Neuronal Cell Surface Molecules Mediate Specific Binding to Rabies Virus Glycoprotein Expressed by a Recombinant Baculovirus on the Surfaces of Lepidopteran Cells

The existence of specific rabies virus (RV) glycoprotein (G) binding sites on the surfaces of neuroblastoma cells is demonstrated. Spodoptera frugiperda (Sf21) cells expressing G of the RV strain CVS (Gcvs-Sf21 cells) bind specifically to neuroblastoma cells of different species but not to any other cell type (fibroblast, myoblast, epithelial, or glioma). Attachment to mouse neuroblastoma NG108-15 cells is abolished by previous treatment of Gcvs-Sf21 cells with anti-G antibody. Substitutions for lysine at position 330 and for arginine at position 333 in RV G greatly reduce interaction between Gcvs-Sf21 cells and NG108-15 cells. These data are consistent with in vivo results: an avirulent RV mutant bearing the same double mutation is not able to infect sensory neurons or motoneurons (P. Coulon, J.-P. Ternaux, A. Flamand, and C. Tuffereau, J. Virol. 72:273–278, 1998) after intramuscular inoculation into a mouse. Furthermore, infection of NG108-15 cells by RV but not by vesicular stomatitis virus leads to a reduction of the number of binding sites at the neuronal-cell surface. Our data strongly suggest that these specific attachment sites on neuroblastoma cells represent a neuronal receptor(s) used by RV to infect certain types of neurons in vivo.     

Thioredoxin reductase-1 knock down does not result in thioredoxin-1 oxidation

The active site of thioredoxin-1 (Trx1) is oxidized in cells with increased reactive oxygen species (ROS) and is reduced by thioredoxin reductase-1 (TrxR1). The purpose of the present study was to determine the extent to which the redox state of Trx1 is sensitive to changes in these opposing reactions. Trx1 redox state and ROS generation were measured in cells exposed to the TrxR1 inhibitors aurothioglucose (ATG) and monomethylarsonous acid (MMA(III)) and in cells depleted of TrxR1 activity by siRNA knock down. The results showed that all three treatments inhibited TrxR1 activity to similar extents (90% inhibition), but that only MMA(III) exposure resulted in oxidation of Trx1. Similarly, ROS levels were elevated in response to MMA(III), but not in response to ATG or TrxR1 siRNA. Therefore, TrxR1 inhibition alone was not sufficient to oxidize Trx1, suggesting that Trx1-independent pathways should be considered when evaluating pharmacological and toxicological mechanisms involving TrxR1 inhibition.     

Identification of Mutations in TMEM5 and ISPD as a Cause of Severe Cobblestone Lissencephaly

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a “cobblestone” brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.     

Combined chlorhexidine and PVP-I decontamination of human donor eyes prior to corneal preservation

The aim of this study was to report the efficacy of adding chlorhexidine to the protocol for decontamination of human donor globes prior to excision of corneo-scleral rims for future keratoplasty procedures. In 2005, chlorhexidine was introduced by our eye bank as an additional step in the protocol for decontaminating human donor globes. After 5?years, we prospectively evaluated the number of contaminations. Out of 2,891 globes included in our study, 2,663 globes were processed, of which 36 (1.4%) were considered contaminated. Seventeen contaminations (0.6%) were detected by culturing limbal swabs, directly after decontamination, eight (0.3%) by visible discoloration of the culture medium carrying a corneo-scleral rim, and eleven (0.4%) after inoculation of the culture medium on blood agar plates. Importantly, after 4?weeks of incubation, none of the aerobic and anaerobic cultures taken from the secondary ??transport medium?? (dextran containing medium used to transport corneal tissue to the transplantation centre) showed microbiological growth. In conclusion, the combined use of 0.02% chlorhexidine and 0.5% povidone-iodine may allow decontamination of donor globes to a level at which the risk of tissue contamination at the time of transplantation is minimized, while corneal viability is preserved.     

Immunomagnetic separation as a final purification step of liver endothelial cells

Summary We describe a fast and reproducible method that can be used as a final step in obtaining pure populations of liver endothelial cells. This method employs endothelial cell specific lectin covalently bound to magnetic polystyrene beads (Dynabeads). Evonymus europaeus agglutinin (EEA)-coated Dynabeads were used to purify monkey liver endothelium from Percoll gradient separated nonparenchymal cells. EEA-coated beads were also successfully used to purify monkey aortic endothelial cells. The endothelial cells grew to confluence as a cobblestonelike monolayer, expressed Factor VIII related antigen, and incorporated acetylated-low density lipoprotein. The magnetic beads seemed not to modify the normal properties of the isolated endothelium, thus facilitating their use in experimental studies. This immunomagnetic separation technique may be applicable for purification of endothelial cells from a wide variety of tissue sources.     

