Evidence for a dual mechanism of chick embryo fibroblast adhesion on fibronectin and laminin substrata

Eight-day-old chick embryo fibroblasts were shown to adhere specifically to fibronectin and laminin substrata. Moreover, the Scatchard analysis reveals 540,000 binding sites per cell for the fibronectin with a dissociation constant (Kd) of 1.35 microM and 5,500 binding sites per cell for laminin with a Kd of 1.5 nM. Furthermore, cell-fibronectin interactions are mediated by plasma membrane proteins of high molecular weight (HMW) (150K and 125K) insensitive to trypsin treatment and low molecular weight (LMW) proteins (95K, 80K, 65K and 45K) sensitive to trypsin treatment. Adhesion of 8-day-old chick embryo fibroblasts on laminin is mediated by plasma membrane proteins highly sensitive to trypsin treatment. Regarding the paucity of laminin-binding sites, the identification of laminin receptor could not be achieved. Nevertheless, this study provides quantitative and qualitative evidences for different mechanisms of 8-day-old chick embryo fibroblasts on laminin and fibronectin.     

Export and secretion of overproduced OmpA-beta-lactamase in Escherichia coli

The export of beta-lactamase to the periplasm of Escherichia coli can be directed by the OmpA signal peptide in the secretion cloning vector pIN-III. The overproduction of the hybrid precursor specifically induces a delay in the onset of processing of newly synthesized polypeptide chains. However, when the processing starts, no alteration in the rate of cleavage itself is observed. Our results suggest that the temporal mode of processing (which reflects translocation) does not depend on the nature of the signal peptide but rather depends on the nature of the polypeptide chain exported.     

Differential inactivation of inotropic and toxic digitalis receptors in ischemic dog heart. Molecular basis of the deleterious effects of digitalis

When applied to ischemic hearts digitalis exhibits depressed inotropic effect and increased toxicity. The molecular basis of these effects was investigated at the level of the digitalis receptors characterized by Na,K-ATPase assays and [3H]ouabain-binding measurements. In sarcolemma obtained from dog hearts rendered ischemic for 15, 30, and 60 min (left anterior descending), two populations (high and low affinity) of digitalis receptors were detected. The apparent affinity (KD, 300 nM) and the binding capacity of the low-affinity sites (responsible for toxicity) remained constant and similar to those found in normal hearts. The KD value of the high-affinity sites, "responsible for inotropy," remained unchanged (2 nM), but the site number sharply decreased (up to 90%). These inotropic sites that account for 66% of the total binding in normals are gradually inactivated, as the duration of ischemia increases. This inactivation would occur in situ since it was detectable in homogenates and was not depressed by the isolation procedure per se. The loss of function of the inotropic sites and the increased contribution of the low-affinity toxic sites represent the setting of a new distribution of the digitalis receptors in the ischemic heart before reperfusion is instituted. This constitutes the molecular basis of the deleterious pharmacological effects observed with digitalis.     

Link protein interactions with hyaluronate and proteoglycans. Characterization of two distinct domains in bovine cartilage link proteins

Hyaluronic acid-binding region and trypsin-link protein were prepared from bovine nasal cartilage proteoglycan complex after trypsin digestion. Binary complexes were reformed between trypsin-link protein and hyaluronic acid-binding region or hyaluronate. Upon trypsin treatment of these complexes, two fragments deriving from trypsin-link protein were characterized. One of them, of 20 kDa, corresponds in fact to a 140-amino acid long fragment and bears the glycosylated site of trypsin-link protein; it appears to be involved in proteoglycan/link protein interaction. The other, of 22 kDa, corresponds to the 200 C-terminal amino acids of trypsin-link protein; it appears to be involved in the binding of link protein with hyaluronic acid. A structural model of bovine trypsin-like protein depicting two distinct domains involved in hyaluronate and proteoglycan subunit interactions is proposed.     

