Time Course Of mRNA Expression Research Articles

Concurrent changes in thermal tolerance thresholds and cellular heat stress response reveals novel molecular signatures and markers of high temperature acclimation in rainbow trout.

The objective of this study was to better understand the molecular mechanisms which regulate acclimatory responses and thermal safety margins of rainbow trout (Oncorhynchus mykiss) at temperatures above physiological optimum. For this, we investigated the time course of changes in critical thermal tolerance thresholds and associated hepatic and renal transcript abundance of molecular markers related to cellular stress response, during high temperature acclimation. The experimental fish were initially acclimated to 17°C and later exposed to a gradually raised elevated temperature regime (22°C) for a period of 30 days. CTmax, CTmin and mRNA expression of candidate markers were examined before the thermal challenge (T0) and over the time-course (days) of high temperature exposure (T1, T3, T7, T15 and T30). With respect to organismal response, CTmax was significantly elevated at T3, but the degree of gain in heat tolerance was not persistent. Contrarily, we observed a gradual loss in cold tolerance with highest CTmin estimate at T30. Based on the time-course of mRNA expression, the studied markers could be categorized into those which were persistently elevated (hsp70a, hsp70b, hspa5, hsp90a, hsp90b, stip1 and serpinh1 in kidney and hsp90b in liver); those which concurred with changes in CTmin (hspbp1, hsp90b, stip1, gr1, hif1a, hyou1, tnfa and tlr5 in kidney); and those which concurred with changes in CTmax (hsp90a, serpinh1, tlr5 and lmo2 in liver). Apparently, transcriptional changes in kidney and liver reflected CTmin and CTmax trend, respectively. Expression profile of stip1 and tlr5 suggest that they are potential novel markers which could reflect thermal limits in rainbow trout. Hepatic metabolic markers were either initially elevated (alt, glud, g6pase1) or down-regulated at different time-points (ast2, gls1, fas, cpt1b, mtor), linked to gluconeogenesis and metabolic depression, respectively. Whereas, growth-axis markers showed no significant differences. Overall, this time-course analysis has revealed potential associations in organismal and tissue-specific cellular response to high temperature acclimation in a thermally sensitive coldwater ectotherm.

A high-resolution mRNA expression time course of embryonic development in zebrafish.

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.

Upregulation of cytokine mRNA in circulating leukocytes during human endotoxemia.

Endotoxemia induces pronounced changes in leukocyte count and enhances the release of many cytokines. However, the molecular regulation of this cytokine release is poorly characterized in humans. The time course of mRNA expression of 24 cytokines in circulating leukocytes was studied in a well-standardized model of human endotoxemia (2ng/kg). Real-time polymerase chain reaction (RT-PCR) was used to quantify the lipopolysaccharide (LPS)-inducible mRNA levels of leukocytes from 16 healthy volunteers in a randomized, placebo-controlled trial. Baseline mRNA levels of interleukins including IL-1α, IL-3, IL-5, IL-6, IL-12p40, IL-13, IL-15, IL-17, granulocyte colony-stimulating factor (G-CSF) and granulocyte monocyte CSF (GM-CSF) were below detectable levels in normal blood of the healthy participants. After 2h, LPS infusion increased median mRNA levels of IL-1α by >1100-fold and IL-1β and IL-8 by 33-fold and 46-fold, respectively. In contrast, levels of tumor necrosis factor (TNF-α) and IL-10 mRNA increased by only 7-fold, whereas changes in mRNA expression of other cytokines showed either a more than two fold increase or were undetectable. In vitro incubation of whole blood with 50pg/mL LPS for 2h enhanced transcription levels of IL-1α mRNA by >10,000-fold, IL-6 and IL-12p40 by >1000-fold, IL-1β by 400-fold, TNF-α by 100-fold, IL-8, IL-18, interferon γ (IFN-γ) and G-CSF by >10-25-fold, and IL-10, IL-12p35, TNF-β, and IL-13 by 10-25-fold. Only half of the 24 evaluated cytokines were expressed at the mRNA level in circulating leukocytes under basal conditions and after an LPS challenge. Only IL-1α, IL-1β, IL-10, IL-8, and TNF-α were upregulated in the circulating leukocytes, whereas several other cytokines (including IL-6 and G-CSF), were expressed on the mRNA level following in vitro incubation of blood with LPS. In addition, IL-1α and IL-1β might be potential diagnostic targets in inflammatory diseases.

