Translocation of RNA-coated gold particles through the nuclear pores of oocytes

In the present study, various sized gold particles coated with tRNA, 5S RNA, or poly(A) were used to localize and characterize the pathways for RNA translocation to the cytoplasm. RNA-coated gold particles were microinjected into the nucleus of Xenopus oocytes. The cells were fixed after 15, 60 min, or 6 h, and the particle distribution was later observed by electron microscopy. Similar results were obtained with all classes of RNA used. After nuclear injection, particles ranging from 20-230 A in diameter were observed within central channels of the nuclear pores and in the cytoplasm immediately adjacent to the pores. Particles of this size would not be expected to diffuse through the pores, suggesting that some form of mediated transport occurred. In addition, it was found that the translocation process is saturable. At least 97% of the pores analyzed appeared to be involved in the translocation process. Gold coated with nonphysiological polynucleotides (poly[I] or poly[dA]) were also translocated. When nuclei were injected with either BSA-, ovalbumin-, polyglutamic acid-, or PVP-coated gold, the particles were essentially excluded from the pores. These results indicate that the accumulation of RNA-gold within the pores and adjacent cytoplasm was not due to non-specific effects. We conclude that the translocation sites for gold particles coated with different classes of RNA are located in the centers of the nuclear pores and that particles at least 230 A in diameter can cross the envelope. Tracer particles injected into the cytoplasm were observed within the nuclear pores in areas near the site of injection. However, only a small percentage of the particles actually entered the nucleus. It was also determined, by performing double injection experiments, that individual pores are bifunctional, that is, capable of transporting both proteins and RNA.     

Ion currents through pores. The roles of diffusion and external access steps in determining the currents through narrow pores.

External access steps, which may include restricted aqueous diffusion, are introduced into a kinetic model for ion transport through narrow pores. The conductance-concentration relation and the concentration dependence of the biionic permeability are calculated using two alternative assumptions: (a) access to the mouth of the pore is allowed only when no ion is within the lumen or at either mouth; (b) ions remain at the mouth only very transiently. With either assumption the concentration dependence of the fluxes is the same as in previous treatments in which all steps in access were lumped into a single process. Also as before, the biionic permeability ratio is independent of concentration so long as the lumen is never doubly occupied. For narrow pores, such as those formed by gramicidin A, the slowest external portion of the access process must occur close to the pore's mouth, and thus the region an ion must occupy to gain access is small. As a consequence, the probability of finding an ion within this region is also small. On this basis, it is argued that the second assumption is appropriate for these pores. The kinetic equations that result are identical to those used by Urban, B., S.B. Hladky, and D.A. Haydon (1980, Biochim. Biophys. Acta. 602:331-354).     

Theory of the rotational contribution to facilitated diffusion

Gros and others have recently shown experimentally that the facilitated diffusion of protons carried by a form of haemoglobin is enhanced by rotational diffusion of the carrier, whereas facilitated diffusion of oxygen by the same carrier is not. In this paper the theory of facilitated transport by rotating carriers is developed from first principles. The theory confirms Gros's findings that (i) the rotational contribution appears only when the angle of rotational diffusion over the average time the proton remains bound is small and (ii) under these conditions rotation enhances the normal translational contribution by a factor 1/2 at the lowest carrier concentrations. The theory also shows that there must be a rotational boundary layer.     

Transport of the substance through thin membranes with narrow pores after the preceding stage of surface diffusion

A model of the substance transport through thin membranes is discussed. Existence of a step of the surface diffusion of permeable particles is assumed preceding its entrance into membrane channel. An expression is given for the stationary flow, which has earlier been obtained for other transport models. It is shown that the permeant affinity to the membrane surface is the main condition for the validity of the scheme in question.     

