Active versus Passive Hard Disks against a Membrane: Mechanical Pressure and Instability.

We experimentally study the mechanical pressure exerted by a set of respectively passive isotropic and self-propelled polar disks onto two different flexible unidimensional membranes. In the case of the isotropic disks, the mechanical pressure, inferred from the shape of the membrane, is identical for both membranes and follows the equilibrium equation of state for hard disks. On the contrary, for the self-propelled disks, the mechanical pressure strongly depends on the membrane in use and thus is not a state variable. When self-propelled disks are present on both sides of the membrane, we observe an instability of the membrane akin to the one predicted theoretically for active Brownian particles against a soft wall. In that case, the integrated mechanical pressure difference across the membrane cannot be computed from the sole knowledge of the packing fractions on both sides, further evidence of the absence of an equation of state.

Crystallization of Self-Propelled Hard Discs.

We experimentally study the crystallization of a monolayer of vibrated discs with a built-in polar asymmetry, a model system of active liquids, and contrast it with that of vibrated isotropic discs. Increasing the packing fraction ϕ, the quasicontinuous crystallization reported for isotropic discs is replaced by a transition, or a crossover, towards a "self-melting" crystal. Starting from the liquid phase and increasing the packing fraction, clusters of dense hexagonal-ordered packed discs spontaneously form, melt, split, and merge, leading to a highly intermittent and heterogeneous dynamics. For a packing fraction larger than ϕ^{*}, a few large clusters span the system size. The cluster size distribution is monotonically decreasing for ϕ<ϕ^{*}, nonmonotonic for ϕ>ϕ^{*}, and is a power law at the transition. The system is, however, never dynamically arrested. The clusters permanently melt from place to place, forming droplets of an active liquid which rapidly propagate across the system. This self-melting crystalline state subsists up to the highest possible packing fraction, questioning the stability of the crystal for active discs unless it is at ordered close packing.

[Muscle biopsy in children: Usefulness in 2012]. / Intérêt de la biopsie musculaire chez l'enfant en 2012.

Muscle biopsy is a mainstay diagnostic tool for investigating neuromuscular disorders in children. We report the yield of pediatric muscle biopsy in a population of 415 children by a retrospective study of 419 biopsies performed between 1/01/2000 and 31/12/2009 in a neuropediatric department, including mitochondrial respiratory chain analysis for 87 children. Two hundred and fifty-five biopsies were from boys (61%) 164 from girls (39%). Their mean age at biopsy was 6.5years; 155 (37%) biopsies were obtained before the child was 5years old. Final histopathological diagnoses were: congenital myopathy (n=193, including 15 structural congenital myopathies); progressive muscular dystrophy (n=75 [18%] including 57 dystrophinopathies); congenital muscular dystrophy (n=17, including six primary merosinopathies); dermatomyositis (n=11); spinal muscular atrophy (n=9, including six atypical spinal muscular atrophies); metabolic myopathy (n=32, including 19 mitochondrial myopathies); encephalomyopathy (n=53 [13%], including 27 with a mitochondrial respiratory chain defect). Pathological diagnosis remained undetermined in 16 cases. In 184 patients (44%), the muscle biopsy revealed specific histopathological anomalies (dystrophic process; specific ultrastructural abnormalities; perifascicular atrophy; neurogenic atrophy; metabolic anomalies) enabling a precise etiological diagnosis. For 85% of progressive muscular dystrophies, the biopsy resulted in a genetic diagnosis after identification of the protein defect. In 15% of the congenital myopathies, histopathological anomalies focused attention on one or several genes. Concerning dystrophinopathies, quantification of dystrophin deficiency on the biopsy specimen contributed to the definition of the clinical phenotype: Duchenne, or Becker. In children with a myopathy, muscle biopsy is often indispensable to establish the etiological diagnosis. Based on the results from this series, muscle biopsy can provide a precise orientation in 45% of patients, leading to a genetic hypothesis.

[Present possibilities and future development of clinical proteomics]. / Présent et futur de la Protéomique clinique.

