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.     

Interspecific Introgression in Cetaceans: DNA Markers Reveal Post-F1 Status of a Pilot Whale

Visual species identification of cetacean strandings is difficult, especially when dead specimens are degraded and/or species are morphologically similar. The two recognised pilot whale species (Globicephala melas and Globicephala macrorhynchus) are sympatric in the North Atlantic Ocean. These species are very similar in external appearance and their morphometric characteristics partially overlap; thus visual identification is not always reliable. Genetic species identification ensures correct identification of specimens. Here we have employed one mitochondrial (D-Loop region) and eight nuclear loci (microsatellites) as genetic markers to identify six stranded pilot whales found in Galicia (Northwest Spain), one of them of ambiguous phenotype. DNA analyses yielded positive amplification of all loci and enabled species identification. Nuclear microsatellite DNA genotypes revealed mixed ancestry for one individual, identified as a post-F1 interspecific hybrid employing two different Bayesian methods. From the mitochondrial sequence the maternal species was Globicephala melas. This is the first hybrid documented between Globicephala melas and G. macrorhynchus, and the first post-F1 hybrid genetically identified between cetaceans, revealing interspecific genetic introgression in marine mammals. We propose to add nuclear loci to genetic databases for cetacean species identification in order to detect hybrid individuals.     

Interactions of human lymphoblasts with targeted vesicles containing Sendai virus envelope proteins

We have studied the internalization of targeted fusogenic liposome content to leukemic T cells (CEM) in vitro. We describe a method for the covalent coupling of T101 antibody to the surface of liposomes and the incorporation of fusogenic viral protein into the liposome membrane. Hygromycin B, an impermeant inhibitor of protein synthesis, was encapsulated in the targeted fusogenic liposomes and delivered directly to the cytoplasm of leukemic T cells by fusion between the two membranes. The cytotoxic effect was measured by [3H]thymidine incorporation. We show that CEM are rapidly and specifically killed by the drug encapsulated in the targeted fusogenic liposomes. This effect is due to the binding of the liposome by means of the antibody and then to the fusion of the liposome with the targeted cell membrane, mediated by F protein.     

Evidence for bidirectional transverse diffusion of spin-labeled phospholipids in the plasma membrane of guinea pig blood cells

The distribution and transverse diffusion kinetics of four spin-labeled phospholipid analogues (two with choline heads: phosphatidylcholine (PC) and sphingomyelin (SM); two with amino heads: phosphatidylserine (PS) and phosphatidylethanolamine (PE) were studied in the plasma membrane of guinea pig blood cells: erythrocytes, reticulocytes, and leukemic lymphocytes. Nitroxide reduction by the internal content of the cells was used as an indicator to determine the phospholipids that penetrated the cells. The reduction rates were in the order, PS greater than PE greater than PC greater than SM in all cells. Reoxidation of phospholipids extracted by serum albumin revealed the distribution of the phospholipids at a given time. In all cells, the distribution equilibrium was reached in less than 2 h and the amounts left in the external leaflet were in the following proportional order: PS less than PE less than PC less than SM. In the erythrocytes and especially in the reticulocytes, the shape change induced by adding phospholipids relaxed partially or completely at a lower speed but kept the same proportional order as at equilibrium. All the results were analyzed quantitatively with a simple kinetic model including the rates of transverse diffusion (flip and flop), the exchange between plasma membrane and internal membranes, and the reduction rate of free radicals (determined in either the internal or external membrane leaflet). The calculated rate constants of transverse diffusion varied from 2 x 10(-3) to 1.2 x 10(-1) min-1 for the flip and from 4 x 10(-3) to 1.2 x 10(-1) for the flop, depending on the polar head and the cell type. Possible interpretations of the external phospholipid reduction mechanism and cell deformation are discussed.     

Internalization of low-density-lipoprotein-specific receptors in leukemic guinea pig lymphocytes

Leukemic guinea pig lymphocytes (L2C) have ten times as many low-density lipoprotein (LDL) receptors as healthy lymphocytes, but LDL accounts for only 38% of the cholesterol in L2C cells, compared to more than 95% in normal cells. Our data show that LDL fails to regulate cholesterol biosynthesis and that there is a defect in LDL internalization and receptor turnover in L2C cells. We also demonstrate that the degradation of LDL is not a limiting process. By discriminating between binding and internalization, we show that internalization in L2C is much slower than in normal cells and that the decrease in metabolism is related to the slow turnover of the LDL receptors.     

The effect of temperature on the acetylcholinesterase activity of muscle microsomes

Membrane vesicles which constitute the sarcotubular system were separated and the fraction enriched in T-tubules purified by a calcium loading procedure. The preparations of unfractioned microsomes and T-tubules have been analyzed for their relative content of enzyme markers and acetylcholinesterase. The amount of this enzyme in the T-tubule fraction was higher than in mixed microsomes but less than two-fold the value of vesicles derived from sarcoplasmic reticulum. Arrhenius plots of membrane-bound and soluble acetylcholinesterase from either mixed microsomes or fractions enriched in T-tubules show an anomalous behaviour as two break points were obtained. The first discontinuity was found at about 17 degrees C for membrane-bound, and 12-14 degrees C for soluble acetylcholinesterase. The second one being at about 25 degrees C for both particulate and detergent-solubilized enzyme. The changes in activity with temperature suggest that lipid-protein, detergent-protein and protein-protein interactions might be involved in the stabilization of the enzyme both in the natural membrane and in the soluble state.     

A new synaptic anomaly: irregular synaptonemal complexes

Summary In this paper we describe a new synaptic anomaly characterized by the presence of irregular synaptonemal complexes (SCs) in two sterile patients.     

Interaction of concanavalin A with sugar residues of collagen from different tissues

Topochemical characteristics of reactions of different types of collagen-containing structures with Concanavalin A (Con A) have not been considered up to now. In this study the presence and availability of glucose residues of collagen molecules from intestine, liver, cartilage and tendon are detected using Con A and horseradish peroxidase (HRP). In intestine, cartilage and tendon sections, the Con A-HRP method was only significantly positive when the sections were first submitted to treatment with papain. This suggested the presence of glycoproteins and proteoglycans of the extracellular matrix (ECM), which might interfere either interacting with lateral sugar residues of the collagen molecules, or causing some steric blockade or even masking as occurs in regions with a high state of compactness.     

A murine sequence-specific DNA binding protein shows extensive local similarities to the amyloid precursor protein.

Microinjection experiments suggested previously that protein binding to the DNA nucleotide sequence GTCACATG, identical to the CDEI element of the yeast centromere, plays an important role in the early development of the mouse. We established from a series of overlapping mouse cDNA clones the sequence of a candidate CDEI-binding protein. Synthesis in Escherichia coli of a fusion protein which binds specifically the CDEI box in vitro confirmed its identification. On the other hand, the translated 511 amino acid sequence shows two regions with high degrees of similarity to the protein precursor (APP) of the beta-protein (amyloid) that accumulates in the brain and blood vessels of Alzheimer patients. A continuous stretch of 195 amino acids includes 133 residues identical to part of the extracellular domain of APP, and 48 of the 70 C-terminal residues of the open reading frame are identical to the APP transmembrane and cytoplasmic domains.