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PubMed1983

High-affinity binding of agonists to beta-adrenergic receptors on intact cells.

The interactions of agonists and antagonists with beta-adrenergic receptors on intact 1321N1 human astrocytoma and C62B rat glioma cells were studied by using the radioligand (-)-[125I]iodopindolol. Competition binding assays were performed at 37 degrees C under equilibrium conditions and in short-time nonequilibrium assays that approximated initial velocity conditions for binding of the radioligand. The theoretical basis and experimental validation of the initial velocity approach for determining binding affinities of rapidly equilibrating ligands are presented. For the agonists isoproterenol and epinephrine, high binding affinities that approximated their apparent affinities for binding in membranes and for increase of cyclic AMP concentrations in intact cells could be demonstrated only in short-time assays; in contrast, much lower affinities were observed with equilibrium (60-min) assays as reported previously for various cell lines. High-affinity binding of isoproterenol to 1321N1 cells also was observed in equilibrium (6-hr) binding assays carried out on ice. These results indicate that in the native state the intact cell beta-adrenergic receptor has a high binding affinity for agonists and suggest that incubation at 37 degrees C in the presence of an agonist converts the receptors to a form with low affinity for agonists.

Adrenergic beta-Agonists↗

PubMed1998

Stiffness control of balance in quiet standing.

Our goal was to provide some insights into how the CNS controls and maintains an upright standing posture, which is an integral part of activities of daily living. Although researchers have used simple performance measures of maintenance of this posture quite effectively in clinical decision making, the mechanisms and control principles involved have not been clear. We propose a relatively simple control scheme for regulation of upright posture that provides almost instantaneous corrective response and reduces the operating demands on the CNS. The analytic model is derived and experimentally validated. A stiffness model was developed for quiet standing. The model assumes that muscles act as springs to cause the center-of-pressure (COP) to move in phase with the center-of-mass (COM) as the body sways about some desired position. In the sagittal plane this stiffness control exists at the ankle plantarflexors, in the frontal plane by the hip abductors/adductors. On the basis of observations that the COP-COM error signal continuously oscillates, it is evident that the inverted pendulum model is severely underdamped, approaching the undamped condition. The spectrum of this error signal is seen to match that of a tuned mass, spring, damper system, and a curve fit of this "tuned circuit" yields omega n the undamped natural frequency of the system. The effective stiffness of the system, Ke, is then estimated from Ke = I omega n2, and the damping B is estimated from B = BW X I, where BW is the bandwidth of the tuned response (in rad/s), and I is the moment of inertia of the body about the ankle joint. Ten adult subjects were assessed while standing quietly at three stance widths: 50% hip-to-hip distance, 100 and 150%. Subjects stood for 2 min in each position with eyes open; the 100% stance width was repeated with eyes closed. In all trials and in both planes, the COP oscillated virtually in phase (within 6 ms) with COM, which was predicted by a simple 0th order spring model. Sway amplitude decreased as stance width increased, and Ke increased with stance width. A stiffness model would predict sway to vary as Ke-0.5. The experimental results were close to this prediction: sway was proportional to Ke(-0.55). Reactive control of balance was not evident for several reasons. The visual system does not appear to contribute because no significant difference between eyes open and eyes closed results was found at 100% stance width. Vestibular (otolith) and joint proprioceptive reactive control were discounted because the necessary head accelerations, joint displacements, and velocities were well below reported thresholds. Besides, any reactive control would predict that COP would considerably lag (150-250 ms) behind the COM. Because the average COP was only 4 ms delayed behind the COM, reactive control was not evident; this small delay was accounted for by the damping in the tuned mechanical system.

Adult↗

PubMed2026

Integrative computational analysis combining network pharmacology, regulatory network modeling, and molecular dynamics reveals the mechanisms of Quanshen compound in ITP.

