Specific Stress Conditions Research Articles

CAJAL BODIES: A Long History of Discovery

This review surveys what is known about the structure and function of the subnuclear domains called Cajal bodies (CBs). The major focus is on CBs in mammalian cells but we provide an overview of homologous CB structures in other organisms. We discuss the protein and RNA components of CBs, including factors recently found to associate in a cell cycle-dependent fashion or under specific metabolic or stress conditions. We also consider the dynamic properties of both CBs and their molecular components, based largely on recent data obtained thanks to the advent of improved in vivo detection and imaging methods. We discuss how these data contribute to an understanding of CB functions and highlight major questions that remain to be answered. Finally, we consider the interesting links that have emerged between CBs and alterations in nuclear structure apparent in a range of human pathologies, including cancer and inherited neurodegenerative diseases. We speculate on the relationship between CB function and molecular disease.

Characterization of a cyclooxygenase-2-765G???C promoter polymorphism in human neural cells

Direct sequencing of the human cyclooxygenase-2 gene promoter revealed a common single nucleotide substitution, cyclooxygenase-2-765G-->C, in 24.5% of the populations analyzed. This change introduced a 20 base pair polypyrimidine/polypurine element and a partial recognition feature for RXRalpha, the 9-cis retinoic acid receptor, into the polymorphic promoter. Cyclooxygenase-2-765G-->C constructs, when transfected into human neural cells, exhibited a 1.4-fold higher level of basal expression, while the proinflammatory factors interleukin-1beta and 9-cis retinoic acid synergistically induced polymorphic promoter activity 2.4-fold over wild type. These results suggest that under specific conditions of cellular stress, a common variation in cyclooxygenase-2 promoter structure may enhance cyclooxygenase-2 transcription, and this may contribute to the proliferation of an inflammatory response in brain cells.

Deficiency of Pkc1 activity affects glycerol metabolism in

Protein kinase C is apparently involved in the control of many cellular systems: the cell wall integrity pathway, the synthesis of ribosomes, the appropriated reallocation of transcription factors under specific stress conditions and also the regulation of N-glycosylation activity. All these observations suggest the existence of additional targets not yet identified. In the context of the control of carbon metabolism, previous data had demonstrated that Pkc1p might play a central role in the control of cellular growth and metabolism in yeast. In particular, it has been suggested that it might be involved in the derepression of genes under glucose-repression by driving an appropriated subcellular localization of transcriptional factors, such as Mig1p. In this work, we show that a pkc1Delta mutant is unable to grow on glycerol because it cannot perform the derepression of the GUT1 gene that encodes glycerol kinase. Additionally, active transport is also partially affected. Using this phenotype, we were able to isolate a new pkc1Delta revertant. We also isolated two transformants identified as the nuclear exportin Msn5 and the histone deacetylase Hos2 extragenic suppressors of this mutation. Based on these results, we postulate that Pkc1p may be involved in the control of the cellular localization and/or regulation of the activity of nuclear proteins implicated in gene expression.

P53CSV, a Novel p53-Inducible Gene Involved in the p53-Dependent Cell-Survival Pathway

Although a number of p53 target genes have been identified, the mechanisms of p53-dependent activities that determine cellular survival or death are still not fully understood. Here we report isolation of a novel p53 target gene, designated p53-inducible cell-survival factor (p53CSV). p53CSV contains a p53-binding site within its second exon and the reduction of expression by small interfering RNA enhanced apoptosis, whereas overexpression protected cells from apoptosis caused by DNA damage. p53CSV is induced significantly when cells have a low level of genotoxic stresses, but not when DNA damage is severe. p53CSV can modulate apoptotic pathways through interaction with Hsp70 that probably inhibits activity of apoptosis protease activating factor-1. Our results imply that under specific conditions of stress, p53 regulates transcription of p53CSV and that p53CSV is one of the important players in the p53-mediated cell survival.

EIF2 and the control of cell physiology

Eukaryotic initiation factor eIF2 and its 'exchange factor' eIF2B play a key role in the regulation of protein synthesis in eukaryotes from yeast to mammals. Phosphorylation of eIF2 inhibits eIF2B and thus translation initiation. Four eIF2 kinases are now known in mammalian cells and these are activated in response to specific stress conditions. While phosphorylation of eIF2 serves to impair general protein synthesis, it causes upregulation of the translation of certain specific mRNAs that encode transcription factors. It can, therefore, exert effects on gene expression at multiple levels. The importance of correct control of eIF2 and eIF2B for normal physiology is exemplified by data from transgenic mice carrying knock-in or knock-out mutations and by the fact that mutations in the genes for the eIF2 kinase PERK or for eIF2B give rise to serious human diseases.

