Actin, cofilin and cognition.
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Actin Depolymerizing Factors↗
Biomedical subjects
J Rosenblatt
Publications and source records attributed to J Rosenblatt.
The mathematics behind modeling.
The purpose of this chapter has been to furnish insight into the theoretical background on which compartmental modeling software packages are based. To accomplish this goal, only the basic ideas were stressed, avoiding discussion of the intricacies required for efficiency. The object was to remove the mystery from these powerful programs by examining the fundamental ideas which make them tick. The first section was concerned with how compartmental models are built up and how to obtain information concerning the system behavior described by these models. The cornerstone here is to describe a system by determining how it behaves over (typically) very short time periods. This leads to a differential equation description of a compartmental system. Information can be extracted from these equations by returning to their basic meaning, illustrated in their derivation. Computers are ideal for obtaining this information by piecing together the results obtained over short time periods, to find the behavior over long time periods. In an actual situation governed by a compartmental model, it's often the case that we may know only the form of the model, but not the values of the rate constants which must be known for its effective use. The second section of the chapter was devoted to the practical problem of determining the rate constants of the model, based on observed data. This is a matter of searching for those values which, in some sense, best fit the data. To attack this problem we need a reasonable criterion to judge how well a proposed model fits the data. We chose to use the total squared deviation, psi, which is the most common such criterion--but not the only reasonable one. The search technique we examined--steepest descent--is based on a simple idea: looking at the total squared deviation criterion geometrically. In graphical terms, the best fit corresponds to finding the low point on a surface, whose height above any point at sea level is computable. If we could imagine the view of someone trying to find the low point from some arbitrarily chosen initial position on this mountain-like surface, we would look around and find the direction where (close by) the mountain drops off most steeply. We would go in that specific direction until we reach a low point, moving only along this initially chosen direction. At this new low point, we could change again to a direction of steepest descent, and keep repeating this process until we make no further effective downward progress. No guarantee in general is made for this process, but it often works. Finally, having found the best values for the rate constants, we must recognize that if the data is affected by factors not explicitly taken into account in the model, the variability induced by these factors precludes a perfect fit. For this reason, it is finally necessary to determine how good the model is, as a description of the data, and how accurate are the fitted rate constants. For the model fit, the sample RMS error (simply related to the total squared deviation, and often the same as the sample standard deviation) may be used. For determining the accuracy of the fitted rate constants, practical methods based on computer software simulations are recommended.
Computer Simulation↗
High-speed, three-dimensional kinematic analysis of the normal wrist.
Carpal kinematics during a wrist flexion/extension motion using high-speed videodata acquisition was investigated. A cadaver forearm was stabilized, allowing unconstrained excursion of the wrist for passive range of motion (ROM). The extensor and flexor pairs of the wrist were looped together and a 1-lb weight was attached to each pair, simulating synergistic muscle tension. Capitate/radius and third metacarpal/radius angles were calculated to determine which measurement would be best for determining global wrist angle. The average difference in capitate/radius and third metacarpal/radius angles at each respective flexion/extension wrist angle for all wrists was 1.1 degrees +/- 1.6 degrees (the maximum difference was 4 degrees). Hence, the capitate-third metacarpal joint can be considered rigid. Capitate/lunate motion as described by capitate-radius Euler angles ranged from -16.9 to 23.5 with total capitate/lunate motion of 40.5 (35%) in the 114 degrees total global wrist ROM measured. Radius/lunate motion as described by lunate-radius angle ranged from -8.2 to 48.4 with total radius/lunate motion of 56.5 (49%) in the 114 degrees total global wrist ROM measured. During global wrist motion, the radiolunate joint contributes more motion in flexion than the capitolunate joint and the capitolunate joint contributes more motion in extension than the radiolunate joint. The instantaneous screw axes (ISAs) were calculated for each third metacarpal position with respect to the radius. The average distance difference between ISAs for the 4 wrists tested was -1.23 +/- 14.97 pixels. The maximum distance was 56.51 pixels and the minimum was -24.09 pixels. This new combination of motion analysis and 3-dimensional reconstructions of computed tomography images affords a high-speed, dynamic analysis of kinematics. It shows that during wrist flexion/extension, normal carpal kinematics does not have an ISA fixed in or limited to the capitate. In addition, the ISA data provide evidence that translational motion is a real and measurable component of normal carpal motion. These findings alter the understanding of carpal kinematics obtained from the results of previous studies which suggested that the center of rotation was fixed in the capitate.