UNLABELLED: Immune thrombocytopenia (ITP) is a hemorrhagic disorder caused by immune dysfunction. Quanshen Compound (QSC) is an in-house preparation developed by the Uyghur Hospital in Hotan Prefecture. This study primarily investigates and validates the potential pharmacological basis and mechanism of action of QSC in modulating immune thrombopoiesis. Based on the multi-database screening of the QSC and the related targets of ITP, the intersection was obtained to construct a protein-protein interaction (PPI) network and screen the core targets; the intersection targets were analyzed for gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using R packages; a component-target-pathway network was constructed to screen the key active components and their mechanisms of action. At the same time, the TF-mRNA-miRNA regulatory network of the core targets was constructed, and chromosome localization and subcellular localization analysis were performed; further, the binding stability of key components and core targets was verified through molecular docking and molecular dynamics simulation. A total of 227 potential target sites were screened out, among which TNF, IL6, AKT1, TP53 and IL1B were the core targets. The enrichment results indicated that these intersecting target sites mainly participated in inflammatory responses, immune regulation and hemostasis-related biological processes, and were significantly enriched in the PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, Th17 cell differentiation and PD-1/PD-L1 signaling pathway. The core target TF-mRNA-miRNA regulatory network contained 184 nodes and 200 edges, suggesting that the core targets were subject to multi-level regulation. Molecular docking results showed that the main active components had good binding activity with the core targets, and molecular dynamics simulation further verified the stability of the complex. QSC may improve ITP through a multi-component, multi-target, and multi-pathway synergistic mechanism involving key targets such as TNF, IL6, AKT1, TP53, and IL1B, as well as the PI3K-Akt signaling pathway. These findings provide new insights into the potential therapeutic mechanisms of QSC against ITP and warrant further experimental validation. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at https://doi.org/10.1007/s40203-026-00718-0.

Immune thrombocytopenia↗

PubMed2005

Decreased virus population diversity in p53-null mice infected with weakly oncogenic Abelson virus.

The Abelson murine leukemia virus (Ab-MLV), like other retroviruses that contain v-onc genes, arose following a recombination event between a replicating retrovirus and a cellular oncogene. Although experimentally validated models have been presented to address the mechanism by which oncogene capture occurs, very little is known about the events that influence emerging viruses following the recombination event that incorporates the cellular sequences. One feature that may play a role is the genetic makeup of the host in which the virus arises; a number of host genes, including oncogenes and tumor suppressor genes, have been shown to affect the pathogenesis of many murine leukemia viruses. To examine how a host gene might affect an emerging v-onc gene-containing retrovirus, we studied the weakly oncogenic Ab-MLV-P90A strain, a mutant that generates highly oncogenic variants in vivo, and compared the viral populations in normal mice and mice lacking the p53 tumor suppressor gene. While variants arose in both p53+/+ and p53-/- tumors, the samples from the wild-type animals contained a more diverse virus population. Differences in virus population diversity were not observed when wild-type and null animals were infected with a highly oncogenic wild-type strain of Ab-MLV. These results indicate that p53, and presumably other host genes, affects the selective forces that operate on virus populations in vivo and likely influences the evolution of oncogenic retroviruses such as Ab-MLV.

Abelson murine leukemia virus↗

PubMed2026

Comparative chloroplast genomics of six Bupleurum (Apiaceae) accessions: candidate barcodes, phylogeny based on available plastomes, and candidate RNA-editing sites.

