Effect of Continuous Phorbol Ester Treatment on Muscarinic Receptor-Mediated Calmodulin Redistribution in SK-N-SH Neuroblastoma Cells

Abstract: Stimulation of muscarinic receptors by carbachol and activation of protein kinase C elicits the translocation of calmodulin (CaM) from membranes to cytosol in the human neuroblastoma cell line SK-N-SH. Our previous studies have suggested a role for protein kinase C in the regulation of CaM redistribution. To explore further the role of protein kinase C in carbachol-induced calmodulin translocation, we treated cells for 17 h with 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate protein kinase C isozymes or 72 h to differentiate the cells. Treatment of SK-N-SH cells for 17 h with 70 nM TPA nearly abolished the effect of carbachol on CaM redistribution. After 72 h of TPA, however, the cells appeared differentiated, and the ability of carbachol to increase cytosolic CaM levels was restored. In untreated control cells, the carbachol-mediated increase in cytosolic CaM content was mimicked by TPA and blocked by pretreatment with the selective protein kinase C inhibitor Ro 31-8220 at 10 µM. In the 72-h TPA-treated cells, however, the ability of TPA to increase cytosolic CaM levels was significantly reduced, and the action of carbachol was no longer blocked by Ro 31-8220. The effect of prolonged TPA treatment on select protein kinase C isozymes was examined by immunoblotting. Treatment of cells for either 17 or 72 h abolished the α-isozyme in the cytosol and reduced (17 h) or abolished (72 h) the content in the membranes. In both 17- and 72-h TPA-treated cells, the ε-isozyme was nearly abolished in the cytosol and slightly reduced in the membranes. Some protein kinase C activity may have been maintained during TPA treatment because the basal level of phosphorylation of the protein kinase C substrate myristoylated alanine-rich C kinase substrate was enhanced in cells treated for either 17 or 72 h with TPA. The potential dissociation of carbachol and protein kinase C in eliciting increases in cytosolic CaM content was a function of prolonged TPA treatment and not differentiation per se because carbachol-mediated increases in cytosolic CaM levels were inhibited by Ro 31-8220 in retinoic acid-differentiated SK-N-SH cells. This study demonstrates that continuous TPA treatment, although initially down-regulating the protein kinase C-mediated effect of carbachol on CaM redistribution, uncouples carbachol and protein kinase C at longer times.     

Cytoplasmic streaming in the root cortex and its role in the delivery of potassium to the shoot

The influence of cytochalasin B (CB), a potent inhibitor of cytoplasmic streaming, on ⁸⁶Rb-labelled K⁺ translocation by detopped Lycopersicon esculentum Mill., Cucumis sativus L. and Zea mays L. plants was examined by measuring the radioactivity in xylem exudate before and after the addition of CB to the medium bathing the roots. CB caused complete cessation of cytoplasmic streaming in root segments within 15 min but was without effect on either total ⁸⁶Rb uptake or exudation. Thus factors other than cytoplasmic streaming limit the movement of K⁺ across the symplast of the root of higher plants.     

‘Nucleolar dominance’ as observed in barley translocation lines with specifically reconstructed SAT chromosomes

Summary Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.     

Molecular cytogenetic characterization of Thinopyrum intermedium-derived wheat germplasm specifying resistance to wheat streak mosaic virus

Thinopyrum intermedium is a promising source of resistance to wheat streak mosaic virus (WSMV), a devastating disease of wheat. Three wheat germplasm lines possessing resistance to WSMV, derived from Triticum aestivum×Th. intermedium crosses, are analyzed by C-banding and genomic in situ hybridization (GISH) to determine the amount and location of alien chromatin in the transfer lines. Line CI15092 was confirmed as a disomic substitution line in which wheat chromosome 4A was replaced by Th. intermedium chromosome 4Ai?2. The other two lines, CI17766 and A29-13-3, carry an identical Robertsonian translocation chromosome in which the complete short arm of chromosome 4Ai?2 was transferred to the long arm of wheat chromosome 4A. Fluorescence in situ hybridization (FISH) using ABD genomic DNA from wheat as a probe and S genomic DNA from Pseudoroegneria stipifolia as the blocker, and vice versa, revealed that the entire short arm of the translocation was derived from the short arm of chromosome 4Ai?2 and the breakpoint was located at the centromere. Chromosomal arm ratios (L/S) of 2.12 in CI17766 and 2.15 in A29-13-3 showed that the translocated chromosome is submetacentric. This translocated chromosome is designated as T4AL?? 4Ai?2S as suggested by Friebe et al. (1991).     

