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Cross-reinnervation of rabbit soleus muscle by the peroneal nerve induces a 90% transformation of slow into fast fibres. These changes are reflected in corresponding transformations of the enzyme activity pattern of energy metabolism, the isozyme pattern of lactate dehydrogenase and, in confirmation of previous results (Srihari et al. 1981), transitions from a slow to a fast type myosin light chain pattern. The transformation process appears to be complete after 6 months. Similar changes, although less extensive are also found in the soleus muscle of the contralateral leg. Fibre type transitions in the contralateral muscle are not accompanied by fibre type grouping, as seen in the cross-reinnervated muscle and therefore these changes appear to result from a transformation of the motor units themselves. This phenomenon is interpreted as a compensatory process in maintaining symmetry within the neuromotor system.

The alterations in activity patterns of representative enzymes in energy metabolism were investigated in the superficial (white) and deep (red) portions of the fast vastus lateralis muscle of the adult rat in response to prolonged endurance training. It was found that following 15 weeks of extreme training (final running duration: 210 min per day, 27 m/min at 15 degree grade), increases in the activities of marker enzymes of the citric acid cycle (citrate synthase), beta-oxidation (3-hydroxyacyl CoA dehydrogenase), and ketone body utilization (3-ketoacid CoA transferase) as well as of glutamate pyruvate transaminase occurred in both regions of the muscle, with the greatest increase being observed in the superficial portion (2.6-4.2-fold). Pronounced increases were also seen for hexokinase which showed highest activities after 7 weeks of training. Conversely, decreases were noted for various glycogenolytic, glycolytic and gluconeogenic enzymes (phosphorylase, glyceraldehydephosphate dehydrogenase, pyruvate kinase, lactate dehydrogenase and fructose-1,6-diphosphatase). Reduction in the activities of these enzymes was most pronounced in the deep portion of the muscle. These results demonstrate a fundamental rearrangement of the energy metabolism of the muscle in response to prolonged, high intensity training. In the case of the deep portion of the vastus lateralis muscle, which has been shown to be composed of a large percentage of fast oxidative-glycolytic fibres (FOG), the enzyme profile becomes similar to the slow oxidative (SO) fibre. In the superficial portion which contains predominantly fast glycolytic fibres (FG), the enzyme profile becomes similar to FOG fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

It has been suggested that muscle wasting in muscular dystrophy is causally related to a perturbation of Ca2+ -homeostasis in the muscle fiber (8,525–530) due to an increased permeability of the sarcolemmal membrane. An elevated sarcoplasmic Ca2+ concentration would result from an increased Ca2+ -influx and would thus lead to an activation of Ca2+ -dependent proteases. Ca2+ has also been shown to play an important role in the regulation of glucose-1,6-biphosphate (531,532) a powerful effector of several enzymes in glucose metabolism (533). This modulator has been shown to be significantly reduced in muscles of dystrophic mice (534,535).

The present study compares the time courses of the early changes in parvalbumin content, in the properties of the sarcoplasmic reticulum (SR) and in activity and isozyme patterns of metabolic enzymes in chronically (12 h/day) stimulated fast twitch tibialis anterior (TA) muscle of the rabbit. Under the chosen conditions of stimulation, the first significant changes appeared after 6 days. Except for the delayed reduction in pyruvate kinase, the time course of the changes were the same. After 14 days of stimulation, parvalbumin decreased to 37% and Ca2+-ATPase activity of the SR to 29% of normal values. The transformation of the SR was also reflected by a 64% decrease of the 115000-Mr Ca2+-pumping peptide and a 5-fold increase in a 30000-Mr peptide. Following an identical time course, the mitochondrial activities of citrate synthase, 3-hydroxyacyl-CoA dehydrogenase and ketoacid-CoA transferase increased 2.9, 3.0 and 3.7-fold respectively. A similar time course was observed in the M to H-type transition of the lactate dehydrogenase isozymes. The cause of these changes is discussed as it relates to altered transcriptional and/or translational activities. It is suggested that an increase in free intracellular Ca2+ caused by increased contractile activity, which is then perpetuated by the decrease in Ca2+-binding and sequestering capacities, might be the signal for such altered synthetic activities.

Single-fiber analyses using a kinetic microphotometric method were performed on three patients with chronic progressive external ophthalmoplegia and proximal myopathy accompanied by a partial deficiency of cytochrome c oxidase. Two patients had subsarcolemmal accumulation of mitochondria (ragged-red fibers). Qualitative histochemical demonstration of cytochrome c oxidase showed a mosaic of fibers without detectable cytochrome c oxidase activity. Quantitative single fiber measurements in the patients9 biopsies showed that the majority of the muscle fibers had decreased cytochrome c oxidase activity without selective involvement of a specific fiber type. Succinate dehydrogenase was measured and the ratio of the activities (succinate dehydrogenase/cytochrome c oxidase) was calculated. Normal muscle showed a ratio of about 2, whereas diseased muscle showed values between 10 and 20, due to a decrease in cytochrome c oxidase activity. Ragged-red fibers showed very low or undetectable cytochrome c oxidase activity.

