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PubMed1992

Responsiveness of the intestinal 1,25-dihydroxyvitamin D3 receptor to magnesium depletion in the rat.

In contrast to man, the rat exhibits hypercalcemia during the course of magnesium depletion. To investigate the role of the vitamin D (D) endocrine system in the induction of hypercalcemia, circulating D metabolites, the binding properties of the duodenal 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] receptor (VDR), and 45Ca transport studies were undertaken in magnesium-replete rats or after 10 days of magnesium depletion in animals presenting the following D status: D depletion and hypo- or normocalcemia (achieved by oral calcium supplementation), D3 or 1,25-(OH)2D3 repletion. Magnesium depletion did not influence serum calcium in hypo- or normocalcemic D depleted rats, but increased serum calcium in animals receiving D3 (P less than 0.002) or 1,25-(OH)2D3 (P less than 0.0001), suggesting that the D3 endocrine system is necessary to mediate the rise in extracellular calcium and that dietary calcium alone is not sufficient to significantly increase extracellular calcium in the hypomagnesemic rat. The data also show that 25-hydroxyvitamin D formation was not perturbed, but circulating 1,25-(OH)2D3 concentrations were reduced by 10 days of magnesium depletion (P less than 0.0001) even in animals infused with 1,25-(OH)2D3, suggesting increased clearance of the hormone. The kinetic data of the duodenal VDR revealed maximum binding sites ranging from 1018-1500 fmol/mg DNA and Kd ranging from 0.17-0.38 nM, with no significant between-group difference in magnesium-sufficient animals. Ten days of magnesium depletion did not significantly influence VDR affinity in any of the groups, but significantly increased receptor number in hypocalcemic D-depleted rats from 1190 +/- 154 to 2748 +/- 430 fmol/mg DNA (P less than 0.004). Calcium transport studies in D-replete animals indicate that intestinal calcium transport is influenced by the progressive depletion in magnesium, with time-related increases coinciding with the in vivo increase in circulating ionized calcium (day 6 of magnesium depletion). However, despite persistent elevated serum ionized calcium, calcium transport declined only to predepletion levels on days 8 and 10 of magnesium depletion. To investigate the influence of the D3 endocrine system on 45Ca absorption, D-depleted rats sufficient or depleted in magnesium were injected with 1,25-(OH)2D3, either acutely (to reveal its membrane effects) or 16 and 5 h before death (to reveal its genomic effect). The data reveal a reduced response in magnesium-depleted rats to acute 1,25-(OH)2D3 injection (P less than 0.0002), but similar responses when the hormone was injected 16 and 5 h before the experiment.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of Variance↗
PubMed1997

Prospects for the direct electron crystallographic determination of zeolite structures.

Recently two successful zeolite structures based on experimental electron crystallographic data have been published. Diffraction and image data based on the silicate portion of the zeolite, mordenite, which are perturbed by dynamical (as well as secondary) scattering, have been simulated by a multiple-beam dynamical scattering program. Structure analyses with these data show that the above claims are not unreasonable, given a high enough accelerating voltage for the electron beam. If, for example, 2.9 A resolution micrographs are taken from a 120 A thick crystal in a 200 or 400 kV electron microscope, the crystallographic phases found by image analysis (Fourier filtration) are accurate enough to be extended by the Sayre equation to the (atomic) resolution limit of the electron diffraction pattern (for example from a 105 A thick crystal illuminated by a 1,200 kV electron source). The resultant potential map can be interpreted to find most of the atomic positions and the remaining ones will appear during the progress of a Fourier refinement.

Aluminum Silicates↗
PubMed2002

Conformational changes in chemically modified Escherichia coli thioredoxin monitored by H/D exchange and electrospray ionization mass spectrometry.

