Pseudomonas aeruginosa contains an inducible methylcitrate synthase

Assaying for enzymes of propionyl-CoA catabolism in crude extracts ofPseudomonas aeruginosa was complicated by the presence of a short-chain acyl-CoA hydrolase. Interference by the hydrolase was avoided by using permeabilized whole cells. Cells treated this way exhibited low or undetectable levels of propionyl-CoA carboxylase, propionyl-CoA dehydrogenase, and α-hydroxyglutarate synthase, enzymes initiating propionyl-CoA catabolism in many prokaryotes, but did contain high levels of methylcitrate synthase, an enzyme of the methylcitrate cycle for propionyl-CoA catabolism. Gel filtration experiments revealed a peak of methylcitrate synthase distinct from the citrate synthase of the bacterium. The enzyme was induced by growth on propionate and on propanol and heptanoate, precursors of propionate. These results suggest thatP. aeruginosa may utilize the methylcitrate cycle to metabolize propionate, a pathway heretofore only described in the yeastCandida lipolytica.     

Localization of β-oxidation enzymes in peroxisomes isolated from nonfatty plant tissues

Peroxisomes from spinach leaves, mungbean hypocotyls, and potato tubers catalyze a palmitoyl-CoA-dependent, KCN-insensitive O₂ uptake. In the course of this reaction O₂ is reduced to H₂O₂ in a 1:1 stoichiometry and palmitoyl-CoA oxidized, in a 1:1 stoichiometry, to a product serving as substrate for enoyl-CoA hydratase. These findings demonstrate the existence of a peroxisomal acyl-CoA oxidase in these tissues. Enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35), and thiolase (EC 2.3.1.9) are also associated with the peroxisomes from mung-bean hypocotyls and potato tubers (as well as with spinach leaf peroxisomes as recently reported; Gerhardt 1981, FEBS Lett. 126, 71). The low activities of these enzymes in mitochondrial fractions seem to be due to contaminating peroxisomes since the ratio of β-oxidation enzyme activities to catalase activity did not significantly differ between peroxisomal and mitochondrial fractions isolated on sucrose density gradients. The proof of localization of β-oxidation enzymes in peroxisomes without glyoxysomal function leads to the concept that fatty-acid oxidation is a consistent basic function of the peroxisome in cells of higher plants.     

Isoenzymes of hydroxycinnamate: CoA ligase from poplar stems properties and tissue distribution

Three different forms of hydroxycinnamate: CoA ligase (EC 6.2.1-) have been separated by chromatofocusing from poplar stems. These three forms exhibit different substrate specificities and tissue distribution. A correlation was established between the monomeric composition of lignins isolated from xylem and sclerenchyma and the pattern of hydroxycinnamate: CoA ligase isoforms in these tissues. The results obtained indicate that, in poplar, the hydroxycinnamate: CoA ligase isoenzymes could play an important role in the control of the monomeric composition of lignins.     

Functional identification of ELO-like genes involved in very long chain fatty acid synthesis in Arabidopsis thaliana

Very long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh. that AtELO1, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELO1, AtELO3, and AtELO4 were localized in the endoplasmic reticulum (ER), where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10–20% in seeds of atelo1 T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and might lay the foundation for further genetic manipulations to generate oil crops with the high oil content.     

Quercetin Potentiates the Antitumor Activity of Rituximab in Diffuse Large B-Cell Lymphoma by Inhibiting STAT3 Pathway

STAT3 pathway plays an important role in the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of Quercetin, a flavonoid compound, in combination with rituximab in DLBCL cell lines in vitro. We found that Quercetin synergistically enhanced rituximab-induced growth inhibition and apoptosis in DLBCL cell lines. Moreover, we found Quercetin exerted inhibitory activity against STAT3 pathway and downregulated the expression of survival genes. These results suggest that combining the Quercetin with rituximab may present an attractive and potentially effective way for the treatment of DLBCL.     

The synthesis of short- and long-chain acylarnitine by etio-chloroplasts of greening barley leaves

Etio-chloroplasts of barley, purified on sucrose density gradients were shown to possess carnitine long-chain acyltransferase (carnitine palmitoyltransferase, EC 2.3.1.21) activity and carnitine short-chain acyltransferase (carnitine acetyltransferase EC 2.3.1.7) activity. These enzymes may play a role in the transport of acyl groups as acylcarnitines through the membrane barrier of barley etio-chloroplasts and also ‘or alternatively’ may spare CoA by transferring short- and long-chain acyl groups from short-and long-chain acyl CoA to carnitine.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

Primary alveolar macrophages exposed to diesel particulate matter increase RAGE expression and activate RAGE signaling