To determine whether respiratory muscles undergo alterations in enzyme activities of energy metabolism as a result of increased mechanical activity, adult male Wistar rats were subjected to a prolonged endurance training program. Analysis off maximal enzyme activity patterns in the diaphragm following 15 weeks of extreme training (final running duration: 210 min per day, 27 m.min-1 at 15 degrees grade, indicated significant reductions in the marker enzymes of the citric acid cycle (citrate synthase), glycolysis (pyruvate kinase, PK; lactate dehydrogenase, LDH), ketone body utilization (3-keto acid: CoA transferase) and transamination (glutamate pyruvate transaminase, GPT). No changes were found for the enzymes of glycogenolysis (phosphorylase, PHOSPH), glycolysis (glyceraldehyde phosphate dehydrogenase, GAPDH), glucose phosphorylation (hexokinase, HK) and beta-oxidation (3-hydroxyacyl: CoA dehydrogenase, HAD) following training. In contrast, in the external intercostal muscle, increases in the range of 57-77% were noted for the enzymes CS and HAD, whereas in the internal intercostal muscles no training induced alteration was evident for these enzymes. For both the intercostal muscles, a consistent trend was noted towards a reduction in all of the glycolytic enzymes investigated, however, significantly lower values were recorded for only PK and LDH in the internal intercostals. GPT was increased in the internal intercostal muscles. These findings indicate that the response pattern observed in the enzyme activities studied following training are to some degree specific to the respiratory muscle investigated.

Parvalbumin content is decreased by 40% in hindlimb muscles of adult (3.5 months) dystrophic mice when compared to normal mice of the same age. The concentration of parvalbumin in the dystrophic muscles resembles that of immature muscles of 4-week-old normal mice. The reduction may contribute to an elevation of sarcoplasmic free CA2+, which could stimulate various Ca2+-dependent processes related to the disease.

Developmental profiles were determined for the activities of eight enzymes involved in fatty acid β-oxida-tion in rat brain. The enzymes studied were the palmitoyl-CoA, octanoyl-CoA, butyryl-CoA, glutaryl-CoA, and 3-hy-droxyacyl-CoA dehydrogenases, the enoyl-CoA hydratase (crotonase), and the C4- and C10-thiolases. With the exception of the thiolases, all of the activities (expressed on the basis of brain weight) increased during the postnatal period of brain maturation. The activity of octanoyl-CoA dehy-drogenase was elevated markedly compared to that of pal-mitoyl-CoA dehydrogenase at all developmental stages and in all brain regions in the rat. A similar relationship between these enzymes was observed in various regions of adult human brain. Comparisons of the activities of the β-oxidation enzymes in human brain versus human skeletal muscle and in cultured neural cell lines (neuroblastoma and glioma) versus cultured skin fibroblasts revealed that the elevated activity of octanoyl-CoA dehydrogenase relative to palmitoyl-CoA dehydrogenase was specific to the neural tissues. This relationship was particularly evident when the enzyme activities were normalized to the activity of crotonase. The data support previous findings with ra-diochemical tracers, indicating that the brain is capable of utilizing fatty acids as substrates for oxidative energy metabolism. The relatively high activity of the medium-chain fatty acyl-CoA dehydrogenase in neural tissue may represent an adaptive mechanism to protect the brain from the known encephalopathic effects of octanoate and other medium-chain fatty acids that readily cross the blood-brain barrier.

Quantitative microphotometric measurements of two mitochondrial flavoproteins, glycerolphosphate oxidase (GP-OX) and succinate dehydrogenase (SDH), were performed on serial sections of mouse and rabbit tibialis anterior (TA) muscles in order to study the distribution of these two enzymes and their activity ratios in IIA and IIB fibres. The measurements showed a large scatter of the two enzyme activities in these two myosin-based fibre types. In rabbit TA, IIA and IIB fibres have similar GP-OX activities, whereas generally IIA fibres have higher SDH activities than IIB fibres. An inverse distribution of the two enzymes exists in mouse muscle. Generally, IIA fibres of mouse TA display low SDH and IIB fibres high SDH activities. The mean activity of GP-OX is slightly higher in IIA than in IIB fibres of mouse TA. Since measurements of both enzymes were taken in the same fibres, the ratio of their activities in each fibre could be evaluated. The SDH/ GP-OX activity ratios vary significantly between the two fibre populations both in rabbit and in mouse. The ratio is high in IIA and low in IIB fibres of rabbit TA, whereas it is low in IIA and high in IIB fibres of mouse TA.