An adaptive phase-locking insight to unravel mRNAs synchrony from microarray experiments.

A bulk mRNA expression time course can slow or advance modulating in phase the averaged expression of each gene within the cell-cycle. This is also a synchronism induced at the cycle start to recognize periodic genes. Consistent with the above, a novel adaptive phase-locking insight into microarray experiment is provided. The budding-yeast expression of the major role in the cell-division-cycle could be clustered in phase by a new amplitude noise immune approach which compares well to meta-analysis of the web Cyclebase and to previous results in the field. The new approach to timing elucidation seems to be well matched to cellular phenomena and might be promising as the most accurate ever applied in Fourier context.

Expression and regulation of reelin and its receptors in the enteric nervous system.

In the central nervous system (CNS), reelin coordinates migration and lamination of neurons and regulates synaptic plasticity, whereas its role in the enteric nervous system (ENS) remains enigmatic. Thus we determined the expression pattern and localization of reelin in the human ENS and monitored the time course of mRNA expression of the reelin signaling system in the rat intestine as well as in GDNF treated ENS cultures. Reelin, its receptors and Dab1 were expressed in all intestinal layers as well as in isolated myenteric ganglia. Enteric ganglia and nerve fibers were immunoreactive for reelin which co-localized with PGP 9.5 and synaptophysin. In the rat small intestine, highest expression levels of reelin were detected at early postnatal stages. Enteric nerve cell cultures treated with GDNF showed marked up-regulation of reelin and its receptors. Reelin and its receptors are strongly expressed in the human ENS. Reelin is specifically localized in enteric neurons with highest expression levels during early postnatal life as well as in neuronal network forming enteric nerve cell cultures pointing to putative functions in the differentiation and maintenance of the ENS. Gene expression of reelin, its receptors and Dab1 were analyzed in the human colon and isolated enteric ganglia. Co-localization of reelin with the pan-neuronal marker PGP 9.5 and the synaptic vesicle marker synaptophysin was studied by dual-label-immunocytochemistry. The time course of reelin expression was monitored in an ontogenetic study of rat intestines as well as in GDNF-treated cultures of enteric neurons.

Expression of c-Type Lysozyme from the Fleshy Shrimp Fenneropenaeus chinensis Is Upregulated Following Vibrio anguillarum and Lipopolysaccharide Injection

Chicken-type lysozyme (c-lysozyme) is present in shrimp and is active against some bacteria. To further understand the regulation of c-lysozyme in the fleshy shrimp Fenneropenaeus chinensis, we determined the tissue-specific gene expression of c-lysozyme and the time-course of mRNA expression in response to Vibrio anguillarum and lipopolysaccharide (LPS) injection by quantitative reverse real-time polymerase chain reaction. The results showed that c-lysozyme was expressed in all tissues tested, including gill, eyestalk, eye, hemocytes, hepatopancreas, intestine, heart, and pleopod. It was most highly expressed in the intestine followed by the eyestalk, gill, hemocytes and hepatopancreas. The mRNA expression level began to decline in a short time after V. anguillarum challenge and was then upregulated by two fold or more at 24 h post injection (hpi) compared to that at 0 h. Expression was suppressed shortly after LPS injection and began to increase with higher levels of 5.8-, 5.2- and 8.4-fold at 24, 48, and 72 hpi, respectively. Higher expression was sustained and showed a gradual increasing trend until the end of the experiment (72 hpi). These results increase our understanding of the regulation of defense mechanisms and facilitate an evaluation of the effects of probiotics or immunostimulants in shrimp culture.

Transcription in Pronuclei and One- to Four-Cell Embryos Drives Early Development in a Nematode