Hydrogen/deuterium (H/D) exchange in combination with electrospray ionization mass spectrometry and near-ultraviolet (UV) circular dichroism (CD) was used to study the conformational properties and thermal unfolding of Escherichia coli thioredoxin and its Cys32-alkylated derivatives in 1% acetic acid (pH 2.7). Thermal unfolding of oxidized (Oxi) and reduced (Red) -thioredoxin (TRX) and Cys-32-ethylglutathionyl (GS-ethyl-TRX) and Cys-32-ethylcysteinyl (Cys-ethyl-TRX), which are derivatives of Red-TRX, follow apparent EX1 kinetics as charge-state envelopes, H/D mass spectral exchange profiles, and near-UV CD appear to support a two-state folding/unfolding model. Minor mass peaks in the H/D exchange profiles and nonsuperimposable MS- and CD-derived melting curves, however, suggest the participation of unfolding intermediates leading to the conclusion that the two-state model is an oversimplification of the process. The relative stabilities as measured by melting temperatures by both CD and mass spectral charge states are, Oxi-TRX, GS-ethyl-TRX, Cys-ethyl-TRX, and Red-TRX. The introduction of the Cys-32-ethylglutathionyl group provides extra stabilization that results from additional hydrogen bonding interactions between the ethylglutathionyl group and the protein. Near-UV CD data show that the local environment near the active site is perturbed to almost an identical degree regardless of whether alkylation at Cys-32 is by the ethylglutathionyl group, or the smaller, nonhydrogen-bonding ethylcysteinyl group. Mass spectral data, however, indicate a tighter structure for GS-ethyl-TRX.

Deuterium↗
PubMed1989

Data labeling and sampling effects in harmonics-to-noise ratios.

The harmonics-to-noise ratio (HNR) was proposed for measurement of noise in sustained vowels. A generalized algorithm is described here for removing the dependence on the data offset and for reducing the influence of jitter and shimmer. In addition, a new algorithm is proposed that accommodates periodic perturbations. Formulations are presented that can be computed in a single pass through the data under certain assumptions, thus simplifying the implementation of an efficient analysis program. A mathematical analysis of the effect of small errors in pitch-period demarcation is presented. The analysis indicates that the sensitivity to demarcation errors depends on the preponderance of high-frequency components, and significant underestimation is predicted for /a/ and /i/ data. A method for correcting quantization and demarcation errors in pitch-period markers is described. The method is shown to be effective at reducing demarcation errors for all but the most severely perturbed waveforms. Examples of the use of this method for HNR estimation are provided. Finally, a comparison with recent work by Milenkovic [J. Speech Hear. Res. 30, 529-538 (1987)] is presented.

Algorithms↗
PubMed1987

Complex spikes in Purkinje cells in the lateral vermis (b zone) of the cat cerebellum during locomotion.

1. Complex spikes (c.s.s) due to climbing fibre input were recorded from forty-one Purkinje cells in the lateral part of the vermis (i.e. the b zone) of lobule V of the cerebellum in cats walking on a moving belt or a horizontal ladder. Most cells were near the tips of the folia making up the lobule and some were shown by antidromic invasion to project to the ipsilateral lateral vestibular nucleus. In all cells c.s.s. could be evoked through mechanical stimuli delivered manually to the neck and/or trunk and/or the limb girdles and/or the proximal parts of the limbs. 2. During walking c.s.s. occurred at rates which ranged in different cells from 0.8 to 2.55/s (i.e. ca. 0.8 to 2/step). When activity was averaged across many successive steps the probability of c.s. occurrence was never completely constant throughout the step cycle, but no tendency was detected for c.s.s to recur at any precisely fixed time during the cycle. 3. When ladder locomotion was perturbed because a rung underwent an unexpected 2 cm descent when stepped on, some cells generated a c.s. at short latency in a proportion of trials. Such responses were well time-locked to the onset of rung movement but not to its cessation (which they often preceded). 4. For perturbations of either forelimb the earliest displacement-related c.s. occurred in different cells between 40 and 64 ms after the onset of rung movement. In different cells c.s.s occurred in from one out of five to three out of four perturbed steps (mean ca. two out of five steps). Eight out of seventeen cells responded to perturbation of the forelimb ipsilateral to the cell and five out of ten responded to contralateral perturbations. 5. Perturbation of the ipsilateral hind limb was accompanied by c.s.s in four out of nine cells and latency was usually longer (by ca. 30-40 ms). One cell showed a decrease in the probability of c.s. occurrence. Insufficient data were obtained for a systematic study of responsiveness to perturbation of the contralateral hind limb. 6. Cells showed different patterns of limb specificity, responding to perturbation of one, two or all of the three limbs studied. In total, c.s.s accompanied perturbation of at least one limb in thirteen out of twenty cells studied (65%).(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed2003

