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PubMed2000

The paradox of alloreactivity and self MHC restriction: quantitative analysis and statistics.

Although 1-24% of T cells are alloreactive, i.e., respond to MHC molecules encoded by a foreign haplotype, it is generally believed that T cells cannot recognize foreign peptides binding foreign MHC molecules. We show using a quantitative model that, if T cell selection and activation are affinity-driven, then an alloreactivity of 1-24% is incompatible with the textbook notion that self MHC restriction is absolute. If an average of 1% of clones are alloreactive, then according to our model, at most 20-fold more clones should, on average, be activated by antigens presented on self MHC than by antigens presented on foreign MHC. This ratio is at best 5 if alloreactivity is 5%. These results describe average properties of the murine immune system, but not the outcome of individual experiments. Using supercomputer technology, we simulated 100,000 MHC restriction experiments. Although the average restriction ratio was 7.1, restriction was absolute in 10% of the simulated experiments, greater than 100, although not absolute, in 29%, and below 6 in 24%. This extreme variability agrees with experimental estimates. Our analysis suggests that alloreactivity and average self MHC restriction both cannot be high, but that a low average restriction level is compatible with high levels in a significant number of experiments.

Animals↗

PubMed1998

Formation and fate of tyrosine. Intracellular partitioning of newly synthesized tyrosine in mammalian liver.

Tyrosine in an hepatocyte is transported from the plasma, synthesized from phenylalanine, or released during protein turnover. Effects of phenylalanine and tyrosine on the formation and fate (partitioning) of tyrosine from the different sources were examined in primary rat hepatocyte cultures. Rates of tyrosine degradation, transport, incorporation into and release from protein, and synthesis from phenylalanine were measured as well as the intracellular dilution of labeled tyrosine and phenylalanine incorporated into protein. We found tyrosine had little effect on phenylalanine hydroxylation over a wide range of conditions, that transported tyrosine and tyrosine from phenylalanine are in different metabolic pools, and that there appears to be channeling of newly synthesized tyrosine during degradation. In addition, under some conditions, intracellular partitioning of tyrosine is determined by tyrosine concentration. Specifically, if extracellular tyrosine is low and phenylalanine is at a normal plasma level, tyrosine use in protein synthesis takes precedence over tyrosine degradation or export. It is proposed that the mechanism controlling this is kinetic, based on relative rates of tyrosyl-tRNA formation and tyrosine degradation and export. A quantitative model of tyrosine and phenylalanine in-flow and out-flow in hepatocytes is given, incorporating tyrosine synthesis, degradation, plasma membrane transport, and tyrosine and phenylalanine use and release during protein turnover.

Animals↗

PubMed2001

Dissection of a nuclear localization signal.

The regulated process of protein import into the nucleus of a eukaryotic cell is mediated by specific nuclear localization signals (NLSs) that are recognized by protein import receptors. This study seeks to decipher the energetic details of NLS recognition by the receptor importin alpha through quantitative analysis of variant NLSs. The relative importance of each residue in two monopartite NLS sequences was determined using an alanine scanning approach. These measurements yield an energetic definition of a monopartite NLS sequence where a required lysine residue is followed by two other basic residues in the sequence K(K/R)X(K/R). In addition, the energetic contributions of the second basic cluster in a bipartite NLS ( approximately 3 kcal/mol) as well as the energy of inhibition of the importin alpha importin beta-binding domain ( approximately 3 kcal/mol) were also measured. These data allow the generation of an energetic scale of nuclear localization sequences based on a peptide's affinity for the importin alpha-importin beta complex. On this scale, a functional NLS has a binding constant of approximately 10 nm, whereas a nonfunctional NLS has a 100-fold weaker affinity of 1 microm. Further correlation between the current in vitro data and in vivo function will provide the foundation for a comprehensive quantitative model of protein import.

Amino Acid Sequence↗

PubMed2002

Design of a novel peptide inhibitor of HIV fusion that disrupts the internal trimeric coiled-coil of gp41.

