Ca2+ regulation of gelsolin activity: binding and severing of F-actin

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Severing of F-actin by the amino-terminal half of gelsolin suggests internal cooperativity in gelsolin

Gelsolin is a Ca2+-regulated actin-binding protein that can sever, cap, and nucleate growth from the pointed ends of actin filaments. In this study we have measured the binding of the amino-terminal half of gelsolin, G1-3, to pyrene-labeled F-actin as a function of Ca2+ concentration. The rate of binding is shown to be dependent on micromolar concentrations of Ca2+. Independent experiments demonstrate that conformational changes in G1-3 are induced by micromolar concentrations of Ca2+. Titrations of pyrene-F-actin with G1-3 and gelsolin show that the quenching of pyrene fluorescence is identical in extent and stoichiometry for both G1-3 and gelsolin. In contrast, severing of F-actin by G1-3 is found to be much less efficient than is severing by gelsolin. In experiments in which F-actin severing is quantitatively measured, the filament number is found to be proportional to the 1.35 power of the G1-3 concentration. This deviation from linearity may be explained by cooperativity; the binding of two G1-3 molecules in close proximity may lead to cooperative severing of the polymer, thus increasing the severing efficiency. This model is supported by experiments that show that the efficiency of G1-3 severing of F-actin increases with increasing G1-3:F-actin ratios. Extrapolating from these results, we conclude that G4-6, the carboxyl-terminal half of gelsolin, has an active role in the severing of F-actin by intact gelsolin. Whereas F-actin severing by G1-3 is enhanced by cooperative binding of two separate G1-3 molecules, cooperativity is inherent to intact gelsolin because the cooperative partners are covalently linked.     

Tracer diffusion through F-actin: effect of filament length and cross-linking

We have determined diffusion coefficients for small (50- to 70-nm diameter) fluorescein-thiocarbamoyl-labeled Ficoll tracers through F-actin as a function of filament length and cross-linking. fx45 was used to regulate filament length and avidin/biotinylated actin or ABP-280 was used to prepare cross-linked actin gels. We found that tracer diffusion was generally independent of filament length in agreement with theoretical predictions for diffusion through solutions of rods. However, in some experiments diffusion was slower through short (< or = 1.0 micron) filaments, although this result was not consistently reproducible. Measured diffusion coefficients through unregulated F-actin and filaments of lengths > 1.0 micron were more rapid than predicted by theory for tracer diffusion through rigid, random networks, which was consistent with some degree of actin bundling. Avidin-induced cross-linking of biotinylated F-actin did not affect diffusion through unregulated F-actin, but in cases where diffusion was slower through short filaments this cross-linking method resulted in enhanced tracer diffusion rates indistinguishable from unregulated F-actin. This finding, in conjunction with increased turbidity of 1.0-micron filaments upon avidin cross-linking, indicated that this cross-linking method induces F-actin bundling. By contrast, ABP-280 cross-linking retarded diffusion through unregulated F-actin and decreased turbidity. Tracer diffusion under these conditions was well approximated by the diffusion theory. Both cross-linking procedures resulted in gel formation as determined by falling ball viscometry. These results demonstrate that network microscopic geometry is dependent on the cross-linking method, although both methods markedly increase F-actin macroscopic viscosity.     

Functional characteristics and the complete primary structure of ascidian gelsolin

DNA methyltransferase is an enzyme responsible for generating and maintaining DNA methylation patterns. DNA methylation patterns control different genome functions, thus they are an important component of the epigenetic information. It has been recently postulated that DNA methyltransferase plays an important role in oncogenesis and that it is a candidate target for anticancer therapy. This commentary discusses the possible mechanisms through which DNA methyltransferase participates in oncogenesis and the rationale for targeting it in cancer.     

Quasi- and nonequivalence in the structure of bacterial flagellar filament

In supercoiled forms of flagellar filaments, which are thought to be produced by combinations of two distinct subunit lattices, the lattices are elastically deformed in 11 different ways, depending on their azimuthal positions on the circumference of a tube with 11 protofilaments. Those two interactions are nonequivalent as opposed to quasiequivalent ones in elastically deformed lattices of otherwise identical interactions. The term nonequivalence is defined to represent different bonding interactions, and quasiequivalent is used to describe deformed but conserved bonding interactions. By using two distinct lattices that were accurately determined by x-ray fiber diffraction, 10 possible supercoiled forms of flagellar filaments were simulated, based on a bistable-subunit packing model. Comparison to the observed forms showed good agreement, indicating that the model and determined lattice parameters effectively represent realistic features of the structure. The simulated quasiequivalent lattices have been compared to the two nonequivalent lattices, revealing an interesting feature: the maximum deviation in the intersubunit distance by elastic deformation is almost three-quarters of the difference between the two distinct lattices, demonstrating a balanced coexistence of a well-defined conformational distinction and extensive adaptability in the molecular structure of flagellin and its packing interactions.     