Concurrent signaling from Galphaq- and Galphai-coupled pathways is essential for agonist-induced alphavbeta3 activation on human platelets.

The integrin alphavbeta3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates alphavbeta3 activity in platelets, we measured the effect of the P2Y1 antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y12 antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y1 and P2Y12 was required to activate alphavbeta3. The reducing agent dithiothreitol and Mn2+ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates alphavbeta3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, alphavbeta3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Galphaq- and Galphai-coupled receptors converge to release cytoskeletal constraints on alphavbeta3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for alphavbeta3-mediated platelet adhesion.

Actins↗
PubMed2006

Parameter estimation in models combining signal transduction and metabolic pathways: the dependent input approach.

Biological complexity and limited quantitative measurements pose severe challenges to standard engineering methodologies for modelling and simulation of genes and gene products integrated in a functional network. In particular, parameter quantification is a bottleneck, and therefore parameter estimation, identifiability, and optimal experiment design are important research topics in systems biology. An approach is presented in which unmodelled dynamics are replaced by fictitious 'dependent inputs'. The dependent input approach is particularly useful in validation experiments, because it allows one to fit model parameters to experimental data generated by a reference cell type ('wild-type') and then test this model on data generated by a variation ('mutant'), so long as the mutations only affect the unmodelled dynamics that produce the dependent inputs. Another novel feature of the approach is in the inclusion of a priori information in a multi-objective identification criterion, making it possible to obtain estimates of parameter values and their variances from a relatively limited experimental data set. The pathways that control the nitrogen uptake fluxes in baker's yeast (Saccharomyces cerevisiae) have been studied. Well-defined perturbation experiments were performed on cells growing in steady-state. Time-series data of extracellular and intracellular metabolites were obtained, as well as mRNA levels. A nonlinear model was proposed and was shown to be structurally identifiable given data of its inputs and outputs. The identified model is a reliable representation of the metabolic system, as it could correctly describe the responses of mutant cells and different perturbations.

Algorithms↗
PubMed1978

Local perturbation of the double helical DNA structure near single breaks.

The influences of DNA composition and solvent modification on the stability of the native DNA structure near a single break have been studied. The value of the magnification parameter h of the double helix breakdown probability in a haplotomic degradation process is used as an index of the perturbation range. Methodological and technical details are discussed and comments are made on previously reported data on the h value. From this study it may be concluded that the width of the perturbation zone next to a nick is influenced by solvent-induced changes in the general stability of the native DNA molecule, and by compositional-dependent local interactions. Our results point to entropic effects as the more important contributions to stability perturbation near a discontinuity in the native DNA molecule.

DNA, Bacterial↗
PubMed1995

Hypoxia and modification of the endothelium: implications for regulation of vascular homeostatic properties.

Hypoxia is a common denominator of ischemic microenvironments. Endothelium subjected to oxygen deprivation maintains cell viability and basic biosynthetic mechanisms, but displays multiple changes in properties relevant to vascular homeostasis, including suppression of the anticoagulant cofactor thrombomodulin, decreased barrier function, and generation of proinflammatory cytokines. Diminished intracellular cAMP during the period of hypoxia and lowered nitric oxide/cGMP in the subsequent reperfusion period are proposed as fundamental mechanisms driving vascular dysfunction impacting on coagulation, permeability, vasomotor tone and leukocyte adhesivity. The period of organ preservation for transplantation, recognized to be associated with hypoxia, primes mechanisms leading to subsequent vascular dysfunction which can be ameliorated by buttressing cAMP and nitric oxide/cGMP intra- and intercellular second messenger systems. A mechanism likely to contribute to hypoxia-mediated generation of cytokines, such as interleukin 6, is activation of the transcription factor NF-IL-6, which occurs in oxygen deprivation. These data indicate that study of cellular mechanisms of endothelial perturbation in hypoxia is likely to provide insights ultimately applicable to ischemia-induced vascular damage.