The pre-hairpin intermediate of gp41 from the human immunodeficiency virus (HIV) is the target for two classes of fusion inhibitors that bind to the C-terminal region or the trimeric coiled-coil of N-terminal helices, thereby preventing formation of the fusogenic trimer of hairpins. Using rational design, two 36-residue peptides, N36(Mut(e,g)) and N36(Mut(a,d)), were derived from the parent N36 peptide comprising the N-terminal helix of the gp41 ectodomain (residues 546-581 of HIV-1 envelope), characterized by analytical ultracentrifugation and CD, and assessed for their ability to inhibit HIV fusion using a quantitative vaccinia virus-based fusion assay. N36(Mut(e,g)) contains nine amino acid substitutions designed to disrupt interactions with the C-terminal region of gp41 while preserving contacts governing the formation of the trimeric coiled-coil. N36(Mut(a,d)) contains nine substitutions designed to block formation of the trimeric coiled-coil but retains residues that interact with the C-terminal region of gp41. N36(Mut(a,d)) is monomeric, is largely random coil, does not interact with the C34 peptide derived from the C-terminal region of gp41 (residues 628-661), and does not inhibit fusion. The trimeric coiled-coil structure is therefore a prerequisite for interaction with the C-terminal region of gp41. N36(Mut(e,g)) forms a monodisperse, helical trimer in solution, does not interact with C34, and yet inhibits fusion about 50-fold more effectively than the parent N36 peptide (IC(50) approximately 308 nm versus approximately 16 microm). These results indicate that N36(Mut(e,g)) acts by disrupting the homotrimeric coiled-coil of N-terminal helices in the pre-hairpin intermediate to form heterotrimers. Thus N36(Mut(e,g)) represents a novel third class of gp41-targeted HIV fusion inhibitor. A quantitative model describing the interaction of N36(Mut(e,g)) with the pre-hairpin intermediate is presented.

Amino Acid Sequence↗

PubMed2002

Reaction of reduced flavins and flavoproteins with diphenyliodonium chloride.

The reaction of diphenyliodonium chloride with free reduced flavins has been studied by stopped flow spectrophotometry under anaerobic conditions, and second order rate constants were determined as a function of pH. The reactive flavin species was identified as the reduced anion, based on an observed reaction pK of 6.7. The product mixture was independent of the initial concentration of reactant and contained approximately 20% oxidized flavin. The results can be modeled quantitatively on a modification of the mechanism proposed by Tew (Tew, D. G. (1993) Biochemistry 32, 10209-10215). The composition of the complex reaction mixture has been analyzed, and four flavin-phenyl adducts with distinctive absorbance and fluorescence characteristics have been identified, involving substitution at the flavin C4a, N5, and C8 positions. Inactivation of flavoprotein enzymes by diphenyliodonium has also been studied, and several examples were found where inactivation occurs readily, despite noninvolvement of radical intermediates in their reaction mechanisms. It can be concluded that inactivation by phenyliodonium species is not a valid indicator of catalytic mechanism involving radical intermediates. One of the several factors determining inactivation is maintenance of the enzyme flavin in the reduced form in the steady state of catalysis, the other factors being redox potential and accessibility of the inhibitor to the flavin active site.

4-Hydroxybenzoate-3-Monooxygenase↗

PubMed2005

Mechanism of action of myosin X, a membrane-associated molecular motor.

We have performed a detailed biochemical kinetic and spectroscopic study on a recombinant myosin X head construct to establish a quantitative model of the enzymatic mechanism of this membrane-bound myosin. Our model shows that during steady-state ATP hydrolysis, myosin X exhibits a duty ratio (i.e. the fraction of the cycle time spent strongly bound to actin) of around 16%, but most of the remaining myosin heads are also actin-attached even at moderate actin concentrations in the so-called "weak" actin-binding states. Contrary to the high duty ratio motors myosin V and VI, the ADP release rate constant from actomyosin X is around five times greater than the maximal steady-state ATPase activity, and the kinetic partitioning between different weak actin-binding states is a major contributor to the rate limitation of the enzymatic cycle. Two different ADP states of myosin X are populated in the absence of actin, one of which shows very similar kinetic properties to actomyosin.ADP. The nucleotide-free complex of myosin X with actin shows unique spectral and biochemical characteristics, indicating a special mode of actomyosin interaction.

Actins↗

PubMed1995

Digestive adaptations of rats given white bread and cooked haricot beans (Phaseolus vulgaris): large-bowel fermentation and digestion of complex carbohydrates.

