Modified Hill Equation Research Articles

Selective discrimination of VOCs gases at ppb-level using MOS gas sensor in temperature-pulsed operation mode with modified Hill equation

Due to the complicated gas-sensing process on the surface of metal oxide semiconductors (MOS), it is hard to accurately characterize the transient changes of MOS gas sensor. This paper introduces a modified Hill equation to quantitatively analyze the instantaneous resistance of MOS gas sensors operating in temperature-pulsed operation mode, to extract the gas-sensing dynamic features and key parameters for the selective identification of various volatile organic compounds (VOCs) gases (such as ethanol, formaldehyde, toluene and acetone) by machine learning algorithms. The sensors were tested with the pulse-mode at different temperatures for multiple gases. The characteristic features of the sensing process during the pulse-on period were investigated. Key parameters were extracted via the modified Hill equation, which have shown obvious dependence on the gas type and concentration. Excellent selectivity for the VOC gases was attained at low concentrations of 500 ppb thanks to the equation's further integration with the principal component analysis (PCA) approach. The incorporation of this modified Hill equation in temperature-pulsed operation mode provides the vital features for the possible applications of MOS gas sensors with the emerging machine learning algorithm.

A proposal to model the pasting curve of gluten‐free bakery formulations

AbstractThis research aimed to propose a comprehensive mathematical model that allows simulation of the viscosity profile achieved during the pasting analysis of gluten‐free bakery product formulations. The model divides the pasting curve into three parts, utilizing the modified Hill equation for gelatinization, the Gompertz equation for breakdown and retrogradation, and introduces novel parameters (Krheo1, Krheo2, Krheo3) to quantify the areas under each curve section. Validation involved viscoamylographic profiles of gluten‐free muffins with potato starch and Andean grain flours, with and without adding xanthan gum (XG). Results indicate XG increases resistance to gelatinization and decreases swelling rate in potato starch, while Andean grain flours with XG increase swelling rate. Gompertz equation parameters reveal improved heat stability and reduced retrogradation with Andean grain flours and XG. The proposed model serves as a versatile tool for interpreting ingredient effects, aiding in the optimization of starch‐containing food formulations.Practical applicationsThe viscosity profile obtained in Rapid Visco Analyzer (RVA) test is of utmost importance for the development of gluten‐free bakery products because it allows us to understand the viscosity behavior of the system during the baking process, which is essential to achieve the proper consistency in the dough. The absence of gluten in gluten‐free formulations can make the dough less stable during baking; therefore, the mathematical modeling of the viscosity profile obtained during the pasting analysis helps to understand how the ingredients affect the system's stability, providing information about the gelatinization, breakdown, and retrogradation rates of starches. It also aids in understanding the consistency of the system at the end of the analysis, enabling adjustments to formulations to prevent dough stability issues during the baking process.

Fitting Parameters of a Modified Hill’s Equation and Their Influence on the Shape of the Model Hemoglobin Oxygenation Curve

Oxygen binds to hemoglobin cooperatively, and a correct description of this binding is relevant not only for understanding the mechanisms of involved molecular processes but also for clinical purposes. Hill’s equation, derived over a hundred years ago, is still the simplest and most efficient way to perform this description. However, in terms of accuracy, it is inferior to Adair´s equation, which contains more parameters. We proposed to use a modified Hill equation and showed that it allows a more accurate description of the binding of oxygen to hemoglobin than Adair’s equation. At the same time, unlike Adair’s equation, our model retains the physical meaning given to the original Hill equation. We considered and analyzed the influence of the equation parameters on the course of the oxygenation curve and presented the relationship between the fitting parameters and other parameters derived from them in the form of a diagram-graph, which, in our opinion, simplifies the perception of these estimates and can be useful in solving a number of problems for which the traditional way of analyzing the degree of cooperative interaction was via the Hill equation. We suggest that the newly proposed parameter hmax introduced in our model should be regarded as crucial for a better description of the oxygenation curve.

