C. Numbers of the low affinity binding sites were approximately 34,000, and those with high affinity were 5,200/PMN when estimated at 4 degrees C. Binding of iodinated NAF to PMN was specific since it could be competed by an excess of nonlabeled NAF but not by two other activators of PMN function, formylmethionyl-leucyl-phenylalanine or human recombinant granulocyte-macrophage colony-stimulating factor. In addition to human PMN, NAF also bound specifically to two human monocytic cell lines; however, only the low affinity binding site could be detected on these cells.« less

Identification of two H3-histamine receptor subtypes

DOE Office of Scientific and Technical Information (OSTI.GOV)

West, R.E. Jr.; Zweig, A.; Shih, N.Y.

The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-(3H)methylhistamine ((3H)NAMHA). The results of (3H)NAMHA saturation binding and NAMHA inhibition of (3H)NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of (3H)NAMHA binding by the specific high affinity H3 antagonist thioperamide revealedmore » two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for (3H) NAMHA was reduced less than 2-fold, whereas (3H)NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.« less

Arrays of membrane isolated yttrium-barium-copper-oxide kinetic inductance bolometers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lindeman, M. A., E-mail: mark.a.lindeman@jpl.nasa.gov; Bonetti, J. A.; Bumble, B.

We are developing of arrays of membrane isolated resonator-bolometers, each with a kinetic inductance device (KID) to measure the temperature of the membrane. The KIDs are fabricated out of the high temperature superconductor YBCO to allow operation at relatively high temperatures. The bolometers are designed to offer higher sensitivity than sensors operating at 300 K, but they require less expensive and lighter weight cooling than even more sensitive conventional superconducting detectors operating at lower temperatures. The bolometer arrays are applicable as focal planes in infrared imaging spectrometers, such as for planetary science missions or earth observing satellites. We describe the devicesmore » and present measurements of their sensitivity.« less

Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Sun-Joo; Ren, Feifei; Zangerl-Plessl, Eva-Maria

2016-08-15

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP 2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL -) with a distinct second site is required for high PIP 2sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP 2sensitivity, even in the absence of PL -. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP 2(2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domainmore » (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL -binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP 2site and explaining the positive allostery between PL -binding and PIP 2sensitivity.« less

A highly sensitive and selective aptamer-based colorimetric sensor for the rapid detection of PCB 77.

PubMed

Cheng, Ruojie; Liu, Siyao; Shi, Huijie; Zhao, Guohua

2018-01-05

A highly sensitive, specific and simple colorimetric sensor based on aptamer was established for the detection of polychlorinated biphenyls (PCB 77). The use of unmodified gold nanoparticles as a colorimetric probe for aptamer sensors enabled the highly sensitive and selective detection of polychlorinated biphenyls (PCB 77). A linear range of 0.5nM to 900nM was obtained for the colorimetric assay with a minimum detection limit of 0.05nM. In addition, by the methods of circular dichroism, UV and naked eyes, we found that the 35 base fragments retained after cutting 5 bases from the 5 'end of aptamer plays the most significant role in the PCB 77 specific recognition process. We found a novel way to truncated nucleotides to optimize the detection of PCB 77, and the selected nucleotides also could achieve high affinity with PCB 77. At the same time, the efficient detection of the PCB 77 by our colorimetric sensor in the complex environmental water samples was realized, which shows a good application prospect. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids.

PubMed

Lee, Sun-Joo; Ren, Feifei; Zangerl-Plessl, Eva-Maria; Heyman, Sarah; Stary-Weinzinger, Anna; Yuan, Peng; Nichols, Colin G

2016-09-01

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL(-)) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL(-) Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL(-) binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL(-) binding and PIP2 sensitivity. © 2016 Lee et al.

Trichoplax adhaerens reveals a network of nuclear receptors sensitive to 9-cis-retinoic acid at the base of metazoan evolution

PubMed Central

Novotný, Jan Philipp; Chughtai, Ahmed Ali; Kostrouchová, Markéta; Kostrouchová, Veronika; Kostrouch, David; Kaššák, Filip; Kaňa, Radek; Schierwater, Bernd; Kostrouchová, Marta

2017-01-01

Trichoplax adhaerens, the only known species of Placozoa is likely to be closely related to an early metazoan that preceded branching of Cnidaria and Bilateria. This animal species is surprisingly well adapted to free life in the World Ocean inhabiting tidal costal zones of oceans and seas with warm to moderate temperatures and shallow waters. The genome of T. adhaerens (sp. Grell) includes four nuclear receptors, namely orthologue of RXR (NR2B), HNF4 (NR2A), COUP-TF (NR2F) and ERR (NR3B) that show a high degree of similarity with human orthologues. In the case of RXR, the sequence identity to human RXR alpha reaches 81% in the DNA binding domain and 70% in the ligand binding domain. We show that T. adhaerens RXR (TaRXR) binds 9-cis retinoic acid (9-cis-RA) with high affinity, as well as high specificity and that exposure of T. adhaerens to 9-cis-RA regulates the expression of the putative T. adhaerens orthologue of vertebrate L-malate-NADP+ oxidoreductase (EC 1.1.1.40) which in vertebrates is regulated by a heterodimer of RXR and thyroid hormone receptor. Treatment by 9-cis-RA alters the relative expression profile of T. adhaerens nuclear receptors, suggesting the existence of natural ligands. Keeping with this, algal food composition has a profound effect on T. adhaerens growth and appearance. We show that nanomolar concentrations of 9-cis-RA interfere with T. adhaerens growth response to specific algal food and causes growth arrest. Our results uncover an endocrine-like network of nuclear receptors sensitive to 9-cis-RA in T. adhaerens and support the existence of a ligand-sensitive network of nuclear receptors at the base of metazoan evolution. PMID:28975052

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa.

PubMed

Schneidereit, D; Vass, H; Reischl, B; Allen, R J; Friedrich, O

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules [Formula: see text] is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence.

Alternate working fluids for solar air conditioning applications

NASA Technical Reports Server (NTRS)

Evans, R. D.; Beck, J. K.

1978-01-01

An experimental investigation of sixteen different refrigerant-absorbent fluid pairs has been carried out in order to determine their suitability as the working fluid in a solar-powered absorption cycle air conditioner. The criteria used in the initial selection of a refrigerant-absorbent pair included: high affinity (large negative deviation from Raoult's Law), high solubility, low specific heat, low viscosity, stability, corrosive properties, safety, and cost. For practical solar considerations of a fluid pair, refrigerants were selected with low boiling points whereas absorbent fluids were selected with a boiling point considerably above that of the refrigerant. Additional restrictions are determined by the operating temperatures of the absorber and the generator; these temperatures were specified as 100 F (39 C) and 170 F (77 C). Data are presented for a few selected pressures at the specified absorber and generator temperatures.

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

Reasonable Temperature Schedules for Cold or Hot Charging of Continuously Cast Steel Slabs

NASA Astrophysics Data System (ADS)

Li, Yang; Chen, Xin; Liu, Ke; Wang, Jing; Wen, Jin; Zhang, Jiaquan

2013-12-01

Some continuously cast steel slabs are sensitive to transverse fracture problems during transportation or handling away from their storage state, while some steel slabs are sensitive to surface transverse cracks during the following rolling process in a certain hot charging temperature range. It is revealed that the investigated steel slabs with high fracture tendency under room cooling condition always contain pearlite transformation delayed elements, which lead to the internal brittle bainitic structure formation, while some microalloyed steels exhibit high surface crack susceptibility to hot charging temperatures due to carbonitride precipitation. According to the calculated internal cooling rates and CCT diagrams, the slabs with high fracture tendency during cold charging should be slowly cooled after cutting to length from hot strand or charged to the reheating furnace directly above their bainite formation temperatures. Based on a thermodynamic calculation for carbonitride precipitation in austenite, the sensitive hot charging temperature range of related steels was revealed for the determination of reasonable temperature schedules.

Affinity purification of the voltage-sensitive sodium channel from electroplax with resins selective for sialic acid

DOE Office of Scientific and Technical Information (OSTI.GOV)

James, W.M.; Emerick, M.C.; Agnew, W.S.

1989-07-11

The voltage-sensitive sodium channel present in the eel (Electrophorus electricus) has an unusually high content of sialic acid, including {alpha}-(2{yields}8)-linked polysialic acid, not found in other electroplax membrane glycopeptides. Lectins from Limax flavus (LFA) and wheat germ (WGA) proved the most effective of 11 lectin resins tried. The most selective resin was prepared from IgM antibodies against Neisseria meningitidis {alpha}-(2{yields}8)-polysialic acid which were affinity purified and coupled to Sepharose 4B. The sodium channel was found to bind to WGA, LFA, and IgM resins and was readily eluted with the appropriate soluble carbohydrates. Experiments with LFA and IgM resins demonstrated bindingmore » and unbinding rates and displacement kinetics, which suggest highly specific binding at multiple sites on the sodium channel protein. In preparative-scale purification of protein previously fractionated by anion-exchange chromatography, without stabilizing TTX, high yields were reproducibly obtained. Further, when detergent extracts were prepared from electroplax membranes fractionated by low-speed sedimentation, a single step over the IgM resin provided a 70-fold purification, yielding specific activities of 3,200 pmol of ({sup 3}H)TTX-binding sites/mg of protein and a single polypeptide of {approximately}285,000 Da on SDS-acrylamide gels. No small peptides were observed after this 5-h isolation. The authors describe a cation-dependent stabilization with millimolar levels of monovalent and micromolar levels of divalent species.« less

A Novel High-Sensitivity, Low-Power, Liquid Crystal Temperature Sensor

PubMed Central

Algorri, José Francisco; Urruchi, Virginia; Bennis, Noureddine; Sánchez-Pena, José Manuel

2014-01-01

A novel temperature sensor based on nematic liquid crystal permittivity as a sensing magnitude, is presented. This sensor consists of a specific micrometric structure that gives considerable advantages from other previous related liquid crystal (LC) sensors. The analytical study reveals that permittivity change with temperature is introduced in a hyperbolic cosine function, increasing the sensitivity term considerably. The experimental data has been obtained for ranges from −6 °C to 100 °C. Despite this, following the LC datasheet, theoretical ranges from −40 °C to 109 °C could be achieved. These results have revealed maximum sensitivities of 33 mVrms/°C for certain temperature ranges; three times more than of most silicon temperature sensors. As it was predicted by the analytical study, the micrometric size of the proposed structure produces a high output voltage. Moreover the voltage's sensitivity to temperature response can be controlled by the applied voltage. This response allows temperature measurements to be carried out without any amplification or conditioning circuitry, with very low power consumption. PMID:24721771

AmPMS: Detection of Ammonia and Amines in Particle Formation and Growth Experiments

NASA Astrophysics Data System (ADS)

Hanson, D. R.; McMurry, P. H.; Jiang, J.; Huey, L. G.; Tanner, D.

2010-12-01

Ammonia and amine compounds in the atmosphere can be a significant component of atmospheric aerosol. Theoretical work shows that these compounds have a potentially large affinity for the particulate phase if strong acids are present. The co-accumulation of amines/ammonia with acids on atmospheric particles can be important for growth of atmospheric particles. Also, the role of nitrogen bases in nucleation is believed to be important. While proton transfer mass spectrometry (MS) has been deployed to detect a wide variety of volatile organic compounds in the atmosphere using H3O+ as the ionizing agent, they are generally operated at reduced pressures of 0.002 to 0.01 atm, which can limit the ability to detect pptv levels of amines. Use of this technique at atmospheric pressure can increase its sensitivity, as demonstrated by the efficient detection of ammonia via proton transfer at ambient pressures and relative humidities in the lab [1]. An instrument based on this system was deployed in the field (NCCN 2009, Atlanta) and was recently connected to a chamber at the University of Minnesota where nucleation experiments involving sulfuric acid and amines were carried out. This instrument, Ambient pressure Proton transfer Mass Spectrometer (AmP-MS), combines the specificity of chemical ionization with the high sensitivity of atmospheric pressure ionization techniques. It works for species that have high proton affinities and it is relatively insensitive to highly abundant VOCs such as methanol, acetaldehyde, acetone, etc. Water-proton clusters are electrostatically drawn across a flow of analyte gas resulting in ion-molecule reaction times of ~0.5-to-1 ms, and sensitivities in the few Hz per pptv are possible. In the laboratory, ion-molecule reactions of water proton and water ammonium clusters with various amine species are facile [2] and Sunner et al. [3] showed that species with high gas-phase basicities, and thus high PAs, also react fast with highly hydrated H3O+ and NH4+ ions. Amines have large proton affinities. The basics of the AmP-MS construction and operation will be presented as well as data from its deployment in the field and from the laboratory chamber experiments. Focus will be on the veracity of the technique and on correlations of measurements with environmental conditions, particle size distributions, and sulfuric acid cluster measurements. Candidates for important roles in nucleation will be discussed. [1] Hanson, D.R., E. Kosciuch, The NH3 mass accommodation coefficient for uptake onto sulfuric acid solutions, J. Phys. Chem. A, 2003, 107, 2199-2208. [2] Viggiano, A. A., Dale, F., and Paulson, J. F.: Proton transfer reactions of H+(H2O)n=2-11 with methanol, ammonia, pyridine, acetonitrile and acetone, J. Chem. Phys., 88, 2469-2477, 1988. [3] Sunner J., G. Nicol, and P. Kebarle, Factors Determining Relative Sensitivity of Analytes in Positive Mode Atmospheric Pressure Ionization Mass Spectrometry, Anal. Chem. 1988, 60, 1300-1307.

The crystal structures of the psychrophilic subtilisin S41 and the mesophilic subtilisin Sph reveal the same calcium-loaded state.

PubMed

Almog, Orna; González, Ana; Godin, Noa; de Leeuw, Marina; Mekel, Marlene J; Klein, Daniela; Braun, Sergei; Shoham, Gil; Walter, Richard L

2009-02-01

We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures. Copyright 2008 Wiley-Liss, Inc.

Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

PubMed

Nilsson, J; Bosnes, M; Larsen, F; Nygren, P A; Uhlén, M; Lundeberg, J

1997-04-01

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

High Temperature Stable Separator for Lithium Batteries Based on SiO2 and Hydroxypropyl Guar Gum

PubMed Central

Carvalho, Diogo Vieira; Loeffler, Nicholas; Kim, Guk-Tae; Passerini, Stefano

2015-01-01

A novel membrane based on silicon dioxide (SiO2) and hydroxypropyl guar gum (HPG) as binder is presented and tested as a separator for lithium-ion batteries. The separator is made with renewable and low cost materials and an environmentally friendly manufacturing processing using only water as solvent. The separator offers superior wettability and high electrolyte uptake due to the optimized porosity and the good affinity of SiO2 and guar gum microstructure towards organic liquid electrolytes. Additionally, the separator shows high thermal stability and no dimensional-shrinkage at high temperatures due to the use of the ceramic filler and the thermally stable natural polymer. The electrochemical tests show the good electrochemical stability of the separator in a wide range of potential, as well as its outstanding cycle performance. PMID:26512701

Highly sensitive chemiluminescent aptasensor for detecting HBV infection based on rapid magnetic separation and double-functionalized gold nanoparticles.

PubMed

Xi, Zhijiang; Gong, Quan; Wang, Chao; Zheng, Bing

2018-06-21

Hepatitis B virus (HBV) infection is a major global public health problem and one of the leading causes of chronic liver disease. HBsAg is the first serological marker to appear in the blood and is the most important marker of HBV infection. Detection of HBsAg in serum samples is commonly carried out using an immunoassay such as an enzyme-linked immunosorbent assay (ELISA), which is complex to perform, time-consuming, and unsatisfactory for testing sensitivity. Therefore, new methods for highly sensitive detection of HBV infection are urgently needed. Aptamers are specific recognition molecules with high affinity and specificity toward their targets. Biosensors that employ aptamers as biorecognition elements are known as aptasensors. In this study, we select an HBsAg-specific aptamer and use it to develop a new chemiluminescent aptasensor based on rapid magnetic separation and double-functionalized gold nanoparticles. This sensor enables rapid magnetic separation and highly sensitive detection of HBsAg in HBV-positive serum. The detection limit of this HBsAg-detecting chemiluminescent aptasensor is as low as 0.05 ng/mL, which is much lower than the 0.5 ng/mL limit of a typical ELISA used in hospitals. Furthermore, this aptasensor works well and is highly specific to HBV infection.

Resonance analysis of a high temperature piezoelectric disc for sensitivity characterization.

PubMed

Bilgunde, Prathamesh N; Bond, Leonard J

2018-07-01

Ultrasonic transducers for high temperature (200 °C+) applications are a key enabling technology for advanced nuclear power systems and in a range of chemical and petro-chemical industries. Design, fabrication and optimization of such transducers using piezoelectric materials remains a challenge. In this work, experimental data-based analysis is performed to investigate the fundamental causal factors for the resonance characteristics of a piezoelectric disc at elevated temperatures. The effect of all ten temperature-dependent piezoelectric constants (ε 33 , ε 11 , d 33 , d 31 , d 15 , s 11 , s 12 , s 13 , s 33 , s 44 ) is studied numerically on both the radial and thickness mode resonances of a piezoelectric disc. A sensitivity index is defined to quantify the effect of each of the temperature-dependent coefficients on the resonance modes of the modified lead zirconium titanate disc. The temperature dependence of s 33 showed highest sensitivity towards the thickness resonance mode followed by ε 33 , s 11 , s 13 , s 12 , d 31 , d 33 , s 44 , ε 11 , and d 15 in the decreasing order of the sensitivity index. For radial resonance modes, the temperature dependence of ε 33 showed highest sensitivity index followed by s 11 , s 12 and d 31 coefficient. This numerical study demonstrates that the magnitude of d 33 is not the sole factor that affects the resonance characteristics of the piezoelectric disc at high temperatures. It appears that there exists a complex interplay between various temperature dependent piezoelectric coefficients that causes reduction in the thickness mode resonance frequencies which is found to be agreement in with the experimental data at an elevated temperature. Copyright © 2018 Elsevier B.V. All rights reserved.

Media composition and incubation temperature affect Congo red dye affinity of Shiga toxin-producing Escherichia coli

USDA-ARS?s Scientific Manuscript database

Background: Escherichia coli biofilm formation is dependent on curli fimbriae and cellulose, and the expression of both varies among Shiga toxin-producing E. coli (STEC). Curli and cellulose expression are often identified by their affinity for Congo red dye (CR) but media composition and incubation...

Thermo-optical characterization of fluorescent rhodamine B based temperature-sensitive nanosensors using a CMOS MEMS micro-hotplate☆

PubMed Central

Chauhan, Veeren M.; Hopper, Richard H.; Ali, Syed Z.; King, Emma M.; Udrea, Florin; Oxley, Chris H.; Aylott, Jonathan W.