Receptors for advanced glycation end-products (RAGE) are members of the immunoglobulin superfamily of cell-surface receptors implicated in mechanisms of pulmonary inflammation. In the current study, we test the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) exposed to diesel particulate matter (DPM). Quantitative RT-PCR and immunoblotting revealed that RAGE was up-regulated in Raw264.7 cells, an immortalized murine macrophage cell line and primary AMs exposed to DPM for 2 h. Because DPM increased RAGE expression, we exposed Raw264.7 cells and primary AMs isolated from RAGE null and wild-type (WT) mice to DPM prior to the assessment of inflammatory signaling intermediates. DPM led to the activation of Rat sarcoma GTPase (Ras), p38 MAPK and NF-κB in WT AMs and, when compared to WT AMs, these intermediates were diminished in DPM-exposed AMs isolated from RAGE null mice. Furthermore, cytokines implicated in inflammation, including IL-4, IL-12, IL-13 and TNFα, were all significantly decreased in DPM-exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that diesel-induced inflammatory responses by primary AMs are mediated, at least in part, via RAGE signaling mechanisms. Further work may show that RAGE signaling in both alveolar epithelial cells and resident macrophages is a potential target in the treatment of inflammatory lung diseases exacerbated by environmental pollution.     

Coenzyme A-transferase-independent butyrate re-assimilation in Clostridium acetobutylicum—evidence from a mathematical model

The hetero-dimeric CoA-transferase CtfA/B is believed to be crucial for the metabolic transition from acidogenesis to solventogenesis in Clostridium acetobutylicum as part of the industrial-relevant acetone-butanol-ethanol (ABE) fermentation. Here, the enzyme is assumed to mediate re-assimilation of acetate and butyrate during a pH-induced metabolic shift and to faciliate the first step of acetone formation from acetoacetyl-CoA. However, recent investigations using phosphate-limited continuous cultures have questioned this common dogma. To address the emerging experimental discrepancies, we investigated the mutant strain Cac-ctfA398s::CT using chemostat cultures. As a consequence of this mutation, the cells are unable to express functional ctfA and are thus lacking CoA-transferase activity. A mathematical model of the pH-induced metabolic shift, which was recently developed for the wild type, is used to analyse the observed behaviour of the mutant strain with a focus on re-assimilation activities for the two produced acids. Our theoretical analysis reveals that the ctfA mutant still re-assimilates butyrate, but not acetate. Based upon this finding, we conclude that C. acetobutylicum possesses a CoA-tranferase-independent butyrate uptake mechanism that is activated by decreasing pH levels. Furthermore, we observe that butanol formation is not inhibited under our experimental conditions, as suggested by previous batch culture experiments. In concordance with recent batch experiments, acetone formation is abolished in chemostat cultures using the ctfa mutant.     

Glycosylation and subsequent malonylation of isoflavonoids in E. coli: strain development,production and insights into future metabolic perspectives

Genistin and daidzein exhibit a protective effect on DNA damage and inhibit cell proliferation. Glycosylation and malonylation of the compounds increase water solubility and stability. Constructed pET15b-GmIF7GT and pET28a-GmIF7MAT were used for the transformation of Escherichia coli and bioconversion of genistein and daidzein. To increase the availability of malonyl-CoA, a critical precursor of GmIF7MAT, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinia were also introduced. Thus, the isoflavonoids were glycosylated at position 7 by 7-O-glycosyltranferase and were further malonylated at position 6^″ of glucose by malonyl-CoA: isoflavone 7-O-glucoside-6^″-O-malonyltransferase both from Glycine max. Engineered E. coli produced 175.7 µM (75.90 mg/L) of genistin and 14.2 µM (7.37 mg/L) genistin 6″-O-malonate. Similar conditions produced 162.2 µM (67.65 mg/L) daidzin and 12.4 µM (6.23 mg/L) daidzin 6″-O-malonate when 200 µM of each substrate was supplemented in the culture. Based on our findings, we speculate that isoflavonoids and their glycosides may prove useful as anticancer drugs with added advantage of increased solubility, stability and bioavailability.     

Effect of Recombinant hPTEN Gene Expression on PDGF Induced VSMC Proliferation

Human phosphatase and tensin homolog (hPTEN) gene was expressed in vascular smooth muscle cells (VSMCs) to study its effect on VSMC proliferation induced in platelet-derived growth factor (PDGF) conditioned medium. After G418 selection, MTT assay was conducted to examine transfected VSMC proliferation induced in human PDGF conditioned medium. We successfully constructed eukaryotic expression vector pcDNA4/myc-His-PTEN and transferred into VSMC cells. We report that in vitro proliferation of VSMC was inhibited in PTEN transfected VSMCs induced in PDGF conditioned medium. RT-PCR and Western blot results indicated significantly high levels of protein kinase B-PKB and nuclear factor kappa B mRNA and protein, respectively, in PDGF group as compared with the control group.     

