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.

[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.

[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.

Up-regulation of a novel mRNA (NY-CO-1) involved in the methyl 4-methoxy-3-(3-methyl-2-butenoyl) benzoate (VT1)-induced proliferation arrest of a non-small-cell lung carcinoma cell line (NSCLC-N6).

It is now well known that treatment of tumors, especially non-small-cell lung cancer (NSCLC), remains limited and it is urgent to develop strategies that target tumor cells and their genetic features. In this regard, our work is about genetic modifications arising in an in vitro NSCLC cell line after treatment with a chemical substance, methyl 4-methoxy-3-(3-methyl-2-butenoyl) benzoate (VT1). First, we showed that VT1 induces arrest of proliferation by blocking cells in the G1 phase of the cell cycle. Second, we use "differential display" strategy to clarify the genetic mechanisms involved in this proliferation arrest. A novel mRNA, NY-CO-1 (New-York Colon 1), of unknown function showed up-regulated expression after treatment. Application of "antisense" strategy confirmed this novel mRNA induction was effectively linked to growth arrest. Therefore, these data provide new information about mechanisms participating in arrest of proliferation of tumor cells and open new ways of treatment to target tumor growth.

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.

Characterization of specificity of subtilisin Carlsberg towards peptide T by high-performance liquid chromatography and electrospray mass spectrometry.

Peptide T has a sequence (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) belonging to HIV envelope that is involved in the interaction with CD(4) receptor of T lymphocytes. Research of protease activities towards this peptide is very relevant for AIDS therapy. Characterization of specificity of subtilisin Carlsberg towards this very hydrophilic peptide is proposed by using high-performance liquid chromatography and mass spectrometry. Peptide T was totally hydrolysed by the protease after 24 h. Separation of hydrophilic fragments was perfected with an hydrophilic stationary phase and a reversed acetonitrile gradient. Peptide masses were determined by ion spray mass spectrometry. Four primary and one secondary hydrolysis products were found, corresponding to cleavage at the carboxylic side of threonine. Specifities of subtilisin Carlsberg towards the Segments 19 to 26 of bovine pancreatic ribonuclease A, an homologous fragment of peptide T, and peptide T were compared.

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.

Capillary zone electrophoresis and MALDI-mass spectrometry for the monitoring of in vitro O-glycosylation of a threonine/serine-rich MUC5AC hexadecapeptide.

The in vitro N-acetylgalactosaminylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases was assessed using the peptide motif GTTPSPVPTTSTTSAP, which is found naturally in the tandem repeat domains of the apomucin encoded by the gene MUC5AC. This peptide appeared to be an excellent tool for obtaining an insight into the extensive O-glycosylation processes of apomucins. Up to six N-acetylgalactosamines were added and the given glycopeptide species were well separated by capillary zone electrophoresis. Moreover, the degree of glycosylation (number of monosaccharide O-linked attachments) could be determined by MALDI-mass spectrometry without prior separation. Using different incubation times, we evidenced the accumulation of various glycopeptides, suggesting that the total glycosylation of an apomucin-peptide requires orderly N-acetylgalactosaminylation processing. This information was completed by experimental data showing that N-acetylgalactosaminylated octapeptides (the peptide backbones of which are part of GTTPSPVPTTSTTSAP) were able to selectively inhibit some N-acetylgalactosaminyltransferases. Our results suggest that this inhibition may influence the quality of the intermediate products appearing during the in vitro O-glycosylation process.

Quantitative analysis of human serum corticosterone by high-performance liquid chromatography coupled to electrospray ionization mass spectrometry.

An original method based upon high-performance liquid chromatography coupled to electrospray ionization mass spectrometry has been developed for corticosterone (B) quantification in human serum. After extraction by diethyl ether using triamcinolone (T) as an internal standard, solutes are separated on a C18 microbore column (250 X 1.0 mm, I.D.), using acetonitrile-water-formic acid (40:59.9:0.1, v/v/v) as the mobile phase (flow-rate 40 microl/min). Detection is performed on an API 1 single quadrupole mass spectrometer equipped with a ESI interface and operated in positive ionization mode. Corticosterone quantifications were realized by computing peak area ratios (B/T) of the serum extracts analyzed in SIM mode (m/z 347 and m/z 395 for B and T. respectively), and comparing them with the calibration curve (r=0.998).

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.

Antimicrobial activities of synthetic bismuth compounds against Clostridium difficile.

