pHAST (pH-Driven Aptamer Switch for Thrombin) Catch-and-Release of Target Protein.

A pH-driven DNA nanomachine based on the human α-thrombin binding aptamer was designed for the specific catch-and-release of human α-thrombin at neutral and acidic pH, respectively. In neutral conditions, the thrombin aptamer component of the nanomachine is exposed and exists in the G-quadruplex conformation required to bind to the target protein. At slightly acidic pH, the polyadenine tail of the nanomachine becomes partially protonated and A+(anti)•G(syn) mispairing results in a conformational change, causing the target protein to be released. Förster resonance energy transfer (FRET) was used to monitor conformational switching over multiple pH cycles. Electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy were used to show pH dependent protein binding and release by the nanomachine. This approach could be applied generally to existing G-rich aptamers to develop novel biosensors, theranostics, and nanoswitches.

Effects of training at mild exercise intensities on quadriceps muscle energy metabolism in patients with chronic obstructive pulmonary disease.

AIM: To study the effects of physical training at mild intensities on skeletal muscle energy metabolism in eight patients with chronic obstructive pulmonary disease (COPD) and eight paired healthy sedentary subjects. METHODS: Energy metabolism of patients and controls vastus lateralis muscle was studied before and after 3 months of cycling training at mild exercises intensities. RESULTS: The total amount of work accomplished was about 4059 ± 336 kJ in patients with COPD and 7531 ± 1693 kJ in control subjects. This work corresponds to a mechanical power set at 65.2 ± 7.5% of the maximum power for patients with COPD and 52 ± 3.3% of the maximum power in control group. Despite this low level of exercise intensities, we observed an improvement in mitochondrial oxidative phosphorylation through the creatine kinase system revealed by the increased apparent K(m) for ADP (from 105.5 ± 16.1 to 176.9 ± 26.5 µm, P < 0.05 in the COPD group and from 126.9 ± 16.8 to 177.7 ± 17.0, P > 0.05 in the control group). Meanwhile, maximal mechanical and metabolic power increased significantly from 83.1 ± 7.1 to 91.3 ± 7.4 Watts (P < 0.05) and from 16 ± 0.8 to 18.7 ± 0.98 mL O(2) kg(-1) min(-1) (P < 0.05) only in the COPD group. CONCLUSION: This study shows that physical training at mild intensity is able to induce comparable changes in skeletal muscles oxidative energy metabolism in patients with COPD and sedentary healthy subjects, but different changes of maximal mechanical and metabolic power.

Synthesis, cellular localization, and quantification of penton-dodecahedron in serotype 3 adenovirus-infected cells.

Adenovirus penton is a non-covalent complex composed of the penton base and fiber proteins, localized at the twelve vertices of the icosahedral virus capsid. In cells infected by adenovirus serotype 3 (Ad3), penton is found not only in the virus capsid but also self-assembled in dodecahedra formed through interactions between the twelve penton bases. In this study, the intracellular trafficking of penton proteins from the cytoplasm to the nucleus has been followed, and the nuclear re-arrangement induced by viral infection has been observed by electron microscopy of ultrathin sections. The amount of dodecahedra has been assessed in relation to the number of Ad3 infectious virions produced during the Ad3 replication cycle. It appears that dodecahedra are produced in a large excess over viral infectious particles and that they are located intranuclearly along the nuclear membrane of Ad3-infected cells at late times of infection.

Toxicity of the Clitocybe amoenolens mushroom in the rat.

Ingestion of Clitocybe amoenolens, a mushroom collected in Savoie (France), induced erythermalgia in 5 patients. To assess C. amoenolens toxicity, increasing doses were given randomly to 4 rats corresponding to 1 to 25 fold the dose eaten by the most severely poisoned patient. The 2 rats receiving the highest doses had loss of bodyweight, locomotor disability and erythema of the toes. Examination of sciatic nerves showed decreased axon density and neuronal fiber degeneration. Oral administration of C. amoenolens to rats led to lesions that might be explained by the presence of acromelic acid A, a kainate analogue.

Histological support for the difference between malignant hyperthermia susceptible (MHS), equivocal (MHE) and negative (MHN) muscle biopsies.

The aim of this study was to find, using modern techniques, any histological differences in muscle biopsies between malignant hyperthermia (MH) susceptible (MHS), MH equivocal (MHE) and MH negative (MHN) patients. On the basis of the European MH contracture test carried out in 83 patients, 23 were shown to be MHS, nine MHE and 51 MHN. Four lesions were found with a significantly high frequency in MHS and MHE biopsies: muscle fibre hypertrophy and atrophy, internal nuclei and myofibrillar necrosis. These four lesions were observed together in 35% of MHS but in none of the MHE or MHN biopsies. Three of these lesions occurred together in 57% of MHS, 33% of MHE and 4% of MHN biopsies. Our results support a histological difference between MHE, MHS and MHN biopsies and attempt to contribute towards a better definition of MHE status.

