Psychometric properties of the Aggression Questionnaire: A replication in a sample of partner-assaultive men in psychological treatment / Propiedades psicométricas del Cuestionario de Agresión: una replicación en una muestra de maltratadores en tratamiento psicológico

Background: The Aggression Questionnaire (Buss & Perry, 1992) is one of the most widely used validated self-report measures to assess aggression. This study analyzes the factorial structure, internal consistency, and convergent validity of the AQ in a Spanish sample with high levels of violence. Method: The questionnaire was administered to 767 partner-assaultive men undergoing psychological treatment. Results: The results of the confirmatory factor analysis supported the four-factor structure. Also, positive correlations between the AQ and other instruments measuring aggression were found, finding that aggression and anger decrease with age in this sample. Conclusions: The results obtained showed that the AQ is a suitable instrument to assess aggression, anger, and hostility in partner-assaultive men (AU)

Antecedentes: el Cuestionario de Agresión (Buss & Perry, 1992) constituye una de las medidas de auto-informe más utilizadas para medir agresión. Este estudio analiza la estructural factorial, consistencia interna y validez convergente del AQ en una muestra española con altos niveles de violencia. Método: el cuestionario fue aplicado a 767 maltratadores que se encontraban bajo tratamiento psicológico. Resultados: los resultados del análisis factorial confirmatorio apoyan la estructura de cuatro factores. Asimismo, se encontraron correlaciones positivas y significativas entre el AQ y otros instrumentos que miden agresión, encontrándose que la agresión y la ira disminuyen con la edad en esta muestra. Conclusiones: los resultados obtenidos permiten concluir que el AQ es un instrumento adecuado para evaluar agresión, ira y hostilidad en hombres que maltratan a sus parejas (AU)

Triazolopyrimidines Are Microtubule-Stabilizing Agents that Bind the Vinca Inhibitor Site of Tubulin.

Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.

A Polymerization-Associated Structural Switch in FtsZ That Enables Treadmilling of Model Filaments.

Bacterial cell division in many organisms involves a constricting cytokinetic ring that is orchestrated by the tubulin-like protein FtsZ. FtsZ forms dynamic filaments close to the membrane at the site of division that have recently been shown to treadmill around the division ring, guiding septal wall synthesis. Here, using X-ray crystallography of Staphylococcus aureus FtsZ (SaFtsZ), we reveal how an FtsZ can adopt two functionally distinct conformations, open and closed. The open form is found in SaFtsZ filaments formed in crystals and also in soluble filaments of Escherichia coli FtsZ as deduced by electron cryomicroscopy. The closed form is found within several crystal forms of two nonpolymerizing SaFtsZ mutants and corresponds to many previous FtsZ structures from other organisms. We argue that FtsZ's conformational switch is polymerization-associated, driven by the formation of the longitudinal intersubunit interfaces along the filament. We show that such a switch provides explanations for both how treadmilling may occur within a single-stranded filament and why filament assembly is cooperative.IMPORTANCE The FtsZ protein is a key molecule during bacterial cell division. FtsZ forms filaments that organize cell membrane constriction, as well as remodeling of the cell wall, to divide cells. FtsZ functions through nucleotide-driven filament dynamics that are poorly understood at the molecular level. In particular, mechanisms for cooperative assembly (nonlinear dependency on concentration) and treadmilling (preferential growth at one filament end and loss at the other) have remained elusive. Here, we show that most likely all FtsZ proteins have two distinct conformations, a "closed" form in monomeric FtsZ and an "open" form in filaments. The conformational switch that occurs upon polymerization explains cooperativity and, in concert with polymerization-dependent nucleotide hydrolysis, efficient treadmilling of FtsZ polymers.