Abstract 12713: Increased Calcium Sensitivity in Hypoplastic Left Heart Syndrome Hearts With Systolic Failure

Introduction: Systolic failure of the systemic right ventricle (RV) in children with hypoplastic left heart syndrome (HLHS) is a significant cause of death and a growing indication for heart transplantation. However, the underlying etiology of RV systolic failure in this population remains ill-defined. Hypothesis: We hypothesize that cardiomyocyte autonomous defects in contractility, characterized by increased calcium sensitivity, may contribute to systolic failure in HLHS. Methods: Explanted RV tissue was collected and flash frozen at the time of heart transplant in pediatric subjects with HLHS. HLHS subjects were classified by their primary indication for transplant: HLHS subjects with normal systolic function (HLHS-NF, N=2) were transplanted secondary to refractory protein losing enteropathy, and HLHS-F subjects (N=9) had end-stage heart failure due to decreased systolic function. Cardiomyocytes were isolated from frozen RV tissue, permeabilized, and glued to a force transducer and length controller. Force generated by the cardiomyocyte at different calcium concentrations was measured and force-calcium dose response curves were generated (modified Hill equation). pCa50 denotes the negative log of calcium concentration required to generate 50% maximal tension. Results: Less calcium is required to generate 50% maximal tension in HLHS-F cardiomyocytes (n=33 cardiomyocytes) when compared to HLHS-NF (n=5 cardiomyocytes), demonstrated by a higher pCa50 (5.62 vs. 5.46, p=0.027). Maximal force generation is similar between HLHS-NF and HLHS-F cardiomyocytes (55.5 mN/mm2 vs. 54.8 mN/mm2, p=0.96). Conclusions: Cardiomyocytes isolated from HLHS-F demonstrate increased calcium sensitivity when compared to those from HLHS-NF. Although future studies are needed, the presence of intrinsic alterations in sarcomeric properties in HLHS-F supports the hypothesis that cardiomyocyte autonomous defects may contribute to systolic failure in HLHS.

Blocking SARS-CoV-2 Delta Variant (B.1.617.2) Spike Protein Receptor-Binding Domain Binding with the ACE2 Receptor of the Host Cell and Inhibiting Virus Infections Using Human Host Defense Peptide-Conjugated Graphene Quantum Dots.

The emergence of double mutation delta (B.1.617.2) variants has dropped vaccine effectiveness against SARS-CoV-2 infection. Although COVID-19 is responsible for more than 5.4 M deaths till now, more than 40% of infected individuals are asymptomatic carriers as the immune system of the human body can control the SARS-CoV-2 infection. Herein, we report for the first time that human host defense neutrophil α-defensin HNP1 and human cathelicidin LL-37 peptide-conjugated graphene quantum dots (GQDs) have the capability to prevent the delta variant virus entry into the host cells via blocking SARS-CoV-2 delta variant (B.1.617.2) spike protein receptor-binding domain (RBD) binding with host cells’ angiotensin converting enzyme 2 (ACE2). Experimental data shows that due to the binding between the delta variant spike protein RBD and bioconjugate GQDs, in the presence of the delta variant spike protein, the fluorescence signal from GQDs quenched abruptly. Experimental quenching data shows a nonlinear Stern–Volmer quenching profile, which indicates multiple binding sites. Using the modified Hill equation, we have determined n = 2.6 and the effective binding affinity 9 nM, which is comparable with the ACE2–spike protein binding affinity (8 nM). Using the alpha, beta, and gamma variant spike-RBD, experimental data shows that the binding affinity for the delta B.1.617.2 variant is higher than those for the other variants. Further investigation using the HEK293T-human ACE2 cell line indicates that peptide-conjugated GQDs have the capability for completely inhibiting the entry of delta variant SARS-CoV-2 pseudovirions into host cells via blocking the ACE2–spike protein binding. Experimental data shows that the inhibition efficiency for LL-37 peptide- and HNP1 peptide-attached GQDs are much higher than that of only one type of peptide-attached GQDs.

Bent DNA Bows as Sensing Amplifiers for Detecting DNA-Interacting Salts and Molecules.

Due to the central role of DNA, its interactions with inorganic salts and small organic molecules are important. For example, such interactions play important roles in various fundamental cellular processes in living systems and are involved in many DNA-damage related diseases. Strategies to improve the sensitivity of existing techniques for studying DNA interactions with other molecules would be appreciated in situations where the interactions are too weak. Here we report our development and demonstration of bent DNA bows for amplifying, sensing, and detecting the interactions of 14 inorganic salts and small organic molecules with DNA. With the bent DNA bows, these interactions were easily visualized and quantified in gel electrophoresis, which were difficult to measure without bending. In addition, the strength of the interactions of DNA with the various salts/molecules were quantified using the modified Hill equation. This work highlights the amplification effects of the bending elastic energy stored in the DNA bows and the potential use of the DNA bows for quantitatively measuring DNA interactions with small molecules as simple economic methods; it may also pave the way for exploiting the bent DNA bows for other applications such as screening DNA-interacting molecules and drugs.

