Induced thermotolerance and associated expression of the heat-shock protein Hsp70 in adult Drosophila melanogaster

1. Inducible heat-shock proteins are synthesized when temperatures are increased to levels substantially above normal. The functional role of these proteins is well known at the cellular level. Today increasing interest has been directed towards the importance of heat-shock proteins for resistance of whole organisms to high-temperature stress and other environmental stressors.
2. Here the functional relationship between the heat-shock protein, Hsp70, and thermal resistance in adult Drosophila melanogaster was examined by comparing thermal resistance, i.e. survival at 39 °C for 85 min, and levels of Hsp70 at various times elapsed (2, 4, 8, 16, 32 and 64 h) after thermotolerance was induced by short-term acclimation/heat hardening at 37 °C for 55 min.
3. Levels of Hsp70 in both males and females were highest 2 h after heat hardening and declined with longer times elapsed. The rate of decrease initially was very fast but diminished with increasing time. After 32 h the level of Hsp70 approached the level in flies that were not hardened. Levels of Hsp70 in males exceeded that of females during the entire period.
4. Survival of both sexes increased with increasing time after heat hardening and reached an optimum between 8 and 32 h. Thereafter resistance decreased with longer times elapsed. Survival of females generally exceeded that of males except after 16 and 64 h.
5. Regression analysis applied to the data on Hsp70 levels revealed that the model describing these data could not explain the data for survival. Also, higher levels of Hsp70 in males compared with females were not associated with greater survival in males. However, statistical analysis on paired measurements of Hsp70 and survival revealed a positive association between Hsp70 level and survival at each time elapsed after induction of thermotolerance.     

Effects of aluminium on growth and kinetics of K+(86Rb) uptake in two cultivars of wheat (Triticum aestivum) with different sensitivity to aluminium

Two cultivars of wheat (Triticum aestivum L. cvs Kadett and WW 20299) were grown for 9 days with 20% relative increase in nutrient supply per day at pH 4.1. Aluminium at 50 μ M retarded the growth of roots more than that of shoots in both cultivars, thus decreasing the root/shoot ratio. The inhibition was largest in WW 20299. With long term Al treatment (9 days), K_m for K⁺(⁸⁶Rb) influx increased five times in both cultivars and V_max decreased in WW 20299. Efflux of K⁺(⁸⁶Rb) was little affected. When the roots were treated with aluminium for two days, only relative growth rate of roots was retarded, while growth of shoots was unaffected and influx of K⁺(⁸⁶Rb) adjusted to the actual K⁺ demand of the plants. It is concluded that the effects of aluminium on K⁺ uptake in these wheat cultivars are not primary factors contributing to aluminium sensitivity. However, in soil with Al the demand for a comparatively high concentration of K⁺ to maintain an adequate K⁺ uptake rate, in combination with a slow growth rate of the roots, may secondarily lead to K⁺ deficiency in the plants.     

The effect of nutrition on the seasonal course of needle respiration in Norway spruce stands

 Respiration of 1-year-old needles of 30-year-old Norway spruce trees [Picea abies (L.) Karst.] was studied in a nutrient optimisation experiment in northern Sweden. Respiration rates of detached needles, from ten control (C) and ten irrigated-fertilised (IL) trees, were measured on 16 occasions from June 1992 to June 1993. The aim of the study was to determine the influence of temperature on the seasonal course of needle maintenance respiration, and the effect of nitrogen concentration [N] and carbohydrate content on needle respiration in young Norway spruce trees subjected to long-term fertilisation. The IL treatment significantly affected needle size, in terms of dry mass and length, but not specific needle length (SNL). There was, however, a strong tree-specific effect on SNL (P<10^–9, R ² = 0.75). Needle starch content varied markedly with season (0–25% of total dry mass). This, unless accounted for, would cause erroneous estimates of nutrient concentrations, and of rates of needle respiration, within and between treatments. There was considerable seasonal variation in needle respiration, both in terms of maintenance respiration and temperature dependence (Q₁₀). Q₁₀ had its highest value (2.8) during winter and its lowest (2.0) in the middle of summer. In early autumn (August, September), respiration rate and needle [N] were significantly related (C: P = 0.001, IL: P<0.0005). There was no significant difference in the slope between the two regression lines, but a difference in intercept. At the same needle [N], needles from IL-plots always had a lower respiration rate than needles from control plots. No obvious explanation for the observed difference in intercept was found, but some plausible assumptions are put forward and discussed. Received: 24 January 1997 / Accepted: 1 July 1997     

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease.     

