Identification of epitopes associated with different biological activities on the glycoprotein of vesicular stomatitis virus by use of monoclonal antibodies

Summary Thirteen monoclonal antibodies (MAbs) to the glycoprotein (G) of vesicular stomatitis virus (VSV) serotype Indiana were prepared and examined for their effects on various biological activities of VSV, including in vitro infection, hemagglutination, adsorption to cells, and mediation of cell fusion. Competitive binding assays with these MAbs revealed the presence of at least seven distinct antigenic determinants (epitopes) on the G protein. In some cases, overlappings among epitopes to various degrees were observed as partial inhibition or binding enhancement. The MAbs to all the epitopes but one (epitopes 1–6) reacted with the denatured G protein in a Western immunoblot analysis. Four of the epitopes (epitopes 2, 4, 5, and 7) were involved in neutralization and two (epitopes 1 and 2) in hemagglutination inhibition. None of the MAbs inhibited the adsorption of radiolabeled VSV to BHK-21 cells; the MAbs to epitope 2 slightly enhanced the virus adsorption. All neutralization epitopes except epitope 2 (epitopes 4, 5, and 7) were associated with inhibition of VSV-mediated cell fusion. These results show a direct spatial relationship between the epitopes recognized by the MAbs and functional sites on G protein and further insights into the structure and function of G protein.     

Loss of Hrs in the Central Nervous System Causes Accumulation of Ubiquitinated Proteins and Neurodegeneration

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs^flox/flox;SynI-cre mice. Notably, the hrs^flox/flox;SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs^flox/flox;SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.     

Identification of neural genes using Xenopus DNA microarrays.

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.     

Somatic mosaicism of CAG repeat in dentatorubral-pallidoluysian atrophy (DRPLA)

An unstable expansion of CAG repeat in the coding region ofthe DRPLA gene on chromosome 12p is the mutation specific forhereditary dentatorubralpallidoluysian atrophy (DRPLA). We studiedthe CAG expansion in brain and other tissues from six unre latedDRPLA patients. The CAG repeat lengths showed distinct difterencesbetween tissues. The sizes of the CAG expansion in various regionsof the brain except the cerebellum were generally larger byseveral repeats than in other peripheral tissues. Brain samplesshowed greater variation of the expansion compared with othertissues, but neither the size of the CAG expansion nor the degreeof CAG repeat variation parallels the detailed findings of neuropathologicalinvolvement. We conclude that somatic instabilities of the CAGrepeat cause tissue variability of the CAG repeat size in DRPLAbut other region or cell type-specific factors would be involvedto explain the selectivity of cell damage in DRPLA.     

Chemiluminescence of whole blood. II. Application to clinical examination of phagocytic functions of whole blood from various types of disease

Measurements of whole blood chemiluminescence (CL) were carried out by using 0.1 ml of blood to evaluate phagocytic functions from various kinds of pediatric patients. Patients with definite bacterial infections showed an elevated background CL and high peak CL per 1000 granulocytes after stimulation with zymosan. These results suggest that granulocytes of these patients had been preactivated in vivo, their phagocytic and/or metabolic activity being enhanced. Contrary to these results, blood from the patients with viral infections showed almost no abnormalities. Patients with chronic granulomatous disease showed no significant CL by this method similar to conventional ones. This method was also shown to be a useful technique for monitoring the phagocytic functions of blood after granulocyte transfusion.     

Effect of interferon treatment on serum 2',5'-oligoadenylate synthetase levels in hepatitis C-infected patients

