The wheat multidomain cystatin TaMDC1 displays antifungal, antibacterial, and insecticidal activities in planta.

KEY MESSAGE: Expression of the TaMDC1 in transgenic tomato plants confer resistance to bacterial and fungal pathogens, as well as an insect pest and thus prove in planta function of the wheat cystatin. Cystatins are the polypeptides with cysteine proteinase inhibitory activities. Plant cystatins or phytocystatins are known to contribute to plant resistance against insect pests. Recently, increasing data proved that some of the phytocystatins also have antifungal activities in vitro. Here, we functionally characterized a wheat multidomain cystatin, TaMDC1, using in planta assays. Expression of TaMDC1 in wheat seedlings is up-regulated in response to methyl jasmonate and salicylic acid, indicating that TaMDC1 is involved in biotic stress responses mediated by these plant hormones. The TaMDC1 cDNA was integrated in tomato genome and expressed under cauliflower mosaic virus 35S promoter. Four transgenic plants that show high level of the transgene expression were selected by RNA gel blot and immunoblot analysis and utilized to assess biotic stress resistance against the bacterial pathogen Pseudomonas syringae, the fungal pathogens Botrytis cinerea and Alternaria alternata, and the insect pest Colorado potato beetle (CPB, Leptinotarsa decemlineata). Detached leaf inoculation assays revealed that the tomato plants expressing TaMDC1 showed high levels of resistance against P. syringae and A. alternata, and elevated tolerance against B. cinerea. Sustenance of L. decemlineata larvae to the transgenic plants demonstrated inhibition of CPB larvae growth. Inhibitory activity of TaMDC1 against selected pathogens was also demonstrated by in vitro assays with total protein extracted from transgenic tomato plants. Taken together, the presented data suggest that TaMDC1 is involved in a broad spectrum biotic stress resistance in planta.

True associations between resting fMRI time series based on innovations.

We calculated voxel-by-voxel pairwise crosscorrelations between prewhitened resting-state BOLD fMRI time series recorded from 60 cortical areas (30 per hemisphere) in 18 human subjects (nine women and nine men). Altogether, more than a billion-and-a-quarter pairs of BOLD time series were analyzed. For each pair, a crosscorrelogram was computed by calculating 21 crosscorrelations, namely at zero lag ± 10 lags of 2 s duration each. For each crosscorrelogram, in turn, the crosscorrelation with the highest absolute value was found and its sign, value, and lag were retained for further analysis. In addition, the crosscorrelations at zero lag (irrespective of the location of the peak) were also analyzed as a special case. Based on known varying density of anatomical connectivity, we distinguished four general brain groups for which we derived summary statistics of crosscorrelations between voxels within an area (group I), between voxels of paired homotopic areas across the two hemispheres (group II), between voxels of an area and all other voxels in the same (ipsilateral) hemisphere (group III), and voxels of an area and all voxels in the opposite (contralateral) hemisphere (except those in the homotopic area) (group IV). We found the following. (a) Most of the crosscorrelogram peaks occurred at zero lag, followed by ± 1 lag; (b) over all groups, positive crosscorrelations were much more frequent than negative ones; (c) average crosscorrelation was highest for group I, and decreased progressively for groups II-IV; (d) the ratio of positive over negative crosscorrelations was highest for group I and progressively smaller for groups II-IV; (e) the highest proportion of positive crosscorrelations (with respect to all positive ones) was observed at zero lag; and (f) the highest proportion of negative crosscorrelations (with respect to all negative ones) was observed at lag = 2. These findings reveal a systematic pattern of crosscorrelations with respect to their sign, magnitude, lag and brain group, as defined above. Given that these groups were defined along a qualitative gradient of known overall anatomical connectivity, our results suggest that functional interactions between two voxels may simply reflect the density of such anatomical connectivity between the areas to which the voxels belong.

[Is the Gail model for breast cancer risk assessment valid for the Bulgarian women?].