Propiedades psicométricas del «Inventario breve de afrontamiento» (COPE-28) en una muestra de adolescentes / Psychometric properties of the Spanish version of the Brief COPE Inventory (COPE-28) in a sample of teenage

El objetivo del presente estudio fue analizar las propiedades psicométricas de la versión española (el COPE-28) del "Inventario breve de afrontamiento" (Brief COPE Inventory; Carver, 1997), con 1204 adolescentes (M= 15,3 años; DT= 1,8). El análisis factorial exploratorio mostró una solución de ocho factores que explican el 54,19% de la varianza (KMO= 0,752 y prueba de esfericidad de Bartlett significativa, χ2= 6625,262; gl= 378; p< 0,001). La consistencia interna para la puntuación total del COPE-28 fue de 0,78. Se obtuvo la validez concurrente con el "Cuestionario de agresión" (Aggression Questionnaire, AQ; Buss y Perry, 1992) y el "Cuestionario de agresión reactiva y proactiva" (Reactive and Proactive Aggression Questionnaire, RPQ; Raine et al., 2006). Las correlaciones más significativas fueron entre Autoinculpación (COPE-28) y Hostilidad (AQ) (r= 0,414), Desconexión (COPE-28) y Agresión proactiva (RPQ) (r= 0,315) y, finalmente, Consumo de sustancias (COPE-28) con Agresión reactiva (RPQ) (r= 0,360), Agresión proactiva (RPQ) (r= 0,455) y Agresión física (AQ) (r= 0,392). Estos datos apoyan la fiabilidad y validez del COPE-28 pero señalan a su vez la necesidad de nuevos estudios

The aim of this study was to analyze the psychometric properties of the Spanish version (COPE-28) of the Brief Cope Inventory (Carver, 1997) with 1204 adolescents (M= 15.3 years, SD= 1.8). An exploratory factor analysis showed a solution of eight factors that explains 54.19% of the variance (KMO= .752 and Bartlett's test was significant, χ2= 6625.262, df= 378, p< .001). The internal consistency for the total score of the COPE-28 was .78. Concurrent validity was obtained with the Aggression Questionnaire (AQ; Buss & Perry, 1992) and Reactive and Proactive Aggression Questionnaire (RPQ; Raine et al., 2006). The most significant correlations were between Self-incrimination (COPE-28) and Hostility (AQ) (r= 0.414), Disconnection (COPE-28) and Proactive aggression (RPQ) (r= .315), and finally Substance use (COPE-28) with Reactive aggression (RPQ) (r= .360), Proactive aggression (RPQ) (r= .455) and Physical aggression (AQ) (r= .392). These data support the reliability and validity of COPE-28 but point to the need for further studies

Creencias desadaptativas, estilos de afrontamiento y apoyo social como factores predictores de la vulnerabilidad psicopatológica en mujeres víctimas de agresión sexual / Maladaptive beliefs, coping strategies, and social support as predictive factors of psychopathological vulnerability in female victims of sexual assault

Se han recogido los principales factores de vulnerabilidad descritos en la literatura con objeto de comprobar su efecto sobre la sintomatología desarrollada en víctimas de agresión sexual. Las participantes fueron 77 mujeres víctimas de agresiones sexuales en la edad adulta que acudieron al Centro de Atención a Víctimas de Agresiones Sexuales (C.A.V.A.S.) de Madrid, entre los años 2010 y 2013. Los factores que mayor valor predictivo han presentado han sido: elevadas creencias desadaptativas sobre uno mismo, la creencia de control futuro o la realización de conductas preventivas y, como estrategias de afrontamiento, la planificación, el apoyo social instrumental y el uso de sustancias. En cuanto a los factores que han resultado ser predictores de un mejor ajuste destacan el control presente o control sobre la recuperación, la estrategia de autodistracción y la satisfacción con el apoyo social. Finalmente, se describen las implicaciones de los resultados y se sugieren futuras líneas de investigación

The main vulnerability factors subsequent to rape described in the literature have been included in this research to check their effects on the symptomatology developed in sexual assault victims. The participants were 77 women victims of sexual assaults when they were adults, attending the Victms of Sexual Assaults Centre (C.A.V.A.S.) in Madrid between 2010 and 2013. The factors with a higher predictive value were high maladaptive beliefs about one’s self, the belief about future control, or developing preventative behaviors; coping strategies include planning, instrumental social support, and substance use. Factors standing out as predictors of a better adjustment include present control or control over the recovery, self-distraction (as coping strategy), and satisfaction with the social support. Finally, implications of results are described and future directions of research are suggested

Understanding nucleotide-regulated FtsZ filament dynamics and the monomer assembly switch with large-scale atomistic simulations.