A long-standing view of development is that transcription is silenced in the oocyte until early divisions in the embryo. The point at which major transcription is reactivated varies between organisms, but is usually after the two-cell stage. However, this model may not be universal. We used RNA-seq and exploited the protracted development of the parasitic nematode Ascaris suum to provide a comprehensive time course of mRNA expression, degradation, and translation during early development. Surprisingly, we find that ∼4,000 genes are transcribed prior to pronuclear fusion and in the one- to four-cell embryos. Intriguingly, we do not detect maternal contribution of many orthologs of maternal C. elegans mRNAs, but instead find that these are newly transcribed in the A. suum zygote prior to pronuclear fusion. Ribosome profiling demonstrates that, in general, early embryonic mRNAs are not stored for subsequent translation, but are directly translated after their synthesis. The role of maternally contributed and zygotically transcribed genes differs between the nematodes A. suum and C. elegans despite the fact that the two nematodes appear to exhibit highly similar morphological patterns during early development. Our study indicates that major transcription can occur immediately after fertilization and prior to pronuclear fusion in metazoa, suggesting that newly transcribed genes appear to drive A. suum early development. Furthermore, the mechanisms used for controlling the timing of the expression of key conserved genes has been altered between the two nematodes, illustrating significant plasticity in the regulatory networks that play important roles in developmental outcomes in nematodes.

The effect of aryl hydrocarbon receptor ligands on the expression of polymerase (DNA directed) kappa (Polκ), polymerase RNA II (DNA directed) polypeptide A (PolR2a), CYP1B1 and CYP1A1 genes in rat liver

The aryl hydrocarbon receptor (AhR) mediates a variety of biological responses to ubiquitous environmental pollutants. AhR is ligand activated transcription factor with high affinities for aromatic planar compounds such as β-naphthoflavone (BNF), 3-methylcholanthrene (3-MC), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding appropriate ligand, AhR trigger induction of expression of some phase I and phase II drug metabolizing genes together with numerous other genes. One of such gene appear to be polymerase (DNA directed) kappa (Polκ). Polκ gene encodes newly identified low fidelity DNA polymerase. The enzyme bypasses benzo[a]pyrene-N2-dG lesions in a mostly error free manner by incorporating predominantly dC opposite the bulky lesions. It was demonstrated that AhR activation increases expression of the mouse Polκ gene and probably human POLK gene.In this study we examined the effect of i.p. administration of different AhR ligands on the expression of Polκ, RNA polymerase II polypeptide A (PolR2a) and cytochrome P450 1B1 (CYP1B1), the genes controlled by AhR in Sprague-Dawley rat liver.Quantitative real-time RT-PCR analysis revealed significant induction in the mRNA expression levels of Polκ and PolR2a following BNF treatment. Time courses of mRNA expression after treatment with BNF were similar in both genes, with maximal increases at 8h after treatment. The maximal induction of CYP1B1 and CYP1A1 expression was observed after 24 and 8h after BNF injection, respectively.TCDD treatment caused the significant increase in the mRNA level of CYP1B1 at 72h after administration of the ligand but no effect on Polκ and PolR2a mRNA expression was observed.These results confirm connection between AhR and Polκ, and strongly suggest that AhR up-regulates the mRNA transcription of PolR2a as well. However physiological importance of AhR dependent regulation of PolR2a expression must be further elucidated.

Priming with synthetic oligonucleotides attenuates pressure overload-induced inflammation and cardiac hypertrophy in mice

Inflammation and Toll-like receptor (TLR) signalling have been linked to the development of cardiac hypertrophy following transverse aortic constriction (TAC). In the present study, we investigated whether pre-treatment with the synthetic TLR9 ligands 1668-thioate or 1612-thioate modulates the progression of TAC-induced cardiac inflammation and hypertrophy. C57BL/6N-mice were pre-treated with 1668-thioate, 1612-thioate (0.25 nmol/g, i.p.), or phosphate-buffered saline 16 h prior to TAC or sham surgery. Heart-weight/body-weight ratio (HW/BW), cardiomyocyte cell size, cellular macrophage accumulation, myofibroblast differentiation, and collagen deposition were investigated for up to 28 days. Cardiac function was monitored using a pressure-volume catheter and M-mode echocardiography. Inflammatory gene expression in the heart was analysed via gene array, while the time course of mRNA expression of key inflammatory mediators was assessed via RT-qPCR. TAC increased the HW/BW ratio and cardiomyocyte cell size and induced macrophage accumulation, myofibroblast differentiation, and collagen deposition. These changes were accompanied by cardiac inflammation and a significant loss of left ventricular function. Pre-treatment with cytosine-phosphate-guanine (CpG)-containing 1668-thioate attenuated the inflammatory response, the progression of cardiac hypertrophy, and cardiac remodelling, which resulted in a prolonged preservation of left ventricular function. These changes were induced to a smaller extent by the use of the non-CG-containing oligodeoxynucleotide 1612-thioate. Pre-treatment with 1668-thioate attenuated cardiac hypertrophy following pressure overload, possibly by modifying the hypertrophy-induced inflammatory response, thereby reducing cardiac growth and fibrosis as well as delaying loss of cardiac function.

