Ross G. Johnson

The timing of high sea levels over the past 200,000 years.

The 230Th ages and 234U/238U ratios were determined for Barbados corals that grew during periods of high sea level within the last 200,000 years. The similarity of the initial 234U/238U ratios of some of the corals to the modern marine value suggests that these samples are pristine and that the marine 234U/238U ratio 83,000 and 200,000 years ago was within 2 per mil of the modern value. The accuracies of the 230Th ages are evaluated on the basis of the 234U/238U values and a model of the behavior of uranium and thorium isotopes during diagenesis. For the last three interglacial and two intervening interstadial periods, sea level peaked at or after peaks in summer insolation in the Northern Hemisphere. This overall pattern supports the idea that glacial-interglacial cycles are caused by changes in Earths orbital geometry. The sea-level drop at the end of the penultimate interglacial, the last interglacial, and a subsequent interstadial period lagged behind the decrease in insolation by 5,000 to 10,000 years.

Ser364 of connexin43 and the upregulation of gap junction assembly by cAMP

The assembly of gap junctions (GJs) is a process coordinated by growth factors, kinases, and other signaling molecules. GJ assembly can be enhanced via the elevation of cAMP and subsequent stimulation of connexon trafficking to the plasma membrane. To study the positive regulation of GJ assembly, fibroblasts derived from connexin (Cx)43 knockout (KO) and wild-type (WT) mice were transfected with WT Cx43 (WTCx43) or mutant Cx43. GJ assembly between untransfected WT fibroblasts or stably transfected WTCx43/KO fibroblasts was increased two- to fivefold by 8Br-cAMP, and this increase could be blocked by inhibition of cAMP-dependent protein kinase (PKA) or truncation of the Cx43 COOH terminus (CT). Although serine 364 (S364) of the Cx43 CT was determined to be a major site of phosphorylation, the molar ratio of Cx43 phosphorylation was not increased by 8Br-cAMP. Importantly, GJ assembly between either S364ECx43/KO or S364ECx43/WT fibroblasts was stimulated by 8Br-cAMP, but that between S364ACx43/KO or S364PCx43/KO fibroblasts was not stimulated, indicating that phosphorylation or a negative charge at S364 is required for enhancement of GJ assembly by cAMP. Furthermore, GJ assembly between S364ACx43/WT fibroblasts could be stimulated by 8Br-cAMP, but could not be between S364PCx43/WT fibroblasts. Thus, S364PCx43 interferes with enhanced GJ assembly when coexpressed with WTCx43.

Chicken embryo lens cultures mimic differentiation in the lens

Embryonic chicken lenses, which had been disrupted by trypsin, were grown in culture. These cultures mimic lens development as it occurred in vivo, forming lens-like structures known as lentoids. Using a variety of techniques including electron microscopic analysis, autoradiography, immunofluorescence, and polyacrylamide gel electrophoresis, it was shown that the lentoid cells had many characteristics in common with the differentiated cells of the intact lens, the elongated fiber cells. These characteristics included a shut off of DNA synthesis, a loss of cell organelles, an increase in cell volume, an increase in delta-crystallin protein, and the development of extensive intercellular junctions. The cultures began as a simple epithelial monolayer but then underwent extensive morphogenesis as they differentiated. This morphogenesis involved three distinctive morphological types which appeared in sequence as an epithelial monolayer of polygonal shaped cells with pavement packing, elongated cells oriented end to end, and the multilayered, multicellular lentoids. These distinct morphological stages of differentiation in culture mimic morphogenesis as it occurs in the lens.

Formation of low-resistance junctions in vitro in the absence of protein synthesis and ATP production☆

Abstract Formation of low-resistance junctions between Novikoff hepatoma cells was studied in the presence of inhibitors of protein synthesis or energy production. Cells were dissociated for 30 min in the presence of cycloheximide (CHX, 100 μg/ml), reaggregated by pelleting, and incubated for 60 min in the presence of CHX. This treatment (which inhibited protein synthesis by 99% over 30 min and prevented cell growth over 12 h) produced no significant difference in % of coupled cells or in coupling coefficient between control (0.346 ± 0.030) and treated cells (0.297 ± 0.034). Cells were treated for 12 h in CHX before dissociation and reaggregation with some decrease in % of coupled cells, but with no substantial reduction in coupling coefficient (0.298 ± 0.057). Multiple dissociations (and reaggregations) of the cells in the presence of CHX produced a decrease in 1 coupled cells and a slight reduction in coupling coefficients between treated (0.234 ± 0.029) and control preparations (0.312 ± 0.035). Cells were dissociated, treated with iodoacetate (IAA, 1 mM) for 30 min and reaggregated for 30 min. There was a small reduction in % coupled cells. Also the mean coupling coefficient was lower in treated cells (0.203 ± 0.041) compared to controls (0.393 ± 0.047), but this may reflect a general lowering of non-junctional membrane resistance. IAA resulted in a reduction of ATP to 5% of the control values. Thus, neither inhibition of protein synthesis nor severe reduction of ATP levels prevent or substantially reduce junction formation by Novikoff cells.

