The tight junctions of rabbit choroid plexus and ciliary body epithelia. A comparative study using the double replica freeze-fracture technique

The spatial arrangement of tight junctions in choroid plexus and ciliary body rabbit epithelia has been determined by studying freeze-fracture complementary replicas. In the choroid plexus epithelium, the interruptions of the junctional P-face fibrils were measured to be 14% of their total length. In the ciliary body epithelium, where the fibrils were found to be more fragmented than in the choroid plexus, the P-face fibril interruptions accounted for 12 % of the total length of the zonulae occludentes sealing the non-pigmented cells and 30% in the focal linear tight junctions connecting the non-pigmented and pigmented cells at their apices. In both epithelia, the interruptions of the ridges are precisely complemented by particles or short bars of similar length found in the E-face furrows. Consequently, it is possible to conclude that the junctional fibrils are continuous in these two epithelia. For the zonulae occludentes, this continuity appears to be inconsistent with the ‘leaky’ properties of these epithelia shown by some physiological investigations.     

‘Endogenous yolk’ as the precursor of a possible fertilization envelope in a crab (Carcinus maenas)

After the egg attachment to a maternal ovigerous seta, the Carcinus maenas embryo is enclosed in a tripartite capsule. The innermost layer (envelope 2) which is also the main part of this capsule, is generally detected after egg-laying and is most probably closely related to the fecondation phenomenon. The precursor material of envelope 2, arising from the egg by a massive and very fast exocytosis process, appears as numerous ring-shaped granules. These granules, originated from numerous cortical vesicles perhaps intercommunicating with each others, are observed early in the ooplasm during oogenesis, These so-called ring-shaped granules seem very identical in form with the disc-shaped granules which are classically described as composing the endogenous or intracysternal yolk of many Decapoda crustacean oocytes. In view of our results the role of these granules, in endogenous yolk formation, is re-examined and discussed.     

Prey capture by the cuttlefish (Sepia officinalis L): An experimental study of two strategies

This study shows that the size of the prey ($\text{Carcinus maenas}$) relative to the predator ($\text{Sepia officinalis}$) is of importance in the choice between two types of attack: either capture by ejection of the two extensible tentacles, or capture by jumping on the prey. Small crabs are preferentially captured by the first method and large crabs by the second. Other factors which may explain the observed variations, include previous experience of the predator and the behaviour of the prey.     

Visualization of chromatin distribution in living PTO cells by Hoechst 33342 fluorescent staining

Chromatin distribution was visualized in living cells with the selective DNA fluorochrome Hoechst 33342. This dye was shown to be non-toxic on the rat kangaroo PTO cell line by measuring the labelled cell growth rate. The aim of this work was firstly to visualize chromatin distribution without fixation or dehydration and secondly to demonstrate that quantitative determination of DNA content was possible under these non-toxic labelling conditions. During interphase, condensed, decondensed and thin network chromatin configurations were visualized. In nucleolar regions the fluorochrome revealed well-defined chromocentres. During mitosis, fluorescent chromosome banding was observed in vital conditions and chromocentres on fixed chromosomes. Chromatin segregation was visualized after micronucleation, which induced chromosomal set distribution in individual micronuclei. By this means, we demonstrated that the chromocentres observed in interphase nuclei were part of nuclear organizer region (NOR)-bearing chromosomes. This vital staining of chromatin was shown to be compatible with the quantitative determination of DNA content, both in living PTO cells and in isolated nuclei.     

Cell multiplication and type II collagen production by rabbit articular chondrocytes cultivated in a defined medium

The complexity and the variations in the efficiency of different batches of serum stimulated the preparation of a serum-free medium which could promote not only growth, but also the differentiation properties of rabbit articular chondrocytes in culture. The serum-free medium (SFM) developed in this study contained insulin, transferrin, Na-selenite, human fibronectin bovine serum albumin (BSA), brain growth factor (BGF) or fibroblast growth factor (FGF), hydrocortisone and multiplication stimulating activity (MSA). Primary or secondary cultures of chondrocytes in such a medium attained a proliferation rate equal to 70-80% of that obtained with chondrocytes grown in a serum control medium. The deletion of various factors from SFM indicates that BGF or FGF are the most stimulating of growth factors. Insulin was beneficial when used individually; when combined with BGF or FGF, they had a synergistic effect on cell proliferation. MSA seemed not to play any role in chondrocyte growth in culture. The SFM medium did not modify either the morphology or the progression of cells into the cell cycle. It moreover allowed the maintenance of the specific function of chondrocytes to synthesize type II collagen.     