Effects of Dimethyl Sulphoxide and Dexamethasone on mRNA Expression of Myogenesis- and Muscle Proteolytic System-related Genes in Mouse Myoblastic C2C12 Cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.

Comparison of Inducibility of Sulfotransferase and UDP-Glucuronosyltransferase mRNAs by Prototypical Microsomal Enzyme Inducers in Primary Cultures of Human and Cynomolgus Monkey Hepatocytes

We investigated the change of the mRNA levels of sulfotransferase and UDP-glucuronosyltransferase isoforms by the prototypical microsomal enzyme inducers rifampicin (Rif), dexamethasone (Dex), and omeprazole (Ome) in primary cultures of cryopreserved human and cynomolgus monkey hepatocytes. Real-time RT-PCR analysis was performed using primers and TaqMan probes. Rif, Dex, and Ome increased SULT2A1 mRNA level in both human and cynomolgus monkey hepatocytes in dose-dependent manner, but not SULT1A1 mRNA level. Rif, Dex, and Ome increased the mRNA level of UGT1A1 in both human and cynomolgus monkey hepatocytes, Ome more potently in humans and Rif and Ome more potently in monkeys. They also increased the mRNA levels of UGT1A6 and UGT1A9 in cynomolgus monkey hepatocytes, though the extent of elevation of UGT1A6 and UGT1A9 mRNA levels was smaller than that of UGT1A1 mRNA level. Furthermore, these inducers scarcely affected UGT1A6 and UGT1A9 in human hepatocytes. Rif, Dex, and Ome also showed no remarkable effect on the mRNA levels of UGT2Bs in human or cynomolgus monkey hepatocytes. We also studied in detail the time course of mRNA expression of these enzymes in primary cultures of hepatocytes. In conclusion, the results of the present study show that primary cultures of hepatocytes isolated from the cynomolgus monkey liver are as useful as human hepatocytes for evaluating the induction of drug-metabolizing enzymes in preclinical studies.

Lipopolysaccharide‐induced transcriptional upregulation of interleukin‐6 and tumor necrosis factor‐α precedes muscle specific expression of the ubiquitin ligases, MAFbx and MuRF1 in the rat extensor digitorum longus muscle

The cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (Il-6), are capable of inducing skeletal muscle catabolism. Here, we characterized the time course of Lipopolysaccharide (LPS)-induced expression of TNF-α and Il-6 mRNA in the rat fast-twitch skeletal muscle, the extensor digitorum longus (EDL), and compared it to the time course of mRNA expression of the muscle specific ubiquitin(Ub)-ligases, MuRF1 and MAFbx, of the Ub-proteasome system. Conscious, male, Sprague-Dawley rats were infused i.v. with either saline (0.4 ml h-1) or LPS (150 μg kg-1 h-1) for 2h, 6h or 24h (n = 6-8 per group), after which the EDL was removed. Realtime PCR was used to quantify the TNF-α, Il-6, MAFbx and MuRF1 mRNA transcripts. Levels of TNF-α and IL-6 mRNA expression were elevated as soon as 2h after onset of LPS-infusion (10.9 ± 2.7 and 175 ± 51 fold increase over controls, respectively; P<0.01), and thereafter levels declined (4.4 ± 1.4 and 97 ± 56 fold increase at 24h). In contrast, significant elevation in the Ub-ligases did not occur until after 6h of LPS infusion, with maximal levels of MAFbx and MuRF1 expression seen after 24h LPS infusion (3.6 ± 0.7, and 19.5 ± 1.9 fold increase; respectively, P<0.05). These data are consistent with muscle-derived TNF-α and Il-6 contributing to the increase in the atrophy-inducing Ub-ligases, MAFbx and MuRF1 in sepsis. This work was supported by the Medical Research Council UK.