INTRODUCTION: The quantitave assessment of the breast cancer risk is a fundamental for the process of preventation and early diagnosis. AIMS: The aim of present study is to determinate the validity of the Gail model used in USA for the Bulgarian population, and to assess the relative influence of each risk factor over the common breast cancer risk. MATERIALS AND METHODS: The object of our study were 315 women with histologically proved breast cancer. Retrospectively breast cancer rick was assessed for all patients by using Gail model. The following data were used: family history, previous benign biopsies, proved atypia, age of menarche, age of first birth and patient's age at the moment of detecting the breast cancer. RESULTS: In 186 (59%) patients no high risk is present after Gail model assessment and in the others 129 (41%) patients high risk for development of breast cancer is detected (> 1.7% for 5 years). Statistical reliability for the influence of each factor was found except for the previous benign biopsies. CONCLUSION: The results give us a reason to recommend Gail model as routine method for breast cancer risk assessment for the Bulgarian population for patients between 35 and 70 years old.

Detection of elastin derived peptides in cerebrospinal fluid of patients with first ever ischaemic stroke.

OBJECTIVE: We have previously reported the optimized methods for the detection of elastin derived peptides (EDP) in the serum, synovial fluid, and bronchoalveolar lavage. The aim of the present study was to investigate whether EDP are detectable in cerebrospinal fluid (CSF) of patients with acute brain ischaemia. PATIENTS AND METHODS: Twenty-seven first ever ischaemic stroke patients (mean age 61.5+/-10.8 years; age range 47-70 years; 12 women) were studied in acute phase (1-15 days after the onset) with clinical evaluations, radiological assessments, and the analysis of serum and CSF based on Western blot and ELISA for the detection and quantification of EDP. RESULTS: None of the serum EDP concentrations are significantly higher in stroke patients compared with 25 healthy control individuals. However, EDP levels in CSF are strongly (p<0.0001) elevated compared with healthy subjects. They correlated with total cholesterol (r=0.53; p=0.02), triglycerides (r=0.67; p=0.004) and retinopathy (r=0.24; p=0.03), and with the interval between the stroke onset and the time of lumbar puncture (r=0.35; p=0.02). CONCLUSION: EDPs are detectable in CSF of healthy subjects and patients with ischaemic stroke. Acute brain infarction is followed by increased levels of EDP in CSF.

Increased levels of elastin-derived peptides in cerebrospinal fluid of patients with lacunar stroke.

OBJECTIVE: To investigate whether elastin-derived peptides (EDP) are detectable in the cerebrospinal fluid (CSF) of healthy controls and of patients with acute brain ischemia and if so to assess possible trends in EDP levels in different groups of ischemic stroke patients (small-vessel disease vs. other ischemic strokes; first-ever vs. recurrent stroke). PATIENTS AND METHODS: Levels of EDP were determined by ELISA in blood sera and CSF of 80 patients with acute ischemic stroke (mean age 61.5+/-10.8; age range 47-70; 22 women) and in 15 healthy age- and sex-matched controls (mean age 57.3+/-13.4; age range 50-65). The patients were divided into a group with first ever lacunar stroke (27); first ever non-lacunar ischemic stroke (27) and recurrent stroke (26). EDP were measured early (mean 7 days, range 1-15) after stroke onset. RESULTS: Serum EDP levels were mildly higher in recurrent strokes as compared to first ever lacunar lesion and controls. However, in the CSF the concentrations of EDP in stroke patients were strongly elevated (from 2 up to 30 times depending on subgroup) as compared with healthy subjects. The highest level of EDP in CSF and in the serum was found in recurrent strokes. Subgroup analysis revealed a trend for significantly higher EDP concentrations in CSF in lacunar and recurrent stroke as compared with non-lacunar. CONCLUSIONS: This study is the first application of elastin peptide measurement to human CSF and stroke patients. The increased levels of EDP were detected in CSF of patients with lacunar and recurrent strokes.

Detection of free elastin-derived peptides among diabetic children.

