Expression of c-jun and Egr-1 genes in rat liver in response to partial hepatectomy, and injection of turpentine.

In rat liver stimulated by partial hepatectomy a significant expression of Egr-1 and to less extent c-jun genes was observed within 15-30 min after the surgery. The induction is transient and vanishes within one hour. Egr-1 and c-jun were also induced in liver after subcutaneous injection of turpentine, a strong inducer of acute phase response. A maximum activation of these genes was observed within 4-9 hours after administration of turpentine. Both Egr-1 and c-jun extend the list of "immediate early" genes involved in proliferative response and constitute a group of genes inducible at the beginning of acute phase response.     

Effect of Tenofovir on Nucleotidases and Cytokines in HIV-1 Target Cells

Tenofovir (TFV) has been widely used for pre-exposure prophylaxis of HIV-1 infection with mixed results. While the use of TFV in uninfected individuals for prevention of HIV-1 acquisition is actively being investigated, the possible consequences of TFV exposure for the HIV-target cells and the mucosal microenvironment are unknown. In the current study, we evaluated the effects of TFV treatment on blood-derived CD4⁺ T cells, monocyte-derived macrophages and dendritic cells (DC). Purified HIV-target cells were treated with different concentrations of TFV (0.001-1.0 mg/ml) for 2 to 24hr. RNA was isolated and RT-PCR was performed to compare the levels of mRNA expression of nucleotidases and pro-inflammatory cytokine genes (MIP3α, IL-8 and TNFα) in the presence or absence of TFV. We found that TFV increases 5’-ecto-nucleotidase (NT5E) and inhibits mitochondrial nucleotidase (NT5M) gene expression and increases 5’ nucleotidase activity in macrophages. We also observed that TFV stimulates the expression and secretion of IL-8 by macrophages, DC, and activated CD4⁺ T cells and increases the expression and secretion of MIP3α by macrophages. In contrast, TFV had no effect on TNFα secretion from macrophages, DC and CD4⁺ T cells. Our results demonstrate that TFV alters innate immune responses in HIV-target cells with potential implications for increased inflammation at mucosal surfaces. As new preventive trials are designed, these findings should provide a foundation for understanding the effects of TFV on HIV-target cells in microbicide trials.     

Induction of heme oxygenase-1 and heat shock protein 70 in rat hepatocytes: The role of calcium signaling

Stress response genes including heat shock proteins are induced under a variety of conditions to confer cellular protection. This study investigated the role of calcium signaling in the induction of two stress response genes, heme oxygenase-1/hsp32 and hsp70, in isolated rat hepatocytes. Both genes were induced by cellular glutathione depletion. This induction could be inhibited by BAPTA-AM. Culturing in a calcium-free medium prevented the induction of hsp70 gene expression after glutathione depletion without affecting heme oxygenase-1 gene expression. Thapsigargin increased the gene expression of heme oxygenase-1 but not that of hsp70. Thapsigargin-induced heme oxygenase-1 induction was completely inhibited by BAPTA-AM. Incubation with the Ca²⁺-ionophore A23187 augmented heme oxygenase-1 (two-fold) and hsp70 (5.2-fold) mRNA levels. Our data suggests a significant role of Ca²⁺-dependent pathways in the induction of the two stress genes. An increase in the cytoplasmic Ca²⁺ activity seems to play a key role in the cascade of signaling leading to the induction of the two genes. However, the source of Ca²⁺ that fluxes into the cytoplasm seems to be different. Our data provides evidence for a compartmentalization of calcium fluxes, i.e. the Ca²⁺ flux from intracellular stores (e.g. the endoplasmic reticulum) plays a major role in the induction of heme oxygenase-1. By contrast, Ca²⁺ flux from the extracellular medium seems to be a mechanism initiating the cellular signaling cascade leading to hsp70 gene induction.     

Initial phases of indoleacetic acid induced growth in coleoptile segments of Avena sativa L.

The intial phases of auxin-induced growth in coleoptile segments of Avena sativa L. were investigated using a high resolution growth recording technique, based on an angular position sensing transducer. The first response to the hormone is a slight, transient reduction of the growth rate lasting about 5 min. After this phase growth rate increases to a maximum. The duration of the increase and the maximum clearly depend on the concentration of the hormone. With increasing auxin concentration the duration of the growth rate increase is reduced from about 80 min in 10^-9 M indoleacetic acid (IAA) to about 14 min in 10^-4 M IAA. After the maximum the growth rate declines. Looking at the maximum of the growth rate, we obtained a dose-response curve with a sharp increase between 10^-9 M and 10^-6 M IAA and a slight decline between 10^-6 M and 10^-4 M IAA. This result is confirmed by growth rates measured one and two hours after the application of the hormone.Abbreviations IAA indoleacetic acid     

