Icl Expression Research Articles

Mechanism of lnRNA-ICL involved in lung cancer development in COPD patients through modulating microRNA-19-3p/NKRF/NF-κB axis

The incidence of lung cancer (LC) in chronic obstructive pulmonary disease (COPD) patients is dozens of times higher than that in patients without COPD. Elevated activity of nuclear factor-k-gene binding (NF-κB) was found in lung tissue of patients with COPD, and the continuous activation of NF-κB is observed in both malignant transformation and tumor progression of LC, suggesting that NF-κB and its regulators may play a key role in the progression of LC in COPD patients. Here, we report for the first time that a key long non-coding RNA (lncRNA)-ICL involved in the regulation of NF-κB activity in LC tissues of COPD patients. The analyses showed that the expression of ICL significantly decreased in LC tissues of LC patients with COPD than that in LC tissues of LC patients without COPD. Functional experiments in vitro showed that exogenous ICL only significantly inhibited the proliferation, invasion and migration in primary tumor cells of LC patients with COPD compared to LC patients without COPD. Mechanism studies have shown that ICL could suppress the activation of NF-κB by blocking the hsa-miR19-3p/NKRF/NF-κB pathway as a microRNA sponge. Furthermore, In vivo experiments showed that exogenous ICL effectively inhibited the growth of patient-derived subcutaneous tumor xenografts (PDX) of LC patients with COPD and significantly prolonged the survival time of tumor-bearing mice. In a word, our study shows that the decrease of ICL is associated with an increased risk of LC in patients with COPD, ICL is not only expected to be a new therapeutic target for LC in COPD patients, but also has great potential to be used as a new marker for evaluating the occurrence, severity stratification and prognosis of LC in patients with COPD.

Hypothetical protein FoDbp40 influences the growth and virulence of Fusarium oxysporum by regulating the expression of isocitrate lyase.

Fungal growth is closely related to virulence. Finding the key genes and pathways that regulate growth can help elucidate the regulatory mechanisms of fungal growth and virulence in efforts to locate new drug targets. Fusarium oxysporum is an important plant pathogen and human opportunistic pathogen that has research value in agricultural and medicinal fields. A mutant of F. oxysporum with reduced growth was obtained by Agrobacterium tumefaciens-mediated transformation, the transferred DNA (T-DNA) interrupted gene in this mutant coded a hypothetical protein that we named FoDbp40. FoDbp40 has an unknown function, but we chose to explore its possible functions as it may play a role in fungal growth regulatory mechanisms. Results showed that F. oxysporum growth and virulence decreased after FoDbp40 deletion. FOXG_05529 (NCBI Gene ID, isocitrate lyase, ICL) was identified as a key gene that involved in the reduced growth of this mutant. Deletion of FoDbp40 results in a decrease of more than 80% in ICL expression and activity, succinate level, and energy level, plus a decrease in phosphorylated mammalian target of rapamycin level and an increase in phosphorylated 5'-adenosine monophosphate activated protein kinase level. In summary, our study found that the FoDbp40 regulates the expression of ICL at a transcriptional level and affects energy levels and downstream related pathways, thereby regulating the growth and virulence of F. oxysporum.

Inhibitory Effects of Nitrogenous Metabolites from a Marine-Derived Streptomyces bacillaris on Isocitrate Lyase of Candida albicans.

Two nitrogenous metabolites, bacillimide (1) and bacillapyrrole (2), were isolated from the culture broth of the marine-derived actinomycete Streptomyces bacillaris. Based on the results of combined spectroscopic and chemical analyses, the structure of bacillimide (1) was determined to be a new cyclopenta[c]pyrrole-1,3-dione bearing a methylsulfide group, while the previously reported bacillapyrrole (2) was fully characterized for the first time as a pyrrole-carboxamide bearing an alkyl sulfoxide side chain. Bacillimide (1) and bacillapyrrole (2) exerted moderate (IC50 = 44.24 μM) and weak (IC50 = 190.45 μM) inhibitory effects on Candida albicans isocitrate lyase, respectively. Based on the growth phenotype using icl-deletion mutants and icl expression analyses, we determined that bacillimide (1) inhibits the transcriptional level of icl in C. albicans under C2-carbon-utilizing conditions.

Mycobacterium smegmatis GlnR Regulates the Glyoxylate Cycle and the Methylcitrate Cycle on Fatty Acid Metabolism by Repressing icl Transcription.