Adult↗
Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails.
In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly. Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell. Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration. Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails. Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts. Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization. Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate. Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length. Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent. These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts. We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog. This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.
Actin Cytoskeleton↗
Actin dynamics in vivo.
Actin dynamics in lamellipodia are driven by continuous cycles of actin polymerization, retrograde flow, and depolymerization. In the past year, advances have been made in identifying signaling pathways that regulate actin-filament uncapping and polymerization, in determining the role of myosin motor proteins in retrograde flow, and in evaluating the role of severing proteins in actin depolymerization. Both Listeria monocytogenes and Saccharomyces cerevisiae have emerged as powerful model organisms for studying actin dynamics in cells.
Actins↗
Familial nontoxic multinodular thyroid goiter locus maps to chromosome 14q but does not account for familial nonmedullary thyroid cancer.
Thyroid goiter is a common condition that is often associated with iodine deficiency. Familial forms of goiter in areas not known to feature iodine deficiency are much less common. We have performed a genomic search on a single large Canadian family with 18 cases of nontoxic multinodular goiter in which 2 individuals also had papillary lesions highly suggestive of papillary carcinoma. A locus on chromosome 14q (MNG1 [multinodular goiter 1]) has been identified, with a maximal two-point LOD score of 3.8 at D14S1030 and a multipoint LOD score of 4.88 at the same marker, defined by D14S1062 (upper boundary) and D14S267 (lower boundary). The gene encoding thyroid-stimulating hormone receptor (TSHR), which is located on chromosome 14q, is outside the linked region. To determine the role of this gene in familial nonmedullary thyroid cancer (NMTC), we studied 37 smaller pedigrees each containing at least two cases of NMTC. Analysis by both parametric and nonparametric methods indicates that only a very small proportion of familial NMTC (point estimate 0.001, support intervals 0-.6 under a dominant model) is attributable to MNG1.
Canada↗
Glucose kinetics in interstitial fluid can be predicted by compartmental modeling.
The purposes of this study were to define in sheep a compartmental model for glucose kinetics in the basal condition and to test the hypothesis that interstitial fluid obtained by sampling thoracic duct lymph (TDL) represents one or more peripheral compartments of the glucose model. A bolus of [6,6-(2)H]glucose was injected in nine animals, followed by frequent sampling of blood and TDL. Linear kinetic modeling has been applied to plasma data, indicating that a three-compartment model adequately describes glucose kinetics. Both catenary and mammillary models were identified, and their predictions for the tracer behavior in nonaccessible pools were evaluated. In all experiments, regardless of the model structure, predictions in the compartment most rapidly exchanging with blood (pool 2) well matched the measured tracer-to-tracee ratio in TDL. Furthermore, modeling analysis showed that 90-95% of the tracer in TDL comes from this pool. This supported the physiological identification of a peripheral pool of glucose kinetics as extracellular fluid represented by TDL measurements.
Animals↗
Normal wrist kinematics and the analysis of the effect of various dynamic external fixators for treatment of distal radius fractures.
The purpose of this study was to evaluate the effect that external fixation devices used for distal radius fractures have on normal carpal kinematics, using high speed video data acquisition. Cadaver forearms were stabilized, allowing free excursion of the wrist for passive range of motion. Synergistic muscle tension was simulated by looping the extensor and flexor pairs of the wrist. Global wrist flexion and extension, intercarpal angles, and the instantaneous screw axis (ISA) were studied. These parameters allowed a quantification of normal carpal kinematics and comparison to the kinematics of the carpal bones with external fixators attached. This study shows that normal carpal kinematics during wrist flexion and extension do not have an ISA fixed in or limited to the capitate. In addition, the ISA data provide evidence that translational motion is a real and measurable component of normal carpal motion. This would change the understanding of carpal kinematics in previous studies that suggested that the center of rotation was fixed in the capitate. The proximal carpal (radiolunate) joint contributes more motion (has a greater rotation angle) in normal global wrist flexion and the midcarpal (capitolunate) joint contributes more motion in normal global wrist extension. In addition, the global range of motion and intercarpal joint angle of the wrist are limited with the application of any of the external fixators tested. None of the external fixators allowed normal ISA vector surfaces during wrist motion.