An X ray diffraction study of frog sciatic nerve myelin in dimethyl sulfoxide.

Reversible structure modification of frog sciatic nerve myelin bathed in Ringer's solution containing dimethyl sulfoxide (DMSO) at a concentration of 33% has been studied by low-angle X-ray diffraction using a linear position-sensitive counter. Fourier images of native myelin layers, derived using low-order reflections measured at various stages of the DMSO treatment, reveal that the bilayer profile of native myelin membrane undergoes a specific asymmetric change prior to the phase transformation: The high-density peak on the extracellular side of the central lipid hydrocarbon layer decreases reversibly as the nerve is permeated by DMSO, while the internal peak and the central layer remain virtually unaltered. The dynamic process by which the contracted phase of myelin is derived from native myelin is speculated on the basis of the observed profile change.     

Role of magnesium cations in the yeast orotate phosphoribosyltransferase catalyzed reaction. Mechanism of the inhibition by Cu++ and Ni++ ions

The magnesium chelate of the N(3)H tautomer of orotate, L₃Mg, is the true substrate in the biosynthesis of orotidine 5′-monophosphate (OMP) catalyzed by yeast orotate phosphoribosyltransferase (OPRTase, E.C. 2.4.210) with a Michaelis constant K_m^L₃Mg equal to 12(2) μM. It is postulated that Mg⁺⁺ cations activate the transport of orotate to the active site by neutralizing the orotate charges; the ligand N(3)H is then exchanged between the incoming cation and the cation bound to the enzyme, thus ensuring the stabilization of the appropriate isomeric structure of orotate. This scheme, together with kinetic and thermodynamic data on orotate complexation by Mg⁺⁺ and Ca⁺⁺, accounts for the role of Ca⁺⁺ cations that neither activate nor inhibit OMP synthesis.Cu⁺⁺ and Ni⁺⁺ inhibiting properties arise from the formation of inert complexes of orotate. Ni⁺⁺ complexes have a poor affinity for the protein, whereas Cu⁺⁺ complexes have a Michaelis constant similar to that of the L₃Mg active species. The inertness of these complexes is tentatively understood in terms of low phosphoribosyl transfer rates as postulated from the kinetic study of the protonation of the complexes in water.     

Fine structure of bovine lens epithelial cells in vitro in relation to modifications induced by a retinal extract (EDGF)

Cultured bovine lens epithelial cells are polygonal in shape. In confluent and multilayer cultures they exhibit elaborate arrays of 6 nm filaments, bundles of intermediate-sized filaments, and a fibrous meshwork of subcellular and intercellular material. Cells grown in the presence of a retinal extract (RE) have a higher growth rate, and are smaller and more regular in shape. In them the 6 nm filaments are mostly aligned in sheets, the intermediate-sized filaments form a fine network, and the cells are closely apposed to the plastic substratum. Some homogeneous material is formed intercellularly in older cultures. Cellular elongation, induced in the former cultures by the addition of RE, is accompanied by an alignment of cytoskeletal elements, including microtubules, parallel to the long axis. Other structural features are similar in all cell types. The response to RE is discussed in terms of shape modulations associated with the restricted expression of structural characteristics acquired in vitro.     

Complement inhibitor(s) released from leukocytes. II. Evidence that lymphocytes release and produce an inhibitor of C2 activation.

We have previously reported that mouse spleen and thymus cells in short term culture release an hitherto undescribed complement inhibitor that prevents the activation of C2. Here it is shown that the inhibitor is released by B lymphocytes and may be produced by lymphocytes. Release of the inhibitor takes place at 37 °C and in the presence of metabolic inhibitors, but not at 4 °C. Thus the release of the inhibitor could be related to the shedding of structures located on the lymphocyte membrane.     

