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.     

Activation of GABAB Receptors Ameliorates Cognitive Impairment via Restoring the Balance of HCN1/HCN2 Surface Expression in the Hippocampal CA1 Area in Rats With Chronic Cerebral Hypoperfusion

Hyperpolarization-activated cyclic-nucleotide-gated cation nonselective (HCN) channels are involved in the pathology of nervous system diseases. HCN channels and γ-aminobutyric acid (GABA) receptors can mutually co-regulate the function of neurons in many brain areas. However, little is known about the co-regulation of HCN channels and GABA receptors in the chronic ischemic rats with possible features of vascular dementia. Protein kinase A (PKA) and TPR containing Rab8b interacting protein (TRIP8b) can modulate GABA_B receptors cell surface stability and HCN channel trafficking, respectively, and adaptor-associated kinase 1 (AAK1) inhibits the function of the major TRIP8b-interacting protein adaptor protein 2 (AP2) via phosphorylating the AP2 μ2 subunit. Until now, the role of these regulatory factors in chronic cerebral hypoperfusion is unclear. In the present study, we evaluated whether and how HCN channels and GABA_B receptors were pathologically altered and investigated neuroprotective effects of GABA_B receptors activation and cross-talk networks between GABA_B receptors and HCN channels in the hippocampal CA1 area in chronic cerebral hypoperfusion rat model. We found that cerebral hypoperfusion for 5 weeks by permanent occlusion of bilateral common carotid arteries (two-vessel occlusion, 2VO) induced marked spatial and nonspatial learning and memory deficits, significant neuronal loss and decrease in dendritic spine density, impairment of long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses, and reduction of surface expression of GABA_B R1, GABA_B R2, and HCN1, but increase in HCN2 surface expression. Meanwhile, the protein expression of TRIP8b (1a-4), TRIP8b (1b-2), and AAK1 was significantly decreased. Baclofen, a GABA_B receptor agonist, markedly improved the memory impairment and alleviated neuronal damage. Besides, baclofen attenuated the decrease of surface expression of GABA_B R1, GABA_B R2, and HCN1, but downregulated HCN2 surface expression. Furthermore, baclofen could restore expression of AAK1 protein and significantly increase p-PKA, TRIP8b (1a-4), TRIP8b (1b-2), and p-AP2 μ2 expression. Those findings suggested that, under chronic cerebral hypoperfusion, activation of PKA could attenuate baclofen-induced decrease in surface expression of GABA_B R1 and GABA_B R2, and activation of GABA_B receptors not only increased the expression of TRIP8b (1a-4) and TRIP8b (1b-2) but also regulated the function of TRIP8b via AAK1 and p-AP2 μ2, which restored the balance of HCN1/HCN2 surface expression in rat hippocampal CA1 area, and thus ameliorated cognitive impairment.     

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.     

Medium components for shoot cultures of chlorophyll-deficient mutants of petunia inflata

Selected medium components were tested for 30 day growth promotion of shoot tips of Petunia inflata wild type, a cytoplasmic and a nuclear inherited chlorophyll-deficient mutant. Experiments were conducted independently with iron, sucrose, thiamine-HCl, indole-3-acetic acid (IAA), Kinetin (K), 6-benzylaminopurine (BA), coconut milk (CM), casein hydrolysate (CH), and plant extract (PE) an aqueous leaf extract, added to modified Murashige and Skoog (MS) salts and vitamins medium, and pH between 4.0 and 7.0 was also compared. The optimum concentrations of all test components were used to formulate revised MS media especially designed for in vitro shoot growth of chlorophyll-deficient petunia mutants. The optimum medium for the nuclear albino was: MS salts+1.5 mg/l thiamine-HCl+100 mg/l myo-inositol+3.5% sucrose+1% PE+5.0 mg/l IAA+0.3 mg/l K at pH 6.0; for the wild type: MS salts+0.6 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+1.0 mg/l IAA+0.3 mg/l K at pH 5.0 and for the cytoplasmic albino: MS salts+0.4 mg/l thiamine-HCl+100 mg/l myo-inositol+4% sucrose+20% CM+3% PE+1.0 mg/l K at pH 5.0. On the revised MS media a 3-, 4- and 5- fold increase in 30 day plant fresh weight occurred for the nuclear, wild type and cytoplasmic chlorophyll-deficient plants, respectively.     