INTRODUCTION: Bupleurum L. (Apiaceae), a taxonomically intricate genus of about 190 species and a source of Radix Bupleuri (Chai Hu), is difficult to discriminate because of convergent morphology, infraspecific variation, and limited genomic sampling. This study aimed to characterize plastome variation, identify and validate candidate molecular markers, reconstruct plastid phylogenetic relationships, and assess candidate plastid RNA-editing sites in Bupleurum. METHODS: We assembled six plastomes from subgenus Bupleurum, screened 51 Bupleurum plastomes for diagnostic loci, reconstructed whole-plastome and partitioned protein-coding-sequence phylogenies, and predicted plastid C-to-U RNA-editing candidates across the six newly assembled plastomes using a PREP-Cp-compatible workflow. Candidate barcode performance was evaluated against the reference plastome phylogenies, and codon-based models were used to test for positive selection. RESULTS: The plastomes were 154,496-155,778 bp with the canonical quadripartite structure and GC contents of 37.67-37.73%. Gene content was stable (131-132 genes; 86-87 protein-coding genes); B. falcatum subsp. cernuum lacked ycf15 but contained an additional inverted-repeat-associated ycf1 annotation. A/U-ending synonymous codons were favoured. Finite pairwise Ka/Ks estimates were below 1 for most genes, and site-specific codon models detected no positive selection. Each plastome contained 55-61 pure microsatellites, dominated by A/T mononucleotide motifs. MarkerSeek ranked 265 features and identified atpF-atpH, petA-psbJ, rpl32-trnL-UAG, and ycf1 as leading candidate barcodes. ycf1 recovered 38 of 41 nodes strongly supported by both reference trees, whereas a partitioned four-locus analysis recovered 40 of 41 and distinguished all 51 accession sequences. However, only one of seven multi-accession operational binomial groups was monophyletic, and only one showed a positive local barcode gap. The whole-plastome phylogeny recovered Bupleurum as monophyletic relative to Chamaesium. The two sampled Penninervia accessions occupied early-diverging positions without forming an exclusive clade. B. falcatum subsp. cernuum was sister to B. ranunculoides, with B. ranunculoides subsp. telonense sister to that pair. A partitioned 74-CDS analysis recovered the same key relationships and 45 of 50 internal bipartitions. Across the six newly assembled plastomes, 57-63 nonsynonymous C-to-U candidates were predicted per accession (367 total) in 21-22 genes; 269 affected the second codon position and 98 the first. DISCUSSION: Bupleurum plastomes are structurally conservative but retain localised divergence useful for marker development. Concordant whole-plastome and CDS genealogies support genus monophyly, whereas sparse Penninervia sampling and maternal plastid inheritance preclude rejecting traditional subgeneric classification. The predicted RNA-editing sites represent candidates for future experimental validation rather than an established Bupleurum editome. These genomic resources support authentication, conservation, and evolutionary research in Bupleurum.

Apiaceae↗

PubMed1996

Modifications of the follicle-associated epithelium by short-term exposure to a non-intestinal bacterium.

This study was undertaken in order to study the effects of short-term exposure of the follicle-associated epithelium (FAE) of rabbit Peyer's patches to a non-intestinal, Gram-positive bacterium. Isolated ileal loops, each containing one Peyer's patch (PP), were stimulated for short periods of time (30 and 60 min) with Streptococcus pneumoniae R36a, a micro-organism normally not present in the intestinal area. Samples from antigen-stimulated and control Peyer's patches were analysed by light (LM), transmission electron (TEM), and scanning electron microscopy (SEM). Stimulation with living pneumococci induced dramatic changes in FAE architecture and morphology. A massive passage of cells from lymphoid tissue to the FAE was rapidly detectable, accompanied by alterations of the FAE surface, with a marked increase of M-cell area. Furthermore, TEM analysis revealed that M cells were able to internalize living pneumococci. S. pneumoniae R36a is a valid experimental model for the further study of the unique antigen sampling function which characterizes the highly specialized FAE in Peyer's patches.

Animals↗

PubMed1998

Rational engineering of the TOL meta-cleavage pathway

The meta-cleavage pathway of Pseudomonas putida mt-2 was simulated using a biochemical systems simulation developed by Regan (1996). A non-competitive inhibition term for catechol-2,3-dioxygenase (C23O) by 2-OH-pent-2,4-dienoate (Ki = 150 μM) was incorporated into the model. The simulation predicted steady state accumulation levels in the μM range for metabolites pre-meta-cleavage, and in the mM range for metabolites post-meta-cleavage. The logarithmic gains L[V-i, Xj] and L[X-i, Xj] clearly indicated that the pathway was most sensitive to the concentration of the starting substrate, benzoate, and the first enzyme of the pathway, toluate-1, 2-dioxygenase (TO). The simulation was validated experimentally; it was found that the amplification of TO increased the steady state flux from 0.024 to 0.091 (mmol/g cell dwt)/h. This resulted in an increased accumulation of a number of the pathway metabolites (intra- and extracellularly), especially cis-diol, 4-OH-2-oxovalerate, and 4-oxalocrotonate. Metabolic control analysis indicated that C23O was, in fact, the major controling enzymic step of the pathway with a scaled control coefficient of 0.83. The amplification of TO resulted in a shift of some of the control away from C23O. Catechol-2,3-dioxygenase, however, remained as the major controling element of the pathway. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

PubMed1998

Growth and energy metabolism in aerobic fed-batch cultures of Saccharomyces cerevisiae: simulation and model verification.

