Electron microscopic observations on isolated mitochondrial membranes,prepared by various histological methods

Summary The pictures of isolated mitochondrial membranes, as seen on the electron-microscope, depend very much on the method of specimen preparation. Subunits of linear dimensions of about 25 m, (electron transport particles) are observed in carbon-replicas of the membranes and in specimens treated with trypsin or pepsin (0.02% for 30 mins) and shadowed with platinum. A three-layered structure of the unit membrane is seen in sections of specimens fixed with osmium tetroxide or formalin followed by post-fixation with osmium tetroxide. But fixation with potassium permanganate or with formalin, followed by post-fixation with potassium permanganate reveals an electron-dense globular structural element in the unit membrane. An electron-transparent ultrastructural element of the unit membrane is observed after treatment with trypsin (0.2% for 5 mins) and fixation with osmium tetroxide. Unsectioned specimens treated with 0.02% trypsin for 30 mins show a honeycomb-like structure of the membrane. Thus, part of the results appear to support the concept of a mosaic-like structure of the unit membrane, whereas other results are in agreement with the classical concept of a three-layered structure.The authors wish to express their gratitude to Dr. Sina Rosenthal, Department of Physiological Chemistry, Humboldt University, Berlin, who prepared the isolated membranes, to Mr. E. Fischer, Head Technician of the Department of Electron Microscopy, Greifswald University, who took most of the electron micrographs, to Mr. G. Bartsch, Department of Electron Microscopy, Greifswald University, and especially to Prof. W. Bargmann and to Doz. E. Lindner, Department of Anatomy, Kiel University, for many valuable suggestions.     

STUDIES ON SHELL FORMATION : IX. An Electron Microscope Study of Crystal Layer Formation in the Oyster

Details of crystal growth in the calcitostracum of Crassostrea virginica have been studied with the purpose of analyzing the formation of the overlapping rows of oriented tabular crystals characteristic of this part of the shell. Crystal elongation, orientation, and dendritic growth suggest the presence of strong concentration gradients in a thin layer of solution in which crystallization occurs. Formation of the overlapping rows can be explained by three processes observed in the shell: a two-dimensional tree-like dendritic growth in which one set of crystal branchings creeps over an adjacent set of branchings; three-dimensional dendritic growth; and growth by dislocation of crystal surfaces. Multilayers of crystals may thus be formed at one time. This is favored by infrequent secretion of a covering organic matrix which would inhibit crystal growth. The transitional zone covering the outer part of the calcitostracum and the inner part of the prismatic region is generally characterized by aggregates of small crystals with definite orientation. Growth in this zone appears to take place in a relatively homogeneous state of solution without strong concentration gradients. Thin membranes and bands of organic matrix were commonly observed in the transitional zone bordering the prismatic region. The membrane showed a very fine oriented network pattern.     

How does inactivation change timing of replication in the human X chromosome?

Summary The kinetics of replication of the inactive (late replicating) X chromosome (LRX) were studied in karyotypically normal lymphocytes and human amniotic fluid cells. Both cell types were successively pulse labeled with 1-h or 1/2-h thymidine pulses in an otherwise BrdU-substituted S phase after partial synchronization of the cultures at G₁/S. For the first time with this technique, the entire sequence of replication was analyzed for the LRX from the beginning to the end of the S phase, with special reference to mid S (R-band to G-band transition replication). The inactive X is the last chromosome of the metaphase to start replication, with a delay of 1 or 2h, after which time a thymidine pulse results in R-type patterns. In mid S, the inactive X is the first chromosome to switch to G-type replication (without overlapping of both types and without any detectable replication pause). Until the end of S, a thymidine pulse results in G-type patterns. To rule out artifacts that might arise by the synchronization of cultures in these experiments, controls were carried out with BrdU pulses and the BrdU antibody technique without synchronization. In the course of replication, no fundamental difference was seen between the two different cell types examined. In contrast to studies using continuos labeling, this study did not reveal an interindividual difference of replication kinetics in the LRXs of the seven individuals studied; thus it is concluded that the inactive X chromosome shows only one characteristic course of replication.     

Molecular evolution of pteridophytes and their relationship to seed plants: Evidence from complete 18S rRNA gene sequences

Complete 18S ribosomal RNA sequence data from representatives of all extant pteridophyte lineages together with RNA sequences from different seed plants were used to infer a molecular phylogeny of vascular plants that included all major land plant lineages. The molecular data indicate that lycopsids are monophyletic and are the earliest diverging group within the vascular land plants, whereasPsilotum nudum is more closely related to the seed plants than to other pteridophyte lineages. The phylogenetic trees based on maximum likelihood, parsimony and distance analyses show substantial agreement with the evolutionary relationships of land plants as interpreted from the fossil record.     

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B in associated with distinct changes in F-actin and ZO-1

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 M) and higher concentrations (1.5 M and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 g/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.