The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.

In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.     

GTP analogues interact with the tubulin exchangeable site during assembly and upon binding

The question of whether nonhydrolyzable nucleotide analogues and other nucleoside triphosphates support tubulin assembly was addressed. Tubulin which contained residual GTP at the exchangeable site polymerized in the absence of added GTP in the presence of DMSO or glycerol. After maximum absorbance was reached, disassembly occurred at a slow rate. When 0.5 mM GMPPCP, GMPPNP, or ATP was included in the assembly reaction, disassembly did not occur, and about 0.1 mol of these nucleotides per mole of tubulin was incorporated into the protein. When 5 mM nucleotide was used or alkaline phosphatase was included in the case of the nonhydrolyzable analogues, a greater amount of assembly occurred and about 0.7-0.8 mol of analogue was incorporated. The products of the assembly reaction were cold-labile microtubules and protofilament ribbons. After cold-depolymerization of the microtubules and ribbons, a second cycle of assembly produced some microtubules, but cold-stable amorphous polymers were the major product. In addition, when GTP at the exchangeable site was first removed by a cycle of assembly, followed by depolymerization, assembly in the presence of GMPPCP, GMPPNP, or ATP produced a mixture of microtubules and cold-stable polymers, both of which contained bound analogue. Incorporation of GMPPCP, GMPPNP, or ATP into polymerized tubulin always occurred at the expense of GDP at the exchangeable site, the content of which decreased correspondingly. Incubation of tubulin with 5 mM GMPPCP, GMPPNP, or ATP under nonassembly conditions also displaced GDP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide stereochemistry in the formyltetrahydrofolate synthetase reaction

In a recent study, we have shown that N10-formyltetrahydrofolate synthetase prefers (Sp)-MgATP beta S over the Rp isomer in the forward reaction. In this report the stereochemistry of ATP beta S produced from prochiral ADP beta S in the reverse reaction was determined. The ATP beta S product was purified and tested as a substrate for hexokinase (preference for the Rp isomer), adenylate kinase (preference for the Sp isomer) and N10-formyltetrahydrofolate synthetase. A comparison of kinetic constants for the product and the authentic Sp and Rp isomers shows that the product is the Sp diastereomer. 31P NMR was also used to identify the product as (Sp)-ATP beta S.     

Exercise training in childhood-onset systemic lupus erythematosus: a controlled randomized trial

Introduction

Exercise training has emerged as a promising therapeutic strategy to counteract physical dysfunction in adult systemic lupus erythematosus. However, no longitudinal studies have evaluated the effects of an exercise training program in childhood-onset systemic lupus erythematosus (C-SLE) patients. The objective was to evaluate the safety and the efficacy of a supervised aerobic training program in improving the cardiorespiratory capacity in C-SLE patients.

Methods

Nineteen physically inactive C-SLE patients were randomly assigned into two groups: trained (TR, n = 10, supervised moderate-intensity aerobic exercise program) and non-trained (NT, n = 9). Gender-, body mass index (BMI)- and age-matched healthy children were recruited as controls (C, n = 10) for baseline (PRE) measurements only. C-SLE patients were assessed at PRE and after 12 weeks of training (POST). Main measurements included exercise tolerance and cardiorespiratory measurements in response to a maximal exercise (that is, peak VO₂, chronotropic reserve (CR), and the heart rate recovery (ΔHRR) (that is, the difference between HR at peak exercise and at both the first (ΔHRR1) and second (ΔHRR2) minutes of recovery after exercise).

Results

The C-SLE NT patients did not present changes in any of the cardiorespiratory parameters at POST (P > 0.05). In contrast, the exercise training program was effective in promoting significant increases in time-to-exhaustion (P = 0.01; ES = 1.07), peak speed (P = 0.01; ES = 1.08), peak VO₂ (P = 0.04; ES = 0.86), CR (P = 0.06; ES = 0.83), and in ΔHRR1 and ΔHRR2 (P = 0.003; ES = 1.29 and P = 0.0008; ES = 1.36, respectively) in the C-SLE TR when compared with the NT group. Moreover, cardiorespiratory parameters were comparable between C-SLE TR patients and C subjects after the exercise training intervention, as evidenced by the ANOVA analysis (P > 0.05, TR vs. C). SLEDAI-2K scores remained stable throughout the study.

Conclusion

A 3-month aerobic exercise training was safe and capable of ameliorating the cardiorespiratory capacity and the autonomic function in C-SLE patients.