Adult↗
A purified Drosophila septin complex forms filaments and exhibits GTPase activity.
Septin proteins are necessary for cytokinesis in budding yeast and Drosophila and are thought to be the subunits of the yeast neck filaments. To test whether septins actually form filaments, an immunoaffinity approach was used to isolate a septin complex from Drosophila embryos. The purified complex is comprised of the three previously identified septin polypeptides Pnut, Sep2, and Sep1. Hydrodynamic and sequence data suggest that the complex is composed of a heterotrimer of homodimers. The complex copurifies with one molecule of bound guanine nucleotide per septin polypeptide. It binds and hydrolyzes exogenously added GTP. These observations together with conserved sequence motifs identify the septins as members of the GTPase superfamily. We discuss a model of filament structure and speculate as to how the filaments are organized within cells.
Actin Cytoskeleton↗
Response of phosphoenolpyruvate cycle activity to fasting and to hyperinsulinemia in human subjects.
We have used a new isotopic tracer technique to investigate the physiological role of the phosphoenolpyruvate (PEP) cycle in metabolic adaptation to fasting and to hyperinsulinemia. The forward direction of the PEP cycle is the conversion of oxaloacetate (OAA) to PEP, and the net flux of the cycle is the rate at which PEP from OAA goes on to form glucose or glycogen, as opposed to being recycled to pyruvate and then OAA. Normal volunteers (n = 6) were studied after an overnight fast and then again after 3 days of fasting, and five additional subjects were studied during a hyperinsulinemic clamp (insulin concentration = 568 +/- 25 microU/ml, glucose infusion = 14.2 +/- 0.55 mg.kg-1.min-1). After an overnight fast, 35.4 +/- 6.7% of PEP from OAA was recycled to pyruvate-lactate. Short-term fasting caused a significant increase in the conversion of OAA to PEP and also a drop in the percentage of PEP from OAA that went to pyruvate-lactate to 15.2 +/- 4.0%. The principal response to hyperinsulinemia was a decrease in the recycling of OAA to lactate, because there was no significant change in the conversion of OAA to PEP. We conclude that changes in both directions of the PEP cycle are important in regulating gluconeogenic-glyconeogenic flux.
Fasting↗
Comparison of mass isotopomer dilution methods used to compute VLDL production in vivo.
The recent development of mass isotopomer distribution methods represents an important new tool for quantifying synthetic rates. These methods allow precursor enrichment to be determined indirectly from the enrichment of the product, thus sidestepping the often difficult problem of measuring the precursor enrichment. Two different methods have been described to compute synthetic rates by this general approach in the laboratories of M. K. Hellerstein and J. K. Kelleher, and variations of these basic approaches have also been presented by W. N. Lee and by ourselves. A comparison between the different methods has never been reported. In this paper, we take a specific application, calculation of the fractional rate of incorporation of acetyl-CoA into very low density lipoprotein-bound palmitate, and compare the results obtained from all of the mass isotopomer methods using the same data set obtained in vivo in human subjects. We found that it is critical that the measured background isotopomer distribution of palmitate is used rather than the theoretical background isotopomer distribution. We also found that the different methods give comparable precursor enrichments and comparable fractional synthesis rates, provided that the enrichments in Kelleher's method are properly weighted. Thus the choice of method to use is a matter of personal preference.
Humans↗
The effect of electrostatic charge interactions on release rates of gentamicin from collagen matrices.