Elastin breakdown products are found in the serum of all human subjects. The presence of these elastin-derived peptides (EDP) and the corresponding antibodies in circulation leads to formation of circulating immune complexes (CIC). The aim of this study was to determine if serum level of free-EDP (unbound in CIC) correlate with the development of microvascular complications in children with Type 1 (insulin-dependent) diabetes mellitus. To this end we used a method for detecting immune complexes (CIF-ELISA) in combination with an ELISA for detection of EDP. The levels of free EDP were studied in sera of 81 diabetic children (mean age 13.46+/-3.51 years, diabetes duration 5.17+/-4.21 years). Forty-two of the children had vascular complications (group 1) and 39 were without vascular complications (group 2). Twenty-one healthy children (mean age 12.6+/-2.47 years) were used as controls. Diabetics showed significantly higher levels of free EDP (68.1+/-25 ng/ml versus 51+/-12.5 ng/ml; p=0.003) compared to the control group. In group 1, free EDP showed significantly higher levels than controls (78.9+/-25.6 ng/ml versus 51+/-12.5 ng/ml; p=0.0001). About 38 of 81 (47%) patients were positive for free EDP (30/42--71% in group 1 and 8/39--21% in group 2). Free EDP levels in all diabetics showed a correlation with insulin dose (r=0.23; p=0.041), and microalbuminuria (r=0.57; p=0.0001). Patients who had vascular pathology showed a correlation of free EDP with microalbuminuria (r=0.41; p=0.0081), retinopathy (r=0.32; p=0.041), insulin dose (r=0.37; p=0.02), HbA1c (r=0.35; p=0.03), systolic blood pressure (r=0.30; p=0.045) and total cholesterol (r=0.36; p=0.02). These findings suggest that elevated levels of free EDP are associated with the development of diabetic vascular complications in children.

Treatment of vitiligo with local khellin and UVA: comparison with systemic PUVA.

Vitiligo is an idiopathic leukoderma often with a progressive course causing destruction of melanocytes. The best methods for achieving cosmetically acceptable re-pigmentation of affected skin appear to be both local and systemic PUVA. They may, however, cause serious side effects, which is an argument for conducting research into new, equally effective photo-chemotherapeutic agents. One of these agents is khellin. We conducted a pilot study in 33 patients to evaluate the effectiveness of local KUVA and systemic PUVA therapy for vitiligo and to compare them in terms of the degree of re-pigmentation, duration of treatment, number of procedures, total UVA dose and side effects. Local KUVA required longer duration of treatment and higher UVA doses. KUVA-induced re-pigmentation depended on the age of the patients (r = -0.61, P = 0.001), and better results were achieved with younger individuals [% re-pigmentation = 81.76 - (1.48 x age in years)]. No side effects were observed in cases of local KUVA treatment. Erythema, itching and gastro-intestinal disturbances occurred with some patients treated with PUVA. The results demonstrate that local KUVA may effectively induce re-pigmentation of vitiligo-affected skin areas to a degree comparable to that achieved when using systemic PUVA, provided that treatment duration is long enough.

pH-dependent stability of sperm whale myoglobin in water-guanidine hydrochloride solutions.

An experimental-theoretical approach for the elucidation of protein stability is proposed. The theoretical prediction of pH-dependent protein stability is based on the macroscopic electrostatic model for calculation of the pH-dependent electrostatic free energy of proteins. As a test of the method we have considered the pH-dependent stability of sperm whale metmyoglobin. Two theoretical methods for evaluation of the electrostatic free energy and p K values are applied: the finite-difference Poisson-Boltzmann method and the semiempirical approach based on the modified Tanford-Kirkwood theory. The theoretical results for electrostatic free energy of unfolding are compared with the experimental data for guanidine hydrochloride unfolding under equilibrium conditions over a wide pH range. Using the optical parameters of the Soret absorbance to monitor conformational equilibrium and Tanford's method to estimate the resulting data, it was found that the conformational free energy of unfolding of metmyoglobin is 16.3 kcal mol(-1) at neutral pH values. The total unfolding free energies were calculated on the basis of the theoretically predicted electrostatic unfolding free energies and the experimentally measured midpoints (pH(1/2)) of acidic and alkaline denaturation transitions. Experimental data for alkaline denaturation were used for the first time in theoretical analysis of the pH-dependent unfolding of myoglobin. The present results demonstrate that the simultaneous application of appropriate theoretical and experimental methods permits a more complete analysis of the pH-dependent and pH-independent properties and stability of globular proteins.

Serum antibodies to collagen type IV and development of diabetic vascular complications in children with type 1 (insulin-dependent) diabetes mellitus. A longitudinal study.

Thickening of basement membrane in capillaries and small vessels is a well-known finding and important in the progression of diabetic microangiopathy. To monitor the metabolism of the basement membrane protein collagen type IV (CIV) in diabetes mellitus, serum levels of IgG, IgM and IgA to CIV were measured using an ELISA method in 28 children with Type 1 diabetes mellitus over a period of 6 years. These values were compared to serum antibodies to CIV in 24 age- and sex-matched controls. At the end of the study, 11 children had diabetic microangiopathy. IgG to CIV was associated with age (r = .33, P = .026), diabetes duration (r = .32, P = .021), HbA1c (r = .31, P = .019), microalbuminuria (r = .32, P = .022) and anti-AGE antibodies (r = .47, P = .0007). IgM to CIV correlated with age (r = .46, P = .001), diabetes duration (r = .45, P = .001), HbA1c (r = .26, P = .038) and anti-AGE antibodies (r = .26, P = .038) and IgA to CIV with triglycerides (r = .29, P = .038) and anti-AGE antibodies (r = .44, P = .0025). We suggest that serum levels of IgG to CIV can be used as a marker for the development of diabetic microalbuminuria.