Mass tag-assisted identification of naturally processed HLA class II-presented meningococcal peptides recognized by CD4+ T lymphocytes

The meningococcal class I outer membrane protein porin A plays an important role in the development of T cell-dependent protective immunity against meningococcal serogroup B infection and is therefore a major component of candidate meningococcal vaccines. T cell epitopes from porin A are poorly characterized because of weak in vitro memory T cell responses against purified Ag and strain variation. We applied a novel strategy to identify relevant naturally processed and MHC class II-presented porin A epitopes, based on stable isotope labeling of Ag. Human immature HLA-DR1-positive dendritic cells were used for optimal uptake and MHC class II processing of (14)N- and (15)N-labeled isoforms of the neisserial porin A serosubtype P1.5-2,10 in bacterial outer membrane vesicles. HLA-DR1 bound peptides, obtained after 48 h of Ag processing, contained typical spectral doublets in mass spectrometry that could easily be assigned to four porin A regions, expressed at diverging densities ( approximately 30-4000 copies/per cell). Epitopes from two of these regions are recognized by HLA-DR1-restricted CD4(+) T cell lines and are conserved among different serosubtypes of meningococcal porin A. This mass tag-assisted approach provides a useful methodology for rapid identification of MHC class II presented bacterial CD4(+) T cell epitopes relevant for vaccine development.     

Variation in alpha-L-fucosidase properties among 28 inbred mouse strains: Six strains have high enzyme activity and heat-stabile enzyme with a variant pH-activity curve; twenty-two strains have low activity and heat-labile enzyme

Alpha-L-fucosidase in tissues of 28 inbred mouse strains varied with respect to three properties: high or low heat stability, a pH-activity curve with high or low relative activity at pH 2.8, and high or low activity. Alpha-L-fucosidase from six strains (A/J, BDP/J, LP/J, P/J, SEA/GNJ, and 129/J) had high heat stability, high pH 2.8 relative activity, and high activity, whereas the other 22 strains all had low heat stability, low pH 2.8 relative activity, and low activity. The heat-stability difference was seen in all organs tested (brain, liver, kidney, spleen, heart, skeletal muscle, lung, and testis) for two heat-stabile strains (P/J and 129/J) and four heat-labile strains (C57 BL/6J, C3H/HeJ, DBA/2J, and BALB/cJ) studied in detail. The findings suggested that two structural variants of alpha-L-fucosidase, probably genetically determined, exist in these 28 inbred mouse strains, although the presence of linkage disequilibrium between alleles of tightly linked structural and regulatory genes could not be excluded.This work was supported by grants from the National Institutes of Health (NS-15281 and NS-11766), the Muscular Dystrophy Association (H. Houston Merritt Clinical Center for Muscular Dystrophy and Related Diseases), the March of Dimes Birth Defects Foundation, and a generous gift from the Alexander Rapaport Foundation.     

Long‐term response of temperate canopy trees to removal of browsing from an invasive arboreal herbivore in New Zealand

Defoliation of forest tree canopies by herbivores and other agents, leading to tree mortality and reduced productivity, threatens the ecological stability of forests globally. This study shows that long‐term control of a mammalian arboreal folivore (brushtail possums; Trichosurus vulpecula Phalangeridae) reduces crown dieback and increases foliage cover in browsing‐damaged canopy trees. We monitored indices of possum density, possum browsing, tree foliage cover and crown dieback for 20 years following initiation of possum control in 1994 that repeatedly reduced possum densities to near zero every 5–6 years and kept the population below 35% of pre‐control levels over the entire period. Observable possum browsing was recorded on 20–49% of individuals of three palatable tree species at the time of first control. Those percentages fell to zero after control and never exceeded 2–10% for individual species over the next 19 years. We recorded significant increases in foliage cover attributable to recovery from defoliation by possums for all three species during the first 10 years. Large increases in foliage cover occurred on individuals that were heavily browsed in 1994 (mean increases: 36–89%), but mean population increases were modest (3–19%) because only 10–19% of trees were initially heavily browsed. Twenty‐year mortality rates were similar for plants with, or without, initial possum browsing, indicating no residual impact of pre‐control browsing on tree mortality. Times for full recovery of crown foliage cover varied from 10 years for the youngest trees and faster growing species to more than 20 years for mature individuals of the slowest growing species.