Response of root mRNA population to sulphate shortage in maize seedlings

Roots of ten-days-old seedlings obtained from a maize hybrid grown in complete or in sulphate-deprived medium were used to extract Poly(A)+RNA. The response to sulphate deprivation, which is known to increase the uptake capacity up to ten times, was manifested also by the expression of three mRNA species, as shown by the in vitro translation of the mRNA population. One hour after transfer to complete nutrient medium all three mRNAs were still present.Abbreviations BSA Bovine Serum Albumine - DTT 1,4-Dithio-DL-Threitol - EDTA Ethylenediaminetetraacetic-acid - MES 2-(N-Morpholino) ethane sulfonic acid - SDS Sodium Dodecyl Sulfate - TCA Trichloracetic acid - TRIS Tris(hydroxy-methyl)-aminomethane     

The evolution of pathways for aromatic hydrocarbon oxidation inPseudomonas

The organisation and nucleotide sequences coding for the catabolism of benzene, toluene (and xylenes), naphthalene and biphenylvia catechol and the extradiol (meta) cleavage pathway inPseudomonas are reviewed and the various factors which may have played a part in their evolution are considered. The data suggests that the complete pathways have evolved in a modular way probably from at least three elements. The commonmeta pathway operons, downstream from the ferredoxin-like protein adjacent to the gene for catechol 2,3-dioxygenase, are highly homologous and clearly share a common ancestry. This common module may have become fused to a gene or genes the product(s) of which could convert a stable chemical (benzoate, salicylate, toluene, benzene, phenol) to catechol, thus forming the lower pathway operons found in modern strains. The upper pathway operons might then have been acquired as a third module at a later stage thus increasing the catabolic versatility of the host strains.     

Altered free calcium transients in pig kidney cells (LLC-PK1) cultured with penicillin/streptomycin

Summary The effect of a conventional antibiotic (penicillin/streptomycin) mixture on the widely used kidney epithelial cell line, LLC-PK₁, was investigated by measuring growth and intracellular free calcium. Free calcium concentration was the same in cells cultured for 3 to 7 wk with (“plus”) and without (“minus”) antibiotics both at rest and when challenged with high (14 mM) external calcium. When exposed to vasopressin, minus cells exhibited significantly smaller calcium transients than plus cells. A similar difference existed for transients elicited by a calcium ionophore, 4-br-A23187. After longer periods of culture (>20 wk), minus cells grew slower than plus cells but on reaching confluence (minus cells took 1 day longer) the morphologies and viabilities were indistinguishable. The finding that culture with penicillin/streptomycin reversibly modified some properties of LLC-PK₁ cells, at least partly through altered calcium homeostasis, is of importance for workers using this cell model to study drug effects and raises the general possibility of similar effects on other cultured cells.     

Activity of mushroom polyphenol oxidase in organic medium

A kinetic study of the activity of mushroom polyphenol oxidase in an organic system was carried out to obtain detailed enzyme kinetic data in relation to optimization of reaction conditions and substrate specificity. A simple method for consistent measurement of reaction rates in the heterogeneous enzyme/organic solvent system (consisting of immobilized polyphenol oxidase and a hydrated solution of the substrate in chloroform) was designed. The aqueous content of the system was optimized using p-cresol as the substrate. With this system, a crude extract of Agaricus bisporus was used to hydroxylate and oxidize a range of selected p-substituted phenolic substrates, yielding o-quinone products. Michaelis-Menten kinetics were used to obtain apparent K(M) and V(max) values with respect to each of these substrates. Results from this analysis indicated a correlation between the enzymic kinetic parameters obtained and the steric requirements of the substrates, which could be rationalized in terms of the restricted flexibility of the enzyme when it is in chloroform and also in terms of substrate and solvent hydrophobicity. In the course of the investigation UV molar absorption coefficients of several o-quinones were measured by a novel method: (1)H nuclear magnetic resonance (NMR) spectroscopy was employed to determine component concentrations in reaction mixtures resulting from the transformation of phenols by polyphenol oxidase in chloroform. Thus the UV molar absorption coefficients could be obtained directly, avoiding the necessity to isolate the water-sensitive, unstable o-quinones. (c) 1993 John Wiley & Sons, Inc.     