Clostridium difficile is a major nosocomial pathogen responsible for pseudomembranous colitis and many cases of antibiotic-associated diarrhea. Because of potential relapse of disease with current antimicrobial therapy protocols, there is a need for additional and/or alternative antimicrobial agents for the treatment of disease caused by C. difficile. We have synthesized a systematic series of 14 structurally simple bismuth compounds and assessed their biological activities against C. difficile and four other gastrointestinal species, including Helicobacter pylori. Here, we report on the activities of six compounds that exhibit antibacterial activities against C. difficile, and some of the compounds have MICs of less than 1 microgram/ml. Also tested, for comparison, were the activities of bismuth subcitrate and ranitidine bismuth citrate obtained from commercial sources. C. difficile and H. pylori were more sensitive both to the synthetic bismuth compounds and to the commercial products than were Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis, and the last three species were markedly resistant to the commercial bismuth salts. Testing with human foreskin fibroblast cells revealed that some of the synthetic compounds were more cytotoxic than others. Killing curves for C. difficile treated with the more active compounds revealed rapid death, and electron microscopy showed that the bismuth of these compounds was rapidly incorporated by C. difficile. Energy dispersive spectroscopy X-ray microanalysis of C. difficile cells containing electron-dense material confirmed the presence of internalized bismuth. Internalized bismuth was not observed in C. difficile treated with synthetic bismuth compounds that lacked antimicrobial activity, which suggests that the uptake of the metal is required for killing activity. The nature of the carrier would seem to determine whether bismuth is transported into susceptible bacteria like C. difficile.

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.

Substitution of an alanine residue for glycine 146 in TMP kinase from Escherichia coli is responsible for bacterial hypersensitivity to bromodeoxyuridine.

The wild-type TMP kinases from Escherichia coli and from a strain hypersensitive to 5-bromo-2'-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type tmk gene from Escherichia coli or Bacillus subtilis, but not the defective tmk gene, restored the resistance to bromodeoxyuridine of an E. coli mutant strain.

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.

Dipeptidyl aminotransferase activity and in vitro O-glycosylation of MUC5AC mucin motif peptides by human gastric microsomal preparations.

The in vitro O-glycosylation reaction of the MUC5AC mucin motif peptide, TTSAPTTS (in one-letter code), was achieved with human gastric microsomal homogenates. The analyses using capillary electrophoresis online coupled with electrospray mass spectrometry and further Edman degradation of the purified products (obtained by capillary electrophoresis at preparative scale) allowed us to distinguish two components at close masses: the addition of a mass of 202 corresponded to an N-terminal elongation of the peptide TTSAPTTS with the dipeptide (TT) and the addition of a mass of 203 corresponded to an N-acetylgalactosamine O-linkage. Using different peptidase inhibitors, a dipeptidyl peptidase/transferase activity was further characterized. A thiol dependence and an inhibition by H-Gly-PheCHN2 (specific to cathepsin C activity) were found. Moreover, besides TTSAPTTS, other MUC5AC motif peptides (GTTPSPVP, TSAPTTS) were also dipeptide donors (GT and TS, respectively) and our results suggested the involvement of a single dipeptidyl peptidase/transferase activity. Finally, this latter activity modified the in vitro GalNAc incorporation rates when using our selected MUC5AC motif peptides. Our study therefore shows that caution must be taken to prevent peptidic substrate elongation while performing in vitro O-glycosylation with microsomal preparations as the enzyme source. In fact, the results of the N-acetylgalactosamine incorporation rates and thus the microsomal N-acetylgalactosamine transferase affinity can be misinterpreted if dipeptidyl peptidase/transferase activity is not inhibited by the thiol inhibitor E-64 or the cathepsin C inhibitor H-Gly-PheCHN2.

Influence of the amino acid sequence on the MUC5AC motif peptide O-glycosylation by human gastric UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase(s).

The present work was carried out to study the role of the peptide moiety in the addition of O-linked N-acetylgalactosamineto human apomucin using human crude microsomal homogenates from gastric mucosa (as enzyme source) and a series of peptide acceptors representative of tandem repeat domains deduced from the MUC5AC mucin gene (expressed in the gastric mucosa). Being rich in threonine and serine placed in clusters, these peptides provided several potential sites for O-glycosylation. The glycosylated products were analysed by a combination of electrospray mass spectrometry and capillary electrophoresis in order to isolate the glycopeptides and to determine their sequence by Edman degradation. The O-glycosylation of our MUC5AC motif peptides gave information on the specificity and activity of the gastric microsomal UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase(s). The proline residues and the induced-conformations are of great importance for the recognition of MUC5AC peptides but they are not the only factors for the choice of the O-glycosylation sites. Moreover, for the di-glycosylated peptides, the flanking regions of the proline residues strongly influence the site of the second O-glycosylation.