Mycobacterium smegmatis (Msm), along with its pathogenic counterpart Mycobacterium tuberculosis (Mtb), utilizes fatty acids and cholesterol as important carbon and energy sources during the persistence within host cells. As a dual-functional enzyme in the glyoxylate cycle and the methylcitrate cycle, isocitrate lyase (ICL, encoded by icl or MSMEG_0911) is indispensable for the growth of Msm and Mtb on short-chain fatty acids. However, regulation of icl in mycobacteria in response to nutrient availability remains largely unknown. Here, we report that the global nitrogen metabolism regulator GlnR represses icl expression by binding to an atypical binding motif in the icl promoter region under nitrogen-limiting conditions. We further show that GlnR competes with PrpR, a transcriptional activator of icl, and dominantly occupies the co-binding motif in the icl promoter region. In the absence of GlnR or in response to the excess nitrogen condition, Msm cells elongate and exhibit robust growth on short-chain fatty acids due to the PrpR-mediated activation of icl, thereby inducing enhanced apoptosis in infected macrophages. Taken together, our findings reveal the GlnR-mediated repression of icl on fatty acid metabolism, which might be a general strategy of nutrient sensing and environmental adaptation employed by mycobacteria.

Inhibitory Effects of Diketopiperazines from Marine-Derived Streptomyces puniceus on the Isocitrate Lyase of Candida albicans.

The glyoxylate cycle is a sequence of anaplerotic reactions catalyzed by the key enzymes isocitrate lyase (ICL) and malate synthase, and plays an important role in the pathogenesis of microorganisms during infection. An icl-deletion mutant of Candida albicans exhibited reduced virulence in mice compared with the wild type. Five diketopiperazines, which are small and stable cyclic peptides, isolated from the marine-derived Streptomyces puniceus Act1085, were evaluated for their inhibitory effects on C. albicans ICL. The structures of these compounds were elucidated based on spectroscopic data and comparisons with previously reported data. Cyclo(L-Phe-L-Val) was identified as a potent ICL inhibitor, with a half maximal inhibitory concentration of 27 μg/mL. Based on the growth phenotype of the icl-deletion mutants and icl expression analyses, we demonstrated that cyclo(L-Phe-L-Val) inhibits the gene transcription of ICL in C. albicans under C2-carbon-utilizing conditions.

The Role of the Glyoxylate Shunt in the Acclimation to Iron Limitation in Marine Heterotrophic Bacteria

Iron (Fe) is an essential element for marine microbial growth but is present in trace amounts (<0.1 nM) in surface waters of the ocean. In heterotrophic bacteria, Fe-limitation particularly impacts ATP production as Fe is an essential co-factor of enzymes involved in the electron-transport chain as well as the tricarboxylic acid (TCA) cycle. Fe-limitation can therefore drastically reduce both bacterial growth and respiration, consequently affecting the efficiency of organic carbon remineralization. Heterotrophic bacteria possess various strategies to cope with Fe-limitation. In the present study we tested the hypothesis that the induction of the glyoxylate shunt can represent one such strategy. Genetic approaches were used to gain insight into the potential role the glyoxylate shunt may have in alleviating Fe-stress using the gammaproteobacterium Photobacterium angustum S14. A recombinant bioluminescent reporter of P. angustum S14 (icl-luc) revealed a strong and significant increase in icl expression when cells were subjected to strong Fe-limitation. Although both the growth and respiration rates decreased for the wildtype and an isocitrate lyase knockout mutant (∆icl) under strong Fe-limitation, they were ±30% lower for ∆icl as compared to the wildtype. Complementation of ∆icl restored the growth and respiration rates to those observed in the wild type, further confirming the importance of the glyoxylate shunt under strong Fe-limitation. Due to the ubiquitous nature of the glyoxylate shunt within marine bacteria, our results lead us to propose this pathway as an important acclimation strategy for marine heterotrophic bacteria that are subjected to Fe-limitation.

BactImAS: a platform for processing and analysis of bacterial time-lapse microscopy movies.