Response to severe water stress of the almond (Prunus amygdalus) ’Ferragnès’ grafted on eight rootstocks

The behaviour of almond (Prunus amygdalus) ’Ferragnès’ grafted on eight rootstocks (GF‐677, Stylianidis C., GN‐15, GN‐22, M‐51, 3–7, 3–8, and Drepanoto), as regards certain agronomical and physiological responses, was studied under water stress conditions. From the results it is concluded that scions on GN‐22 were better adapted to water stress conditions as they had higher growth, higher pre‐dawn leaf water potential, and lower leaf shedding. However for some parameters measured, GN‐22 was not significantly different to GN‐15, GF 677, and Drepanoto. A suitable rootstock adapted to water stress may also have the characteristic for high nutrient uptake. Regarding nutrient status, under the specific experimental conditions of water stress and low fertiliser input, scions on Drepanoto maintained most nutrients in leaves in the adequate range (except nitrogen, potassium, and boron) followed by GF 677, 3–8, and M‐51. Furthermore, scions on Drepanoto showed a tendency for higher uptake of manganese (Mn) and zinc (Zn) and therefore this rootstock is appropriate for soils with low Mn and Zn availability.

Differential gene expression of ARGININE DECARBOXYLASE ADC1 and ADC2 in Arabidopsis thaliana: characterization of transcriptional regulation during seed germination and seedling development.

• In plants, polyamines can generally be synthesized by the ornithine decarboxylase and arginine decarboxylase pathways. However, the model plant Arabidopsis thaliana appears to possess only the arginine decarboxylase pathway. As two paralogous ARGININE DECARBOXYLASE (ADC) genes are present in Arabidopsis, we investigated differential expression and potential differences of promoter activity during seedling development and under specific stress conditions. • Promoter activities were studied in stable homozygotic transformants harbouring promoter::reporter gene fusions. • Under temperate conditions, ADC2 promoter activity was strongly associated with seed germination, root and leaf development, whereas ADC1 promoter activity was low during vegetative development. Light, sucrose and ethylene were shown to be important regulators of ADC2 promoter activity. By contrast, in roots and leaves of plantlets subjected to chilling treatment the ADC1 paralogue showed high promoter activity whereas ADC2 promoter activity was considerably decreased. • In situations of seed germination, root development and response to chilling, the modifications of promoter activities were associated with changes in mRNA levels, emphasizing the involvement of transcriptional regulation in ARGININE DECARBOXYLASE gene expression.

Yield response of rice ( Oryza sativa L.) genotypes to drought under rainfed lowlands : 4. Vegetative stage screening in the dry season

Screening for drought resistance of rainfed lowland rice using drought score (leaf death) as a selection index has a long history of use in breeding programs. Genotypic variation for drought score during the vegetative stage in two dry season screens was examined among 128 recombinant inbred lines from four biparental crosses. The genotypic variation detected for drought score in the dry season was used to examine the reliability of the dry season screening method to estimate relative grain yield of genotypes under different types of drought stress in the wet season. Large genotypic variation for drought score existed in two experiments (A and B). However, there was no relationship between the drought scores of genotypes determined in these two experiments. Different patterns of development and severity of drought stress in these two experiments, i.e . slow development and mild plant water deficit in experiment A and fast development and severe plant water deficit in experiment B, were identified as the major factors contributing to the genotypes responding differently. Larger drought score in the dry season experiments was associated with lower grain yield under specific drought stress conditions in the wet season, but the association was weak to moderate and significant only in particular drought conditions. In most cases, a significant phenotypic and moderate genetic correlation between drought score in the dry season and grain yield in the wet season existed only when both drought score and grain yield of genotypes were affected by similar patterns and severity of drought stress in their respective experimental environments. The dry season environments used to measure genotypic variation for drought score should be managed to correspond to relevant types of drought environment that are frequent in the wet season. The efficiency of using the drought score as an indirect selection criterion for improving grain yield for drought conditions was lower than the direct selection for grain yield, and hence wet season screening with grain yield as a selection criterion would be more efficient. However, using drought score as a selection index, a larger number of genotypes can be evaluated than for wet season grain yield. Therefore, it is possible to apply higher selection intensities using the drought score system, and the selected lines can be further tested for grain yield in the wet season.