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.     

Composition of the cell wall and other fractions of the autolyzed yeast form of Histoplasma capsulatum

Comparison of two strains ofHistoplasma capsulatum yielded data differing only in quantification, and the constituents observed and identified were galactose, glucose, mannose, glucosamine and amino acids. A comparison of hydrochloric acid and formic acid hydrolyses ofH. capsulatum fractions indicated hydrochloric acid to be of more value than 88 per cent formic acid hydrolysis for composition analyses. The removal of formyl esters from formic acid hydrolysates was found necessary and was accomplished byN HCl hydrolysis for 30 min. Two derivative artifacts were observed with formic acid hydrolysis; D-1, which was refractory to subsequent HCl hydrolysis, and D-2, which disappeared after HCl hydrolysis. Another artifact, D-3, was observed with 6N HCl hydrolysis of histoplasma cell wall fractions. The following conditions of hydrolysis were found to be useful: (1) glucose release was measured after hydrolysis inN HCl for 4 hr; (2) glucosamine release was measured after hydrolysis in 6N HCl for 9 hr; (3) amino acid release was accomplished by 6N HCl hydrolysis for 18 hr; and (4), hexoses released were determined by gas liquid chromatography (GLC) after hydrolysis in bothN HCl and in 88 per cent formic acid for 24 hr, followed byN HCl for 30 min. Several different types of carbohydrate polymers have been reported in the parasitic yeast form ofH. capsulatum. There is general agreement on the occurrence of amino acids as protein (8, 12, 13), chitin (7, 19) and several hexoses, including glucose and glucosamine, which are found in cell wall polymers (7, 8, 11–16, 19, 20, 24). The presence of uronic acid was also reported (14, 15), but not confirmed, by Domer, Hamilton & Harkin (8), and mannose was not found by all investigators (12). We undertook a study of graded acid hydrolyses and of composition analysis of the autolysis products of the yeast form by various procedures in order to add further to the above information.     

Age-Related Differences in NMDA Receptor Subunits of Prenatally Methamphetamine-Exposed Male Rats

There is accumulating evidence that methamphetamine (MA) is a widely abused drug popular among pregnant women. MA exposure is associated with changes in the function of neurotransmitter systems, namely the dopaminergic, serotonergic and glutamatergic systems. Since N-methyl-d-aspartate receptors (NMDA) are affected by MA-induced glutamate release, we assessed the expression of NMDAR subunits (NR1, NR2A, and NR2B) and postsynaptic density protein 95 (PSD-95), which is connected with NMDAR. We measured the expression of these proteins in adolescent (30 days old) and adult (60 days old) rat males exposed to MA during the entire prenatal period and compared them with the same parameters in age matched saline-exposed rats. There was a significant increase in the NR1 and NR2B subunits in the hippocampus of adult males, but not in adolescent males. We identified a significant change in adult MA-induced rats when compared to adult controls for NR2A and NR2B, while in adolescent MA rats this change was close to the boundary of significance. In summary, our study suggests that prenatal MA exposure is connected with changes in NMDAR subunit expression in adult rats but not in adolescent rats.     

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.     

Factors affecting the determination of total mercury in biological samples by continuous-flow cold vapor atomic absorption spectrophotometry

