Fast tracer efflux from membrane vesicles; investigation by controlled elution

A method has been developed to study tracer efflux from membrane vesicles. Vesicles are trapped on filters, but in contrast to conventional filtration techniques tracer efflux is measured as such. A functional integration of a commercial fraction collector, a pulse generator, and a programmable repetitive pipet with a simple electronic circuit allows monitoring of complete efflux kinetics from seconds onward. The automatic control of discrete elution steps guarantees reproducible results and makes possible a correction for background release of tracer from the filter. The dependence of efflux data on selected combinations of filters is demonstrated. These experiments also shed some light on problems intrinsic to conventional filtration procedures. The performance of the controlled elution technique is shown with ²²Na efflux from Na channel-rich membrane vesicles.     

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.     

Potassium and veratrine-stimulated l-[3H]cysteine sulfinate and l-[3H]glutamate release from rat brain slices

The release of l-[³H]cysteine sulfinic acid, l-[³H]glutamatic acid and [³H]GABA from preloaded slices of various rat brain regions in response to either 30 mM K⁺ or veratrin was investigated. All these aminoacids were released by both depolarizing agents, which did not produce any changes in the spontaneous efflux of [³H]lysine. The K⁺ stimulated cysteine sulfinate release from superfused slices was found partly Ca²⁺-dependent in the subiculum, and mainly Ca²⁺-independent in the hippocampus whereas the K⁺-elicited glutamate release was partly Ca²⁺-dependent in both regions. The veratrine-induced release of both cysteine sulfinate and glutamate was blocked by verapamil in a dose-dependent way, although a small verapamil concentration independent release remained. The release pattern of both amino acids was heterogeneous, but roughly correlated among brain regions, except in the subiculum and hypothalamus.These findings demonstrate the releasability of both substances from various brain regions and suggest that those releases occur from different pools, being probably mainly of neuronal origin. They give further evidence that cysteine sulfinate as well as glutamate may serve a neurotransmitter role in the CNS.     

State of differentiation of bovine epithelial lens cells in vitro : Modulation of the synthesis and of the polymerization of specific proteins (crystallins) and non-specific proteins in relation to cell divisions

Maintenance of the state of differentiation in serially cultured bovine epithelial lens cells has been investigated. The radioactive labelled soluble proteins were studied by gel filtration and gel electrophoresis. 1. In the lens epithelium on its capsule, preferential synthesis of alpha B2 vs alpha A2 crystallin subunits and synthesis of beta-crystallins (mainly beta Bp) were observed. 2. Epithelial lens cells cultured on plastic Petri dishes for up to 35 divisions still synthesized alpha B2 and beta Bp, but no longer alpha A2. Conversely, the same cells injected into nude mice synthesized alpha B and alpha A, but no beta-crystallin could be detected. 3. The ratio of non-crystallin proteins to crystallin polypeptides increased drastically with the number of cell divisions. Among these proteins, both Mr 45 000 and Mr 57 000 proteins are probably constituents of the water-soluble cytoskeletal proteins, respectively actin and vimentin. A Mr 17 000 polypeptide was observed and its relationship with a metabolic product of alpha-crystallin is proposed. 4. The polymerization process of crystallin polypeptides in these cells was studied and compared with crystallin aggregates found in the lens. Newly synthesized alpha crystallins were readily involved in high molecular aggregates. This process does not seem to require alpha A, since only alpha B was detected. Interestingly, non-crystallin-soluble proteins form the bulk of proteins found in high molecular weight (HMW) polymers. The time course of crystallin aggregate formation, in long-term culture cells, seems to be different for alpha- vs beta-polypeptides. These results allowed us to conclude that bovine epithelial lens cells in vitro, although they do not undergo terminal differentiation into fibers, are not dedifferentiated, since they still express specific features of the epithelium in situ.     