2014-01-01

A custom designed microelectromechanical systems (MEMS) micro-hotplate, capable of operating at high temperatures (up to 700 °C), was used to thermo-optically characterize fluorescent temperature-sensitive nanosensors. The nanosensors, 550 nm in diameter, are composed of temperature-sensitive rhodamine B (RhB) fluorophore which was conjugated to an inert silica sol–gel matrix. Temperature-sensitive nanosensors were dispersed and dried across the surface of the MEMS micro-hotplate, which was mounted in the slide holder of a fluorescence confocal microscope. Through electrical control of the MEMS micro-hotplate, temperature induced changes in fluorescence intensity of the nanosensors was measured over a wide temperature range. The fluorescence response of all nanosensors dispersed across the surface of the MEMS device was found to decrease in an exponential manner by 94%, when the temperature was increased from 25 °C to 145 °C. The fluorescence response of all dispersed nanosensors across the whole surface of the MEMS device and individual nanosensors, using line profile analysis, were not statistically different (p < 0.05). The MEMS device used for this study could prove to be a reliable, low cost, low power and high temperature micro-hotplate for the thermo-optical characterisation of sub-micron sized particles. The temperature-sensitive nanosensors could find potential application in the measurement of temperature in biological and micro-electrical systems. PMID:25844025

Engineering an antibody with picomolar affinity to DOTA chelates of multiple radionuclides for pretargeted radioimmunotherapy and imaging

PubMed Central

Orcutt, Kelly Davis; Slusarczyk, Adrian L; Cieslewicz, Maryelise; Ruiz-Yi, Benjamin; Bhushan, Kumar R; Frangioni, John V; Wittrup, K Dane

2014-01-01

Introduction In pretargeted radioimmunotherapy (PRIT), a bifunctional antibody is administered and allowed to pre-localize to tumor cells. Subsequently, a chelated radionuclide is administered and captured by cell-bound antibody while unbound hapten clears rapidly from the body. We aim to engineer high-affinity binders to DOTA chelates for use in PRIT applications. Methods We mathematically modeled antibody and hapten pharmacokinetics to analyze hapten tumor retention as a function of hapten binding affinity. Motivated by model predictions, we used directed evolution and yeast surface display to affinity mature the 2D12.5 antibody to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), reformatted as a single chain variable fragment (scFv). Results Modeling predicts that for high antigen density and saturating bsAb dose, a hapten binding affinity of 100 picomolar (pM) is needed for near-maximal hapten retention. We affinity matured 2D12.5 with an initial binding constant of about 10 nanomolar (nM) to DOTA-yttrium chelates. Affinity maturation resulted in a 1000-fold affinity improvement to biotinylated DOTA-yttrium, yielding an 8.2 ± 1.9 picomolar binder. The high-affinity scFv binds DOTA complexes of lutetium and gadolinium with similar picomolar affinity and indium chelates with low nanomolar affinity. When engineered into a bispecific antibody construct targeting carcinoembryonic antigen (CEA), pretargeted high-affinity scFv results in significantly higher tumor retention of a 111In-DOTA hapten compared to pretargeted wild-type scFv in a xenograft mouse model. Conclusions We have engineered a versatile, high-affinity DOTA-chelate-binding scFv. We anticipate it will prove useful in developing pretargeted imaging and therapy protocols to exploit the potential of a variety of radiometals. PMID:21315278

Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Xinxin; Zhang, Min; Takano, Tetsuo

Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membranemore » and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.« less

Main immunogenic region structure promotes binding of conformation-dependent myasthenia gravis autoantibodies, nicotinic acetylcholine receptor conformation maturation, and agonist sensitivity

PubMed Central

Luo, Jie; Taylor, Palmer; Losen, Mario; de Baets, Marc H.; Shelton, G. Diane; Lindstrom, Jon

2009-01-01

The main immunogenic region (MIR) is a conformation-dependent region at the extracellular apex of α1 subunits of muscle nicotinic acetylcholine receptor (AChR) that is the target of half or more of the autoantibodies to muscle AChRs in human myasthenia gravis and rat experimental autoimmune myasthenia gravis. By making chimeras of human α1 subunits with α7 subunits, both MIR epitopes recognized by rat mAbs and by the patient-derived human mAb 637 to the MIR were determined to consist of two discontiguous sequences, which are adjacent only in the native conformation. The MIR, including loop α1 67–76 in combination with the N-terminal α helix α1 1–14, conferred high-affinity binding for most rat mAbs to the MIR. However, an additional sequence corresponding to α1 15–32 was required for high-affinity binding of human mAb 637. A water soluble chimera of Aplysia acetylcholine binding protein with the same α1 MIR sequences substituted was recognized by a majority of human, feline, and canine MG sera. The presence of the α1 MIR sequences in α1/α7 chimeras greatly promoted AChR expression and significantly altered the sensitivity to activation. This reveals a structural and functional, as well as antigenic, significance of the MIR. PMID:19890000

Diadenosine polyphosphates induce intracellular Ca2+ mobilization in human neutrophils via a pertussis toxin sensitive G-protein.

PubMed Central

Gasmi, L; McLennan, A G; Edwards, S W

1997-01-01

The diadenosine polyphosphates diadenosine 5',5"'-P1,P3-triphosphate (Ap3A), diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A), diadenosine 5',5"'-P1,P5-pentaphosphate (Ap5A) and diadenosine 5',5"'-P1,P6-hexaphosphate (Ap6A) all stimulated increases in intracellular Ca2+ in human neutrophils. Maximal increases in intracellular Ca2+ of 650 nM were obtained at dinucleotide concentrations of 500-700 microM. These increases in intracellular, Ca2+ were completely abolished by pre-treatment of the neutrophils with pertussis toxin and were hardly affected when the extracellular buffer was devoid of Ca2+. On the other hand, adenosine triphosphate (ATP) could stimulate much greater increases in intracellular Ca2+ (up to 1.1 microM) at much lower concentrations (half maximal responses obtained at around 5 microM ATP). Receptor de-sensitization experiments indicate that human neutrophils may possess two types of P2-purinoceptors. The first of these may bind ATP (but not the dinucleotides) with high affinity whilst the second may bind the dinucleotides with lower affinity and also bind ATP. PMID:9038726

Derivatized graphitic nanofibres (GNF) as a new support material for mass spectrometric analysis of peptides and proteins.

PubMed

Greiderer, Andreas; Rainer, Matthias; Najam-ul-Haq, Muhammad; Vallant, Rainer M; Huck, Christian W; Bonn, Günther K

2009-07-01

Graphitic nanofibres (GNFs), 100-200 nm in diameter and 5-20 microm in length have been modified in order to yield different affinities (Cu2+ and Fe3+ loaded immobilized metal affinity chromatography (IMAC) as well as cation and anion exchange materials) for the extraction of a range of biomolecules by their inherited hydrophobicity and the hydrophilic chemical functionalities, obtained by derivatization. Modified GNFs have for the first time been employed as carrier materials for protein profiling in material-enhanced laser desorption/ionization (MELDI) for the enrichment and screening of biofluids. For that purpose, the derivatized GNF materials have comprehensively been characterized regarding surface area, structural changes during derivatization, IMAC, as well as ion exchange and protein-loading capacity and recovery. GNF derivatives revealed high protein-binding capacity (2,000 microg ml(-1) for insulin) and ideal sensitivities, resulting in a detection limit of 50 fmol microl(-1) (for insulin), which is crucial for the detection of low abundant species in biological samples. Compared to other MELDI carrier materials, sensitivity was enhanced on GNF derivatives, which might be ascribed to the fact that GNFs support desorption and ionization mechanisms and by absorbing laser energy in addition to matrix.

Principles of Toxicological Interactions Associated with Multiple Chemical Exposures.

DTIC Science & Technology

1980-12-01

chemicals from sites of activation or deactivation , the agent possessing the higher binding affinity would also be expected to antagonize or act...kcal/mol. Because of their high binding energy, covalent bonds are essentially irreversible at ordinary body temperature unless a catalytic agent such...determining the toxicity of chemicals is the route or routes by which such agents gain entry into the body. The inhalation and dermal routes of absorption

Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants.

PubMed Central

Wang, R; Crawford, N M

1996-01-01

Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake. These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate. Progency from one of the backcrossed mutants showed no constitutive uptake of nitrate below 0.5 mM at pH 7.0 in liquid culture (that is, within 30 min of initial exposure to nitrate). All other uptake activities measured (high-affinity phosphate and sulfate uptake, inducible high-affinity nitrate uptake, and constitutive low-affinity nitrate uptake) were present or nearly normal in the backcrossed mutant. Electrophysiological analysis of individual root cells showed that the nrt2 mutant showed little response to 0.25 mM of nitrate, whereas NRT2 wild-type cells showed an initial depolarization followed by recovery. At 10 mM of nitrate both the mutant and wild-type cells displayed similar, strong electrical responses. These results indicate that NRT2 is a critical and perhaps necessary gene for constitutive, high-affinity nitrate uptake in Arabidopsis, but not for inducible, high-affinity nor constitutive, low-affinity nitrate uptake. Thus, these systems are genetically distinct. PMID:8799195

Reaction of Unalloyed and Cr-Mo Alloyed Steels with Nitrogen from the Sintering Atmosphere

NASA Astrophysics Data System (ADS)

Dlapka, Magdalena; Gierl-Mayer, Christian; Calderon, Raquel de Oro; Danninger, Herbert; Bengtsson, Sven; Dudrova, Eva

2016-12-01

Nitrogen is usually regarded as an inert sintering atmosphere for PM steels; however, this cannot be taken for granted in particular for steels alloyed with nitride forming elements. Among those elements, chromium has become more and more important as an alloying element in sintered low alloy structural steels in the last decade due to the moderate alloying cost and the excellent mechanical properties obtainable, in particular when sinter hardening is applied. The high affinity of Cr to oxygen and the possible ways to overcome related problems have been the subject of numerous studies, while the fact that chromium is also a fairly strong nitride forming element has largely been neglected at least for low alloy steel grades, although frequently used materials like steels from Cr and Cr-Mo prealloyed powders are commonly sintered in atmospheres consisting mainly of nitrogen. In the present study, nitrogen pickup during sintering at different temperatures and for varying times has been studied for Cr-Mo prealloyed steel grades as well as for unalloyed carbon steel. Also the effect of the cooling rate and its influence on the properties, of the microstructure and the composition have been investigated. It showed that the main nitrogen uptake occurs not during isothermal sintering but rather during cooling. It could be demonstrated that a critical temperature range exists within which the investigated CrM-based steel is particularly sensitive to nitrogen pickup.

Inferring Lower Boundary Driving Conditions Using Vector Magnetic Field Observations

NASA Technical Reports Server (NTRS)

Schuck, Peter W.; Linton, Mark; Leake, James; MacNeice, Peter; Allred, Joel

2012-01-01

Low-beta coronal MHD simulations of realistic CME events require the detailed specification of the magnetic fields, velocities, densities, temperatures, etc., in the low corona. Presently, the most accurate estimates of solar vector magnetic fields are made in the high-beta photosphere. Several techniques have been developed that provide accurate estimates of the associated photospheric plasma velocities such as the Differential Affine Velocity Estimator for Vector Magnetograms and the Poloidal/Toroidal Decomposition. Nominally, these velocities are consistent with the evolution of the radial magnetic field. To evolve the tangential magnetic field radial gradients must be specified. In addition to estimating the photospheric vector magnetic and velocity fields, a further challenge involves incorporating these fields into an MHD simulation. The simulation boundary must be driven, consistent with the numerical boundary equations, with the goal of accurately reproducing the observed magnetic fields and estimated velocities at some height within the simulation. Even if this goal is achieved, many unanswered questions remain. How can the photospheric magnetic fields and velocities be propagated to the low corona through the transition region? At what cadence must we observe the photosphere to realistically simulate the corona? How do we model the magnetic fields and plasma velocities in the quiet Sun? How sensitive are the solutions to other unknowns that must be specified, such as the global solar magnetic field, and the photospheric temperature and density?

[125I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y1 and Y4 receptors

PubMed Central

Dumont, Yvan; Quirion, Rémi

2000-01-01

GR231118 (also known as 1229U91 and GW1229), a purported Y1 antagonist and Y4 agonist was radiolabelled using the chloramine T method. [125I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [125I]-GR231118 binds with very high affinity (Kd of 0.09–0.24 nM) in transfected HEK293 cells with the rat Y1 and Y4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y2 or Y5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [125I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y1, but not rat Y4, receptor cDNA. Autoradiographic studies demonstrated that [125I]-GR231118 binding sites were fully inhibited by the Y1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [125I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [125I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y4 receptor subtype. In summary, [125I]-GR231118 is a new radiolabelled probe to investigate the Y1 and Y4 receptors; its major advantage being its high affinity. Using highly selective Y1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [125I]-GR231118 to the Y1 receptor allowing for the characterization and visualization of the purported Y4 subtype. PMID:10694200

Mutation of a single residue in the S2-S3 loop of CNG channels alters the gating properties and sensitivity to inhibitors.

PubMed

Crary, J I; Dean, D M; Maroof, F; Zimmerman, A L

2000-12-01

We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.

Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV).

PubMed

Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

2016-10-12

Zr(IV) can form phosphate and Zr(IV) (-PO₃ 2- -Zr 4+ -) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A 520nm / A 650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP.

The influence of reducing fever on blood oxygen saturation in children.

PubMed

Goldberg, Shmuel; Heitner, Shmuel; Mimouni, Francis; Joseph, Leon; Bromiker, Reuben; Picard, Elie

2018-01-01

Laboratory-based studies on the oxyhemoglobin dissociation curve (ODC) suggest that high blood temperature decreases the affinity of hemoglobin for oxygen. The aim of the study was to evaluate the influence of pyrexia on oxygen saturation (SpO 2 ) in children presenting to the emergency department. Normoxemic children with body temperature at or above 38.5 °C were included. Patients with a dynamic respiratory disease were excluded. SpO 2 was measured before and after antipyretic treatment. The changes in body temperature and SpO 2 were assessed and compared to the changes predicted from the ODC. Thirty-four children completed the study. Mean temperature at presentation was 39.17 ± 0.549 °C and mean SpO 2 was 96.15 ± 2.21%. The mean decrease in temperature after antipyretic treatment was 1.71 ± 0.67 °C and mean increase in SpO 2 was 0.95 ± 1.76%. Among children in whom pyrexia decreased by 1.5 °C or more, the mean increase in SpO 2 was 1.45 ± 1.57%. The measured increase in SpO 2 was close to the increase anticipated from the ODC. Pyrexia was associated with decreased SpO 2 in normoxemic children. The influence of pyrexia in children with low-normal oxygen saturation is expected to be much higher because of the non-linear shape of the ODC. Physicians treating patients with fever should be aware of this effect, especially in patients with borderline hypoxia. What is Known: • High blood temperature decreases the affinity of oxygen to hemoglobin. • It is not known whether fever would decrease SpO 2 . What is New: • Fever is associated with decreased SpO 2 .

Endoprotease profiling with double-tagged peptide substrates: a new diagnostic approach in oncology.

PubMed

Peccerella, Teresa; Lukan, Nadine; Hofheinz, Ralf; Schadendorf, Dirk; Kostrezewa, Markus; Neumaier, Michael; Findeisen, Peter

2010-02-01

The measurement of disease-related proteolytic activity in complex biological matrices like serum is of emerging interest to improve the diagnosis of malignant diseases. We developed a mass spectrometry (MS)-based functional proteomic profiling approach that tracks degradation of artificial endoprotease substrates in serum specimens. The synthetic reporter peptides that are cleaved by tumor-associated endopeptidases were systematically optimized with regard to flanking affinity tags, linkers, and stabilizing elements. Serum specimens were incubated with reporter peptides under standardized conditions and the peptides subsequently extracted with affinity chromatography before MS. In a pilot study an optimized reporter peptide with the cleavage motif WKPYDAADL was added to serum specimens from colorectal tumor patients (n = 50) and healthy controls (n = 50). This reporter peptide comprised a known cleavage site for the cysteine-endopeptidase "cancer procoagulant." Serial affinity chromatography using biotin- and 6xHis tags was superior to the single affinity enrichment using only 6xHis tags. Furthermore, protease-resistant stop elements ensured signal accumulation after prolonged incubation. In contrast, signals from reporter peptides without stop elements vanished completely after prolonged incubation owing to their total degradation. Reporter-peptide spiking showed good reproducibility, and the difference in proteolytic activity between serum specimens from cancer patients and controls was highly significant (P < 0.001). The introduction of a few structural key elements (affinity tags, linkers, d-amino acids) into synthetic reporter peptides increases the diagnostic sensitivity for MS-based protease profiling of serum specimens. This new approach might lead to functional MS-based protease profiling for improved disease classification.

Hierarchy and Assortativity as New Tools for Binding-Affinity Investigation: The Case of the TBA Aptamer-Ligand Complex.

PubMed

Cataldo, Rosella; Alfinito, Eleonora; Reggiani, Lino

2017-12-01

Aptamers are single stranded DNA, RNA, or peptide sequences having the ability to bind several specific targets (proteins, molecules as well as ions). Therefore, aptamer production and selection for therapeutic and diagnostic applications is very challenging. Usually, they are generated in vitro, although computational approaches have been recently developed for the in silico production. Despite these efforts, the mechanism of aptamer-ligand formation is not completely clear, and producing high-affinity aptamers is still quite difficult. This paper aims to develop a computational model able to describe aptamer-ligand affinity. Topological tools, such as the conventional degree distribution, the rank-degree distribution (hierarchy), and the node assortativity are employed. In doing so, the macromolecules tertiary-structures are mapped into appropriate graphs. These graphs reproduce the main topological features of the macromolecules, by preserving the distances between amino acids (nucleotides). Calculations are applied to the thrombin binding aptamer (TBA), and the TBA-thrombin complex produced in the presence of Na + or K + . The topological analysis is able to detect several differences between complexes obtained in the presence of the two cations, as expected by previous investigations. These results support graph analysis as a novel computational tool for testing affinity. Otherwise, starting from the graphs, an electrical network can be obtained by using the specific electrical properties of amino acids and nucleobases. Therefore, a further analysis concerns with the electrical response, revealing that the resistance is sensitively affected by the presence of sodium or potassium, thus suggesting resistance as a useful physical parameter for testing binding affinity.

Research on high-temperature sensing characteristics based on modular interference of single-mode multimode single-mode fiber

NASA Astrophysics Data System (ADS)

Peng, Zhaozhuang; Wang, Li; Yan, Huanhuan

2016-11-01

Application of high temperature fiber sensing system is very extensive. It can be mainly used in high temperature test aerospace, such as, materials, chemicals, and energy. In recent years, various on-line optical fiber interferometric sensors based on modular interference of single-mode-multimode-single-mode(SMS) fiber have been largely explored in high temperature fiber sensor. In this paper we use the special fiber of a polyimide coating, its sensor head is composed of a section of multimode fiber spliced in the middle of Single-mode fiber. When the light is launched into the multimode fiber(MMF) through the lead-in single-mode fiber(SMF), the core mode and cladding modes are excited and propagate in the MMF respectively. Then, at the MMF-SMF spliced point, the excited cladding modes coupled back into the core of lead-out SMF interfere with SMF core mode. And the wavelength of the interference dip would shift differently with the variation of the temperature. By this mean, we can achieve the measurement of temperature. The experimental results also show that the fiber sensor based on SMS structure has a highly temperature sensitivity. From 30° to 300°, with the temperature increasing, the interference dip slightly shifts toward longer wavelength and the temperature sensitivity coefficient is 0.0115nm/°. With high sensitivity, simple structure, immunity to electromagnetic interferences and a good linearity of the experimental results, the structure has an excellent application prospect in engineering field.

Cluster analyses of 20th century growth patterns in high elevation Great Basin bristlecone pine in the Snake Mountain Range, Nevada, USA

NASA Astrophysics Data System (ADS)

Tran, T. J.; Bruening, J. M.; Bunn, A. G.; Salzer, M. W.; Weiss, S. B.