The Aspergillus fumigatus transcriptional activator CpcA contributes significantly to the virulence of this fungal pathogen.

We have cloned and characterized the Aspergillus fumigatus cpcA gene encoding the transcriptional activator of the cross-pathway control system of amino acid biosynthesis. cpcA encodes a functional orthologue of Saccharomyces cerevisiae Gcn4p. The coding sequence of the 2.2 kb transcript is preceded by two short upstream open reading frames, the larger one being well conserved among Aspergilli. Deletion strains in which either the coding sequence or the entire locus are replaced by a bifunctional dominant marker are impaired in their cross-pathway control response upon amino acid starvation, as demonstrated by analyses of selected reporter genes and specific enzymatic activities. In a murine model of pulmonary aspergillosis, cpcAdelta strains display attenuated virulence. Pathogenicity is restored to wild-type levels in strains with reconstitution of the genomic locus. Competitive mixed infection experiments additionally demonstrate that cpcAdelta strains are less able to survive in vivo than their wild-type progenitor. Our data suggest that specific stress conditions are encountered by A. fumigatus within the mammalian host and that the fungal cross-pathway control system plays a significant role in pulmonary aspergillosis.

Effect of Water‐Deficit Stress on Germination and Early Seedling Growth in Sugar Beet

AbstractSugar beet progeny lines screened for both high water use efficiency and high sugar yield under drought stress conditions in the field were assessed for the rate of seed germination and early seedling growth in water deficit stress, induced by mannitol solutions. Seeds of nine different sugar beet progeny lines were grown in three experimental conditions using filter paper, perlite and water agar as substrate. Three levels of 0.0, 0.2 and 0.3 m mannitol concentrations were applied in each experiment. A factorial design was used with three replications. Germination percentage was determined in all experiments. Seedling growth parameters such as cotyledon fresh weight, cotyledon dry weight, root fresh weight, root dry weight (RDW) and root length (RL) were measured experimentally. Abnormality was only recorded in the filter paper experiment. The results showed that drought stress could be simulated by mannitol solution and significant differences were found between stress levels for seedling characteristics. Distinct genetic variances were found among progeny lines with respect to germination and early seedling growth characteristics, except for cotyledons and RDW. Seedling growth and germination rates severely declined at the highest concentration of mannitol. The rate of abnormality was increased progressively at the germination stage with an increase in mannitol concentration but it was more pronounced in the drought‐susceptible progeny lines. The highest values of relative germination % and relative growth % of RL were obtained for the most tolerant line. In conclusion, seedling characteristics, in addition to other physiological components involved in the seed germination process under specific stress conditions, may be considered for breeding purposes.

Development and application of molecular DNA markers in Africa: a South African view

The development of molecular DNA markers for genetic analysis has greatly increased our understanding of the structure and behaviour of plant genomes. Different DNA marker technologies exist, however, information suggests that restriction fragment polymorphisms, amplified fragment length polymorphisms and also microsatellites are currently the most effective techniques for the detection of polymorphisms in plant genomes. Several other technologies, such as cDNA-amplified fragment length polymorphism, representational difference analysis or suppression subtractive hybridisation analysis, and cDNA microarrays, belong to a range of novel techniques very useful for the analysis of transcriptome variation, under specific stressful environmental conditions. However, the application of these technologies especially in Africa is not trivial, as execution of these techniques is largely dependent upon the availability of a technological advanced infrastructure, technical skills in plant molecular biology and substantial funding of the research groups involved in this type of research. Different molecular DNA-based marker techniques and their application to genome analysis and molecular breeding, and suitability for use in Africa, are discussed in this review.

Redundancy in the function of mitochondrial phosphate transport in Saccharomyces cerevisiae and Arabidopsis thaliana.

Most cellular ATP is produced within the mitochondria from ADP and Pi which are delivered across the inner-membrane by specific nuclearly encoded polytopic carriers. In Saccharomyces cerevisiae, some of these carriers and in particular the ADP/ATP carrier, are represented by several related isoforms that are distinct in their pattern of expression. Until now, only one mitochondrial Pi carrier (mPic) form, encoded by the MIR1 gene in S. cerevisiae, has been described. Here we show that the gene product encoded by the YER053C ORF also participates in the delivery of phosphate to the mitochondria. We have called this gene PIC2 for Pi carrier isoform 2. Overexpression of PIC2 compensates for the mitochondrial defect of the double mutant Deltamir1 Deltapic2 and restores phosphate transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPic-like proteins are also able to complement the double mutant Deltamir1 Deltapic2. Finally, we demonstrate that Pic2p is a mitochondrial protein and that its steady state level increases at high temperature. We propose that Pic2p is a minor form of mPic which plays a role under specific stress conditions.