Density dependent selection incorporating intraspecific competition 1. A haploid model

A haploid model is introduced and analyzed in which intraspecific competition is incorporated within a density dependent framework. It is assumed that each genotype has a unique carrying capacity corresponding to the equilibrium population size when fixed for that type. Each genotypic fitness at a single multi-allelic locus is a function of a distinctive effective population size formed by adding the numbers of each genotype present, weighted by an intraspecific competition coefficient. As a result, the fitnesses depend upon the relative frequencies of the various genotypes as well as the total population size. Intergenotypic interactions can have a profound effect upon the outcome of the population. In particular, when the density effect of one individual upon another depends upon their respective genotypes, a unique stable interior equilibrium is possible in which all alleles are present. This stands in contrast to the purely density dependent haploid system in which the only possible stable state corresponds to fixation for the type with the highest carrying capacity. In the present model selective advantage is determined by a balance between carrying capacity and sensitivity to density pressures from other genotypes. Fixation for the genotype with the highest carrying capacity, for instance, will not be stable if it exerts a sufficiently weak competitive effect upon the other genotypes. In the diallelic case, maintenance of both alleles at a stable equilibrium requires that the net intragenotypic competition between individuals of like genotype be stronger than that between unlike types. As for purely density regulated systems, there may be no stable equilibria and/or regular and chaotic cycling may occur. The results may also be interpreted in terms of a discrete time model of interspecific competition with each haplotype representing a different species.     

The use of fluorescence anisotropy decay of poly d(A-T) ethidium bromide complex to estimate the unwinding angle of the double helix.

We measured the fluorescence decay under polarized light, of ethidium bromide bound to the poly d(A-T) isolated from Cancer Pagurus. The decay of the whole fluorescence is a single exponential function revealing a good homogeneity of the binding sites. The anisotropy decay due to energy transfers between the ethidium bromide molecules bound to a same poly d(A-T) molecule has been analysed, with a Monte Carlo calculation. We found the dye unwinds the poly d(A-T) duplex by an angle of 17 degrees plus or minus 2 degrees. This result is in agreement with the value previously found in the case of calf thymus DNA-ethidium bromide complex, although the base compositions of the two nucleic acids are different.     

Energy migration in the poly(ra-rU)-ethidium complex. determination of the unwinding angle of the polyribonucleic helix

The polarized fluorescence of the ethidium bromide (EB)-poly(rA-rU) complex has been studied by pulse fluorometry. As expected for a polynucleotide snowing one single kind of intercalation site, the decay of the whole emission is a single exponential (time constant 27 ns). The anisotropy decay is analysed as follows: (1) A brownian contribution having two correlation times, one of which characterizes local motions and the other a macromolecular motion. (2) A contribution due to transfers between EB molecules fixed to the same polynucleotide molecule, is analysed by a method analogous to the method used in previous work on EB-DNA complexes. That method consists in choosing a molecular model of the complex depending on geometrical parameters, and in simulating the energy migration on that model with a Monte Carlo calculation. Poly(rA-rU) is assumed here to adopt the structure A of RNA. Intercalated EB molecules modify the anale between two consecutive base pairs by δ. The angular position of the EB transition moment is defined by an angle φ. One finds that the angle φ is situated between 0° and 30°, which corresponds to a whole intercalation of the chroniophore as opposed to the semi-intercalation which has been proposed for certain dyes. The angle δ is negative; therefore there is an unwinding of the polyribonucleotide helix. Its absolute value is about 38°, sensibly greater than The value previously found for EB-DNA complexes.     

The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, we find that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu and the extracellular protein Snsl. In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. Our data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.     

Isolation and characterization of faithful and altered clones of the genomes of cauliflower mosaic virus isolates Cabb B-JI, CM4-184, and Bari I

Full-length genomes of cauliflower mosaic virus (CaMV) isolates Cabb B-JI, CM4-184, and Bari I have been cloned in the SalGI site of plasmid pAT 153. The cloned DNAs were characterized by restriction mapping and infectivity assays. All the sites present in the virion DNAs were found in the cloned DNAs. Comparison of restriction maps with those of DNA from two other isolates which have been recently completely sequenced revealed a close relationship among the different isolates. Some of the clones appear to be faithful copies of the viral genomes and these viral inserts are infectious when inoculated into turnip plants. Various clones with deletions in the CaMV DNA have been isolated and characterized. Some of them may correspond to deletions naturally occurring in a subpopulation of the virus whereas others occurred during cloning. None of the deleted fragments are infectious when inoculated into plants. Strikingly, all the deletions overlap one or two of the specific single-stranded breaks characteristic of caulimoviruses, suggesting that sequences surrounding the breaks are not dispensable.     

