Intermediate filament expression and lifespan potential in human somatic cell hybrids

Summary Limited lifespan human diploid fibroblast cells have been fused with the HeLa derived cell line HEB 7A which possesses transformed growth characteristics and unlimited division potential. HEB 7A expresses keratin intermediate filaments, while the fibroblast cells express only vimentin intermediate filaments. Independently arising clones of hybrids were examined for the presence of keratin by indirect immunofluorescence. Of 11 limited lifespan hybrids, all were keratin negative and possessed the growth characteristics of the fibroblast parent. Of 8 transformed hybrids, 6 arising early after fusion and 2 arising late, all were keratin-positive and simultaneously expressed the transformed growth characteristics of loss of density dependent growth inhibition, low serum dependence, and anchorage independence. It is concluded that the growth properties of these hybrids are associated with the type of intermediate filament expressed. The intermediate filament expression is therefore a marker of proliferative potential in these hybrids. This work was supported by grant no. AG 02664 from NIA (to C.L.B.) and by grant nos. 1R01 HD 18129-01 from NIH and PCM83-09068 from NSF (to R.H.S.). Editor’s Statement The tight correlation between the expression of the intermediate filaments of the immortal parent in hybrids of limited lifespan fibroblasts and HeLa cells with the transformed phenotype is of interest. It may offer important clues to the mechanism involved in cellular senescence. Gordon H. Sato     

Electrical and adaptive properties of rod photoreceptors in bufo marinus. I. Effects of altered extracellular Ca(2+) levels

The effects of altering extracellular Ca(2+) levels on the electrical and adaptive properties of toad rods have been examined. The retina was continually superfused in control (1.6 mM Ca(2+)) or test ringer’s solutions, and rod electrical activity was recorded intracellularly. Low-calcium ringer’s (10(-9)M Ca(2+)) superfused for up to 6 min caused a substantial depolarization of the resting membrane potential, an increase in light-evoked response amplitudes, and a change in the waveform of the light-evoked responses. High Ca(2+) ringer’s (3.2 mM) hyperpolarized the cell membrane and decreased response amplitudes. However, under conditions of either low or high Ca(2+) superfusion for up to 6 min, in both dark-adapted and partially light-adapted states, receptor sensitivity was virtually unaffected; i.e., the V-log I curve for the receptor potential was always located on the intensity scale at a position predicted by the prevailing light level, not by Ca(2+) concentration. Thus, we speculate that cytosol Ca(2+) concentration is capable of regulating membrane potential levels and light-evoked response amplitudes, but not the major component of rod sensitivity. Low Ca(2+) ringer’s also shortened the period of receptor response saturation after a bright but nonbleaching light flash, hence accelerating the onset of both membrane potential and sensitivity recovery during dark adaptation.

Exposure of the retina to low Ca(2+) (10(-9)M) ringer’s for long periods (7-15 min) caused dark-adapted rods to lose responsiveness. Response amplitudes gradually decreased, and the rods became desensitized. These severe conditions of low Ca(2+) caused changes in the dark-adapted rod that mimic those observed in rods during light adaptation. We suggest that loss of receptor sensitivity during prolonged exposure to low Ca(2+) ringer’s results from a decrease of intracellular (intradisk) stores of Ca(2+); i.e., less Ca(2+) is thereby released per quantum catch.

     

Growth and cell survival are unevenly impaired in pixie mutant wing discs

It is largely unknown how growth slows and then stops in vivo. Similar to most organs, Drosophila imaginal discs undergo a fast, near-exponential growth phase followed by a slow growth phase before final target size is reached. We have used a genetic approach to study the role of an ABC-E protein, Pixie, in wing disc growth. pixie mutants, like mutants in ribosomal proteins genes (known as Minutes), show severe developmental delay with relatively mild alterations in final body size. Intriguingly, pixie mutant wing imaginal discs show complex regional and temporal defects in growth and cell survival that are compensated to result in near-normal final size. In S2 cells, Pixie, like its yeast homolog RLI1, is required for translation. However, a comparison of the growth of eukaryotic translation initiation factor eIF4A and pixie mutant clones in wing discs suggests that only a subset of translation regulators, including pixie, mediate regional differences in growth and cell survival in wing discs. Interestingly, some of the regional effects on pixie mutant clone growth are enhanced in a Minute background. Our results suggest that the role of Pixie is not merely to allow growth, as might be expected for a translation regulator. Instead, Pixie also behaves as a target of putative constraining signals that slow disc growth during late larval life. We propose a model in which a balance of growth inhibitors and promoters determines tissue growth rates and cell survival. An alteration in this balance slows growth before final disc size is reached.     

Is insulin an anabolic agent in bone? Dissecting the diabetic bone for clues

Diabetic osteoporosis is increasingly recognized as a significant comorbidity of type 1 diabetes mellitus. In contrast, type 2 diabetes mellitus is more commonly associated with modest increases in bone mineral density for age. Despite this dichotomy, clinical, in vivo, and in vitro data uniformly support the concept that new bone formation as well as bone microarchitectural integrity are altered in the diabetic state, leading to an increased risk for fragility fracture and inadequate bone regeneration following injury. In this review, we examine the contribution that insulin, as a potential anabolic agent in bone, may make to the pathophysiology of diabetic bone disease. Specifically, we have assimilated human and animal data examining the effects of endogenous insulin production, exogenous insulin administration, insulin sensitivity, and insulin signaling on bone. In so doing, we present evidence that insulin, acting as an anabolic agent in bone, can preserve and increase bone density and bone strength, presumably through direct and/or indirect effects on bone formation.     