Presence of zinc and calcium permeant channels in the inner membrane of the nuclear envelope.

The nuclear envelope possesses specific ion channels that regulate the ionic traffic between the cytoplasm or the perinuclear space and the nucleoplasm. Using the patch-clamp technique to isolated rat nuclei exhibiting only the inner membrane of the nuclear envelope, we report the existence of calcium and zinc permeant channels. These channels displayed similar characteristics (conductance : 8 and 11 pS respectively, open time constant (3.5 ms and 3.7 ms) and close time constant (5.1 ms and 4.8 ms)) and were insensitive to different types of calcium channels blockers and to calcium concentration in the bathing solution. The exact role of these channels remains to define, but they may contribute to the regulation of intranuclear Ca++ or Zn++ dependent processes as important as cell proliferation or programmed cell death. Moreover, this work demonstrates that our nuclei preparation provides a way to study the inner membrane of the nuclear envelope.

In vitro 31P-magnetic resonance spectroscopy of muscle extracts in malignant hyperthermia-susceptible patients.

BACKGROUND: It was recently suggested that malignant hyperthermia-susceptible (MHS) patients could have an elevated peak of phosphodiesters in leg muscles using in vivo phosphorus magnetic resonance spectroscopy. In the current study, analysis of the phosphodiesters of muscle extracts of MHS and malignant hyperthermia-negative patients was performed using in vitro phosphorus magnetic resonance spectroscopy to chemically identify and to compare the muscle concentrations of water-soluble compounds between the two groups with respect to the muscle fiber type composition. METHODS: Perchloric acid extracts of the vastus medialis muscle of seven MHS patients and ten malignant hyperthermia-negative patients on the basis of the European malignant hyperthermia contracture test were subjected to in vitro phosphorus magnetic resonance spectroscopy carried out at 9.4 T. In addition, chemical identification of the phosphodiester region and histologic examination of the muscle specimens were performed. RESULTS: The peak in the phosphodiester region was assigned to glycerophosphorylcholine. Muscle perchloric acid extracts of MHS patients had a significantly (P < 0.05) higher glycerophosphorylcholine to the sum of phosphocreatine and inorganic phosphate (glycerophosphorylcholine/ [phosphocreatine +inorganic phosphate]) value than those of malignant hyperthermia-negative patients. Neither a difference in the fiber type composition between the two groups nor any specific myopathy were found. CONCLUSIONS: In the absence of histologic differences between muscle specimens of MHS and malignant hyperthermia-negative patients, these results could suggest that glycerophosphorylcholine could be a marker of an impairment in the phospholipid metabolism in the skeletal muscle of MHS patients.

Contribution of apoptosis to the phenotypic changes of adrenocortical cells in primary culture.

The aim of this study was to evaluate the occurrence and physiological consequences of apoptosis in primary cultures of bovine adrenocortical cells (of fasciculata-reticularis origin). Under ACTH-free culture conditions, we observed apoptotic cells in the cell layer and the accumulation of apoptotic bodies in the culture medium. These were hardly detectable in ACTH-supplemented cultures. Under ACTH-free conditions, the DNA content of apoptotic bodies collected over 48 h represented up to 10-15% of that of the cell layer at the onset of the culture (as compared to 3% in ACTH-supplemented cultures). Past the fourth day of culture in the absence of ACTh, most cells lacked several markers of their originating fasciculata-reticularis phenotype and progressively evolved to an undifferentiated phenotype. The vast majority of the apoptotic bodies released during the first 4 days of culture were immunoreactive for P450 17 alpha. Inversely, during the same period of time, the proliferating cells (PCNA-positive) did not appear to express P450 17 alpha. Therefore, apoptosis could contribute, together with dedifferentiation, to the phenotype shift observed in ACTH-depleted cultures of adrenal fasciculata-reticularis cells. These observations also characterize this endocrine cell system as an in vitro model for the study of hormone-repressed apoptosis.

Morphometric analysis of the peripheral nerve: advantages of the semi-automated interactive method.

A semi-automated interactive method (BIOCOM) was developed for the morphometric analysis of the peripheral nerve in routine histopathological examination. The human superficial peroneal nerve was used for myelinated fibre measurements. The precision and reproducibility of the results were validated using a manual morphometric method on a digitalizing table. Having compared the two methods, we believe that the semi-automated method constitutes an interesting alternative to traditional methods.

[Churg-Strauss syndrome with rectal localization and exudative enteropathy]. / Syndrome de Churg et Strauss avec localisation rectale et entéropathie exsudative.