Circadian changes in carrageenin-induced edema: the anti-inflammatory effect and bioavailability of phenylbutazone in rats

The circadian variations in paw-edema produced by carrageenin and the anti-inflammatory effect of phenylbutazone were studied, in rats kept under a 12 light-12 dark regimen, in comparison with the variations of plasma phenylbutazone and oxyphenbutazone levels. When the experiment was performed during the light span (08.00 and 14.00 h), the rats were highly sensitive to the phlogistic effect of carrageenin, the plasma levels of phenylbutazone and oxyphenbutazone were lower, and the anti-inflammatory effect of phenylbutazone, weaker. Opposite results were obtained when the experiment was performed during the dark span (02.00 and 20.00 h). The results indicate that the chronoeffectiveness of phenylbutazone is influenced by both its chronokinetics and the chronesthesy of the biosystem involved.     

Effects of SH group reagents on creatine kinase interaction with the mitochondrial membrane

Solubilization of the specific mitochondrial isoenzyme of creatine kinase (CKm) from rabbit heart mitochondria by treatment with SH group reagents has been studied. From the various compounds tested only the negatively charged organomercurials are able to induce an extensive solubilization of the enzyme. This effect is fully reversible since the solubilized enzyme readily reassociates with the membrane when the bound organomercurial is removed by treatment of the homogenate by an excess of dithiothreitol. Solubilization by negatively charged organomercurials can be partly prevented by pretreatment of mitochondria with either disulfide or uncharged organomercurials. No clear-cut relationship has been pointed out when the amount of SH titrated by various reagents has been compared with the extent of CKm solubilization. More detailed studies with para-chloromercuribenzoate (pCMB) show that extensive CKm solubilization (about 75%) occurs for pCMB concentration as low as 25 microM, whereas pronounced inhibition of the enzyme is observed only for concentrations greater than 200 microM. By cross-reassociation of enzyme solubilized either by para-hydroxymercuribenzoate (pHMB) or by 20 mM sodium phosphate (NaPi) with mitochondria depleted of CKm by pHMB or by NaPi treatment, SH groups whose titration impedes CKm reassociation with the mitochondrial membrane have been tentatively located on the enzyme. Thus, negatively charged organomercurials, could induce a reversible conformational modification of the enzyme which is no longer able to bind on the inner mitochondrial membrane. Furthermore, our results show that the binding of an excess of mitochondrial CK, which has been previously reported, could reflect unspecific binding since it occurs only on mitoplasts incubated in very hypotonic medium, but not in isotonic medium.     

Response to the amoebocyte-producing organ of sensitized Biomphalaria glabrata after exposure to Echinostoma caproni miracidia

Stimulation by exposure to Echinostoma caproni miracidia triggered the activity of the amoebocyte-producing organ (APO) of sensitized Biomphalaria glabrata snails. This organ is the site of formation of cells which subsequently migrate to all parts of the body. Amoebocyte production started very soon after exposure, and was maximal at 3 or 4 days; it then declined very fast and, at 6–7 days, the organ had almost returned to its initial state.     

Formation of copper-bleomycin complexes: evidence of a three-step process

The formation of Cu(II)-bleomycin complexes as a function of pH has been studied using circular dichroism, absorption, electron paramagnetic resonance spectroscopy, and potentiometric titration. Our data support the following points: the formation of Cu(II)-bleomycin complexes occurs in a three-step process: a first complex (I) is formed at pH 1.2, which most probably involves the pyrimidine nitrogen, the secondary amine nitrogen, and two water molecules as the four in-plane ligands of copper. A second complex (II) is formed at pH 2.5, through the further coordination of the peptide nitrogen of histidine residue, and histidine imidazole nitrogen giving rise to the release of two protons. The fixation, in apical position, of the alpha-amino nitrogen of beta-aminoalanine occurs in a last step through the release of one additional proton. A value of 2.7 has been obtained for the pK of formation of this third complex, which is the species present at physiological pH. In the Cu(II)-depbleomycin system only one complex (II') has been detected.