This review focuses on "clinical proteomics" which represents an emerging discipline in biomedical research. "Clinical proteomics" relies on the analysis of the proteome, i.e. the entire set of peptides and proteins present in a biological sample, to provide relevant data for diagnosis, prognosis or therapeutic strategies of human pathologies. This new type of approach has tremendous potential for the diagnosis of complex pathologies or for the early detection of cancers. This article reports the conclusions of a workgroup of the French Society for Clinical Biology (SFBC) 2004-2006 which evaluated the status, the impact and the future development of proteomics in the clinical field. It provides therefore a broad view going from the methods already present in the clinical laboratories (multiplex technologies...), to the tools for clinical and basis research including bioinformatics.

Location of the globular region in chicken erythrocyte histone H5.

Chicken erythrocyte histone H5 has been cleaved by acetic acid hydrolysis at the two aspartic acid residues 65 and 99 and the 4 peptides (1-65), (66-185) (1-99) (100-185) recovered in a pure form. 270 MHz magnetic resonance and circular dichroic studies show that the two C-terminal peptides are unable to form secondary or tertiary structure. The N-terminal peptides however, form both secondary and tertiary structure. In particular, the peptide (1-99) at high ionic strength possesses a similar number of helical residues to intact histone H5 and also had a closely related nuclear magnetic resonance spectrum. It is concluded that the peptide (1-99) contains most, but not quite all of the residues that are included in the globular segment of histone H5.

A corrected primary structure for dog-fish Scylliorhinus caniculus protamine Z3.

We have redetermined the primary structure for dog-fish protamine using automated amino-acid sequencing associated to mass spectrometry techniques and report, on the basis of these findings, that the previously published amino-acid sequence is incorrect. The correct protamine sequence is 37 amino acids long and differs from the original published sequence by the C-terminal hexapeptide Arg32-Gly-Arg-Arg-Ser-Arg37.

Identification of insulin domains important for binding to and degradation by endosomal acidic insulinase.

The endosomal compartment of hepatic parenchymal cells contains an acidic endopeptidase, endosomal acidic insulinase (EAI), which hydrolyzes internalized insulin at a limited number of sites. Although the positions of these cleavages are partially known, the residues of insulin important in its binding to and proteolysis by EAI have not been defined. To this end, we have studied the degradation over time of native human insulin and three insulin-analog peptides using a soluble endosomal extract from rat liver parenchyma followed by purification of the products by HPLC and determination of their structure by mass spectrometry. We found variable rates of ligand processing, i.e. high ([Asp(B10)]- and [Glu(A13),Glu(B10)]-insulin), moderate (insulin) and low (the H2-analog). On the basis of IC(50) values, competition studies revealed that human and mutant insulins display nearly equivalent affinity for the EAI. Proteolysis of human and mutant insulins by EAI resulted in eight cleavages in the B-chain which occurred in the central region (Glu(B13)-Leu(B17)) and at the C-terminus (Arg(B22)-Thr(B27)), the latter region comprising the initial cleavages at Phe(B24)-Phe(B25) (major pathway) and Phe(B25)-Tyr(B26) (minor pathway) bonds. Except for the [Glu(A13),Glu(B10)]-insulin mutant, only one cleavage on the A-chain was observed at residues Gln(A15)-Leu(A16). Analysis of the nine cleavage sites showed a preference for hydrophobic and aromatic amino acid residues on both the carboxyl and amino sides of a cleaved peptide bond. Using the B-chain alone as a substrate resulted in a 30-fold increase in affinity for EAI and a 6-fold increase in the rate of hydrolysis compared with native insulin. A similar role for the C-terminal region of the B-chain of insulin in the high-affinity recognition of EAI was supported by the use of the corresponding B(22)-B(30) peptide, which displayed an increase in EAI affinity similar to the entire B-chain vs. wild-type insulin. Thus, we have identified a highly specific molecular interaction of insulin with EAI at the aromatic locus Phe(B24)-Phe(B25)-Tyr(B26). Analytical subfractionation of a postmitochondrial supernatant fraction showed that a pulse of internalized [(125)I]Tyr(A14)-H2-analog, a protease-resistant insulin analog, undergoes a greater lysosomal transfer and lesser degradation than [(125)I]Tyr(A14)-insulin, confirming that endosomal sorting is regulated directly or indirectly by endosomal proteolysis.