Trial registration

NCT01515163.     

Automated Entire Thrombus Density Measurements for Robust and Comprehensive Thrombus Characterization in Patients with Acute Ischemic Stroke

Background and Purpose

In acute ischemic stroke (AIS) management, CT-based thrombus density has been associated with treatment success. However, currently used thrombus measurements are prone to inter-observer variability and oversimplify the heterogeneous thrombus composition. Our aim was first to introduce an automated method to assess the entire thrombus density and then to compare the measured entire thrombus density with respect to current standard manual measurements.

Materials and Method

In 135 AIS patients, the density distribution of the entire thrombus was determined. Density distributions were described using medians, interquartile ranges (IQR), kurtosis, and skewedness. Differences between the median of entire thrombus measurements and commonly applied manual measurements using 3 regions of interest were determined using linear regression.

Results

Density distributions varied considerably with medians ranging from 20.0 to 62.8 HU and IQRs ranging from 9.3 to 55.8 HU. The average median of the thrombus density distributions (43.5 ± 10.2 HU) was lower than the manual assessment (49.6 ± 8.0 HU) (p<0.05). The difference between manual measurements and median density of entire thrombus decreased with increasing density (r = 0.64; p<0.05), revealing relatively higher manual measurements for low density thrombi such that manual density measurement tend overestimates the real thrombus density.

Conclusions

Automatic measurements of the full thrombus expose a wide variety of thrombi density distribution, which is not grasped with currently used manual measurement. Furthermore, discrimination of low and high density thrombi is improved with the automated method.     

Full-contact domain labeling: identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein

Gelsolin, an actin and phosphoinositide binding protein, was photoaffinity labeled using a variety of benzophenone-containing phosphoinositide polyphosphate analogues. The N-terminal half and the C-terminal half of gelsolin showed synergy in the binding of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. Competitive displacement experiments with dibutyryl, dioctanoyl, or dipalmitoyl derivatives of PtdIns(4,5)P(2) suggested that, in addition to the inositol headgroup, a diacylglyceryl moiety was important for binding; these analogues also inhibited the gelsolin-severing activity of F-actin. In addition to the previously identified PtdIns(4,5)P2 binding site in the N-terminal half of gelsolin, a new binding site was identified in the C-terminal half by mapping the photocovalently modified peptide fragments. Moreover, increasing concentrations of Ca(2+) decreased the binding of the photolabile analogues to the C-terminal phosphoinositide binding site on gelsolin. A molecular model of the binding of PtdIns(4,5)P2 within two folded repeats of gelsolin has been calculated using these data.     

The calcium activation of gelsolin: insights from the 3A structure of the G4-G6/actin complex

Gelsolin participates in the reorganization of the actin cytoskeleton that is required during such phenomena as cell movement, cytokinesis, and apoptosis. It consists of six structurally similar domains, G1-G6, which are arranged at resting intracellular levels of calcium ion so as to obscure the three actin-binding surfaces. Elevation of Ca(2+) concentrations releases latches within the constrained structure and produces large shifts in the relative positioning of the domains, permitting gelsolin to bind to and sever actin filaments. How Ca(2+) is able to activate gelsolin has been a major question concerning the function of this protein. We present the improved structure of the C-terminal half of gelsolin bound to monomeric actin at 3.0 A resolution. Two classes of Ca(2+)-binding site are evident on gelsolin: type 1 sites share coordination of Ca(2+) with actin, while type 2 sites are wholly contained within gelsolin. This structure of the complex reveals the locations of two novel metal ion-binding sites in domains G5 and G6, respectively. We identify both as type 2 sites. The absolute conservation of the type 2 calcium-ligating residues across the six domains of gelsolin suggests that this site exists in each of the domains. In total, gelsolin has the potential to bind eight calcium ions, two type 1 and six type 2. The function of the type 2 sites is to facilitate structural rearrangements within gelsolin as part of the activation and actin-binding and severing processes. We propose the novel type 2 site in G6 to be the critical site that initiates overall activation of gelsolin by releasing the tail latch that locks calcium-free gelsolin in a conformation unable to bind actin.     

Towards a framework for the evolutionary genomics of Kinetoplastids: what kind of data and how much?

The current status of kinetoplastids phylogeny and evolution is discussed in view of the recent progresses on genomics. Some ideas on a potential framework for the evolutionary genomics of kinetoplastids are presented.