Hydrophobia, lectin binding, and molecular order in the stratum corneum of adult and neonatal rats and in human breast cells in culture.

A search for differences due to ANS staining (hydrophobia), Con A and PNA binding capacity, and birefringence was carried out on stratified epithelia of rat skin and human breast cells (HBC) in culture. Microfluorimetric measurements confirm that the ANS fluorescence of the stratum corneum from adults is higher than that of newborns. HBC exhibited an unexpected deep ANS-fluorescence. Differences in the binding capacity of the epithelial layers to Con A and PNA were detected with advancing age. Retardation measurements revealed that the form birefringence of the stratum corneum is higher in adult animals specially as revealed by the fact that its form birefringence curve branch from n = 1.414 to n = 1.479 is steeper, i.e. depict higher values. The strong birefringence of the cytoplasmic tonofilaments presented by cultured human breast cells was considered an unexpected finding and attributed to changes that the cells underwent following the in vitro conditions.     

Molecular analysis of yellow fever virus 17DD vaccine strain.

The Oswaldo Cruz Foundation produces most of the yellow fever (YF) vaccine prepared worldwide. As part of a broader approach to determine the genetic variability in YF 17D seeds and vaccines and its relevance to viral attenuation the 17DD virus was purified directly from chick embryo homogenates which is the source of virus used for vaccination of millions of people in Brazil and other countries for half a century. Neutralization and hemagglutination tests showed that the purified virus is similar to the original stock. Furthermore, radioimmune precipitation of 35S-methionine-labeled viral proteins using mouse hyperimmune ascitic fluid revealed identical patterns for the purified 17DD virus and the YF 17D-204 strain except for the 17DD E protein which migrated slower on SDS-PAGE. This difference is likely to be due to N-linked glycosylation. Finally, comparison by northern blot hybridization of virion RNAs of purified 17DD with two other strains of YF 17D virus revealed only genome-sized molecules for all three viruses. These observations suggest that the vaccine phenotype is primarily associated with the accumulation of mutations.     

Resialylation of sialidase-treated sheep and human erythrocytes by Trypanosoma cruzi trans-sialidase: restoration of complement resistance of desialylated sheep erythrocytes.

Trypanosoma cruzi trans-sialidase (TS) is a recently described enzyme which transfers alpha(2-3)-linked sialic acid from host-derived sialylated glycoconjugates to parasite surface molecules [Schenkman et al. (1991) Cell, 65, 1117]. We report here on the ability of TS to transfer sialic acid from donor sialyl-alpha(2-3)lactose to sialidase-treated sheep and human erythrocytes. Up to approximately 50% resialylation of both desialylated red cells could be attained. Resialylation of desialylated sheep erythrocytes restores their resistance to lysis by human complement. This ascribes a possible biological role for T. cruzi TS and demonstrates directly that sialic acid is solely responsible for preventing alternative pathway activation of human complement by sheep erythrocytes.     

Antibodies specific for VB8 receptor peptide suppress experimental autoimmune encephalomyelitis.

Recent studies from our laboratory have shown, for the first time, that a synthetic peptide from that TCR VB chain used preferentially by encephalitogenic T cells induced the formation of protective, MHC class I-restricted T cells and prevented the development of EAE in Lewis rats. In this report we 1) demonstrate that immunization with the TCR-VB8-39-59 peptide generated peptide-specific antibodies that protect against experimental autoimmune encephalomyelitis induced by either of the two distinct encephalitogenic epitopes of basic protein, and 2) characterize the production and biologic functions of rat and rabbit antibody responses to the TCR peptide. The antibodies in both species increased in titer over time, were highly specific for the immunogen by direct reaction and inhibition assays, stained only VB8+ T cells, and suppressed clinical signs and to lesser extent the number of histologic lesions of experimental autoimmune encephalomyelitis mediated by VB8+ T cells. Coupled with our previous work, these results indicate that both humoral and cellular responses to the TCR-VB8-39-59 peptide can contribute independent immunoregulatory effects on encephalitogenic T lymphocytes that use common V region genes in response to epitopes of myelin basic protein.     

