Engineering a camelid antibody fragment that binds to the active site of human lysozyme and inhibits its conversion into amyloid fibrils

A single-domain fragment, cAb-HuL22, of a camelid heavy-chain antibody specific for the active site of human lysozyme has been generated, and its effects on the properties of the I56T and D67H amyloidogenic variants of human lysozyme, which are associated with a form of systemic amyloidosis, have been investigated by a wide range of biophysical techniques. Pulse-labeling hydrogen-deuterium exchange experiments monitored by mass spectrometry reveal that binding of the antibody fragment strongly inhibits the locally cooperative unfolding of the I56T and D67H variants and restores their global cooperativity to that characteristic of the wild-type protein. The antibody fragment was, however, not stable enough under the conditions used to explore its ability to perturb the aggregation behavior of the lysozyme amyloidogenic variants. We therefore engineered a more stable version of cAb-HuL22 by adding a disulfide bridge between the two beta-sheets in the hydrophobic core of the protein. The binding of this engineered antibody fragment to the amyloidogenic variants of lysozyme inhibited their aggregation into fibrils. These findings support the premise that the reduction in global cooperativity caused by the pathogenic mutations in the lysozyme gene is the determining feature underlying their amyloidogenicity. These observations indicate further that molecular targeting of enzyme active sites, and of protein binding sites in general, is an effective strategy for inhibiting or preventing the aberrant self-assembly process that is often a consequence of protein mutation and the origin of pathogenicity. Moreover, this work further demonstrates the unique properties of camelid single-domain antibody fragments as structural probes for studying the mechanism of aggregation and as potential inhibitors of fibril formation.     

Rapid internalization and recycling of the human neuropeptide Y Y(1) receptor.

Desensitization of G protein-coupled receptors (GPCRs) involves receptor phosphorylation and reduction in the number of receptors at the cell surface. The neuropeptide Y (NPY) Y(1) receptor undergoes fast desensitization. We examined agonist-induced signaling and internalization using NPY Y(1) receptors fused to green fluorescent protein (EGFP). When expressed in HEK293 cells, EGFP-hNPY Y(1) receptors were localized at the plasma membrane, desensitized rapidly as assessed using calcium responses, and had similar properties compared to hNPY Y(1) receptors. Upon agonist challenge, the EGFP signal decreased rapidly (t(1/2) = 107 +/- 3 s) followed by a slow recovery. This decrease was blocked by BIBP3226, a Y(1) receptor antagonist, or by pertussis toxin, in agreement with Y(1) receptor activation. Internalization of EGFP-hNPY Y(1) receptors to acidic endosomal compartments likely accounts for the decrease in the EGFP signal, being absent after pretreatment with monensin. Concanavalin A and hypertonic sucrose, which inhibit clathrin-mediated endocytosis, blocked the decrease in fluorescence. After agonist, intracellular EGFP signals were punctate and co-localized with transferrin-Texas Red, a marker of clathrin-associated internalization and recycling, but not with LysoTracker Red, a lysosomal pathway marker, supporting receptor trafficking to recycling endosomes rather than the late endosomal/lysosomal pathway. Pulse-chase experiments revealed no receptor degradation after internalization. The slow recovery of fluorescence was unaffected by cycloheximide or actinomycin D, indicating that de novo synthesis of receptors was not limiting. Use of a multicompartment model to fit our fluorescence data allows simultaneous determination of internalization and recycling rate constants. We propose that rapid internalization of receptors via the clathrin-coated pits recycling pathway may largely account for the rapid desensitization of NPY Y(1) receptors.     

Selective inhibition of ADAMTS-1, -4 and -5 by catechin gallate esters.

Three mammalian ADAMTS enzymes, ADAMTS-1, -4 and -5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS-1, -4 and -5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan-degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM-10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.     

Motor and psycho-cognitive clinical types in adult metachromatic leukodystrophy: genotype/phenotype relationships?

Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is biochemically characterized by an accumulation of sulfatides (sulfogalactosylceramides) mainly in oligodendrocytes and macrophages/microglia. The deficient enzyme is a lysosomal hydrolase, cerebroside sulfate sulfatase (arylsulfatase A). MLD is both a dysmyelinating and a demyelinating disease. The main clinical forms are infantile or juvenile, but some forms appear at adulthood. This disease involves also neuronal cells as sulfatides are also present in neurons in which the defect in degradation occurs also. We have studied 12 cases of adult MLD and clearly distinguished two clinical forms. One of them was characterized by mainly central nervous system motor signs (pyramidal, cerebellar, and seldom dystonia) and a peripheral neuropathy. The other form always started by behavioural abnormalities with modifications of mood, peculiar social reactions; a progressive mental deterioration occurred also. The diagnosis of schizophrenia was often mentioned. Most of these patients remained for many years without any neurological symptoms, and the diagnosis was only made when neurological signs appeared, or when Magnetic Resonance Imaging (MRI) was performed. MRI showed a diffuse demyelination, bilateral and often symmetrical, which could be temporarily limited to the periventricular areas. The diagnosis of adult MLD was biochemical, evidencing the low activity of arylsulfatase A (ASA) and sulfatide accumulation. To determine the respective participation of neurons and glial cells in the physiopathology of both the motor forms and the psycho-cognitive forms, our first approach was to search for mutations differing according to the clinical status. Motor forms involved the major adult ASA mutation P426L in a homozygote form in contrast to psycho-cognitive forms which involved as a compound heterozygote a specific I179S mutation.     

Evaluation of the potential of Aspergillus niger species for the bioconversion of L-phenylalanine into 2-phenylethanol

Aspergillus niger was explored, for the first time, for the production of 2-phenylethanol (a rose-like aroma) using L-phenylalanine as precursor. Among the strains screened, A. niger CMICC 298302 was shown to produce, in a culture medium containing 6 g L-phenylalanine l^–1 and 60 g glucose l^–1, 1375 mg 2-phenylethanol l^–1 with a productivity of 153 mg l^–1 day^–1 and a molar yield of 74%. 2-Phenylethanol concentrations of 1 to 2 g l^–1 led to a two-fold and ten-fold decrease, respectively, in the mycelial radial growth rate. However, 2-phenylethanol was synthesized as the sole aromatic product and accumulated in the culture broth.     

Kinetics of partly diffusion controlled reactions. VII — Pyrene excimer formation in erythrocyte membranes

Intermolecular excimer forming systems such as pyrene have been proposed as probes for the microviscosity measurement of model and biological membranes. However, our experimental study of pyrene excimer formation in erythrocyte membranes and the theoretical calculations show that it is quite difficult to get the diffusion coefficient of pyrene in membranes and therefore the microviscosity of its environment.     

Microhabitat characteristics of <Emphasis Type="Italic">Stegastes planifrons</Emphasis> and <Emphasis Type="Italic">S. adustus</Emphasis> territories

Stegastes adustus and Stegastes planifrons are two species of damselfishes commonly found in the Caribbean. These territorial fishes have been widely studied due to their major ecological role on coral reef in controlling the growth of macroalgae that compete with corals for space and, inversely, on their deleterious role in destroying coral tissues to impulse the development of algae. However, few studies were conducted on the biotic and abiotic components of their territories. In the present study, territory size and surfaces of benthic components (macroalgae, algal turf, massive corals, branching corals, Milleporidae, sponges, sand and rubbles) were estimated for the two species at two contrasted sites. At Ilet Pigeon site (IP), the two damselfishes were found at different depth and exhibited different territory sizes. S. adustus defended a larger territory characterized by massive corals, sand and Milleporidae, while S. planifrons territories were smaller, deeper and characterized by branching corals, sponges and rubble. At Passe-à-Colas site (PC), the two fish species coexisted in the same depth range and defended territories of similar size. Their territories presented higher proportions of macroalgae, but smaller surfaces of Milleporidae than at IP. At PC, the main difference between the two species was a higher surface of massive corals inside S. planifrons territories than S. adustus territories. Differences in microhabitat characteristics between the two Stegastes seemed mostly site related. This resulted from the high plasticity of two species, allowing them to persist on Caribbean coral reefs after the decline of most branching acroporids, their former favorite habitats.     

