Biochemical characterization of plasma membrane isolated from human skeletal muscle

AbstractSpecific components of ion translocation systems were studied in excitable plasma membranes isolated from normal human muscle. Na+ - K+ ATPase and ouabain-sensitive K+ phosphatase activities were 8.9 ± 1 μmol Pih per mg protein and 96 ± 9 nmolmin per mg protein, respectively. Scatchard analysis of equilibrium binding assays with [3H]ouabain showed non-linear curves consistent with high- and low-affinity sites (estimated Kd 3 nM and 0.22 μM). Two families of receptors with different affinities for a tritiated TTX derivative (estimated Kd 0.4 and 4 nM) were also identified suggesting the existence in human muscle of at least two classes of voltage-dependent Na+ channels. In addition (+)-[methyl-3H]PN200-110, a potent Ca2+ antagonist used for labeling voltage-dependent Ca2+ channels, was observed to bind to a homogeneous population of receptors in the plasma membrane (Kd = 0.2 nM)

Поздняя форма болезни Помпе: диагностические и терапевтические подходы

Болезнь Помпе — клинически гетерогенное заболевание, в основе патогенеза которого лежит избыточное накопление гликогена лизосомами в результате мутации гена GAA и потери активности фермента кислой α‑глюкозидазы. Неонатальная форма заболевания, проявляющаяся сердечной, дыхательной и печеночной недостаточностью, имеет неблагоприятный прогноз. Поздние, медленно прогрессирующие хронические формы, проявляющиеся в возрасте после 1 года жизни с преимущественным поражением скелетной и дыхательной мускулатуры, имеют более благоприятное течение. Понимание и уточнение тонкостей патофизиологических процессов при болезни Помпе, а также разработка заместительной энзимотерапии (ЗЭТ) вызвали новую волну интереса к данной патологии. При ранних формах ЗЭТ имеет достаточно высокую эффективность в отличие от пациентов с поздними формами, что объясняется уже имеющимися значительными нарушениями клеточных и тканевых функций в результате избыточного накопления гликогена. С целью улучшения прогноза при поздних формах заболевания требуется как можно более раннее назначение ЗЭТ, что возможно только при своевременной постановке диагноза. Основным и самым простым способом диагностики болезни Помпе является определение активности фермента по сухому кровяному пятну. Вопрос о том, следует ли проводить ЗЭТ всем пациентам с поздней формой заболевания, остается открытым

A study on the safety and efficacy of reveglucosidase alfa in patients with late-onset Pompe disease.

BackgroundLate-onset Pompe disease is a rare genetic neuromuscular disorder caused by lysosomal acid alpha-glucosidase (GAA) deficiency that ultimately results in mobility loss and respiratory failure. Current enzyme replacement therapy with recombinant human (rh)GAA has demonstrated efficacy in subjects with late-onset Pompe disease. However, long-term effects of rhGAA on pulmonary function have not been observed, likely related to inefficient delivery of rhGAA to skeletal muscle lysosomes and associated deficits in the central nervous system. To address this limitation, reveglucosidase alfa, a novel insulin-like growth factor 2 (IGF2)-tagged GAA analogue with improved lysosomal uptake, was developed. This study evaluated the pharmacokinetics, safety, and exploratory efficacy of reveglucosidase alfa in 22 subjects with late-onset Pompe disease who were previously untreated with rhGAA.ResultsReveglucosidase alfa plasma concentrations increased linearly with dose, and the elimination half-life was <1.2 h. Eighteen of 22 subjects completed 72 weeks of treatment. The most common adverse events were hypoglycemia (63%), dizziness, fall, headache, and nausea (55% for each). Serious adverse events included hypersensitivity (n = 1), symptomatic hypoglycemia (n = 2), presyncope (n = 1), and acute cardiac failure (n = 1). In the dose-escalation study, all treated subjects tested positive for anti-reveglucosidase alfa, anti-rhGAA, anti-IGF1, and anti-IGF2 antibodies at least once. Subjects receiving 20 mg/kg of reveglucosidase alfa demonstrated increases in predicted maximum inspiratory pressure (13.9%), predicted maximum expiratory pressure (8.0%), forced vital capacity (-0.4%), maximum voluntary ventilation (7.4 L/min), and mean absolute walking distance (22.3 m on the 6-min walk test) at 72 weeks.ConclusionsAdditional studies are needed to further assess the safety and efficacy of this approach. Improvements in respiratory muscle strength, lung function, and walking endurance in subjects with LOPD may make up for the risk of hypersensitivity reactions and hypoglycemia. Reveglucosidase alfa may provide a new treatment option for patients with late-onset Pompe disease.Trial registrationISRCTN01435772 and ISRCTN01230801 , registered 27 October 2011

