Porosity of the Yeast Cell Wall and Membrane

The limiting sizes of molecules that can permeate the intact cell wall and protoplast membrane of Saccharomyces cerevisiae were determined from the inflection points in a triphasic pattern of passive equilibrium uptake values obtained with a series of inert probing molecules varying in molecular size. In the phase identified with the yeast protoplast, the uptake-exclusion threshold corresponded to a monodisperse ethylene glycol of molecular weight = 110 and Einstein-Stokes hydrodynamic radius (r(ES)) = 0.42 nm. In the cell wall phase, the threshold corresponded to a polydisperse polyethylene glycol of number-average molecular weight ( M(n)) = 620 and average radius (r(ES)) = 0.81 nm. The third phase corresponded to complete exclusion of larger molecules. The assessment of cell wall porosity was confirmed by use of a second method involving analytical gel chromatographic analyses of the molecular weight distribution for a single polydisperse polyglycol before and after uptake by the cells, which indicated a quasi-monodisperse threshold for the cell wall of M(n) = 760 and r(ES) = 0.89 nm. The results were reconciled with two situations in which much larger protein molecules previously have been reported able to penetrate the yeast cell wall.     

Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems.

Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.     

Permeability of cyanobacterial mucous surface structures for macromolecules

The space of diffusive distribution of neutral hydrophilic macromolecules (dextrans with molecular sizes of 1.5 to 9 nm in the Stokes’ radius values) in the mucous surface structures (MSS) of intact bacterial cells has been studied for the first time on cyanobacteria. Cyanobacterial species and strains under study belong to different taxonomic groups, the members of which form MSS of various morphology and ultrastructure and can grow in association with plants and animals, inter alia as mucous microcolonies. The range of permeability has been determined by the fractionation of polydisperse dextrans method, previously applied for plants, in combination with electron microscopy. Dextrans are supposedly distributed in the MSS polysaccharide matrix in accordance with their sizes, in much the same way as in a macroporous unitary gel. The similarity of the chemical composition and macromolecular organization of cyanobacterial MSS with pectins of plant cell walls and the role of MSS and the intercellular matrix as permeability barriers in associative interactions of microorganisms are under consideration.     

Permeability of cyanobacterial mucous surface structures for macromolecules

The space of diffusive distribution of neutral hydrophilic macromolecules (dextrans with molecular sizes of 1.5 to 9 nm in the Stokes radius values) in the mucous surface structures (MSS) of intact bacterial cells has been studied for the first time on cyanobacteria. Cyanobacterial species and strains under study belong to different taxonomic groups, the members of which form MSS of various morphology and ultrastructure and can grow in association with plants and animals, inter alia as mucous microcolonies. The range of permeability has been determined by the fractionation of polydisperse dextrans method, previously applied for plants, in combination with electron microscopy. Dextrans are supposedly distributed in the MSS polysaccharide matrix in accordance with their sizes, in much the same way as in a macroporous unitary gel. The similarity of the chemical composition and macromolecular organization of cyanobacterial MSS with pectins of plant cell walls and the role of MSS and the intercellular matrix as permeability barriers in associative interactions of microorganisms are under consideration.     

Compositional changes in cell wall polymers during mango fruit ripening

Softening of mango fruit has been investigated by analysis of ripening related changes in the composition of the fruit cell walls. There is an apparent overall loss of galactosyl and deoxyhexosyl residues during ripening, the latter indicating degradation of the pectin component of the wall. The loss of galactose appears to be restricted to the chelator soluble fraction of the wall pectin, whilst loss of deoxyhexose seems to be more evenly distributed amongst the pectin. The chelator soluble pectin fraction is progressively depolymerised and becomes more polydisperse during ripening. These changes are similar to those occurring in other fruit and are related to the action of wall hydrolases during ripening.     

A DNA glycosylase from Escherichia coli that releases free urea from a polydeoxyribonucleotide containing fragments of base residues.