The influence of soil cover organization on the floristic and structural heterogeneity of a Guianan rain forest

The impact of soil cover organization on the forest community has been studied in a 19-ha tract at Piste de St Elie station in French Guiana. 195 species each represented by at least 10 individuals were chosen from records of the position, diameter at breast height (dbh) and precise identification by botanical sampling of 12104 ligneous plants (dbh 10 cm).Spatial variations in the soil were mapped using the method proposed by Boulet et al. (1982). The soil mapping units correspond to the successive stages of evolution of a currently unbalanced ferralitic cover. These stages describe firstly the thinning by erosion of the microaggregated upper horizon and secondly the mineralogical changes under more or less extended hydromorphic conditions. The degree of evolution of ferralitic cover is also related to the hydrodynamic functioning and chemical properties of the soil. Geological substrate, topography and slope have also been taken into account.Analysis of the influence of environmental variables on plant cover has been performed using the Ecological Profiles method and Correspondence Analysis (CA) of the table of ecological profiles.The forest community seems to be dependent on the soil and the topographical features that govern it. There are significant, exclusive soil-species links for each soil functioning mapping unit. However, the highest proportion of significant positive links is connected with a thick microaggregated horizon (25%). Several species are of real value as indicators and more particularly enable differentiation between the forest stands of typical ferralitic soil and the ones of thinned out, transformed and hydromorphic soils. The CA of the species by environmental variables matrices reveals two significant factorial axes. The first can be associated with the drainage mainly related to the thinning of the soil and the second with the hydromorphic conditions related to the topography. The vegetation ordination of the stands ( 0.25 ha) delimited in the various soil domains clearly shows that changes in ferralitic cover and in particular the transition from soil with deep vertical drainage to soil with superficial lateral drainage is accompanied by substantial changes in the forest community.     

Refinement of genetic localization of the Alström syndrome on chromosome 2p12-13 by linkage analysis in a North African family

Alström syndrome is a rare autosomal recessive disorder characterized by retinal pigment degeneration, neurogenic deafness, infantile obesity, hyperlipidemia, and non-insulin-dependent diabetes mellitus. While the disease-related gene remains unknown, studies of the genetic isolate of French Acadians provisionally locate the Alström syndrome on chromosome 2p12-13 within a 14.9-cM interval. To confirm this finding in another ethnic population and refine the candidate region we investigated by linkage analysis a consanguineous family of North African origin, in which three of seven siblings displayed all major neurological and metabolic features of Alström syndrome. Genotyping was performed on an ABI377 DNA automatic sequencer and LOD scores were obtained with the Fastlink program. Five markers previously investigated in French Acadians confirmed the involvement of the candidate region, although pairwise LOD scores were of poor significance (Z _max=2.9). To further confirm homogeneity and refine the candidate region, 20 additional markers were investigated. Haplotype analysis and allele segregation revealed that affected children shared a single haplotype and were homozygous for the eight most centromeric markers (D2S291–D2S2114), over a 6.1-cM interval. Significative multipoint LOD scores (Z _max=3.96) were obtained between markers D2S2110/145 and D2S286. Two clusters of known genes are present in this refined region of chromosome 2p, the most attractive candidate being the hexokinase II gene. However, except for several known polymorphisms, no mutations were detected in the coding region of this gene. In conclusion, the location of Alström syndrome on chromosome 2p12-13 is confirmed, reducing the genetic interval to 6.1 cM.