A kinetic model of overflow metabolism in Saccharomyces cerevisiae was used for simulation of aerobic fed-batch cultivations. An inhibitory effect of ethanol on the maximum respiration of the yeast was observed in the experiments and included in the model. The model predicts respiration, biomass, and ethanol formation and the subsequent ethanol consumption, and was experimentally validated in fed-batch cultivations. Oscillating sugar feed with resulting oscillating carbon dioxide production did not influence the maximum respiration rate, which indicates that the pyruvate dehydrogenase complex is not involved as a bottleneck causing aerobic ethanol formation.

Aerobiosis↗

PubMed2000

Correction for B(1) and B(0) variations in quantitative T(2) measurements using MRI.

A new method is described for compensating for the bias introduced by variations in radiofrequency (RF) field strength and main magnetic field strength when making quantitative T(2) measurements using MRI. Field measurements made during the MRI study are used in combination with a signal model for off-resonance and imperfect RF pulses to correct the estimated T(2) value at every voxel. Applicable to both multicomponent and conventional single-component T(2) studies, the method has been validated experimentally using paramagnetic salt solutions in a multicompartment phantom. Studies of the human head are used to demonstrate the method in practice.

Artifacts↗

PubMed1993

The VCOP Scale: a measure of overprotection in parents of physically vulnerable children.

A scale is developed for measuring the overprotecting vs. optimal developmental stimulation tendencies for parents of physically "vulnerable" children. A series of items were administered to parents whose parenting techniques had been rated as either highly overprotective or as optimal by a group of MDs and other professionals. Correlations were estimated between each of the items and parental tendencies as rated by professionals. Twenty-eight items were selected that provided maximum prediction of over-protection. The resulting R2 was extraordinarily high (.94). Coefficient alpha and test-retest coefficients were acceptable. It is hoped that release of the new instrument (VCOPS) at this time will allow others to join in determining the clinical and experimental validity of this scale.

Adolescent↗

PubMed2000

Estimation of pressure gradients in pulsatile flow from magnetic resonance acceleration measurements.

A method for estimating pressure gradients from MR images is demonstrated. Making the usual assumption that the flowing medium is a Newtonian fluid, and with appropriate boundary conditions, the inertial forces (or acceleration components of the flow) are proportional to the pressure gradients. The technique shown here is based on an evaluation of the inertial forces from Fourier acceleration encoding. This method provides a direct measurement of the total acceleration defined as the sum of the velocity derivative vs. time and the convective acceleration. The technique was experimentally validated by comparing MR and manometer pressure gradient measurements obtained in a pulsatile flow phantom. The results indicate that the MR determination of pressure gradients from an acceleration measurement is feasible with a good correlation with the true measurements (r = 0.97). The feasibility of the method is demonstrated in the aorta of a normal volunteer. Magn Reson Med 44:66-72, 2000.

Adult↗

PubMed2002

Design and optimization of on-chip capillary electrophoresis.

We present a systematic, experimentally validated method of designing electrokinetic injections for on-chip capillary electrophoresis applications. This method can be used to predict point-wise and charge-coupled device (CCD)-imaged electropherograms using estimates of species mobilities, diffusivities and initial sample plug parameters. A simple Taylor dispersion model is used to characterize electrophoretic separations in terms of resolution and signal-to-noise ratio (SNR). Detection convolutions using Gaussian and Boxcar detector response functions are used to relate optimal conditions for resolution and signal as a function of relevant system parameters including electroosmotic mobility, sample injection length, detector length scale, and the length-to-detector. Analytical solutions show a tradeoff between signal-to-noise ratio and resolution with respect to dimensionless injection width and length to the detector. In contrast, there is no tradeoff with respect to the Peclet number as increases in Peclet number favor both SNR and separation solution (R). We validate our model with quantitative epifluorescence visualizations of electrophoretic separation experiments in a simple cross channel microchip. For the pure advection regime of dispersion, we use numerical simulations of the transient convective diffusion processes associated with electrokinetics together with an optimization algorithm to design a voltage control scheme which produces an injection plug that has minimal advective dispersion. We also validate this optimal injection scheme using fluorescence visualizations. These validations show that optimized voltage scheme produces injections with a standard deviation less than one-fifth of the width of the microchannel.