Photoprotective effect of vitamins A and E on polyamine and oxygenated free radical metabolism in hairless mouse epidermis.

The purpose of this study was to confirm the photoprotective effect on skin of vitamins A and E, due to inhibition of polyamine synthesis and production of free radicals. These variables were measured in the lumbar epidermis of the female hairless mouse subjected to UVA + B irradiation. Polyamines were assayed in epidermal homogenate by HPLC, and production of oxygenated free radicals was determined by spectrofluorometric assay of malonyl dialdehyde. It was determined that butyl-hydroxy-toluene and vitamin E inhibited production of free radicals (56% and 60%, respectively) and caused a significant reduction in polyamine biosynthesis (P less than 0.01), whereas the inhibitory effect of malonyl dialdehyde induced by vitamin A (30%) had no associated effect on polyamine metabolism.

Polypeptide:N-acetylgalactosaminyltransferase activities towards the mucin MUC5AC peptide motif using microsomal preparations of normal and tumoral digestive mucosa.

The selected-acceptor substrate peptide (TTSAPTTS), deduced from the human mucin gene MUC5AC (expressed essentially in the human gastric and tracheobronchial mucosa), was used to assay polypeptide:N-acetylgalactosaminyltransferases (GalNAc transferases) of different microsomal preparations, obtained from gastric and colonic mucosa in normal and tumoral situations. The O-glycosylated products, analyzed by capillary electrophoresis and electrospray mass spectrometry, showed a variable number of GalNAc O-linked to the different hydroxy amino acids of TTSAPTTS, depending on the tissue studied. Our observations were consistent with the existence of more than one form of GalNAc transferases which were expressed differentially in the gastrointestinal tract (stomach and/or colon). The levels of enzyme activities showed a tissue-specific pattern as they were high in normal colonic tissue and low in colon cancer. On the other hand, in the tumoral gastric tissue (displaying intestinal metaplasia) a high level of GalNAc transferase activities was obtained, similar to that found in the normal colon. Moreover, slight discrepancies (activities and number of O-linked GalNAc) were only detected between normal gastric and tumoral colonic preparations. Thus, the data indicated that the dedifferentiation of the gastric cancer tissue may induce GalNAc transferase activities similar to those in the normal colonic, tissue and that colonic and gastric tissues may contain families of glycosyltransferases involved specifically in reaction towards particular peptide or protein substrates. In addition, the analysis by capillary electrophoresis and electrospray mass spectrometry revealed, in tumoral gastric as well as in normal colonic tissues, a high dipeptidylaminotransferase activity inducing an elongation of TTSAPTTS by dithreonine. This activity was low in normal gastric and tumoral colonic tissues.

Partial determination of the primary structure of a variant surface glycoprotein from Trypanosoma equiperdum. Composition and location of a carbohydrate moiety.

Salivarian trypanosomes have the ability to evade the immune response of their hosts by the sequential expression of different cell surface glycoproteins. Among the isolated specific antigens from cloned variants of Trypanosoma equiperdum, a structural study was undertaken on two immunologically cross-reacting variant surface glycoproteins, and results concerning the basic antigenic type are reported. The glycoprotein was cleaved by cyanogen bromide, and amino acids of several purified fractions obtained by gel filtration chromatography of this cleavage mixture were sequenced by automated Edman degradation. Sequencing in particular allowed the identification of the N-terminal portion of the molecule (residues 1-74). Sugar compositions of the fractions have demonstrated the presence of at least two carbohydrate moieties in the glycoprotein. Using a subsequent enzymatic subcleavage we were able to locate the first glycosylation site in position 57. An important observation was that the first oligosaccharide identified was rich in mannose and devoid of galactose.

Metabolic studies in twin brothers with 2-methylacetoacetyl-CoA thiolase deficiency.

