Leucine supplementation improves muscle protein synthesis in elderly men independently of hyperaminoacidaemia

The present study was designed to assess the effects of dietary leucine supplementation on muscle protein synthesis and whole body protein kinetics in elderly individuals. Twenty healthy male subjects (70 ± 1 years) were studied before and after continuous ingestion of a complete balanced diet supplemented or not with leucine. A primed (3.6 μmol kg⁻¹) constant infusion (0.06 μmol kg⁻¹ min⁻¹) of l -[1-¹³C]phenylalanine was used to determine whole body phenylalanine kinetics as well as fractional synthesis rate (FSR) in the myofibrillar fraction of muscle proteins from vastus lateralis biopsies. Whole body protein kinetics were not affected by leucine supplementation. In contrast, muscle FSR, measured over the 5-h period of feeding, was significantly greater in the volunteers given the leucine-supplemented meals compared with the control group (0.083 ± 0.008 versus 0.053 ± 0.009% h⁻¹, respectively, P < 0.05). This effect was due only to increased leucine availability because only plasma free leucine concentration significantly differed between the control and leucine-supplemented groups. We conclude that leucine supplementation during feeding improves muscle protein synthesis in the elderly independently of an overall increase of other amino acids. Whether increasing leucine intake in old people may limit muscle protein loss during ageing remains to be determined.     

Multilaboratory Evaluation of a Viability Assay for Measurement of Opsonophagocytic Antibodies Specific to the Capsular Polysaccharides of Streptococcus pneumoniae

Opsonophagocytosis is a correlate of protection that measures the functional activity of vaccine-induced antibodies. A standardized opsonophagocytosis assay (OPA) should be used as part of the evaluation of current and future pneumococcal (Pnc) polysaccharide (Ps)-based vaccines. We enrolled five laboratories to evaluate a previously standardized viability OPA. Each laboratory was provided with a detailed OPA protocol, seven target Pnc strains (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F), two quality control sera and 12 paired sera (blinded) from adult donors who received one dose of the 23-valent Pnc Ps vaccine. Laboratories sent their results to the Centers for Disease Control and Prevention for analysis. Sera were tested in duplicate (single run), and the results were averaged to yield a single OPA titer (≥50% killing) for each serum sample. The percentage of sera within one or two dilutions of the calculated median OPA titer was determined for each laboratory and for each serotype. In general, laboratories were capable of detecting OPA titers within one or two dilutions of the median for at least 75 and 88%, respectively, of the sera tested. The level of agreement with the median OPA titers varied depending on the participating laboratory (overall agreement = 0.8 [99% confidence interval = 0.75 to 0.85]). All OPA median titers reported for quality control sera were within one dilution of the expected titer. We conclude that this OPA can be done in multiple laboratories with a high degree of interlaboratory reproducibility.     

Anti-TWEAK monoclonal antibodies reduce immune cell infiltration in the central nervous system and severity of experimental autoimmune encephalomyelitis

TWEAK is a member of the TNF family, constitutively expressed in the central nervous system (CNS), with pro-inflammatory, proliferative or apoptotic effects depending upon cell types. Its receptor, Fn14, is expressed in CNS by endothelial cells, reactive astrocytes and neurons. We showed that TWEAK and Fn14 mRNA expression increased in spinal cord during experimental autoimmune encephalomyelitis (EAE). We investigated the role of TWEAK during EAE using neutralizing anti-TWEAK antibody in myelin oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice. We observed a reduction of disease severity and leukocyte infiltration when mice were treated after the priming phase.     