Significant sparse polygenic risk scores across 813 traits in UK Biobank

We present a systematic assessment of polygenic risk score (PRS) prediction across more than 1,500 traits using genetic and phenotype data in the UK Biobank. We report 813 sparse PRS models with significant (p < 2.5 x 10⁻⁵) incremental predictive performance when compared against the covariate-only model that considers age, sex, types of genotyping arrays, and the principal component loadings of genotypes. We report a significant correlation between the number of genetic variants selected in the sparse PRS model and the incremental predictive performance (Spearman’s ⍴ = 0.61, p = 2.2 x 10⁻⁵⁹ for quantitative traits, ⍴ = 0.21, p = 9.6 x 10⁻⁴ for binary traits). The sparse PRS model trained on European individuals showed limited transferability when evaluated on non-European individuals in the UK Biobank. We provide the PRS model weights on the Global Biobank Engine (https://biobankengine.stanford.edu/prs).     

Role of HSF activation for resistance to heat, cold and high-temperature knock-down

Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance of Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression. In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF, such as Hsps, although occurring following LTCA, are not of major importance for cold stress resistance and neither for RCH nor high-temperature knock-down resistance in D. melanogaster.     

Full genome gene expression analysis of the heat stress response in Drosophila melanogaster

The availability of full genome sequences has allowed the construction of microarrays, with which screening of the full genome for changes in gene expression is possible. This method can provide a wealth of information about biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening (36 degrees C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved in the stress response. We identified 3 main groups of stress responsive genes that were early-upregulated, early-downregulated, and late-upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis, which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in general. The majority of stress responsive genes identified in this and other studies were downregulated, and the degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an expected exception, heat shock genes were generally found to be upregulated by stress in general.     

Guinea pig (Cavio cambayo) 5S rRNA genes map to 7q2, 20q2 and 30q2 shown by an R-banded karyotype with PNA-FISH

A karyotype for the guinea pig (Cavio cambayo) is proposed based on an R-banding pattern. R-bands were obtained by BrdU incorporation into the cells followed by a combined DAPI and propidium iodide staining of the fixed metaphase spreads. In situ hybridization was performed with two biotinylated 18-mer PNA (peptide nucleic acid) probes complementary to sequences within the 5S rRNA gene. The 5S rRNA gene repeats map to chromosomes 7q2. 20q2 and 30q2, respectively.     

Class-1 translation termination factors: invariant GGQ minidomain is essential for release activity and ribosome binding but not for stop codon recognition

Previously, we have shown that all class-1 polypeptide release factors (RFs) share a common glycine-glycine-glutamine (GGQ) motif, which is critical for RF activity. Here, we subjected to site-directed mutagenesis two invariant amino acids, Gln185 and Arg189, situated in the GGQ minidomain of human eRF1, followed by determination of RF activity and the ribosome binding capacity for mutant eRF1. We show that replacement of Gln185 with polar amino acid residues causes partial inactivation of RF activity; Gln185Ile, Arg189Ala and Arg189Gln mutants are completely inactive; all mutants that retain partial RF activity respond similarly to three stop codons. We suggest that loss of RF activity for Gln185 and Arg189 mutants is caused by distortion of the conformation of the GGQ minidomain but not by damage of the stop codon recognition site of eRF1. Our data are inconsistent with the model postulating direct involvement of Gln185 side chain in orientation of water molecule toward peptidyl-tRNA ester bond at the ribosomal peptidyl transferase centre. Most of the Gln185 mutants exhibit reduced ability to bind to the ribosome, probably, to rRNA and/or (peptidyl)-tRNA(s). The data suggest that the GGQ motif is implicated both in promoting peptidyl-tRNA hydrolysis and binding to the ribosome.     

Specific inhibitors prevent proteolytic degradation of recombinant proteins expressed in High Five cells

The insect cell line BTI-TN-5B1-4 (High Five) is frequently used to express recombinant proteins in large amounts using the baculovirus expression system. However, extensive proteolytic degradation of recombinant proteins is often encountered. Furthermore, we have observed that recombinant proteins migrate in SDS-PAGE in agreement with poly-ubiquitinated forms of the protein, suggesting a ubiquitin/proteasome degradation pathway. Here, we describe a systematic study unraveling the effect of adding proteasome inhibitors or specific protease inhibitors to the growth medium of High Five insect cells infected with recombinant baculovirus. Furthermore, protease inhibitors were added to the lysis buffer to establish the most efficient way to inhibit proteolytic activity after lysis of baculovirus-infected cells expressing recombinant proteins. We conclude that a combination of adding protease inhibitors to the growth medium and to the lysis buffer minimizes the proteolytic activity in High Five cells. The most efficient protease inhibitors were E-64 in the growth medium together with Leupeptin in the lysis buffer at concentrations higher than with available cocktails of inhibitors. The optimal treatment of High Five cells is different from the optimal treatment of Sf9 cells. For proteins susceptible to ubiquitinylation, a treatment of insect cell cultures with the proteasome inhibitor MG132 (LLL) leads to a considerable reduction of the yield of production of recombinant protein.