The acidic reduction of Hg using a continuous-flow analytical system was evaluated. With 25% SnCl₂ as the reductant, characteristic concentrations (sensitivities) of 0.44 μg/L (open cell) and 0.29 μg/L (flow-through cell) were obtained using inorganic Hg²⁺ standards in 1.5% HCl. When CH₃Hg⁺ standards were used, absorption signals were an order of magnitude lower, indicating that Sn(II) is incapable of producing Hg° from organic Hg in this acidic reduction system. Addition of CdCl₂ to the SnCl₂ reductant, as suggested by Magos (1) for the reduction of organomercurials under alkaline conditions, was without beneficial effect. Similarly, combining Sn, with another reducing agent (hydroxylamine hydrochloride), or a strong alkaline solution (40% NaOH), in the reaction coil of the flow-through system did not significantly enhance the Hg absorption signal for either inorganic or organic Hg. Recovery of Hg from spiked liver homogenates digested at 70–80°C using a HNO₃/H₂SO₄/HCl procedure and stabilized with 0.5 mM K₂Cr₂O₇ was >85%, using either inorganic Hg²⁺ or CH₃Hg⁺, indicating that this digestion procedure successfully breaks the C-Hg bond to form readily reducible Hg species. Usingl-cysteine to stabilize standards of inorganic Hg²⁺ in HCl caused significant depressions of the Hg absorption signal atl-cysteine concentrations >0.001% (≈0.5 mM); 0.1%l-cysteine caused total suppression of the Hg signal. These results indicate that: (1) acidic reduction of Hg by Sn in this continuous-flow system requires breakdown of organomercurials prior to analysis; (2) tissue digestion using HNO₃/H₂SO₄/HCl followed by the addition of K₂Cr₂O₇ to stabilize Hg²⁺ achieves this breakdown and allows good recovery of total Hg; and (3) use ofl-cysteine to complex and prevent losses of Hg should be avoided in systems using acidic reduction of Hg. Concentrations of endogenous tissue sulfhydryls are generally lower than those associated with depressed absorbance signals during the acidic reduction of Hg.     

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.     

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.     

Analysis of γ-Aminobutyric Acid (GABA) Type A Receptor Subtypes Using Isosteric and Allosteric Ligands

The GABA_A receptors (GABA_ARs) play an important role in inhibitory transmission in the brain. The GABA_ARs could be identified using a medicinal chemistry approach to characterize with a series of chemical structural analogues, some identified in nature, some synthesized, to control the structural conformational rigidity/flexibility so as to define the ‘receptor-specific’ GABA agonist ligand structure. In addition to the isosteric site ligands, these ligand-gated chloride ion channel proteins exhibited modulation by several chemotypes of allosteric ligands, that help define structure and function. The channel blocker picrotoxin identified a noncompetitive channel blocker site in GABA_ARs. This ligand site is located in the transmembrane channel pore, whereas the GABA agonist site is in the extracellular domain at subunit interfaces, a site useful for low energy coupled conformational changes of the functional channel domain. Also in the trans-membrane domain are allosteric modulatory ligand sites, mostly positive, for diverse chemotypes with general anesthetic efficacy, namely, the volatile and intravenous agents: barbiturates, etomidate, propofol, long-chain alcohols, and neurosteroids. The last are apparent endogenous positive allosteric modulators of GABA_ARs. These binding sites depend on the GABA_AR heteropentameric subunit composition, i.e., subtypes. Two classes of pharmacologically very important allosteric modulatory ligand binding site reside in the extracellular domain at modified agonist sites at other subunit interfaces: the benzodiazepine site, and the low-dose ethanol site. The benzodiazepine site is specific for certain subunit combination subtypes, mainly synaptically localized. In contrast, the low-dose (high affinity) ethanol site(s) is found at a modified benzodiazepine site on different, extrasynaptic, subtypes.     

Nicotine inhibits the proliferation by upregulation of nitric oxide and increased HDAC1 in mouse neural stem cells