2015-12-01

Great Basin bristlecone pine (Pinus longaeva) is a useful climate proxy because of the species' long lifespan (up to 5000 years) and the climatic sensitivity of its annually-resolved rings. Past studies have shown that growth of individual trees can be limited by temperature, soil moisture, or a combination of the two depending on biophysical setting at the scale of tens of meters. We extend recent research suggesting that trees vary in their growth response depending on their position on the landscape to analyze how growth patterns vary over time. We used hierarchical cluster analysis to examine the growth of 52 bristlecone pine trees near the treeline of Mount Washington, Nevada, USA. We classified growth of individual trees over the instrumental climate record into one of two possible scenarios: trees belonging to a temperature-sensitive cluster and trees belonging to a precipitation-sensitive cluster. The number of trees in the precipitation-sensitive cluster outnumbered the number of trees in the temperature-sensitive cluster, with trees in colder locations belonging to the temperature-sensitive cluster. When we separated the temporal range into two sections (1895-1949 and 1950-2002) spanning the length of the instrumental climate record, we found that most of the 52 trees remained loyal to their cluster membership (e.g., trees in the temperature-sensitive cluster in 1895-1949 were also in the temperature sensitive cluster in 1950-2002), though not without exception. Of those trees that do not remain consistent in cluster membership, the majority changed from temperature-sensitive to precipitation-sensitive as time progressed. This could signal a switch from temperature limitation to water limitation with warming climate. We speculate that topographic complexity in high mountain environments like Mount Washington might allow for climate refugia where growth response could remain constant over the Holocene.

Advancing Peptide-Based Biorecognition Elements for Biosensors Using in-Silico Evolution.

PubMed

Xiao, Xingqing; Kuang, Zhifeng; Slocik, Joseph M; Tadepalli, Sirimuvva; Brothers, Michael; Kim, Steve; Mirau, Peter A; Butkus, Claire; Farmer, Barry L; Singamaneni, Srikanth; Hall, Carol K; Naik, Rajesh R

2018-05-25

Sensors for human health and performance monitoring require biological recognition elements (BREs) at device interfaces for the detection of key molecular biomarkers that are measurable biological state indicators. BREs, including peptides, antibodies, and nucleic acids, bind to biomarkers in the vicinity of the sensor surface to create a signal proportional to the biomarker concentration. The discovery of BREs with the required sensitivity and selectivity to bind biomarkers at low concentrations remains a fundamental challenge. In this study, we describe an in-silico approach to evolve higher sensitivity peptide-based BREs for the detection of cardiac event marker protein troponin I (cTnI) from a previously identified BRE as the parental affinity peptide. The P2 affinity peptide, evolved using our in-silico method, was found to have ∼16-fold higher affinity compared to the parent BRE and ∼10 fM (0.23 pg/mL) limit of detection. The approach described here can be applied towards designing BREs for other biomarkers for human health monitoring.

Pseudotargeted MS Method for the Sensitive Analysis of Protein Phosphorylation in Protein Complexes.

PubMed

Lyu, Jiawen; Wang, Yan; Mao, Jiawei; Yao, Yating; Wang, Shujuan; Zheng, Yong; Ye, Mingliang

2018-05-15

In this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence. The development of this targeted approach is very easy as the same sample and the same LC-system were used for the discovery and the targeted analysis phases. No sample fractionation or enrichment was required for the discovery phase which allowed this method to analyze minute amount of sample. We applied this pseudotargeted MS method to quantitatively examine phosphopeptides in affinity purified endogenous Shc1 protein complexes at four temporal stages of EGF signaling and identified 82 phospho-sites. To our knowledge, this is the highest number of phospho-sites identified from the protein complexes. This pseudotargeted MS method is highly sensitive in the identification of low abundance phosphopeptides and could be a powerful tool to study phosphorylation-regulated assembly of protein complex.

Analytic implementation of the GRAAL model: Application to a R7T7-type glass package in a geological disposal environment

NASA Astrophysics Data System (ADS)

Minet, Y.; Bonin, B.; Gin, S.; Frugier, P.

2010-09-01

The Glass Reactivity with Allowance for the Alteration Layer Model (GRAAL) was proposed in 2008 to describe borosilicate nuclear glass alteration based on coupling an affinity law with the formation and dissolution of a passivating reactive interface. It is examined here in a simplified form in which only the affinity with respect to silicon is taken into account with a concentration at saturation Csat, and the precipitation of neoformed phases is described by an affine relation for silicon above a precipitation threshold Csat'. This simplified "analytical GRAAL" model is capable of predicting the quantities of altered glass and the silicon and boron concentration variations in analytical or semi-analytical form, and thereby identify the main characteristic quantities of the system. The model was tested against a series of laboratory experiments lasting from a few days to a few years; its sensitivity to the parameter values was examined, and the model was validated with respect to SON68 glass alteration in initially pure water. It was then applied to the alteration of a glass package in a repository over periods of up to a million years, by means of exploratory calculations comprising a sensitivity study of the internal model parameters and extrapolation to the temperatures expected in a geological repository in order to identify the parameters and mechanisms having the greatest impact on the residual alteration rate. Alteration is controlled by the precipitation of neoformed phases in every case. The transient conditions are of very limited duration with respect to either silicon or boron (no more than a 100 years, with less than 0.01% alteration of the package). In the precipitation law used in the model, the residual alteration rate and total package lifetime are determined primarily by two parameters: k' (the precipitation kinetics) and σ' (the precipitate surface area per unit volume in the geological barrier). The package lifetime is about 3 × 10 5 years at 30 °C assuming a reasonable value for σ' (10 6 m -1), and would be increased by a factor 3-6 if precipitation in the barrier were disregarded. This cursory description of precipitation will be validated and refined through specific laboratory tests at 50 °C and lower temperatures, coordinated with the development of the "geochemical GRAAL" model and with integral tests in contact with clay and canister corrosion products.

A comparison of blood nitric oxide metabolites and hemoglobin functional properties among diving mammals.

PubMed

Fago, Angela; Parraga, Daniel Garcia; Petersen, Elin E; Kristensen, Niels; Giouri, Lea; Jensen, Frank B

2017-03-01

The ability of marine mammals to hunt prey at depth is known to rely on enhanced oxygen stores and on selective distribution of blood flow, but the molecular mechanisms regulating blood flow and oxygen transport remain unresolved. To investigate the molecular mechanisms that may be important in regulating blood flow, we measured concentration of nitrite and S-nitrosothiols (SNO), two metabolites of the vasodilator nitric oxide (NO), in the blood of 5 species of marine mammals differing in their dive duration: bottlenose dolphin, South American sea lion, harbor seal, walrus and beluga whale. We also examined oxygen affinity, sensitivity to 2,3-diphosphoglycerate (DPG) and nitrite reductase activity of the hemoglobin (Hb) to search for possible adaptive variations in these functional properties. We found levels of plasma and red blood cells nitrite similar to those reported for terrestrial mammals, but unusually high concentrations of red blood cell SNO in bottlenose dolphin, walrus and beluga whale, suggesting enhanced SNO-dependent signaling in these species. Purified Hbs showed similar functional properties in terms of oxygen affinity and sensitivity to DPG, indicating that reported large variations in blood oxygen affinity among diving mammals likely derive from phenotypic variations in red blood cell DPG levels. The nitrite reductase activities of the Hbs were overall slightly higher than that of human Hb, with the Hb of beluga whale, capable of longest dives, having the highest activity. Taken together, these results underscore adaptive variations in circulatory NO metabolism in diving mammals but not in the oxygenation properties of the Hb. Copyright © 2016 Elsevier Inc. All rights reserved.

Lithospheric structure of the Arabian Shield from the joint inversion of receiver functions and surface-wave group velocities

NASA Astrophysics Data System (ADS)

Julià, Jordi; Ammon, Charles J.; Herrmann, Robert B.

2003-08-01

We estimate lithospheric velocity structure for the Arabian Shield by jointly modeling receiver functions and fundamental-mode group velocities from events recorded by the 1995-1997 Saudi Arabian Portable Broadband Deployment. Receiver functions are primarily sensitive to shear-wave velocity contrasts and vertical travel times, and surface-wave dispersion measurements are sensitive to vertical shear-wave velocity averages, so that their combination bridge resolution gaps associated with each individual data set. Our resulting models correlate well with the observed surface geology; the Asir terrane to the West consists of a 10-km-thick upper crust of 3.3 km/s overlying a lower crust of 3.7-3.8 km/s; in the Afif terrane to the East, the upper crust is 20 km thick and has an average velocity of 3.6 km/s, and the lower crust is about 3.8 km/s; separating the terranes, the Nabitah mobile belt is made of a gradational upper crust up to 3.6 km/s at 15 km overlying an also gradational lower crust up to 4.0 km/s. The crust-mantle transition is found to be sharp in terranes of continental affinity (east) and gradual in terranes of oceanic affinity (west). The upper mantle shear velocities range from 4.3 to 4.6 km/s. Temperatures around 1000 °C are obtained from our velocity models for a thin upper mantle lid observed beneath station TAIF, and suggest that the lithosphere could be as thin as 50-60 km under this station.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

Multifunctional Core–Shell Nanoparticles: Discovery of Previously Invisible Biomarkers

PubMed Central

2011-01-01

Many low-abundance biomarkers for early detection of cancer and other diseases are invisible to mass spectrometry because they exist in body fluids in very low concentrations, are masked by high-abundance proteins such as albumin and immunoglobulins, and are very labile. To overcome these barriers, we created porous, buoyant, core–shell hydrogel nanoparticles containing novel high affinity reactive chemical baits for protein and peptide harvesting, concentration, and preservation in body fluids. Poly(N-isopropylacrylamide-co-acrylic acid) nanoparticles were functionalized with amino-containing dyes via zero-length cross-linking amidation reactions. Nanoparticles functionalized in the core with 17 different (12 chemically novel) molecular baits showed preferential high affinities (KD < 10–11 M) for specific low-abundance protein analytes. A poly(N-isopropylacrylamide-co-vinylsulfonic acid) shell was added to the core particles. This shell chemistry selectively prevented unwanted entry of all size peptides derived from albumin without hindering the penetration of non-albumin small proteins and peptides. Proteins and peptides entered the core to be captured with high affinity by baits immobilized in the core. Nanoparticles effectively protected interleukin-6 from enzymatic degradation in sweat and increased the effective detection sensitivity of human growth hormone in human urine using multiple reaction monitoring analysis. Used in whole blood as a one-step, in-solution preprocessing step, the nanoparticles greatly enriched the concentration of low-molecular weight proteins and peptides while excluding albumin and other proteins above 30 kDa; this achieved a 10,000-fold effective amplification of the analyte concentration, enabling mass spectrometry (MS) discovery of candidate biomarkers that were previously undetectable. PMID:21999289

Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity.

PubMed

Jin, Rongsheng; Rummel, Andreas; Binz, Thomas; Brunger, Axel T

2006-12-21

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the neuroparalytic syndrome of botulism. With a lethal dose of 1 ng kg(-1), they pose a biological hazard to humans and a serious potential bioweapon threat. BoNTs bind with high specificity at neuromuscular junctions and they impair exocytosis of synaptic vesicles containing acetylcholine through specific proteolysis of SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors), which constitute part of the synaptic vesicle fusion machinery. The molecular details of the toxin-cell recognition have been elusive. Here we report the structure of a BoNT in complex with its protein receptor: the receptor-binding domain of botulinum neurotoxin serotype B (BoNT/B) bound to the luminal domain of synaptotagmin II, determined at 2.15 A resolution. On binding, a helix is induced in the luminal domain which binds to a saddle-shaped crevice on a distal tip of BoNT/B. This crevice is adjacent to the non-overlapping ganglioside-binding site of BoNT/B. Synaptotagmin II interacts with BoNT/B with nanomolar affinity, at both neutral and acidic endosomal pH. Biochemical and neuronal ex vivo studies of structure-based mutations indicate high specificity and affinity of the interaction, and high selectivity of BoNT/B among synaptotagmin I and II isoforms. Synergistic binding of both synaptotagmin and ganglioside imposes geometric restrictions on the initiation of BoNT/B translocation after endocytosis. Our results provide the basis for the rational development of preventive vaccines or inhibitors against these neurotoxins.

Comparative transcriptome profiling of a thermal resistant vs. sensitive silkworm strain in response to high temperature under stressful humidity condition

PubMed Central

Xiao, Jinshu; Wang, La; Liu, Taihang; Wu, Yunfei; Dong, Feifan; Jiang, Yaming; Pan, Minhui; Zhang, Youhong; Lu, Cheng

2017-01-01

Thermotolerance is important particularly for poikilotherms such as insects. Understanding the mechanisms by which insects respond to high temperatures can provide insights into their adaptation to the environment. Therefore, in this study, we performed a transcriptome analysis of two silkworm strains with significantly different resistance to heat as well as humidity; the thermo-resistant strain 7532 and the thermos-sensitive strain Knobbed. We identified in total 4,944 differentially expressed genes (DEGs) using RNA-Seq. Among these, 4,390 were annotated and 554 were novel. Gene Ontology (GO) analysis of 747 DEGs identified between RT_48h (Resistant strain with high-temperature Treatment for 48 hours) and ST_48h (Sensitive strain with high-temperature Treatment for 48 hours) showed significant enrichment of 12 GO terms including metabolic process, extracellular region and serine-type peptidase activity. Moreover, we discovered 12 DEGs that may contribute to the heat-humidity stress response in the silkworm. Our data clearly showed that 48h post-exposure may be a critical time point for silkworm to respond to high temperature and humidity. These results provide insights into the genes and biological processes involved in high temperature and humidity tolerance in the silkworm, and advance our understanding of thermal tolerance in insects. PMID:28542312

Comparative transcriptome profiling of a thermal resistant vs. sensitive silkworm strain in response to high temperature under stressful humidity condition.

PubMed

Xiao, Wenfu; Chen, Peng; Xiao, Jinshu; Wang, La; Liu, Taihang; Wu, Yunfei; Dong, Feifan; Jiang, Yaming; Pan, Minhui; Zhang, Youhong; Lu, Cheng

2017-01-01

Thermotolerance is important particularly for poikilotherms such as insects. Understanding the mechanisms by which insects respond to high temperatures can provide insights into their adaptation to the environment. Therefore, in this study, we performed a transcriptome analysis of two silkworm strains with significantly different resistance to heat as well as humidity; the thermo-resistant strain 7532 and the thermos-sensitive strain Knobbed. We identified in total 4,944 differentially expressed genes (DEGs) using RNA-Seq. Among these, 4,390 were annotated and 554 were novel. Gene Ontology (GO) analysis of 747 DEGs identified between RT_48h (Resistant strain with high-temperature Treatment for 48 hours) and ST_48h (Sensitive strain with high-temperature Treatment for 48 hours) showed significant enrichment of 12 GO terms including metabolic process, extracellular region and serine-type peptidase activity. Moreover, we discovered 12 DEGs that may contribute to the heat-humidity stress response in the silkworm. Our data clearly showed that 48h post-exposure may be a critical time point for silkworm to respond to high temperature and humidity. These results provide insights into the genes and biological processes involved in high temperature and humidity tolerance in the silkworm, and advance our understanding of thermal tolerance in insects.

Thermal effects on nonlinear vibration of a carbon nanotube-based mass sensor using finite element analysis

NASA Astrophysics Data System (ADS)

Kang, Dong-Keun; Kim, Chang-Wan; Yang, Hyun-Ik

2017-01-01

In the present study we carried out a dynamic analysis of a CNT-based mass sensor by using a finite element method (FEM)-based nonlinear analysis model of the CNT resonator to elucidate the combined effects of thermal effects and nonlinear oscillation behavior upon the overall mass detection sensitivity. Mass sensors using carbon nanotube (CNT) resonators provide very high sensing performance. Because CNT-based resonators can have high aspect ratios, they can easily exhibit nonlinear oscillation behavior due to large displacements. Also, CNT-based devices may experience high temperatures during their manufacture and operation. These geometrical nonlinearities and temperature changes affect the sensing performance of CNT-based mass sensors. However, it is very hard to find previous literature addressing the detection sensitivity of CNT-based mass sensors including considerations of both these nonlinear behaviors and thermal effects. We modeled the nonlinear equation of motion by using the von Karman nonlinear strain-displacement relation, taking into account the additional axial force associated with the thermal effect. The FEM was employed to solve the nonlinear equation of motion because it can effortlessly handle the more complex geometries and boundary conditions. A doubly clamped CNT resonator actuated by distributed electrostatic force was the configuration subjected to the numerical experiments. Thermal effects upon the fundamental resonance behavior and the shift of resonance frequency due to attached mass, i.e., the mass detection sensitivity, were examined in environments of both high and low (or room) temperature. The fundamental resonance frequency increased with decreasing temperature in the high temperature environment, and increased with increasing temperature in the low temperature environment. The magnitude of the shift in resonance frequency caused by an attached mass represents the sensing performance of a mass sensor, i.e., its mass detection sensitivity, and it can be seen that this shift is affected by the temperature change and the amount of electrostatic force. The thermal effects on the mass detection sensitivity are intensified in the linear oscillation regime and increase with increasing CNT length; this intensification can either improve or worsen the detection sensitivity.

Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathryn L.

2012-11-15

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A-G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the present study, we have developed an ELISA-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotype A, B, C, D, E and F. With engineered high-affinity antibodies, the assays have sensitivities in buffer of 8 fM (1.2 pg/mL)more » for serotypes A and B, and 32 fM (4.9 pg/mL) for serotypes C, D, E, and F. Using clinical and environmental samples (serum and milk), the microarray is capable of detecting BoNT/A-F to the same levels as in standard buffer. Cross reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical or environmental samples.« less

Non-destructive Testing (NDT) of metal cracks using a high Tc rf-SQUID and eddy current method

NASA Technical Reports Server (NTRS)

Lu, D. F.; Fan, Chang-Xin; Ruan, J. Z.; Han, S. G.; Wong, K. W.; Sun, G. F.

1995-01-01

A SQUID is the most sensitive device to detect change in magnetic field. A nondestructive testing (NDT) device using high temperature SQUID's and eddy current method will be much more sensitive than those currently used eddy current systems, yet much cheaper than one with low temperature SQUID's. In this paper, we present our study of such a NDT device using a high temperature superconducting rf-SQUID as a gradiometer sensor. The result clearly demonstrates the expected sensitivity of the system, and indicates the feasibility of building a portable HTS SQUID NDT device with the help from cryocooler industry. Such a NDT device will have a significant impact on metal corrosion or crack detection technology.

MODIS and GIMMS Inferred Northern Hemisphere Spring Greenup in Responses to Preseason Climate

NASA Astrophysics Data System (ADS)

Xu, X.; Riley, W. J.; Koven, C.; Jia, G.

2017-12-01

We compare the discrepancies in Normalized Difference Vegetation Index (NDVI) inferred spring greenup (SG) between Terra Moderate Resolution Imaging Spectroradiometer (MODIS) and Advanced Very High Resolution Radiometer (AVHRR) instruments carried by the Global Inventory Monitoring and Modeling Studies (GIMMS) in North Hemisphere. The interannual variation of SG inferred by MODIS and GIMMS NDVI is well correlated in the mid to high latitudes. However, the presence of NDVI discrepancies leads to discrepancies in SG with remarkable latitudinal characteristics. MODIS NDVI inferred later SG in the high latitude while earlier SG in the mid to low latitudes, in comparison to GIMMS NDVI inferred SG. MODIS NDVI inferred SG is better correlated to preseason climate. Interannual variation of SG is only sensitive to preseason temperature. The GIMMS SG to temperature sensitivity over two periods implied that the inter-biome SG to temperature sensitivity is relatively stable, but SG to temperature sensitivity decreased over time. Over the same period, MODIS SG to temperature sensitivity is much higher than GIMMS. This decreased sensitivity demonstrated the findings from previous studies with continuous GIMMS NDVI analysis that vegetation growth (indicated by growing season NDVI) to temperature sensitivity is reduced over time and SG advance trend ceased after 2000s. Our results also explained the contradictive findings that SG advance accelerated after 2000s according to the merged GIMMS and MODIS NDVI time series. Despite the found discrepancies, without ground data support, the quality of NDVI and its inferred SG cannot be effectively evaluated. The discrepancies and uncertainties in different NDVI products and its inferred SG may bias the scientific significance of climate-vegetation relationship. The different NDVI products when used together should be first evaluated and harmonized.