Animals↗
PubMed2004

Aperiodic flow-induced oscillations of collapsible tubes: a critical reappraisal.

The evidence for the aperiodic self-excited oscillations of flow-conveying collapsible tubes being mathematically chaotic is re-examined. Many cases which powerfully suggest nonlinear deterministic behaviour have not been recorded over time-spans which allow their exhaustive examination. The present investigation centred on a previously recorded robust and generic oscillation, but more recent and more discerning tests were applied. Despite hints that a low embedding dimension might suffice, the data appeared on most indices high-dimensional. A U-shaped return map was found and modelled using both radial basis functions and polynomials, but lack of detailed structure in the map prevented effective parameter estimation. On the basis of power-law rather than exponential divergence of nearby trajectories, and of inability to discriminate against behaviour which would also be manifested by a surrogate consisting of a noise-perturbed nonlinear periodic oscillator, it is concluded that the data do not support the idea that the aperiodicity in the particular oscillation examined is caused by deterministic chaos. There was evidence that the distributed nature of the physical system might underlie aspects of the high dimensionality. We advocate equally searching testing of any future candidate chaotic oscillations in the investigation of collapsed-tube flows.

Biomedical Engineering↗
PubMedDate not supplied

Chemiluminescence emission during reactions between superoxide and selected aliphatic and aromatic halocarbons in aprotic media.

The reactions between superoxide free radical anion (.O2-) with the halocarbons CCl4. CHCl3, BrCH2CH2Br(EDB), decachlora-biphenyl (DCBP), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in dimethyl sulphoxide (DMSO) results in the emission of chemiluminescence (CL). The chemiluminescence reactions are characterized as having biphasic second order kinetics, CL wavelengths between 350 nm and 650 nm, and exhibiting perturbation by chemicals reactive with singlet oxygen. These data suggest that singlet oxygen species are the excited state responsible for the light emissions. Polarographic studies confirm .O2- consumption and halide release in the reactions, while gas liquid chromatography and NBT reduction demonstrate the decomposition of the halocarbons into products. A chemiluminescent reaction mechanism is proposed involving reductive dehalogenation of the halocarbons and the generation of singlet oxygen. The significance of singlet oxygen generation is discussed with respect to a general mechanism for explaining the rapid initiation of lipid peroxidative membrane damage in halocarbon toxigenicity in animal and plant tissues.

Animals↗
PubMed1991

Inhibition of axonal development after injection of neurofilament antibodies into a Xenopus laevis embryo.

The ability to target specific cytoskeletal components in axons for disruption within intact developing embryos would provide a valuable tool for studying neuronal development. Neurofilaments are an attractive target for such an approach, because they are neuron specific and are expressed late in embryogenesis principally beginning during axon outgrowth. No pharmacological agents are currently available that disrupt neurofilaments without also affecting general development. One approach that has been used successfully to affect proteins in vivo is to inject specific antibodies into living cells. We employed this approach in Xenopus laevis embryos by injecting two antibodies directed against the middle molecular weight neurofilament protein (NF-M) into a single blastomere of a two-cell stage embryo. Injected antibodies could be detected for as long as 3.5 days in cells descended from the injected blastomere. Only cell bodies of neurons descended from anti-NF-M-injected blastomeres contained abnormal accumulations of intermediate filament proteins, and peripheral nerve development was unilaterally retarded in these neurofilament antibody-injected tadpoles. Such accumulations and peripheral nerve defects were not seen in neurons derived from uninjected blastomeres or from blastomeres injected with control antibodies. These data demonstrate the usefulness of specific antibodies to perturb neuronal development in intact frog embryos and, in addition, suggest a role for neurofilaments in axon elongation.