Rheological studies on nanocrystalline cellulose/alginate suspensions

Rheological behavior of mixtures containing nanocrystalline cellulose (CNC) and alginate was studied. A shear-thinning behavior was observed in all samples, which was progressively enhanced as a function of CNC and alginate concentrations. Electrostatic interactions between anionic alginate chains and anionic CNC rods promote physical gelation at small CNC concentrations (4wt%), resulting in soft gels with pronounced shear-thinning and solid-like behavior. Next, hydrogels were obtained using the internal gelation method, and the crosslinking kinetics were monitored by time sweep tests. The experimental data were well described by a modified Hill equation. Fourier transform infrared spectroscopy (FTIR) was performed in order to analyze the hydrogen bonding interactions that occur between CNC rods and alginate chains, and the alginate ionic crosslinking.

Abstract A063: Identification of an integrated stress response-inducing agent (NH125) that modulates DR5-dependent TRAIL synergy and ATF3-driven cell death in gliomaspheres

Abstract Purpose: The high incidence of glioblastoma recurrence necessitates additional therapeutic strategies to improve outcomes. One potential strategy targets treatment resistant cells via the integrated stress response (ISR) pathway as a means of sensitizing these cells to TNF-related apoptosis-inducing ligand (TRAIL). Methods: We generated a novel cell-based death receptor assay and used this assay to screen compounds from three commercially available libraries (total of 223 compounds) at 2 or 10 μM (Z prime = 0.64). A single hit was identified that was further characterized by immunoblotting and immunofluorescence. A compound dilution series was applied to a panel of cell lines including patient-derived glioma stem cells (GSC). IC50 values were calculated by fitting normalized viability data to a modified Hill Equation. TRAIL synergy was assessed using the combination index method following treatment with compound and TRAIL. Knockout of C/EBP Homologous Protein (CHOP) was used to determine the role of integrated stress in TRAIL synergy. GSC were also treated with temozolomide followed by assessment of cell viability. RNA-seq analysis was performed on compound treated GSC, U251, and Normal Human Astrocytes. Short hairpin RNAs were used to establish the role of ATF3 in compound-treated GSC. Results: Using a novel cell-based death receptor we identified 1-Benzyl-3-cetyl-2-methylimidazolium iodide (NH125) as a potent inducer of DR5 expression. NH125 signals through the EIF2α-ATF4-CHOP axis, culminating in DR5 expression at low micromolar doses. CHOP is required for NH125-mediated TRAIL synergy. Compared to other cancer cell lines, GSC demonstrate an increased sensitivity to NH125 treatment, and synergize with TRAIL at lower doses than U251. NH125 produced a significant decrease in GSC viability when compared to temozolomide treatment (p < 0.05). This enhanced sensitivity of GSC is partially due to increased expression of ATF3 following NH125 treatment. Conclusions: NH125 generates an integrated stress response leading to CHOP-mediated TRAIL synergy. Patient-derived glioma stem cells demonstrate increased sensitivity to NH125 treatment that is partially driven by increased ATF3 expression. Engaging the ISR to enhance the efficacy of TRAIL represents a promising strategy to target the treatment refractory GSC that have been implicated in glioblastoma recurrence. IC50 valuesCell LineIC50 (μM)Adj R2T4213-GSC0.89 ± 0.110.99NS039-GSC1.95 ± 0.350.99HK2962.51 ± 0.410.97U2513.30 ± 0.160.99Ovcar34.68 ± 0.340.97A5496.06 ± 0.230.99GL2616.20 ± 0.400.97U87-MG7.15 ± 0.160.98H129911.02 ± 0.430.99 Citation Format: Saad Sheikh, Xiaobing Tian, Deeksha Saxena, Xiangsheng Xu, Gary Kao, Jay F. Dorsey. Identification of an integrated stress response-inducing agent (NH125) that modulates DR5-dependent TRAIL synergy and ATF3-driven cell death in gliomaspheres [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A063.

Unconventional Coupling between Ligand Recognition and Allosteric Control in the Multidrug Resistance Gene Regulator, BmrR.