The vestibulo-ocular reflex and velocity storage in spinocerebellar ataxia 8.

The autosomal dominant spinocerebellar ataxias (SCAs) are a group of neurodegenerative diseases characterized by progressive instability of posture and gait, incoordination, ocular motor dysfunction, and dysarthria due to degeneration of cerebellar and brainstem neurons. Among the more than 20 genetically distinct subtypes, SCA8 is one of several wherein clinical observations indicate that cerebellar dysfunction is primary, and there is little evidence for other CNS involvement. The aim of the present work was to study the decay of the horizontal vestibulo-ocular reflex (VOR) after a short period of constant acceleration to understand the pathophysiology of the VOR due to cerebellar Purkinje cell degeneration in SCA8. The VOR was recorded in patients with genetically defined SCA8 during rotation in the dark. Moderate to severely affected patients had a qualitatively intact VOR, but there were quantitative differences in the gain and dynamics compared to normal controls. During angular velocity ramp rotations, there was a reversal in the direction of the VOR that was more pronounced in SCA8 compared to controls. Modeling studies indicate that there are significant changes in the velocity storage network, including abnormal feedback of an eye position signal into the network that contributes to this reversal. These and other results will help to identify features that are diagnostic for SCA subtypes and provide new information about selective vulnerability of neurons controlling vestibular reflexes.

Antibodies to advanced glycation end products in children with diabetes mellitus.

The tissue accumulation of advanced glycation end products (AGE) alters the structure and function of long-lived proteins. A number of studies have shown that tissue accumulation of AGE correlates with the severity of diabetic complications. Proteins containing AGE are highly immunogenic and anti-AGE antibodies were found in sera of diabetic rats and human. Considering the potential use of anti-AGE antibodies as a marker of AGE deposition during diabetes, we have investigated, by competitive ELISA, the presence of anti-AGE antibodies in sera of 58 children with Type 1 diabetes mellitus. The patients were studied for the period of 5 years. Positive for anti-AGE antibodies were 19 children with diabetes. Fourteen of them showed initial data for vascular complications. Anti-AGE antibodies were related to age (r = .25, P = .024), duration of diabetes (r = .41, P = .0001), HbA1c (r = .27, P = .016), microalbuminuria (r = .41, P = .0001), retinopathy (r = .35, P = .001), triglycerides (r = .27, P = .016), and total cholesterol (r = .19, P = .05). In conclusion, our study showed that the investigation of the levels and dynamics of anti-AGE antibodies might give the possibility for early diagnosis and prognosis of the severity of diabetic late complications.

Human motor unit recruitment and derecruitment during long lasting intermittent contractions.

Seven healthy subjects were investigated in cyclic ramp-and-hold long lasting isometric contractions. Wire branched electrodes were used for selective recording of single motor unit (MU) potentials from m. biceps brachii. MU behaviour was defined in terms of recruitment/derecruitment thresholds (RT and DT) and the duration of interspike intervals (ISI). A total of 63 MUs was investigated: 40 units were active from the beginning of the task performance and another 23 were recruited later. There were no changes in the recruitment pattern of MUs with fatigue development - a short first ISI followed by a very long second one and an almost constant firing rate after this transient phase. The tendency of RT to gradually decrease dominates the results. Thus, the required constant rate of force increase with fatigue development was maintained mostly by the mechanisms of space coding (i.e., decrease of RT and recruitment of additional MUs). Oppositely, the time behaviour of the DT changes was not uniform and rate coding was an essential mechanism in the adaptation of MU activity to muscle fatigue during relaxation phases. The recruitment pattern and fatigue related behaviour of the additionally recruited MUs were similar to those of MUs active from the first cycle of the motor task performance.

Detection of serum collagen type IV in children with type 1 (insulin-dependent) diabetes mellitus--a longitudinal study.