Keratinocyte growth factor expression by fibroblasts in pulmonary fibrosis: poor response to interleukin-1beta

Keratinocyte growth factor (KGF) is secreted by fibroblasts and protects from pulmonary fibrosis in animal models. Interleukin (IL)-1beta is the most potent inducer of KGF in fibroblasts, acting through the c-Jun pathway. We evaluated in vitro KGF production by human lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF, n = 10) and from control subjects (n = 7) at baseline and after IL-1beta stimulation. Basal KGF secretion by IPF fibroblasts was similar to controls. In fibroblasts from control subjects, IL-1beta increased c-Jun expression, c-Jun activation, and KGF secretion. SP600125, a specific c-Jun N-terminal kinase (JNK) inhibitor, inhibited the effect of IL-1beta. By contrast, in IPF fibroblasts, IL-1beta did not increase c-Jun expression and c-Jun activation, and weakly increased KGF secretion, whereas SP600125 had no effect. IL-1beta similarly increased JunB expression in fibroblasts from patients with IPF and control subjects. Total JNK content was not different in either unstimulated or IL-1beta-stimulated IPF and control fibroblasts. IL-1beta increased phosphorylated JNK in control and IPF fibroblasts, but this increase was weaker and heterogeneous in IPF. Altogether, our results demonstrate a dysregulation of KGF secretion by IPF fibroblasts. The weak response to IL-1beta is associated with a defect of c-Jun expression and activation and a defect of JNK activation.     

Saethre‐Chotzen syndrome: Notable intrafamilial phenotypic variability in a large family with Q28X TWIST mutation

Saethre‐Chotzen syndrome is an autosomal dominant disease characterized by craniosynostosis, ptosis, and limb and external ear abnormalities. Variable expressivity is a well‐known phenomenon in this disorder. A large Indian family has been recently identified as carrying a nonsense TWIST mutation (Q28 X) in 17 members, of whom 16 were examined in detail. Only 4 (25%) of the patients showed patent craniostenosis, namely, oxycephaly. The penetrance of craniosynostosis in this family is lower than previously reported in the literature. Fifteen patients (93%) had moderate to severe ptosis. Minor limb and external ear abnormalities were present in most patients. Eyelid features were the hallmark of the disease for 12 members of the family, suggesting that mutations in TWIST may lead to a phenotype with mainly palpebral features and no craniostenosis. The clinical analysis of this large family clearly illustrates the significant variable expressivity, probably related to haploinsufficiency because of the TWIST mutation. This phenotypic variability remains unclear but could be the result of modifier genes and/or genetic background effect, as noticed previously in the transgenic twist‐null heterozygous mice. © 2002 Wiley‐Liss, Inc.     

Hydrophobically Modified Poly(acrylic acid) Using 3‐Pentadecylcyclohexylamine: Synthesis and Rheology

Summary: Hydrophobically modified poly(acrylic acid) was synthesized using 3‐pentadecylcyclohexylamine (3‐PDCA), which was in turn synthesized from 3‐pentadecylphenol, one of the components of cashew‐nut shell liquid (CNSL), a renewable resource material. ¹H NMR spectra confirmed the incorporation of 3‐PDCA onto PAA and a series of HMPs with three different molar concentrations, viz ? 3, 5 and 7 mol‐% of 3‐PDCA, were synthesized. An increase in viscosity with increasing hydrophobic content was observed by rheological measurements. The critical association concentrations were determined using an Ubbelohde viscometer and a controlled stress rheometer. The stability of HMPs towards temperature and shear was studied. Rheological measurements showed that there was a steady increase in viscosity with increase in hydrophobe content due to the formation of reversible networks. These polymers exhibited gel‐like behavior at low concentrations (≥2 wt.‐%) with an apparent yield stress (ca. 10 Pa) and showed shear thinning properties (non‐Newtonian). However, below a critical concentration, c [η], they showed Newtonian behavior.

η_sp of unmodified and modified PAA‐Na at various polymer concentrations.     