A method was developed for the hemoglobin-free perfusion of the rabbit psoas muscle in situ. This muscle consists almost exclusively of fast-twitch (type IIb) glycolytic fibres and was therefore used as a model of a homogeneous muscle of the glycolytic, metabolic type.

We have developed a spectrophotometric assay to measure carnitine in any tissue. The test is based on the transformation of carnitine to acetyl-carnitine catalyzed by carnitine acetyltransferase. The second reaction product, reduced Coenzyme A, is converted to succinoyl-CoA by adding 2-oxoglutarate. The enzyme which catalyzes this reaction, 2-oxoglutarate dehydrogenase, reduces NAD which is followed spectrophotometrically. Via external standards the carnitine concentration can be determined. In a detailed study we proved the reproducibility, precision and specificity of the assay and its correspondence with the radiochemical test.

We report 2 brothers with hypertrophic cardiomyopathy, lipid storage and skeletal mitochondrial myopathy. Biochemical analyses detected complex II (succinate dehydrogenase) deficiency of the respiratory chain. Single fiber analyses using a microphotometer were performed and revealed reduced succinate dehydrogenase activity in all muscle fibers. This report shows the validity of kinetic microphotometric analysis in revealing complex II defects.

Parkinson's Disease (PD) is a neurodegenerative disorder with early non-motor/motor symptoms that may evade clinical detection for years after the disease onset due to their mildness and slow progression. Digital health tools that process densely sampled data streams from the daily human-mobile interaction can objectify the monitoring of behavioral patterns that change due to the appearance of early PD-related signs. In this context, touchscreens can capture micro-movements of fingers during natural typing; an unsupervised activity of high frequency that can reveal insights for users’ fine-motor handling and identify motor impairment. Subjects' typing dynamics related to their fine-motor skills decline, unobtrusively captured from a mobile touchscreen, were recently explored in-the-clinic assessment to classify early PD patients and controls. In this study, estimation of individual fine motor impairment severity scores is employed to interpret the footprint of specific underlying symptoms (such as brady-/hypokinesia (B/H-K) and rigidity (R)) to keystroke dynamics that cause group-wise variations. Regression models are employed for each fine-motor symptom, by exploiting features from keystroke dynamics sequences from in-the-clinic data captured from 18 early PD patients and 15 healthy controls. Results show that R and B/H-K UPDRS Part III single items scores can be predicted with an accuracy of 78\% and 70\% respectively. The generalization power of these trained regressors derived from in-the-clinic data was further tested in a PD screening problem using data harvested in-the-wild for a longitudinal period of time ($mean\pm std: 7\pm 14$ weeks) via a dedicated smartphone application for unobtrusive sensing of their routine smartphone typing. From a pool of 210 active users, data from 13 self-reported PD patients and 35 controls were selected based on demographics matching with the ones in-the-clinic setting. The results have shown that the estimated index achieve {0.84(R),0.80(B/H-K)ROC AUC, respectively, with {sensitivity/specificity: 0.77/0.8(R), 0.92/0.63(B/H-K), on classifying PD and controls in-the-wild setting. Apparently, the proposed approach constitutes a step forward to unobtrusive remote screening and detection of specific early PD signs from mobile-based human-computer interaction, introduces an interpretable methodology for the medical community and contributes to the continuous improvement of deployed tools and technologies in-the-wild.

In order to study putative genotype phenotype correlations in mitochondrial disorders due to large-scale mtDNA deletions we performed a quantitative analysis of biochemical, morphological, and genetic findings in 20 patients. The size of the mtDNA deletions varied from 2 to 7.5 kb with a degree of heteroplasmy ranging from 16% to 78%. Applying improved methods for measuring respiratory chain enzyme activities, we found highly significant inverse correlations between the percentage of cytochrome c oxidase (COX)- negative fibers and citrate synthase (CS) normalized COX ratios. Significant correlations were also established between CS normalized complex I and complex IV ratios as well as between the degree of heteroplasmy of mtDNA deletions and the percentage of ragged red fibers, COX-negative fibers, and CS normalized complex I and complex IV ratios. Our results indicate that the degree of heteroplasmy of mtDNA deletions is mirrored on the histological as well as the biochemical level. Furthermore, our findings suggest that single large-scale deletions equally influence the activities of all mitochondrially encoded respiratory chain enzymes. Even low degrees of heteroplasmy of mtDNA deletions were found to result in biochemical abnormalities indicating the absence of any well-defined mtDNA deletion threshold in skeletal muscle.