Congenital toxoplasmosis and seroconversion at the end of pregnancy: clinical observations

OBJECTIVE: To determine the knowledge of HIV-disease management and the adherence to contemporary guidelines among British Columbia physicians whose practices focused on HIV/AIDS. DESIGN: Self-administered mail survey. PARTICIPANTS: All 659 physicians registered in a province-wide HIV/AIDS drug treatment program. OUTCOME MEASURES: Data on demographic and personal characteristics of respondents, level of HIV-related experience, use of preventive vaccinations and tests, and preferred approaches to the prophylaxis and treatment of common opportunistic infections. Knowledge scores in 4 areas of patient care, as well as an overall score, were computed by comparing respondents' answers with the therapeutic strategies recommended at the time of the survey. Associations between physician characteristics and knowledge scores were identified by linear regression analysis. RESULTS: Of the 659 physicians surveyed, 65% returned responses: only 38% returned completed surveys while a further 27% returned a follow-up survey that asked nonrespondents about their demographic characteristics and HIV-related experience. Scores for specific areas of patient management ranged from 29% for the treatment of opportunistic infections to 62% for preventive measures, with a mean overall score of 47%. Physician knowledge in all areas of patient care was associated with the number of HIV-positive patients in the practice (p = 0.003 to p < 0.001). Physicians who were younger were more knowledgeable regarding preventive measures (p = 0.001); those whose practice location was in Vancouver had a greater knowledge of prophylaxis (p = 0.047); and those who had medical specialty training were more knowledgeable about the treatment of opportunistic infections (p = 0.009). CONCLUSIONS: There is substantial disparity in how physicians approach the management of HIV and related conditions. Deviations from therapeutic guidelines are common and may be associated with physician characteristics, particularly lack of experience in managing HIV.     

Leadership dynamics: Character and character structure in executives.

While the public and the mass media have continued to uphold and find relevance in the time-honored construct of character, the scientific and professional community are in the process of rediscovering a construct they had essentially relinquished for the past few decades. This paper briefly traces the recent history of character and character structure in psychology and overviews a number of promising theoretical and empirical studies of character and character structure that have particular relevance for consulting psychologists and others involved in executive coaching and consultation. Finally, it describes six commonly noted character structures in executives. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Starting at the beginning, middle, and end: translation initiation in eukaryotes

A method is described for the densitometric determination of the p-hydroxybenzoic esters and p-hydroxybenzoic acid in mixtures or in drugs. This method is compared with the one used in high performance liquid chromatography (HPLC). The calibration curves were linear in interval 0.250-3.60 mumol ml-1 per 200 nl per spot. The limit of detection and the relative standard deviation (RSD) are higher than in HPLC (RSD is 6% in HPTLC. 3% in HPLC; limit of detection about 40 pmol in HPTLC and 25 pmol in HPLC) but HPTLC quantitative determination of parabens in drugs is faster.     

Alpha and omega: studying resuscitation at the beginning and end stages of life

BACKGROUND: To clarify whether or not multiple pulmonary metastases from colorectal cancer are contraindicated for a surgical resection, we retrospectively evaluated the influence of the number of pulmonary metastases on both the postthoracotomy survival and the pattern of the first failure. METHODS: From 1981 to 1993, 36 patients underwent a complete resection for pulmonary metastases from colorectal cancer. RESULTS: Of the various factors investigated including gender, primary site, disease-free interval, tumor size, the number of metastases, type of resection, and the history of hepatic metastases, only the number of pulmonary metastases was found to be significantly related to postthoracotomy survival. The rate of disease-free survival at 5 years was 62% for solitary metastasis (n = 17), 35% for two metastases (n = 8), and 0% for four or more metastases (n = 11). The pattern of failure also differed according to the number of pulmonary metastases. In particular, the incidence of local recurrence at the primary site increased with the number of pulmonary metastases (ie, 1 of 17 patients with a solitary metastasis, 3 of 8 with two metastases, and 6 of 11 with four or more metastases). CONCLUSIONS: These results suggest that multiple metastases might indicate the presence of local recurrence at the primary site; therefore, in cases of multiple pulmonary metastases, the primary site should be thoroughly explored.     