A poly (dA, [2-14C]dT) copolymer has been synthesized using terminal deoxynucleotidyltransferase. Treatment of the polydeoxyribonucleotide with potassium permanganate converts the thymine residues to urea and N-substituted urea derivatives, while the adenine residues are resistant to oxidation. This damaged polymer has been annealed with an equimolar amount of poly (dT) to generate a double-stranded polydeoxyribonucleotide containing scattered fragmented base residues, which are radioactively labeled selectively. On incubation of the latter with crude cell extracts from E. coli, free urea is released by a DNA glycosylase activity. The enzyme has been partly purified, and appears to be different from previously studied DNA glycosylase. It shows a strong preference for a double-stranded substrate, exhibits no cofactor requirement, and has a molecular weight of 20000 - 25000. Since fragmentation of pyrimidine residues is a major type of base lesion introduced in DNA by exposure to ionizing radiation, it seems likely this DNA glycosylase is active in repair of X-ray-induced lesions.     

Microscopic fat cell size measurements on frozen-cut adipose tissue in comparison with automatic determinations of osmium-fixed fat cells

Diameters of fat cells in adipose tissue slices, floating in an isoosmolar solution, were measured under a microscope. The slices were obtained from percutaneous biopsies by freeze-cutting after brief formaldehyde fixation. All cells in a given part of the slice were measured, thus avoiding selection. A normal distribution of fat cell diameters could be demonstrated with this method, as has been found with previously described methods. The error of the method was 2.6% for diameter and 8.0% for weight determinations. Storage of adipose tissue at 4 degrees C for 48 hr had no effects on cell size determinations. Results with this microscopic method were compared with those obtained from a previously described method for automatic determinations of osmium-fixed fat cells. The latter method was slightly modified by using a viscous electrolyte, which prevented sedimentation of large fat cells, and by using sonication to complete cell separation. The methods agreed closely. A method for calculating mean fat cell weight using osmium-fixed fat cells is described, which makes determinations of sample wet weight and ratio of lipid to wet weight unnecessary.     

Modulation of the induction and circumvention of immunological tolerance to human gamma-globulin by interleukin 1

As with agents capable of causing the release of IL 1, IL 1 itself is capable of modulating certain tolerance-inducing events. Under the condition used in the present study, it previously has been firmly established that injection of A/J mice with DHGG induces a state of antigen-specific tolerance in both T helper (Th) and B cells. The tolerance in the B cell is of long duration, whereas that in the B cell is of shorter duration. Recombinant IL 1 (rIL 1) given shortly after the tolerogen DHGG results in the inhibition of the induction of tolerance resulting in antibody production. The induction of tolerance is inhibited at both its antigen-specific Th cell and B cell levels, although the latter may be caused by the former. The inhibition of the induction of tolerance by rIL 1 is not correlated to the generation of antigen-specific T suppressor cells. IL 1 mimics lipopolysaccharide and 8-bromoguanosine, which generate IL 1 production, in its ability to interfere with the in vivo induction of tolerance. However, in contrast to these latter mitogens which cause both terminal differentiation of B cells and IL 1 production, IL 1 itself does not cause in vivo circumvention of long-term tolerant Th cells in the presence of competent B cells and antigen. These latter findings suggest that a signal(s) in addition to those delivered by IL 1 is required for activation of the B cell compartment recovering from tolerance to antibody production. AHGG (immunogen) is a potent generator of IL 1 release, whereas DHGG has no effect on IL 1 release from macrophages and AHGG inhibits the induction of tolerance by DHGG. These latter results suggest that the lack of an IL 1 signal may be responsible for the deliverance of a tolerogenic rather than an immunogenic signal to the Th cell.     

A template matching model for pattern recognition: Self-organization of templates and template matching by a disinhibitory neural network

A template matching model for pattern recognition is proposed. By following a previouslyproposed algorithm for synaptic modification (Hirai, 1980), the template of a stimulus pattern is selforganized as a spatial distribution pattern of matured synapses on the cells receiving modifiable synapses. Template matching is performed by the disinhibitory neural network cascaded beyond the neural layer composed of the cells receiving the modifiable synapses. The performance of the model has been simulated on a digital computer. After repetitive presentations of a stimulus pattern, a cell receiving the modifiable synapses comes to have the template of that pattern. And the cell in the latter layer of the disinhibitory bitory neural network that receives the disinhibitory input from that cell becomes electively sensitive to that pattern. Learning patterns are not restricted by previously learned ones. They can be subset or superset patterns of the ones previously learned. If an unknown pattern is presented to the model, no cell beyond the disinhibitory neural network will respond. However, if previously learned patterns are embedded in that pattern, the cells which have the templates of those patterns respond and are assumed to transmit the information to higher center. The computer simulation also shows that the model can organize a clean template under a noisy environment.     

Studies on polydisperse systems using an air-driven ultracentrifuge: application to phosphatidylcholine vesicles.

A high-speed air-driven ultracentrifuge (Airfuge) has been used to determine the molecular weight and effective specific volume of phosphatidylcholine vesicles. The method used to determine the effective specific volume involved varying the solution density until zero sedimentation of the vesicles occurred. The value obtained for the effective specific volume of 0.9885 ml/g agrees well with previously reported values. The determination of the molecular weight of the vesicles is based on a method in which the fraction of vesicles remaining in an upper fraction of the solution column is compared with the values obtained using standard proteins. The values obtained for the molecular weight of the vesicles range from 1.7 X 10(6) to 2.3 X 10(6) and are in good agreement with results obtained using the analytical ultracentrifuge and with previously reported results. Possible effects due to the polydispersity of the solute are assessed using theoretical calculations and the possibility of using the Airfuge for the study of other polydisperse systems is discussed.     

Biological invasions of Southern Ocean islands: the Collembola of Marion Island as a test of generalities

It has been suggested previously that the presence and abundance of indigenous species have a marked influence on the likelihood of invasion of a community. It has also been suggested that such biotic resistance has a negligible influence on the outcome of an invasion, but that the abiotic characteristics of the environment being invaded are more important. The latter has been claimed to be especially important on the islands of the Southern Ocean. In order to test these competing hypotheses we examined the distribution and abundance of indigenous and introduced springtails across 13 habitats, which differ considerably in the properties of their soils, and soil temperature, on the eastern quarter of sub‐Antarctic Marion Island. There was no evidence of negative abundance covariation or species associations within habitats, nor were there significant relationships between species richness or abundance of the indigenous as opposed to the introduced collembolans across habitats. Interspecific interactions thus seem to have played no readily identifiable role in the outcome of invasions by Collembola on Marion island. In contrast, the indigenous and introduced species responded very differently to abiotic variables. The indigenous Collembola prefer drier, more mineral soils with a low organic carbon content, and species richness tends to be highest in cold, fellfield areas. On the other hand, the introduced springtails prefer moist, warm sites, with organically enriched soils, introduced species richness was negligible in cold, fellfield areas. Disturbance also appeared to influence positively the species richness and abundance of introduced species at a site. These results provide independent support for the idea that abiotic factors, especially temperature, significantly influence the likelihood of biological invasions on Southern Ocean islands. They also suggest that predicting the outcome of climate change on community structure in this region is likely to be problematic, especially in the case of the Collembola.     

Oblique dipole layer potentials applied to electrocardiology

We study the properties of the potential field generated by an oblique dipole layer. This field arises, for instance, in describing the potential elicited by a depolarization wavefront spreading in the myocardium when a dependence of the potential on the cardiac fiber orientation is introduced. The representation of cardiac bioelectric sources by means of an oblique dipole layer leads to a mathematical structure which generalizes the classical solid angle theory used in electrocardiology, which has been challenged by recent experimental evidence, and links models previously proposed with a view to adequately reproduce the potential observed in experiments. We investigate also the relationship between our model and an intracellular current model and we derive potential jump formulae for some models which account for the anisotropic structure of the myocardium. The potential generated by an oblique dipole layer is considered both for unbounded and bounded domains. In the latter case an integral boundary equation is derived and we study its solvability. A numerical procedure for solving this integral equation by means of the finite element method with collocation is outlined.     

Restriction analysis of chromosomal sequences homologous to single-copy fragments cloned from small polydisperse circular DNA (spcDNA)

Summary Restriction fragments from the fraction of small polydisperse circular DNA (spcDNA) were cloned in pBR322. The spcDNA was prepared from cell cultures derived from an angiofibroma of a patient with tuberous sclerosis (TS). Such cultures have been shown previously to contain increased amounts of spcDNA. Four cloned spcDNA fragments containing single-copy sequences were chosen to characterize the homologous chromosomal DNA segments by restriction analysis. When used as hybridization probes, these four fragments generate well-defined nonvariable patterns in the chromosomal DNA from healthy donors. The restriction patterns obtained with one of the fragments (D-C4) can best be interpreted by assuming the presence of two copies of the homologous sequences in chromosomal DNA. A second sequence, A-B4, occurs at least 30–50 times in the haploid human genome. In both cases the duplicated regions span relatively large segments of DNA.     

Presence of a large β(1-3)glucan linked to chitin at the Saccharomyces cerevisiae mother-bud neck suggests involvement in localized growth control

Previous results suggested that the chitin ring present at the yeast mother-bud neck, which is linked specifically to the nonreducing ends of β(1-3)glucan, may help to suppress cell wall growth at the neck by competing with β(1-6)glucan and thereby with mannoproteins for their attachment to the same sites. Here we explored whether the linkage of chitin to β(1-3)glucan may also prevent the remodeling of this polysaccharide that would be necessary for cell wall growth. By a novel mild procedure, β(1-3)glucan was isolated from cell walls, solubilized by carboxymethylation, and fractionated by size exclusion chromatography, giving rise to a very high-molecular-weight peak and to highly polydisperse material. The latter material, soluble in alkali, may correspond to glucan being remodeled, whereas the large-size fraction would be the final cross-linked structural product. In fact, the β(1-3)glucan of buds, where growth occurs, is solubilized by alkali. A gas1 mutant with an expected defect in glucan elongation showed a large increase in the polydisperse fraction. By a procedure involving sodium hydroxide treatment, carboxymethylation, fractionation by affinity chromatography on wheat germ agglutinin-agarose, and fractionation by size chromatography on Sephacryl columns, it was shown that the β(1-3)glucan attached to chitin consists mostly of high-molecular-weight material. Therefore, it appears that linkage to chitin results in a polysaccharide that cannot be further remodeled and does not contribute to growth at the neck. In the course of these experiments, the new finding was made that part of the chitin forms a noncovalent complex with β(1-3)glucan.     

Homologues of c-hairy1 (her9) and lunatic fringe in zebrafish are expressed in the developing central nervous system, but not in the presomitic mesoderm

A number of genes that are involved in somitogenesis in vertebrates are cyclically expressed in the presomitic mesoderm. These include homologues of the Drosophila genes fringe and hairy. We have analysed here two genes that belong to these classes in the zebrafish, namely the apparent orthologues of lunatic fringe (l-fng) and of c-hairy1 (called her9). However, unlike the respective mouse and chicken genes, they are not expressed cyclically in the presomitic mesoderm. Instead, both genes are mainly expressed in the central nervous system. her9 is predominantly expressed in the fore- and midbrain, and transiently in the hindbrain. Thus, the previously identified and only very distantly related her1 gene of zebrafish has more similarities to the expression of the c-hairy1 gene than its apparent orthologue her9, indicating that sequence similarity and similarity of function are not necessarily linked in this case. l-fng expression is found in alternating pre-rhombomeres, comparable to the equivalent mouse gene expression and in the anterior compartments of the mature somites, which was also shown for the chicken l-fng gene. The latter expression indicates that it might be involved in boundary definition and cell fate decision processes, rather than in pre-patterning of the somites. Interestingly, a similar role has previously been inferred for the grasshopper homologue of l-fng. This suggests that the function of l-fng in boundary definition of the somites might be ancestral, while its recruitment to the pre-patterning process of the somites might be a derived feature in higher vertebrates.     

Actin protofilament orientation at the erythrocyte membrane.

The short actin filaments in the erythrocyte's membrane skeleton are shown to be largely oriented tangent to the lipid bilayer. Actin "proto"-filaments have previously been described as junctional centers intertriangulated by spectrin; however, the protofilaments may simultaneously serve as pinning centers between the network and the overlying bilayer. The latter function now seems of particular importance because near-normal network assembly has been reported with transgenic mouse sphero-erythrocytes that lack the primary linkage protein Band 3. To assess possible physical constraints on actin protofilaments in intact membranes, fluorescence polarization microscopy (FPM) has been used to study rhodamine phalloidin-labeled red cell ghosts. A basis for interpreting FPM images of cells is provided by FPM applied to isolated actin filaments. These are labeled with the same rhodamine probes and imaged at various orientations with respect to the polarizers, including filament orientations perpendicular to the image plane. High aperture and fluorophore conjugation effects are found to be minimal, enabling development of a simple, semi-empirical model which indicates that protofilaments are generally within approximately 20 degrees of the membrane tangent plane.     

Subcellular distribution and properties of poly(A)-containing RNA from cultured plant cells.

Cultured sycamore cells rapidly incorporate [3H]uridine or [32P]orthophosphate into rRNA precursors and polydisperse RNA. Mature rRNA accumulates only after a lag period of approximately 40 min. Fractionation of pulse-labelled cells and analysis of the RNA shows that after 30 min the rRNA precursors, together with some polydisperse RNA, are confined to the nucleus. In consequence radioactive polydisperse RNA can be isolated from polyribosomes in the complete absence of labelled rRNA. Approximately 40% of this RNA is retained by an oligo(dT)-cellulose column and by this criterion is judged to contain poly(A) sequences. A smaller proportion of nuclear polydisperse RNA also contains poly(A). The tendency for poly(A)-containing RNA to aggregate complicates molecular weight determinations. Denaturation of poly(A)-containing RNA in 8 M urea prior to gel electrophoresis produces a broad peak of RNA with an average Mr = 10(6). Analysis of the nucleotide composition of total cell poly(A)-containing RNA shows that it contains 41% AMP. Roughly 6% of this RNA is resistant to digestion by ribonuclease A and T1. AMP is the only nucleotide detectable in these fragments. From their mobility during electrophoresis in 8 M urea at 60 degrees C with 5.8-S, 5-S and tRNA as molecular weight markers it is concluded that the poly(A) regions contain an average of 160 nucleotides.     

Free fatty acids and esters can be immobilized by receptor rich membranes from Torpedo marmorata but not phospholipid acyl chains.

A long chain spin labeled fatty acid and the corresponding ester have been introduced into receptor rich membranes from Torpedo Marmorata. Superimposed to a mobile component, typical of the lipid phase, a strongly immobilized component is seen on the ESR spectra, both at low temperature (?4°C) and at room temperature. An estimation of the amount of immobilized signal as a function of the concentration of spin label in the membrane shows that a saturation is reached which corresponds to approximately twice the concentration of receptor protein. In the same membranes, a spin labeled phosphatidylcholine was introduced by the release of the phosphatidylcholine analog from purified phosphatidylcholine exchange protein, preloaded with this spin label. No immobilized component is seen in this latter case even at low temperatures. Therefore the immobilized component seen with the fatty acid cannot be considered as reporting on an immobilized boundary layer of phospholipids surrounding the proteins. We attribute the immobilized signal seen with fatty acids and esters to a particular interaction of amphiphilic molecules with the cholinergic receptor protein. Very likely this effect can be associated with the local anaesthetic effect detected previously with this fatty acid.