Dynamic control of Dps protein levels by ClpXP and ClpAP proteases in Escherichia coli.

The Escherichia coli starvation-induced DNA protection protein Dps was observed to be degraded rapidly during exponential growth. This turnover is dependent on the clpP and clpX genes. The clpA gene is not required for Dps proteolysis, suggesting that Dps is a substrate for ClpXP protease but not for ClpAP protease. Dps proteolysis was found to be highly regulated. Upon carbon starvation, Dps is stabilized, which together with increased Dps synthesis allows strong accumulation of Dps in the stationary phase. The addition of glucose to starving cells results in rapid resumption of Dps proteolysis by ClpXP. Oxidative stress also leads to efficient stabilization of Dps. After hyperosmotic shift, however, proteolysis remains unaffected. Thus, regulated proteolysis of Dps strongly contributes to controlling Dps levels under very specific stress conditions. In contrast to the regulated degradation of RpoS by ClpXP, Dps proteolysis is independent of the recognition factor RssB. In addition, during starvation, clpP and, to a somewhat lesser extent, clpA are involved in maintaining ongoing Dps synthesis (acting at the level of Dps translation), which is required for strong Dps accumulation in long-term stationary phase cells. In summary, both ClpXP and ClpAP exert significant control of Dps levels by affecting log phase stability and stationary phase synthesis of Dps respectively.

The Psychobiology of Stress

Stressful life experience can have significant effects on a variety of physiological systems, including the autonomic nervous system, the hypothalamic-pituitary-adrenal axis, and the immune system. These relationships can be bidirectional; for example, immune cell products can act on the brain, altering mood and cognition, potentially contributing to depression. Although acute physiological alterations may be adaptive in the short term, chronic or repeated provocation can result in damage to health. The central dogma in the field of stress research assumes a stereotyped physiological response to all stressors (the generality model). However, increasing evidence suggests that specific stressful conditions and the specific way an organism appraises these conditions can elicit qualitatively distinct emotional and physiological responses (the integrated specificity model). For example, appraisals of threat (vs. challenge), uncontrollability, and negative social evaluation have been shown to provoke specific psychobiological responses. Emotional responses appear to have specific neural substrates, which can result in differentiated alterations in peripheral physiological systems, so that it is incorrect to presume a uniform stress response.

HtrA is a key factor in the response to specific stress conditions in Lactococcus lactis

We investigated the physiological role of Lactococcus lactis housekeeping surface protease HtrA. It is involved in surface properties under regular growth conditions, as the htrA mutant strain forms longer chains in liquid medium. It participates in cellular defence against environmental stress conditions: compared to the wild-type strain, the htrA mutant strain exhibited increased sensitivity to heat, ethanol, puromycin, and NaCl, but not to pH, H 2O 2, bile salts or to carbon or nitrogen starvation. htrA transcription in the wild-type strain showed a transient increase under stress conditions determined as requiring htrA, but not under overexpression of a secreted heterologous protein. Our results demonstrate that in L. lactis, htrA is a key factor in the response to specific stress conditions.

PAC1 phosphatase is a transcription target of p53 in signalling apoptosis and growth suppression

p53 has a role in many cellular processes through the transcriptional regulation of target genes. PAC1 (phosphatase of activated cells 1; also known as dual specificity phosphatase 2, DUSP2) is a dual threonine/tyrosine phosphatase that specifically dephosphorylates and inactivates mitogen-activated protein (MAP) kinases. Here we show that during apoptosis, p53 activates transcription of PAC1 by binding to a palindromic site in the PAC1 promoter. PAC1 transcription is induced in response to serum deprivation and oxidative stress, which results in p53-dependent apoptosis, but not in response to gamma-irradiation, which causes cell cycle arrest. Reduction of PAC1 transcription using small interfering RNA inhibits p53-mediated apoptosis, whereas overexpression of PAC1 increases susceptibility to apoptosis and suppresses tumour formation. Moreover, activation of p53 significantly inhibits MAP kinase activity. We conclude that, under specific stress conditions, p53 regulates transcription of PAC1 through a new p53-binding site, and that PAC1 is necessary and sufficient for p53-mediated apoptosis. Identification of a palindromic motif as a p53-binding site may reveal a novel mechanism whereby p53 regulates its target genes.