BackgroundThe software available to date for analyzing image sequences from time-lapse microscopy works only for certain bacteria and under limited conditions. These programs, mostly MATLAB-based, fail for microbes with irregular shape, indistinct cell division sites, or that grow in closely packed microcolonies. Unfortunately, many organisms of interest have these characteristics, and analyzing their image sequences has been limited to time consuming manual processing.ResultsHere we describe BactImAS – a modular, multi-platform, open-source, Java-based software delivered both as a standalone program and as a plugin for Icy. The software is designed for extracting and visualizing quantitative data from bacterial time-lapse movies. BactImAS uses a semi-automated approach where the user defines initial cells, identifies cell division events, and, if necessary, manually corrects cell segmentation with the help of user-friendly GUI and incorporated ImageJ application. The program segments and tracks cells using a newly-developed algorithm designed for movies with difficult-to-segment cells that exhibit small frame-to-frame differences. Measurements are extracted from images in a configurable, automated fashion and an SQLite database is used to store, retrieve, and exchange all acquired data. Finally, the BactImAS can generate configurable lineage tree visualizations and export data as CSV files. We tested BactImAS on time-lapse movies of Mycobacterium smegmatis and achieved at least 10-fold reduction of processing time compared to manual analysis. We illustrate the power of the visualization tool by showing heterogeneity of both icl expression and cell growth atop of a lineage tree.ConclusionsThe presented software simplifies quantitative analysis of time-lapse movies overall and is currently the only available software for the analysis of mycobacteria-like cells. It will be of interest to the community of both end-users and developers of time-lapse microscopy software.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2105-15-251) contains supplementary material, which is available to authorized users.

Changes of swelling-activated chloride channels in atrial myocardium of rabbits with heart failure

ObjectiveHeart failure is associated with an increased incidence of atrial fibrillation in patients with chronic heart failure. The potential mechanism has not been well explored. Recent study has demonstrated that...

Inhibition to excitation ratio regulates visual system responses and behavior in vivo

The balance of inhibitory to excitatory (I/E) synaptic inputs is thought to control information processing and behavioral output of the central nervous system. We sought to test the effects of the decreased or increased I/E ratio on visual circuit function and visually guided behavior in Xenopus tadpoles. We selectively decreased inhibitory synaptic transmission in optic tectal neurons by knocking down the γ2 subunit of the GABA(A) receptors (GABA(A)R) using antisense morpholino oligonucleotides or by expressing a peptide corresponding to an intracellular loop of the γ2 subunit, called ICL, which interferes with anchoring GABA(A)R at synapses. Recordings of miniature inhibitory postsynaptic currents (mIPSCs) and miniature excitatory PSCs (mEPSCs) showed that these treatments decreased the frequency of mIPSCs compared with control tectal neurons without affecting mEPSC frequency, resulting in an ∼50% decrease in the ratio of I/E synaptic input. ICL expression and γ2-subunit knockdown also decreased the ratio of optic nerve-evoked synaptic I/E responses. We recorded visually evoked responses from optic tectal neurons, in which the synaptic I/E ratio was decreased. Decreasing the synaptic I/E ratio in tectal neurons increased the variance of first spike latency in response to full-field visual stimulation, increased recurrent activity in the tectal circuit, enlarged spatial receptive fields, and lengthened the temporal integration window. We used the benzodiazepine, diazepam (DZ), to increase inhibitory synaptic activity. DZ increased optic nerve-evoked inhibitory transmission but did not affect evoked excitatory currents, resulting in an increase in the I/E ratio of ∼30%. Increasing the I/E ratio with DZ decreased the variance of first spike latency, decreased spatial receptive field size, and lengthened temporal receptive fields. Sequential recordings of spikes and excitatory and inhibitory synaptic inputs to the same visual stimuli demonstrated that decreasing or increasing the I/E ratio disrupted input/output relations. We assessed the effect of an altered I/E ratio on a visually guided behavior that requires the optic tectum. Increasing and decreasing I/E in tectal neurons blocked the tectally mediated visual avoidance behavior. Because ICL expression, γ2-subunit knockdown, and DZ did not directly affect excitatory synaptic transmission, we interpret the results of our study as evidence that partially decreasing or increasing the ratio of I/E disrupts several measures of visual system information processing and visually guided behavior in an intact vertebrate.