We report clinical and biological investigations in two patients (twin brothers) with 2-methylacetoacetyl-CoA thiolase deficiency. Main clinical features included important staturo-ponderal delay, frequent infectious rhinopharyngitis episodes and an acute metabolic acidosis at the age of 4 years, this metabolic decompensation being adequately halted by bicarbonate supplementation. Since that age, patients developed rather favorably, however, with persistence of the staturo-ponderal delay. Organicaciduria typical of 2-methylacetoacetyl-CoA thiolase deficiency was recorded consisting of excessive excretion of tiglylglycine, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-methylglutaconate, adipate and 2-methylacetoacetate. Blood carnitine levels were altered in patients with increased total and esterified carnitine concentrations and enhanced acyl/free carnitine ratios. Determination of acylcarnitine profiles showed that patients excreted excessive amounts of several acylcarnitines in urine including propionyl, butyryl, isobutyryl, isovaleryl, 2-methylbutyryl and tiglyl-carnitine, the latter acylcarnitine being prominent with, in one of the patients, occurrence of a previously undescribed isomer of this carnitine ester, possibly 2-ethylacrylyl-carnitine. Excretion of these acylcarnitines in urine was increased in response to L-carnitine although, as a whole, this therapy resulted in a less important stimulation of esterified carnitine removal in urine from patients than in the case of supplemented controls. Biochemical investigations on cultured skin fibroblasts confirmed 2-methylacetoacetyl-CoA thiolase deficiency. Through the present report on this rare disease in two siblings, we would like to underline that acylcarnitines can be used in the diagnosis of 2-methylacetoacetyl-CoA thiolase deficiency, a view supported by acylcarnitine profiles further determined in another patient with proven oxothiolase deficiency, adding this pathology to the list of beta-oxidation disorders that may be screened successfully through determination of acylcarnitine profiles in body fluids.

Acylcarnitine removal in a patient with acyl-CoA beta-oxidation deficiency disorder: effect of L-carnitine therapy and starvation.

Carnitine levels and acylcarnitine profiles in a patient with mild multiple acyl-CoA dehydrogenase deficient beta-oxidation were compared with control results. Whereas blood and urine total carnitine levels were moderately decreased, blood esterified carnitine levels in the patient were about 2-fold higher than in controls. Urinary acylcarnitine profiles presented with a larger variety of carnitine esters than in controls and included propionylcarnitine, butyrylcarnitine, 2-methylbutyrylcarnitine, hexanoylcarnitine and octanolycarnitine. Total carnitine levels in body fluids were similarly affected by chronic oral L-carnitine administration in patient and controls. By contrast, esterified carnitine level increase was 2-fold more important in controls than in patient. Whereas no qualitative changes in urinary acylcarnitine profiles were induced by L-carnitine therapy in controls, several alterations of these profiles were observed in the patient. The effect of starvation on metabolites was also studied, especially beta-oxidation rates assessed by free fatty acids to 3-hydroxybutyric acid ratios in blood from the patient in the untreated and L-carnitine treated states. In the L-carnitine-supplemented patient, the effect of starvation on the time course of carnitine levels and acylcarnitine profiles could also be documented. The ability of chronic oral L-carnitine administration to remove relatively less important amounts of acylcarnitines in the patient than in controls is further discussed, as well as qualitative alterations of acylcarnitine profiles induced by this therapy in the pathological condition.

Metabolic studies in a patient with severe carnitine palmitoyltransferase type II deficiency.

Here we report on a patient with severe ("non-classic") carnitine palmitoyltransferase type II (CPT II) deficiency. Hypoglycemia prompted by an infectious episode and associated with non-ketotic dicarboxylic aciduria orientated diagnosis towards beta-oxidation deficiency disorders. Blood carnitine levels revealed a secondary carnitine deficiency that was responsive to oral L-carnitine supplementation. Blood acylcarnitine profiles were abnormal and included acetyl (C2:0), butyryl/isobutyryl (C4:0), isovaleryl/2-methylbutyryl (C5:0), hexanoyl (C6:0), myristoyl (C14:0), palmitoyl (C16:0), hexadecenoyl (C16:1), oleyl (C18:1) and stearoyl (C18:0) carnitine. In urine, excess excretion of dicarboxylylcarnitines, mainly dodecanedioylcarnitine, was noticed. Upon carnitine supplementation, C8 to C12 fatty acylcarnitines, with decanoylcarnitine as well as C10 to C14 dicarboxylylcarnitines being prominent, were observed in urine. Biochemical measurements disclosed a severe reduction of mitochondrial CPT II activity (7% of normal values). Correlations of metabolic findings in the patient and physiological roles of CPT II are briefly discussed.

