Lactate levels affect motor performance in MD 1

Myotonic Dystrophy type 1 (DM1) is a dominantly inherited disease comprehending multiple features. Fatigue and exhaustion during exercise often represent significant factors able to negatively influence their compliance to rehabilitation programs. Mitochondrial abnormalities and a significant increase in oxidative markers, previously reported, suggest the hypothesis of a mitochondrial functional impairment. The study aims at evaluating oxidative metabolism efficiency in 18 DM1 patients and in 15 healthy subjects, through analysis of lactate levels at rest and after an incremental exercise test. The exercise protocol consisted of a submaximal incremental exercise performed on an electronically calibrated treadmill, maintained in predominantly aerobic condition. Lactate levels were assessed at rest and at 5, 10 and 30 minutes after the end of the exercise. The results showed early exercise-related fatigue in DM1 patients, as they performed a mean number of 9 steps, while controls completed the whole exercise. Moreover, while resting values of lactate were comparable between the patients and the control group (p=0.69), after the exercise protocol, dystrophic subjects reached higher values of lactate, at any recovery time (p<0,05). These observations suggest an early activation of anaerobic metabolism, thus evidencing an alteration in oxidative metabolism of such dystrophic patients. As far as intense aerobic training could be performed in DM1 patients, in order to improve maximal muscle oxidative capacity and blood lactate removal ability, then, this safe and validate method could be used to evaluate muscle oxidative metabolism and provide an efficient help on rehabilitation programs to be prescribed in such patients

4'-Deoxypyridoxine disrupts vitamin B6 homeostasis in Escherichia coli K12 through combined inhibition of cumulative B6 uptake and PLP-dependent enzyme activity

Pyridoxal 5 '-phosphate (PLP) is the active form of vitamin B6 and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4'-deoxypyridoxine (4dPN) is a long known B6 antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism Escherichia coli K12, we showed that 4dPN cannot be used as a source of vitamin B6 as previously claimed and that it is toxic in several conditions where vitamin B6 homeostasis is affected, such as in a B6 auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, yggS. In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4'-deoxypyridoxine phosphate (4dPNP) and inhibi-tion of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK)

Isolation of a Complex Formed Between Acinetobacter baumannii HemA and HemL, Key Enzymes of Tetrapyrroles Biosynthesis

Plants, algae and most bacteria synthesize 5-aminolevulinic acid (ALA), the universal precursor of tetrapyrroles such as heme, chlorophyll and coenzyme B12, by a two-step transformation involving the NADPH-dependent glutamyl-tRNA reductase (HemA), which reduces tRNA-bound glutamate to glutamate-1-semialdehyde (GSA), and the pyridoxamine 5′-phosphate-dependent glutamate-1-semialdehyde-2,1-aminomutase (HemL), responsible for the isomerization of GSA into ALA. Since GSA is a very unstable compound at pH values around neutrality, the formation of a HemA-HemL complex has been proposed to occur, allowing for direct channeling of this intermediate from HemA to HemL. Experimental evidence of the formation of this complex has been obtained with the enzymes from Escherichia coli and Chlamydomonas reinhardtii. However, its isolation has never been attained, probably because HemA is degraded when intracellular heme accumulates. In this work, we devised a co-expression and co-purification strategy of HemA and HemL from Acinetobacter baumannii, which allowed the isolation of the HemA-HemL complex. Our results indicate that HemA is stabilized when co-expressed with HemL. The addition of citrate throughout the expression and purification procedure further promotes the formation of the HemA-HemL complex, which can be isolated in fair amount for functional and structural studies. This work lays the bases for a rational design of HemA-HemA inhibitors to be developed as antibacterial agents against A. baumannii, a multidrug resistant opportunistic pathogen responsible for a broad range of severe nosocomial infections

Valutazione dell'efficacia di una terapia antiossidante in pazienti affetti da Distrofia Miotonica tipo 1