The basement membrane is a major focus of scientific interest because of its role in a variety of diseases. In diabetes mellitus, a thickening of the capillary basement membrane results in microangiopathic lesions. To monitor the metabolism of the basement membrane protein collagen type IV (CIV) in diabetes mellitus, serum levels of CIV were measured using an enzyme-linked immunosorbent assay (ELISA) method in 28 children with type 1 diabetes mellitus over a period of 6 years. These values were compared to serum CIV levels in 24 age and sex matched controls. In the first 3 years, serum CIV levels were normal. In the 4th year, 1 patient and in years 5 and 6, 4 patients had increased CIV serum levels. At the end of the investigation, 3 children had developed retinopathy, 6 microalbuminuria, and 2 both microalbuminuria and retinopathy. Only those patients with microalbuminuria had increased CIV serum levels. In conclusion, we suggest that CIV serum levels can be used as a marker for the development of diabetic microalbuminuria.

Human motor unit activity during concentric and eccentric movements.

Single motor units (MUs) activity was investigated in human m. biceps brachii during movements against an elastic load. A total of sixty-five MUs were studied by means of subcutaneously placed fine-wire branched electrodes. Subjects were asked to perform active shortening and lengthening of the muscle with approximately constant velocities at two different speeds--slow and fast. Both recruitment (RT) and decruitment (DT) thresholds of MU were found to be lower in movement with higher velocity. The recruitment order of MUs was approximately one and the same during concentric movements with a different but constant velocity. The firing onset of MUs is organized so that the peak of the first twitch contraction occurs at approximately the same force level irrespective of how fast the movement is. In contrast, during the eccentric movements the peak of the last twitch contraction of MU occurs at different torque levels depending on the velocity. The decruitment of the MUs during eccentric movement was in a reverse order to their recruitment during concentric movements. Generally, at one and the same velocity the RT of a given MU was lower than DT. Nevertheless, the peaks of the first and the last twitch contractions during concentric and eccentric movements with one and the same velocity occurred at approximately one and the same torque level.

Thermal unfolding and conformational stability of the recombinant domain II of glutamate dehydrogenase from the hyperthermophile Thermotoga maritima.

Domain II (residues 189-338, M(r) = 16 222) of glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima was used as a model system to study reversible unfolding thermodynamics of this hyperthermostable enzyme. The protein was produced in large quantities in E.COLI: using a T7 expression system. It was shown that the recombinant domain is monomeric in solution and that it comprises secondary structural elements similar to those observed in the crystal structure of the hexameric enzyme. The recombinant domain is thermostable and undergoes reversible and cooperative thermal unfolding in the pH range 5.90-8.00 with melting temperatures between 75.1 and 68.0 degrees C. Thermal unfolding of the protein was studied using differential scanning calorimetry and circular dichroism spectroscopy. Both methods yielded comparable values. The analysis revealed an unfolding enthalpy at 70 degrees C of 70.2 +/- 4.0 kcal/mol and a DeltaC(p) value of 1.4 +/- 0.3 kcal/mol K. Chemical unfolding of the recombinant domain resulted in m values of 3.36 +/- 0.10 kcal/mol M for unfolding in guanidinium chloride and 1.46 +/- 0.04 kcal/mol M in urea. The thermodynamic parameters for thermal and chemical unfolding equilibria indicate that domain II from T.MARITIMA: glutamate dehydrogenase is a thermostable protein with a DeltaG(max) of 3.70 kcal/mol. However, the thermal and chemical stabilities of the domain are lower than those of the hexameric protein, indicating that interdomain interactions must play a significant role in the stabilization of T. MARITIMA: domain II glutamate dehydrogenase.

Ion-induced conformational and stability changes in Nereis sarcoplasmic calcium binding protein: evidence that the APO state is a molten globule.

Nereis sarcoplasmic Ca(2+)-binding protein (NSCP) is a calcium buffer protein that binds Ca(2+) ions with high affinity but is also able to bind Mg(2+) ions with high positive cooperativity. We investigated the conformational and stability changes induced by the two metal ions. The thermal reversible unfolding, monitored by circular dichroism spectroscopy, shows that the thermal stability is maximum at neutral pH and increases in the order apo < Mg(2+) < Ca(2+). The stability against chemical denaturation (urea, guanidinium chloride) studied by circular dichroism or intrinsic fluorescence was found to have a similar ion dependence. To explore in more detail the structural basis of stability, we used the fluorescent probes to evaluate the hydrophobic surface exposure in the different ligation states. The apo-NSCP exhibits accessible hydrophobic surfaces, able to bind fluorescent probes, in clear contrast with denatured or Ca(2+)/Mg(2+)-bound states. Gel filtration experiments showed that, although the metal-bound NSCP has a hydrodynamic volume in agreement with the molecular mass, the volume of the apo form is considerably larger. The present results demonstrate that the apo state has many properties in common with the molten globule. The possible factors of the metal-dependent structural changes and stability are discussed.