Internalization of surface anionic sites and phagosome-lysosome fusion during interaction of Toxoplasma gondii with macrophages

The participation of cell surface anionic sites on the interaction between tachyzoites of Toxoplasma gondii and macrophages and the process of phagosome-lysosome fusion were analyzed using cationized ferritin as a marker of cell surface anionic sites and albumin-colloidal gold as a marker for secondary lysosomes. Incubation of either the macrophages or the parasites with cationized ferritin before the interaction increased the ingestion of parasites by macrophages. Anionic sites of the macrophage's surface, labeled with cationized ferritin before the interaction, were internalized together with untreated parasites. However, after interaction with glutaraldehyde-fixed or specific antibody-coated parasites, the cationized ferritin particles were observed in endocytic vacuoles which did not contain parasites. Macrophages previously labeled with albumin-gold at 37 degrees C, were incubated in the presence of cationized ferritin at 4 degrees C and then incubated with untreated or specific antibody-coated parasites. After interaction with opsonized parasites, the colloidal gold particles were observed in the parasitophorous vacuoles while the cationized ferritin particles were observed in cytoplasmic vesicles. However, when the interaction was carried out with untreated parasites, the parasitophorous vacuoles exhibited ferritin particles while the colloidal gold particles were observed in cytoplasmic vesicles. These observations, in association with studies previously reported, suggest that the state of the parasite surface determines the mechanism of parasite entry into the macrophage, the composition of the membrane lining the parasitophorous vacuole and the ability of lysosomes to fuse with the vacuoles.     

Properties of Chicken Lens MIP Channels Reconstituted into Planar Lipid Bilayers

Membrane fractions highly enriched in chicken lens MIP (MIP28) were found to form ion channels when incorporated into planar lipid bilayers. The channels displayed prominent unitary conductances of about 60 and 290 pS in symmetric 150 mm KCl solution and were slightly anion selective. For both depolarizing and hyperpolarizing voltages, voltage sensitivity of the MIP28-induced conductance could be fit by a Boltzmann relation, symmetric around zero mV, with V ₀ = 18.5 mV, n= 4.5 and g _min/g _max= 0.17. Channel properties were not appreciably altered by pH in the range of 5.8 to 7, although channel incorporation was observed to occur more frequently at lower pH values. Calcium, at millimolar concentrations, decreased the channel mean open time. Partial proteolysis of MIP28 to yield MIP21 did not appreciably affect single-channel conductance or voltage sensitivity of the reconstituted channels. MIP28 was not phosphorylated by cAMP dependent protein kinase (PKA). Although unitary conductance and selectivity of the chicken MIP channel are similar to those reported for the bovine MIP (MIP26), the voltage sensitivity of MIP28 was higher than that of the bovine homologue, and voltage sensitivity of MIP28 was not modulated by treatments previously shown to affect MIP26 voltage gating (partial proteolysis and protein phosphorylation by PKA: (Ehring et al., 1990). The existence of such strikingly different functional properties in highly homologous channel isoforms may provide a useful system for exploration of the structure-function relations of MIP channels. Received: 27 March 1996/Revised: 5 August 1996     

Solvent effects on the catalytic activity of subtilisin suspended in compressed gases

We studied a model transesterification reaction catalyzed by subtilisin Carlsberg suspended in carbon dioxide, propane, and mixtures of these solvents under pressure. To account for solvent effects due to differences in water partitioning between the enzyme and the bulk solvents, we measured water sorption isotherms for the enzyme in each solvent. We measured catalytic activity as a function of enzyme hydration and obtained bell-shaped curves with maxima at the same enzyme hydration (12%) in all the solvents. However, the activity maxima were different in all media, being much higher in propane than in either CO(2) or the mixtures with 50 and 10% CO(2). Considerations based on the solvation ability of the solvents did not offer an explanation for the differences in catalytic activity observed. Our results suggest that CO(2) has a direct adverse effect on the catalytic activity of subtilisin. (c) 1996 John Wiley & Sons, Inc.