Cigarette smoking (CS) is considered one of the major risk factors to cause neurodegenerative disorders. Nicotine is the main chemical in CS which is responsible for dysfunction of the brain as a neuroteratogen. Also, nicotine dependency is a real mental illness and disease. Recently, chronic nicotine exposure has been shown to cause oxidative/nitrosative stress leading to a deleterious condition to cellular death in different brain regions. However, little is known about the effects of nicotine on mouse neural stem cells (mNSCs). The aim of this study is to investigate the effects of nicotine on mNSCs and elucidate underlying mechanisms involved in expression of a diversity of genes regulated by nicotine. When mNSCs were isolated from the whole brain of embryonic day 16 mice treated with nicotine at vehicle, 100, 400, and 800 μM for 5 d, nicotine significantly decreased the number and size of neurospheres. In immunocytochemistry, nicotine-exposed mNSCs expressing nestin showed the shortened filaments and condensed nuclei. In RT-PCR, messenger RNA (mRNA) levels of proliferating cell nuclear antigen (PCNA) and sirtuin1 (SIRT1) were significantly decreased, while the production of nitric oxide and mRNA levels of cyclooxygenase2 (COX-2), tumor necrosis factor-alpha TNF-α, and histone deacetylase 1 (HDAC1) were increased in a dose-dependent manner. In addition, sodium butyrate and valproic acid, HDAC inhibitors, partially rescue proliferation of mNSCs via inhibition of HDAC1 expression and NO production. Taken together, these data demonstrate that prolonged exposure of nicotine decreased proliferation of mNSCs by increased NO and inflammatory cytokine through increased HDAC1. Furthermore, this study could help in the development of a therapy for nicotine-induced neurodegenerative disorder and drug abuse.     

The effect of resveratrol and its methylthio-derivatives on EGFR and Stat3 activation in human HaCaT and A431 cells

Epidermal growth factor receptor (EGFR) interacting with Stat3 is considered to be an attractive therapeutic target. In the current study, we investigated the effect of resveratrol and its two 4′-methylthio-trans-stilbene derivatives (3-M-4′-MTS; S2) (3,5-DM-4″-MTS; S5) on EGFR and Stat3 activation in human immortalized HaCaT keratinocytes and epidermoid carcinoma A431 cells. In the HaCaT cells both derivatives, similarly as resveratrol, decreased the total level of the EGFR receptor. In the A431 cells, resveratrol in the higher dose significantly (p < 0.05) reduced Y1173 and Y1068 EGFR residue phosphorylation, while S2 affected only the phosphorylation of the Y1068 residue. In this cell line, resveratrol in both tested doses and the S2 derivative in the lower concentration significantly diminished Stat3 binding capacity to the DNA consensus site. The effect of the tested compounds on Stat3 activation in HaCaT cells was only slightly affected. These results indicate that methylthiostilbenes are not more potent modulators of the EGFR/Stat3 complex than resveratrol and that introducing an additional methoxy group makes them less effective.     

Pyramiding Bt genes for increasing resistance of cotton to two major lepidopteran pests: Spodoptera litura and Heliothis armigera

To more effectively control two major cotton insects (cotton bollworm and Spodoptera litura) and improve the efficacy of the pest resistance management, novel transgenic plants expressing Bacillus thuringiensis Cry9C gene were generated, and gene stacking strategy was incorporated. Initially, a binary plasmid vector harboring Cry9C gene was introduced into an elite cotton cultivar Simian-3 by Agrobacterium-mediated transformation. Integration and expression of the Cry9C genes in three transgenic lines were confirmed by PCR and RT-PCR. Among these transgenic lines, T0 generation of line 16 (L-16) with normal phenotypes were selected by ELISA assays for its highest expression level of Cry9C. In T1 population of L-16, the expression level of Cry9C ranged from 29 to 45 μg/g fresh leaf. The following insect bioassays demonstrated that transgenic S3-35S::Cry9C cotton plants exhibited moderate toxicity to Heliothis armigera but strong toxicity to S. litura compared with the transgenic plants expressing Cry 1Ac gene. For incorporation of gene staking strategy, Cry9C gene and Cry 2A or Cry 1Ac were pyramided, respectively by sexual crossing. The expression of Cry9C protein in all F1 progenies had a similar level as the parent plants indicating the high heritability of Bt genes in transgenic progenies. Progenies from both Cry9C × Cry 2A and Cry9C × Cry 1Ac exhibited higher resistance to S. litura compared with their parents. Together our data demonstrated that our newly generated transgenic plants represent a reservoir of novel insect-resistant materials in cotton breeding, and the successful incorporation of gene pyramiding technology can provide a new solution of developing multiple resistance management strategies.