Diagnostic Imaging↗

PubMedDate not supplied

Studies on the chemiexcitation step in peroxyoxalate chemiluminescence using steroid-substituted activators.

The peroxyoxalate reaction is utilized in a wide variety of analytical applications; however, its mechanism is still not very well understood, especially with respect to the excitation step, where the 'chemical energy' is transformed into 'excitation energy'. This base-catalysed reaction of activated oxalic phenyl esters with hydrogen peroxide in the presence of highly fluorescent aromatic hydrocarbons with low oxidation potentials is the only known chemiluminescence system for which exists experimental evidence for the occurrence of the intermolecular chemically initiated electron exchange luminescence (CIEEL) mechanism of proven high efficiency for excited state formation. We report here the singlet quantum yields and relative rate constants of the excitation step (k(CAT)/k(D)), obtained in the peroxyoxalate reaction, utilizing steroid-substituted oxazolinylidenes as activators. In agreement with the CIEEL mechanism, a linear correlation of ln(k(CAT)/k(D)) with the oxidation potential of the activators is obtained, and the singlet quantum yields can be rationalized in terms of the free energy balance of the back electron transfer, leading to the formation of the activator's excited state. Thus, these results contribute to the experimental validation of the widely employed, thus still controversial, CIEEL mechanism.

Fluorescence↗

PubMed2003

Atomistic protein folding simulations on the submillisecond time scale using worldwide distributed computing.

Atomistic simulations of protein folding have the potential to be a great complement to experimental studies, but have been severely limited by the time scales accessible with current computer hardware and algorithms. By employing a worldwide distributed computing network of tens of thousands of PCs and algorithms designed to efficiently utilize this new many-processor, highly heterogeneous, loosely coupled distributed computing paradigm, we have been able to simulate hundreds of microseconds of atomistic molecular dynamics. This has allowed us to directly simulate the folding mechanism and to accurately predict the folding rate of several fast-folding proteins and polymers, including a nonbiological helix, polypeptide alpha-helices, a beta-hairpin, and a three-helix bundle protein from the villin headpiece. Our results demonstrate that one can reach the time scales needed to simulate fast folding using distributed computing, and that potential sets used to describe interatomic interactions are sufficiently accurate to reach the folded state with experimentally validated rates, at least for small proteins.

Algorithms↗

PubMed2003

Metabolic model for glycogen-accumulating organisms in anaerobic/aerobic activated sludge systems.

Glycogen-accumulating organisms (GAO) have the potential to directly compete with polyphosphate-accumulating organisms (PAO) in EBPR systems as both are able to take up VFA anaerobically and grow on the intracellular storage products aerobically. Under anaerobic conditions GAO hydrolyse glycogen to gain energy and reducing equivalents to take up VFA and to synthesise polyhydroxyalkanoate (PHA). In the subsequent aerobic stage, PHA is being oxidised to gain energy for glycogen replenishment (from PHA) and for cell growth. This article describes a complete anaerobic and aerobic model for GAO based on the understanding of their metabolic pathways. The anaerobic model has been developed and reported previously, while the aerobic metabolic model was developed in this study. It is based on the assumption that acetyl-CoA and propionyl-CoA go through the catabolic and anabolic processes independently. Experimental validation shows that the integrated model can predict the anaerobic and aerobic results very well. It was found in this study that at pH 7 the maximum acetate uptake rate of GAO was slower than that reported for PAO in the anaerobic stage. On the other hand, the net biomass production per C-mol acetate added is about 9% higher for GAO than for PAO. This would indicate that PAO and GAO each have certain competitive advantages during different parts of the anaerobic/aerobic process cycle.

Acyl Coenzyme A↗

PubMed2005

Inferring gene regulatory relationships by combining target-target pattern recognition and regulator-specific motif examination.