Two experiments were carried out to examine the short (1-3 d) and medium (14 d) term adaptations of rat large-bowel (LB) fermentation to alterations in substrate supply brought about by including cooked haricot beans (Phaseolus vulgaris) in diets based on white bread. Changes in organic matter (OM) flow from the ileum occurred within 1 d and were stable for 14 d but the pattern of caecal short-chain fatty acids took much longer to stabilize with considerable increases in butyrate between 1 and 3 d and up to 14 d. This suggests that the metabolic activity of the LB microflora may take a considerable time to stabilize after an abrupt change in substrate supply. Despite an almost fourfold change in OM supply to the LB, the proportion of this OM apparently fermented in that organ (0.46) remained fairly constant. None of the apparent resistant starch measured in the diets could be detected in faeces. Dietary non-starch polysaccharides (NSP) were extensively fermented with similar values (0.82) for both bread and bean NSP and there was little indication of any interaction between the two diet components on NSP fermentation. An attempt was made to fractionate ileal and faecal OM to provide a basis for a quantitative model of LB stoichiometry.

Adaptation, Physiological↗

PubMed1996

The effects of condensed tannins from Desmodium intortum and Calliandra calothyrsus on protein and carbohydrate digestion in sheep and goats.

A factorial experiment was conducted to study the effects of condensed tannins (CT) from the tropical legumes Desmodium intortum and Calliandra calothyrsus on the digestion and utilization of protein and carbohydrate in sheep and goats. CT-free Centrosema pubescens was also fed for comparison with the CT legumes, and each legume was included (300 g/kg DM) in a basal diet of pangola grass (Digitaria decumbens). Pangola grass alone was used as a control diet. There were no significant (P > 0.05) differences between sheep and goats for the efficiency of digestion of N (0.574, SE 0.013), organic matter (OM; 0.519, SE 0.010), neutral-detergent fibre (NDF; 0.524, SE 0.011) and acid-detergent fibre (ADF; 0.407, SE 0.016). Diets containing desmodium and calliandra were digested less well in the rumen (64 and 62% of total OM digested) when compared with the pangola and centrosema diets (74 and 73% of total OM digested in rumen). There was an apparent net gain of 30% in ADF across the digestive tract of sheep and goats given calliandra, and this gain was ascribed to the formation of 'artifact' fibre as a result of fibre-tannin interaction. Overall, inclusion of legume at 300 g/kg in the diet significantly increased (P < 0.05) the concentration of acetic acid and decreased butyric acid concentration in the rumen fluid of sheep and goats. Significantly higher proportions of dietary N apparently reached the abomasum of animals given the diets containing desmodium (50%) and calliandra (56%) when compared with animals given the centrosema and pangola diets (35%). Sheep and goats given the CT diets also had higher excretions of faecal N. This increment of faecal N (14%) did not affect post-rumen N digestion (P > 0.05) since animals given CT diets absorbed more N (19%) per kg total OM digested than those given the control diets. It was concluded that whilst the low levels of CT provided in desmodium (1.0%) and calliandra (2.3%) diets protected dietary protein from degradation in the rumen, there were no overall beneficial or detrimental effects of CT in these diets for sheep or goats. A method was developed to categorize CT into fractions representative of their forms (free, protein-bound, and fibre-bound) during the digestion process. A quantitative model of CT metabolism during passage through the digestive tract was developed from the measured exchanges of CT between free, protein-bound and fibre-bound pools in the rumen and lower digestive tract. CT interchange mainly occurred in the reticulo-rumen of both animal species. Desmodium and calliandra free CT showed net losses of 68 and 78% in the rumen respectively and 57 and 68% of the fibre-bound CT was lost in the same site for sheep and goats respectively. However, protein-bound CT increased across the rumen by 73 and 56% for both animal species. Post-rumen losses of the total CT abomasal flow were 86 and 83% (free CT) for sheep and goats respectively, 70 and 66% (protein-bound CT), whilst 28% loss of fibre-bound CT occurred in sheep and goats respectively.

Animals↗

PubMed2001

How do tympanic-membrane perforations affect human middle-ear sound transmission?