La Distrofia Miotonica tipo 1 (DM-1) è una malattia a trasmissione autosomica dominante caratterizzata da impegno multisistemico. Il difetto genico causale consiste in un'abnorme ripetizione di una tripletta trinucleotidica CTG (citosina-timina-guanina) a livello del locus 19q13.3; il gene coinvolto codifica per una protein-chinasi, la cui funzione non è ancora nota. Il meccanismo patogenetico della malattia non è chiaro, ma precedenti studi hanno evidenziato aumentati livelli di radicali liberi ed una riduzione delle difese antiossidanti, suggerendo un possibile ruolo dello stress ossidativo nella DM-1. SCOPO DELLA TESI: Scopo della presente tesi è quello di valutare alcuni parametri ematici, indicatori di aumentato stress ossidativo, "in vivo" in soggetti affetti da DM-1, sia in condizioni di riposo, sia in relazione ad esercizio muscolare, e di verificare come questi possano modificarsi dopo una terapia a base di un integratore alimentare ad attività antiossidante, in uno studio condotto in doppio cieco. MATERIALI E METODI: Sono stati selezionati 20 pazienti, randomizzati e suddivisi in due bracci di studio, rispettivamente trattati con integratore donatore di cisteina (10 g/die), oppure con placebo, per trenta giorni. I pazienti hanno inoltre eseguito, prima e dopo terapia, un protocollo di esercizio intermittente incrementale ai muscoli dell'avambraccio, ed i prelievi sono stati praticati prima, durante e dopo sforzo. RISULTATI: La determinazione dei livelli dei prodotti di ossidazione avanzata delle proteine (AOPP) nei pazienti trattati ha evidenziato una riduzione significativa in condizioni di riposo (valore A: p= 0,008) e durante l'esercizio (tempo B: p= 0,02); la capacità ferro-riducente ha subito soltanto un non significativo aumento. Si è rilevato inoltre un aumento significativo dopo terapia del glutatione totale, in condizioni di riposo (valore A: p=0,0007) e durante lo sforzo (tempo B: p= 0,003; tempo C: p=0,004; tempo D: p=0,004). DISCUSSIONE: Questo studio evidenzia i potenziali benefici della terapia antiossidante sui marcatori biochimici di stress ossidativo, e consente di ipotizzare lo sviluppo di un minor danno ossidativo generato sia in condizioni di riposo che durante l'attività muscolare aerobica

Efficacia di un training riabilitativo di tipo task-oriented nei disturbi deambulatori in pazienti affetti da Sclerosi Multipla.