Bacterial cytoskeletal protein FtsZ assembles in a head-to-tail manner, forming dynamic filaments that are essential for cell division. Here, we study their dynamics using unbiased atomistic molecular simulations from representative filament crystal structures. In agreement with experimental data, we find different filament curvatures that are supported by a nucleotide-regulated hinge motion between consecutive FtsZ monomers. Whereas GTP-FtsZ filaments bend and twist in a preferred orientation, thereby burying the nucleotide, the differently curved GDP-FtsZ filaments exhibit a heterogeneous distribution of open and closed interfaces between monomers. We identify a coordinated Mg(2+) ion as the key structural element in closing the nucleotide site and stabilizing GTP filaments, whereas the loss of the contacts with loop T7 from the next monomer in GDP filaments leads to open interfaces that are more prone to depolymerization. We monitored the FtsZ monomer assembly switch, which involves opening/closing of the cleft between the C-terminal domain and the H7 helix, and observed the relaxation of isolated and filament minus-end monomers into the closed-cleft inactive conformation. This result validates the proposed switch between the low-affinity monomeric closed-cleft conformation and the active open-cleft FtsZ conformation within filaments. Finally, we observed how the antibiotic PC190723 suppresses the disassembly switch and allosterically induces closure of the intermonomer interfaces, thus stabilizing the filament. Our studies provide detailed structural and dynamic insights into modulation of both the intrinsic curvature of the FtsZ filaments and the molecular switch coupled to the high-affinity end-wise association of FtsZ monomers.

Endowing indole-based tubulin inhibitors with an anchor for derivatization: highly potent 3-substituted indolephenstatins and indoleisocombretastatins.

Colchicine site ligands with indole B rings are potent tubulin polymerization inhibitors. Structural modifications at the indole 3-position of 1-methyl-5-indolyl-based isocombretastatins (1,1-diarylethenes) and phenstatins endowed them with anchors for further derivatization and resulted in highly potent compounds. The substituted derivatives displayed potent cytotoxicity against several human cancer cell lines due to tubulin inhibition, as shown by cell cycle analysis, confocal microscopy, and tubulin polymerization inhibitory activity studies and promoted cell killing mediated by caspase-3 activation. Binding at the colchicine site was confirmed by means of fluorescence measurements of MTC displacement. Molecular modeling suggests that the tropolone-binding region of the colchicine site of tubulin can adapt to hosting small polar substituents. Isocombretastatins accepted substitutions better than phenstatins, and the highest potencies were achieved for the cyano and hydroxyiminomethyl substituents, with TPI values in the submicromolar range and cytotoxicities in the subnanomolar range. A 3,4,5-trimethoxyphenyl ring usually afforded more potent derivatives than a 2,3,4-trimethoxyphenyl ring.

[Should we add antiplatelet therapy to oral anticoagulation in patients with atrial fibrillation and vascular disease? Review of available evidence]. / Debe añadirse terapia antiagregante plaquetaria a la anticoagulación oral en los pacientes con fibrilación auricular y enfermedad vascular? Revisión de la evidencia disponible.

Current recommendation is to add antiplatelet drug to oral anticoagulation in patients with atrial fibrillation (AF) and vascular disease. However, it is debatable to join both antithrombotic drugs in stable vascular disease.

¿Debe añadirse terapia antiagregante plaquetaria a la anticoagulación oral en los pacientes con fibrilación auricular y enfermedad vascular? Revisión de la evidencia disponible / Should we add antiplatelet therapy to oral anticoagulation in patients with atrial fibrillation and vascular disease? Review of available evidence

La práctica común consiste en añadir tratamiento antiagregante a la terapia anticoagulante, en los pacientes con fibrilación auricular (FA) y patología vascular establecida. Sin embargo, la necesidad de combinar ambas estrategias es un tema controvertido actualmente.

Current recommendation is to add antiplatelet drug to oral anticoagulation in patients with atrial fibrillation (AF) and vascular disease. However, it is debatable to join both antithrombotic drugs in stable vascular disease.

Septin C-terminal domain interactions: implications for filament stability and assembly.