Transcriptional control mechanisms of genes of lipid and fatty acid metabolism in the Atlantic salmon (Salmo salar L.) established cell line, SHK-1

The regulatory control mechanisms of lipid and fatty acid metabolism were investigated in Atlantic salmon. We identified sterol regulatory element binding protein (SREBP) genes in salmon and characterised their response, and the response of potential target and other regulatory genes including liver X receptor (LXR), to cholesterol and long-chain polyunsaturated fatty acids (LC-PUFA) in the salmon established cell line, SHK-1. Two cDNAs for SREBPs homologous to mammalian SREBP-1 and SREBP-2 were characterised. We identified three groups of genes whose expression responded differently to the treatments. One group of genes, including cholesterol biosynthetic genes, showed increased expression in response to lipid depletion but supplementary cholesterol or LC-PUFA had no further effect. The expression of a second group of genes belonging to fatty acid biosynthetic pathways, included fatty acid synthase, Δ6 and Δ5 fatty acyl desaturases, also increased after lipid depletion but this was negated by cholesterol or by LC-PUFA supplementation. The expression of a third group of genes including acyl-CoA oxidase, HMG-CoA reductase and Elovl5 elongase was increased by cholesterol treatment but was not affected by lipid depletion or by LC-PUFA. This same pattern of expression was also shown by liver X receptor (LXR), indicating that acyl-CoA oxidase, HMG-CoA reductase and Elovl5 are possible direct targets of LXR. This suggests that salmon Elovl5 may be regulated differently from mammalian Elovl5, which is an indirect target of LXR, responding to LXR-dependent increases in SREBP-1.     

Plasticity of Migrating CD1b+ and CD1b- Lymph Dendritic Cells in the Promotion of Th1, Th2 and Th17 in Response to Salmonella and Helminth Secretions

Dendritic cells (DCs) are pivotal in the development of specific T-cell responses to control pathogens, as they govern both the initiation and the polarization of adaptive immunity. To investigate the capacities of migrating DCs to respond to pathogens, we used physiologically generated lymph DCs (L-DCs). The flexible polarization of L-DCs was analysed in response to Salmonella or helminth secretions known to induce different T cell responses. Mature conventional CD1b⁺ L-DCs showed a predisposition to promote pro-inflammatory (IL-6), pro-Th1 (IL-12p40) and anti-inflammatory (IL-10) responses which were amplified by Salmonella, and limited to only IL-6 induction by helminth secretions. The other major population of L-DCs did not express the CD1b molecule and displayed phenotypic features of immaturity compared to CD1b⁺ L-DCs. Salmonella infection reduced the constitutive expression of TNF-α and IL-4 mRNA in CD1b^- L-DCs, whereas this expression was not affected by helminth secretions. The cytokine response of T cells promoted by L-DCs was analysed in T cell subsets after co-culture with Salmonella or helminth secretion-driven CD1b⁺ or CD1b^- L-DCs. T cells preferentially expressed the IL-17 gene, and to a lesser extent the IFN-γ and IL-10 genes, in response to Salmonella-driven CD1b⁺ L-DCs, whereas a preferential IL-10, IFN-γ and IL-17 gene expression was observed in response to Salmonella-driven CD1b^- L-DCs. In contrast, a predominant IL-4 and IL-13 gene expression by CD4⁺ and CD8⁺ T cells was observed after stimulation of CD1b⁺ and CD1b^- L-DCs with helminth secretions. These results show that mature conventional CD1b⁺ L-DCs maintain a flexible capacity to respond differently to pathogens, that the predisposition of CD1b^- L-DCs to promote a Th2 response can be oriented towards other Th responses, and finally that the modulation of migrating L-DCs responses is controlled more by the pathogen encountered than the L-DC subsets.     