Interferon (IFN) is widely used for patients with hepatitis C. Less than half of treated patients respond to IFN therapy, however, and increased resistance to IFN is particularly observed in genotype 1b patients. Recently, genotype 1b patients with the wild type sequence in the NS5A gene were shown to be resistant to therapy, suggesting that the NS5A protein may be involved to IFN resistance. Thus, we investigated the serum 2',5'-oligoadenylate synthetase (2',5'-OAS) levels before and during IFN treatment. In addition, other biochemical markers and NS5A mutations were also examined in 30 HCV genotype 1b-positive patients. Before IFN treatment, 2',5'-OAS activity in sera was significantly lower in wild type patients than in mutant type patients. All patients were subsequently enrolled in IFN therapy, and 2',5'-OAS activity was elevated both in wild and mutant type patients, irrespective of the number of mutations in NS5A. Logistic regression analysis revealed that clearance of serum HCV RNA was independently related to the pretreatment viral load and NS5A mutations, but not to serum 2',5'-OAS activity. We concluded that the NS5A protein, that is associated with the outcome of IFN therapy, affects the kinetics of IFN-induced molecules, such as 2', 5'-OAS. 2',5'-OAS activity does not, however, seem to be related to long-term virological response to IFN therapy.     

The Serum Factor from Patients with Ulcerative Colitis that Induces T Cell Proliferation in the Mouse Thymus Is Interleukin-7

The disturbance of immune regulatory T cells is related to the pathogenesis of ulcerative colitis. Here we demonstrated and characterized the serum factor from ulcerative colitis patients that induced proliferation of intrathymic T cells. The factor isolated from the patient sera by a combination of gel filtration and anion-exchange chromatography induced proliferation of CD4⁺CD8^– intrathymic T cells in the organ-cultured embryonic mouse thymus. Purification and amino acid sequence analysis of the serum factor demonstrated that the N-terminal 12 sequence was homologous to that of interleukin-7. SDS-PAGE and Western blot confirmed that purified serum factor was interleukin-7. Enzyme immunoassay demonstrated that the serum interleukin-7 concentration was significantly increased in the patients. PCR and Southern blot hybridization demonstrated that interleukin-7 mRNA expression was increased in the thymus tissues from patients but decreased in the colonic mucosa. Since interleukin-7 is a crucial cytokine for proliferation and differentiation of T cells in the thymus, the present study indicates that interleukin-7 may contribute to the disturbance of immune regulatory T cells in ulcerative colitis.     

Concentration quenching of photoluminescent Ru(bpy) dispersed in a polysiloxane film containing 2,2′-bipyridine pendant groups in dependence of molecular distribution

The photoluminescent Ru(bpy) complex was dispersed in a polysiloxane film containing 2,2′-bipyridine (bpy) pendant groups. The unusually long photoluminescence lifetime of the Ru(bpy) (1,94 μs at 25°C) and the blue-shifted photoluminescent wavelength suggest a rigid polymer matrix. The fluorescence yield becomes lower with higher probe concentration, indicating concentration quenching. According to the analysis based on Stern-Volmer plots, the quenching obeys a mechanism composed of both static and dynamic processes. A statistical intermolecular distance distribution between the probes was used to interpret the results in terms of static and dynamic quenching. It is shown that in the present system the dispersed complexes diffuse slightly during the excited state.     

Protection of hepatocytes from apoptosis by a novel substance from actinomycetes culture medium

A novel substance, #675, found from an Streptomyces sp. SM675 culture medium, dose-dependently stimulates the proliferation of human functional liver cell 4 (FLC4). When FLC4 cells were incubated under conditions without fetal bovine serum (FBS), typical features of apoptotic cell death such as shrinkage and nuclear condensation appeared; high molecular weight (HMW) DNA fragments were found; and caspase-3 and poly (ADP-ribose) polymerase (PARP) proteins were cleaved. When FLC4 cells were incubated with #675 and without FBS, the cells grew healthy, no HMW DNA fragments were found, and caspase-3 and PARP cleavage weakened, suggesting that #675 protects FLC4 cells from apoptosis induced by FBS-deprivation. The quantitative reverse-transcribed polymerase chain reaction did not show differences in PARP or Bcl-2 mRNA expression in FLC4 cells incubated with or without #675, indicating other genes may be involved in this anti-apoptosis effect. These results show that #675 enhances FLC4 proliferation via an apoptosis-inhibition pathway, implying potential pharmacological and clinical applications.