Analysis of the experimental compressibility of deep water marine sediments from the Gulf of Guinea

This study strives to analyze the compressibility of deep water marine sediments originating from the Gulf of Guinea. Our approach is multi-scaled and, on the macroscopic level, based on two equations which link the overburden effective stress with the liquidity index, the Biarez & Favre equation on the one hand, and the Burland equation on the other hand (after calculation of the ICL expression as a function of the I L parameter). On the local level, the evolution of the microstructures is analysed and compared with GoG remoulded clay. Two additional observation techniques have been used, the scanning electron microscope and the mercury intrusion porosimetry.

Overexpression of isocitrate lyase—glyoxylate bypass influence on metabolism in Aspergillus niger

In order to improve the production of succinate and malate by the filamentous fungus Aspergillus niger the activity of the glyoxylate bypass pathway was increased by over-expression of the isocitrate lyase ( icl) gene. The hypothesis was that when isocitrate lyase was up-regulated the flux towards glyoxylate would increase, leading to excess formation of malate and succinate compared to the wild-type. However, metabolic network analysis showed that an increased icl expression did not result in an increased glyoxylate bypass flux. The analysis did show a global response with respect to gene expression, leading to an increased flux through the oxidative part of the TCA cycle. Instead of an increased production of succinate and malate, a major increase in fumarate production was observed. The effect of malonate, a competitive inhibitor of succinate dehydrogenase (SDH), on the physiological behaviour of the cells was investigated. Inhibition of SDH was expected to lead to succinate production, but this was not observed. There was an increase in citrate and oxalate production in the wild-type strain. Furthermore, in the strain with over-expression of icl the organic acid production shifted from fumarate towards malate production when malonate was added to the cultivation medium. Overall, the icl over-expression and malonate addition had a significant impact on metabolism and on organic acid production profiles. Although the expected succinate and malate formation was not observed, a distinct and interesting production of fumarate and malate was found.

Ca2+-activated Cl- current reduces transmural electrical heterogeneity within the rabbit left ventricle.

Various cationic membrane channels contribute to the heterogeneity of action potential configuration between the transmural layers of the left ventricle. The role of anionic membrane channels is less intensively studied. We investigated the role of the Ca2+-activated Cl- current, ICl(Ca), in transmural electrical heterogeneity. We determined the density of ICl(Ca) and its physiological role in subepicardial and subendocardial ventricular myocytes of rabbit using the patch-clamp technique. ICl(Ca) was measured as the 4,4'diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) sensitive current. The current-voltage relationships and the densities of ICl(Ca) were similar in subepicardial and subendocardial myocytes. However, the functional role of ICl(Ca) exhibited striking differences. In subendocardial myocytes, blockade of ICl(Ca) by DIDS increased action potential duration (APD) significantly at all measured stimulus frequencies (3.33-0.2 Hz). In subepicardial myocytes, ICl(Ca) blockade increased APD only at 3.33 Hz, but not at the lower stimulus frequencies. At 1 Hz, ICl(Ca) blockade in subepicardial myocytes only caused an APD increase when the transient outward K+ current, Ito1, was blocked. The densities and gating properties of ICl(Ca) are similar in subepicardial and subendocardial myocytes. ICl(Ca) contributes to APD shortening in subendocardial, but not in subepicardial myocytes except at 3.33 Hz. These differences in functional expression of ICl(Ca) reduce the electrical heterogeneity in rabbit left ventricle.

Axotomy-induced expression of calcium-activated chloride current in subpopulations of mouse dorsal root ganglion neurons.

Whole cell patch-clamp recordings of calcium-activated chloride current [ICl(Ca)] were made from adult sensory neurons of naive and axotomized mouse L4-L6 lumbar dorsal root ganglia after 1 day of culture in vitro. A basal ICl(Ca) was specifically expressed in a subset of naive medium-diameter neurons (30-40 microm). Prior nerve injury, induced by sciatic nerve transection 5 days before experiments, increased both ICl(Ca) amplitude and its expression in medium-diameter neurons. Moreover, nerve injury also induced ICl(Ca) expression in a new subpopulation of neurons, the large-diameter neurons (40-50 microm). Small-diameter neurons (inferior to 30 microm) never expressed ICl(Ca). Regulated ICl(Ca) expression was strongly correlated with injury-induced regenerative growth of sensory neurons in vitro and nerve regeneration in vivo. Cell culture on a substrate not permissive for growth, D,L-polyornithine, prevented both elongation growth and ICl(Ca) expression in axotomized neurons. Regenerative growth and the induction of ICl(Ca) expression take place 2 days after injury, peak after 5 days of conditioning in vivo, slowly declining thereafter to control values. The selective expression of ICl(Ca) within medium- and large-diameter neurons conditioned for rapid, efficient growth suggests that these channels play a specific role in postinjury behavior of sensory neuron subpopulations such as neuropathic pain and/or axonal regeneration.

