N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Phorbol myristate acetate stimulation of lymphocyte guanylate cyclase

Human lymphocyte guanylate cyclase activities are increased in a dose-dependent fashion by incubation of intact cells with phorbol myristate acetate, a tumor promoter and lymphocyte mitogen. Increased activity is detectable after 1 minute, and peak membrane-bound and soluble forms of guanylate cyclase occur after 10- and 30-minute exposure to phorbol myristate acetate, respectively. The soluble form is stimulated much more than the membrane form. Enzyme activities measured in the presence of either Ca²⁺, Mg²⁺, or Mn²⁺ are elevated to similar degrees. Comparisons of phorbol and a series of its diesters revealed a good correlation between the capacities for guanylate cyclase stimulation, lymphocyte mitogenesis, and tumor promotion.     

Aldehyde-modified proteins as mediators of early inflammation in atherosclerotic disease

Inflammation is widely accepted to play a major role in atherosclerosis and other cardiovascular diseases. However, the exact mechanism(s) by which inflammation exerts its pathogenic effect remains poorly understood. A number of oxidatively modified proteins have been associated with cardiovascular disease. Recently, attention has been given to the oxidative compound of malondialdehyde and acetaldehyde, two reactive aldehydes known to covalently bind and adduct macromolecules. These products have been shown to form stable malondialdehyde–acetaldehyde (MAA) adducts that are reactive and induce immune responses. These adducts have been found in inflamed and diseased cardiovascular tissue of patients. Antibodies to these adducted proteins are measurable in the serum of diseased patients. The isotypes involved in the immune response to MAA (i.e., IgM, IgG, and IgA) are predictive of atherosclerotic disease progression and cardiovascular events such as an acute myocardial infarction or coronary artery bypass grafting. Therefore, it is the purpose of this article to review the past and current knowledge of aldehyde-modified proteins and their role in cardiovascular disease.     

Biomechanical mechanism of lateral trunk lean gait for knee osteoarthritis patients

The biomechanical mechanism of lateral trunk lean gait employed to reduce external knee adduction moment (KAM) for knee osteoarthritis (OA) patients is not well known. This mechanism may relate to the center of mass (COM) motion. Moreover, lateral trunk lean gait may affect motor control of the COM displacement. Uncontrolled manifold (UCM) analysis is an evaluation index used to understand motor control and variability of the motor task. Here we aimed to clarify the biomechanical mechanism to reduce KAM during lateral trunk lean gait and how motor variability controls the COM displacement. Twenty knee OA patients walked under two conditions: normal and lateral trunk lean gait conditions. UCM analysis was performed with respect to the COM displacement in the frontal plane. We also determined how the variability is structured with regards to the COM displacement as a performance variable. The peak KAM under lateral trunk lean gait was lower than that under normal gait. The reduced peak KAM observed was accompanied by medially shifted knee joint center, shortened distance of the center of pressure to knee joint center, and shortened distance of the knee–ground reaction force lever arm during the stance phase. Knee OA patients with lateral trunk lean gait could maintain kinematic synergy by utilizing greater segmental configuration variance to the performance variable. However, the COM displacement variability of lateral trunk lean gait was larger than that of normal gait. Our findings may provide clinical insights to effectively evaluate and prescribe gait modification training for knee OA patients.     

A dual fluorescent/MALDI chip platform for analyzing enzymatic activity and for protein profiling

Being able to rapidly and sensitively detect specific enzymatic products is important when screening biological samples for enzymatic activity. We present a simple method for assaying protease activity in the presence of protease inhibitors (PIs) by measuring tryptic peptide accumulation on copolymer pMALDI target chips using a dual fluorescence/MALDI‐TOF‐MS read‐out. The small platform of the chip accommodates microliter amounts of sample and allows for rapid protein digestion. Fluorescamine labeling of tryptic peptides is used to indicate the proteolytic activity and is shown to be an affordable, simple process, yielding a strong fluorescence signal with a low background. Subsequent MALDI‐TOF‐MS analysis, performed in the same sample well, or in a parallel well without adding fluorescamine, detects the specific tryptic peptides and provides confidence in the assay. The dual read‐out method was applied to screen the inhibition activity of plant PIs, components of plant defense against herbivores and pathogens. Extracts of PIs from Solanum nigrum and trypsin were applied together to a pMALDI chip on which a suitable substrate was adsorbed. The fluorescence and MALDI‐TOF‐MS signal decrease were associated with the inhibitory effect of the PIs on trypsin. The developed platform can be modified to screen novel protease inhibitors, namely, those potentially useful for treating or preventing infection by viruses, including HIV and hepatitis C.     