Structure and Function of Cardiac Troponin C (TNNC1): Implications for Heart Failure, Cardiomyopathies, and Troponin Modulating Drugs

PubMed Central

Li, Monica X.; Hwang, Peter M.

2015-01-01

In striated muscle, the protein troponin complex turns contraction on and off in a calcium-dependent manner. The calcium-sensing component of the complex is troponin C, which is expressed from the TNNC1 gene in both cardiac muscle and slow-twitch skeletal muscle (identical transcript in both tissues) and the TNNC2 gene in fast-twitch skeletal muscle. Cardiac troponin C (cTnC) is made up of two globular EF-hand domains connected by a flexible linker. The structural C-domain (cCTnC) contains two high affinity calcium-binding sites that are always occupied by Ca2+ or Mg2+ under physiologic conditions, stabilizing an open conformation that remains anchored to the rest of the troponin complex. In contrast, the regulatory N-domain (cNTnC) contains a single low affinity site that is largely unoccupied at resting calcium concentrations. During muscle activation, calcium binding to cNTnC favors an open conformation that binds to the switch region of troponin I, removing adjacent inhibitory regions of troponin I from actin and allowing muscle contraction to proceed. Regulation of the calcium binding affinity of cNTnC is physiologically important, because it directly impacts the calcium sensitivity of muscle contraction. Calcium sensitivity can be modified by drugs that stabilize the open form of cNTnC, post-translational modifications like phosphorylation of troponin I, or downstream thin filament protein interactions that impact the availability of the troponin I switch region. Recently, mutations in cTnC have been associated with hypertrophic or dilated cardiomyopathy. A detailed understanding of how calcium sensitivity is regulated through the troponin complex is necessary for explaining how mutations perturb its function to promote cardiomyopathy and how post-translational modifications in the thin filament affect heart function and heart failure. Troponin modulating drugs are being developed for the treatment of cardiomyopathies and heart failure. PMID:26232335

Temperature effect on affinity chromatography of two lectins from the seeds of Ricinus communis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, H.W.; Davis, D.S.; Wei, C.H.

1976-06-01

Specific adsorption capacity of Sepharose 4B in affinity chromatography for two purified galactose-binding lectins, designated as III/sub L/ and III/sub H/, from the seed of Ricinus communis (castor bean) was measured from 7 to 24/sup 0/C. The adsorption coefficients for these two protein fractions as a function of temperature were also obtained. It was found that there is a characteristic transition of adsorption coefficient at 18/sup 0/C for both lectins. Adsorption coefficients between Sepharose 4B and these two lectins were also expressed in terms of ..delta..G, ..delta..H, and ..delta..S. It is suggested that the difference in the temperature dependence ofmore » the binding energy of these two lectins may be used for their separation at selected temperature.« less

Force generation and temperature-jump and length-jump tension transients in muscle fibers.

PubMed Central

Davis, J S; Rodgers, M E

1995-01-01

Muscle tension rises with increasing temperature. The kinetics that govern the tension rise of maximally Ca(2+)-activated, skinned rabbit psoas fibers over a temperature range of 0-30 degrees C was characterized in laser temperature-jump experiments. The kinetic response is simple and can be readily interpreted in terms of a basic three-step mechanism of contraction, which includes a temperature-sensitive rapid preequilibrium(a) linked to a temperature-insensitive rate-limiting step and followed by a temperature-sensitive tension-generating step. These data and mechanism are compared and contrasted with the more complex length-jump Huxley-Simmons phases in which all states that generate tension or bear tension are perturbed. The rate of the Huxley-Simmons phase 4 is temperature sensitive at low temperatures but plateaus at high temperatures, indicating a change in rate-limiting step from a temperature-sensitive (phase 4a) to a temperature-insensitive reaction (phase 4b); the latter appears to correlate with the slow, temperature-insensitive temperature-jump relaxation. Phase 3 is absent in the temperature-jump, which excludes it from tension generation. We confirm that de novo tension generation occurs as an order-disorder transition during phase 2slow and the equivalent, temperature-sensitive temperature-jump relaxation. PMID:7612845

Passive potassium transport in LK sheep red cells. Modification by N- ethyl maleimide

PubMed Central

1983-01-01

Passive K transport, as modified by N-ethyl maleimide (NEM), was studied in erythrocytes of the low-K (LK) phenotype of sheep. Brief (5- min) treatment with NEM at less than 0.5 mM caused inhibition of passive K influx; NEM at concentrations greater than 0.5 mM caused stimulation of K influx. NEM had similar effects on K efflux. The treatments with NEM did not affect cell volumes (passive K transport in LK cells is sensitive to changes in cell volume). The stimulation of K transport by high [NEM] was also not a consequence of an effect on the metabolic state of the cells. Passive K transport in LK cells is dependent on Cl (it is inhibited in Cl-free media; it may be K/Cl cotransport). NEM had no effect on K influx in Cl-free (NO3- substituted) media. Pretreatment of the cells with anti-L antiserum (L antigen is found on LK cells and not on HK cells) prevented stimulation of K influx by NEM, but did not prevent inhibition. Therefore, NEM modifies the Cl-dependent K transport pathway at two separate sites, a low-affinity site, at which it stimulates, and a high-affinity site, at which it inhibits. Anti-L antibody prevents NEM's action, but only at the low-affinity site. PMID:6875508

Changing body temperature affects the T2* signal in the rat brain and reveals hypothalamic activity.

PubMed

Vanhoutte, G; Verhoye, M; Van der Linden, A

2006-05-01

This study was designed to determine brain activity in the hypothalamus-in particular the thermoregulatory function of the hypothalamic preoptic area (PO). We experimentally changed the body temperature in rats within the physiological range (37-39 degrees C) and monitored changes in blood oxygenation level-dependent (BOLD) MR signal. To explore PO activity we had to deal with general signal changes caused by temperature-dependent alterations in the affinity of oxygen for hemoglobin, which contributes to BOLD contrast because it is partly sensitive to the amount of paramagnetic deoxyhemoglobin in the voxel. To reduce these overall temperature-induced effects, we corrected the BOLD data using brain-specific correction algorithms. The results showed activity of the PO during body warming from 38 degrees C to 39 degrees C, supported by an increased BOLD signal after correction. This is the first fMRI study on the autonomous nervous system in which hypothalamic activity elicited by changes in the internal environment (body temperature) was monitored. In this study we also demonstrate 1) that any fMRI study of anesthetized small animals should guard against background BOLD signal drift, since animals are vulnerable to body temperature fluctuations; and 2) the existence of a link between PO activity and the sympathetically-mediated opening of the arteriovenous anastomoses in a parallel study on the rat tail, a peripheral thermoregulatory organ.

Interactions Between Mineral Surfaces, Substrates, Enzymes, and Microbes Result in Hysteretic Temperature Sensitivities and Microbial Carbon Use Efficiencies and Weaker Predicted Carbon-Climate Feedbacks

NASA Astrophysics Data System (ADS)

Riley, W. J.; Tang, J.

2014-12-01

We hypothesize that the large observed variability in decomposition temperature sensitivity and carbon use efficiency arises from interactions between temperature, microbial biogeochemistry, and mineral surface sorptive reactions. To test this hypothesis, we developed a numerical model that integrates the Dynamic Energy Budget concept for microbial physiology, microbial trait-based community structure and competition, process-specific thermodynamically ­­based temperature sensitivity, a non-linear mineral sorption isotherm, and enzyme dynamics. We show, because mineral surfaces interact with substrates, enzymes, and microbes, both temperature sensitivity and microbial carbon use efficiency are hysteretic and highly variable. Further, by mimicking the traditional approach to interpreting soil incubation observations, we demonstrate that the conventional labile and recalcitrant substrate characterization for temperature sensitivity is flawed. In a 4 K temperature perturbation experiment, our fully dynamic model predicted more variable but weaker carbon-climate feedbacks than did the static temperature sensitivity and carbon use efficiency model when forced with yearly, daily, and hourly variable temperatures. These results imply that current earth system models likely over-estimate the response of soil carbon stocks to global warming.

From quantum affine groups to the exact dynamical correlation function of the Heisenberg model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bougourzi, A.H.; Couture, M.; Kacir, M.

1997-01-20

The exact form factors of the Heisenberg models XXX and XXZ have been recently computed through the quantum affine symmetry of XXZ model in the thermodynamic limit. The authors use them to derive an exact formula for the contribution of two spinons to the dynamical correlation function of XXX model at zero temperature.

Effect of Temperature and Dynamic Loading on the Mechanical Properties of Copper-Alloyed High-Strength Interstitial-Free Steel

NASA Astrophysics Data System (ADS)

Rana, R.; Singh, S. B.; Bleck, W.; Mohanty, O. N.

2009-04-01

Crash resistance and formability relevant mechanical properties of a copper-alloyed interstitial-free (IF) steel processed under various conditions of batch annealing (BA), continuous annealing (CA), and postcontinuous annealing aging have been studied in a wide range of strain rate (3.33 × 10-4 to 200 s-1) and temperature (-100 °C to +20 °C). These properties have been compared with similarly processed traditional mild and high-strength IF steels. Assessment of various parameters such as strength, elongation, strain rate sensitivity of stress, strain-hardening capacity, temperature sensitivity of stress, activation volume, and specific energy absorption of all these steels implies that copper-alloyed IF steel is soft and formable in CA condition. It can be made stronger and more crash resistant than the conventional mild- or high-strength IF steels when aged to peak strength after CA. Room-temperature strain rate sensitivity of stress of the investigated steels exhibits a two-stage behavior. Copper in solution in ferrite causes solid solution softening at low temperatures (≤20 °C) and at high strain rates (200 s-1).

Ccl22/MDC, is a prostaglandin dependent pyrogen, acting in the anterior hypothalamus to induce hyperthermia via activation of brown adipose tissue.

PubMed

Osborn, Olivia; Sanchez-Alavez, Manuel; Dubins, Jeffrey S; Gonzalez, Alejandro Sanchez; Morrison, Brad; Hadcock, John R; Bartfai, Tamas

2011-03-01

CC Chemokine ligand 22 (Ccl22) is a selective, high affinity ligand at the CC chemokine receptor 4 (Ccr4). We have identified cDNAs encoding both ligand and receptor of the Ccl22-Ccr4 pair in cDNA libraries of the anterior hypothalamus/pre-optic area (AH/POA) by PCR. The AH/POA is the key brain region where endogenous pyrogens have been shown to act on warm sensitive neurons to affect thermogenesis in brown adipose tissue (BAT) and other thermogenically responsive tissues. We show that functional Ccr4 receptors are present in the AH/POA neurons as injection of Ccl22 into the POA but not to other hypothalamic nuclei induces an increase in core body temperature as measured by radiotelemetry. Indomethacin (5 mg/kg s.c) pre-treatment markedly reduced the hyperthermia evoked by POA injection of Ccl22 (10 ng/0.5 ul) and thus suggests that this hyperthermia is mediated through cyclooxygenase activation and thus likely through the formation and action of the pyrogen prostaglandin E2. The temperature elevation involves a decrease in the respiratory exchange ratio and increased activation of the brown adipose tissue as demonstrated by ¹⁸F-FDG-PET imaging. We describe a novel role to the ligand Ccl22 and its receptor Ccr4 in the anterior hypothalamus in temperature regulation that depends on the synthesis of the endogenous pyrogen, prostaglandin E2. Copyright © 2010 Elsevier Ltd. All rights reserved.

Rare Earth Doped High Temperature Ceramic Selective Emitters

NASA Technical Reports Server (NTRS)

Chubb, Donald L.; Pal, AnnaMarie; Patton, Martin O.; Jenkins, Phillip P.

1999-01-01

As a result of their electron structure, rare earth ions in crystals at high temperature emit radiation in several narrow bands rather than in a continuous blackbody manner. This study develops a spectral emittance model for films of rare earth containing materials. Although there are several possible rare earth doped high temperature materials, this study was confined to rare earth aluminum garnets. Good agreement between experimental and theoretical spectral emittances was found for erbium, thulium and erbium-holmium aluminum garnets. Spectral emittances of these films are sensitive to temperature differences across the film. Emitter efficiency is also a sensitive function of temperature. For thulium aluminum garnet the efficiency is 0.38 at 1700 K but only 0.19 at 1262 K.

Compact and cost-effective multi-channel optical spectrometer for fine FBG sensing in IoT technology

NASA Astrophysics Data System (ADS)

Konishi, Tsuyoshi; Yamasaki, Yu

2018-02-01

Optical fiber sensor networks have attracted much attention in IoT technology and a fiber Bragg grating is one of key sensor devices there because of their advantages in a high affinity for optical fiber networks, compactness, immunity to electromagnetic interference and so on. Nevertheless, its sensitivity is not always satisfactory so as to be usable together with widespread cost-effective multi-channel spectrometers. In this paper, we introduce a new cost-effective approach for a portable multi-channel spectrometer with high spectral resolution and demonstrates some preliminary experimental results for fine FBG sensing.

In situ XAS of Pt monolayer model fuel cell catalysts: balance of na-nostructure and bimetallic interactions

NASA Astrophysics Data System (ADS)

Friebel, Daniel; Viswanathan, Venkat; Larsen, Ask; Miller, Daniel J.; Ogasawara, Hirohito; Anniyev, Toyli; O'Grady, Christopher P.; Nørskov, Jens; Nilsson, Anders

2012-02-01

The mechanism of the electrochemical oxygen reduction reaction (ORR) has been well understood based on DFT calculations, but there has been a lack of supporting experimental data, due to the difficulties of probing the electrocatalyst surface in situ. Our new approach using Pt monolayer model catalysts provides true surface sensitivity for - originally bulk sensitive - x-ray absorption spectroscopy (XAS) and, owing to the high resolution of the Bragg analyzer at SSRL beamline 6-2, allows for in situ detection of chemisorbed O and OH, whose stability can be used as a descriptor in predicting the activity of new ORR catalyst materials. Our ability to control the growth mode in the Pt/Rh(111) model system allows us to generate Pt nanostructures with highly different O affinities from identical starting materials.

Multiple environmental factors regulate the expression of the carbohydrate-selective OprB porin of Pseudomonas aeruginosa.

PubMed

Adewoye, L O; Worobec, E A

1999-12-01

In response to low extracellular glucose concentration, Pseudomonas aeruginosa induces the expression of the outer membrane carbohydrate-selective OprB porin. The promoter region of the oprB gene was cloned into a lacZ transcriptional fusion vector, and the construct was mobilized into P. aeruginosa OprB-deficient strain, WW100, to evaluate additional environmental factors that influence OprB porin gene expression. Growth temperature, pH of the growth medium, salicylate concentration, and carbohydrate source were found to differentially influence porin expression. This expression pattern was compared to those of whole-cell [14C]glucose uptake under conditions of high osmolarity, ionicity, variable pH, growth temperatures, and carbohydrate source. These studies revealed that the high-affinity glucose transport genes are down-regulated by salicylic acid, differentially regulated by pH and temperature, and are specifically responsive to exogenous glucose induction.

Aptamer-functionalized nano-biosensors.

PubMed

Chiu, Tai-Chia; Huang, Chih-Ching

2009-01-01

Nanomaterials have become one of the most interesting sensing materials because of their unique size- and shape-dependent optical properties, high surface energy and surface-to-volume ratio, and tunable surface properties. Aptamers are oligonucleotides that can bind their target ligands with high affinity. The use of nanomaterials that are bioconjugated with aptamers for selective and sensitive detection of analytes such as small molecules, metal ions, proteins, and cells has been demonstrated. This review focuses on recent progress in the development of biosensors by integrating functional aptamers with different types of nanomaterials, including quantum dots, magnetic nanoparticles (NPs), metallic NPs, and carbon nanotubes. Colorimetry, fluorescence, electrochemistry, surface plasmon resonance, surface-enhanced Raman scattering, and magnetic resonance imaging are common detection modes for a broad range of analytes with high sensitivity and selectivity when using aptamer bioconjugated nanomaterials (Apt-NMs). We highlight the important roles that the size and concentration of nanomaterials, the secondary structure and density of aptamers, and the multivalent interactions play in determining the specificity and sensitivity of the nanosensors towards analytes. Advantages and disadvantages of the Apt-NMs for bioapplications are focused.

A highly sensitive and selective diagnostic assay based on virus nanoparticles

NASA Astrophysics Data System (ADS)

Park, Jin-Seung; Cho, Moon Kyu; Lee, Eun Jung; Ahn, Keum-Young; Lee, Kyung Eun; Jung, Jae Hun; Cho, Yunjung; Han, Sung-Sik; Kim, Young Keun; Lee, Jeewon

2009-04-01

Early detection of the protein marker troponin I in patients with a higher risk of acute myocardial infarction can reduce the risk of death from heart attacks. Most troponin assays are currently based on the conventional enzyme linked immunosorbent assay and have detection limits in the nano- and picomolar range. Here, we show that by combining viral nanoparticles, which are engineered to have dual affinity for troponin antibodies and nickel, with three-dimensional nanostructures including nickel nanohairs, we can detect troponin levels in human serum samples that are six to seven orders of magnitude lower than those detectable using conventional enzyme linked immunosorbent assays. The viral nanoparticle helps to orient the antibodies for maximum capture of the troponin markers. High densities of antibodies on the surfaces of the nanoparticles and nanohairs lead to greater binding of the troponin markers, which significantly enhances detection sensitivities. The nickel nanohairs are re-useable and can reproducibly differentiate healthy serum from unhealthy ones. We expect other viral nanoparticles to form similar highly sensitive diagnostic assays for a variety of other protein markers.

Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions

PubMed Central

Geng, Steven B.; Cheung, Jason K.; Narasimhan, Chakravarthy; Shameem, Mohammed; Tessier, Peter M.

2014-01-01

A limitation of using monoclonal antibodies as therapeutic molecules is their propensity to associate with themselves and/or with other molecules via non-affinity (colloidal) interactions. This can lead to a variety of problems ranging from low solubility and high viscosity to off-target binding and fast antibody clearance. Measuring such colloidal interactions is challenging given that they are weak and potentially involve diverse target molecules. Nevertheless, assessing these weak interactions – especially during early antibody discovery and lead candidate optimization – is critical to preventing problems that can arise later in the development process. Here we review advances in developing and implementing sensitive methods for measuring antibody colloidal interactions as well as using these measurements for guiding antibody selection and engineering. These systematic efforts to minimize non-affinity interactions are expected to yield more effective and stable monoclonal antibodies for diverse therapeutic applications. PMID:25209466

Synthesis of methoxy-X04 derivatives and their evaluation in Alzheimer's disease pathology.

PubMed

Boländer, Alexander; Kieser, Daniel; Scholz, Christoph; Heyny-von Haußen, Roland; Mall, Gerhard; Goetschy, Valérie; Czech, Christian; Schmidt, Boris

2014-01-01

Alzheimer's disease is characterized by two notorious protein aggregates in the brain: extracellular senile plaques mainly consisting of amyloid-β peptides and tau-protein-derived intracellular paired helical filaments. The diagnosis of Alzheimer's disease is impaired by insufficient sensitivity and specificity of diagnostic methods to visualize these pathological hallmarks over all disease stages. The established fluorescence marker methoxy-X04 stains plaques, tau tangles and amyloid-derived angiopathies with good specificity, yet it is limited by slow elimination in vivo. Since the need for new markers is high, we prepared methoxy-X04 derivatives and evaluated their potential as imaging agents in Alzheimer's disease pathology. In this study, we describe an improved synthesis for methoxy-X04 and its derivatives and their affinity determination for the respective protein targets by immunohistology and a displacement assay. This resulted in the identification of new derivatives of methoxy-X04 with improved binding affinity.