PURPOSE: This work studied the effect of changes in the magnitude of electrostatic charge interactions on the release kinetics of gentamicin from collagen matrices. METHODS: The charge distribution on collagen was altered by specific charge chemistries to yield net negative charges which exhibited binding interactions with positively charged gentamicin. The adsorption isotherms were measured to characterize binding interactions and release of gentamicin from modified matrices were measured. The release rates were compared to a mathematical model based on an instantaneous desorption coupled with diffusion mechanism. RESULTS: Ninety percent of the gentamicin loaded was released from native collagen matrices in 2.5 days (one-sided slab geometry in-vitro). Succinylated collagen matrices released 70% in 2.5 days and phosphonylated collagen matrices released 50% in 2.5 days. Excellent agreement between model predictions and experiment results were obtained. CONCLUSIONS: Modified collagen can be much more effective in antibiotic therapy in sustaining release rates compared to native collagen for charged antibiotics like gentamicin.
Adsorption↗
The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.
Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.
Actins↗
Evaluation of enzyme-linked immunoassay for serological diagnosis of cysticercosis.
We evaluated a commercially available enzyme-linked immunoassay (ELISA) from LMD Laboratories, Inc., Carlsbad, Calif., for the detection of antibodies in serum to the cysticercus of Taenia solium. The ELISA was performed on 308 serum samples; 198 from a pool of healthy individuals, 42 from patients who had antibodies against a variety of parasites other than T. solium, and 68 from patients suspected of having cysticercosis. All of these 68 specimens were tested both by the ELISA and by an immunoblot method (enzyme-linked immunoelectrotransfer blot assay [EITB]) developed at the Parasitic Serology Laboratory of the Centers for Disease Control and Prevention. Twenty-seven of the 68 serum samples from patients suspected of having cysticercosis were positive by both EITB and ELISA, while 31 were negative by both assays. ELISA results for three and two samples were considered false positive and false negative, respectively, when compared with the results of EITB. Results for an additional five samples were considered equivocal but were technically positive because their optical density readings were slightly above the cutoff value. Three of the 198 serum samples from the bank of serum samples from healthy individuals were also false positive by ELISA (the EITB result for the samples was negative). Six other serum samples from healthy individuals which had equivocal results and the five serum samples from patients with equivocal results were EITB negative. Serum samples containing antibodies against Echinococcus spp. frequently cross-reacted with the cysticercus ELISA antigen (13 of 16 specimens), but serum samples with antibodies against other parasites did not (2 of 26 specimens); all of these serum samples were EITB negative. The commercially available ELISA that we describe is a simple and rapid test. Considering all 308 specimens, the ELISA had a specificity of 93% (when samples with equivocal results were considered negative) or 89% (when samples with equivocal results were considered positive); the sensitivity was 93%.
Animals↗
Practice tip. Simple removal of xanthoma.
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Eyelid Diseases↗
Facial edema in a woman with a history of histiocytosis X.
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Adult↗
Hormone replacement therapy.
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Estrogen Replacement Therapy↗
PITALRE, a nuclear CDC2-related protein kinase that phosphorylates the retinoblastoma protein in vitro.
Members of the cell division cycle 2 (CDC2) family of kinases play a pivotal role in the regulation of the eukaryotic cell cycle. In this communication, we report the isolation of a cDNA that encodes a CDC2-related human protein kinase temporarily designated PITALRE for the characteristic Pro-Ile-Thr-Ala-Leu-Arg-Glu motif. Its deduced amino acid sequence is 47% identical to that of the human cholinesterase-related cell division controller (CHED) kinase, which is required during hematopoiesis, and 42% identical to the Saccharomyces cerevisiae SGV1 gene product, a putative kinase involved in the response to pheromone via its guanine nucleotide-binding protein alpha subunit. PITALRE expression is ubiquitous, but its expression levels are different in various human tissues. PITALRE is an approximately 43-kDa protein that associates with three cellular polypeptides of 80, 95, and 155 kDa. PITALRE is localized primarily to the nucleus. In addition, we have identified a retinoblastoma protein kinase activity associated with PITALRE immunocomplexes that cannot phosphorylate histone H1, suggesting that the target phosphorylation site of PITALRE differs from that of CDC2 kinase. Interestingly, the retinoblastoma kinase activity associated with PITALRE does not oscillate during the cell cycle.
Amino Acid Sequence↗