Comparison of EMG activity on abdominal muscles during plank exercise with unilateral and bilateral additional isometric hip adduction

The aim of this study was to investigate the effects of additional isometric hip adduction during the plank exercise on the abdominal muscles. Twenty healthy young men participated in this study. Surface electromyography (EMG) was used to monitor the activity of the bilateral rectus abdominis (RA), the internal oblique (IO), and the external oblique (EO) muscles. The participants performed three types of plank exercise; the standard plank exercise, the plank exercise with bilateral isometric hip adduction, and the plank exercise with unilateral isometric hip adduction. All abdominal muscle activity was significantly increased during the plank exercise combined with the bilateral and unilateral isometric hip adduction compared with the standard plank exercise (p < 0.05). Bilateral IO, EO, and left RA muscle activity was significantly increased during the unilateral isometric hip adduction compared with the bilateral isometric hip adduction (p < 0.05). These findings suggest that additional isometric hip adduction during the plank exercise could be a useful method to enhance abdominal muscle activity. In particular, the unilateral isometric hip adduction is a more beneficial exercise than the bilateral isometric hip adduction.     

Reference database of the gait cycle for young healthy Tunisian adults

BackgroundQuantified gait analysis is a rising technology used increasingly to assess motor disorders. Normal reference data are required in order to evaluate patients, but there are no reference data available for the Tunisian healthy population.AimTo assess the features of normal Tunisian gait pattern, and examine the intrinsic reliability of spatio-temporal, kinematic and kinetic parameters within a new specific reference database.MethodsEighteen healthy active-young adults (age: 23.30 ± 2.54 years, height: 1.78 ± 0.04 m and, weight: 70.00 ± 4.80 kg) have participated to five trials of step gait where the dominant lower limb were recorded. Two over the five trials were randomly selected to be further analyzed. Twenty-three spatio-temporal, kinematic and kinetic parameters determined from 3-dimensional gait analysis. The intrinsic reliability was examined for each variable and our results were compared with those available in the literature.ResultsTwelve over 23 parameters have an excellent intrinsic reliability (P > 0.05, ICC > 0.9 and SEM < 5% of the grand mean). There are similarities with other studies (P < 0.05) but we noticed the existence of some specificity (the height of hip extension peak and the low cadence of gait) that could characterize the Tunisian population.ConclusionA specific reference database of the gait cycle has been established for healthy Tunisian active-young adults and excellent inter-trial reliability may be observed for different variables.     

A conundrum in molecular toxicology: Molecular and biological changes during neoplastic transformation of human cells