An activator of protein kinase C inhibits gap junction communication between cultured bovine lens cells

Currently little is known about the regulation of gap junction communication in the lens. We report here on the effects of the protein kinase C activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on cultured bovine lens cells which appeared to be epithelial in nature. Dramatically reduced intercellular transfer of the fluorescent dye Lucifer yellow was observed when the cultured lens cells were treated with octanol, a known inhibitor of gap junction communication. TPA (4 beta isomer) was also shown to reduce intercellular permeability within these cultures. In contrast, an inactive form of TPA, 4 alpha-TPA, did not decrease dye transfer. Permeability was evaluated in terms of both the number of cells receiving dye and the rate of decrease in fluorescence intensity in the injected cell. The maximum decreases in dye transfer occurred at 2 h of TPA treatment and dye transfer gradually increased to control levels over a time course of many hours. Incubation of cultures with 32Pi and immunoprecipitation using antibodies to the N- and C-terminal regions of connexin43 demonstrated a gap junction phosphoprotein of 43,000 Da. Phosphorylation of connexin43 increased during the first 2 h of TPA treatment. These results suggest that protein kinase C has a direct or indirect effect on gap junction communication in cultured lens cells.

Lipids in gap junction assembly and function

Gap junctions (GJ) are important regulators of cellular function. They provide channels for the direct movement of small molecules between cells and thus control cell-to-cell transfer of metabolites and the transmission of various stimuli. Gap junctions have been shown to be involved in a multitude of cellular processes ranging from cell synchronization and neuronal function to cell differentiation and carcinogenesis. Much knowledge has been gained in recent years concerning the structure and molecular organization of GJ proteins; yet, the mechanisms that control and modulate gap junction assembly and function are still not well understood. Although it is quite apparent that the GJ proteins assemble in the lipid milieu of the plasma membrane, and that the cluster of proteins assembled in the junction do function in a lipid environment, there is a general paucity of information on the role of lipids in the gap junction assembly process and in the function of gap junctions.The present review is a comprehensive account of current knowledge on gap junction lipids. We also discuss what is known to date on the involvement of lipids in gap junction formation. Special emphasis is being placed on the potential role of membrane cholesterol in gap junction assembly and function.

Homocellular and heterocellular gap junctions in Limulus: A thin-section and freeze-fracture study

Gap junctions between three different cell combinations in Limulus are studied with thin-section, lanthanum-tracer, and freeze-fracture techniques. Midgut epithelial (E) cells form E—E gap junctions. Reserve (R) cells adjacent to the outer surface of the midgut form R—R gap junctions. E—R junctions are formed when R-cell processes extend through a basement membrane and contact E-cells. All junctions possess parallel membranes with 30 A gaps, polygonal substructure when treated with lanthanum and sectioned en face , and electron-dense 25 A dots in subunit centers. In freeze-fracture replicas only E—E and R—R junctions can be identified. All junctions possess aggregates of 125 A particles in each membrane. Particles are observed only on outer fracture faces and, like the complementary pits, are not highly patterned. In both particle centers and pits, 30 A spots of platinum are seen. The substructure of gap junctions between cells of the same type appears indistinguishable from that between different cell types.

Enhanced gap junction formation with LDL and apolipoprotein B

Gap junctions are plasma membrane specializations involved in direct cell-cell communication. Intercellular communication is dependent upon the assembly of gap junction structures and would be influenced by agents which alter the assembly process. We investigated the effects of low density lipoprotein (LDL) on gap junction assembly between cultured Novikoff cells using quantitative dye transfer and freeze-fracture electron microscopic methods. We observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms/ml) and a sixfold increase in the number of aggregated gap junction particles per cell. Immunoblots of Novikoff cells probed with anti-connexin43 antibody revealed no detectable increase in gap junction protein (connexin) levels. The influence of the different components of LDL on junction formation was also examined. First, we treated cells with cholesterol (0-150 microM) in serum-free BSA media and observed a decrease in junction assembly. Second, we added apolipoprotein-B (apo-B) in phosphatidyl choline vesicles to the cells and observed a concentration-dependent increase in dye transfer (maximum effect at 2.5 micrograms protein/ml) and a fivefold increase in the number of aggregated gap junction particles per cell. The addition of phosphatidyl choline vesicles without apo-B had no effect on gap junction formation. Thus, we demonstrated that gap junction assembly can be modulated by LDL and apo-B treatments.

Gap junctions and rhombic particle arrays in planaria

Freshwater planaria ( Dugesia ) were found to have three distinct forms of gap junctions. The three forms differ from one another in morphology and in the kinds of cells that they are found between. Utilizing freeze-fracture and lanthanum tracer preparations, the three types were recognized by differences in the spacing and pattern of their particles or subunits and in the membrane faces to which the particles adhere. One of the three types is associated with gastrodermal, parenchymal, and excretory cells; another is found on muscles and nerves; and the third is localized apparently on secretory cells. The possibility of the three junctional forms being correlated with functional differences is discussed. Muscle and nerve cells also have a nonjunctional type of specialization consisting of intramembranous particles arranged in a rhombic pattern.

Arrangement of MP26 in lens junctional membranes: Analysis with proteases and antibodies

SummaryThe major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21–22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2–3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately fourfifths of the primary sequence “protected” by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.