Structural investigations of the regio- and enantioselectivity of lipases

Although lipases are widely applied for the stereospecific resolution of racemic mixtures of esters, the atomic details of the factors that are responsible for their stereospecificity are largely obscure. We determined the X-ray structures of Pseudomonas cepacia lipase in complex with two enantiopure triglyceride analogues, that closely mimic natural substrates. This allowed an unambiguous view of how the two wings of the boomerang-shaped active site accommodate the acyl and alcohol parts of the triglyceride. The binding groove for the hydrophobic sn-3 fatty acid chain is large and hydrophobic. The cleft for the alcohol moiety is divided in two parts, one tightly binding the sn-2 acyl chain with hydrophilic and hydrophobic interactions, the other more weakly binding the sn-1 fatty acid. The enantioselectivity of Pseudomonas cepacia lipase seems therefore to be predominantly determined by the size and interactions of the sn-2 chain and by the size of the sn-3 chain.

Peptic hydrolysis of ovalbumin modified by acetylation,

1. 1. Two types of acetylated ovalbumin derivatives designated as N-and N,O-acetylovalbumin were prepared by treatment with ketene at pH's 5.6 and 9.0, respectively.2. 2. Both limited and extensive acetylation decreases the digestibility of ovalbumin by crystalline pepsin.3. 3. The limited digestion of the N,O-acetylovalbumin may be attributed to three influences: the acetyl groups on the seven amino groups unsubstituted in the N-acetyl derivative, those on the tyrosyl residues, and those in unidentified positions, rather than to any one of these.4. 4. The results are in agreement with the view that pepsin is of broad specificity toward protein substrates.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32500/1/0000587.pd

Chain length affects pancreatic lipase activity and the extent and pH–time profile of triglyceride lipolysis

Triglycerides (TG) are one of the most common excipients used in oral lipid-based formulations. The chain length of the TG plays an important role in the oral bioavailability of the co-administered drug. Fatty acid (FA) chain-length specificity of porcine pancreatic lipase was studied by means of an in vitro lipolysis model under bio-relevant conditions at pH 6.80. In order to determine the total extent of lipolysis, back-titration experiments at pH 11.50 were performed. Results suggest that there is a specific chain length range (C2–C8) for which pancreatic lipase shows higher activity. This specificity could result from a combination of physicochemical properties of TGs, 2-monoglycerides (2-MGs) and FAs, namely the droplet size of the TGs, the solubility of 2-MGs within mixed micelles, and the relative stability of the FAs as leaving groups in the hydrolysis reaction. During experimentation, it was evident that an optimisation of lipolysis conditions was needed for tighter control over pH levels so as to better mimic in vivo conditions. 1 M NaOH, 3.5 mL/min maximum dosing rate, and 3 μL/min minimum dosing rate were the optimised set of conditions that allowed better pH control, as well as the differentiation of the lipolysis of different lipid loads

In vitro digestion models to assess lipolysis: The impact of the simulated conditions of gastric and intestinal pH, bile salts and digestive fluids

[EN] In vitro digestion models are a valid methodology to study nutrient hydrolysis by simulating standard physiological gastrointestinal conditions. However, there are pathologies in which some conditions are affected, which should be considered in the design of an in vitro digestion study. Our work aims at elucidating the role of different gastrointestinal conditions on lipolysis. In the context of exocrine pancreatic insufficiency, gastric pH, intestinal pH, bile salts composition, bile salts concentration, fat concentration in the digestion medium and volumetric ratio digestion fluid/food were the selected study parameters. The pH-stat method was applied to assess lipolysis extent and kinetics. Descriptive results were summarised in digestibility curves and beta regression models were used to explain the effect (odds ratio, OR) of the studied conditions on lipolysis. Additionally, distribution of emulsion droplets was measured and optical microscopy images of fat globules were taken in a selection of experimental conditions. Results showed that intestinal pH was the variable with the highest effect on lipolysis (OR 22.86, p < 0.001), followed by fat concentration in the digestion medium (OR 6.76, p < 0.001) and bile salts concentration (OR 1.56, p < 0.001). Overall, lipolysis was significantly associated with particle size (OR - 6.98, p < 0.001). We conclude that the assessment of lipolysis by means of in vitro digestion models is sensitive to the simulated gastrointestinal conditions, which should be adapted to the real physiological conditions occurring in altered health conditions.The authors of this paper, on behalf of MyCyFAPP consortium, acknowledge the European Union and the Horizon 2020 Research and Innovation Framework Programme for funding the project (ref. 643806)Calvo-Lerma, J.; Fornes-Ferrer, V.; Heredia Gutiérrez, AB.; Andrés Grau, AM. (2019). In vitro digestion models to assess lipolysis: The impact of the simulated conditions of gastric and intestinal pH, bile salts and digestive fluids. Food Research International. 125:1-10. https://doi.org/10.1016/j.foodres.2019.108511S11012