Animals↗
PubMed1997

Impaired spontaneous endocytosis of HLA-G.

HLA-G is a class Ib (non-classical) major histocompatibility complex (MHC) protein expressed at the maternal-fetal interface that inhibits natural killer (NK) cell-mediated lysis in an allotype-independent manner. Here we report that the spontaneous endocytosis of HLA-G is severely reduced because of its short cytoplasmic tail. Class I (classical) MHC proteins on the surface of B cell transfectants detected by primary and secondary antibodies underwent endocytosis at a moderate rate, whereas HLA-G, chimeric proteins consisting of the extracellular domains of HLA-C with the C-terminal sequence of HLA-G, or glycophosphatidylinositol-tailed HLA-C proteins, were not efficiently internalized. In addition, a mutant of beta 2-microglobulin (Ser88Cys) that could be specifically labeled with Texas red (or other fluorescent probes) and exchanged into class I or class Ib MHC proteins was employed to study spontaneous internalization of MHC proteins by a non-perturbative method independent of an antibody ligand. These data are discussed in terms of both the role of HLA-G expressed on the fetal trophoblast and the function of the cytoplasmic tail in class I MHC proteins.

B-Lymphocytes↗
PubMed1975

Drug-biomolecule interactions: mechanism of ligand interactions with carbonic anhydrase studied by magnetic resonance relaxation and rapid reaction methods.

Kinetcs of interaction between the metalloenzyme carbonic anhydrase and either monovalent anions or aromatic sulfonamides were examined by three distinct fast reaction techniques: stopped flow, equilibrium perturbation, and magentic resonance relaxation. By correlating spectroscopic data on conformational and ionization equilibria of the complex and free species with the reaction kinetics, a relatively complete description of the mechanism can be presented. A proton-dependent equilibrium between two coordination forms of the free enzyme can be demonstrated spectroscopically. Anions selectively combine with the form predominating at low pH. For a series of carboxylate ligands, formate and substituted acetates, anion association is found to be three orders of magnitude greater than similar ligand substitution processes known in inorganic chemistry. For sulfonamide association, the attacking species are the form of carbonic anhydrase predominating at high pH and the neutral sulfonamide. Combination involving the neutral species is followed by loss of a proton to form the sulfonamido anion in the stabilized complex. This obligate proton transfer offers a probable explanation for the unique specificity of sulfonamides in inhibiting this enzyme. The anionic sulfonamido moiety can be shown to resemble closely a transition intermediate in the catalytic step of substrate hydration.

Acetates↗
PubMed2000

Separation of neural induction and neurulation in Xenopus.

Cellular interactions with laminin are important for numerous morphogenetic events. In Xenopus, the first of these is neurulation. The integrin alpha6 subunit mediates an attachment of the cells of the neural plate to the underlying basal lamina. A disruption of this interaction results in embryos that fail to neurulate (T. E. Lallier et al., 1996, Development 122, 2539-2554). Here we provide evidence supporting the specificity of this phenomenon and characterize developmental events as either disrupted or unaffected by a perturbation of alpha6 integrin expression. First, reduction of alpha6 integrin expression does not halt mitotic division throughout the embryo, indicating that the neural defects observed are not simply a global perturbation of all developmental processes. Second, a gene associated with dorsal mesoderm formation, brachyury, is expressed normally in alpha6 integrin-perturbed embryos. Third, the expression of BMP4, noggin, chordin, and follistatin, all of which are critical for neural induction, are at near normal levels. In addition, several genes expressed shortly after neural induction (N-CAM, nrp1, and Xanf1) are not perturbed in nonneurulating embryos. Interestingly, expression of one neural-specific gene (synaptobrevin), which is normally detectable late in neurulation, is abolished in these alpha6 integrin-perturbed embryos. Furthermore, the spatial expression of several transcripts is expanded in alpha6 integrin-perturbed embryos (orthodenticle and engrailed). Taken together, these data indicate that while alpha6 integrin-mediated interactions with laminin are required for neurulation, they are not required for the initial processes of neural induction. However, these cell-extracellular matrix interactions appear to be important in later inductive events and rostrocaudal patterning of the neural tube.