Effect on solute size on diffusion rates through the transmembrane pores of the outer membrane of Escherichia coli

Nutrients usually cross the outer membrane of Escherichia coli by diffusion through water-filled channels surrounded by a specific class of protein, porins. In this study, the rates of diffusion of hydrophilic nonelectrolytes, mostly sugars and sugar alcohols, through the porin channels were determined in two systems, (a) vesicles reconstituted from phospholipids and purified porin and (b) intact cells of mutant strains that produce many fewer porin molecules than wild-type strains. The diffusion rates were strongly affected by the size of the solute, even when the size was well within the "exclusion limit" of the channel. In both systems, hexoses and hexose disaccharides diffused through the channel at rates 50-80% and 2-4%, respectively, of that of a pentose, arabinose. Application of the Renkin equation to these data led to the estimate that the pore radius is approximately 0.6 nm, if the pore is assumed to be a hollow cylinder. The results of the study also show that the permeability of the outer membrane of the wild-type E. coli cell to glucose and lactose can be explained by the presence of porin channels, that a significant fraction of these channels must be functional or "open" under our conditions of growth, and that even 10(5) channels per cell could become limiting when E. coli tries to grow at a maximal rate on low concentrations of slowly penetrating solutes, such as disaccharides.     

A contribution to the study ofChenopodium album agg

Plants gathered in Staré Brno (1979)—classified under the genusChenopodium L. and having K (2n)=54—are hybrids.Chenopodium strictum Roth (n=18) andChenopodium suecicum J. Murr (n=9) are probably parental species. Hybridization may have been linked with amphidiploidy.     

A hydrodynamic theory of ion conductance through Ohmic pores

A method of calculating the size of membrane pores lacking strong ionic selectivity is presented. By treating the flow of ions through a small channel as a hydrodynamic phenomenon, the electrical conductance becomes a function of the ratio of ion radius to channel radius. Thus when both the channel conductance and the ion size are known, the radius of the channel may be estimated. The method gives good agreement among radii predicted from conductances of four different alkali cations in alamethicin pores.     

Fluid mechanical and electrostatic study on the osmotic flow through cylindrical pores

When two solutions differing in solute concentration are separated by a porous membrane, the osmotic pressure will generate a net volume flux of the suspending fluid across the membrane; this is termed osmotic flow. We consider the osmotic flow across a membrane with circular cylindrical pores when the solute and the pore walls are electrically charged, and the suspending fluid is an electrolytic solution containing small cations and anions. Under the condition in which the radius of the pores and that of the solute molecules greatly exceed those of the solvent as well as the ions, a fluid mechanical and electrostatic theory is introduced to describe the osmotic flow in the presence of electric charge. The interaction energy, including the electrostatic interaction between the solute and the pore wall, plays a key role in determining the osmotic flow. We examine the electrostatic effect on the osmotic flow and discuss the difference in the interaction energy determined from the nonlinear Poisson-Boltzmann equation and from its linearized equation (the Debye-Hückel equation).     

Translocon pores in the endoplasmic reticulum are permeable to a neutral, polar molecule

The pore of the translocon complex in the endoplasmic reticulum (ER) is large enough to be permeated by small molecules, but it is generally believed that permeation is prevented by a barrier at the luminal end of the pore. We tested the hypothesis that 4-methylumbelliferyl alpha-d-glucopyranoside (4MalphaG), a small, neutral dye molecule, cannot permeate an empty translocon pore by measuring its activation by an ER resident alpha-glucosidase, which is dependent on entry into the ER. The basal entry of dye into the ER of broken Chinese hamster ovary-S cells was remarkably high, and it was increased by the addition of puromycin, which purges translocon pores of nascent polypeptides, creating additional empty pores. The basal and puromycin-dependent entries of 4MalphaG were mediated by a common, salt-sensitive pathway that was partially blocked by spermine. A similar activation of 4MalphaG was observed in nystatin-perforated cells, indicating that the entry of 4MalphaG into the ER did not result simply from the loss of cytosolic factors in broken cells. We reject the hypothesis and conclude that a small, neutral molecule can permeate the empty pore of a translocon complex, and we propose that translationally inactive, ribosome-bound translocons could provide a pathway for small molecules to cross the ER membrane.     