Fiber optic evanescent wave biosensor

NASA Astrophysics Data System (ADS)

Duveneck, Gert L.; Ehrat, Markus; Widmer, H. M.

1991-09-01

The role of modern analytical chemistry is not restricted to quality control and environmental surveillance, but has been extended to process control using on-line analytical techniques. Besides industrial applications, highly specific, ultra-sensitive biochemical analysis becomes increasingly important as a diagnostic tool, both in central clinical laboratories and in the doctor's office. Fiber optic sensor technology can fulfill many of the requirements for both types of applications. As an example, the experimental arrangement of a fiber optic sensor for biochemical affinity assays is presented. The evanescent electromagnetic field, associated with a light ray guided in an optical fiber, is used for the excitation of luminescence labels attached to the biomolecules in solution to be analyzed. Due to the small penetration depth of the evanescent field into the medium, the generation of luminescence is restricted to the close proximity of the fiber, where, e.g., the luminescent analyte molecules combine with their affinity partners, which are immobilized on the fiber. Both cw- and pulsed light excitation can be used in evanescent wave sensor technology, enabling the on-line observation of an affinity assay on a macroscopic time scale (seconds and minutes), as well as on a microscopic, molecular time scale (nanoseconds or microseconds).

DNA sensing by a Eu-binding peptide containing a proflavine unit.

PubMed

Ancel, Laetitia; Gateau, Christelle; Lebrun, Colette; Delangle, Pascale

2013-01-18

Synthesis of a lanthanide-binding peptide (LBP) for the detection of double-stranded DNA is presented. A proflavine moiety was introduced into a high affinity LBP involving two unnatural chelating amino acids in the Ln ion coordination. The Eu(3+)-LBP complex is demonstrated to bind to ct-DNA and to sensitize Eu luminescence. The DNA binding process is effectively detected via the Eu-centered luminescence thanks to the intimate coupling between the LBP scaffold and DNA intercalating unit.

Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles.

PubMed

Erster, Oran; Eisenstein, Miriam; Liscovitch, Mordechai

2007-05-01

The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 beta-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).

The High Strain Rate Deformation Behavior of High Purity Magnesium and AZ31B Magnesium Alloy

NASA Astrophysics Data System (ADS)

Livescu, Veronica; Cady, Carl M.; Cerreta, Ellen K.; Henrie, Benjamin L.; Gray, George T.

The deformation in compression of pure magnesium and AZ31B magnesium alloy, both with a strong basal pole texture, has been investigated as a function of temperature, strain rate, and specimen orientation. The mechanical response of both metals is highly dependent upon the orientation of loading direction with respect to the basal pole. Specimens compressed along the basal pole direction have a high sensitivity to strain rate and temperature and display a concave down work hardening behavior. Specimens loaded perpendicularly to the basal pole have a yield stress that is relatively insensitive to strain rate and temperature and a work hardening behavior that is parabolic and then linearly upwards. Both specimen orientations display a mechanical response that is sensitive to temperature and strain rate. Post mortem characterization of the pure magnesium was conducted on a subset of specimens to determine the microstructural and textural evolution during deformation and these results are correlated with the observed work hardening behavior and strain rate sensitivities were calculated.

Aptamer-Immobilized Surface Plasmon Resonance Biosensor for Rapid and Sensitive Determination of Virulence Determinant.

PubMed

Song, Myeong-Sub; Sekhon, Simranjeet Singh; Shin, Woo-Ri; Rhee, Sung-Keun; Ko, Jung Ho; Kim, Sang Yong; Min, Jiho; Ahn, Ji-Young; Kim, Yang-Hoon

2018-05-01

Shigella sonnei isolate invasion plasmid antigen protein, IpaH, was successfully expressed in recombinant overexpression bacterial system. The soluble expression IpaH was enhanced with molecular chaperon co-expressed environment. Specific aptamer IpaH17 was isolated through the SELEX process and showed fM binding affinity. IpaH17-SPR biosensor platform was involved to verify the binding sensitivity and specificity. The IpaH concentration dependent IpaH17-SPR sensor response was highly linear with a linear regression constant of 99.4% in the range between 0 and 100 ng/mL. In addition, S. sonnei revealed the specific RU value and detected in a real-time manner within 1 hour. Our study indicated that IpaH17-SPR sensor can allow for rapid, sensitive and specific determination of Shigella sonnei virulent factor.

Synthesis of hapten and preparation of specific polyclonal antibody with high affinity for lenalidomide, the potent drug for treatment of multiple myeloma.

PubMed

Darwish, Ibrahim A; Alzoman, Nourh Z; Abuhejail, Reem M; El-Samani, Tilal E

2012-10-26

For therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM), a specific antibody was required for the development of a sensitive immunoassay system for the accurate determination of LND in plasma. In this study, a hapten of LND (N-glutaryl-LND) was synthesized by introducing the glutaryl moiety, as a spacer, into the primary aromatic amine site of the LND molecular structure. The structure of the hapten (G-LND) was confirmed by mass, 1H-NMR, and 13C spectrometric techniques. G-LND was coupled to each of bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH) proteins by ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling reagent. LND-KLH conjugate was used as an immunogen. Four female 2-3 months old New Zealand white rabbits were immunized with an emulsion of LND-KLH with Freund`s adjuvant. The immune response of the rabbits was monitored by direct enzyme-linked immunosorbent assay (ELISA) using LND-BSA immobilized onto microwell plates as a solid phase. The rabbit that showed the highest antibody titer and affinity to LND was scarified and its sera were collected. The IgG fraction was isolated and purified by affinity chromatography on protein A column. The specificity of the purified antibody for LND was evaluated by indirect competitive ELISA using dexamethasone as a competitor as it is used with LND in a combination therapy. The high affinity of the antibody (IC50 = 10 ng/mL) will be useful in the development of an immunoassay system for the determination of plasma LND concentrations. Current research is going to optimize the assay conditions and validate the procedures for the routine application in clinical laboratories.

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody.

PubMed

Lago, Sara; Nadai, Matteo; Rossetto, Monica; Richter, Sara N

2018-06-01

G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens. SPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures. The use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability. Optimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells. The heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Frontal affinity chromatography: A unique research tool for biospecific interaction that promotes glycobiology

PubMed Central

KASAI, Kenichi

2014-01-01

Combination of bioaffinity and chromatography gave birth to affinity chromatography. A further combination with frontal analysis resulted in creation of frontal affinity chromatography (FAC). This new versatile research tool enabled detailed analysis of weak interactions that play essential roles in living systems, especially those between complex saccharides and saccharide-binding proteins. FAC now becomes the best method for the investigation of saccharide-binding proteins (lectins) from viewpoints of sensitivity, accuracy, and efficiency, and is contributing greatly to the development of glycobiology. It opened a door leading to deeper understanding of the significance of saccharide recognition in life. The theory is also concisely described. PMID:25169774

IgG1 memory B cells keep the memory of IgE responses.

PubMed

He, Jin-Shu; Subramaniam, Sharrada; Narang, Vipin; Srinivasan, Kandhadayar; Saunders, Sean P; Carbajo, Daniel; Wen-Shan, Tsao; Hidayah Hamadee, Nur; Lum, Josephine; Lee, Andrea; Chen, Jinmiao; Poidinger, Michael; Zolezzi, Francesca; Lafaille, Juan J; Curotto de Lafaille, Maria A

2017-09-21

The unique differentiation of IgE cells suggests unconventional mechanisms of IgE memory. IgE germinal centre cells are transient, most IgE cells are plasma cells, and high affinity IgE is produced by the switching of IgG1 cells to IgE. Here we investigate the function of subsets of IgG1 memory B cells in IgE production and find that two subsets of IgG1 memory B cells, CD80 + CD73 + and CD80 - CD73 - , contribute distinctively to the repertoires of high affinity pathogenic IgE and low affinity non-pathogenic IgE. Furthermore, repertoire analysis indicates that high affinity IgE and IgG1 plasma cells differentiate from rare CD80 + CD73 + high affinity memory clones without undergoing further mutagenesis. By identifying the cellular origin of high affinity IgE and the clonal selection of high affinity memory B cells into the plasma cell fate, our findings provide fundamental insights into the pathogenesis of allergies, and on the mechanisms of antibody production in memory B cell responses.IgE is an important mediator of protective immunity as well as allergic reaction, but how high affinity IgE antibodies are produced in memory responses is not clear. Here the authors show that IgE can be generated via class-switch recombination in IgG1 memory B cells without additional somatic hypermutation.

Modulation of Cardiac Ryanodine Receptor Channels by Alkaline Earth Cations

PubMed Central

Diaz-Sylvester, Paula L.; Porta, Maura; Copello, Julio A.

2011-01-01

Cardiac ryanodine receptor (RyR2) function is modulated by Ca2+ and Mg2+. To better characterize Ca2+ and Mg2+ binding sites involved in RyR2 regulation, the effects of cytosolic and luminal earth alkaline divalent cations (M2+: Mg2+, Ca2+, Sr2+, Ba2+) were studied on RyR2 from pig ventricle reconstituted in bilayers. RyR2 were activated by M2+ binding to high affinity activating sites at the cytosolic channel surface, specific for Ca2+ or Sr2+. This activation was interfered by Mg2+ and Ba2+ acting at low affinity M2+-unspecific binding sites. When testing the effects of luminal M2+ as current carriers, all M2+ increased maximal RyR2 open probability (compared to Cs+), suggesting the existence of low affinity activating M2+-unspecific sites at the luminal surface. Responses to M2+ vary from channel to channel (heterogeneity). However, with luminal Ba2+or Mg2+, RyR2 were less sensitive to cytosolic Ca2+ and caffeine-mediated activation, openings were shorter and voltage-dependence was more marked (compared to RyR2 with luminal Ca2+or Sr2+). Kinetics of RyR2 with mixtures of luminal Ba2+/Ca2+ and additive action of luminal plus cytosolic Ba2+ or Mg2+ suggest luminal M2+ differentially act on luminal sites rather than accessing cytosolic sites through the pore. This suggests the presence of additional luminal activating Ca2+/Sr2+-specific sites, which stabilize high Po mode (less voltage-dependent) and increase RyR2 sensitivity to cytosolic Ca2+ activation. In summary, RyR2 luminal and cytosolic surfaces have at least two sets of M2+ binding sites (specific for Ca2+ and unspecific for Ca2+/Mg2+) that dynamically modulate channel activity and gating status, depending on SR voltage. PMID:22039534

Does ecophysiology mediate reptile responses to fire regimes? Evidence from Iberian lizards

PubMed Central

Ferreira, Catarina C.; Santos, Xavier

2016-01-01

Background. Reptiles are sensitive to habitat disturbance induced by wildfires but species frequently show opposing responses. Functional causes of such variability have been scarcely explored. In the northernmost limit of the Mediterranean bioregion, lizard species of Mediterranean affinity (Psammodromus algirus and Podarcis guadarramae) increase in abundance in burnt areas whereas Atlantic species (Lacerta schreiberi and Podarcis bocagei) decrease. Timon lepidus, the largest Mediterranean lizard in the region, shows mixed responses depending on the locality and fire history. We tested whether such interspecific differences are of a functional nature, namely, if ecophysiological traits may determine lizard response to fire. Based on the variation in habitat structure between burnt and unburnt sites, we hypothesise that Mediterranean species, which increase density in open habitats promoted by frequent fire regimes, should be more thermophile and suffer lower water losses than Atlantic species. Methods. We submitted 6–10 adult males of the five species to standard experiments for assessing preferred body temperatures (Tp) and evaporativewater loss rates (EWL), and examined the variation among species and along time by means of repeated-measures AN(C)OVAs. Results. Results only partially supported our initial expectations, since the medium-sized P. algirus clearly attained higher Tp and lower EWL. The two small wall lizards (P. bocagei and P. guadarramae) displayed low Tp and high EWL while the two large green lizards (T. lepidus and L. schreiberi) displayed intermediate values for both parameters. Discussion. The predicted differences according to the biogeographic affinities within each pair were not fully confirmed. We conclude that ecophysiology may help to understand functional reptile responses to fire but other biological traits are also to be considered. PMID:27330864

Temperature insensitive curvature sensor based on cascading photonic crystal fiber

NASA Astrophysics Data System (ADS)

Fu, Guangwei; Li, Yunpu; Fu, Xinghu; Jin, Wa; Bi, Weihong

2018-03-01

A temperature insensitive curvature sensor is proposed based on cascading photonic crystal fiber. Using the arc fusion splicing method, this sensor is fabricated by cascading together a single-mode fiber (SMF), a three layers air holes structure of photonic crystal fiber (3PCF), a five layers air holes structure of photonic crystal fiber (5PCF) and a SMF in turn. So the structure SMF-3PCF-5PCF-SMF can be obtained with a total length of 20 mm. During the process of fabrication, the splicing machine parameters and the length of each optical fiber are adjusted to obtain a high sensitivity curvature sensor. The experimental results show that the curvature sensitivity is -8.40 nm/m-1 in the curvature variation range of 0-1.09 m-1, which also show good linearity. In the range of 30-90 °C, the temperature sensitivity is only about 3.24 pm/°C, indicating that the sensor is not sensitive to temperature. The sensor not only has the advantages of easy fabricating, simple structure, high sensitivity but also can solve the problem of temperature measurement cross sensitivity, so it can be used for different areas including aerospace, large-scale bridge, architectural structure health monitoring and so on.

Fecal Immunochemical Test (FIT) for Colon Cancer Screening: Variable Performance with Ambient Temperature

PubMed Central

Doubeni, Chyke A.; Jensen, Christopher D.; Fedewa, Stacey A.; Quinn, Virginia P.; Zauber, Ann G.; Schottinger, Joanne E.; Corley, Douglas A.; Levin, Theodore R.

2017-01-01

Introduction Fecal immunochemical tests (FITs) are widely used in colorectal cancer (CRC) screening, but hemoglobin degradation, due to exposure of the collected sample to high temperatures, could reduce test sensitivity. We examined the relation of ambient temperature exposure with FIT positivity rate and sensitivity. Methods This was a retrospective cohort study of patients 50 to 75 years in Kaiser Permanente Northern California’s CRC screening program, which began mailing FIT kits annually to screen-eligible members in 2007. Primary outcomes were FIT positivity rate and sensitivity to detect CRC. Predictors were month, season, and daily ambient temperatures of test result dates based on US National Oceanic and Atmospheric Administration data. Results Patients (n =472,542) completed 1,141,162 FITs. Weekly test positivity rate ranged from 2.6% to 8.0% (median, 4.4%) and varied significantly by month (June/July vs December/January rate ratio [RR] =0.79, 95% confidence interval [CI], 0.76 to 0.83) and season. FIT sensitivity was lower in June/July (74.5%; 95% CI, 72.5 to 76.6) than January/December (78.9%; 95% CI, 77.0 to 80.7). Conclusions FITs completed during high ambient temperatures had lower positivity rates and lower sensitivity. Changing kit design, specimen transportation practices, or avoiding periods of high ambient temperatures may help optimize FIT performance, but may also increase testing complexity and reduce patient adherence, requiring careful study. PMID:28076249

Acyl-gelatins for cell-hybrid biomaterials: preparation of gelatins with high melting point and affinity for hydrophobic surfaces.

PubMed

Miyamoto, Keiichi; Chinzei, Hiroko; Komai, Takashi

2002-12-01

In the development of cell-hybrid biomaterials, the functional activity of cells depends on the selective binding of cells to artificial ligands on the biomaterials. The extracellular matrix (ECM) is the most important ligand for cell activity. ECM is known to contain collagen, one of whose constituents is gelatin. Although natural gelatin has good cell attachment properties, the melting point of gelatin hydrogel is lower than body temperature. Thus, non-chemically cross-linked gelatin hydrogel is not a biomaterial that is used for prostheses. In the present study, we report the preparation of acyl-gelatin hydrogels with high melting point (>37 degrees C) and high affinity for hydrophobic surfaces for easy handling for transportation and adhesion activities on the hydrophobic surfaces. In addition, the doubling time of endothelial cells on the coated cell culture plate was faster than that of natural gelatin owing to the higher adhesion activity of acyl-gelatin. The results clearly demonstrated that the acyl-gelatin acted as an interface that enabled cell adhesion to artificial materials surfaces.

Thermionic Properties of Carbon Based Nanomaterials Produced by Microhollow Cathode PECVD

NASA Technical Reports Server (NTRS)

Haase, John R.; Wolinksy, Jason J.; Bailey, Paul S.; George, Jeffrey A.; Go, David B.

2015-01-01

Thermionic emission is the process in which materials at sufficiently high temperature spontaneously emit electrons. This process occurs when electrons in a material gain sufficient thermal energy from heating to overcome the material's potential barrier, referred to as the work function. For most bulk materials very high temperatures (greater than 1500 K) are needed to produce appreciable emission. Carbon-based nanomaterials have shown significant promise as emission materials because of their low work functions, nanoscale geometry, and negative electron affinity. One method of producing these materials is through the process known as microhollow cathode PECVD. In a microhollow cathode plasma, high energy electrons oscillate at very high energies through the Pendel effect. These high energy electrons create numerous radical species and the technique has been shown to be an effective method of growing carbon based nanomaterials. In this work, we explore the thermionic emission properties of carbon based nanomaterials produced by microhollow cathode PECVD under a variety of synthesis conditions. Initial studies demonstrate measureable current at low temperatures (approximately 800 K) and work functions (approximately 3.3 eV) for these materials.

Optical fiber evanescent absorption sensors for high-temperature gas sensing in advanced coal-fired power plants

NASA Astrophysics Data System (ADS)

Buric, Michael P.; Ohodnicky, Paul R.; Duy, Janice

2012-10-01

Modern advanced energy systems such as coal-fired power plants, gasifiers, or similar infrastructure present some of the most challenging harsh environments for sensors. The power industry would benefit from new, ultra-high temperature devices capable of surviving in hot and corrosive environments for embedded sensing at the highest value locations. For these applications, we are currently exploring optical fiber evanescent wave absorption spectroscopy (EWAS) based sensors consisting of high temperature core materials integrated with novel high temperature gas sensitive cladding materials. Mathematical simulations can be used to assist in sensor development efforts, and we describe a simulation code that assumes a single thick cladding layer with gas sensitive optical constants. Recent work has demonstrated that Au nanoparticle-incorporated metal oxides show a potentially useful response for high temperature optical gas sensing applications through the sensitivity of the localized surface plasmon resonance absorption peak to ambient atmospheric conditions. Hence, the simulation code has been applied to understand how such a response can be exploited in an optical fiber based EWAS sensor configuration. We demonstrate that interrogation can be used to optimize the sensing response in such materials.

Accelerated solvent extraction followed by on-line solid-phase extraction coupled to ion trap LC/MS/MS for analysis of benzalkonium chlorides in sediment samples

USGS Publications Warehouse

Ferrer, I.; Furlong, E.T.

2002-01-01

Benzalkonium chlorides (BACs) were successfully extracted from sediment samples using a new methodology based on accelerated solvent extraction (ASE) followed by an on-line cleanup step. The BACs were detected by liquid chromatography/ion trap mass spectrometry (LC/MS) or tandem mass spectrometry (MS/MS) using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the high sensitivity offered by the ion trap MS/MS. The effects of solvent type and ASE operational variables, such as temperature and pressure, were evaluated. After optimization, a mixture of acetonitrile/water (6:4 or 7:3) was found to be most efficient for extracting BACs from the sediment samples. Extraction recoveries ranged from 95 to 105% for C12 and C14 homologues, respectively. Total method recoveries from fortified sediment samples, using a cleanup step followed by ASE, were 85% for C12BAC and 79% for C14-BAC. The methodology developed in this work provides detection limits in the subnanogram per gram range. Concentrations of BAC homologues ranged from 22 to 206 ??g/kg in sediment samples from different river sites downstream from wastewater treatment plants. The high affinity of BACs for soil suggests that BACs preferentially concentrate in sediment rather than in water.