A dual participation of ZAP-70 and scr protein tyrosine kinases is required for TCR-induced tyrosine phosphorylation of Sam68 in Jurkat T cells

Sam68 has been initially described as a substrate of src kinases during mitosis in fibroblasts. Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction between Sam68 and the two src kinases involved in T cell activation, p59^fyn and p56^lck, as well as a partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cγ-1 and Grb2, has been shown. In this study we analyze the contribution of p56^lck, as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using the src inhibitor PP1 [4-amino-5-(4-methylphenyl)7-(t-butyl) pyrazolo [3,4-d] pyrymidine] and cell variants with defective expression of p56^lck or expressing a dominant negative form of ZAP-70, we demonstrate that, while both p56^lck and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase its rapid phosphorylation observed in vivo after CD3 stimulation. We also show that recombinant forms of both p56^lck and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, we observe that p56^lck activation by itself does not induce Sam68 tyrosine phosphorylation. We conclude that p59^lck and p56^lck differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation.     

Evaluation of the VITEK 2 System for Rapid Identification of Medically Relevant Gram-Negative Rods

The new VITEK 2 system (bioMérieux) was evaluated at two independent sites with the identification card for gram-negative bacilli (ID-GNB card). Of the 845 strains tested, which represented 70 different taxa belonging to either the family Enterobacteriaceae or the nonenteric bacilli, 716 (84.7%) were correctly identified at the species level. Thirty-two (3.8%) additional strains were identified to the species level after the performance of simple, rapid manual tests (oxidase, hemolysis, indole reaction, motility, and pigmentation). For 80 (9.5%) strains, these additional tests did not lead to an identification at the species level but the correct species identification was given among the organisms listed. Only 7 (0.8%) strains were misidentified, and 10 (1.2%) were not identified. Mistakes were randomly distributed over different taxa. Due to the new, more sensitive fluorescence-based technology of the VITEK 2 system, final results were available after 3 h. Since our evaluation was mainly a stress test, it is predicted that the VITEK 2 system in conjunction with the ID-GNB card would perform well under conditions of a routine clinical laboratory in identifying members of the family Enterobacteriaceae and selected species of nonenteric bacteria. This system is a promising, highly automated new tool for the rapid identification of gram-negative bacilli from human clinical specimens.     

Fourteen novel OPA1 mutations in autosomal dominant optic atrophy including two de novo mutations in sporadic optic atrophy

The OPA1 gene, encoding a dynamin-related GTPase that plays a role in mitochondrial biogenesis, is implicated in most cases of autosomal dominant optic atrophy (ADOA). Sixty-nine pathogenic OPA1 mutations have been reported so far. Most of these are truncating mutations located in the GTPase domain coding region (exons 8-16) and at the 3'-end (exons 27-28). We screened 44 patients with typical ADOA using PCR-sequencing. We also tested 20 sporadic cases of bilateral optic atrophy compatible with ADOA. Of the 18 OPA1 mutations found, 14 have never been previously reported. The novel mutations include one nonsense mutation, 3 missense mutations, 6 deletions, one insertion and 3 exon-skipping mutations. Two of these are de novo mutations, which were found in 2 patients with sporadic optic atrophy. The recurrent c.2708_2711delTTAG mutation was found in 2 patients with a severe congenital presentation of the disease. These results suggest that screening for OPA1 gene mutations may be useful for patients with optic atrophy who have no affected relatives, or when the presentation of the disease is atypical as in the case of early onset optic atrophy.     

Measurement of 5-HIAA levels in ventricular CSF (by LCEC) and in striatum (byin vivo voltammetry) during pharmacological modifications of serotonin metabolism in the rat

Summary The relationship between the concentrations of 5-hydroxyindoleacetic acid (5-HIAA) in the CSF and in the striatum has been evaluated in the rat by measuring the levels of this metabolite in ventricular CSF (by liquid chromatography coupled with electrochemical detection) and in the striatal extracellular fluid (byin vivo voltammetry) after administration of inhibitors of serotonin synthesis or degradation. Pargyline, NSD 1015 and-propyldopacetamide all caused an exponential decline of 5-HIAA in both CSF and striatum. For a given drug, the rate constants for 5-HIAA disappearance were identical in the CSF and in the striatal extracellular fluid. These results confirm the view that CSF 5-HIAA may serve as a good index of brain serotonin turnover.