Although microarray data have been successfully used for gene clustering and classification, the use of time series microarray data for constructing gene regulatory networks remains a particularly difficult task. The challenge lies in reliably inferring regulatory relationships from datasets that normally possess a large number of genes and a limited number of time points. In addition to the numerical challenge, the enormous complexity and dynamic properties of gene expression regulation also impede the progress of inferring gene regulatory relationships. Based on the accepted model of the relationship between regulator and target genes, we developed a new approach for inferring gene regulatory relationships by combining target-target pattern recognition and examination of regulator-specific binding sites in the promoter regions of putative target genes. Pattern recognition was accomplished in two steps: A first algorithm was used to search for the genes that share expression profile similarities with known target genes (KTGs) of each investigated regulator. The selected genes were further filtered by examining for the presence of regulator-specific binding sites in their promoter regions. As we implemented our approach to 18 yeast regulator genes and their known target genes, we discovered 267 new regulatory relationships, among which 15% are rediscovered, experimentally validated ones. Of the discovered target genes, 36.1% have the same or similar functions to a KTG of the regulator. An even larger number of inferred genes fall in the biological context and regulatory scope of their regulators. Since the regulatory relationships are inferred from pattern recognition between target-target genes, the method we present is especially suitable for inferring gene regulatory relationships in which there is a time delay between the expression of regulating and target genes.

Algorithms↗

PubMed2006

Engineering aromatic L-amino acid transaminase for the asymmetric synthesis of constrained analogs of L-phenylalanine.

An enzymatic asymmetric synthesis was carried out for the preparation of enantiomerically pure L-diphenylalanine using the rationally engineered aromatic L-amino acid transaminase (eAroATEs) obtained from Enterobacter sp. BK2K-1. To rationally redesign the enzyme, structural model was constructed by the homology modeling. The structural model was experimentally validated by the site-directed mutagenesis of the predicted pyridoxal-5'-phosphate (PLP) binding site and the substrate-recognition region, and the cell-free protein synthesis of mutated enzymes. It was suggested that Arg281 and Arg375 were the key residues to recognize the distal carboxylate and alpha-carboxylate group of the substrates, respectively. The model also predicted that Tyr66 forms hydrogen bond with the phosphate moiety of PLP and interacts with the side chain attached to beta-carbon of the amino acid substrate. Among the various site-directed mutants, Y66L variant was able to synthesize L-diphenylalanine with 23% conversion yield for 10 h, whereas the wild-type AroATEs was inactive for the transamination between diphenylpyruvate and L-phenylalanine as amino acceptor and amino donor, respectively.

Amino Acids, Aromatic↗

PubMedDate not supplied

A central role of eNOS in the protective effect of wine against metabolic syndrome.

The positive health effects derived from moderate wine consumption are pleiotropic. They appear as improvements in cardiovascular risk factors such as plasma lipids, haemostatic mechanisms, endothelial function and antioxidant defences. The active principles would be ethanol and mainly polyphenols. Results from our and other laboratories support the unifying hypothesis that the improvements in risk factors after red wine consumption are mediated by endothelial nitric oxide synthase (eNOS). Many genes are involved, but the participation of eNOS would be a constant feature. The metabolic syndrome is a cluster of metabolic risk factors associated with high risk of cardiovascular disease (CVD). The National Cholesterol Education Programmmes Adult Treatment Panel III (NCEPATP III) clinical definition of the metabolic syndrome requires the presence of at least three risk factors, from among abdominal obesity, high plasma triacylglycerols, low plasma HDL, high blood pressure and high fasting plasma glucose. The molecular mechanisms responsible for the metabolic syndrome are not known. Since metabolic syndrome apparently affects 10-30% of the population in the world, research on its pathogenesis and control is needed. The recent finding that eNOS knockout mice present a cluster of cardiovascular risk factors comparable to those of the metabolic syndrome suggests that defects in eNOS function may cause human metabolic syndrome. These mice are hypertensive, insulin resistant and dyslipidemic. Further support for a pathogenic role of eNOS comes from the finding in humans that eNOS polymorphisms associate with insulin resistance and diabetes, with hypertension, with inflammatory and oxidative stress markers and with albuminuria. So, the data sustain the hypothesis that eNOS enhancement should reduce metabolic syndrome incidence and its consequences. Therefore red wine, since it enhances eNOS function, should be considered as a potential tool for the control of metabolic syndrome. This hypothesis is supported by epidemiological observations and needs experimental validation in human intervention studies.

Animals↗