BmrR is a multidrug resistance (MDR) regulator that responds to diverse ligands. To obtain insight into signal recognition, allosteric control, and cooperativity, we used a quantitative in vitro transcription assay to determine the ligand-dependent activation profiles for a diverse set of cations, zwitterions, and uncharged ligands. As for many other biological switch systems, the data are well described by a modified Hill equation. Parameters extracted from curve fits to the data include L50 , RMAX and N. We found that L50 values correlate directly with ΔGBIND values, suggesting that the parameter reflects binding, whereas RMAX and N reflect allosteric control and cooperativity, respectively. Our results suggest unconventional coupling between ligand binding and allosteric control, with weakly interacting ligands exhibiting the highest levels of activation. Such properties are in stark contrast to those often exhibited by biological switch proteins, whereby ligand binding and allostery are tightly coupled, yielding both high selectivity and ultrasensitivity. We propose that weakened coupling, as observed for BmrR, may be important for providing robust activation responses to unrelated ligands. We also propose that other MDR proteins and other polyspecific switch systems will show similar features.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

β-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca(2+)] relationships were determined at two sarcomere lengths (SL = 1.9 μm and SL = 2.3 μm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca(2+)] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67 and ~33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.

Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP.

Systematic classification of tablet disintegrants by water uptake and force development kinetics.

Water uptake and force development of disintegrating tablets provide a high degree of information about the disintegration mechanisms and process itself. An apparatus for the simultaneous measurement of water uptake and force development of tablets is presented, and the gathered data are analysed. Flat faced, 10 mm, dibasic calcium phosphate tablets containing 2% disintegrant are investigated with the newly constructed apparatus. The force is determined with a texture analyser, whereas the water uptake is recorded by a balance. Different measurement regimes are compared with each other. Measured curves are analysed according to their shape and fitted with a modified Hill equation. Known disintegration mechanisms can be confirmed with the newly constructed apparatus - swelling for sodium starch glycolate and croscarmellose sodium, and shape recovery for crospovidone disintegrants. Different brands of polacrilin potassium act by different mechanisms. All curves could be fitted successfully with a modified Hill equation. A novel classification to facilitate the appropriate disintegrant selection is presented on basis of the fit parameters. The new apparatus allows the acquisition of water uptake and force data simultaneously. Parameters calculated with the modified Hill equation provide a simple way to classify disintegrants according to their behaviour.

Methylation by DNMT1 is more Efficient in Chronic Lymphocytic Lymphoma Cells than in Normal Cells

Binding of activators and repressors plays a significant role in transcriptional regulation of gene expression. The systems biology approach has been applied to predict and determine the efficiency of this process; however, most of the work is done in prokaryotic systems. Epigenetic regulation provides another dimension of transcriptional regulation, which does not involve changes in the coding region of genes. Methylation of specific CpG residues in the CpG islands of DNA by the enzyme DNA methyl transferase 1 (DNMT1) causes gene silencing. In cancer cells, the enzyme DNMT1 is highly expressed, and the methylation level at specific sites of genes, including tumor suppressor genes, is elevated. This results in the silencing of tumor suppressor genes for carcinogenesis. However, it is not known whether the increase in DNMT1 expression is proportional to the increase in the level of methylation. This paper developed a systems biology approach to determine whether DNMT1 acts more efficiently in cancer cells than in normal cells. The cooperativity of DNMT1 binding to eight selected genes was determined in chronic lymphocytic leukemia (CLL) cells using a modified Hill equation and compared these values with those obtained from normal lymphocytes and granulocytes. The cooperativity of DNMT1 was found to be positive in four cancer-associated genes, whereas their values were negative in both normal lymphocytes and granulocytes. These results show for the first time how to apply systems biology in the analysis of transcriptional gene regulation. This approach will be beneficial in determining the efficiency of genome-wide methylation in development and in diverse types of diseases.