Trusting the Health System and COVID 19 Restriction Compliance

We examine the extent to which exposure to higher relative COVID-19 mortality (RM), influences health system trust (HST), and whether changes in HST explain the perceived ease of compliance with pandemic restrictions during the COVID-19 pandemic. Drawing on evidence from two representative surveys covering all regions of 28 European countries before and after the first COVID-19 wave, and using a difference in differences strategy together with Coarsened Exact Matching (CEM), we document that living in a region with higher RM during the first wave of the pandemic increased HST. However, the positive effect of RM on HST is driven by individuals over 45 years of age, and the opposite effect is found among younger cohorts. Furthemore, we find that a higher HST reduces the costs of complying with COVID-19 restrictions, but only so long as excess mortality does not exceed the average by more than 20%, at which point the ease of complying with COVID-19 restrictions significantly declines, offsetting the positive effect of trust in the healthcare system. Our interpretation of these estimates is that a higher RM is interpreted as a risk signal among those over 45, and as a signal of health-care system failure among younger age individuals.     

Tyrosine protein kinase assays

Protein kinases form a large family of enzymes that play a major role in a number of live processes. The study of their action is important for the understanding of the transformation mechanisms and of the normal and pathological growth events. The quality of an enzyme assay is often the key point of an enzymatic study. It must be flexible and compatible with various experimental conditions, such as those for the purification process, the screening of inhibitors and the substrate specificity studies. As will be shown in the present review, two categories of substrates, peptidic and proteic, should be distinguished. The use of peptide substrates facilitates the determination of the recognition requirements of the enzyme and of the kinetic effects of even minute variations in their sequence. These linear peptide structures are assumed to mimic a complex interaction between the enzyme and a proteic substrate in which distant amino acids in the sequence are vicinal in the folded substrate. Less amenable to a systematic study, but probably more adequate to investigate the natural substrate of a given kinase, are the proteic substrates. Obviously the tools to measure protein kinase activities are not the same in these two cases. The main difficulty in assaying protein kinases is the use of labelles γ-ATP, mostly at large excess concentration, since the final product of the reaction has to be separated from the non-reacted labelled ATP. In the case of peptide substrates, the difficulty is to separate them from ATP basing on differences of molecular mass. Despite the efforts of many investigators to rely upon differences in solubility, in charges or in “affinity”, this separation, which is crucial for the assay, is still an unsolved experimental problem. Chromatographic, as well as electrophoretic assays appeared relatively late in this domain, and more work in assessing new methodologies might bring new breakthroughs in the next few years. Specific, simple and reliable kinase assays are still a major challenge. Their improvement will help to conduct specificity studies, to elucidate complex growth mechanisms in which they are involved and to discover more selective potent inihibitors.     

Conformation of P22 tailspike folding and aggregation intermediates probed by monoclonal antibodies.

The partitioning of partially folded polypeptide chains between correctly folded native states and off-pathway inclusion bodies is a critical reaction in biotechnology. Multimeric partially folded intermediates, representing early stages of the aggregation pathway for the P22 tailspike protein, have been trapped in the cold and isolated by nondenaturing polyacrylamide gel electrophoresis (PAGE) (speed MA, Wang DIC, King J. 1995. Protein Sci 4:900-908). Monoclonal antibodies against tailspike chains discriminate between folding intermediates and native states (Friguet B, Djavadi-Ohaniance L, King J, Goldberg ME. 1994. J Biol Chem 269:15945-15949). Here we describe a nondenaturing Western blot procedure to probe the conformation of productive folding intermediates and off-pathway aggregation intermediates. The aggregation intermediates displayed epitopes in common with productive folding intermediates but were not recognized by antibodies against native epitopes. The nonnative epitope on the folding and aggregation intermediates was located on the partially folded N-terminus, indicating that the N-terminus remained accessible and nonnative in the aggregated state. Antibodies against native epitopes blocked folding, but the monoclonal directed against the N-terminal epitope did not, indicating that the conformation of the N-terminus is not a key determinant of the productive folding and chain association pathway.     

Induction of autonomous endosperm in Lupinus luteus,Helleborus niger and Melandrium album by in vitro culture of unpollinated ovaries

Autonomous division of the endosperm was induced by in vitro culture of unpollinated ovaries or placenta-attached ovules in Helleborus niger, Lupinus luteus and Melandrium album. The induction frequencies for the three species were 50%, 10–20% and 0.1%, respectively. The endosperms contained up to 20 free nuclei; only a few ovules with 80–420 endosperm nuclei were found. Induction of autonomous division of the endosperm, which is unusual in amphimictic plants, was observed in three new species. No embryos appeared in the ovules. This suggests a developmental independence of the endosperm from the embryo in the culture of unpollinated ovaries or ovules.