La Sclerosi Multipla (SM) è una patologia neurologica cronica e degenerativa che colpisce comunemente giovani adulti in tutto il mondo, rappresentando una delle principali cause di disabilità nel tempo. Le difficoltà nella deambulazione sono molto comuni in queste persone, ed è stato dimostrato che circa l’80% dei pazienti presenta una perdita della capacità deambulatoria, e quindi di conseguenza della propria indipendenza ed autonomia. La progressiva disabilità che si accumula durante il corso della patologia, associata all’alta prevalenza di questi deficit nella SM, rende ragione del forte impatto sociale, economico ed in termini di qualità di vita provocato da questi disturbi. Il panorama attuale offre una moltitudine di approcci riabilitativi efficaci nel migliorare la performance motoria e la qualità di vita di queste persone. Sebbene, ad oggi, sia consigliata la preferenza di protocolli riabilitativi intensivi e personalizzati sulla base delle esigenze del paziente, non esistono delle linee guida condivise e specifiche e manca l’identificazione di precisi criteri di trattamento in termini di modalità, tempistiche e dosaggi. Si evidenzia, quindi, la necessità di conoscere dei fattori prognostici di miglioramento funzionale in maniera da poter proporre trattamenti riabilitativi altamente personalizzati. In questo studio abbiamo quindi somministrato un intervento riabilitativo intensivo e task-specifico per i disturbi deambulatori in pazienti con uno specifico grado di disabilità motoria. Abbiamo quindi cercato di individuare dei fattori prognostici di miglioramento funzionale, attraverso l’utilizzo di estensive indagini cliniche, strumentali e neurofisiologiche. Lo scopo di questo tipo di approccio è quello di fornire dei marker clinici e strumentali con cui testare i pazienti, con il fine ultimo di strutturare trattamenti riabilitativi centrati sulla specificità della singola persona. Nel primo studio, abbiamo valutato gli effetti di un protocollo riabilitativo di tipo task-oriented (TOCT) sulla biomeccanica del cammino in 19 pazienti affetti da SM di grado lieve-moderato. Inoltre, abbiamo studiato le eventuali correlazioni tra l’entità del recupero motorio e le modifiche della dinamica del cammino. I risultati dell’analisi chinesiologica del cammino hanno evidenziato un miglioramento dell’escursione dinamica del ginocchio in seguito al training. Inoltre, la performance funzionale ai test clinici prima del trattamento era correlata alle modifiche nei parametri spazio-temporali e della qualità di vita. Infine, la miglior performance ai test clinici in seguito al TOCT era associata ad una miglior cinematica della caviglia. Questi dati suggeriscono, quindi, la centralità di uno specifico inquadramento clinico-funzionale e strumentale nell’impostazione dei trattamenti riabilitativi. Nella seconda parte dello studio, ci siamo invece focalizzati sulla valutazione meccanica ed elettrofisiologica della forza e della fatica e sulle possibili modificazioni in seguito all’esecuzione del circuito TOCT. I risultati hanno evidenziato come, ad un miglioramento funzionale delle scale cliniche, corrispondesse un aumento dell’output di forza in condizione isometriche durante la prova di estensione del ginocchio. Dall’altro lato, sebbene ci fosse un miglioramento della qualità di vita e della fatica percepita dai soggetti, non si è riscontrata alcuna modifica nella fatica meccanica ed elettrica in seguito al training riabilitativo. Le analisi delle correlazioni hanno rivelato come l’entità del miglioramento della qualità di vita percepita dai pazienti fosse associato al grado di incremento dell’output di forza esercitato alla prova di estensione del ginocchio. Presi nell’insieme, questi dati confermano quindi la centralità di alcune indagini clinico-funzionali in fase di “screening” dei pazienti, cui devono essere associate valutazioni strumentali di forza segmentale. Nell’ultima parte di esperimenti, abbiamo invece strutturato uno studio con indagini neurofisiologiche e strumentali, al fine di valutare i correlati neurali delle modificazioni cliniche indotte dal training task-oriented. Abbiamo, quindi, sottoposto 16 pazienti alla registrazione di un elettroencefalogramma (EEG) a 64 canali prima e dopo il trattamento riabilitativo, al fine di verificare le modificazioni della connettività funzionale sia a livello inter- che intra-emisferico tra diverse regioni cerebrali. Contestualmente, abbiamo collezionato una Risonanza Magnetica (RM) basale prima dell’inizio della riabilitazione, quantificando l’entità del carico lesionale encefalico ed il grado di atrofia cerebrale. Le analisi delle correlazioni tra le variabili cliniche, neurofisiologiche e strumentali hanno evidenziato l’importanza della connettività funzionale studiata con due indici specifici: il weighted Phase Lag Index (wPLI) è un indice predittivo dell’entità del recupero funzionale, mentre il weighted Simbolic Mutual Information (wSMI) rappresenta una stima delle riorganizzazione funzionale corticale indotta dal TOCT. Dall’altro lato, il carico lesionale stimato con la RM non sembra essere determinante nel predire l’entità del recupero funzionale in seguito al training. Questi risultati suggeriscono un nuovo approccio metodologico: gli indici neurofisiologici possono rappresentare dei buoni marker in grado di stimare il recupero motorio e di seguire i fenomeni di plasticità corticale, fornendo degli ottimi strumenti nel processo decisionale di personalizzazione degli interventi