Evidence for negative regulation of T cell growth by low affinity interleukin 2 receptors

Two experimental situations have been studied, and the results provide evidence for a negative regulatory role for the low affinity interleukin 2 receptor (LA-IL 2R). The IL 2-dependent T helper cell line L-14, deprived of IL 2, becomes quiescent and expresses comparable numbers of high affinity IL 2R (HA-IL 2R) and LA-IL 2R. After activation by recombinant IL 2, this cell line preferentially expresses LA-IL 2R. The IL 2 responsiveness of the L-14 cell line was found to vary according to the ratio of LA-IL 2R to HA-IL 2R: the relative predominance of the LA-IL 2R coincides with a hyporeactivity of cells to IL 2. In contrast, a predominance of HA-IL 2R is accompanied by an increase in cellular IL 2 reactivity. Treatment of three IL 2-dependent T cell lines (L-14, HT-2, and C30.1) with limited amounts of recombinant IL 2 and moderate concentrations of anti-IL 2R monoclonal antibodies stimulates T cell growth. This treatment was shown to selectively diminish the expression of membrane LA-IL 2R. The stimulation was attributed to the decrease of expression of LA-IL 2R.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

Complementarity of particles and pits in freeze-fractured hepatic and cardiac gap junctions

Summary Particles and pits of freeze-fractured gap junctions are considered as complementary structures despite the frequent observations of more regular and closer spacings of pits, ascribed to plastic deformation of particle arrays. Recently, however, the noncomplementarity of pits and particles in Purkinje fibers has been reported. To ascertain the relationship between both structures, gap junctions from fixed, cryoprotected liver and myocardium were investigated using spacing and density measurements and complementary replicas.In hepatocyte gap junctions, the center-to-center distances (mean±sd) among pits, 9.57±1.49 nm, and particles, 9.70±1.77 nm, are not significantly different. Density determinations yielded a slightly higher value for the pits, (11,510±830)/m², than for the particles, (11,230±950)/m². In the myocardium, the spacing of the regularly arrayed pits, 9.55±1.33 nm barely exceeds the value of 9.44±1.62 nm for the particles, which show some clustering. However, the packing density for the pits, (10,090±740)/m², appears a little higher than that of the particles (9,890±920)/m². As density and spacing measurements provided no decisive answers, the positions of individual pits and particles of complementary junctional faces were recorded on transparent sheets and compared. In this fashion, a one-to-one correspondence between particles and pits could be established, while small discrepancies may be attributed to plastic deformation. Moreover, the collinearity of pits and particles may be suggested by the observation of a platinum grain in the center of many pits.     

Meiofauna associated with a Pacific coral reef in Costa Rica

The meiofauna of two coral reef habitats at Isla del Naño, Costa Rica was studied over a one year period. The dominant groups were: Foraminifera (21.2%), Copepoda (19.7%), Nematoda (19.1%), Gastropoda (16.5%), Polychaeta (7.2%) and Bivalvia (6.6%). The highest diversity was found in coarse, heterogeneous sands with the highest percentage of carbonates. The meiofauna showed a high degree of horizontal aggregation, which is a characteristic pattern for macro- and meiofauna in sediments of variable composition. No vertical variation in distribution was evident, probably due to the deep location of the Redox Potential Discontinuity layer. The total densities of organisms found in this study (99 to 575 ind/10 cm²) are low compared with densities in similar non-reefal sands (7 to 6116), and from fine sediments (80 to 17 000), but are comparable to densities found in other reef areas (39 to 609.5 ind/10 cm²). This is the first report on meiofauna from the eastern Pacific, and the first time that foraminiferans are the dominant group.     

Circumferential analysis of digitalized gamma angiocardiography by assessment of regional left ventricular contraction

After acquisition of a digital equilibrium gamma-angiocardiographie, circumferential analysis of end-diastolic and end-systolic frames gives 120 points diastolic and systolic curves. Their difference represents systolic volume and leads to regional left ventricular ejection fraction assessment at the considered radius level. The circumferential analysis evolute gives the regional left ventricular ejection fraction representative curves which allows especially differential diagnosis between left ventricular akinesia and dyskinesia.     

Binding of anti-fibronectin to early amphibian ectoderm does not result in inhibition of neural induction under in vitro conditions

Summary Antibodies directed to fibronectin (anti-FN) were injected into the blastocoel of late blastulae of Xenopus laevis. Two animal caps (ectoderm) were isolated, when control embryos reached the early gastrula stage, and were combined with untreated upper blastopore lip in the sandwich method. In two control series fibronectin or Holtfreter solution was injected into the blastocoel. The results of the experiments suggest that neural induction cannot be prevented by binding anti-FN to fibronectin, which covers the blastocoelic side of the ectoderm. The data support the view that extracellular matrix proteins are not themselves responsible for neural induction. However, in comparison with the control series a slight shift of the differentiation pattern in the spinocaudal direction could be observed in the anti-FN series. The possible role of extracellular proteins in the formation of a close juxtaposition of mesodermal and ectodermal target cells as a prerequisite for shortdistance transmission of neural inducers is discussed.