Natural killer activity: Effector cells in purified human T or null cells have different phenotypes defined by monoclonal antibodies

The present report demonstrates that NK effector cells present in the T-cell population and the null cell population of human peripheral blood mononuclear cells (PBMC) possess distinct phenotypes as defined by monoclonal antibodies. All the E^? null cell-type effectors are OKM1⁺ OKT3^?. Conversely the E⁺ T-cell-type effectors are OKM1^? OKT3^?. These results were obtained by fractionation of PBMC by means of anti-F(ab′)₂ immunoabsorbent combined with E-rosette sedimentation. Each cell subset obtained was then treated with monoclonal antibody OKT3 or OKM1, and C. The NK activity of each cell population was quantitatively titrated against ⁵¹Cr-labeled JM target cells.     

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.     

Biosynthesis of proteoglycans in organ cultures of developing kidney mesenchyme

The biosynthesis of proteoglycans was studied in organ cultures of differentiating metanephric mesenchymes. When triggered by a contact-mediated inductive interaction, this tissue undergoes transition from a mesenchyme to an epithelium. In the present study, proteoglycans were extracted by guanidinium hydrochloride in the presence of protease inhibitors. We found that, as a response to induction, the differentiating mesenchyme begins to synthesize large size proteoglycans with an apparent molecular weight (MW) of 1 X 10(6) D. The major glycosaminoglycans detected were chondroitin sulfates. Heparan sulfate proteoglycans were also detected, constituting 20% of the proteoglycans. An inhibitor of glucosamine synthesis, 6-diazo-5-oxo-norleucine (DON) was found to inhibit glycosaminoglycan synthesis by approx. 60%, and the size of the proteoglycans was also diminished. Our studies suggest that the transition of the mesenchyme to epithelium is associated with initiation of synthesis of large size proteoglycans.     

Interaction of bovine epithelial lens (BEL) cells with extracellular matrix (ECM) and eye-derived growth factor (EDGF): I. Effects on short-term adhesiveness and on long-term organization of the culture

A growth factor (EDGF) derived from the retina controls the proliferation and shape of adult bovine epithelial lens (BEL) cells in vitro as well as extracellular matrix (ECM) assembly. In order to analyse this mechanism and the specificity of the interactions between BEL cells and the extracellular matrix we have investigated the adhesion and growth of BEL cells on various substrata (fibronectin, laminin, ECM). BEL cells treated with EDGF adhered more slowly to plastic Petri dishes than untreated cells, in part due to EDGF inhibition of fibronectin deposition. The untreated BEL cells spread less well on ECM or laminin than on fibronectin-coated plastic. The preferential adhesiveness of BEL cells on fibronectin vs laminin was confirmed by attachment experiments performed on replicas of SDS-PAGE of these proteins. However, in long-term cultures, 8 days after seeding, BEL cells were very differently arranged on plastic or on ECM. ECM by itself did not increase the proliferation rate but helped to restore an organized cell monolayer. BEL cells stimulated to grow on ECM by treatment with EDGF exhibited at least transiently contact inhibition producing a perfectly organized epithelium similar to the one observed in vivo. These results suggest specific interactions between ECM or ECM components with BEL cell that restrain excessive cell spreading and restore an original polarized phenotype of the cells seen in vivo.     

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.     

Modification of the aggregation state of the multifunctional enzyme complex catalyzing the first steps in pyrimidine biosynthesis in the course of development of Drosophila melanogaster