Phosphorylation of Cardiac Myosin Binding Protein-C Acts as On/Off Switch to Modulate cMyBP-C's Inhibition of Actomyosin Power Generation in the Load-Clamp Laser Trap Assay

Cardiac myosin binding protein-C (cMyBP-C), a sarcomeric protein with 11 domains- C0-C10, binds myosin LMM via its C-terminus, while its N-terminus binds both myosin S2 and actin. These N-terminal interactions can be attenuated by phosphorylation of serines within the C1-C2 linker. In vivo, cMyBP-C exists in a range of phosphorylation states, which may alter its regulation of actomyosin. Therefore, N-terminal fragments (C0-C3) of cMyBP-C were bacterially expressed with one or more phosphorylatable serines (S273, S282, S302) mutated to either alanines to mimic dephosphorylation or aspartic acids as phosphomimetics. Using C0-C3 fragments that were effectively mono-, di- or tri-phosphorylated, we characterized how the extent of cMyBP-C phosphorylation modulated the the force:velocity relationship generated by a small ensemble (∼8 motors) of skeletal muscle myosin in the load-clamped laser trap assay. Mono-phosphorylated C0-C3 inhibited unloaded velocity by 70% as did unphosphorylated C0-C3, while di- and tri-phosphorylated constructs were indistinguishable and less inhibitory (30%). Maximal force was unaffected by the extent of phosphorylation. Thus, the un- and mono-phosphorylated C0-C3 constructs depressed the force-velocity relationship, which was well fitted by a modified Hill equation containing an assumed viscous load due to C0-C3 binding to actin (Weith et al., 2011). In addition, both un- and mono- phosphorylated C0-C3 reduced peak power by ∼40%, while neither di- nor tri-phosphorylated C0-C3 had any effect on peak power. cMyBP-C phosphorylation appears to modulate cMyBP-C's inhibition of actomyosin power generation in an “on/off” manner.

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1

The zebrafish, Danio rerio, is emerging as an important model organism for the pathophysiological study of some human kidney diseases, but the sites of expression and physiological roles of a number of protein orthologues in the zebrafish nephron remain mostly undefined. Here we show that a zebrafish potassium channel is orthologous to the mammalian kidney potassium channel, ROMK. The cDNA (kcnj1) encodes a protein (Kcnj1) that when expressed in Xenopus laevis oocytes displayed pH- and Ba2+-sensitive K+-selective currents, but unlike the mammalian channel, was completely insensitive to the peptide inhibitor tertiapin-Q. In the pronephros, kcnj1 transcript expression was restricted to a distal region and overlapped with that of sodium–chloride cotransporter Nkcc, chloride channel ClC-Ka, and ClC-Ka/b accessory subunit Barttin, indicating the location of the diluting segment. In a subpopulation of surface cells, kcnj1 was coexpressed with the a1a.4 isoform of the Na+/K+-ATPase, identifying these cells as potential K+ secretory cells in this epithelium. At later stages of development, kcnj1 appeared in cells of the developing gill that also expressed the a1a.4 subunit.Morpholino antisense-mediated knockdown of kcnj1 was accompanied by transient tachycardia followed by bradycardia, effects consistent with alterations in extracellular K+ concentration in the embryo.Our findings indicate that Kcnj1 is expressed in cells associated with osmoregulation and acts as a K+ efflux pathway that is important in maintaining extracellular levels of K+ in the developing embryo.

Constitutive Activation of the N-Methyl-d-aspartate Receptor via Cleft-spanning Disulfide Bonds

Although the N-methyl-D-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure.

Molecular Basis for Zinc Potentiation at Strychnine-sensitive Glycine Receptors

The divalent cation Zn(2+) is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn(2+). Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn(2+) potentiation site on GlyRs and account for the differential sensitivity of GlyR alpha(1) and GlyR alpha(2) to Zn(2+) potentiation. In addition, juxtaposed to this Zn(2+) site, which is located externally on the N-terminal domain of the alpha subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn(2+) potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn(2+) binds to a site on the extracellular outer face of the GlyR alpha subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.

A TASK‐like pH‐ and amine‐sensitive ‘leak’ K+ conductance regulates neonatal rat facial motoneuron excitability in vitro