芸香苷对家蚕谷胱甘肽-S-转移酶部分基因的诱导表达

谷胱甘肽-S-转移酶(glutathione-S-transferases, GSTs) 是对一种机体的解毒代谢起重要作用并可以被诱导的酶系。为了从分子水平上探究家蚕Bombyx mori GSTs 基因与植物次生物质芸香苷代谢的关系, 本实验采用双跟踪标定定量PCR (dual-spike-in qPCR)方法, 用 5×10^-1, 5×10^-2, 5×10^-3 ng/μL 3 个浓度芸香苷溶液处理家蚕5龄幼虫, 并对各组织中 GSTs Epsilon 家族不同基因的转录水平进行检测。结果显示, 浓度为 5×10^-2 ng/μL 的芸香苷溶液能诱导GSTs 基因的转录水平发生显著变化。在中肠中, BmGSTe1, BmGSTe2 和 BmGSTe6的诱导转录水平较高, 在诱导后24 h达到最大值; 在脂肪体中BmGSTe1, BmGSTe6和BmGSTe7的转录水平相对较高且分别在诱导后 2 h, 2 h和4 h 达到最大值; 而GSTs基因在马氏管中表达量均很低或者检测不到表达。与 5×10^-2 ng/μL 浓度相比, 5×10^-1 ng/μL 的芸香苷溶液诱导后各基因的转录水平上升的幅度较小并且达到峰值的时间不同, 而 5×10^-3 ng/μL 浓度的芸香苷溶液并不能使GSTs基因的诱导转录水平发生变化。结果提示, 家蚕 GSTs Epsilon 家族基因对芸香苷的代谢具有重要作用。     

Mutations in the Ca2+/H+ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis

Transient increases in cytosolic free calcium concentration ([Ca2+]cyt) are essential for plant responses to a variety of environmental stimuli, including low temperature. Subsequent reestablishment of [Ca2+]cyt to resting levels by Ca2+ pumps and antiporters is required for the correct transduction of the signal [corrected]. C-repeat binding factor/dehydration responsive element binding factor 1 (Ca2+/H+) antiporters is required for the correct transduction of the signal. We have isolated a cDNA from Arabidopsis that corresponds to a new cold-inducible gene, rare cold inducible4 (RCI4), which was identical to calcium exchanger 1 (CAX1), a gene that encodes a vacuolar Ca2+/H+ antiporter involved in the regulation of intracellular Ca2+ levels. The expression of CAX1 was induced in response to low temperature through an abscisic acid-independent pathway. To determine the function of CAX1 in Arabidopsis stress tolerance, we identified two T-DNA insertion mutants, cax1-3 and cax1-4, that display reduced tonoplast Ca2+/H+ antiport activity. The mutants showed no significant differences with respect to the wild type when analyzed for dehydration, high-salt, chilling, or constitutive freezing tolerance. However, they exhibited increased freezing tolerance after cold acclimation, demonstrating that CAX1 plays an important role in this adaptive response. This phenotype correlates with the enhanced expression of CBF/DREB1 genes and their corresponding targets in response to low temperature. Our results indicate that CAX1 ensures the accurate development of the cold-acclimation response in Arabidopsis by controlling the induction of CBF/DREB1 and downstream genes.     

Repeated resistance exercise training induces different changes in mRNA expression of MAFbx and MuRF-1 in human skeletal muscle

The gain in muscle mass as a result of resistance training is dependent on changes in both anabolic and catabolic reactions. A frequency of two to three exercise sessions per week is considered optimal for muscle gain in untrained individuals. Our hypothesis was that a second exercise session would enlarge the anabolic response and/or decrease the catabolic response. Eight male subjects performed resistance exercise on two occasions separated by 2 days. Muscle biopsies were taken from the vastus lateralis before and 15 min, 1 h, and 2 h after exercise. Exercise led to severalfold increases in phosphorylation of mTOR at Ser2448, p70 S6 kinase (p70S6k) at Ser424/Thr421 and Thr389, and ribosomal protein S6, which persisted for up to 2 h of recovery on both occasions. There was a tendency toward a larger effect of the second exercise on p70S6k and S6, but the difference did not reach statistical significance. The mRNA expression of MuRF-1, which increased after exercise, was 30% lower after the second exercise session than after the first one. MAFbx expression was not altered after exercise but downregulated 30% 48 h later, whereas myostatin expression was reduced by 45% after the first exercise and remained low until after the second exercise session. The results indicate that 1) changes in expression of genes involved in protein degradation are attenuated as a response to repetitive resistance training with minor additional increases in enzymes regulating protein synthesis and 2) the two ubiquitin ligases, MuRF-1 and MAFbx, are differently affected by the exercise as well as by repeated exercise.     