Mutations at the bithoraxoid (bxd) and postbithorax (pbx) loci cause a transformation of posterior haltere to posterior wing. It has previously been shown that pbx and $\text{pbx}\text{Ubx}{}^{101}$ cause this transformation by affecting the maintenance (or cell heredity function) of determination so that the transformed cells are indistinguishable from normal wing cells, and have no “memory” of having been part of a haltere disk (Adler, 1978a). I report here that Tp(3) $\text{bxd}{}^{100}\text{Ubx}{}^{101}$ and bxd¹, pbx, e^w both cause the transformation of posterior haltere to posterior wing in the same way as pbx. On the other hand, bxd¹, $\text{bxd}{}^1\text{Ubx}{}^{101}$, bxd^51j, $\text{bxd}{}^{\mathrm{<mtext>51</mtext><mtext>j</mtext>}}\text{Ubx}{}^{101}$, and $\text{bxd}{}^{\mathrm{<mtext>51</mtext><mtext>j</mtext>}}\text{red pbx}$ cause this same transformation of posterior haltere to posterior wing by interfering with the expression of the determined state so that the developmental information of posterior haltere is “misread” as posterior wing. The transformed cells in these disks retain the memory of having been part of a haltere disk; that is, these posterior cells that would secrete wing cuticle during metamorphosis regenerate anterior haltere structures. Thus it appears clear that it is possible to uncouple the expression and cell heredity functions of determination in the haltere disk of Drosophila.     

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.     

Phosphorus-31 nuclear magnetic resonance study of the thiamine diphosphate-magnesium interaction

The results obtained from a phosphorus NMR study of the interactions of Mg²⁺ with thiamine diphosphate confirm the existence of a Mg-thiamine diphosphate complex with a $\text{1}\text{1}$ stoichiometry in which the α phosphorus seems to be the most influenced by the interaction. The variations of δ and J_αβ with various concentrations of Mg²⁺ are described.     

Modulation of the immune response by passive antibodies. III. Effects of IgG1 and IgG2 anti-carrier antibodies.

The effects of IgG₁ and IgG₂ anti-carrier antibodies were studied on cellular and humoral reactions induced by immunization with a hapten-carrier complex. IgG₁ was shown to depress both delayed hypersensitivity reactions (DHR) to the carrier and anaphylaxis to the hapten whereas IgG₂ had no activity. A mixture of IgG₁ and IgG₂ depressed only DHR to the carrier. The modulating effects of passive anti-carrier antibodies were shown to depend on their immunoglobulin class and the concentration used.     

Changes in the polyadenylated messenger RNA population during development of Dictyostelium discoideum

The program of gene expression during the life cycle of Dictyostelium discoideum has been assessed by molecular hybridization of cDNA probes with polysomal RNA extracted at the following different stages of development: vegetative growth, interphase (2.5 hr), aggregation (8 hr), postaggregation (12 hr), and preculmination (18 hr). Several different cDNA probes were used. Two probes were prepared from vegetative stage poly(A⁺) RNA, one representing all species present and the other enriched for abundant species. A third cDNA probe was prepared from preculmination stage polysomal RNA and a fourth probe consisted of the preculmination stage cDNA depleted in those species also present at the vegetative stage. Hybridization of the various probes with the different polysomal RNA preparations has revealed developmental changes in the mRNA populations. These changes were not detected in an aggregation less mutant under similar conditions of starvation. Abundant RNA species of vegetative cells were found to drop to low levels, especially during the aggregation period. Fifty percent by mass of the RNA present in polysomes at 18 hr is not present during vegetative growth. Some of the new RNA species appeared during interphase and the remaining during the postaggregation period. A gradual increase in the number of copies per cell of certain RNA species comprising both new species as well as some shared with vegetative cells was observed throughout development. Other results indicated that the composition of polysomal and cytoplasmic RNA is similar during vegetative growth but differs markedly at 18 hr of development. Also, cytoplasmic RNA at 18 hr contained, in addition to polysomal RNA, a large proportion by mass of nonpolysomal RNA similar to vegetative RNA. The number of polysomal RNA species detected by this analysis during vegetative growth and during the preculmination stage were estimated to be 3000 and 3700, respectively. The number of copies of these RNA species ranged between 30 and 2000 per cell during vegetative growth and 3 to 300 per cell in polysomes at 18 hr. Developmentally induced RNAs which were preferentially distributed among abundant and intermediate classes were estimated to number 700–900 species.