The cold-inducible icl gene encoding thermolabile isocitrate lyase of a psychrophilic bacterium, Colwellia maris.

The gene encoding isocitrate lyase (ICL; EC 4.1.3.1) of a psychrophilic bacterium, Colwellia maris, was cloned and sequenced. The ORF of the gene (icl) was 1584 bp long, and the predicted gene product consisted of 528 aa (molecular mass 58150 Da) and showed low homology with the corresponding enzymes from other organisms. The analyses of amino acid content and primary structure of the C. maris ICL suggested that it possessed many features of a cold-adapted enzyme. Primer extension and Northern blot analyses revealed that two species of the icl mRNAs with differential lengths of 5'-untranslated regions (TS1 and TS2) were present, of which the 5' end (TS1 and TS2 sites) were G and A, located at 130 and 39 bases upstream of the translation start codon, respectively. The levels of TS1 and TS2 mRNAs were increased by both acetate and low temperature. The induction of icl expression by low temperature took place in the C. maris cells grown on succinate as the carbon source but not acetate. Furthermore, a similar manner of inductions was also found in the levels of the translation and the enzyme activity in cell-free extract. These results suggest that the icl gene, encoding thermolabile isocitrate lyase, of C. maris is important for acetate utilization and cold adaptation.

Expression of glyoxylate cycle genes in cucumber roots responds to sugar supply and can be activated by shading or defoliation of the shoot.

When cucumber roots are excised and incubated without a carbon source, isocitrate lyase (ICL) and malate synthase (MS) mRNAs increase significantly in amount. However, if sucrose is added to the excised roots, the mRNAs do not accumulate. Hairy roots obtained by transformation with Agrobacterium rhizogenes show the same response. Transgenic hairy roots containing the Icl and Ms gene promoters fused to the GUS reporter gene, have very low GUS activity which increases dramatically when roots are incubated in the absence of sugar, indicating regulation at the transcriptional level. Staining of sugar-deprived roots shows that GUS activity is concentrated mainly in root tips and lateral root primordia, where demand for carbohydrate is greatest. In order to determine if Icl and Ms genes are expressed in roots of whole plants under conditions which may occur in nature, cucumber plants were subjected to reduced light intensity or defoliation. In both cases increases were observed in ICL and MS mRNAs. These treatments also reduced root sugar content, consistent with the hypothesis that sugar supply could control expression of Icl and Ms genes in roots of whole plants.

Molecular cloning of cucumber phosphoenolpyruvate carboxykinase and developmental regulation of gene expression.

A cDNA library from RNA of senescing cucumber cotyledons was screened for sequences also expressed in cotyledons during post-germinative growth. One clone encodes ATP-dependent phosphoenolpyruvate carboxykinase (PCK; EC 4.1.1.49), an enzyme of the gluconeogenic pathway. The sequence of a full-length cDNA predicts a polypeptide of 74,397 Da which is 43%, 49% and 57% identical to bacterial, trypanosome and yeast enzymes, respectively. The cDNA was expressed in Escherichia coli and antibodies raised against the resultant protein. The antibody recognises a single polypeptide of ca. 74 kDa, in extracts of cotyledons, leaves and roots. The cucumber genome contains a single pck gene. In the seven-day period after seed imbibition, PCK mRNA and protein steady-state levels increase in amount in cotyledons, peaking at days 2 and 3 respectively, and then decrease. Both accumulate again to a low level in senescing cotyledons. This pattern of gene expression is similar to that of isocitrate lyase (ICL) and malate synthase (MS). When green cotyledons are detached from seedlings and incubated in the dark, ICL and MS mRNAs increase rapidly in amount but PCK mRNA does not. Therefore it seems unlikely that the glyoxylate cycle serves primarily a gluconeogenic role in starved (detached) cotyledons, in contrast to post-germinative and senescing cotyledons where PCK, ICL and MS are coordinately synthesised. While exogenous sucrose greatly represses expression of icl and ms genes in dark-incubated cotyledons, it has a smaller effect on the level of PCK mRNA.