Low penetrance in facioscapulohumeral muscular dystrophy type 1 with large pathological D4Z4 alleles: a cross-sectional multicenter study.

BackgroundFacioscapulohumeral muscular dystrophy type 1(FSHD1) is an autosomal dominant disorder associated with the contraction of D4Z4 less than 11 repeat units (RUs) on chromosome 4q35. Penetrance in the range of the largest alleles is poorly known. Our objective was to study the penetrance of FSHD1 in patients carrying alleles ranging between 6 to10 RUs and to evaluate the influence of sex, age, and several environmental factors on clinical expression of the disease. Methods A cross-sectional multicenter study was conducted in six French and one Swiss neuromuscular centers. 65 FSHD1 affected patients carrying a 4qA allele of 6¿10 RUs were identified as index cases (IC) and their 119 at-risk relatives were included. The age of onset was recorded for IC only. Medical history, neurological examination and manual muscle testing were performed for each subject. Genetic testing determined the allele size (number of RUs) and the 4qA/4qB allelic variant. The clinical status of relatives was established blindly to their genetic testing results. The main outcome was the penetrance defined as the ratio between the number of clinically affected carriers and the total number of carriers. Results Among the relatives, 59 carried the D4Z4 contraction. At the clinical level, 34 relatives carriers were clinically affected and 25 unaffected. Therefore, the calculated penetrance was 57% in the range of 6¿10 RUs. Penetrance was estimated at 62% in the range of 6¿8 RUs, and at 47% in the range of 9¿10 RUs. Moreover, penetrance was lower in women than men. There was no effect of drugs, anesthesia, surgery or traumatisms on the penetrance. Conclusions Penetrance of FSHD1 is low for largest alleles in the range of 9¿10 RUs, and lower in women than men. This is of crucial importance for genetic counseling and clinical management of patients and families

Primary subcutaneous cyst hydatic disease in proximal thigh: an unusual localisation: a case report

BACKGROUND: Musculoskeletal hydatidosis is very rare and represents 1% – 5.4% of all cases of echinococcosis. On clinical basis, infection mimics a soft-tissue tumor, and the preoperative radiological diagnosis is very important to avoid biopsy. CASE PRESENTATION: We report an unusual case of primary subcutaneous hydatidosis in proximity to vastus lateralis muscle. It was diagnosed according to the computed tomography appearance, clinical and pathological findings. A 43 year old female patient was admitted with a history of pain at proximal thigh for the last 30 days. On physical examination, a mass which was 4 × 5 cm in diameter, painful and erythamatous, was palpated over greater trochanter. Sedimentation rate was 40 mm in the first hour. CT (Computed Tomography) scan demonstrated, a soft tissue mass with central cystic component in the subcutaneous tissue near vastus lateralis muscle. Histopathological examination of the specimen revealed a pericystic structure, which consisted of connective tissue and scattered hyaline cells showing a necrotic basophilic structure that resembled a cuticular membrane. Treatment with high dose albendazole was conducted for 4 weeks. CONCLUSIONS: This case illustrates that echinococcal disease should be considered in the differential diagnosis of every cystic mass in every anatomic location, especially when they occur in areas where the disease is endemic

Determination of acyl-CoA concentrations using pancreatic lipase

A rapid and sensitive assay for acyl-CoA thiol esters has been devised using pancreatic lipase as a hydrolytic agent in the presence of dithionitrobenzoic acid to detect the liberated CoA. Acyl thiol esters containing 12-22 carbon atoms and 0-6 double bonds are measurable in this system whereas acetyl-CoA and malonyl-CoA were not cleaved.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33530/1/0000029.pd