TILAPIA'S HYPEROSMOTIC TRANSCRIPTION FACTORS

Salt-tolerant fish like tilapia, Orechromis mossambicus, revert from actively absorbing salt in freshwater to secreting salt across their gill epithelium in seawater. This requires a fast remodelling of the cellular gill epithelium upon transfer from freshwater to seawater and is accompanied by changes in mRNA levels of several ATPases, ion transporters and water channels. Given what we know about mammalian osmotic regulation, it seems reasonable that transcriptional regulation plays a role in inducing these changes. So far, only one transcription factor (TonEBP) is known to be involved in vertebrate osmoregulation. Diego Fiol and colleagues from the University of California Davis now have evidence for two novel transcription factors that are specifically induced in response to hyperosmotic stress in fish.Fiol's team reasoned that any transcriptional changes observed after they moved tilapia from freshwater to seawater would reflect a response to help the fish cope with increased saltiness. They got their first glimpse of the transcription factors that potentially mediate this response when they screened a cDNA library obtained by subtracting cDNA of a freshwater fish from cDNA of a fish that the authors had exposed to seawater for four hours. Among the induced transcripts they found two novel transcription factors. One of these they called osmotic stress transcription factor 1 (OSTF1), and to find some clues regarding its function they compared its gene sequence to other vertebrate gene sequences. They found that OSTF1 was similar to a splice variant of a glucocorticoid-induced leucine zipper (GILZ) transcription factor that responds to glucocorticoid treatment. This suggests that cortisol, a glucocorticoid hormone that plays a major role in the acclimation of fish to high salinities, may have a similar effect on OSTF1. The second novel factor was a homologue of TFIIB, a general transcription factor found in vertebrates. Tilapia TFIIB showed high sequence identity (89.6%) with other vertebrate TFIIBs and, thus, is likely to be very similar in function to these TFIIB homologues. Importantly, the team found that OSTF1 and TFIIB possess similar phosphorylation sites for stress-signalling kinases, indicating that these two novel factors mediate stress responses and appear to be affected by similar stress signals.But were these two transcription factors really induced by hyperosmotic stress caused by a move from freshwater to seawater? The authors used quantitative PCR to follow the time course of mRNA expression before and after they transferred fish from freshwater to seawater, and found that OSTF1 and TFIIB levels significantly increased between one and four hours after the transfer. So these two factors are indeed co-induced when saltiness increases. Since there are many stress-induced genes, the authors took great care to show that these transcription factors were specifically linked to osmotic stress. Exposure to oxidative stress (H2O2) and heat-shock led to strong induction of heat-shock protein70, a general stress protein, but did not affect OSTF1 and TFIIB levels. So OSTF1 and TFIIB are not part of the general stress response, but are induced specifically by hyperosmotic stress.Although the exact roles of these two novel transcription factors and the genes they affect need to be elucidated, homologies and sites of post-translational regulation provide insights into their possible functions. Their rapid induction suggests a role in the transduction of the osmosensory signal to the acclimation response to hyperosmotic conditions. Since TFIIB is known to interact with other transcription factors, its co-induction with OSTF1 in tilapia gill cells may indicate that these two transcription factors interact during osmotic stress.

Integrative analysis of genome-scale data by using pseudoinverse projection predicts novel correlation between DNA replication and RNA transcription

We describe an integrative data-driven mathematical framework that formulates any number of genome-scale molecular biological data sets in terms of one chosen set of data samples, or of profiles extracted mathematically from data samples, designated the "basis" set. By using pseudoinverse projection, the molecular biological profiles of the data samples are least-squares-approximated as superpositions of the basis profiles. Reconstruction of the data in the basis simulates experimental observation of only the cellular states manifest in the data that correspond to those of the basis. Classification of the data samples according to their reconstruction in the basis, rather than their overall measured profiles, maps the cellular states of the data onto those of the basis and gives a global picture of the correlations and possibly also causal coordination of these two sets of states. We illustrate this framework with an integration of yeast genome-scale proteins' DNA-binding data with cell cycle mRNA expression time course data. Novel correlation between DNA replication initiation and RNA transcription during the yeast cell cycle, which might be due to a previously unknown mechanism of regulation, is predicted.