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

Mutation at Tyrosine in AMLRY (GILRY Like) Motif of Yeast eRF1 on Nonsense Codons Suppression and Binding Affinity to eRF3

PubMed Central

Akhmaloka; Susilowati, Prima Endang; Subandi; Madayanti, Fida

2008-01-01

Termination translation in Saccharomyces cerevisiae is controlled by two interacting polypeptide chain release factors, eRF1 and eRF3. Two regions in human eRF1, position at 281-305 and position at 411-415, were proposed to be involved on the interaction to eRF3. In this study we have constructed and characterized yeast eRF1 mutant at position 410 (correspond to 415 human eRF1) from tyrosine to serine residue resulting eRF1(Y410S). The mutations did not affect the viability and temperature sensitivity of the cell. The stop codons suppression of the mutant was analyzed in vivo using PGK-stop codon-LACZ gene fusion and showed that the suppression of the mutant was significantly increased in all of codon terminations. The suppression on UAG codon was the highest increased among the stop codons by comparing the suppression of the wild type respectively. In vitro interaction between eRF1 (mutant and wild type) to eRF3 were carried out using eRF1-(His)6 and eRF1(Y410S)-(His)6 expressed in Escherichia coli and indigenous Saccharomyces cerevisiae eRF3. The results showed that the binding affinity of eRF1(Y410S) to eRF3 was decreased up to 20% of the wild type binding affinity. Computer modeling analysis using Swiss-Prot and Amber version 9.0 programs revealed that the overall structure of eRF1(Y410S) has no significant different with the wild type. However, substitution of tyrosine to serine triggered the structural change on the other motif of C-terminal domain of eRF1. The data suggested that increasing stop codon suppression and decreasing of the binding affinity of eRF1(Y410S) were probably due to the slight modification on the structure of the C-terminal domain. PMID:18463713

[Interaction of human factor X with thromboplastin].

PubMed

Kiselev, S V; Zubairov, D M; Timarbaev, V N

2003-01-01

The binding of 125I-labeled human factor X to native and papaine-treated tissue tromboplastin in the presence of CaCl2 or EDTA was studied. The Scatchard analysis suggests the existence of high (Kd=l,8 x10(-9) M) and low affinity binding sites on the thromboplastin surface. The removal of Ca2+ reduced affinity of factor X to the high affinity sites. This was accompanied by some increase of their number. Proteolysis by papaine decreased affinity of high affinity sites and caused the increase of their number in the presence of Ca2+. In the absence of Ca2+ the affinity remained unchanged, but the number of sites decreased. At low concentrations of factor X positive cooperativity for high affinity binding sites was observed. It did not depend on the presence of Ca2+. The results indirectly confirm the role of hydrophobic interactons in Ca2+ dependent binding of factor X to thromboplastin and the fact that heterogeneity of this binding is determined by mesophase structure of the thromboplastin phospholipids.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low Affinity Memory CD8+ T Cells During Heterologous Immunity

PubMed Central

Krummey, Scott M.; Chen, Ching-Wen; Guasch, Sara A.; Liu, Danya; Wagener, Maylene; Larsen, Christian P; Ford, Mandy L.

2016-01-01

The affinity of a T cell receptor (TCR) binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic antigen and mediate graft rejection. We found that low affinity primed memory CD8+ T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared to high affinity primed memory T cells. Low affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2hi proliferating cells. Low affinity primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low but not high affinity primed animals. These data establish a functional connection between TNF signaling and TCR priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation. PMID:27481849

Static gas expansion cooler

DOEpatents

Guzek, J.C.; Lujan, R.A.

1984-01-01

Disclosed is a cooler for television cameras and other temperature sensitive equipment. The cooler uses compressed gas ehich is accelerated to a high velocity by passing it through flow passageways having nozzle portions which expand the gas. This acceleration and expansion causes the gas to undergo a decrease in temperature thereby cooling the cooler body and adjacent temperature sensitive equipment.

Sensitivity of Support Vector Machine Predictions of Passive Microwave Brightness Temperature Over Snow-covered Terrain in High Mountain Asia

NASA Astrophysics Data System (ADS)

Ahmad, J. A.; Forman, B. A.

2017-12-01

High Mountain Asia (HMA) serves as a water supply source for over 1.3 billion people, primarily in south-east Asia. Most of this water originates as snow (or ice) that melts during the summer months and contributes to the run-off downstream. In spite of its critical role, there is still considerable uncertainty regarding the total amount of snow in HMA and its spatial and temporal variation. In this study, the NASA Land Information Systems (LIS) is used to model the hydrologic cycle over the Indus basin. In addition, the ability of support vector machines (SVM), a machine learning technique, to predict passive microwave brightness temperatures at a specific frequency and polarization as a function of LIS-derived land surface model output is explored in a sensitivity analysis. Multi-frequency, multi-polarization passive microwave brightness temperatures as measured by the Advanced Microwave Scanning Radiometer - Earth Observing System (AMSR-E) over the Indus basin are used as training targets during the SVM training process. Normalized sensitivity coefficients (NSC) are then computed to assess the sensitivity of a well-trained SVM to each LIS-derived state variable. Preliminary results conform with the known first-order physics. For example, input states directly linked to physical temperature like snow temperature, air temperature, and vegetation temperature have positive NSC's whereas input states that increase volume scattering such as snow water equivalent or snow density yield negative NSC's. Air temperature exhibits the largest sensitivity coefficients due to its inherent, high-frequency variability. Adherence of this machine learning algorithm to the first-order physics bodes well for its potential use in LIS as the observation operator within a radiance data assimilation system aimed at improving regional- and continental-scale snow estimates.

Enantiomers of Single-Wall Carbon Nanotubes Show Distinct Coating Displacement Kinetics.

PubMed

Zheng, Yu; Bachilo, Sergei M; Weisman, R Bruce

2018-06-27

It is known that specific oligomers of single-stranded DNA (ssDNA) can show remarkable selectivity when coating different structural species of single-wall carbon nanotubes (SWCNTs). We report that (ATT) 4 ssDNA coatings strongly distinguish between the two optical isomers of (7,5) SWCNTs. This causes resolvable shifts in their fluorescence spectra and differences of 2 orders of magnitude in the room temperature rates of coating displacement, as monitored through changes in nanotube fluorescence wavelength and intensity on exposure to sodium deoxycholate. During coating displacement, the enantiomer with high affinity for the ssDNA oligomer is deduced to form an intermediate hybrid that is not observed for the low affinity enantiomer. These results reveal that enantiomeric differences in SWCNTs complexed with ssDNA are more diverse and dramatic than previously recognized.

A quartz-based micro catalytic methane sensor by high resolution screen printing

NASA Astrophysics Data System (ADS)

Lu, Wenshuai; Jing, Gaoshan; Bian, Xiaomeng; Yu, Hongyan; Cui, Tianhong

2016-02-01

A micro catalytic methane sensor was proposed and fabricated on a bulk fused quartz substrate using a high resolution screen printing technique for the first time, with reduced power consumption and optimized sensitivity. The sensor was designed by the finite element method and quartz was chosen as the substrate material and alumina support with optimized dimensions. Fabrication of the sensor consisted of two MEMS processes, lift-off and high resolution screen printing, with the advantages of high yield and uniformity. When the sensor’s regional working temperature changes from 250 °C to 470 °C, its sensitivity increases, as well as the power consumption. The highest sensitivity can reach 1.52 mV/% CH4. A temperature of 300 °C was chosen as the optimized working temperature, and the sensor’s sensitivity, power consumption, nonlinearity and response time are 0.77 mV/% CH4, 415 mW, 2.6%, and 35 s, respectively. This simple, but highly uniform fabrication process and the reliable performance of this sensor may lead to wide applications for methane detection.

Strain Sensors with Adjustable Sensitivity by Tailoring the Microstructure of Graphene Aerogel/PDMS Nanocomposites.

PubMed

Wu, Shuying; Ladani, Raj B; Zhang, Jin; Ghorbani, Kamran; Zhang, Xuehua; Mouritz, Adrian P; Kinloch, Anthony J; Wang, Chun H

2016-09-21

Strain sensors with high elastic limit and high sensitivity are required to meet the rising demand for wearable electronics. Here, we present the fabrication of highly sensitive strain sensors based on nanocomposites consisting of graphene aerogel (GA) and polydimethylsiloxane (PDMS), with the primary focus being to tune the sensitivity of the sensors by tailoring the cellular microstructure through controlling the manufacturing processes. The resultant nanocomposite sensors exhibit a high sensitivity with a gauge factor of up to approximately 61.3. Of significant importance is that the sensitivity of the strain sensors can be readily altered by changing the concentration of the precursor (i.e., an aqueous dispersion of graphene oxide) and the freezing temperature used to process the GA. The results reveal that these two parameters control the cell size and cell-wall thickness of the resultant GA, which may be correlated to the observed variations in the sensitivities of the strain sensors. The higher is the concentration of graphene oxide, then the lower is the sensitivity of the resultant nanocomposite strain sensor. Upon increasing the freezing temperature from -196 to -20 °C, the sensitivity increases and reaches a maximum value of 61.3 at -50 °C and then decreases with a further increase in freezing temperature to -20 °C. Furthermore, the strain sensors offer excellent durability and stability, with their piezoresistivities remaining virtually unchanged even after 10 000 cycles of high-strain loading-unloading. These novel findings pave the way to custom design strain sensors with a desirable piezoresistive behavior.

Use Dependence of Heat Sensitivity of Vanilloid Receptor TRPV2

PubMed Central

Liu, Beiying; Qin, Feng

2016-01-01

Thermal TRP channels mediate temperature transduction and pain sensation. The vanilloid receptor TRPV2 is involved in detection of noxious heat in a subpopulation of high-threshold nociceptors. It also plays a critical role in development of thermal hyperalgesia, but the underlying mechanism remains uncertain. Here we analyze the heat sensitivity of the TRPV2 channel. Heat activation of the channel exhibits strong use dependence. Prior heat activation can profoundly alter its subsequent temperature responsiveness, causing decreases in both temperature activation threshold and slope sensitivity of temperature dependence while accelerating activation time courses. Notably, heat and agonist activations differ in cross use-dependence. Prior heat stimulation can dramatically sensitize agonist responses, but not conversely. Quantitative analyses indicate that the use dependence in heat sensitivity is pertinent to the process of temperature sensing by the channel. The use dependence of TRPV2 reveals that the channel can have a dynamic temperature sensitivity. The temperature sensing structures within the channel have multiple conformations and the temperature activation pathway is separate from the agonist activation pathway. Physiologically, the use dependence of TRPV2 confers nociceptors with a hypersensitivity to heat and thus provides a mechanism for peripheral thermal hyperalgesia. PMID:27074678

Non-destructive testing (NDT) of metal cracks using a high Tc rf-SQUID and eddy current method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, D.F.; Fan, C.; Ruan, J.Z.

1994-12-31

A SQUID is the most sensitive device to detect change in magnetic field. A non-destructive testing (NDT) device using high temperature SQUIDs and eddy current method will be much more sensitive than those currently used eddy current systems, yet much cheaper than one with low temperature SQUIDs. In this paper, we present our study of such a NDT device using a high temperature superconducting rf-SQUID as a gradiometer sensor. The result clearly demonstrates the expected sensitivity of the system, and indicates the feasibility of building a portable HTS SQUID NDT device with the help from cryocooler industry. Such a NDTmore » device will have a significant impact on metal corrosion or crack detection technology.« less

Drought-induced weakening of growth-temperature associations in high-elevation Iberian pines

NASA Astrophysics Data System (ADS)

Diego Galván, J.; Büntgen, Ulf; Ginzler, Christian; Grudd, Håkan; Gutiérrez, Emilia; Labuhn, Inga; Julio Camarero, J.

2015-01-01

The growth/climate relationship of theoretically temperature-controlled high-elevation forests has been demonstrated to weaken over recent decades. This is likely due to new tree growth limiting factors, such as an increasing drought risk for ecosystem functioning and productivity across the Mediterranean Basin. In addition, declining tree growth sensitivity to spring temperature may emerge in response to increasing drought stress. Here, we evaluate these ideas by assessing the growth/climate sensitivity of 1500 tree-ring width (TRW) and 102 maximum density (MXD) measurement series from 711 and 74 Pinus uncinata trees, respectively, sampled at 28 high-elevation forest sites across the Pyrenees and two relict populations of the Iberian System. Different dendroclimatological standardization and split period approaches were used to assess the high- to low-frequency behavior of 20th century tree growth in response to temperature means, precipitation totals and drought indices. Long-term variations in TRW track summer temperatures until about 1970 but diverge afterwards, whereas MXD captures the recent temperature increase in the low-frequency domain fairly well. On the other hand summer drought has increasingly driven TRW along the 20th century. Our results suggest fading temperature sensitivity of Iberian high-elevation P. uncinata forest growth, and reveal the importance of summer drought that is becoming the emergent limiting factor of tree ring width formation in many parts of the Mediterranean Basin.

SiC-Based Schottky Diode Gas Sensors

NASA Technical Reports Server (NTRS)

Hunter, Gary W.; Neudeck, Philip G.; Chen, Liang-Yu; Knight, Dak; Liu, Chung-Chiun; Wu, Quing-Hai

1997-01-01

Silicon carbide based Schottky diode gas sensors are being developed for high temperature applications such as emission measurements. Two different types of gas sensitive diodes will be discussed in this paper. By varying the structure of the diode, one can affect the diode stability as well as the diode sensitivity to various gases. It is concluded that the ability of SiC to operate as a high temperature semiconductor significantly enhances the versatility of the Schottky diode gas sensing structure and will potentially allow the fabrication of a SiC-based gas sensor arrays for versatile high temperature gas sensing applications.

Thermodynamic Bounds on the Ultra- and Infra-affinity of Hsp70 for Its Substrates

NASA Astrophysics Data System (ADS)

Nguyen, Basile; Hartich, David; Seifert, Udo; Rios, Paolo De Los

2017-07-01

The 70 kDa Heat Shock Proteins Hsp70 have several essential functions in living systems, such as protecting cells against protein aggregation, assisting protein folding, remodeling protein complexes and driving the translocation into organelles. These functions require high affinity for non-specific amino-acid sequences that are ubiquitous in proteins. It has been recently shown that this high affinity, called ultra-affinity, depends on a process driven out of equilibrium by ATP hydrolysis. Here we establish the thermodynamic bounds for ultra-affinity, and further show that the same reaction scheme can in principle be used both to strengthen and to weaken affinities (leading in this case to infra-affinity). We show that cofactors are essential to achieve affinity beyond the equilibrium range. Finally, biological implications are discussed.

Dephosphorylation and quantification of organic phosphorus in poultry litter by purified phytic-acid high affinity Aspergillus phosphohydrolases.

PubMed

Dao, Thanh H; Hoang, Khanh Q

2008-08-01

Extracellular phosphohydrolases mediate the dephosphorylation of phosphoesters and influence bioavailability and loss of agricultural P to the environment to pose risks of impairment of sensitive aquatic ecosystems. Induction and culture of five strains of Aspergillus were conducted to develop a source of high-affinity and robust phosphohydrolases for detecting environmental P and quantifying bioactive P pools in heterogeneous environmental specimens. Enzyme stability and activity against organic P in poultry litter were evaluated in 71 samples collected across poultry producing regions of Arkansas, Maryland, and Oklahoma of the US Differences existed in strains' adaptability to fermentation medium as they showed a wide range of phytate-degrading activity. Phosphohydrolases from Aspergillus ficuum had highest activity when the strain was cultured on a primarily chemical medium, compared to Aspergillus oryzae which preferred a wheat bran-based organic medium. Kinetics parameters of A. ficuum enzymes (K(m)=210 microM; V(max) of 407 nmol s(-1)) indicated phytic acid-degrading potential equivalent to that of commercial preparations. Purified A. ficuum phosphohydrolases effectively quantified litter bioactive P pools, showing that organic P occurred at an average of 54 (+/-14)% of total P, compared to inorganic phosphates, which averaged 41 (+/-12)%. Litter management and land application options must consider the high water-extractable and organic P concentrations and the biological availability of the organic enzyme-labile P pool. Robustness of A. ficuum enzymes and simplicity of the in situ ligand-based enzyme assay may thus increase routine assessment of litter bioactive P composition to sense for on-farm accumulation of such environmentally-sensitive P forms.

A Eu/Tb-mixed MOF for luminescent high-temperature sensing

NASA Astrophysics Data System (ADS)

Wang, Huizhen; Zhao, Dian; Cui, Yuangjing; Yang, Yu; Qian, Guodong

2017-02-01

Temperature measurements and thermal mapping using luminescent MOF operating in the high-temperature range are of great interest in the micro-electronic diagnosis. In this paper, we report a thermostable Eu/Tb-mixed MOF Eu0.37Tb0.63-BTC-a exhibiting strong luminescence at elevated temperature, which can serve as a ratiometric luminescent thermometer for high-temperature range. The high-temperature operating range (313-473 K), high relative sensitivity and accurate temperature resolution, make such a Eu/Tb-mixed MOF useful for micro-electronic diagnosis.

A multi-core fiber based interferometer for high temperature sensing

NASA Astrophysics Data System (ADS)

Zhou, Song; Huang, Bo; Shu, Xuewen

2017-04-01

In this paper, we have verified and implemented a Mach-Zehnder interferometer based on seven-core fiber for high temperature sensing application. This proposed structure is based on a multi-mode-multi-core-multi-mode fiber structure sandwiched by a single mode fiber. Between the single-mode and multi-core fiber, a 3 mm long multi-mode fiber is formed for lead-in and lead-out light. The basic operation principle of this device is the use of multi-core modes, single-mode and multi-mode interference coupling is also utilized. Experimental results indicate that this interferometer sensor is capable of accurate measurements of temperatures up to 800 °C, and the temperature sensitivity of the proposed sensor is as high as 170.2 pm/°C, which is much higher than the current existing MZI based temperature sensors (109 pm/°C). This type of sensor is promising for practical high temperature applications due to its advantages including high sensitivity, simple fabrication process, low cost and compactness.

SOLID PHASE MICROEXTRACTION SAMPLING OF FIRE DEBRIS RESIDUES IN THE PRESENCE OF RADIONUCLIDE SURROGATE METALS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duff, M; Keisha Martin, K; S Crump, S

2007-03-23

The Federal Bureau of Investigation (FBI) Laboratory currently does not have on site facilities for handling radioactive evidentiary materials and there are no established FBI methods or procedures for decontaminating highly radioactive fire debris (FD) evidence while maintaining evidentiary value. One experimental method for the isolation of FD residue from radionuclide metals involves using solid phase microextraction (SPME) fibers to remove the residues of interest. Due to their high affinity for organics, SPME fibers should have little affinity for most (radioactive) metals. The focus of this research was to develop an examination protocol that was applicable to safe work inmore » facilities where high radiation doses are shielded from the workers (as in radioactive shielded cells or ''hot cells''). We also examined the affinity of stable radionuclide surrogate metals (Co, Ir, Re, Ni, Ba, Cs, Nb, Zr and Nd) for sorption by the SPME fibers. This was done under exposure conditions that favor the uptake of FD residues under conditions that will provide little contact between the SPME and the FD material (such as charred carpet or wood that contains commonly-used accelerants). Our results from mass spectrometric analyses indicate that SPME fibers show promise for use in the room temperature head space uptake of organic FD residue (namely, diesel fuel oil, kerosene, gasoline and paint thinner) with subsequent analysis by gas chromatography (GC) with mass spectrometric (MS) detection. No inorganic forms of ignitable fluids were included in this study.« less

Theoretical determination of the ionization potential and the electron affinity of organic semiconductors

NASA Astrophysics Data System (ADS)

Yanagisawa, Susumu

2017-11-01

Ionization potential and electron affinity of organic semicondutors are important quantities, which are relevant to charge injection barriers. The electrostatic and dynamical contributions to the polarization energies for the injected charges in pentacene polymorphs were investigated. While the dynamical polarization induced narrowing of the energy gap, the electrostatic effect shifted up or down the frontier energy levels, which is sensitive to the molecular orientation at the surface.