An association of anti-elastin IgA antibodies with development of retinopathy in diabetic children.

An important factor in the development of vascular wall alterations is degradation of the elastic fiber major protein-elastin. Elastin peptides derived from this degradation are present in the circulating blood and they are a stimulus for increased production of anti-elastin antibodies (AEAb). The aim of the present study was to examine the possible association between serum elastin AEAb and the development of diabetic vascular complications. Levels of AEAb (IgG, IgM and IgA) were determined by ELISA in sera of 28 children with Type 1 (insulin-dependent) diabetes mellitus (mean age 11.6+/-2.8 years, diabetes duration 5.1+/-2.5 years). None of the children had clinical or laboratory evidence of vascular complications. The children were followed over a period of 7 years, and 24 healthy children of similar age and sex served as a control group. During the study, four diabetics developed retinopathy, six microalbuminuria and two both retinopathy and microalbuminuria. Anti-elastin IgG showed correlation with diabetes duration (r=.48, P=.0007), HbA1c (r=.28, P=.05), triglycerides (r=.28, P=.05) and antibodies to advanced glycation endproducts (AGE) (r=.41, P=.005). Anti-elastin IgM correlated with HbA1c (r=.26, P=.038) and IgA with retinopathy (r=.32, P=.017). Our results suggest an association between the level of anti-elastin IgA antibodies and the development of diabetic retinopathy.

The highly similar TMP kinases of Yersinia pestis and Escherichia coli differ markedly in their AZTMP phosphorylating activity.

Thymidine monophosphate (TMP) kinases are key enzymes in nucleotide synthesis for all living organisms. Although eukaryotic and viral TMP kinases have been studied extensively, little is known about their bacterial counterparts. To characterize the TMP kinase of Yersinia pestis, a chromosomal region encompassing its gene (tmk) was cloned and sequenced; a high degree of conservation with the corresponding region of Escherichia coli was found. The Y. pestis tmk gene was overexpressed in E. coli, where the enzyme represented over 20% of total soluble proteins. The CD spectrum of the purified TMP kinase from Y. pestis was characteristic for proteins rich in alpha-helical structures. Its thermodynamic stability was significantly lower than that of E. coli TMP kinase. However, the most striking difference between the two enzymes was related to their ability to phosphorylate 3'-deoxy-3'-azidothymidine monophosphate (AZTMP). Although the enzymes of both species had comparable Km values for this analogue, they differed significantly in their Vmax for AZTMP. Whereas E. coli used AZTMP as a relatively good substrate, the Y. pestis enzyme had a Vmax 100 times lower with AZTMP than with TMP. This fact explains why AZT, a potent bactericidal agent against E. coli, is only moderately active on Y. enterocolitica. Sequence comparisons between E. coli and Y. pestis TMP kinases along with the three-dimensional structure of the E. coli enzyme suggest that segments lying outside the main regions involved in nucleotide binding and catalysis are responsible for the different rates of AZTMP phosphorylation.

Surface EMG recorded by branched electrodes during sustained muscle activity.

The purpose of the present investigation is to use surface interference EMG recorded by branched electrodes for assessment of muscle fatigue during sustained voluntary isometric contractions at different levels. Level-trigger averaging and turn/amplitude analysis have been applied. The conduction velocity (CV) of excitation was calculated from the time shift of the negative peaks of the averaged potentials (AvPs) derived from the EMG recorded by two electrodes placed along the muscle fibers. The recruitment of new motor units affects the negative amplitude (NA) of AvPs, the number of turns per second and the mean amplitude of turns in a different way depending on the level of sustained contractions. In contrast, the CV declined at all levels of sustained contractions and was the most appropriate parameter for the muscle fatigue assessment. There was a good correlation between CV decrease and torque reduction during sustained maximal efforts. The level-trigger averaging technique of the interference EMG recorded by surface branched electrodes is easy and non-invasive, thus being very convenient for routine application.