Transcriptional Activation of the MUC2 Gene by p53

MUC2 is one of the major components of mucins that provide a protective barrier between epithelial surfaces and the gut lumen. We investigated possible alterations of MUC2 gene expression by p53 and p21(Sdi1/Waf1/Cip1) in a human colon cancer cell line, DLD-1, establishing subclones in which a tetracycline-regulatable promoter controls exogenous p53 and p21 expression. MUC2 mRNA more significantly increased in response to p53 than to p21. Unexpectedly, MUC2 expression was also induced in human osteosarcoma cells, U-2OS and Saos-2, by exogenous p53. We next performed a reporter assay to test the direct regulation of MUC2 gene expression by p53. Deletion and mutagenesis of the MUC2 promoter region showed that it contains two sites for transactivation by p53. Furthermore, an electrophoretic mobility shift assay indicated that p53 binds to those elements. We analyzed MUC2 expression in other cell types possessing a functional p53 after exposure to various forms of stress. In MCF7 breast cancer and A427 lung cancer cells, MUC2 expression was increased along with the endogenous p53 level by actinomycin D, UVC, and x-ray, but not in RERF-LC-MS lung cancer cells carrying a mutated p53. These results suggest that p53 directly activates the MUC2 gene in many cell types.

Kainic acid induces leukemia inhibitory factor mRNA expression in the rat brain: differences in the time course of mRNA expression between the dentate gyrus and hippocampal CA1/CA3 subfields

Leukemia inhibitory factor (LIF) is a pluripotent cytokine which affects the survival and differentiation of various types of cells both in the hematopoietic and nervous systems. In this study, the time course and localization of LIF mRNA expression following kainic acid-induced seizures were examined by northern blot analyses and in situ hybridization. Northern blot analyses demonstrated that intraperitoneal injection of kainic acid at a convulsive dose induced LIF mRNA expression intensely in the hippocampus and moderately to weakly in the cerebral cortex, thalamus and hypothalamus. The expression peaked at 8–24 h after the injection in the hippocampus and cerebral cortex and at 8 h in the thalamus and hypothalamus. In situ hybridization revealed different time courses of LIF mRNA expression depending on the area of the hippocampus; that is, the expression peaked at 10 h in the granule cell layer of the dentate gyrus, then at 12 h in the polymorph and molecular layers of the dentate gyrus, and finally at 12–24 h in the strata oriens and radiatum of the CA1 and CA3 subfields. It is worth noting that the expression of LIF mRNA was intense in the dentate gyrus, the region where neurogenesis and aberrant network reorganization have been shown to be induced by seizures. The upregulation of LIF mRNA expression in the dentate granule cell layer followed by that in the dentate polymorph and molecular layers may be involved in activity-dependent neurogenesis in the granule cell layer and ectopic migration of granule cells to the polymorph and molecular layers in the dentate gyrus.

Sarin causes altered time course of mRNA expression of alpha tubulin in the central nervous system of rats.

Sarin induced neurotoxicity is suspected to be one of the key factors responsible for Gulf-war syndrome. We studied the effect of a single (50 microg/kg/i.m) dose of sarin (0.5 x LD50) on the mRNA expression of alpha tubulin in the central nervous system (CNS) of rats which were sacrificed at different time points i.e. 1 and 2 hrs, as well as, 1, 3 and 7 days post-treatment. Northern data collected from CNS regions indicate differential, spatial, and temporal regulation of alpha tubulin mRNA levels. Immediate induction and persistence of alpha tubulin transcripts in sarin-treated CNS suggest that sarin-induced neurotoxicity is in part mediated by the altered expression of cytoskeletal genes which may be regulated at multiple levels.

Altered time course of mRNA expression of alpha tubulin in the central nervous system of hens treated with diisopropyl phosphorofluoridate (DFP).

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus-ester induced delayed neurotoxicity (OPIDN) in the hen, human and other sensitive species. We studied the effect of single dose of DFP (1.7 mg/kg/s.c.) on the expression of alpha tubulin which is one of the major sub-unit of tubulin polymers that constitute an important constituent of cellular architecture. The hens were sacrificed at different time points i.e. 1, 2, 5, 10, and 20 days. Total RNA was extracted from the following brain regions: cerebrum, cerebellum, and brainstem as well as spinal cord. Northern blots prepared using standard protocols were hybridized with alpha tubulin as well as with beta-actin and 28S RNA cDNA (controls) probes. The results indicate a differential/spatial/temporal regulation of alpha tubulin levels which may be the result of perturbed microtubule dynamics not only in the axons but also in perikarya of neurons in the CNS of DFP treated hens. In the highly susceptible tissues like brainstem and spinal cord the initial down-regulation of mRNA levels could be attributed to DFP induced stress response resulting in inhibited cell metabolism and or cell injury/cell death. Increase in levels of mRNA at 5 days and thereafter coincided with increased tubulin transport which may be due to increased phosphorylation of tubulins in both axons and perikarya and other intraaxonal changes resulting in impaired axonal transport. DFP induced decreased rate of tubulin polymerization resulting in increased levels of free tubulin monomers may be involved in the altered alpha tubulin mRNA expression at different time points by autoregulatory circuits. Cerebellum being the less susceptible tissue showed only a moderate decline at day 2, while the alpha tubulin remained at near control levels at day 1. Delayed down-regulation may be due to the co-ordinated up or down-regulation of different sub-types of alpha and beta tubulins as well as the differential response of specialised cell types in cerebellum. Continuous overexpression of alpha tubulin in cerebrum from the beginning may be its effective protective strategy to safeguard itself from neurotoxicity. Differential expression pattern observed could be due to the differential susceptibility and variability in the rate of axonal transport of different regions besides the tubulin heterogenity of CNS. Hence our results indicte differential expression of alpha tubulin is either one of the reasons for the development of OPIDN or the result of progressive changes taking place during OPIDN.