Animals↗
PubMed1983

The localization of motoneuron pools innervating wing muscles in the chick.

Motoneuron pools supplying principal muscles of the shoulder girdle and wing were localized in the chicks 2-7 days post hatching with the use of retrograde axonic transport of horse radish peroxidase (HRP). HRP was injected into selected muscles and the animals sacrificed after 24 h survival. Labelled motoneuron pools representing individual muscles were found to be clustered in longitudinal columns along the brachial spinal cord segments 13-16. Muscles derived from the dorsal muscle mass were innervated by motoneurons located in the ventro-lateral portion of the horn, while those originating from the ventral muscle mass received innervation from neurons occupying the dorsomedial portion within the ventral horn of the spinal cord. The rostrocaudal extent of the motoneuron pools could be correlated with the proximodistal position of wing muscles. The observed orderly topographical relationships between clusters of motoneurons of the brachial spinal cord and the muscles they innervate will be used as baseline data for experiments where the limb innervation is perturbed.

Animals↗
PubMed1989

Physicochemical characterization of alpha-crystallins from bovine lenses: hydrodynamic and conformational properties.

A detailed investigation of hydrodynamic and conformational behavior has been made of the HM alpha-crystallin and alpha-crystallins of bovine lens. Results from this study indicated that HM alpha (high-molecular-weight alpha-crystallin) and alpha (low-molecular-weight alpha-crystallin) possess considerable size and charge heterogeneities in their native structures and subunit polypeptides, respectively. Sedimentation velocity showed a heterogeneous polydisperse system of HM alpha with an average sedimentation coefficient of about 50S and a more homogeneous system of alpha-crystallin of 20 S. Viscosity and circular dichroism studies pointed to a compact and globular shape of dominant beta-sheet conformation for alpha-crystallin, yet a highly asymmetrical and aggregated form for HM alpha. The conformational stability of alpha-crystallin was investigated in the presence of various denaturants. The evidence presented shows that hydrogen bonding is the main force in maintaining the quaternary structure of compact native alpha-crystallin. Conformational flexibility of alpha-crystallin demonstrated in the equilibrium unfolding study indicated a multistep transition that made the extraction of thermodynamic data from the heat denaturation study difficult. Temperature perturbation on alpha-crystallin suggested the possible involvement of hydrophobic interaction in the aggregation process, leading to the formation of HM alpha from alpha-crystallin. The comparison of conformational properties between HM alpha and alpha-crystallin strongly indicated that HM alpha is a denatured form of alpha-crystallin.

Amino Acid Sequence↗
PubMed1995

A solidification procedure to facilitate kinematic analyses based on video system data.

When video-based motion analysis systems are used to measure segmental kinematics, the major source of error is the displacement of skin-fixed markers relative to the underlying skeletal structure. Such displacements cause the marker representation of the segment to deform, thereby decreasing the accuracy of subsequent three-dimensional kinematic calculations. We have developed a two-step solidification procedure to address this problem. First, the mean rigid shape is computed which best represents the time-varying marker configuration of each segment. Second, a least-squares minimization is used to replace the measured marker coordinates with those corresponding to the best-fit mean rigid shape. Rigid body theory can then be applied unambiguously to perform kinematic analyses. To evaluate this approach, we defined an unperturbed three-dimensional reference movement using kinematic data from the swing phase of gait. After perturbing the marker coordinates with artificial noise, the rotation matrix and translation vector (absolute and relative movement) between each pair of successive images were computed using (1) reference frames fixed directly to the perturbed marker coordinates, (2) a least-squares minimization procedure found in the literature, and (3) the proposed solidification procedure. The least-squares and solidification procedures produced extremely similar results which, relative to the direct calculation, reduced kinematic errors on average by 20-25% when the maximum distance between markers was small (e.g. < 15 cm). The solidification methodology therefore combines the numerical benefits of the least-squares method with the conceptual benefits of a rigid body method.

Algorithms↗