Changes in Gene Expression during Drying in a Desiccation-Tolerant Grass Sporobolus stapfianus and a Desiccation-Sensitive Grass Sporobolus pyramidalis

1997 ◽  
Vol 24 (5) ◽  
pp. 617 ◽  
Author(s):  
D.F. Gaff ◽  
D. Bartels ◽  
J.L. Gaff

For the first time in the grasses, a desiccation-tolerant species (Sporobolus stapfianus) was examined for evidence of drought-induced changes in gene transcription. Desiccation tolerance (the ability of this species to recover from a water potential of –540 MPa) is induced in the resurrection grass during the drying process itself. Specific mRNA was compared in extracts of air-dry, drying and fully hydrated leaves by comparisons of the encoded proteins translated in vitro and partitioned by 2- dimensional electrophoresis. Forty-one genes, that were not expressed in hydrated leaves, were transcribed during drying, whereas only 25 novel polypeptides (translated in vitro) were detected; this suggests that gene expression was controlled mainly at the transcriptional level, but possibly also at the translational level. Leaves of S. stapfianus become desiccation tolerant as they dry on intact plants with mechanically undisturbed roots, whereas leaves on plants whose roots have been disturbed die during drying. Complements of mRNA from live S. stapfianus leaves changed markedly from full hydration to 70% RWC and to air-dryness; they also differed markedly from drought-sensitive leaves (on plants with disturbed roots) at 70% RWC and dead air-dry S. stapfianus leaves and from leaves of the desiccation sensitive grass S. pyramidalis at the same water contents. Drought-induced injury could not be attributed to low abundance of mRNA in either species. Five criteria which might be involved in desiccation tolerance were applied to specific in vitro proteins of S. stapfianus; 12 novel proteins correlated with desiccation tolerance in a least four of the five criteria.

2019 ◽  
Vol 58 (1) ◽  
pp. 30-38
Author(s):  
Patricia Navarro-Rodríguez ◽  
Adela Martin-Vicente ◽  
Loida López-Fernández ◽  
Josep Guarro ◽  
Javier Capilla

AbstractCandida glabrata causes difficult to treat invasive candidiasis due to its antifungal resistance, mainly to azoles. The aim of the present work was to study the role of the genes ERG11, CDR1, CDR2, and SNQ2 on the resistance to voriconazole (VRC) in a set of C. glabrata strains with known in vitro and in vivo susceptibility to this drug. Eighteen clinical isolates of C. glabrata were exposed in vitro to VRC, and the expression of the cited genes was quantified by real time quantitative polymerase chain reaction (q-PCR). In addition, the ERG11 gene was amplified and sequenced to detect possible mutations. Ten synonymous mutations were found in 15 strains, two of them being reported for the first time; however, no amino acid changes were detected. ERG11 and CDR1 were the most expressed genes in all the strains tested, while the expression of CDR2 and SNQ2 was modest. Our results show that gene expression does not directly correlate with the VRC MIC. In addition, the expression profiles of ERG11 and efflux pump genes did not change consistently after exposure to VRC. Although individual analysis did not result in a clear correlation between MIC and gene expression, we did observe an increase in ERG11 and CDR1 expression in resistant strains. It is of interest that considering both in vitro and in vivo results, the slight increase in such gene expression correlates with the observed resistance to VRC.


2006 ◽  
Vol 26 (1) ◽  
pp. 55-67 ◽  
Author(s):  
Céline Chicault ◽  
Bertrand Toutain ◽  
Annabelle Monnier ◽  
Marc Aubry ◽  
Patricia Fergelot ◽  
...  

Regulation of iron absorption by duodenal enterocytes is essential for the maintenance of homeostasis by preventing iron deficiency or overload. Despite the identification of a number of genes implicated in iron absorption and its regulation, it is likely that further factors remain to be identified. For that purpose, we used a global transcriptomic approach, using the CaCo-2 cell line as an in vitro model of intestinal absorptive cells. Pangenomic screening for variations in gene expression correlating with intracellular iron content allowed us to identify 171 genes. One hundred nine of these genes are clustered into five types of expression profile. This is the first time that most of these genes have been associated with iron metabolism. Functional annotation of these five clusters indicates potential links between the immune response, proteolysis processes, and iron depletion. In contrast, iron overload is associated with cellular metabolism, especially that of lipids and glutathione involving redox function and electron transfer.