The authors report the case of a 61-year old patient with a Churg-Strauss syndrome revealed by abdominal pain. Investigations showed a diffuse inflammatory digestive tract involvement, documented by endoscopy associated with protein-losing enteropathy. Deep rectal biopsy revealed vasculitis in an otherwise macroscopically normal rectum. Faced with acute and life-threatening course of disease, emergency medical treatment with steroids and cyclophosphamide led to rapid regression of clinical, biological and radiological abnormalities.

Ultrastructural localization of the major components of the extracellular matrix in normal rat nerve.

In this study we determined the ultrastructural distribution of the various components of the extracellular matrix (laminin, fibronectin, Type I, III, and IV collagens) of the normal peripheral nerve in adult rat. The localization of these macromolecules was investigated in basement membranes as well as in different areas of epi-, peri-, and endoneurium, by use of a pre-embedding immunoperoxidase method.

Chronic intestinal pseudoobstruction with myopathy and ophthalmoplegia. A muscular biochemical study of a mitochondrial disorder.

The association of chronic intestinal pseudoobstruction with ophthalmoplegia has been reported previously in visceral myopathies. We report a case of this association in which muscle mitochondria had a crystalline appearance, a dense core, and decreased cytochrome c oxidase and succinate cytochrome c reductase activities. The absence of evident mitochondrial DNA deletion in the skeletal muscle of this patient does not exclude the possibility of localized deletion or mutation of mitochondrial DNA in digestive muscle.

[Selective laryngeal re-innervations in the dog by nervous microsutures of branches of the recurrent laryngeal nerve]. / Réinnervations laryngées sélectives chez le chien par micro-sutures nerveuses des branches de division du nerf récurrent.

INTRODUCTION: This work has been carried out to investigate in an animal model, the possibility of surgical selective reinnervation of the larynx following destruction of the laryngeal branch of the vagus nerve. MATERIAL AND METHODS: In ten dogs, on the right side of the neck only, the recurrent laryngeal nerve was identified, cut and section of the nerve removed. Following this the hemi-larynx on the operated side was reinnervated in the following manner. Using a microsurgical suture (described in the text) the motor nerves from extra-laryngeal muscles were anastomosed onto the nerves supplying the intrinsic abductors and adductors of the larynx. The nerve to sterno-thyroid was anastomosed onto the nerve supplying the posterior crico-arytenoid muscle (vocal cord abductor). Similarly the nerve supplying thyrohyoid was anastomosed onto the cut distal end of the adductor division of the recurrent laryngeal nerve. Clinical, electromyographical and histological evaluations have been used to prove the reinnervation. RESULTS: In the nine surviving animals successful re-innervations, as defined by the return of normal function, has been achieved for posterior crico-arytenoid and 7 adductor muscles. CONCLUSION: This study has demonstrated the feasibility of laryngeal reinnervation after surgical section of the recurrent laryngeal nerve, and give some cause for optimism for its ultimate application in man.

Ultrastructural changes associated with peripheral neuropathy in HIV/AIDS.

Light and electron microscopic studies were performed on neuromuscular biopsy specimens from 12 neurologically affected seropositive patients, 7 with the acquired immune deficiency syndrome (AIDS), 2 with AIDS-related complex, and 3 with no symptoms except for neuropathy. All patients had an axonal injury associated or unassociated with demyelination and peripheral neurogenic atrophy. Capillary lesions were consistently present, which seems to be a new finding. Moreover, tubuloreticular inclusions (TRIs) were found in endomysial (9 of 12 cases) and endoneurial (7 of 12 cases) vessels. Statistical analysis showed that TRIs in more than 20% of endomysial vessels correlated with a survival time shorter than 12 months (P = 0.028). Thus the quantification of TRIs seems to be one of the vital prognostic elements in this patient population.

Microangiopathy of endoneurial vessels in hypoxemic chronic obstructive pulmonary disease (COPD). A quantitative ultrastructural study.

A peripheral neuropathy has been reported in patients with chronic respiratory insufficiency due to chronic obstructive pulmonary disease (COPD). It is mainly characterized by axonal degeneration, secondary demyelination and abnormal endoneurial vessels. The pathogenesis of these lesions remains obscure. To investigate whether relationships exist between neuritic and vascular lesions, a qualitative and quantitative ultrastructural study was performed on nerve biopsies in 13 patients with chronic respiratory insufficiency due to COPD, and in 9 normal controls without pulmonary lesions. A computer-assisted multiple regression analysis taking into account clinical, electrophysiological, biological and morphometric parameters was performed. Statistically significant differences in the endoneurial structure of microvessels were: (1) thickening of the basement membrane; (2) narrowing of the lumen; (3) mural pericytic debris deposits, occurring in the COPD group. In the latter, hypercapnia correlated positively with nerve fibers lesions (P = 0.03) and endothelial area (P = 0.03). No correlations were found between age and other parameters. These findings highlight the fact that the microangiopathy in peripheral nerves in patients with COPD may be diffuse and essentially due to hypoxia and reduction in blood flow, as in diabetic neuropathy.