The effect of solvent viscosity on the rate-determining step of fatty acid synthetase

The overall activity of an animal fatty acid synthetase at the saturation level of substrate concentration decreased when the solvent viscosity, eta, of the reaction mixture was increased with viscogens such as glycerol, sucrose, and polyethylene glycol. The activity of the enzyme changed roughly proportional to eta-P, where p = 1.0 for glycerol, p = 0.66 for sucrose, and p less than 0.6 for polyethylene glycol with different molecular sizes. The thioesterase activity, which catalyzes the final partial reaction in the multifunctional enzyme, was not affected by 5-fold increase of solvent viscosity with sucrose. These results suggested that the rate-determining step of the enzyme other than the thioesterase reaction involves a microscopic transport step, the rate of which is influenced by the solvent viscosity. The microscopic transport step may be related to the transfer of the reaction intermediate from one active site to another or to the motion of a larger part of the enzyme requisite for the catalytic reaction. In the solution containing glycerol, the rate-determining motion was primarily diffusion limited since the inverse of the initial rate was proportional to eta, i.e., p = 1. Since the substrate concentration was at a saturation level in this experiment, the viscosity-dependent step cannot be the encounter between the enzyme and substrates, but must be intramolecular in origin, most probably the reaction catalyzed by beta-ketoacyl synthetase. In solutions containing other viscogens, however, p was less than 1.0, indicating a significant involvement of chemical steps in the rate-determining step as well. Bovine serum albumin, when used as a proteinic viscogen, also decreased the initial rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and functional characterization of the bacterial translocation inhibitor GE82832

The structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl-tRNA and mRNA movement and with the Pi release occurring after ribosome- and EF-G-dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit “head” which accompanies the ribosome-unlocking step of translocation.     

A stage‐wise approach for the analysis of multi‐environment trials

Plant breeders and variety testing agencies routinely test candidate genotypes (crop varieties, lines, test hybrids) in multiple environments. Such multi‐environment trials can be efficiently analysed by mixed models. A single‐stage analysis models the entire observed data at the level of individual plots. This kind of analysis is usually considered as the gold standard. In practice, however, it is more convenient to use a two‐stage approach, in which experiments are first analysed per environment, yielding adjusted means per genotype, which are then summarised across environments in the second stage. Stage‐wise approaches suggested so far are approximate in that they cannot fully reproduce a single‐stage analysis, except in very simple cases, because the variance–covariance matrix of adjusted means from individual environments needs to be approximated by a diagonal matrix. This paper proposes a fully efficient stage‐wise method, which carries forward the full variance–covariance matrix of adjusted means from the individual environments to the analysis across the series of trials. Provided the variance components are known, this method can fully reproduce the results of a single‐stage analysis. Computations are made efficient by a diagonalisation of the residual variance–covariance matrix, which necessitates a corresponding linear transformation of both the first‐stage estimates (e.g. adjusted means and regression slopes for plot covariates) and the corresponding design matrices for fixed and random effects. We also exemplify the extension of the general approach to a three‐stage analysis. The method is illustrated using two datasets, one real and the other simulated. The proposed approach has close connections with meta‐analysis, where environments correspond to centres and genotypes to medical treatments. We therefore compare our theoretical results with recently published results from a meta‐analysis.     

The use (or misuse) of microsatellite allelic distances in the context of inbreeding and conservation genetics

In line with inbreeding theory, genetic diversity at a set of molecular markers may explain variation in fitness‐associated traits in partially inbred populations, and such associations will appear as ‘genotype–fitness correlations’. An individual genetic diversity index specifically used for microsatellites is ‘mean d²’, i.e. the mean squared distance between alleles. The original hypothesis for mean d²–fitness correlations assumes that mean d² captures fitness effects at both ends of the inbreeding–outbreeding spectrum. This hypothesis received strong criticism from work showing that even a plain diversity estimate such as multi‐locus heterozygosity (MLH) outperforms mean d² as a predictor of the inbreeding coefficient and fitness in most realistic situations. Despite this critique, the mean d²‐approach is still used frequently in ecological and evolutionary research, producing results suggesting that mean d² sometimes provides a stronger prediction of fitness than does MLH. In light of the critique, such results are unexpected, but potential explanations for them may exist (at least hypothetically), including scenarios based on close linkage and recent admixture. Nevertheless, a major caveat is that it is very difficult to predict a priori if mean d² will improve the genotype–fitness correlation, which in turn makes objective interpretations difficult. Mean d²–fitness associations are potentially interesting, but the fact that we cannot easily understand them is problematic and should be thoroughly addressed in each study. Therefore, instead of hastily reached interpretations of mean d²–fitness correlations, conclusions need support from complementary analyses, e.g. verifying admixture of genetically structured populations.     

Temporal autocorrelation functions for movement rates from global positioning system radiotelemetry data

Autocorrelation has been viewed as a problem in telemetry studies because sequential observations are not independent in time or space, therefore violating assumptions for statistical inference. Yet nearly all ecological and behavioural data are autocorrelated in both space and time. We argue that there is much to learn about the structure of ecological and behavioural data from patterns of autocorrelation. Such patterns include periodicity in movement and patchiness in spatial data, which can be characterized by an autocorrelogram, semivariogram or spectrum. We illustrate the utility of temporal autocorrelation functions (ACFs) for analysing step-length data from GPS telemetry of wolves (Canis lupus), cougars (Puma concolor), grizzly bears (Ursus arctos) and elk (Cervus elaphus) in western Alberta. ACFs often differ by season, reflecting differences in foraging behaviour. In wilderness landscapes, step-length ACFs for predators decay slowly to apparently random patterns, but sometimes display strong daily rhythms in areas of human disturbance. In contrast, step lengths of elk are consistently periodic, reflecting crepuscular activity.