The process of multistage carcinogenesis lends itself to the concept that the effects of carcinogens are mediated through dose-related, multi-hit, linear changes. Multiplein vitro model systems have been developed that are designed to examine the cellular changes associated with the progression of cells through the different stages in the process; however, these systems may have inherent limitations due to the cell lines used for these studies, the manner of assessing the effects of the carcinogens, and the subsequent growth and differentiation of the exposed cells. Each of these variables results in increasing levels of uncertainty relative to the correlation of the events with the actual process of human tumor development. Therefore, the prediction of the ultimate effect of any carcinogen is difficult. Moreover, relationships between individual biological endpoints resulting from carcinogen treatment appear at best to be approximations. The presence of an activated carcinogen inside the cell can give rise to multiple outcomes, only some of which may be critical events. For example, site-specific modification of the 12th and 13th codons of H-ras is different than that in the adjacent 14th and 15th codons. It is interesting to speculate what effect these differences might have on a biological outcome, e.g., transformation to anchorage-independent growth. The use of different model systems to examine the effects of activated carcinogens also creates additional problems. Comparisons ofin vitro transformed cells with similar cells isolated from human tumors indicate that the culture environment appears to influence the expression of a particular phenotype, in that human tumor cells in culture express many of the same parameters as those found in cells transformed with carcinogensin vitro. If the process of transformation is linear, then less aggressive phenotypes should progress to a more aggressive transformed stage. However, in carcinogen-transformed human cells, the populations exhibit phenotypic diversity in that many of the transformed cells differentiate and fial to continue to divide in culture. Historically, we have assumed only a limited role for epigenetic modulation of molecular changes that occur during progression; however, our data suggest quite strongly that nonmalignant tumor populations can be converted to a more malignant phenotype without additional mutations taking place and, conversely, malignant populations can be downregulated to a nontumorigenic phenotype. Tumor cell plasticity is not only a fundamental characteristic of diverse types of human tumors, but also appears as an integral characteristic of carcinogen-transformed cellsin vitro.Abbreviations AIG anchorage-independent growth - B[a]P benzo[a]pyrene - BPDE-I benzo[a]pyrene diol epoxide I - I-NP 1-nitrosopyrene - PCR polymerase chain reaction - PDL population doubling(s)     

Asymmetric labeling of amino lipids in liposomes

Fluorescamine and trinitrobenzenesulfonate were used as chemical probes to differentially label amino phospholipids in liposomes. At low concentrations, fluorescamine reacts primarily with amino lipids on the external half of the bilayer. Further increase in fluorescamine concentration resulted in a linear increase of labeling indicating penetration and reaction with the internal half of the bilayer. Because of the pH requirements of the fluorescamine reaction, internal labeling was eliminated with a H⁺ gradient: inside acidic/outside alkaline. Differential labeling was also achieved with trinitrobenzenesulfonate, which is normally not permeable but which can be transported by valinomycin K⁺ complex and react with internal amines. Thus, either half of the bilayer can be labeled with the same or different reagents.When liposomes were double-labeled, the fluorescence of fluorescamine was quenched by the trinitrobenzenesulfonate label. This quenching was reversed by solubilizing the liposomes with acidic ethanol. No quenching occurred when fluorescamine-labeled liposomes were mixed with trinitrobenzenesulfonate-reacted liposomes (or trinitrophenylated methylamine) suggesting close proximity of two labels is required for quenching. Conditions which promoted vesicular fusion promptly produced quenching.These differential labeling procedures can be usefully applied to quantitate aminolipids on internal and external vesicular surfaces, monitor vesicular fusion, and assess liposomal structure.     

Quantitative evaluation of the major determinants of human gait

Accurate knowledge of the isolated contributions of joint movements to the three-dimensional displacement of the center of mass (COM) is fundamental for understanding the kinematics of normal walking and for improving the treatment of gait disabilities. Saunders et al. (1953) identified six kinematic mechanisms to explain the efficient progression of the whole-body COM in the sagittal, transverse, and coronal planes. These mechanisms, referred to as the major determinants of gait, were pelvic rotation, pelvic list, stance knee flexion, foot and knee mechanisms, and hip adduction. The aim of the present study was to quantitatively assess the contribution of each major gait determinant to the anteroposterior, vertical, and mediolateral displacements of the COM over one gait cycle. The contribution of each gait determinant was found by applying the concept of an ‘influence coefficient’, wherein the partial derivative of the COM displacement with respect to a prescribed determinant was calculated. The analysis was based on three-dimensional measurements of joint angular displacements obtained from 23 healthy young adults walking at slow, normal and fast speeds. We found that hip flexion, stance knee flexion, and ankle-foot interaction (comprised of ankle plantarflexion, toe flexion and the displacement of the center of pressure) are the major determinants of the displacements of the COM in the sagittal plane, while hip adduction and pelvic list contribute most significantly to the mediolateral displacement of the COM in the coronal plane. Pelvic rotation and pelvic list contribute little to the vertical displacement of the COM at all walking speeds. Pelvic tilt, hip rotation, subtalar inversion, and back extension, abduction and rotation make negligible contributions to the displacements of the COM in all three anatomical planes.