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-β) and their effect on in vitro development of caprine preantral follicles

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.     

Suppression of PI3K/Akt signaling by synthetic bichalcone analog TSWU-CD4 induces ER stress- and Bax/Bak-mediated apoptosis of cancer cells

Suppression of the activity of pro-apoptotic Bcl-2-family proteins frequently confers chemoresistance to many human cancer cells. Using subcellular fractionation, the ER calcium (Ca⁺⁺) channel inhibitor dantrolene and small interfering RNA (siRNA) against Bax or Bak, we show that the new synthetic bichalcone analog TSWU-CD4 induces apoptosis in human cancer cells by releasing endoplasmic reticulum (ER)-stored Ca⁺⁺ through ER/mitochondrial oligomerization of Bax/Bak. Blockade of the protein kinase RNA-like ER kinase or the unfolded protein response regulator glucose-regulated protein 78 expression by siRNA not only suppressed oligomeric Bax/Bak-mediated pro-caspase-12 cleavage and apoptosis but also resulted in an inhibition of Bcl-2 downregulation induced by TSWU-CD4. Induction of the ER oligomerization of Bax/Bak and apoptosis by TSWU-CD4 were suppressed by Bcl-2 overexpression. Inhibition of lipid raft-associated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling by TSWU-CD4 induced ER stress- and oligomeric Bax/Bak-mediated apoptosis, which were substantially reversed by overexpression of the wt PI3K p85α subunit. Taken together, these results suggest that suppression of lipid raft-associated PI3K/Akt signaling is required for the ER stress-mediated apoptotic activity of Bax/Bak, which is responsible for the ability of TSWU-CD4-treated cancer cells to exit the ER-mitochondrial apoptotic cell death pathway.     

Supplementation Dietary Zinc Levels on Growth Performance,Carcass Traits,and Intramuscular Fat Deposition in Weaned Piglets

This study was conducted to estimate dietary zinc (Zn) levels on growth performance, carcass traits, and intramuscular fat (IMF) deposition in weaned piglets. Sixty piglets were randomly divided into five groups, as follows: control (basal diet), Zn250, Zn380, Zn570, and Zn760 with supplementation of 250, 380, 570, and 760 mg Zn/kg of the basal diet, respectively. The final weight, average daily gain (ADG), gain/feed (G/F), lean meat percentage, fat meat percentage, lean eye area, backfat thickness, and IMF content were dose-dependently increased in all groups of Zn treatment. The serum total triglycerides (TG) and free fatty acid (FFA) were significantly higher in all Zn treatments than in the control. The enzyme activities of lipoprotein lipase (LPL), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) were markedly higher, while enzyme activities of hormone-sensitive lipase (HSL) and carnitine palmitoyltransferase-1 (CPT-1) were significantly lower in all Zn treatments than in the control. The messenger RNA (mRNA) levels of sterol regulatory element-binding protein 1 (SREBP-1), stearoyl-CoA desaturase (SCD), FAS, ACC, peroxisome proliferator-activated receptor γ (PPARγ), LPL, and adipocyte fatty acid-binding protein (A-FABP) were significantly higher, while the mRNA levels of CPT-1 and HSL were significantly lower in all Zn treatments compared with the control. These results indicated that high levels of Zn increased IMF accumulation by up-regulating intramuscular lipogenic and fatty acid transport gene expression and enzyme activities while down-regulating lipolytic gene expression and enzyme activities.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.     

Calcitonin Gene-Related Peptide Inhibits Osteolytic Factors Induced by Osteoblast In Co-Culture System with Breast Cancer

Recently, it was found that α-Calcitonin gene-related peptide (CGRP) was associated with breast cancer metastases, but the role of CGRP in interaction between breast cancer and osteoblast during bone metastases is not clear. Here, we investigated the effect of CGRP on osteoblast in co-culture system with breast cancer. Using a breast cancer–osteoblast co-culture system, we chose MDA-MB-231 for breast cancer and human cell line MG-63 for osteoblast. CGRP was added to this co-culture system. The expression levels of the Runx2, RANK1, and osteoprotegerin (OPG) were analyzed using real-time PCR and western blot. CGRP receptors were investigated by immunofluorescence. We found that breast cancer cells cause osteolysis lesions by upregulating Runx2 expression, decreasing OPG expression, and increasing RANKL expression in osteoblasts. Our data prove that CGRP can regulate osteoclast coupling genes in osteoblast by increasing OPG, and decreasing RANKL and Runx2 expressions in a time-dependent manner; and inhibit those osteolytic factors induced by interaction between breast cancer cells and osteoblast. This inhibition could be abolished by the CGRP antagonist, CGRP8–37. In conclusion, calcitonin receptor-like receptor is the key player for CGRP’s effect in this co-culture system.