Although tympanic-membrane (TM) perforations are common sequelae of middle-ear disease, the hearing losses they cause have not been accurately determined, largely because additional pathological conditions occur in these ears. Our measurements of acoustic transmission before and after making controlled perforations in cadaver ears show that perforations cause frequency-dependent loss that: (1) is largest at low frequencies; (2) increases as perforation size increases; and (3) does not depend on perforation location. The dominant loss mechanism is the reduction in sound-pressure difference across the TM. Measurements of middle-ear air-space sound pressures show that transmission via direct acoustic stimulation of the oval and round windows is generally negligible. A quantitative model predicts the influence of middle-ear air-space volume on loss; with larger volumes, loss is smaller.

Ear, Middle↗

PubMed1996

Interactions between memory scanning and visual scanning in display monitoring.

Many real-world tasks require the simultaneous performance of memory scanning of several memorized items and visual scanning of several physically separated sources of information in the visual field. This paper reports a study that was conducted to quantify the possible interactions between memory scanning and visual scanning. A quantitative model was derived to integrate Sternberg's linear model of memory scanning and Neisser's linear model of visual scanning. The derived model was tested through two experiments. The experiments simulated a process controller's task of monitoring an array of instrument meters to detect if any of them indicated a system error. The subjects were required to keep a number of items in their working memory (the definition of errors) and search through an organized array of instrument meters to decide whether any of the meters carried an item that matched any of the memorized items in their working memory. Two experimental factors were investigated in both experiments: the number of memorized items and the number of circles needed to be searched. These defined memory scanning demand and visual scanning demand respectively. Experiment 1 employed a different set of memorized items for each experimental trial, whereas experiment 2 employed the same set for all the trials that had the same experimental condition. The experiments identified both the strengths and the limitations of the derived model. Implications for human-machine interface design and human performance modelling are discussed.

Humans↗

PubMed1994

Cortical cell assemblies: a possible mechanism for motor programs.

The concept of a motor program has been used to interpret a diverse range of empirical findings related to preparation and initiation of voluntary movement. In the absence of an underlying mechanism, its exploratory power has been limited to that of an analogy with running a stored computer program. We argue that the theory of cortical cell assemblies suggests a possible neural mechanism for motor programming. According to this view, a motor program may be conceptualized as a cell assembly, which is stored in the form of strengthened synaptic connections between cortical pyramidal neurons. These connections determine which combinations of corticospinal neurons are activated when the cell assembly is ignited. The dynamics of cell assembly ignition are considered in relation to the problem of serial order. These considerations lead to a plausible neural mechanism for the programming of movements and movement sequences that is compatible with the effects of precue information and sequence length on reaction times. Anatomical and physiological guidelines for future quantitative models of cortical cell assemblies are suggested. By taking into account the parallel re-entrant loops between the cerebral cortex and basal ganglia, the theory of cortical cell assemblies suggests a mechanism for motor plans that involve longer sequences. The suggested model is compared with other existing neural network models for motor programming.

Journal Article↗

PubMed2004

The number of spectral channels required for speech recognition depends on the difficulty of the listening situation.

Cochlear implants provide a limited number of electrodes, each of which represents a channel of spectral information. Studies have shown that implant recipients are not receiving all of the information from the channels presented to their implant. The present paper provides a quantitative framework for evaluating how many spectral channels of information are necessary for speech recognition. Speech and melody recognition data from previous studies with cochlear implant simulations are compared as a function of the number of spectral channels of information. A quantitative model is applied to the results. Speech recognition performance increases as the number of spectral channels increases. A sigmoid function best describes this increase when plotted as a function of the log number of channels. As speech materials become more difficult, the function shifts to the right, indicating that more spectral channels of information are required. A model proposed by Plomp provides a single index to relate the difficulty of the task to the number of spectral channels needed for moderate recognition performance. In conclusion, simple sentence recognition in quiet can be achieved with only 3-4 channels of spectral information, while more complex materials can require 30 or more channels for an equivalent level of performance. The proposed model provides a single index that not only quantifies the number of functional channels in a cochlear implant, but also predicts the level of performance for different listening tasks.

Audiometry, Speech↗

PubMed1992

Lower extremity applications of functional neuromuscular stimulation.

Functional electrical stimulation (FES) applications in the lower extremity are common in research laboratories, but clinical applications are minimal. This review summarizes current knowledge with respect to clinical application. When electrical stimulation is used in clinical applications for functional movement such as standing and walking, it is typically applied in an open-loop manner; a predetermined stimulus pattern is delivered regardless of the consequences of the actual movement. Few clinical applications of FES involve closed-loop control because of the numerous difficulties involved in its application. As with any volitional muscle contraction, electrically stimulated muscle contractions will exhibit fatigue. Although the dynamics of fatigue may differ, electrically stimulated muscle contractions cannot be continuously sustained, and if the duty cycle is too severe, even alternating periods of rest and contraction cannot be sustained at a constant force level. The exact nature of fatigue is highly specific to the past history of the individual muscle and to the individual subject. Despite their intricate detail, quantitative modeling studies have not yet been applied extensively to clinical applications. Present implantable systems are not yet a viable option for clinical application. It is not clear whether more success with surface or percutaneous systems must first be achieved to justify implantation or whether greater improvements in implantable technology and surgical protocols are needed before implantable systems will become practical. It is clear that almost any reasonably designed stimulation protocol will increase muscle bulk. The existence of other therapeutic benefits and their cost/benefit ratios remain to be fully established. It is possible to stand through bilateral stimulation of the quadriceps. Using surface electrodes, this technique is achievable in any physical therapy clinic having minimal expertise in neuromuscular stimulation. FES-aided standing must be conducted as a research project with a protocol approved by the local institutional review board, as there are currently no FDA-approved stimulation devices for standing. Multichannel FES systems are not currently available for clinical application in the United States. This may change if the "Parastep" system receives FDA approval. Percutaneous and implanted systems are years away from commercialization and clinical availability. Hybrid systems, based primarily on the reciprocating gait orthosis (RGO), are presently the only clinically available form of walking that includes some form of FES assistance. The costs and benefits of adding FES to the RGO and the long-term user acceptance rate for these systems remain to be determined.

Cerebrovascular Disorders↗

PubMedDate not supplied

Evidence for competitive inhibition of iodide uptake by perchlorate and translocation of perchlorate into the thyroid.

Various published data sets that investigate the potential effect of exogenous perchlorate (ClO4-) on the uptake of iodide in the thyroid and subsequent changes in thyroid hormone levels are available. In order to best use the data towards the prediction of human health effects resulting from ClO4- exposure, the available literature data must be integrated into a self-consistent, coherent, and parsimonious quantitative model based on the most likely mode of action of perchlorate effect on thyroid function. We submit that the simplest mode of action for ClO4- in the thyroid that remains consistent with all available data involves competitive inhibition of iodide transport into the thyroid follicle, transport of perchlorate into the thyroid follicle against a concentration gradient, further transport into the thyroid lumen (where it may again interfere with iodide transport), and, finally, passive diffusion back into the blood. We believe this description of perchlorate's kinetic behavior should serve as the foundation for predictive physiologically based pharmacokinetic (PBPK) models and as a working hypothesis for further experimental exploration.

Animals↗

PubMed2005

Spines and neurite branches function as geometric attractors that enhance protein kinase C action.

Ca2+ and diacylglycerol-regulated protein kinase Cs (PKCs; conventional PKC isoforms, such as PKCgamma) are multifunctional signaling molecules that undergo reversible plasma membrane translocation as part of their mechanism of activation. In this article, we investigate PKCgamma translocation in hippocampal neurons and show that electrical or glutamate stimulation leads to a striking enrichment of PKCgamma in synaptic spines and dendritic branches. Translocation into spines and branches was delayed when compared with the soma plasma membrane, and PKCgamma remained in these structures for a prolonged period after the response in the soma ceased. We have developed a quantitative model for the translocation process by measuring the rate at which PKCgamma crossed the neck of spines, as well as cytosolic and membrane diffusion coefficients of PKCgamma. Our study suggests that neurons make use of a high surface-to-volume ratio of spines and branches to create a geometric attraction process for PKC that imposes a delayed enhancement of PKC action at synapses and in peripheral processes.

Animals↗

PubMed2006

Mechanism of transport of IFT particles in C. elegans cilia by the concerted action of kinesin-II and OSM-3 motors.