Septins form a conserved family of filament forming GTP binding proteins found in a wide range of eukaryotic cells. They share a common structural architecture consisting of an N-terminal domain, a central GTP binding domain and a C-terminal domain, which is often predicted to adopt a coiled-coil conformation, at least in part. The crystal structure of the human SEPT2/SEPT6/SEPT7 heterocomplex has revealed the importance of the GTP binding domain in filament formation, but surprisingly no electron density was observed for the C-terminal domains and their function remains obscure. The dearth of structural information concerning the C-terminal region has motivated the present study in which the putative C-terminal domains of human SEPT2, SEPT6 and SEPT7 were expressed in E. coli and purified to homogeneity. The thermal stability and secondary structure content of the domains were studied by circular dichroism spectroscopy, and homo- and hetero-interactions were investigated by size exclusion chromatography, chemical cross-linking, analytical ultracentrifugation and surface plasmon resonance. Our results show that SEPT6-C and SEPT7-C are able to form both homo- and heterodimers with a high α-helical content in solution. The heterodimer is elongated and considerably more stable than the homodimers, with a K(D) of 15.8 nM. On the other hand, the homodimer SEPT2-C has a much lower affinity, with a K(D) of 4 µM, and a moderate α-helical content. Our findings present the first direct experimental evidence toward better understanding the biophysical properties and coiled-coil pairings of such domains and their potential role in filament assembly and stability.

Cyclostreptin derivatives specifically target cellular tubulin and further map the paclitaxel site.

Cyclostreptin is the first microtubule-stabilizing agent whose mechanism of action was discovered to involve formation of a covalent bond with tubulin. Treatment of cells with cyclostreptin irreversibly stabilizes their microtubules because cyclostreptin forms a covalent bond to ß-tubulin at either the T220 or the N228 residue, located at the microtubule pore or luminal taxoid binding site, respectively. Because of its unique mechanism of action, cyclostreptin overcomes P-glycoprotein-mediated multidrug resistance in tumor cells. We used a series of reactive cyclostreptin analogues, 6-chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and [(14)C-acetyl]-8-acetyl-cyclostreptin, to characterize the cellular target of the compound and to map the binding site. The three analogues were cytotoxic and stabilized microtubules in both sensitive and multidrug resistant tumor cells. In both types of cells, we identified ß-tubulin as the only or the predominantly labeled cellular protein, indicating that covalent binding to microtubules is sufficient to prevent drug efflux mediated by P-glycoprotein. 6-Chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and 8-acetyl-cyclostreptin labeled both microtubules and unassembled tubulin at a single residue of the same tryptic peptide of ß-tubulin as was labeled by cyclostreptin (219-LTTPTYGDLNHLVSATMSGVTTCLR-243), but labeling with the analogues occurred at different positions of the peptide. 8-Acetyl-cyclostreptin reacted with either T220 or N228, as did the natural product, while 8-chloroacetyl-cyclostreptin formed a cross-link to C241. Finally, 6-chloroacetyl-cyclostreptin reacted with any of the three residues, thus labeling the pathway for cyclostreptin-like compounds, leading from the pore where these compounds enter the microtubule to the luminal binding pocket.

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach.

The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/ß-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/ß-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/ß-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubules.

Modulation of microtubule interprotofilament interactions by modified taxanes.

Microtubules assembled with paclitaxel and docetaxel differ in their numbers of protofilaments, reflecting modification of the lateral association between αß-tubulin molecules in the microtubule wall. These modifications of microtubule structure, through a not-yet-characterized mechanism, are most likely related to the changes in tubulin-tubulin interactions responsible for microtubule stabilization by these antitumor compounds. We have used a set of modified taxanes to study the structural mechanism of microtubule stabilization by these ligands. Using small-angle x-ray scattering, we have determined how modifications in the shape and size of the taxane substituents result in changes in the interprotofilament angles and in their number. The observed effects have been explained using NMR-aided docking and molecular dynamic simulations of taxane binding at the microtubule pore and luminal sites. Modeling results indicate that modification of the size of substituents at positions C7 and C10 of the taxane core influence the conformation of three key elements in microtubule lateral interactions (the M-loop, the S3 ß-strand, and the H3 helix) that modulate the contacts between adjacent protofilaments. In addition, modifications of the substituents at position C2 slightly rearrange the ligand in the binding site, modifying the interaction of the C7 substituent with the M-loop.