2006 ◽  
Vol 189 (2) ◽  
pp. 437-445 ◽  
Author(s):  
Jenny A. Hyde ◽  
Jerome P. Trzeciakowski ◽  
Jonathan T. Skare

ABSTRACT The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO2, serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO2, 3% H2, 0.087 ppm O2) and microaerophilic (1% CO2, 3.48 ppm O2) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO2. In this study we identified CO2 as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO2, independent of O2 levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO2. Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO2, indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO2 may be an additional cue for borrelial host adaptation and gene regulation.


1996 ◽  
Vol 151 (1) ◽  
pp. 87-96 ◽  
Author(s):  
G Croissandeau ◽  
N Schussler ◽  
D Grouselle ◽  
P Pagesy ◽  
C Rauch ◽  
...  

Abstract TRH gene expression in the anterior pituitary has previously been reported in the human in vivo and in the rat in vitro. Until now, modulation of this synthesis with glucocorticoids and thyroid hormones has been observed in rats. The present study demonstrates for the first time that the TRH gene is also expressed, in vivo, in the rat anterior pituitary and that anterior pituitary TRH-like immunoreactivity (TRH-LI) and elongated forms of the immediate TRH progenitor sequence (TRH-elongated peptide) contents are also modulated by estrogens (E2). To investigate the presence of proTRH mRNA in the rat anterior pituitary, total RNA was reverse transcribed (RT) and the RT products were then amplified by PCR. Treatments with E2 were performed on intact and ovariectomized (OVX) rats for 2 months. TRH-LI was measured by RIA with an antibody which did not recognize the TRH-like peptide, pGlu-Glu-Pro-NH2 (<EEP-NH2) (cross-reactivity <0·1%) and was characterized further as TRH-LI by HPLC. TRH-elongated peptides were measured by EIA and characterized by Sephadex G-50 chromatography and immunoblotting (molecular mass 25–35 kDa). The plasma prolactin levels and the pituitary sizes were increased by E2 treatment in both intact and OVX rats. Anterior pituitary TRH-LI increased in intact E2-treated rats compared with intact rats (82·7 ± 19·0 versus 39·6 ± 3·6 fmol/mg protein; means ± s.e.m.; P<0·001). This increase was greater when E2 was administered to OVX rats (599·0 ± 98·4 after E2 treatment versus 58·6 ± 3·6 fmol/mg protein; P<0·001). In intact rats, anterior pituitary TRH-elongated peptide contents were not modified by E2 treatment while they were significantly decreased in OVX E2-treated rats (144·6 ±8·8 versus 223·7 ± 9·5 fmol/mg protein; P<0·001). These results demonstrate TRH gene expression in the rat anterior pituitary in vivo and suggest that E2 treatment is responsible for an increase in anterior pituitary TRH-LI, together with a decrease in TRH-elongated peptide contents. Journal of Endocrinology (1996) 151, 87–96


2013 ◽  
Vol 641-642 ◽  
pp. 732-735
Author(s):  
Chun Xia Song ◽  
Qian Zhang ◽  
Yu Liang Xiao

Thymidylate synthase (TS) is an important target in cancer therapy, which is a folate-dependent enyme, catalyzing the de novo synthesis of dUMP. In this report, the effect of 5-flurorouracil (5-FU) on the regulation of TS gene expression was estimated in zebrafish. The results showed 5-FU could significantly increase the TS expression in zebrafish embryos. However, TS mRNA level were remained unchanged. To determine the effect of 5-FU and 5-FdUMP on translation of TS mRNA, a rabbit reticulocyte lysate translation system was used. Addition of 5-FU, not inhibited the translation of TS mRNA. While addition of 5-FdUMP, completely repressed the translation of TS mRNA. Therefore, induced expression of thymidylate synthase by 5-FU in zebrafish occurred in translational level, not in transcriptional level. The findings demonstrated that zebrafish TS protein was able to bind to its own cogate mRNA and the 5-FU regulated TS in the translational level. This is the first time to confirm that the regulation of TS is affected by TS and its cognant mRNA interaction in the whole animal level.