The assembly and function of cilia on Caenorhabditis elegans neurons depends on the action of two kinesin-2 motors, heterotrimeric kinesin-II and homodimeric OSM-3-kinesin, which cooperate to move the same intraflagellar transport (IFT) particles along microtubule (MT) doublets. Using competitive in vitro MT gliding assays, we show that purified kinesin-II and OSM-3 cooperate to generate movement similar to that seen along the cilium in the absence of any additional regulatory factors. Quantitative modeling suggests that this could reflect an alternating action mechanism, in which the motors take turns to move along MTs, or a mechanical competition, in which the motors function in a concerted fashion to move along MTs with the slow motor exerting drag on the fast motor and vice versa. In vivo transport assays performed in Bardet-Biedl syndrome (BBS) protein and IFT motor mutants favor a mechanical competition model for motor coordination in which the IFT motors exert a BBS protein-dependent tension on IFT particles, which controls the IFT pathway that builds the cilium foundation.

Animals↗

PubMed1989

The specificity of rejection and the absence of susceptibility of pancreatic islet beta cells to nonspecific immune destruction in mixed strain islets grafted beneath the renal capsule in the rat.

The specificity of rejection of isolated pancreatic islets was examined in the rat using a quantitative model in which syngeneic (DA) or a mixture of syngeneic and allogeneic (DA and LEW or PVG) islets were implanted beneath the capsule of the kidney of nondiabetic normal rats (DA). 3 wk after transplantation total insulin extraction assays of the kidney with its islet implant together with immunohistological examination of the site of transplantation for evidence of syngeneic or allogeneic tissue demonstrated the total destruction of allogeneic islets without any evidence of damage to syngeneic islets either distant or in immediate proximity to allogeneic islets. Pancreatic islets, and especially beta cells, appear to be particularly vulnerable to the effector arm of both autoimmune and alloimmune responses, a vulnerability that has been attributed to the cytotoxic effects of lymphokines, notably IL-1, released in both autoimmune and alloimmune responses. The experiments reported here demonstrate not only the exquisite specificity of the allograft reaction but are not compatible with a hypothesis that B cells within an intact islet are nonspecifically susceptible to destruction by lymphokines.

Animals↗

PubMed1994

Inositol (1,4,5)-trisphosphate (InsP3)-gated Ca channels from cerebellum: conduction properties for divalent cations and regulation by intraluminal calcium.

The conduction properties of inositol (1,4,5)-trisphosphate (InsP3)-gated calcium (Ca) channels (InsP3R) from canine cerebellum for divalent cations and the regulation of the channels by intraluminal Ca were studied using channels reconstituted into planar lipid bilayers. Analysis of single-channel recordings performed with different divalent cations present at 55 mM on the trans (intraluminal) side of the membrane revealed that the current amplitude at 0 mV and the single-channel slope conductance fell in the sequence: Ba (2.2 pA, 85 pS) > Sr (2.0 pA, 77 pS) > Ca (1.4 pA, 53 pS) > Mg (1.1 pA, 42 pS). The mean open time of the InsP3R recorded with Ca (2.9 ms) was significantly shorter than with other divalent cations (approximately 5.5 ms). The "anomalous mole fraction effect" was not observed in mixtures of divalent cations (Mg and Ba), suggesting that these channels are single-ion pores. Measurements of InsP3R activity at different intraluminal Ca levels demonstrated that Ca in the submillimolar range did not potentiate channel activity, and that very high levels of intraluminal Ca (> or = 10 mM) decreased channel open probability 5-10-fold. When InsP3R were measured with Ba as a current carrier in the presence of 110 mM cis potassium, a PBa/PK of 6.3 was estimated from the extrapolated value for the reversal potential. When the unitary current through the InsP3R at 0 mV was measured as a function of the permeant ion (Ba) concentration, the half-maximal current occurred at 10 mM trans Ba. The following conclusions are drawn from these data: (a) the conduction properties of InsP3R are similar to the properties of the ryanodine receptor, another intracellular Ca channel, and differ dramatically from the properties of voltage-gated Ca channels of the plasma membrane. (b) The estimated size of the Ca current through the InsP3R under physiological conditions is 0.5 pA, approximately four times less than the Ca current through the ryanodine receptor. (c) The potentiation of InsP3R by intraluminal Ca in the submillimolar range remains controversial. (d) A quantitative model that explains the inhibitory effects of high trans Ca on InsP3R activity was developed and the kinetic parameters of InsP3R gating were determined.

Animals↗