Functional expression, production, and biochemical characterization of a laccase using yeast surface display technology.

PubMed

Bertrand, Brandt; Trejo-Hernández, María R; Morales-Guzmán, Daniel; Caspeta, Luis; Suárez Rodríguez, Ramón; Martínez-Morales, Fernando

2016-12-01

A Trametes versicolor laccase was functionally expressed on the membrane surface of Saccharomyces cerevisiae EBY100. Laccase expression was increased 6.57-fold by medium optimization and surpassed production by the native strain. Maximal laccase and biomass production reached 19 735 ± 1719 Ug -1 and 6.22 ± 0.53 gL -1 respectively, after 2 d of culture. Optimum oxidization of all substrates by laccase was observed at pH 3. Laccase showed high affinity towards substrates used with Km (mM) and Vmax (μmol min -1 ) values of 0.57 ± 0.0047 and 24.55 ± 0.64, 1.52 ± 0.52 and 9.25 ± 1.78, and 2.67 ± 0.12 and 11.26 ± 0.75, were reported for ABTS, 2, 6-DMP and GUA, respectively. EDTA and NaN 3 displayed none competitive inhibition towards laccase activity. The optimum temperature for activity was 50 °C; however, the enzyme was stable over a wide range of temperatures (25-70 °C). The biologically immobilized laccase showed high reusability towards phenolic substrates and low reusability with non-phenolic substrates. High affinity for a diversity phenolic compounds and great ethanol tolerance substantiates this laccase/yeast biocatalyst potential for application in the production of bioethanol. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Realizing the promise of reverse phase protein arrays for clinical, translational, and basic research: a workshop report: the RPPA (Reverse Phase Protein Array) society.

PubMed

Akbani, Rehan; Becker, Karl-Friedrich; Carragher, Neil; Goldstein, Ted; de Koning, Leanne; Korf, Ulrike; Liotta, Lance; Mills, Gordon B; Nishizuka, Satoshi S; Pawlak, Michael; Petricoin, Emanuel F; Pollard, Harvey B; Serrels, Bryan; Zhu, Jingchun

2014-07-01

Reverse phase protein array (RPPA) technology introduced a miniaturized "antigen-down" or "dot-blot" immunoassay suitable for quantifying the relative, semi-quantitative or quantitative (if a well-accepted reference standard exists) abundance of total protein levels and post-translational modifications across a variety of biological samples including cultured cells, tissues, and body fluids. The recent evolution of RPPA combined with more sophisticated sample handling, optical detection, quality control, and better quality affinity reagents provides exquisite sensitivity and high sample throughput at a reasonable cost per sample. This facilitates large-scale multiplex analysis of multiple post-translational markers across samples from in vitro, preclinical, or clinical samples. The technical power of RPPA is stimulating the application and widespread adoption of RPPA methods within academic, clinical, and industrial research laboratories. Advances in RPPA technology now offer scientists the opportunity to quantify protein analytes with high precision, sensitivity, throughput, and robustness. As a result, adopters of RPPA technology have recognized critical success factors for useful and maximum exploitation of RPPA technologies, including the following: preservation and optimization of pre-analytical sample quality, application of validated high-affinity and specific antibody (or other protein affinity) detection reagents, dedicated informatics solutions to ensure accurate and robust quantification of protein analytes, and quality-assured procedures and data analysis workflows compatible with application within regulated clinical environments. In 2011, 2012, and 2013, the first three Global RPPA workshops were held in the United States, Europe, and Japan, respectively. These workshops provided an opportunity for RPPA laboratories, vendors, and users to share and discuss results, the latest technology platforms, best practices, and future challenges and opportunities. The outcomes of the workshops included a number of key opportunities to advance the RPPA field and provide added benefit to existing and future participants in the RPPA research community. The purpose of this report is to share and disseminate, as a community, current knowledge and future directions of the RPPA technology. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Fluid transition layer between rigid solute and liquid solvent: is there depletion or enrichment?

PubMed

Djikaev, Yuri S; Ruckenstein, Eli

2016-03-21

The fluid layer between solute and liquid solvent is studied by combining the density functional theory with the probabilistic hydrogen bond model. This combination allows one to obtain the equilibrium distribution of fluid molecules, taking into account the hydrogen bond contribution to the external potential whereto they are subjected near the solute. One can find the effective width of the fluid solvent-solute transition layer and fluid average density in that layer, and determine their dependence on temperature, solvent-solute affinity, vicinal hydrogen bond (hb) energy alteration ratio, and solute radius. Numerical calculations are performed for the solvation of a plate and spherical solutes of four different radii in two model solvents (associated liquid and non-associated one) in the temperature range from 293 K to 333 K for various solvent-solute affinities and hydrogen bond energy alteration ratios. The predictions of our model for the effective width and average density of the transition layer are consistent with experiments and simulations. The small-to-large crossover lengthscale for hydrophobic hydration is expected to be about 3-5 nm. Remarkably, characterizing the transition layer with the average density, one can observe that for small hydrophobes, the transition layer becomes enriched with rather than depleted of fluid when the solvent-solute affinity and hb-energy alteration ratio become large enough. The boundary values of solvent-solute affinity and hb-energy alteration ratio, needed for the "depletion-to-enrichment" crossover (in the smoothed density sense), are predicted to decrease with increasing temperature.

cDNA cloning and characterization of an aryl hydrocarbon receptor from the harbor seal (Phoca vitulina): a biomarker of dioxin susceptibility?

PubMed

Kim, Eun-Young; Hahn, Mark E

2002-07-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are found at high concentrations in some marine mammals. Species differences in sensitivity to TCDD and PHAHs are a major limitation in assessing the ecological risk to these animals. Harbor seals accumulate high levels of PHAHs and are thought to be highly sensitive to the toxic effects of these compounds. To investigate the mechanistic basis for PHAH toxicity in harbor seals (Phoca vitulina), we sought to characterize the aryl hydrocarbon receptor (AHR), an intracellular protein that is responsible for PHAH effects. Here we report the cDNA cloning and characterization of a harbor seal AHR. The harbor seal AHR cDNA has an open reading frame of 2529 nucleotides that encodes a protein of 843 amino acids with a predicted molecular mass of 94.6 kDa. The harbor seal AHR protein possesses basic helix-loop-helix (bHLH) and Per-ARNT-Sim (PAS) domains. It is most closely related to the beluga AHR (82%) and human AHR (79%) in overall amino acid identity, indicating a high degree of conservation of AHR structure between terrestrial and some marine mammals. The ligand binding properties of the harbor seal AHR were determined using protein synthesized by in vitro transcription and translation from the cloned cDNA. Velocity sedimentation analysis on sucrose gradients showed that the harbor seal AHR exhibits specific binding of [(3)H]TCDD. The [(3)H]TCDD-binding affinity of the harbor seal AHR was compared with that of the AHR from a dioxin-sensitive mouse strain (C57BL/6) using a hydroxylapatite assay. The equilibrium dissociation constants of seal and mouse AHRs were 0.93+/-0.19 and 1.70+/-0.26 nM, respectively. Thus, the harbor seal AHR bound TCDD with an affinity that was at least as high as that of the mouse AHR, suggesting that this seal species may be sensitive to PHAH effects. The characteristics of the AHR potentially can be used as a biomarker of susceptibility to dioxin-like compounds, contributing to the assessment of the risk of these compounds to marine mammals and other protected animals.

Novel monoclonal antibodies against Plasmodium falciparum histidine-rich protein 2: development and application in rapid diagnostic tests of malaria in hyperendemic regions of China and Myanmar.

PubMed

Kang, Keren; Dzakah, Emmanuel E; Li, Wenmei; Xie, Mingquan; Luo, Xiaochun; Liu, Hui

2015-05-12

Malaria presents a considerable threat to public health. Histidine-rich protein 2 (HRP 2) is the major protein released into human blood upon infection by Plasmodium falciparum. In this study, we aimed to evaluate the immunogenicity of HRP 2 exon II and the efficacy of novel monoclonal antibodies (mAbs) against HRP 2 for Point-of-Care Test (POCT). The recombinant protein was expressed in soluble form in E. coli and used to immunize mice for mAb production. Two IgG1 mAbs (1A5 and 1C10) with high affinity, specificity and sensitivity for both native and recombinant HRP 2 were selected after fusion of mouse spleen with myeloma cells. The affinity constant of 1A5 and 1C10 were 7.15 and 4.91 × 10-7 L/mol, respectively. Subsequently, an immunochromatograhic assay was used for screening of clinical samples in endemic regions of China and Myanmar. The immunochromatographic test retrospectively showed an overall sensitivity of 99.07%, and specificity of 100%. Sensitivity at parasite densities < 200, 200-2000, and > 2000 parasites/μL was 87.5, 98.7, and 100%, respectively. These results suggest that HRP 2 exon II contains immunogenic sites similar to those of the native antigen and can be used for the development of mAbs suitable for malaria diagnosis in endemic communities.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin

PubMed Central

Fago, Angela; Malte, Hans; Storz, Jay F.; Gorr, Thomas A.

2013-01-01

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3−), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3− levels and metabolic rates increase. Examination of α- and β-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: β82Lys→Gln, β143His→Val, and β146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3− binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues. PMID:23720132

The Delicate Balance of Preorganisation and Adaptability in Multiply Bonded Host-Guest Complexes.

PubMed

von Krbek, Larissa K S; Achazi, Andreas J; Schoder, Stefan; Gaedke, Marius; Biberger, Tobias; Paulus, Beate; Schalley, Christoph A

2017-02-24

Rigidity and preorganisation are believed to be required for high affinity in multiply bonded supramolecular complexes as they help reduce the entropic penalty of the binding event. This comes at the price that such rigid complexes are sensitive to small geometric mismatches. In marked contrast, nature uses more flexible building blocks. Thus, one might consider putting the rigidity/high-affinity notion to the test. Multivalent crown/ammonium complexes are ideal for this purpose as the monovalent interaction is well understood. A series of divalent complexes with different spacer lengths and rigidities has thus been analysed to correlate chelate cooperativities and spacer properties. Too long spacers reduce chelate cooperativity compared to exactly matching ones. However, in contrast to expectation, flexible guests bind with chelate cooperativities clearly exceeding those of rigid structures. Flexible spacers adapt to small geometric host-guest mismatches. Spacer-spacer interactions help overcome the entropic penalty of conformational fixation during binding and a delicate balance of preorganisation and adaptability is at play in multivalent complexes. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.

PubMed

Pernomian, Larissa; Gomes, Mayara Santos; Moreira, Josimar Dornelas; da Silva, Carlos Henrique Tomich de Paula; Rosa, Joaquin Maria Campos; Cardoso, Cristina Ribeiro de Barros

2017-01-01

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

MEMS fiber-optic Fabry-Perot pressure sensor for high temperature application

NASA Astrophysics Data System (ADS)

Fang, G. C.; Jia, P. G.; Cao, Q.; Xiong, J. J.

2016-10-01

We design and demonstrate a fiber-optic Fabry-Perot pressure sensor (FOFPPS) for high-temperature sensing by employing micro-electro-mechanical system (MEMS) technology. The FOFPPS is fabricated by anodically bonding the silicon wafer and the Pyrex glass together and fixing the facet of the optical fiber in parallel with the silicon surface by glass frit and organic adhesive. The silicon wafer can be reduced through dry etching technology to construct the sensitive diaphragm. The length of the cavity changes with the deformation of the diaphragm due to the loaded pressure, which leads to a wavelength shift of the interference spectrum. The pressure can be gauged by measuring the wavelength shift. The pressure experimental results show that the sensor has linear pressure sensitivities ranging from 0 kPa to 600 kPa at temperature range between 20°C to 300°C. The pressure sensitivity at 300°C is approximately 27.63 pm/kPa. The pressure sensitivities gradually decrease with increasing the temperature. The sensor also has a linear thermal drift when temperature changes from 20°C - 300°C.

Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element.

PubMed

Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

2016-08-11

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

High-throughput SISCAPA quantitation of peptides from human plasma digests by ultrafast, liquid chromatography-free mass spectrometry.

PubMed

Razavi, Morteza; Frick, Lauren E; LaMarr, William A; Pope, Matthew E; Miller, Christine A; Anderson, N Leigh; Pearson, Terry W

2012-12-07

We investigated the utility of an SPE-MS/MS platform in combination with a modified SISCAPA workflow for chromatography-free MRM analysis of proteotypic peptides in digested human plasma. This combination of SISCAPA and SPE-MS/MS technology allows sensitive, MRM-based quantification of peptides from plasma digests with a sample cycle time of ∼7 s, a 300-fold improvement over typical MRM analyses with analysis times of 30-40 min that use liquid chromatography upstream of MS. The optimized system includes capture and enrichment to near purity of target proteotypic peptides using rigorously selected, high affinity, antipeptide monoclonal antibodies and reduction of background peptides using a novel treatment of magnetic bead immunoadsorbents. Using this method, we have successfully quantitated LPS-binding protein and mesothelin (concentrations of ∼5000 ng/mL and ∼10 ng/mL, respectively) in human plasma. The method eliminates the need for upstream liquid-chromatography and can be multiplexed, thus facilitating quantitative analysis of proteins, including biomarkers, in large sample sets. The method is ideal for high-throughput biomarker validation after affinity enrichment and has the potential for applications in clinical laboratories.

Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

PubMed Central

Ertekin, Özlem; Öztürk, Selma; Öztürk, Zafer Ziya

2016-01-01

This study introduces the use of an IgA isotype aflatoxin (AF) specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM) immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM) formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxy succinimide (EDC/NHS) method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1. PMID:27529243

Precipitation Behavior and Quenching Sensitivity of a Spray Deposited Al-Zn-Mg-Cu-Zr Alloy

PubMed Central

Lei, Qian; Xiao, Zhu; Wang, Mingpu

2017-01-01

Precipitation behavior and the quenching sensitivity of a spray deposited Al-Zn-Mg-Cu-Zr alloy during isothermal heat treatment have been studied systematically. Results demonstrate that both the hardness and the ultimate tensile strength of the studied alloy decreased with the isothermal treatment time at certain temperatures. More notably, the hardness decreases rapidly after the isothermal heat treatment. During isothermal heat treatment processing, precipitates readily nucleated in the medium-temperature zone (250–400 °C), while the precipitation nucleation was scarce in the low-temperature zone (<250 °C) and in the high-temperature zone (>400 °C). Precipitates with sizes of less than ten nanometers would contribute a significant increase in yield strength, while the ones with a larger size than 300 nm would contribute little strengthening effect. Quenching sensitivity is high in the medium-temperature zone (250–400 °C), and corresponding time-temperature-property (TTP) curves of the studied alloy have been established. PMID:28925964

Spatial patterns of stream temperatures and electric conductivity in a mesoscale catchment

NASA Astrophysics Data System (ADS)

Lieder, Ernestine; Weiler, Markus; Blume, Theresa

2017-04-01

Stream temperature and electric conductivity (EC) are both relatively easily measured and can provide valuable information on runoff generation processes and catchment storage.This study investigates the spatial variability of stream temperature and EC in a mesoscale basin. We focus on the mesoscale (sub-catchments and reach scale), and long term (seasonal / annual) stream temperature and EC patterns. Our study basin is the Attert catchment in Luxembourg (288km2), which contains multiple sub-catchments of different geology, topography and land use patterns. We installed 90 stream temperature and EC sensors at sites across the basin in summer 2015. The collected data is complemented by land use and discharge data and an extensive climate data set. Thermal sensitivity was calculated as the slope of daily air temperature-water-temperature regression line and describes the sensitivity of stream temperature to long term environmental change. Amplitude sensitivity was calculated as slope of the daily air and water temperature amplitude regression and describes the short term warming capacity of the stream. We found that groups with similar long term thermal and EC patterns are strongly related to different geological units. The sandstone reaches show the coldest temperatures and lowest annual thermal sensitivity to air temperature. The slate reaches are characterized by comparably low EC and high daily temperature amplitudes and amplitude sensitivity. Furthermore, mean annual temperatures and thermal sensitivities increase exponentially with drainage area, which can be attributed to the accumulation of heat throughout the system. On the reach scale, daily stream temperature fluctuations or sensitivities were strongly influenced by land cover distribution, stream shading and runoff volume. Daily thermal sensitivities were low for headwater streams; peaked for intermediate reaches in the middle of the catchment and then decreased again further downstream with increasing drainage area. Combining spatially distributed time series of stream temperatures and EC with information about geology, landscape and climate provides insight into the underlying hydrological processes and allows for the identification of thermally sensitive regions and reaches.

Reconstitution of high affinity. cap alpha. /sub 2/ adrenergic agonist binding by fusion with a pertussis toxin substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, M.H.; Neubig, R.R.

1986-03-05

High affinity ..cap alpha../sub 2/ adrenergic agonist binding is thought to occur via a coupling of the ..cap alpha../sub 2/ receptor with N/sub i/, the inhibitory guanyl nucleotide binding protein. Human platelet membranes pretreated at pH 11.5 exhibit a selective inactivation of agonist binding and N/sub i/. To further study the mechanism of agonist binding, alkali treated membranes (ATM) were mixed with membranes pretreated with 10 ..mu..M phenoxybenzamine to block ..cap alpha../sub 2/ receptors (POB-M). The combined membrane pellet was incubated in 50% polyethylene glycol (PEG) to promote membrane-membrane fusion and assayed for binding to the ..cap alpha../sub 2/ agonistmore » (/sup 3/H)UK 14,304 (UK) and the antagonist (/sup 3/H) yohimbine. PEG treatment resulted in a 2-4 fold enhancement of UK binding whereas yohimbine binding was unchanged. No enhancement of UK binding was observed in the absence of PEG treatment. The reconstitution was dependent on the addition of POB-M. They found that a 1:1 ratio of POB-M:ATM was optimal. Reconstituted binding was inhibited by GppNHp. Fusion of rat C6 glioma cell membranes, which do not contain ..cap alpha../sub 2/ receptors, also enhanced agonist binding to ATM. Fusion of C6 membranes from cells treated with pertussis toxin did not enhance (/sup 3/H) UK binding. These data show that a pertussis toxin sensitive membrane component, possibly N/sub i/, can reconstitute high affinity ..cap alpha../sub 2/ agonist binding.« less

Visual and Plasmon Resonance Absorption Sensor for Adenosine Triphosphate Based on the High Affinity between Phosphate and Zr(IV)

PubMed Central

Qi, Wenjing; Liu, Zhongyuan; Zhang, Wei; Halawa, Mohamed Ibrahim; Xu, Guobao

2016-01-01

Zr(IV) can form phosphate and Zr(IV) (–PO32−–Zr4+–) complex owing to the high affinity between Zr(IV) with phosphate. Zr(IV) can induce the aggregation of gold nanoparticles (AuNPs), while adenosine triphosphate(ATP) can prevent Zr(IV)-induced aggregation of AuNPs. Herein, a visual and plasmon resonance absorption (PRA)sensor for ATP have been developed using AuNPs based on the high affinity between Zr(IV)with ATP. AuNPs get aggregated in the presence of certain concentrations of Zr(IV). After the addition of ATP, ATP reacts with Zr(IV) and prevents AuNPs from aggregation, enabling the detection of ATP. Because of the fast interaction of ATP with Zr(IV), ATP can be detected with a detection limit of 0.5 μM within 2 min by the naked eye. Moreover, ATP can be detected by the PRA technique with higher sensitivity. The A520nm/A650nm values in PRA spectra increase linearly with the concentrations of ATP from 0.1 μM to 15 μM (r = 0.9945) with a detection limit of 28 nM. The proposed visual and PRA sensor exhibit good selectivity against adenosine, adenosine monophosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate. The recoveries for the analysis of ATP in synthetic samples range from 95.3% to 102.0%. Therefore, the proposed novel sensor for ATP is promising for real-time or on-site detection of ATP. PMID:27754349

Unconventional route to encapsulated ultrasmall gold nanoparticles for high-temperature catalysis.