Extensive Functional Evaluations to Monitor Aerobic Training in Becker Muscular Dystrophy: A Case Report

Low-intensity aerobic training seems to have positive effects on muscle strength, endurance and fatigue in Becker Muscular Dystrophy (BMD) patients. We describe the case of a 33-year old BMD man, who performed a four-week aerobic training. Extensive functional evaluations were executed to monitor the efficacy of the rehabilitative treatment. Results evidenced an increased force exertion and an improvement in muscle contraction during sustained exercise. An improvement of walk velocity, together with agility, endurance capacity and oxygen consumption during exercise was observed. Moreover, an enhanced metabolic efficiency was evidenced, as shown by reduced lactate blood levels after training. Interestingly, CK showed higher levels after the training protocol, revealing possible muscle damage. In conclusion, aerobic training may represent an effective method improving exercise performance, functional status and metabolic efficiency. Anyway, a careful functional assessment should be taken into account as a useful approach in the management of the disease's rehabilitative treatment

Functional assessment and rehabilitative treatment in myotonic dystrophy type 1

Motor impairment in Myotonic Dystrophy type 1 (DM1) involves different aspects of activity of daily living. Muscle weakness leads to impaired walking abilities, with an increased risk of stumbles and falls. While, motor performance can also be limited by the occurrence of precocious fatigability. Therefore the evaluation of functional impairment plays a central role in the chronic management of the disease. Muscle strength can be assessed by subjective clinical scale or by instrumental devices, such as isokinetic dynamometers, in order to limit the operator's test quality. Gait abilities can be assessed with standardized walking test, while computerized 3D gait analysis has been applied in order to quantitatively assess gait kinetics and kinematics. Moreover, instrumental assessment of balance impairment by means of posturography or stabilometric platforms is frequently used, generally accompanied by clinical balance scales. Exercise tolerance and resistance in general is assessed with cycle or treadmill submaximal incremental testing, while creatine kinase (CK) serum level are monitored in order to assess exercise safety. In this review, we will summarize the different rehabilitative approaches that have been proposed in these disease. Strength training and aerobic exercise programs have been proposed in order to improve impaired muscle and cardio-respiratory function and to prevent additional disuse atrophy The effects of aerobic training determines an improvement in terms of oxidative capacity and cardiovascular fitness. Moderateintense strength training seems harmless, even if there is still poor evidence of a real benefit. The effects of a specific resistance hand program was also evaluated, focusing on changes in hand function and in occupational performance. Only one trial evaluated the effects of a more comprehensive group exercise program, focusing on balance, strength and aerobic activities. An interesting approach tested the efficacy of a period of localized neuromuscular electrical stimulation on muscular weakness and the sarcolemmal excitability. In conclusion, patients with DM1 may probably benefit by strengthening exercises or aerobic protocols without any observed deleterious effect, even if the possible benefits in the long-term course are still not completely understood. Neuromuscular electrical stimulation could also be considered in the early management of the disease, as far as it represents a useful tool able to improve motor performance and walking abilities. Nevertheless, controlled trials performed on homogeneous groups and with wider number of patients should be tested. Moreover, specific both clinical and instrumental assessments need to be systematically introduced in everyday clinical practice, in order to evidence functional improvements

The MocR‐like transcription factors: pyridoxal 5′‐phosphate‐dependent regulators of bacterial metabolism

Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds. This is the case of MocR-like bacterial transcription factors (MocR-TFs), a subclass of GntR transcription regulators, whose structure is the outcome of the fusion between DNA-binding proteins and pyridoxal 5'-phosphate (PLP)-dependent enzymes. The resultant chimeras can count on the properties of both protein classes, i.e. the capability to recognize specific DNA sequences and to bind PLP and amino-compounds; it is the modulation of such binding properties to confer to MocR-TFs chimeras the ability to interact with effector molecules and DNA so as to regulate transcription. MocR-TFs control different metabolic processes involving vitamin B6and amino acids, which are canonical ligands of PLP-dependent enzymes. However, MocR-TFs are also implicated in the metabolism of compounds that are not substrates of PLP-dependent enzymes, such as rhizopine and ectoine. Genomic analyses show that MocR-TFs are widespread among eubacteria, implying an essential role in their metabolism and highlighting the scarcity of our knowledge on these important players in microbial metabolism. Although MocR-TFs have been discovered 15 years ago, the research activity on these transcriptional regulators has only recently intensified, producing a wealth of information that needs to be brought back to general principles. This is the main task of this review, which reports and analyses the available information concerning MocR-TFs functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor-binding sites of MocR-based regulatory systems

The MocR-like transcription factors: pyridoxal 5'-phosphate-dependent regulators of bacterial metabolism

Many biological functions played by current proteins were not created by evolution from scratch, rather they were obtained combining already available protein scaffolds. This is the case of MocR-like bacterial transcription factors (MocR-TFs), a subclass of GntR transcription regulators, whose structure is the outcome of the fusion between DNA-binding proteins and pyridoxal 5'-phosphate (PLP)-dependent enzymes. The resultant chimeras can count on the properties of both protein classes, i.e. the capability to recognize specific DNA sequences and to bind PLP and amino-compounds; it is the modulation of such binding properties to confer to MocR-TFs chimeras the ability to interact with effector molecules and DNA so as to regulate transcription. MocR-TFs control different metabolic processes involving vitamin B6and amino acids, which are canonical ligands of PLP-dependent enzymes. However, MocR-TFs are also implicated in the metabolism of compounds that are not substrates of PLP-dependent enzymes, such as rhizopine and ectoine. Genomic analyses show that MocR-TFs are widespread among eubacteria, implying an essential role in their metabolism and highlighting the scarcity of our knowledge on these important players in microbial metabolism. Although MocR-TFs have been discovered 15 years ago, the research activity on these transcriptional regulators has only recently intensified, producing a wealth of information that needs to be brought back to general principles. This is the main task of this review, which reports and analyses the available information concerning MocR-TFs functional role, structural features, interaction with effector molecules and the characteristics of DNA transcriptional factor-binding sites of MocR-based regulatory systems

Salmonella typhimurium PtsJ is a novel MocR-like transcriptional repressor involved in regulating the vitamin B6 salvage pathway

The vitamin B6 salvage pathway, involving pyridoxine 5′-phosphate oxidase (PNPOx) and pyridoxal kinase (PLK), recycles B6 vitamers from nutrients and protein turnover to produce pyridoxal 5′-phosphate (PLP), the catalytically active form of the vitamin. Regulation of this pathway, widespread in living organisms including humans and many bacteria, is very important to vitamin B6 homeostasis but poorly understood. Although some information is available on the enzymatic regulation of PNPOx and PLK, little is known on their regulation at the transcriptional level. In the present work, we identified a new MocR-like regulator, PtsJ from Salmonella typhimurium, which controls the expression of the pdxK gene encoding one of the two PLKs expressed in this organism (PLK1). Analysis of pdxK expression in a ptsJ knockout strain demonstrated that PtsJ acts as a transcriptional repressor. This is the first case of a MocR-like regulator acting as repressor of its target gene. Expression and purification of PtsJ allowed a detailed characterisation of its effector and DNA-binding properties. PLP is the only B6 vitamer acting as effector molecule for PtsJ. A DNA-binding region composed of four repeated nucleotide sequences is responsible for binding of PtsJ to its target promoter. Analysis of binding stoichiometry revealed that protein subunits/DNA molar ratio varies from 4 : 1 to 2 : 1, depending on the presence or absence of PLP. Structural characteristics of DNA transcriptional factor-binding sites suggest that PtsJ binds DNA according to a different model with respect to other characterised members of the MocR subgroup. © 2016 Federation of European Biochemical Societie