Subtractive hybridization and differential screening identified two genes differentially expressed after induction of in vitro (atypical) terminal differentiation in the NSCLC-N6 cell line by a marine substance (bistramide K).

Non-small-cell lung carcinoma is generally refractory to chemotherapy. The difficulties that arise in the treatment of this type of tumor make it necessary to develop new therapeutic strategies. Previous work done in our laboratory showed that a marine substance named bistramide K induced in vitro (atypical) terminal differentiation of NSCLC-N6 cell line. This activity is linked to a growth arrest of NSCLC-N6 cell line and an irreversible block at the G1 phase of the cell cycle (G1DT). In order to identify the genes that could be expressed after the treatment by the drug, we constructed a subtractive cDNA library with enriched mRNA extracted from BK-treated NSCLC-N6. After differential hybridization and DNA sequencing, we identified two sequences. The sequence identified for the clone 8 showed strong homology to the sequence of the ribosomal protein L35A. The sequence identified for the clone 4 did not show any homology with known sequences in official gene data banks.

Antitumor and antiproliferative effects of an aqueous extract from the marine diatom Haslea ostrearia (Simonsen) against solid tumors: lung carcinoma (NSCLC-N6), kidney carcinoma (E39) and melanoma (M96) cell lines.

An aqueous extract of the marine diatom Haslea ostrearia (Simonsen) was studied for its antiproliferative properties against human solid tumors: lung carcinoma (NSCLC-N6), kidney carcinoma (E39) and melanoma (M96). These types of carcinoma are particularly chemoresistant. The extract has a potent cytostatic effect in vitro on the three cell lines and blocks the NSCLC-N6 line in the G1/S phase of the cell cycle. Moreover, the extract strongly inhibits tumor growth of NSCLC-N6 bearing nude mice. These preliminary results indicate that the aqueous extract of Haslea ostrearia exhibits inhibitory effects both in vitro and in vivo against solid carcinoma lines, suggesting the presence of a new potent antitumor agent in the aqueous algal homogenate.

Cytotoxicity against human leukemic cell lines, and the activity on the expression of resistance genes of flavonoids from Platanus orientalis.

The cytotoxic activity of three flavonoids, belonging to the kaempherol series, was evaluated against 15 human leukemic cell lines. Flavonoids bearing acyl substituants, 2 and 3, were found to be the most active compounds. A further compound, 1, was examined for its ability to modulate the expression of MDR-1 and GST-pi resistance genes and compounds 2 and 3 for their effect on the uptake of [3H]-thymidine as a marker of DNA synthesis.

Endogenous glutathione as potential protectant against free radicals in the skin of vitamin A deficient mice.

In order to evaluate the relationships between oxidative degradation of lipids and antioxidant defence systems in the skin, weanling female SKH1 hairless mice were randomly divided into two groups. Each group was fed a well-balanced diet, supplemented, in one group with 5 IU vitamin A/g, and vitamin A free in the other, for 20 wk. Liver and plasma vitamin E were increased in mice fed the vitamin A-free diet. Superoxide dismutases, catalase and Se-glutathione peroxidase were determined in dorsal skin homogenates, as well as the concentration of reduced glutathione (GSH) and of thiobarbituric acid-reactive substances (TBARS); the latter is an index of peroxidation of murine skin cell membranes. Vitamin A deficiency did not alter enzyme activities but enhanced the skin reserve of GSH, which appeared to be the reason for a decrease in endogenous lipid peroxidation. Statistical analysis showed a highly significant negative correlation (R2 > 0.6) between the concentrations of TBARS and GSH in these untreated animals. GSH could play a critical role in maintaining a lower background of lipid alteration in the skin of healthy animals and minimizing individual risk.

Vitamin A status and metabolism of cutaneous polyamines in the hairless mouse after UV irradiation: action of beta-carotene and astaxanthin.

