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.     

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.     

Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC 6.2.1.12), which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation.     

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.     

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.     

Implementation of Transportation Distance for Analyzing FLIM and FRET Experiments

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.     

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.     

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.     

Intromitochondrial IκB/NF-κB signaling pathway is involved in amyloid β peptide-induced mitochondrial dysfunction

Mitochondrial dysfunction is a hallmark of amyloid β peptide (Aβ)-induced neuronal toxicity in Alzheimer’s disease (AD). However, the underlying mechanism (s) of Aβ-induced mitochondrial dysfunction is still not fully understood. There is evidence that nuclear factor-κB (NF-κB) is involved in Aβ-induced neurotoxicity and is present in mitochondria. Using HT22 murine hippocampal neuronal cells and isolated mitochondria, the present study investigated whether intramitochondrial inhibitor of NF-κB (IκB)/NF-κB signaling pathway was involved in mitochondrial dysfunction induced by Aβ. It was found that Aβ impaired mitochondrial function through a NF-κB-dependent signaling pathway. Intramitochondrial IκBα/NF-κB pathway, induced by Aβ, decreased the expression of cytochrome c oxidase subunit (COXIII) and inhibited COX activity. These results provide new insights into the mechanism underlying the neurotoxic effect of Aβ and open up new therapeutic perspectives for AD.     

Response of the population size and community structure of Paenibacillus spp. to different fertilization regimes in a long-term experiment of red soil

Background and aims

Paenibacillus spp. are widely considered to impact the fertility and health of soil. The aim of this study was to evaluate how different fertilization regimes affect the population size and community structure of Paenibacillus spp. over a long period of time in red soil.

Methods

Soil samples were collected from a long-term experiment and were then analyzed using real-time PCR and PCR-DGGE. The correlation analysis, PCA and RDA were used to explore the relationships among Paenibacillus spp. population, community structure and soil properties in different treatments.

Results

The pH was seriously decreased only by the application of chemical fertilizer. The largest population of Paenibacillus spp. was found in the soil treated with organic fertilizer application, while the richest diversity was observed in the soil treated only with the chemical fertilizer. The Paenibacillus spp., Paenibacillus alkaliterrae, Paenibacillus campinasensis, and Paenibacillus xylanilyticus were found in all treatments. Paenibacillus castaneae was found in the soil treated with NPK, and Paenibacillus pabuli was specifically observed in the lime-amended treatment. Paenibacillus taichungensis and Paenibacillus prosopidis were detected in the soil treated with only chemical fertilizer. Except for the ammonium and pH, all the tested soil fertility parameters (total C, total N, nitrate, available K and available P) could significantly affect both the Paenibacillus spp. population number and diversity. The soil pH was significantly correlated with Paenibacillus spp. diversity only.

Conclusions

Our results indicate that the different long-term fertilization regimes have varied impact on both the Paenibacillus spp. population size and the diversity of the community associated with the soil properties tested. These results can help to enrich the information on the response of beneficial soil microbes to different long-term fertilization regimes.     

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.     

Expression of microRNA-1, microRNA-133a and Hand2 protein in cultured embryonic rat cardiomyocytes

In this study, we investigated the expression of the pathway, SRF–microRNA-1/microRNA-133a–Hand2, in the Wistar rat embryonic ventricular cardiomyocytes under conventional monolayer culture. The morphological observation of the cultured cardiomyocytes and the mRNA expression levels of three vital constituent proteins, MLC-2v, N-cadherin, and connexin43, demonstrated the immaturity of these cultured cells, which was featured by less myofibril density, immature sarcomeric structure, and significantly lower mRNA expression of the three constituent proteins than those in neonatal ventricular samples. More importantly, results in this study suggest that the change of SRF–microRNA-1/microRNA-133a–Hand2 pathway results into the attenuation of the Hand2 repression in cultured cardiomyocytes. These outcomes are valuable to understand the cellular state as embryonic cardiomyocytes to be in vitro model and might be useful for the assessment of engineered cardiac tissue and cardiac differentiation of stem cells.     

GDNF is Involved in the Barrier-Inducing Effect of Enteric Glial Cells on Intestinal Epithelial Cells Under Acute Ischemia Reperfusion Stimulation

Acute intestinal ischemia reperfusion (IR) injury is often associated with intestinal epithelial barrier (IEB) dysfunction. Enteric glial cells (EGCs) play an essential role in maintaining the integrity of IEB functions. However, the precise mechanism of EGCs under IR stimulation remains unclear. Here, we report that EGCs are closely involved in the modulation of IEB functions in response to IR challenge. The intestinal IR treatment led to the significant upregulation of the EGC activation marker, glial fibrillary acidic protein, accompanied by the increasing abundance of glial-derived neurotrophic factor (GDNF) and inducible nitric oxidase (iNOS) proteins, which was also confirmed in in vitro hypoxia reoxygenation (HR) tests. Co-culturing with EGCs attenuated the tight junctional abnormalities, blocked the downregulation of ZO-1 and occludin protein expression, and relieved the decrease of permeability of intestinal epithelial cell (IEC) monolayers under HR treatment. Furthermore, exogenous GDNF administration displays the barrier-protective effects similar to EGCs against HR stimulation, while RNA interference-mediated knockdown of GDNF significantly inhibited the protective capability of EGCs. The expression of both GDNF and iNOS proteins of EGCs was significantly upregulated by co-culturing with IECs, which was further increased by HR treatment. Interestingly, through inhibiting iNOS activity, the barrier-protective effect of EGCs was influenced in normal condition but enhanced in HR condition. These results suggest that GDNF plays an important role in the barrier-protective mechanism of activated EGCs under IR stimulation, whereas EGCs (via iNOS release) are also involved in intestinal inflammation response, which may contribute to IEB damage induced by IR injury.