The effects of presumptive morphogens on prestalk and prespore cell gene expression in monolayers of Dictyostelium discoideum

Abstract. The expression of three prestalk cell-specific genes ( ecm A, ecm B and pDd26) was examined during in vitro differentiation in cell monolayers, in an attempt to explain the spatial heterogeneity of the prestalk region of migrating Dictyostelium pseudoplasmodia. Under these conditions ecm A, ecm B and pDd26 mRNAs were expressed sequentially in response to the addition of differentiation inducing factor-1 (DIF)-1, a temporal sequence similar to that observed during normal development. ecm A and ecm B mRNAs reached a maximum level 2–4 h after DIF-1 supplementation and then declined, whereas pDd26 mRNA levels increased more slowly but remained high 24 h after DIF addition. The increases in expression in response to increasing concentrations of either DIF-1 or DIF-2 were identical for the three genes, suggesting that neither alteration in DIF concentration nor species was an important determinant of spatial heterogeneity. Ammonia had the same inhibitory effect on the expression of all three prestalk cell-specific genes and stimulated the expression of the prespore cell-specific gene, D19. These results indicate that ammonia is also not responsible for the spatial heterogeneity of the prestalk cell region. In contrast, cyclic AMP had a differential effect on the expression of the prestalk cell specific genes: ecm A expression was variably stimulated, pDd26 expression was inhibited and ecm B expression was sometimes stimulated and sometimes inhibited. These results are difficult to explain in terms of a gradient of cyclic AMP in the prestalk region. We postulate that temporal responses are more important than spatial responses to cyclic AMP in regulating stalk cell differentiation.     

Plasticity Response in the Contralesional Hemisphere after Subtle Neurotrauma: Gene Expression Profiling after Partial Deafferentation of the Hippocampus

Neurotrauma or focal brain ischemia are known to trigger molecular and structural responses in the uninjured hemisphere. These responses may have implications for tissue repair processes as well as for the recovery of function. To determine whether the plasticity response in the uninjured hemisphere occurs even after a subtle trauma, we subjected mice to a partial unilateral deafferentation of the hippocampus induced by stereotactically performed entorhinal cortex lesion (ECL). The expression of selected genes was assessed by quantitative real-time PCR in the hippocampal tissue at the injured side and the contralesional side at day 4 and 14 after injury. We observed that expression of genes coding for synaptotagmin 1, ezrin, thrombospondin 4, and C1q proteins, that have all been implicated in the synapse formation, re-arrangement and plasticity, were upregulated both in the injured and the contralesional hippocampus, implying a plasticity response in the uninjured hemisphere. Several of the genes, the expression of which was altered in response to ECL, are known to be expressed in astrocytes. To test whether astrocyte activation plays a role in the observed plasticity response to ECL, we took advantage of mice deficient in two intermediate filament (nanofilament) proteins glial fibrillary acidic protein (GFAP) and vimentin (GFAP^−/−Vim^−/−) and exhibiting attenuated astrocyte activation and reactive gliosis. The absence of GFAP and vimentin reduced the ECL-induced upregulation of thrombospondin 4, indicating that this response to ECL depends on astrocyte activation and reactive gliosis. We conclude that even a very limited focal neurotrauma triggers a distinct response at the contralesional side, which at least to some extent depends on astrocyte activation.     

Zinc-Finger Nuclease Knockout of Dual-Specificity Protein Phosphatase-5 Enhances the Myogenic Response and Autoregulation of Cerebral Blood Flow in FHH.1BN Rats

We recently reported that the myogenic responses of the renal afferent arteriole (Af-Art) and middle cerebral artery (MCA) and autoregulation of renal and cerebral blood flow (RBF and CBF) were impaired in Fawn Hooded hypertensive (FHH) rats and were restored in a FHH.1^BN congenic strain in which a small segment of chromosome 1 from the Brown Norway (BN) containing 15 genes including dual-specificity protein phosphatase-5 (Dusp5) were transferred into the FHH genetic background. We identified 4 single nucleotide polymorphisms in the Dusp5 gene in FHH as compared with BN rats, two of which altered CpG sites and another that caused a G155R mutation. To determine whether Dusp5 contributes to the impaired myogenic response in FHH rats, we created a Dusp5 knockout (KO) rat in the FHH.1^BN genetic background using a zinc-finger nuclease that introduced an 11 bp frame-shift deletion and a premature stop codon at AA121. The expression of Dusp5 was decreased and the levels of its substrates, phosphorylated ERK1/2 (p-ERK1/2), were enhanced in the KO rats. The diameter of the MCA decreased to a greater extent in Dusp5 KO rats than in FHH.1^BN and FHH rats when the perfusion pressure was increased from 40 to 140 mmHg. CBF increased markedly in FHH rats when MAP was increased from 100 to 160 mmHg, and CBF was better autoregulated in the Dusp5 KO and FHH.1^BN rats. The expression of Dusp5 was higher at the mRNA level but not at the protein level and the levels of p-ERK1/2 and p-PKC were lower in cerebral microvessels and brain tissue isolated from FHH than in FHH.1^BN rats. These results indicate that Dusp5 modulates myogenic reactivity in the cerebral circulation and support the view that a mutation in Dusp5 may enhance Dusp5 activity and contribute to the impaired myogenic response in FHH rats.