A study of the diffusion of compact particles in polymer solutions using quasi-elastic light scattering

This paper describes an investigation, using quasi-elastic light scattering, of the diffusion of polystyrene spheres through solutions of dextran. The diffusion coefficient, D, of the spheres is shown to vary inversely with the volume fraction, φ, of dextran according to $\text{D =}\text{D}{}_0\text{(1 + νφ + κφ}{}^2\text{)}$. Changes in the molecular weight of dextran are shown to reflect changes in the macromolecular shape parameter, ν, and the interaction parameter, κ. This result differs from previous studies which suggested an $\text{exp}\text{(?}\text{B}\text{φ}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{)}$ dependence and no molecular weight dependence [8].     

A simple model for diffusion in independent, temporally fluctuating pores

A simple model is presented for one-dimensional diffusion in an ensemble of semi-infinite and finite pores or capillaries in which the boundary at one end of each capillary is allowed to fluctuate randomly between a perfectly reflecting barrier and a perfectly absorbing barrier. The model is independent of the spatial distribution of the capillaries; it is only assumed that there are a large number of them and that they are noninteracting. Exact solutions are possible and results are obtained, in terms of the fluctuation parameters, for the total amount per unit area of solute passed through the capillary system in the semi-infinite case, and for a permeability coefficient and time lag to steady state in the finite system. Applications of the model to diffusion in biological membranes are discussed.     

HIV cell-to-cell transmission requires the production of infectious virus particles and does not proceed through env-mediated fusion pores

Direct cell-to-cell transmission of human immunodeficiency virus (HIV) is a more potent and efficient means of virus propagation than infection by cell-free virus particles. The aim of this study was to determine whether cell-to-cell transmission requires the assembly of enveloped virus particles or whether nucleic acids with replication potential could translocate directly from donor to target cells through envelope glycoprotein (Env)-induced fusion pores. To this end, we characterized the transmission properties of viruses carrying mutations in the matrix protein (MA) that affect the incorporation of Env into virus particles but do not interfere with Env-mediated cell-cell fusion. By use of cell-free virus, the infectivity of MA mutant viruses was below the detection threshold both in single-cycle and in multiple-cycle assays. Truncation of the cytoplasmic tail (CT) of Env restored the incorporation of Env into MA mutant viruses and rescued their cell-free infectivity to different extents. In cell-to-cell transmission assays, MA mutations prevented HIV transmission from donor to target cells, despite efficient Env-dependent membrane fusion. HIV transmission was blocked at the level of virus core translocation into the cytosol of target cells. As in cell-free assays, rescue of Env incorporation by truncation of the Env CT restored the virus core translocation and cell-to-cell infectivity of MA mutant viruses. These data show that HIV cell-to-cell transmission requires the assembly of enveloped virus particles. The increased efficiency of this infection route may thus be attributed to the high local concentrations of virus particles at sites of cellular contacts rather than to a qualitatively different transmission process.     

A model for membrane transport through alpha-helical protein pores

In this communication we explore possible mechanisms by which hydrogen-bonded, knobs-into-holes packed side chains from adjoining α-helical segments could function in proton transport through membranes and mechanisms by which proton transport could be coupled to active transport of other substances.     

A mathematical contribution to the study of orientation of organisms

Organisms orient themselves to a stimulus by two general methods. One method is by directed orientation (taxis); the other is by undirected locomotory reaction (kinesis). An equation, and the methods for finding the necessary parameters of this equation, is derived for the distribution of organisms within a container, with the following limitations: (1) the organisms have no accommodation, (2) they are always active, and (3) the stimulus changes slowly with position. Necessary modifications of the equation are then derived, so that the last two limitations may be eliminated. The equation cannot be solved excatly because of its complexity; hence an approximation method must be used. This method is discussed, an approximate solution is found, and a time constant for equilibrium to be established is derived. Applications tovarious experiments in the literature are then made with fairly satisfactory results. A new interpretation of the theory of klino-kinesis with accommodation is found upon application of the equations developed to experimental work. Further limitations and uses of these equations are then discussed. This work was done while the author was Public Health Service Research Fellow of The National Institute of Mental Halth, Federal Security Agency.     

A new contribution to the study of 22 trisomy.

The case of a 2 1/2-month-old male child with intrauterine distrophy features and multiple congenital malformations is presented. Cytogenetic studies of the child and his parents, completed with Q- and G-banding techniques led us to conclude that it is a case of 22 trisomy inherited from his mother.