The Tyrosine Phosphorylation and Cytoskeletal Translocation of Phospholipase Cγ1 in Bovine Adrenal Medullary Chromaffin Cells

The tyrosine phosphatase inhibitor BpV(phen) stimulated a concentration-dependent increase of phospholipase C (PLC) activity in bovine adrenal medullary chromaffin cells. This response was accompanied by an increase in PLCgamma1 tyrosine phosphorylation and its cytosketetal translocation. Insulin, at high concentrations, stimulated PLC activity to a similar extent as BpV(phen), a response that was also accompanied by an increase in PLCgamma1 translocation but not its tyrosine phosphorylation. BpV(phen) strongly enhanced the insulin-stimulated increase in PLC activity and caused a small rise in PLCgamma1 translocation above that seen with insulin alone. Despite the synergistic rise in activity PLCgamma1 tyrosine phosphorylation did not increase beyond that seen with BpV(phen) alone. These results indicate that PLCgamma1 activation in chromaffin cells may be more closely associated with its cytoskeletal translocation than its tyrosine phosphorylation although other factors may also be important for activation of enzyme activity.     

Relative importance of competition and plant–soil feedback,their synergy,context dependency and implications for coexistence

Plants interact simultaneously with each other and with soil biota, yet the relative importance of competition vs. plant–soil feedback (PSF) on plant performance is poorly understood. Using a meta‐analysis of 38 published studies and 150 plant species, we show that effects of interspecific competition (either growing plants with a competitor or singly, or comparing inter‐ vs. intraspecific competition) and PSF (comparing home vs. away soil, live vs. sterile soil, or control vs. fungicide‐treated soil) depended on treatments but were predominantly negative, broadly comparable in magnitude, and additive or synergistic. Stronger competitors experienced more negative PSF than weaker competitors when controlling for density (inter‐ to intraspecific competition), suggesting that PSF could prevent competitive dominance and promote coexistence. When competition was measured against plants growing singly, the strength of competition overwhelmed PSF, indicating that the relative importance of PSF may depend not only on neighbour identity but also density. We evaluate how competition and PSFs might interact across resource gradients; PSF will likely strengthen competitive interactions in high resource environments and enhance facilitative interactions in low‐resource environments. Finally, we provide a framework for filling key knowledge gaps and advancing our understanding of how these biotic interactions influence community structure.     

Histamine-Stimulated Inositol Phospholipid Metabolism in Bovine Adrenal Medullary Cells: A Kinetic Analysis

Abstract: Histamine stimulation of bovine adrenal medullary cells rapidly activated phospholipase C. [³H]Inositol 1,4,5-trisphosphate [[³H]Ins(1,4,5)P₃] levels were transiently increased (200% of basal values between 1 and 5 s) before declining to a new steady-state level of ～140% of basal values. [³H]Inositol 1,4-bisphosphate [[³H]Ins(1,4)P₂] content increased to a maximal and maintained level of 250% of basal values after 1 s, whereas levels of [³H]inositol 1,3,4-trisphosphate [[³H]-Ins(1,3,4)P₃], [³H]inositol 1,3-bisphosphate, and [³H]-inositol 4-monophosphate ([³H]Ins4P) increased more slowly. The rapid responses were not reduced by the removal of extracellular Ca²⁺, but they were no longer sustained over time. The turnover rates of selected inositol phosphate isomers have been estimated in the intact cell. [³H]Ins(1,4,5)P₃ was rapidly metabolized (t_1/2 of 11 s), whereas the other isomers were metabolized more slowly, with t_1/2 values of 113, 133, 104, and 66 s for [³H]Ins(1,3,4)P₃, [³H]Ins(1,4)P₂, an unresolved mixture of [³H]inositol 1- and 3-monophosphate ([³H]Ins1/3P), and [³H]Ins4P, respectively. The calculated turnover rate of [³H]Ins(1,4,5)P₃ was sufficient to account for the turnover of the combination of both [³H]Ins(1,4)P₂ and [³H]Ins(1,3,4)P₃ but not that of [³H]Ins1/3P or [³H]Ins4P. These observations demonstrate that histamine stimulation of these cells results in a complex Ca²⁺-dependent and -independent response that may involve the hydrolysis of inositol phospholipids in addition to phosphatidylinositol 4,5-bisphosphate.     

Dispersal and recruitment of Tasiagma ciliata (Trichoptera: Tasimiidae) in rainforest streams, south-eastern Australia

1. This study examined genetic variation within and among populations of the caddis fly Tasiagma ciliata (Tasimiidae: Trichoptera) from rainforest streams in south-east Queensland, Australia.
2. Very low levels of genetic differentiation at large spatial scales, between subcatchments and between catchments, indicated that dispersal by the winged adults is widespread. However, significant genetic differentiation at the smallest spatial scale examined, within reaches in a single stream, suggested limited movement by larvae within streams.
3. A patchy distribution of deviations from Hardy–Weinberg equilibrium and differences in patterns among allozyme loci suggested that populations in particular reaches were the result of only a few matings.
4. These results are surprising, given the large numbers of larvae present within a single reach. We suggest that stochastic effects of recruitment may underlie much of the spatial and temporal variation in population numbers in these rainforest streams.