PubMed

Zhang, Tingting; Zhao, Hongyu; He, Shengnan; Liu, Kai; Liu, Hongyang; Yin, Yadong; Gao, Chuanbo

2014-07-22

Ultrasmall gold nanoparticles (us-AuNPs, <3 nm) have been recently recognized as surprisingly active and extraordinarily effective green catalysts. Their stability against sintering during reactions, however, remains a serious issue for practical applications. Encapsulating such small nanoparticles in a layer of porous silica can dramatically enhance the stability, but it has been extremely difficult to achieve using conventional sol-gel coating methods due to the weak metal/oxide affinity. In this work, we address this challenge by developing an effective protocol for the synthesis of us-AuNP@SiO2 single-core/shell nanospheres. More specifically, we take an alternative route by starting with ultrasmall gold hydroxide nanoparticles, which have excellent affinity to silica, then carrying out controllable silica coating in reverse micelles, and finally converting gold hydroxide particles into well-protected us-AuNPs. With a single-core/shell configuration that prevents sintering of nearby us-AuNPs and amino group modification of the Au/SiO2 interface that provides additional coordinating interactions, the resulting us-AuNP@SiO2 nanospheres are highly stable at high temperatures and show high activity in catalytic CO oxidation reactions. A dramatic and continuous increase in the catalytic activity has been observed when the size of the us-AuNPs decreases from 2.3 to 1.5 nm, which reflects the intrinsic size effect of the Au nanoparticles on an inert support. The synthesis scheme described in this work is believed to be extendable to many other ultrasmall metal@oxide nanostructures for much broader catalytic applications.

Characterization of a conformationally sensitive murine monoclonal antibody directed to the metal ion-dependent adhesion site face of integrin CD11b.

PubMed

Li, Rui; Haruta, Ikuko; Rieu, Philippe; Sugimori, Takashi; Xiong, Jian-Ping; Arnaout, M Amin

2002-02-01

Integrin binding to physiologic ligands requires divalent cations and an inside-out-driven switch of the integrin to a high-affinity state. Divalent cations at the metal ion-dependent adhesion site (MIDAS) face of the alpha subunit-derived A domain provide a direct bridge between ligands and the integrin, and it has been proposed that activation dependency is caused by reorientation of the surrounding residues relative to the metal ion, forming an optimal binding interface. To gain more insight into the functional significance of the protein movements on the MIDAS face, we raised and characterized a murine mAb 107 directed against the MIDAS face of the A domain from integrin CD11b. We find that mAb 107 behaves as a ligand mimic. It binds in a divalent-cation-dependent manner to solvent-exposed residues on the MIDAS face of CD11b, blocks interaction of 11bA or the holoreceptor with ligands, and inhibits spreading and phagocytosis by human neutrophils. However, in contrast to physiologic ligands, mAb 107 preferentially binds to the inactive low-affinity form of the integrin, suggesting that its antagonistic effects are exerted in part by stabilizing the receptor in the low-affinity state. These data support a functional relevance of the protein movements on the MIDAS face and suggest that stabilizing the A domain in the low-affinity state may have therapeutic benefit.

Affinity-tuning leukocyte integrin for development of safe therapeutics

NASA Astrophysics Data System (ADS)

Park, Spencer

Much attention has been given to the molecular and cellular pathways linking inflammation with cancer and the local tumor environment to identify new target molecules that could lead to improved diagnosis and treatment. Among the many molecular players involved in the complex response, central to the induction of inflammation is intercellular adhesion molecule (ICAM)-1, which is of particular interest for its highly sensitive and localized expression in response to inflammatory signals. ICAM-1, which has been implicated to play a critical role in tumor progression in various types of cancer, has also been linked to cancer metastases, where ICAM-1 facilitates the spread of metastatic cancer cells to secondary sites. This unique expression profile of ICAM-1 throughout solid tumor microenvironment makes ICAM-1 an intriguing molecular target, which holds great potential as an important diagnostic and therapeutic tool. Herein, we have engineered the ligand binding domain, or the inserted (I) domain of a leukocyte integrin, to exhibit a wide range of monovalent affinities to the natural ligand, ICAM-1. Using the resulting I domain variants, we have created drug and gene delivery nanoparticles, as well as targeted immunotherapeutics that have the ability to bind and migrate to inflammatory sites prevalent in tumors and the associated microenvironment. Through the delivery of diagnostic agents, chemotherapeutics, and immunotherapeutics, the following chapters demonstrate that the affinity enhancements achieved by directed evolution bring the affinity of I domains into the range optimal for numerous applications.

Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

PubMed

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.

Evidence for two distinct phosphorylation pathways activated by high affinity immunoglobulin E receptors.

PubMed

Adamczewski, M; Paolini, R; Kinet, J P

1992-09-05

The high affinity receptor for immunoglobulin (Ig) E on mast cells, along with the antigen receptors on T and B cells and Fc receptors for IgG, belongs to a class of receptors which lack intrinsic kinase activity, but activate non-receptor tyrosine and serine/threonine kinases. Receptor engagement triggers a chain of signaling events leading from protein phosphorylation to activation of phosphatidylinositol-specific phospholipase C, an increase in intracellular calcium levels, and ultimately the activation of more specialized functions. IgE receptor disengagement leads to reversal of phosphorylation by undefined phosphatases and to inhibition of activation pathways. Here we show that phenylarsine oxide, a chemical which reacts with thiol groups and has been reported to inhibit tyrosine phosphatases, uncouples the IgE receptor-mediated phosphorylation signal from activation of phosphatidyl inositol metabolism, the increase in intracellular calcium levels, and serotonin release. Phenylarsine oxide inhibits neither the kinases (tyrosine and serine/threonine) phosphorylating the receptor and various cellular substrates nor, unexpectedly, the phosphatases responsible for the dephosphorylation following receptor disengagement. By contrast, it abolishes the receptor-mediated phosphorylation of phospholipase C-gamma 1, but not phospholipase C activity in vitro. Therefore the phosphorylation and activation of phospholipase C likely requires a phenylarsine oxide-sensitive element. Receptor aggregation thus activates at least two distinct phosphorylation pathways: a phenylarsine oxide-insensitive pathway leading to phosphorylation/dephosphorylation of the receptor and of various substrates and a sensitive pathway leading to phospholipase C-gamma 1 phosphorylation.

N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging.

PubMed

Gourni, Eleni; Mansi, Rosalba; Jamous, Mazen; Waser, Beatrice; Smerling, Christiane; Burian, Antje; Buchegger, Franz; Reubi, Jean Claude; Maecke, Helmut R

2014-10-01

Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes.

PubMed

Argyris, Jason; Dahal, Peetambar; Hayashi, Eiji; Still, David W; Bradford, Kent J

2008-10-01

Lettuce (Lactuca sativa 'Salinas') seeds fail to germinate when imbibed at temperatures above 25 degrees C to 30 degrees C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37 degrees C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.

A new fluorescent probe for distinguishing Zn2+ and Cd2+ with high sensitivity and selectivity.

PubMed

Tan, Yiqun; Gao, Junkuo; Yu, Jiancan; Wang, Ziqi; Cui, Yuanjing; Yang, Yu; Qian, Guodong

2013-08-28

A new fluorescence probe for distinguishing Zn(2+) and Cd(2+) is designed and synthesized. For the first time to our knowledge, this probe can recognize similar metal ions by coherently utilizing intramolecular charge transfer (ICT) and different electronic affinities of various metal ions, instead of by selective coordination alone, which may be interfered with and lose its selectivity easily in a complicated environment, providing a distinct recognition even by the naked eye for Zn(2+) and Cd(2+) with the sensitivity at the ppb level. This design strategy may initiate a straightforward approach for the selective detection of various metal ions with similar chemical properties in extensive applications such as environmental, industrial, and bio-science.

Imbibition period as the critical temperature sensitive stage in germination of lima bean seeds.

PubMed

Pollock, B M; Toole, V K

1966-02-01

Lima bean seeds (Phaseolus lunatus L.) and excised embryonic axes can be injured during imbibition at temperatures below 25 degrees . The early imbibitional stage is critical; imbibition at 25 degrees followed by low temperature exposure does not cause injury. Sensitivity to chilling injury is conditioned by the pre-harvest seed history. Low vigor (bleached) seeds are most sensitive to injury, the effects of which can be intensified by restricted oxygen supply during early axis growth. The seed coat, by preventing water uptake, can permit the seed to avoid injury. This protective mechanism is most effective at low temperature and high moisture stress. Immediately following low temperature imbibition, injured axes lose organic materials, probably nucleotides. This organic leachate is a potential influence on soil microorganisms and, together with the temperature sensitivity, vigor, and seed coat effect undoubtedly is important in controlling the potential variability in germination shown by a seed population.

Imbibition Period as the Critical Temperature Sensitive Stage in Germination of Lima Bean Seeds

PubMed Central

Pollock, B. M.; Toole, Vivian K.

1966-01-01

Lima bean seeds (Phaseolus lunatus L.) and excised embryonic axes can be injured during imbibition at temperatures below 25°. The early imbibitional stage is critical; imbibition at 25° followed by low temperature exposure does not cause injury. Sensitivity to chilling injury is conditioned by the pre-harvest seed history. Low vigor (bleached) seeds are most sensitive to injury, the effects of which can be intensified by restricted oxygen supply during early axis growth. The seed coat, by preventing water uptake, can permit the seed to avoid injury. This protective mechanism is most effective at low temperature and high moisture stress. Immediately following low temperature imbibition, injured axes lose organic materials, probably nucleotides. This organic leachate is a potential influence on soil microorganisms and, together with the temperature sensitivity, vigor, and seed coat effect undoubtedly is important in controlling the potential variability in germination shown by a seed population. Images PMID:16656243

Calcium Sensitive Fluorescent Dyes Fluo-4 and Fura Red under Pressure: Behaviour of Fluorescence and Buffer Properties under Hydrostatic Pressures up to 200 MPa

PubMed Central

Vass, H.; Reischl, B.; Allen, R. J.; Friedrich, O.

2016-01-01

The fluorescent Ca2+ sensitive dyes Fura Red (ratiometric) and Fluo-4 (non-ratiometric) are widely utilized for the optical assessment of Ca2+ fluctuations in vitro as well as in situ. The fluorescent behavior of these dyes is strongly depends on temperature, pH, ionic strength and pressure. It is crucial to understand the response of these dyes to pressure when applying calcium imaging technologies in the field of high pressure bioscience. Therefore, we use an optically accessible pressure vessel to pressurize physiological Ca2+-buffered solutions at different fixed concentrations of free Ca2+ (1 nM to 25.6 μM) and a specified dye concentration (12 μM) to pressures of 200 MPa, and record dye fluorescence intensity. Our results show that Fluo-4 fluorescence intensity is reduced by 31% per 100 MPa, the intensity of Fura Red is reduced by 10% per 100 MPa. The mean reaction volume for the dissociation of calcium from the dye molecules Δdv¯ is determined to -17.8 ml mol-1 for Fluo-4 and -21.3 ml mol-1 for Fura Red. Additionally, a model is presented that is used to correct for pressure-dependent changes in pH and binding affinity of Ca2+ to EGTA, as well as to determine the influence of these changes on dye fluorescence. PMID:27764134

The role of CH/π interactions in the high affinity binding of streptavidin and biotin.

PubMed

Ozawa, Motoyasu; Ozawa, Tomonaga; Nishio, Motohiro; Ueda, Kazuyoshi

2017-08-01

The streptavidin-biotin complex has an extraordinarily high affinity (Ka: 10 15 mol -1 ) and contains one of the strongest non-covalent interactions known. This strong interaction is widely used in biological tools, including for affinity tags, detection, and immobilization of proteins. Although hydrogen bond networks and hydrophobic interactions have been proposed to explain this high affinity, the reasons for it remain poorly understood. Inspired by the deceased affinity of biotin observed for point mutations of streptavidin at tryptophan residues, we hypothesized that a CH/π interaction may also contribute to the strong interaction between streptavidin and biotin. CH/π interactions were explored and analyzed at the biotin-binding site and at the interface of the subunits by the fragment molecular orbital method (FMO) and extended applications: PIEDA and FMO4. The results show that CH/π interactions are involved in the high affinity for biotin at the binding site of streptavidin. We further suggest that the involvement of CH/π interactions at the subunit interfaces and an extended CH/π network play more critical roles in determining the high affinity, rather than involvement at the binding site. Copyright © 2017 Elsevier Inc. All rights reserved.

Amine-functionalized PVA-co-PE nanofibrous membrane as affinity membrane with high adsorption capacity for bilirubin.

PubMed

Wang, Wenwen; Zhang, Hao; Zhang, Zhifeng; Luo, Mengying; Wang, Yuedan; Liu, Qiongzhen; Chen, Yuanli; Li, Mufang; Wang, Dong

2017-02-01

In this study, poly(vinyl alcohol-co-ethylene) (PVA-co-PE) nanofibrous membrane was activated by sodium hydroxide and cyanuric chloride, and then the activated membranes were functionalized by 1,3-propanediamine, hexamethylenediamine and diethylenetriamine to be affinity membranes for bilirubin removal, respectively. The chemical structures and morphologies of membranes were investigated by SEM, FTIR and XPS. And the adsorption ability of different amine-functionalized nanofibrous membranes for bilirubin was characterized. Furthermore, the effects of temperature, initial concentration of bilirubin, NaCl concentration and BSA concentration on the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane were studied. Results indicated that the adsorption capacity for bilirubin of diethylenetriamine-functionalized nanofibrous membrane could reach 85mg/g membrane when the initial bilirubin concentration was 200mg/L while the adsorption capacity could be increased to 110mg/g membrane if the initial bilirubin concentration was more than 400mg/L. The dynamic adsorption of diethylenetriamine-functionalized nanofibrous membrane showed that the ligands of amine groups on the membrane surface could be used as far as possible by recirculating the plasma with certain flow rates. Therefore, the diethylenetriamine-functionalized PVA-co-PE nanofibrous membrane possessed high adsorption capacity for bilirubin and it can be candidate as affinity membrane for bilirubin removal. Copyright © 2016. Published by Elsevier B.V.

Self-Limiting Oxides on WSe2 as Controlled Surface Acceptors and Low-Resistance Hole Contacts.

PubMed

Yamamoto, Mahito; Nakaharai, Shu; Ueno, Keiji; Tsukagoshi, Kazuhito

2016-04-13

Transition metal oxides show much promise as effective p-type contacts and dopants in electronics based on transition metal dichalcogenides. Here we report that atomically thin films of under-stoichiometric tungsten oxides (WOx with x < 3) grown on tungsten diselenide (WSe2) can be used as both controlled charge transfer dopants and low-barrier contacts for p-type WSe2 transistors. Exposure of atomically thin WSe2 transistors to ozone (O3) at 100 °C results in self-limiting oxidation of the WSe2 surfaces to conducting WOx films. WOx-covered WSe2 is highly hole-doped due to surface electron transfer from the underlying WSe2 to the high electron affinity WOx. The dopant concentration can be reduced by suppressing the electron affinity of WOx by air exposure, but exposure to O3 at room temperature leads to the recovery of the electron affinity. Hence, surface transfer doping with WOx is virtually controllable. Transistors based on WSe2 covered with WOx show only p-type conductions with orders of magnitude better on-current, on/off current ratio, and carrier mobility than without WOx, suggesting that the surface WOx serves as a p-type contact with a low hole Schottky barrier. Our findings point to a simple and effective strategy for creating p-type devices based on two-dimensional transition metal dichalcogenides with controlled dopant concentrations.

S-Wave Velocity Models Under the Saudi Arabian Portable Broadband Deployment: Evidence for Lithospheric Erosion Beneath the Arabian Shield

NASA Astrophysics Data System (ADS)

Julià, J.; Ammon, C. J.; Herrmann, R. B.

2002-12-01

Models of crustal evolution strongly rely on our knowledge on the mineralogical composition of subsurface rocks, as well as pressure and temperature conditions. Direct sampling of subsurface rocks is often not possible, so that constraints have to be placed from indirect estimates of rock properties. Detailed seismic imaging of subsurface rocks has the potential for providing such constraints, and probe the extent at depth of surface geologic observations. In this study, we provide detailed S-wave velocity profiles for the crust and uppermost mantle beneath the Saudi Arabian Portable Broadband Deployment stations. Seismic velocities have been estimated from the joint inversion of receiver functions and fundamental mode group velocities. Receiver functions are sensitive to S-wave velocity contrasts and vertical travel times, and surface-wave dispersion is sensitive to vertical S-wave velocity averages, so that their combination bridge resolution gaps associated with each individual data set. Our resulting models correlate well with surface geology observations in the Arabian Shield and characterize its terranes at depth: the Asir terrane consists of a 10-km thick upper crust of 3.3~km/s overlying a lower crust with shear-wave velocities of 3.7-3.8 km/s; the Afif terrane is made of a 20-km thick upper crust with average velocity of 3.6 km/s and a lower crust with a shear-velocity of about 3.8~km/s; the Nabitah mobile belt has a gradational, 15-km thick upper crust up to 3.6 km/s overlying a gradational lower crust with velocities up to 4.0 km/s. The crust-mantle transition is sharper in terranes of continental affinity and more gradational beneath terranes of oceanic affinity. In the uppermost mantle, our models suggest a thin lid between up to 50-60 km depth overlying a low velocity zone beneath station TAIF, located close to a region of upwelling mantle material. Temperatures in the lid are estimated to be about 1000 C, which are in good agreement with independent xenolith data, and suggest that the lithosphere could be eroded to a thickness as little as 50~km under this station.

A versatile near-infrared asymmetric tricarbocyanine for zinc ion sensing in water.

PubMed

Menéndez, Guillermo O; López, Cecilia Samaniego; Jares-Erijman, Elizabeth A; Spagnuolo, Carla C

2013-01-01

We have synthesized a near-infrared emissive asymmetric tricarbocyanine conveniently functionalized to improve bioconjugation. The leading structure contains a versatile derivatization point at the meso position for facile radical-nucleophilic aromatic substitution. We have evaluated a DPEN (N,N-di(2-picolyl)ethylendiamine) derivative of this dye as a highly selective sensor for zinc (II) in aqueous medium, which performs in an appropriate sensitivity range for biological studies. The probe was successfully conjugated to a protein-ligand model with high affinity and specificity (biotin-streptavidin technology) rendering an excellent performance of sensing. In a general strategy to obtain sensitive probes combining fluorescent nanoparticles and molecular fluorophores, a preliminary design of a supramolecular assembly derived from the conjugation of the molecular sensor to quantum dots (QDs) was also investigated. The advantages and problems of FRET-based sensors are also discussed. © 2013 The American Society of Photobiology.