Conducta antisocial, impulsividad y creencias justificativas: análisis de sus interrelaciones con la agresión proactiva y reactiva en adolescentes / Antisocial behavior, impulsiveness and justification beliefs: analysis of their interrelationships with proactive and reactive aggression in adolescents

Este estudio analiza las interrelaciones entre creencias justificativas, impulsividad y conducta antisocial en relación con la agresión proactiva y reactiva. Ambos tipos de agresión han sido ampliamente analizados en la infancia, pero en mucha menor medida en la adolescencia. Por ello, en este estudio se utilizó una muestra de adolescentes compuesta por 320 participantes de ambos sexos para analizar las complejas relaciones existentes entre todas estas variables. Los resultados mostraron que, tanto en hombres como en mujeres, la agresión proactiva estuvo muy relacionada con la conducta antisocial, mientras que la agresión reactiva lo estuvo con la impulsividad. Además, a través de un modelo de ecuaciones estructurales, se encontró una relación indirecta entre agresión proactiva y conducta antisocial mediatizada parcialmente por la presencia de creencias justificativas. Los autores concluyen el estudio señalando la importancia de considerar la impulsividad y las creencias justificativas como factores de especial interés para comprender el desarrollo de la agresión en adolescentes (AU)

This study assessed the relationships between reactive and proactive aggression and justification beliefs, impulsivity and antisocial behavior, examined characteristically in the context of children but less often studied in adolescents. For this reason the current study assessed a sample of 320 adolescents of both sexes in order to examine the relationships between reactive and proactive aggression and antisocial behavior, impulsivity and normative believes. Results showed that proactive aggression was significantly related to antisocial behavior and normative believes, in both sexes, whereas impulsivity showed the highest correlation to reactive aggression. Using structural equation modeling, a direct relationship was found between aggression and antisocial behavior partially mediated by normative beliefs. According to these results, the authors conclude by stressing the importance of considering impulsivity and normative beliefs as key factors for understanding the development of aggression in adolescents

3-Hydroxyphenylpropionate and phenylpropionate are synergistic activators of the MhpR transcriptional regulator from Escherichia coli.

The degradation of the aromatic compound phenylpropionate (PP) in Escherichia coli K-12 requires the activation of two different catabolic pathways coded by the hca and the mhp gene clusters involved in the mineralization of PP and 3-hydroxyphenylpropionate (3HPP), respectively. The compound 3-(2,3-dihydroxyphenyl)propionate (DHPP) is a common intermediate of both pathways which must be cleaved by the MhpB dioxygenase before entering into the primary cell metabolism. Therefore, the degradation of PP has to be controlled by both its specific regulator (HcaR) but also by the MhpR regulator of the mhp cluster. We have demonstrated that 3HPP and DHPP are the true and best activators of MhpR, whereas PP only induces no response. However, in vivo and in vitro transcription experiments have demonstrated that PP activates the MhpR regulator synergistically with the true inducers, representing the first case of such a peculiar synergistic effect described for a bacterial regulator. The three compounds enhanced the interaction of MhpR with its DNA operator in electrophoretic mobility shift assays. Inducer binding to MhpR is detected by circular dichroism and fluorescence spectroscopies. Fluorescence quenching measurements have revealed that the true inducers (3HPP and DHPP) and PP bind with similar affinities and independently to MhpR. This type of dual-metabolite synergy provides great potential for a rapid modulation of gene expression and represents an important feature of transcriptional control. The mhp regulatory system is an example of the high complexity achievable in prokaryotes.

Optimization of taxane binding to microtubules: binding affinity dissection and incremental construction of a high-affinity analog of paclitaxel.

The microtubule binding affinities of a series of synthetic taxanes have been measured with the aims of dissecting individual group contributions and obtaining a rationale for the design of novel compounds with the ability to overcome drug resistance. As previously observed for epothilones, the positive and negative contributions of the different substituents to the binding free energies are cumulative. By combining the most favorable substitutions we increased the binding affinity of paclitaxel 500-fold. Insight into the structural basis for this improvement was gained with molecular modeling and NMR data obtained for microtubule-bound docetaxel. Taxanes with affinities for microtubules well above their affinities for P-glycoprotein are shown not to be affected by multidrug resistance. This finding strongly indicates that optimization of the ligand-target interaction is a good strategy to overcome multidrug resistance mediated by efflux pumps.