Background and Aim: It is well accepted that the neurodegenerative process in Parkinson's disease (PD) starts many years before motor symptoms become evident enough to impact patients' life to an extent that will lead to clinical diagnosis. Therefore, the Movement Disorders Society defined new clinical diagnostics, as well as research criteria in the form of risk and prodromal markers, focusing especially on non-motor symptoms (NMS) to diagnose PD earlier or at least to evaluate the risk of transition to clinically evident PD in the future. Earlier diagnosis of PD is a significant need for developing neuroprotective therapies. Objective: The i-PROGNOSIS project developed the "iPrognosis" Android smartphone application for unobtrusive big data collection with the aim of evolving it into an early PD detection tool in the daily living. Methods: The iPrognosis application collects participants' behavioural data arising from the everyday use of their smartphones over time in an unobtrusive way. The types of data, namely general usage data (GData), have been defined based on known motor and non-motor symptoms of PD and include features of voice (recorded during phone calls), movement (by recording accelerometer data during specific gestures), location patterns and touch screen typing as well as NMS-related features such as mood characteristics (via analyses of text messages and selfies). The application can be downloaded for free from the Google Play Store in Germany, Greece and Portugal since May 2017. Further request of ethical approval is in process in the United Kingdom. Healthy people and patients in early stages of PD, aged over 40 years, can participate in the GData collection study after providing electronic informed consent via the application. Results: Preliminary analyses of accelerometer data collected in laboratory conditions yielded a tremor recognition accuracy of 87%, 75% and 72% when holding the phone in rest position, near the ear during calls and while typing, respectively. Differences between PD patients and controls in terms of typing patterns (keystroke dynamics and pressure applied on keys) were also observed, leading to a discrimination accuracy of 91%. The focus is now placed on validating these findings on big data collected "in real life". Two months after the release of the iPrognosis application, 254 participants in total (187 healthy persons with 71.6% male and a mean age of 45 ± 16.1; 67 PD patients with 95.5% male and a mean age of 52 ± 15.9) downloaded it and enrolled in the study. As a result, more than 17 GB of ecologically-valid GData are already available to be used towards validation of the approach in due time. Conclusion: Modern technologies such as smartphones offer the ability of remotely monitoring people's behaviour over time in an unobtrusive way, as opposed to snapshot assessments in the artificial clinical environment. This offers the potential of detecting symptoms and behavioural changes earlier, allowing for earlier disease detection and fostering the development of relevant treatments. Initial evidence of pattern differences between healthy controls and PD patients, produced by the i-PROGNOSIS approach, are promising regarding the potential for early PD detection.

Introduction: We present a family comprising a clinically normal mother and two daughters, each with severe encephalopathy with onset in late childhood. A third daughter had died previously of an earlier onset but neuropathologically similar disease. Methods: Sequence analysis of the entire mtDNA was carried out in muscle, fibroblasts, and lymphocytes of the affected daughters and unaffected mother. Biochemical analysis of individual respiratory chain enzymes was performed on the same tissues, and on several transmitochondrial cybrid clones containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA. Results: Genetic analyses revealed in both daughters and mother the presence of a novel mutation in the tRNAIle gene of mtDNA, which was homoplasmic in fibroblasts, lymphocytes, and skeletal muscle of the two patients. It was also homoplasmic in fibroblast and skeletal muscle samples of the mother, and approximately 97% heteroplasmic in her lymphocytes. Combined defects of complexes I and IV of the mitochondrial respiratory chain were found not only in fibroblasts of the two probands, but surprisingly also in those of their clinically unaffected mother. The respiratory chain defect segregated in transmitochondrial cybrids containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA, indicating that the latter was responsible for the biochemical phenotype. Discussion: Our results support the concept that homoplasmic mutations in tRNA genes can be responsible for mitochondrial disorders characterised by extremely variable penetrance. Albeit still unexplained, this phenomenon has important consequences in the nosological characterisation, clinical management, and genetic counselling of mitochondrial disorders.

0.0055 + 0.0021** 0.0034 + 0.0022** 0.0071 + 0.0027** Succinate 0.0568 + 0.0200 0.0462 + 0.0262* 0.0696 + 0.0125** 0.0700 + 0.0240**

Neurodegenerative diseases are characterized by a progressive dysfunction of the nervous system. Often associated with atrophy of the affected central or peripheral nervous structures, they include diseases such as Parkinson’s Disease (PD), Alzheimer’s Disease and other dementias, Genetic Brain Disorders, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig’s Disease), Huntington’s Disease, Prion Diseases, and others. The prevalence of neurodegenerative diseases has increased over the last years. This has had a major impact both on patients and their families and has exponentially increased the medical bill by hundreds of billions of Euros. Therefore, understanding the role of environmental and genetic factors in the pathogenesis of PD is crucial to develop preventive strategies. While some authors believe that PD is mainly genetic and that the aging of the society is the principal cause for this increase, different studies suggest that PD may be due to an increased exposure to environmental toxins. In this article we review epidemiological, sociological and experimental studies to determine which hypothesis is more plausible. Our conclusion is that, at least in idiopathic PD (iPD), the exposure to toxic environmental substances could play an important role in its aetiology.