Nutritional implications of mould development in feedstuffs and alternatives to reduce the mycotoxin problem in poultry feeds

Fungi may develop in stored grains following field inoculation. In the course of their development fungi utilize plant nutrients, contributing to a reduction in their content. This reduction is obviously related to the degree of fungi development, but may become relevant in extreme cases. During the process of host colonization fungi may also produce toxins that are harmful for humans and animals. This occurs mainly under specific conditions of plant stress, such as those produced by adverse weather conditions, for instance drought has been shown to prompt aflatoxin production in peanuts. Differences in mycotoxin production are also related to plant genotype. Aflatoxin B1 produced by Aspergillus flavus is the most carcinogenic natural substance known. It is frequently charged with the reduction in performance of domestic birds, even when its concentration in the feed is very low. Approaches to eliminate mycotoxins from contaminated grains are not always worthwhile in practical and economical terms. Strategies used to reduce mycotoxin levels in cereal grains include the dilution of infected with healthier grains or the addition of adsorbents to the feeds. Several compounds such as activated charcoal, aluminosilicates and processed cell wall of Saccharomyces sp. (esterified glucomannan) have shown to be effective as mycotoxin adsorbents in poultry feeds. Adsorbent effectiveness has been reported with in vitro and in vivo techniques; however, it is still very difficult to quantify their benefits in commercial operations.

Protein aggregation in recombinant bacteria: biological role of inclusion bodies.

Protein aggregation is an ordinary consequence of thermal stress. In recombinant bacteria, the over-expression of plasmid-encoded genes triggers transcription of heat-shock genes and other stress responses and often results in the aggregation of the encoded protein as inclusion bodies. The formation of these deposits represents a major obstacle for the production of biologically active polypeptides and restricts the spectrum of protein products being available for the industrial-biomedical market. Inclusion body formation was formerly considered to occur passively by the irretrievable deposition of partially-folded intermediates. Increasing evidence, however, indicates that protein aggregation in bacteria occurs as a reversible process deeply integrated in the cell mechanisms for coping with thermal stress, and that inclusion bodies are structurally dynamic structures. Inclusion body formation might actually be supported by the cellular machinery that when operated under specific stress conditions, transiently stores misfolded polypeptides until they could be further processed: either refolded or proteolysed. A better understanding of protein aggregation in cell physiology could allow not only inclusion body formation to be minimized more efficiently for a higher soluble yield, but also to comprehend in detail the intricacy of cell mechanisms committed to handling the misfolding danger.

Functional Expression of Phosphagen Kinase Systems Confers Resistance to Transient Stresses in Saccharomyces cerevisiae by Buffering the ATP Pool

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.

A portable toxicity biosensor using freeze-dried recombinant bioluminescent bacteria

A portable biosensor has been developed to meet the demands of field toxicity analysis. This biosensor consists of three parts, a freeze-dried biosensing strain within a vial, a small light-proof test chamber, and an optic-fiber connected between the sample chamber and a luminometer. Various genetically engineered bioluminescent bacteria were freeze-dried to measure different types of toxicity based upon their modes of action. GC2 ( lac::luxCDABE), a constitutively bioluminescent strain, was used to monitor the general toxicity of samples through a decrease in its bioluminescence, while specific toxicity was detected through the use of strains such as DPD2540 ( fabA::luxCDABE), TV1061 ( grpE::luxCDABE), DPD2794 ( recA::luxCDABE), and DPD2511 ( katG::luxCDABE). These inducible strains show an increase in bioluminescence under specific stressful conditions, i.e. membrane-, protein-, DNA-, and oxidative-stress, respectively. The toxicity of a sample could be detected by measuring the bioluminescence 30 min after addition to the freeze-dried strains. In an attempt to enhance the sensitivity of the freeze-dried cells, glucose and Tween 80 were tested as additives. It was found that the addition of glucose had a negative effect on the viability of the freeze-dried cells, while samples having Tween 80 showed an increase in their viability. On the other hand, the addition of either Tween 80 or glucose decreased the final bioluminescent response of DPD2540 in response to 4-chlorophenol. Using these strains, many different chemicals were tested and characterized. This portable biosensor, with a very simple protocol, can be used for field sample analysis and the monitoring of various water systems on-site.