The bound conformation of microtubule-stabilizing agents: NMR insights into the bioactive 3D structure of discodermolide and dictyostatin.

A protocol based on a combination of NMR experimental data with molecular mechanics calculations and docking procedures has been employed to determine the microtubule-bound conformation of two microtubule-stabilizing agents, discodermolide (DDM) and dictyostatin (DCT). The data indicate that tubulin in assembled microtubules recognizes DDM through a conformational selection process, with minor changes in the molecular skeleton between the major conformer in water solution and that bound to assembled microtubules. For DCT, the deduced bound geometry presents some key conformation differences around certain torsion angles, with respect to the major conformer in solution, and still displays mobility even when bound. The bound conformer of DCT resembles that of DDM and provides very similar contacts with the receptor. Competition experiments indicate that both molecules compete with the taxane-binding site. A model of the binding mode of DDM and DCT to tubulin is proposed.

Probing FtsZ and tubulin with C8-substituted GTP analogs reveals differences in their nucleotide binding sites.

The cytoskeletal proteins, FtsZ and tubulin, play a pivotal role in prokaryotic cell division and eukaryotic chromosome segregation, respectively. Selective inhibitors of the GTP-dependent polymerization of FtsZ could constitute a new class of antibiotics, while several inhibitors of tubulin are widely used in antiproliferative therapy. In this work, we set out to identify selective inhibitors of FtsZ based on the structure of its natural ligand, GTP. We found that GTP analogs with small hydrophobic substituents at C8 of the nucleobase efficiently inhibit FtsZ polymerization, whereas they have an opposite effect on the polymerization of tubulin. The inhibitory activity of the GTP analogs on FtsZ polymerization allowed us to crystallize FtsZ in complex with C8-morpholino-GTP, revealing the binding mode of a GTP derivative containing a nonmodified triphosphate chain.

Energetics and geometry of FtsZ polymers: nucleated self-assembly of single protofilaments.

Essential cell division protein FtsZ is an assembling GTPase which directs the cytokinetic ring formation in dividing bacterial cells. FtsZ shares the structural fold of eukaryotic tubulin and assembles forming tubulin-like protofilaments, but does not form microtubules. Two puzzling problems in FtsZ assembly are the nature of protofilament association and a possible mechanism for nucleated self-assembly of single-stranded protofilaments above a critical FtsZ concentration. We assembled two-dimensional arrays of FtsZ on carbon supports, studied linear polymers of FtsZ with cryo-electron microscopy of vitrified unsupported solutions, and formulated possible polymerization models. Nucleated self-assembly of FtsZ from Escherichia coli with GTP and magnesium produces flexible filaments 4-6 nm-wide, only compatible with a single protofilament. This agrees with previous scanning transmission electron microscopy results and is supported by recent cryo-electron tomography studies of two bacterial cells. Observations of double-stranded FtsZ filaments in negative stain may come from protofilament accretion on the carbon support. Preferential protofilament cyclization does not apply to FtsZ assembly. The apparently cooperative polymerization of a single protofilament with identical intermonomer contacts is explained by the switching of one inactive monomer into the active structure preceding association of the next, creating a dimer nucleus. FtsZ behaves as a cooperative linear assembly machine.

Large scale purification of brain tubulin with the modified Weisenberg procedure.

This method is a modification of the initial procedure employed to purify tubulin from mammalian brain. It consists of tissue homogenization, elimination of cell membranes, ammonium sulfate fractionation, and batch anion exchange, followed by selective precipitation with magnesium chloride. Half gram of electrophoretically homogenous, active, concentrated calf brain tubulin is typically purified in 9 h, dialyzed overnight, and stored under liquid nitrogen. Prior to use the protein is equilibrated in the experimental buffer and its concentration measured. This tubulin preparation has been very extensively characterized. Frozen aliquots have been found to retain microtubule assembly activity after 10 yr of storage.