The effect of F-actin on the binding and hydrolysis of guanine nucleotide by Dictyostelium elongation factor 1A

Indirect evidence implicates actin as a cofactor in eukaryotic protein synthesis. The present study directly examines the effects of F-actin on the biochemical properties of eukaryotic elongation factor 1A (eEF1A, formerly EF1alpha), a major actin-binding protein. The basal mechanism of eEF1A alone is determined under physiological conditions with the critical finding that glycerol and guanine nucleotide are required to prevent protein aggregation and loss of enzymatic activity. The dissociation constants (Kd) for GDP and GTP are 2.5 microM and 0.6 microM, respectively, and the kcat of GTP hydrolysis is 1.0 x 10(-3) s-1. When eEF1A binds to F-actin, there is a 7-fold decrease in the affinity for guanine nucleotide and an increase of 35% in the rate of GTP hydrolysis. Based upon our results and the relevant cellular concentrations, the predominant form of cellular eEF1A is calculated to be GTP.eEF1A.F-actin. We conclude that F-actin does not significantly modulate the basal enzymatic properties of eEF1A; however, actin may still influence protein synthesis by sequestering GTP.eEF1A away from interactions with its known translational ligands, e.g. aminoacyl-tRNA and ribosomes.     

The anthelmintic albendazole affects in vivo the dynamics and the detyrosination-tyrosination cycle of rat brain microtubules

The rate of verapamil-sensitive uptake of 45Ca by rat heart cells stimulated to beat in suspension with 0.2 mM Ca and isoproterenol was increased > 2-fold by cell loading with the chelator Quin-2. No effect of Quin-2 loading was observed on the rate of uptake of trace levels of 54Mn, present in addition to Ca, which was used as an index of Ca channel activity. Quin-2 loading also had little effect on the rate of 45Ca uptake by cells diluted into a high K/low Na medium, where Ca uptake was primarily by Na/Ca exchange. The fast chelator 1,2-bis(o-aminophenoxy)ethane-N,N,-N',N'-tetraacetic acid (BAPTA) was 3-fold more effective than the slow chelator EGTA at preventing Ca efflux. BAPTA loading also caused an increase in sarcoplasmic reticulum (SR) Ca content. These results suggest that chelator loading had little effect on the rate of Ca influx by Ca channels or by Na/Ca exchange, and that the increased rate of 45Ca uptake seen with Quin-2 loading was caused by an inhibition of Ca efflux, either directly by chelation or by increased Ca uptake by the SR or by other intracellular organelles. This further suggests that most of the Ca entering the cell without chelator leaves again within the same beat, and that this may result from Ca efflux from a kinetically limited Ca pool in or around the diad cleft.     

Relative changes in F-actin during the first cell cycle: evidence for two distinct pools of F-actin in the sea urchin egg

The cortical actin cytoskeleton undergoes dramatic rearrangements during fertilization of sea urchin eggs. To characterize these changes further, we quantified the relative changes in filamentous actin (F-actin) during fertilization and the first cell cycle in both intact eggs and in isolated cortices by quantitative fluorescence microscopy. The level of F-actin in the intact egg decreased after fertilization and continued to decrease throughout the first cell cycle. By 60 min after fertilization, the level of F-actin had decreased to 50% of the unfertilized sea urchin egg. By cytokinesis, the level of F-actin had decreased to 30% of the unfertilized egg. After completion of cell division, individual blastomeres had 10% of the F-actin in the unfertilized egg. In contrast, there was an increase in cortical F-actin to 370% of the level in the unfertilized egg after fertilization. This increase corresponded to the formation of microvilli. There was little change in the level of cortical F-actin during the first cell cycle. We draw parallels to other systems that increase the amount of F-actin in the Triton-insoluble cytoskeleton by recruiting actin from a Triton-soluble pool of F-actin.     

How a protein prepares for B12 binding: structure and dynamics of the B12-binding subunit of glutamate mutase from Clostridium tetanomorphum

BACKGROUND: Glutamate mutase is an adenosylcobamide (coenzyme B12) dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S,3S)-3-methylaspartate. The enzyme from Clostridium tetanomorphum comprises two subunits (of 53.7 and 14.8 kDa) and in its active form appears to be an alpha 2 beta 2 tetramer. The smaller subunit, termed MutS, has been characterized as the B12-binding component. Knowledge on the structure of a B12-binding apoenzyme does not exist. RESULTS: The solution structure and important dynamical aspects of MutS have been determined from a heteronuclear NMR study. The global fold of MutS in solution resembles that determined by X-ray crystallography for the B12-binding domains of Escherichia coli methionine synthase and Propionibacterium shermanii methylmalonyl CoA mutase. In these two proteins a histidine residue displaces the endogenous cobalt-coordinating ligand of the B12 cofactor. In MutS, however, the segment of the protein containing the conserved histidine residue forms part of an unstructured and mobile extended loop. CONCLUSIONS: A comparison of the crystal structures of two B12-binding domains, with bound B12 cofactor, and the solution structure of the apoprotein MutS has helped to clarify the mechanism of B12 binding. The major part of MutS is preorganized for B12 binding, but the B12-binding site itself is only partially formed. Upon binding B12, important elements of the binding site appear to become structured, including an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the cofactor.     

Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 A resolution

Using a liquid-helium-cooled superconducting electron cryo-microscope, we obtained low-dose images of negatively stained preparations at 4 K and collected structural data to 1/9.6 -1 for flagellar filaments from the strain SJW117 of Salmonella typhimurium (serotype gt). The subunits of this left-handed, straight filament are non-helically perturbed in a pairwise manner. The perturbation corresponds to an alternating conformation in every other row of subunits. These are the 5-start rows and, necessarily, the resulting structure has a seam. The perturbation is not confined to the outside but extends into the structure. We separated the non-symmetric and symmetric parts of the structural data and generated a three-dimensional reconstruction from the latter. The resulting density map is a structure similar in domain organization to the left-handed filament of S. typhimurium SJW1660. Filtered images generated from the non-symmetric component show an ordered and polar structure. The nature of the perturbation was analyzed by model building using a sphere to represent the subunit at low resolution. A lateral shift of approximately 10 degrees mimics the perturbation.     

Role of the outermost subdomain of Salmonella flagellin in the filament structure revealed by electron cryomicroscopy

A mutant strain of Salmonella typhimurium, SJW46, has flagellar filaments supercoiled in the same form as the wild-type strain, SJW1103, and swims normally. However, its flagellar filaments are mechanically unstable and show anomalous behaviors of polymorphism. Flagellin from SJW46 has a large central deletion from Ala204 to Lys292 of SJW1103 flagellin, which has been thought to be located in the outer surface of the filament. Since the filament structure is determined by intersubunit interactions of the terminal regions in the densely packed core of the filament, no serious involvement of the deleted portion was expected in the filament stability and polymorphism. In order to locate the deleted portion and to understand the underlying mechanism of these anomalous characteristics, we carried out structure analysis of the L-type straight filament reconstituted from a mutant flagellin of SJW46 (SJW46S) and compared the structure with that of the SJW1660 filament, which is also the L-type but composed of flagellin with no deletion. The deleted portion was identified as the outermost subdomain, and the structure in the core region showed no appreciable differences. The structure revealed the previously identified folding of flagellin in further detail, and the significance of intersubunit interactions between outer domains, which are present in the SJW1660 filament but absent in the SJW46 filament. This suggests that these contacts have a significant contribution to the filament stability and polymorphic behavior, despite the fact that the contacting surface area occupies only a minor portion of the whole intersubunit interactions.     

Genetic variability in sensitivity to population density affects the dynamics of simple ecological models

Many 1-dimensional discrete time ecological models contain a sensitivity parameter that does not affect the dynamic complexity of these models. We show that genetic variability in this parameter can have a strong effect on population dynamics. We incorporate ecological dynamics in two different population genetic models with one locus and two alleles. The first is the classical model of a randomly mating population in Hardy-Weinberg equilibrium, and the second is a model of differential selection in males and females. In populations in Hardy-Weinberg equilibrium, variability in the sensitivity parameter can be maintained by overdominance. In this case, the dynamics of the polymorphic population tend to be much simpler than those of monomorphic populations. In the model with different selection in males and females, polymorphisms can be maintained in various ways, e.g., by opposing directional selection in males and females. Polymorphism in the sensitivity parameter tends to simplify population dynamics in the model with different selection in males and females as well. A number of interesting dynamic effects can be observed, e.g., multiple attractors with complicated basins of attraction. Then the final state of the system after a successful invasion by mutant alleles may depend on the mutation rate and on the distribution of mutational steps. In addition, there are situations in which genetic variability destabilizes a stable population dynamic equilibrium in the monomorphic model. There is an analogy between genetic variability and variability imposed by the environment. If differences in sensitivity are caused by the environment, dynamic effects similar to those in the genetic models can be observed. In addition, source-sink structures that are known to occur in spatially structured models can be seen in the genetic model if one of the genotypes is inviable. The results suggest that combining ecological and population genetic models can lead to a number of new insights. More work is needed, e.g., with fertility models, in which fitnesses are not assigned to individuals, but to mating pairs.