Cell death is prevented in thalamic fields but not in injured neocortical areas after permanent focal ischaemia in mice overexpressing the anti-apoptotic protein Bcl-2.

Previous studies have suggested that various apoptotic-related proteins could be involved in the death process induced by cerebral ischaemia. In order to further clarify their role and examine how the anti-apoptotic protein Bcl-2 could influence this process, the time-course of mRNA expression of various cell death genes was studied from 1 to 14 days following permanent occlusion of the middle cerebral artery in wild-type (WT) and Bcl-2 transgenic mice, within and outside the area of infarction. No differences of the infarct sizes were observed between the two groups of mice, showing that the extent of neuronal injury could not have been lowered by the Bcl-2 transgene. Seven days after the ischaemic insult, the mRNA expression of the cell death gene effector cpp32 was dramatically upregulated in the penumbra of WT and Bcl-2 transgenic mice. Interestingly, the cpp32 transcript was markedly induced from 3 days in the ipsilateral thalamus of the two groups of mice. However, apoptotic bodies were observed in the thalamic field of WT but not transgenic mice. This suggests that cpp32 mRNA may be induced in an attempt to kill the injured cells and, in contrast to the penumbra, cell death in the thalamus may be prevented in Bcl-2 transgenic mice. Based on these results, the pathophysiological mechanisms that underly neuronal damage following ischaemia need consideration in order to evaluate the extent of neuroprotection that may be afforded by the Bcl-2 anti-apoptotic protein. Although the present study does not confirm previous data showing a protective role of Bcl-2 in neocortical infarcted areas, it suggests that anti-apoptotic therapies may constitute a possible treatment for areas of the brain remote from those directly affected by ischaemia.

Time Course of Cytokine mRNA Expression in Kidneys of Rats with Unilateral Ureteral Obstruction

The development of renal interstitial fibrosis (RIF) is related to the expression and excretion of cytokines and growth factors. Thus, we investigated the time course of mRNA expression of cytokines known as causative factors in a model of RIF in rats before and on day 10 after unilateral ureteral obstruction (UUO), when first signs of fibrosis were visible, as well as during progressive RIF. UUO causes a fivefold increase in mRNA expression of monocyte chemoattractant protein 1 15 days after surgery as compared with contralateral kidneys. The level remains elevated about three-fold up to day 25. The mRNA of the fibrogenic cytokine transforming growth factor beta 1 (TGF-β1) is increased two- to threefold during the time course, whereas the mRNAs of platelet-derived growth factor B chain (PDGF-B) and its receptor beta (PDGF-Rβ) increase after UUO, reaching their maxima on days 10–15. PDGF-B mRNA increase up to day 15, marking the onset of fibrosis, and decreases thereafter, whereas the expression of the PDGF-Rβ mRNA remains elevated more than threefold over the entire study period. Incubation of cultured renal fibroblasts with TGF-β1 and/or PDGF-B suggests that their specific action on cell growth and proliferation is maintained even when they are used in combination. The sustained elevation of TGF-β1 and PDGF-B/PDGF-Rβ mRNA levels confirms the assumption of a particular involvement of these cytokines in the pathogenesis of RIF. The mRNA expression of the gap junctional protein connexin 43 in ureteral ligated kidneys is increased sixfold already 5 days after UUO. In this way, the increased connexin 43 mRNA levels indicate a possible function in the remodeling of the kidney tissue after tubular damage and fibrosis.