1980 ◽  
Vol 84 (3) ◽  
pp. 803-807 ◽  
Author(s):  
M D Norenberg ◽  
K Dutt ◽  
L Reif-Lehrer

We report here for the first time, in chick retina, Muller cell localization of glutamine synthetase (GS) activity by an immunohistochemical technique, in agreement with previous reports of glial localization of this enzyme in rat brain and retina. Age-dependent changes in the endogenous enzyme activity as well as cortisol-induced changes in GS activity, both in ovo and in vitro, measured biochemically, reflect the changes observed by staining.


Author(s):  
Doaa E. Ahmed ◽  
Fatma B. Rashidi ◽  
Heba K. Abdelhakim ◽  
Amr S. Mohamed ◽  
Hossam M. M. Arafa

Abstract Background Glufosfamide (β-d-glucosylisophosphoramide mustard, GLU) is an alkylating cytotoxic agent in which ifosforamide mustard (IPM) is glycosidically linked to the β-d-glucose molecule. GLU exerted its cytotoxic effect as a targeted chemotherapy. Although, its cytotoxic efficacy in a number of cell lines, there were no experimental or clinical data available on the oncolytic effect of oxazaphosphorine drugs in hepatocellular carcinoma. Therefore, the main objective of the current study is to assess the cytotoxic potential of GLU for the first time in the hepatocellular carcinoma HepG2 cell line model. Methods Cytotoxicity was assayed by the MTT method, and half-maximal inhibitory concentration (IC50) was calculated. Flow cytometric analysis of apoptosis frequencies was measured by using Annexin V/PI double stain, an immunocytochemical assay of caspase-9, visualization of caspase-3, and Bcl2 gene expression were undertaken as apoptotic markers. Mitochondrial membrane potential was measured using the potentiometric dye; JC-1, as a clue for early apoptosis as well as ATP production, was measured by the luciferase-chemiluminescence assay. Results Glufosfamide induced cytotoxicity in HepG2 cells in a concentration- and time-dependent manner. The IC50 values for glufosfamide were significantly lower compared to ifosfamide. The frequency of apoptosis was much higher for glufosfamide than that of ifosfamide. The contents of caspase-9 and caspase-3 were elevated following exposure to GLU more than IFO. The anti-apoptotic Bcl2 gene expression, the mitochondrial membrane potential, and the cellular ATP levels were significantly decreased than in case of ifosfamide. Conclusions The current study reported for the first time cytotoxicity activity of glufosfamide in HepG2 cells in vitro. The obtained results confirmed the higher oncolytic activity of glufosfamide than its aglycone ifosfamide. The generated data warrants further elucidations by in vivo study.


2020 ◽  
Vol 9 (12) ◽  
pp. 1135-1147
Author(s):  
Jessica S Jarmasz ◽  
Yan Jin ◽  
Hana Vakili ◽  
Peter A Cattini

Human (h) growth hormone (GH) production studies are largely limited to effects on secretion. How pituitary hGH gene (hGH-N/GH1) expression is regulated is important in our understanding of the role hGH plays in physiology and disease. Here we assess for the first time the effect of sleep deprivation (SD) and high-fat diet (HFD) on hGH-N expression in vivo using partially humanized 171hGH/CS transgenic (TG) mice, and attempted to elucidate a role for DNA methylation. Activation of hGH-N expression requires interactions between promoter and upstream locus control region (LCR) sequences including pituitary-specific hypersensitive site (HS) I/II. Both SD and diet affect hGH secretion, but the effect of SD on hGH-N expression is unknown. Mice fed a HFD or regular chow diet for 3 days underwent SD (or no SD) for 6 h at Zeitgeber time (ZT) 3. Serum and pituitaries were assessed over 24 h at 6-h intervals beginning at ZT 14. SD and HFD caused significant changes in serum corticosterone and insulin, as well as hGH and circadian clock-related gene RNA levels. No clear association between DNA methylation and the negative effects of SD or diet on hGH RNA levels was observed. However, a correlation with increased methylation at a CpG (cytosine paired with a guanine) in a putative E-box within the hGH LCR HS II was suggested in situ. Methylation at this site also increased BMAL1/CLOCK-related nuclear protein binding in vitro. These observations support an effect of SD on hGH synthesis at the level of gene expression.


Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 829-829
Author(s):  
Fengdong Cheng ◽  
Zi Wang ◽  
Hongwei Wang ◽  
Maritza Lienlaf ◽  
Karrune V. Woan ◽  
...  

Abstract Abstract 829 APCs are critical in T-cell activation and in the induction of T-cell tolerance. Epigenetic modifications of specific genes in the APC play a key role in this process, and among them, histone deacetylases (HDACs) have emerged as key participants. HDAC6 is a 131 KDa protein with preferential cytoplasmic localization where it regulates the acetylation of proteins involved in cytoskeleton, cell-cell interaction and cell migration. Emerging evidence also implicates HDAC6 in regulation of immune responses, in particular at the level of the APC/T cell immune synapse1 and in the suppressive function of regulatory T-cells2. Expanding upon these immunoregulatory properties, here we show for the first time that HDAC6 physically interacts with STAT3, a transcriptional activator of IL-10 gene expression. By co-immunoprecipitation studies and confocal studies we found that HDAC6 co-localize with STAT3 in the cytoplasm and nuclei of macrophages. Furthermore, by using several HDAC6 and STAT3 mutants we have identified that the aminoacids 503–840 of HDAC6 and the STAT3 domain comprising aminoacids 465–585 are required for this interaction. Functionally, knocking down HDAC6 in a macrophage cell line (RAW264.7-HDAC6KD) resulted in inhibition of STAT3 phosphorylation, decreased recruitment of STAT3 to the IL-10 gene promoter and abrogation of IL-10 production by these cells in response to either LPS or IL-10. Similar results were observed in dendritic cells (DCs) or macrophages isolated from HDAC6 knock-out (KO) mice. Furthermore, HDAC6KD clones or APCs from HDAC6 KO mice displayed an enhanced expression of the co-stimulatory molecule B7.2 and are better activators of antigen-specific CD4+ T-cell responses in vitro. More importantly, these APCs are able of restoring the responsiveness of anergic T-cells from lymphoma-bearing mice. Pharmacologic inhibition of HDAC6 in APCs with Tubastatin A, an isotype-selective HDAC6 inhibitor, yielded similar enhancement of APC and T-cell function in vitro. Further support for HDAC6 as an appealing target in cancer immunotherapy has been recently provided by the significant delay in tumor growth observed in either HDAC6 KO mice or in wild type mice treated with Tubastatin A. In summary, we have shown for the first time that HDAC6 interacts physically with STAT3 and such an interaction is necessary for STAT3 phosphorylation and IL-10 gene expression in APCs. Disrupting the HDAC6/STAT3/IL-10 axis in APCs with selective HDAC6 inhibitors represents a novel approach to overcome tolerogenic pathways in these cells and tip the balance towards effective antitumor immune responses. Disclosures: No relevant conflicts of interest to declare.


Parasitology ◽  
1996 ◽  
Vol 112 (1) ◽  
pp. 89-95 ◽  
Author(s):  
R. C. Ko ◽  
L. Fan

SUMMARYHeat shock proteins (HSPs) were documented for the first time in both somatic extracts and excretory/secretory (ES) products of the infective-stage larvae of Trichinella spiralis and T. pseudospiralis. Larvae recovered from muscles of infected mice were heat shocked at 37, 40, 43 and 45 °C in RPMI 1640 medium containing L-[35S]methionine. Somatic extracts and ES products of heat-shocked worms were then analysed by SDS-PAGE, autoradiography and laser densitometry. Prominent bands of HSPs were observed at 43 °C which is the optimal heat shock temperature. The major HSPs in somatic extracts of T. spiralis were 20, 47, 50, 70, 80 and 86 kDa. When the temperature was increased from 37 to 43 °C, the greatest increase in absorbance was observed in HSPs 70 and 86. In vitro translation of mRNA in a nuclease-treated rabbit reticulocyte lysate system showed an increase in the synthesis of the 80 kDa protein. This suggests that the production of HSP 80 is regulated at the transcriptional level. The major HSPs in the ES products were 11, 45, 53 and 64 kDa. In T. pseudospiralis, the major HSPs in the somatic extracts were 20, 26, 31, 50, 53, 70, 80 and 86 kDa, and in the ES products, 11, 35, 37, 41 and 64 kDa.


Sign in / Sign up

Export Citation Format

Share Document