Solar radiations (UV A and B) can cause epidermis photoaging and skin cancers. These frequently irreversible effects result from the in situ generation of free radicals. However, it has been noted that nutritional factors can modulate photochemical damage, in particular the common carotenoids present in food, which can be considered as potential prophylactic agents against carcinogenesis. We investigated the effect of UV A and B radiations on the skin of the SKH1 hairless mouse fed a diet either lacking in vitamin A or supplemented with retinol, beta-carotene or astaxanthin. The latter is an oxygenated carotenoid (like canthaxanthin) without provitamin A activity and with strong singlet oxygen quenching ability. After analysing of vitamin status of each group (plasma retinol concentrations and hepatic reserves), we searched for UV-induced modifications of polyamine metabolism by measuring epidermal ornithine decarboxylase (ODC) activity and free polyamines concentration (putrescine, spermidine and spermine). In the basal state without irradiation, differences in ODC activity between groups were nonsignificant; but after UV stimulation, ODC increased markedly in the skin of vitamin A-deficient animals, much more than in other groups. Curiously, the addition of astaxanthin or beta-carotene to the regimen containing retinol reduced the protective effect of retinol alone. Regarding polyamines after irradiation, putrescine was significantly increased in the skin of deficient animals, in parallel with ODC activity. However, astaxanthin had a stronger inhibitory effect on putrescine accumulation than retinol, and decreased spermidine and spermine concentrations: this suggests a specific action on transglutaminases.

Modulation of ultraviolet light-induced oxidative stress in mice skin related to dietary vitamin A and selenium intake.

Weanling female SKH1 hairless mice were randomly divided into 4 groups. Each group was fed a particular regimen for 20 weeks: 1) normal basal diet with 5 IU vitamin A/g and 0.45 microgram selenium/g, 2) vitamin A deficient, 3) selenium deficient, and 4) vitamin A plus selenium deficient. Three hours before being sacrificed, half of the animals were subjected to UV A + B irradiation (3 J/cm2). Superoxide dismutases (SOD), catalase, Se glutathione peroxidase activities were determined in dorsal skin homogenates, as well as the concentrations of GSH and of thiobarbituric acid-reactive substances (TBARS), the latter being an index of lipid peroxidation. Ultraviolet light altered the antioxidant defense of mouse skin tissue: GSH level, catalase and Se glutathione peroxidase activities were lowered and SOD was unequally enhanced according to the nutritional status. Vitamin A and Se deficiencies did not perceptibly aggravate the UV-induced oxidative stress, although the former enhanced the decline of catalase expression induced by the irradiation. Probably through an adaptive mechanism, both dietary deficiencies increased the skin reserve of antioxidant GSH and thus appear to modulate the effects caused by reactive oxygen species.

[Medium-chain acyl-CoA-dehydrogenase (MCAD) deficiency: French consensus for neonatal screening, diagnosis, and management]. / Déficit en acyl-CoA-déshydrogénase des acides gras à chaîne moyenne (MCAD): consensus français pour le dépistage, le diagnostic, et la prise en charge.

MCAD deficiency is the most common fatty acid oxidation disorder, with the prevalence varying from 1/10,000 to 1/27,000 in the countries adjacent to France. As the High Authority for Health has recently proposed including MCAD deficiency in the panel of diseases neonatally screened for in France, a consensus was written for the management of MCAD deficiency diagnosed either clinically or by neonatal screening. Patients may present acutely with hyperammonemia, hypoglycemia, encephalopathy, and hepatomegaly, mainly after a prolonged fast of intercurrent infection. Sudden death related to heartbeat disorders may also occur. The diagnosis of MCAD deficiency is suspected on the plasma acylcarnitine and/or the urinary organic acid profile. The diagnosis is confirmed by molecular biology and the enzymatic activity for patients who are not homozygous for the main mutation c.985A>G. However, some MCAD-deficient individuals may remain asymptomatic throughout life. The mainstay of treatment consists in avoiding prolonged fast and prescribing l-carnitine for patients who exhibit a deficiency in plasma carnitine. This management has radically modified the natural history of MCAD deficiency. This consensus will allow homogeneous management of these patients once the neonatal screening of MCAD deficiency has been introduced in France.