A 'leak' potassium (K+) conductance (gK(Leak)) modulated by amine neurotransmitters is a major determinant of neonatal rat facial motoneuron excitability. Although the molecular identity of gK(Leak) is unknown, TASK-1 and TASK-3 channel mRNA is found in facial motoneurons. External pH, across the physiological range (pH 6-8), and noradrenaline (NA) modulated a conductance that displayed a relatively linear current/voltage relationship and reversed at the K+ equilibrium potential, consistent with inhibition of gK(Leak). The pH-sensitive current (I(pH)), was maximal around pH 8, fully inhibited near pH 6 and was described by a modified Hill equation with a pK of 7.1. The NA-induced current (I(NA)) was occluded at pH 6 and enhanced at pH 7.7. The TASK-1 selective inhibitor anandamide (10 microM), its stable analogue methanandamide (10 microM), the TASK-3 selective inhibitor ruthenium red (10 microM) and Zn2+ (100-300 microM) all failed to alter facial motoneuron membrane current or block I(NA) or I(pH). Isoflurane, a volatile anaesthetic that enhances heteromeric TASK-1/TASK-3 currents, increased gK(Leak). Ba2+, Cs+ and Rb+ blocked I(NA) and I(pH) voltage-dependently with maximal block at hyperpolarized potentials. 4-Aminopyridine (4-AP, 4 mM) voltage-independently blocked I(NA) and I(pH). In summary, gK(Leak) displays some of the properties of a TASK-like conductance. The linearity of gK(Leak) and an independence of activation on external [K+] suggests against pH-sensitive inwardly rectifying K+ channels. Our results argue against principal contributions to gK(Leak) by homomeric TASK-1 or TASK-3 channels, while the potentiation by isoflurane supports a predominant role for heterodimeric TASK-1/TASK-3 channels.

Length-dependent activation in three striated muscle types of the rat.

The process whereby sarcomere length modulates the sensitivity of the myofilaments to Ca(2+) is termed length-dependent activation. Length-dependent activation is a property of all striated muscles, yet the relative extent of length-dependent activation between skeletal muscle and cardiac muscle is unclear. Although length-dependent activation may be greater in fast skeletal muscle (FSM) than in slow skeletal muscle (SSM), there has not been a well controlled comparison of length-dependent activation between skeletal muscle and cardiac muscle (CM). Accordingly, we measured sarcomere length-dependent properties in skinned soleus (SSM), psoas (FSM) and ventricular trabeculae (CM) of the rat under carefully controlled conditions. The free Ca(2+)-force relationship was determined at sarcomere lengths (SL) of 1.95 microm, 2.10 microm and 2.25 microm and fitted to a modified Hill equation. FSM and SSM were more sensitive to Ca(2+) than CM. Length-dependent activation was ordered as CM > FSM > SSM. Cooperativity as measured by the Hill coefficient of the Ca(2+)-force relationship was not significantly different between CM and FSM, both of which exhibited greater cooperativity than SSM. SL did not significantly alter this parameter in each muscle type. To establish whether the observed differences can be explained by alterations in interfilament spacing, we measured myofilament lattice spacing (LS) by synchrotron X-ray diffraction in relaxed, skinned muscle preparations. LS was inversely proportional to SL for each muscle type. The slope of the SL-LS relationship, however, was not significantly different between striated muscle types. We conclude that (1) length-dependent activation differs among the three types of striated muscle and (2) these differences in the length-dependent properties among the striated muscle types may not solely be explained by the differences in the response of interfilament spacing to changes in muscle length in relaxed, skinned isolated muscle preparations.

The invention and development of blood gas analysis apparatus.

In 1953, the doctor draft interrupted Dr. Severinghaus' anesthesia and physiology training and sent him to the National Institutes of Health as director of anesthesia research at the newly opened Clinical Center. He developed precise laboratory partial pressure of carbon dioxide (PCO(2)) and pH analysis to investigate lung blood gas exchange during hypothermia. Constants for carbon dioxide solubility and pK' were more accurately determined. In August 1954, he heard Richard Stow describe invention of a carbon dioxide electrode and immediately built one, improved its stability, and tested its response characteristics. In April 1956, he also heard Leland Clark reveal his invention of an oxygen electrode. Dr. Severinghaus obtained one and constructed a stirred cuvette in which blood partial pressure of oxygen (PO(2)) could be accurately measured. Technician Bradley and Dr. Severinghaus combined these, making the first blood gas analysis system in 1957 and 1958, and shortly thereafter, they added a pH electrode. Blood gas analyzers rapidly developed commercially. Dr. Severinghaus collaborated with Astrup and other Danes on the Haldane and Bohr effects and their concepts of base excess during two sabbaticals in Copenhagen. Work with both Astrup and Roughton on the oxygen dissociation curve led Dr. Severinghaus to devise a modified Hill equation that closely fit their new, better human oxygen dissociation curve and a blood gas slide rule that solved oxygen dissociation curve, PCO(2), pH, and acid-base questions. Blood gas analysis revolutionized both clinical medicine and cardiorespiratory and metabolic physiology.