Lysosomal integral membrane glycoproteins are expressed at high levels in the inclusion bodies of I-cell disease fibroblasts

The localization, expression, and transport of two lysosomal integral membrane glycoproteins of human cells, hLAMP-1 and hLAMP-2, have been studied in mucolipidosis II (I-cell disease) fibroblasts. These cells are deficient in N-acetylglucosaminylphosphotransferase, one of the enzymes required for addition of the mannose 6-phosphate recognition signal to newly synthesized lysosomal hydrolases and a prerequisite for the sorting and transport of the hydrolases to lysosomes. I-cells analyzed by immunofluorescence microscopy with monoclonal antibodies against hLAMP-1 and hLAMP-2 showed intense staining of the inclusion bodies covering most of the cytoplasm of the cells. Immunoelectron microscopy confirmed this localization and showed that the hLAMP-positive vesicles commonly contained membrane structures or electron-dense homogeneous material characteristic of secondary lysosomes. Studies of the biosynthesis of hLAMP-2 in I-cells pulse-labeled with [35S]methionine indicated that the molecule is glycosylated in the Golgi system, is transported to vesicles with the high density characteristic of lysosomes, and has chemical properties similar to those of the glycoprotein synthesized in normal cells. The concentration of the hLAMP-2 glycoprotein was three- to fourfold greater than that in normal fibroblasts, in sharp contrast to the reduced levels of lysosomal hydrolases seen in I-cells. These experiments demonstrate that the inclusion bodies in I-cells have properties of secondary lysosomes and that the transport and targeting of the lysosomal membrane glycoproteins to the inclusion bodies of these cells is not coupled to the mannose 6-phosphate system for transporting soluble acid hydrolases.     

Insect cell production of a secreted form of human alpha(1)-proteinase inhibitor as a bifunctional protein which inhibits neutrophil elastase and has growth factor-like activities.

alpha(1)-proteinase inhibitor (API) is a potential therapeutic agent in all diseases in which elastase released by neutrophils has to be effectively neutralized. We ligated the cDNA of human API to the C-terminal section of an insulin-like growth factor II analogue (BOMIGF), known to be properly folded and secreted in insect cells using the baculovirus expression system. The BOMIGF-API chimera was recovered from the incubation medium of the infected cells. It shared the properties of both IGFs and API. It inhibited neutrophil elastase and formed SDS-stable complexes with the enzyme. The attachment of the large API protein to the C-terminal end of the 10 kDa IGF analogue did not destroy the IGF-mediated stimulation of thymidine incorporation into bovine fetal erythroid cells. We tested the capacity of the chimera to affect fibronectin-dependent TF-1 cell migration. BOMIGF-API significantly restored TF-1 cell migration in the presence of elastase, which is the enzyme of burn wound fluid most probably involved in fibronectin degradation. Some of the beneficial uses for this chimera may include all instances for which inhibition of elastase-mediated extracellular matrix destruction as well as stimulation of cell migration and proliferation are required for tissue repair.     

Epidemiology of toxoplasmosis in Chile. VI. Prevalence of human infection, investigated by means of an indirect hemagglutination test, in regions VII, VIII and IX

In presecuting the investigations on the epidemiology of toxoplasmosis in Chile, a new series of serological surveys has been performed during 1982-1989 in 10 urban and 25 periurban-rural localities from the regions VII, VIII and IX of the country (34 degrees 41'-39 degrees 38' South lat.). In 9,758, randomly selected apparently healthy persons, and indirect hemagglutination test (IHAT) for toxoplasmosis was carried out. The age of these individuals (4,203 males and 5,555 females) varied between 4 and 84 years. The examined persons represent a 0.33% of the total population of the three studied regions. IHAT titers of 1: > or = 16 were regarded as positive. The global prevalence for positive IHAT was 45.5% (50.5% in men and 41.7% in women). A higher proportion of positive tests was observed in urban areas (47.0%) than in periurban-rural sections (33.3%). An increasing prevalence with age was also observed. Only 5 (0.05%) persons had IHAT titers higher than 1:1000.     

Chagasic infection in blood donors from hospitals in endemic regions of Chile (1982-1987). Epidemiological impact of the problem]

Chagas' disease, produced by Trypanosoma cruzi and transmitted by hematophagous triatomine bugs, exists in the Western Hemisphere from the south-western United States to central Chile and Argentina. It exists in rural and periurban sections of the northern half of Chile, with a prevalence of 16.9%. Constant rural-urban migrations have contributed to its spreading to urban sections. In order to investigate the impact of these migrations on the population susceptible of being blood donors and the probable increasing of the risk of T. cruzi transmission by blood transfusion, epidemiological surveys were carried out in donors from 22 hospitals located in the northern half of Chile. By means of an indirect hemagglutination test for Chagas' disease 16,841 blood donors were examined, arising a 2.7% of positivity, percentage that permitted to estimate that 126,477 potential blood donors infected with T. cruzi should be in the urban sections studied. These facts strengthen the need that serology for Chagas' disease must be routinely performed in endemic regions of the country, to adopt or reinforce the pertinent preventive measures.     

Flow cytometry analysis of cell population dynamics and cell cycle during HIV-1 envelope-mediated formation of syncytia in vitro

Cell fusion occurs in physiological and pathological conditions and plays a role in regulation of cell fate. The analysis of cell population dynamics and cell cycle in cell–cell fusion experiments is necessary to determine changes in the quantitative equilibrium of cell populations and to identify potential bystander effects. Here, using cocultures of Jurkat HIV-1 envelope expressing cells and CD4⁺ cells as a model system and flow cytometry for the analysis, the number, viability, and cell cycle status of the populations participating in fusion were determined. In 3-day cocultures, a sustained reduction of the number of CD4⁺ cells was observed while they showed high viability and normal cell cycle progression; fusion, but not inhibition of proliferation or death, accounted for their decrease. In contrast, the number of Env⁺ cells decreased in cocultures due to fusion, death, and an inherent arrest at G1. Most of syncytia formed in the first 6 h of coculture showed DNA synthesis activity, indicating that the efficient recruitment of proliferating cells contributed to amplify the removal of CD4⁺ cells by syncytia formation. Late in cocultures, approximately 50% of syncytia were viable and a subpopulation still underwent DNA synthesis, even when the recruitment of additional cells was prevented by the addition of the fusion inhibitor T-20, indicating that a population of syncytia may progress into the cell cycle. These results show that the quantitative analysis of cellular outcomes of cell–cell fusion can be performed by flow cytometry.