Zeroing of six-component handrim dynamometer for biomechanical studies of manual wheelchair locomotion

A six-component handrim dynamometer (HRD) is a dynamometer that rotates around the wheel axle during measurements. For this kind of dynamometer, static zero level calibration is insufficient because the proportion of the forces (i.e. handrim weight and centrifugal force) measured by each sensor varies according to the angular position and velocity of the dynamometer. The dynamic calibration presented in this paper is based on the direct correction of the sensor signals using Fourier's polynomials that take into account the influences of both the handrim weight distribution on the sensors with respect to the wheel's angular position and the effect of the wheel's angular velocity. When these corrections were applied to the signals produced by the sensors while the HRD was rotating and no effort was being exerted on the handrim, the calculated forces and torques remained close to zero, as expected. Based on these results, the wheel dynamometer can be confidently used for studying manual wheelchair locomotion under various real conditions. The method could also be applied in other situations in which a dynamometer rotates during measurements.     

Mechanical Properties of β-Catenin Revealed by Single-Molecule Experiments

β-catenin is a central component of the adaptor complex that links cadherins to the actin cytoskeleton in adherens junctions and thus, it is a good candidate to sense and transmit mechanical forces to trigger specific changes inside the cell. To fully understand its molecular physiology, we must first investigate its mechanical role in mechanotransduction within the cadherin system. We have studied the mechanical response of β-catenin to stretching using single-molecule force spectroscopy and molecular dynamics. Unlike most proteins analyzed to date, which have a fixed mechanical unfolding pathway, the β-catenin armadillo repeat region (ARM) displays low mechanostability and multiple alternative unfolding pathways that seem to be modulated by its unstructured termini. These results are supported by steered molecular dynamics simulations, which also predict its mechanical stabilization and unfolding pathway restrictions when the contiguous α-helix of the C-terminal unstructured region is included. Furthermore, simulations of the ARM/E-cadherin cytosolic tail complex emulating the most probable stress geometry occurring in vivo show a mechanical stabilization of the interaction whose magnitude correlates with the length of the stretch of the cadherin cytosolic tail that is in contact with the ARM region.     

Ribosome changes during translation

Studies on the quantitative binding of [³H]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their affinity for [³H]anisomycin.Free ribosomes, either in vivo run-off ribosomes (1 mm-sodium azide treatment or 8 °C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity defined by K_d = 3.3 to 3.6 μm.Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by K_d,H = 0.81 μm and K_d,L = 12 μm). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of K_d^CHX = 12 μm and post-translocated polysomes (polysomes blocked with doxycycline) have an affinity of K_d^DC = 0.82 μm. These dissociation constants are identical to K_d,L and K_d,H obtained with control untreated polysomes, respectively.Moreover, a new ribosome conformation defined by K_d^DT = 1.5 μm and K_d^FA = 1.8 μm was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidic acid.We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974a) being a direct consequence of the high-salt washing treatment of ribosomes.     

A Comparative Statistical Analysis of Genetic Distances. II–Confidence Intervals and Tests of Comparison

We present methods for constructing the confidence intervals of the estimations of absolute genetic distance and Nei's minimum and standard genetic distances. Two different estimators of the standard distance are considered: jack-knife and corrected. A Monte-Carlo simulation analysis permitted to choose the best adapted confidence interval type for each distance estimator and for each size of the samples of loci and of gametes, taking into account the magnitude of the estimation value. Simulated true confidence levels showed that the best results are obtained using abolute genetic distance. We also give rules for testing the significance of the difference of two distance estimations. Monte-Carlo simulations were performed in order to obtain the distributions of the difference of two distance estimations under the null hypothesis and consequently the true type I errors of tests. Test powers, obtained by simulation under the alternative hypothesis, also showed the superiority of absolute genetic distance.     

Chromosome and blood marker studies in families of patients affected by xeroderma pigmentosum and trichothiodystrophy

Chromosome and blood marker studies were performed in the families of 4 patients in which the association of 2 rare recessive Mendelian disorders, xeroderma pigmentosum (XP-D) and trichothiodystrophy (TTD), was present. Blood genotypes did not indicate any linkage with the pathologic condition, nor any segregation anomaly. Cytogenetic analysis using high-resolution banding techniques showed a normal karyotype both in the heterozygous and in the homozygous individuals. These findings lead us to exclude a cytologically detectable chromosome rearrangement, such as a microdeletion, as a possible cause of the association of XP-D and TTD in our patients.     

Leukemia-inhibitory factor and neuroblastoma cell lines: Study on the expression of this citokine and on its efficacy as differentiating agent

Supported by grants from AIRC, Regione Emilia Romagna and MURST     

Molecular Basis of Filtering Carbapenems by Porins from β-Lactam-resistant Clinical Strains of Escherichia coli

Integral membrane proteins known as porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by “trapping” the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria.