Free Edges in Epithelial Cell Sheets Stimulate Epidermal Growth Factor Receptor Signaling

The ability of epithelia to migrate and cover wounds is essential to maintaining their functions as physical barriers. Wounding induces many cues that may affect the transition to motility, including the immediate mechanical perturbation, release of material from broken cells, new interactions with adjacent extracellular matrix, and breakdown of physical separation of ligands from their receptors. Depending on the exact nature of wounds, some cues may be present only transiently or insignificantly. In many epithelia, activation of the epidermal growth factor receptor (EGFR) is a central event in induction of motility, and we find that its continuous activation is required for progression of healing of wounds in sheets of corneal epithelial cells. Here, we examine the hypothesis that edges, which are universally and continuously present in wounds, are a cue. Using a novel culture model we find that their presence is sufficient to cause activation of the EGFR and increased motility of cells in the absence of other cues. Edges that are bordered by agarose do not induce activation of the EGFR, indicating that activation is not due to loss of any specific type of cell–cell interaction but rather due to loss of physical constraints.     

Interactions between actin and myosin filaments in skeletal muscle visualized in frozen-hydrated thin sections.

For the purpose of determining net interactions between actin and myosin filaments in muscle cells, perhaps the single most informative view of the myofilament lattice is its averaged axial projection. We have studied frozen-hydrated transverse thin sections with the goal of obtaining axial projections that are not subject to the limitations of conventional thin sectioning (suspect preservation of native structure) or of equatorial x-ray diffraction analysis (lack of experimental phases). In principle, good preservation of native structure may be achieved with fast freezing, followed by low-dose electron imaging of unstained vitrified cryosections. In practice, however, cryosections undergo large-scale distortions, including irreversible compression; furthermore, phase contrast imaging results in a nonlinear relationship between the projected density of the specimen and the optical density of the micrograph. To overcome these limitations, we have devised methods of image restoration and generalized correlation averaging, and applied them to cryosections of rabbit psoas fibers in both the relaxed and rigor states. Thus visualized, myosin filaments appear thicker than actin filaments by a much smaller margin than in conventional thin sections, and particularly so for rigor muscle. This may result from a significant fraction of the myosin S1-cross-bridges averaging out in projection and thus contributing only to the baseline of projected density. Entering rigor incurs a loss of density from an annulus around the myosin filament, with a compensating accumulation of density around the actin filament. This redistribution of mass represents attachment of the fraction of cross-bridges that are visible above background. Myosin filaments in the "nonoverlap" zone appear to broaden on entering rigor, suggesting that on deprivation of ATP, cross-bridges in situ move outwards even without actin in their immediate proximity.     

Chromosome distribution of intracisternal A-particle sequences in the Syrian hamster and mouse

Metaphase chromosomes of Syrian hamster and BALB/c mice were hybridized in situ with radiolabeled probes derived from cloned intracisternal A-particle (IAP) genes of the corresponding species. The DNAs of these species are known to contain about 900 and 1,000 copies, respectively, of the retrovirus-like IAP sequence elements per haploid genome. Multiple IAP sequences were found on all chromosomes of both hamster and mouse. In the hamster, more than half of the IAP sequences were located in regions of non-centromeric constitutive heterochromatin, at an average concentration per unit chromosome length 5 times greater than in the euchromatic regions. The other dispersed sequences showed marked local variations in concentration along the chromosome lengths; both discrete foci and large grain clusters were observed as well as regions apparently lacking IAP sequences. Within the resolution of the techniques, IAP sequences appeared to be more evenly distributed over the mouse chromosomes; however, some prominent variations in concentration were seen. The number of potentially active IAP genes in the Syrian hamster, and by extension in the mouse, may be restricted by the preferential location of IAP sequences in genetically inert regions of the genome.     

Distribution of mass in relaxed frog skeletal muscle and its redistribution upon activation.

Five orders of equatorial reflection were recorded from both relaxed and fully activated intact frog sartorius muscle using synchrotron x-ray radiation. Electron density maps of the myofilament lattice in axial projection were calculated from the integrated intensities by Fourier synthesis, using all possible phase combinations. These maps were evaluated systematically in terms of their compatibility with electron microscopically and biochemically derived properties of the lattice structure and with the minimum wavelength principle. For the relaxed state, one phase combination emerged as most consistent with these constraints: it shows a thick filament with a compact core surrounded by an annular shell of density. The distribution of mass suggests that the S-2 moiety of the myosin molecule is an integral part of the thick-filament backbone and the S-1 moiety makes up the shell and is tilted or slewed around the backbone. For the active state, there are two feasible maps, which differ according to whether or not the activation process is associated with phase inversion in two of the reflections. Both maps represent patterns of redistribution of mass upon activation in which the thick-filament backbone is practically unaffected and there is movement of density from the annular shell towards the thin filaments. In addition to this outward radial flux of density from the thick-filament periphery, the pattern of net mass transfer involves a pronounced azimuthal component in both cases. The total net mass transfer is equivalent to approximately 20% (no phase change) or approximately 40% (with phase change) of the S-1 mass. From the observed systematic increase in peak widths of the higher orders, the size of the crystalline domain in the myofilament lattice in the relaxed sartorius is estimated to be greater than 650 nm and the variations in myofilament lattice spacing among different myofibrils to be about +/- 3%. Furthermore, in the activated state, the equilibrium positions of the myofilaments are no longer well ordered, but are distributed statistically about the lattice points with a standard deviation of approximately 3 nm.     

Oligosaccharide structures of human colonic mucin

Purified human colonic mucin was separated into six distinct components by DEAE-cellulose chromatography, and the structures of oligosaccharide side chains from the three most abundant species were determined. Oligosaccharide side chains were isolated from colonic mucin species III, IV, and V after alkaline borohydride reductive cleavage in the presence of sodium borotritide. After initial separation of acidic and neutral oligosaccharides by ion exchange chromatography, individual oligosaccharides were isolated by sequential chromatography on Bio-Gel P-4 and Bio-Gel P-2 resins followed by preparative normal phase high performance liquid chromatography. Composition and structure of individual oligosaccharides were determined by combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Collectively, 21 discrete oligosaccharide structures were identified in the major human colonic mucin species including 10 acidic oligosaccharides and 11 neutral structures which ranged in size from 2 to 12 sugar residues. Although detailed structures were defined for each oligosaccharide, the majority of the structures identified were variations of a relatively small number of "basic" structures, and several generalizations pertained. First, many oligosaccharides represented variations of a biantennary structure in which branch chains arise in N-acetylglucosaminyl residues linked to C3 and C6 of a galactosyl residue linked in turn to a GlcNAc beta (1-3)GalNAc core; second, non-branched oligosaccharides appeared to be linear chain derivatives of the same core structure; third, all acidic oligosaccharides could be derived from neutral structures present in the mucin species; fourth, sialic acid substitution was limited to few sites and always included substitution in alpha 2-6 linkage to the reducing terminal N-acetylgalactosamine, and finally several structures contained both sialic acid and fucose residues. Individually, mucin species III, IV, and V were found to contain unique mixtures of 13, 14, and 10 oligosaccharide structures, respectively. These data demonstrate that human colonic mucin contain a wide range of oligosaccharides reflecting variations of common core oligosaccharide structures. The major chromatographically defined constituents of normal colonic mucin appear to possess characteristic and distinguishable combinations of oligosaccharide structures. These findings support the concept that colonic mucin contains structurally and functionally distinct subpopulations.     

X-ray diffraction evidence for cross-bridge formation in relaxed muscle fibers at various ionic strengths

Equatorial x-ray diffraction patterns from single skinned rabbit psoas fibers were studied at various ionic strengths to obtain structural information regarding cross-bridge formation in relaxed muscle fibers. At ionic strengths between 20 and 50 mM, the intensity of the 11 reflection, I11, of the relaxed state was close to that of the rigor state, whereas the intensity of the 10 reflection, I10, was approximately twice that of rigor reflection. Calculations by two-dimensional Fourier synthesis indicated that substantial extra mass was associated with the thin filaments under these conditions. With increasing ionic strength between 20 and 100 mM, I10 increased and I11 decreased in an approximately linear way, indicating net transfer of mass away from the thin filaments towards the thick filaments. These results provided evidence that cross-bridges were formed in a relaxed fiber at low ionic strengths, and that the number of cross-bridges decreased as ionic strength was raised. Above mu = 100 mM, I10 and I11 both decreased, indicating the onset of increasing disorder within the filament lattice.     

A survey of toxicity of cyanobacterial blooms in Lake Ladoga and adjacent water bodies

Twentyfive cyanobacterial blooms in Lake Ladoga and adjacent water bodies were studied in the summer of 1990–1992. Toxicity of the water bloom material for mice was detected in 9 cases. The maximal tolerable doses (MTD) of the material extracted from biomass varied within 3–30 mg kg^–1 mouse body weight; 50% lethal doses (LD₅₀) were within 45–125 mg kg^–1. Toxic water blooms were registered in Karelian lakes and in the Neva Bay, Gulf of Finland. Cyanobacterial samples collected on the eastern coast of Lake Ladoga proved to be non-toxic. The species identified in toxic bloom material included Anabaena circinalis, A. flos-aquae, A. lemmermannii, Anabaena sp., Aphanizomenonflos-aquae, Gloeotrichia echinulata, G. pisum, Microcystis aeruginosa and Oscillatoria sp. These data suggest that toxic forms of cyanobacteria are widespread in Karelian lakes belonging to the drainage basin of Lake Ladoga.     

Frank Macfarlane Burnet and the immune self

This essay is excerpted from Alfred I. Tauber,The Immune Self: Theory or Metaphor? (New York and Cambridge: Cambridge University Press, 1994).     

REPRODUCTIVE ISOLATION BY HOST SPECIFICITY IN A PAIR OF PHYTOPHAGOUS LADYBIRD BEETLES

Crossing experiments and food-choice tests show that two sympatric species of phytophagous ladybird beetles, Epilachna niponica and E. yasutomii, are reproductively isolated by host-plant specificity. Adult beetles selected their natural hosts when given choices, though some accepted the host of the other species when no choice was offered. In each species, survival of larvae to the second instar was significantly higher on their own host plant. No evidence for sexual isolation, gametic isolation, hybrid inviability, or reduced hybrid fertility was detected. Reproductive isolation by host specificity is an important prerequisite for certain models of sympatric speciation. Although the present example supports the plausibility of such models, an allopatric origin of host-plant specificity cannot be discounted.