Molecular forces in ribosomes and polynucleotide complexes.

The stability constant of the complex of tRNA with 50S subunits of ribosomes was compared in ordinary and heavy water. A considerable effect (about fourfold) was observed, showing the importance of hydrogen bonds in this interaction. In addition, the isotope effect of complementary polynucleotide interaction was measured for two examples. In the case of the binary complex of heptainosinic acid oligomers with poly(C) in the presence of 10^?3 M MgCl₂, the transfer from ordinary to heavy water gave an increase of the stability constant of about 5%. But in the case of a ternary complex of hexaadenylic acid with poly(U) under the same conditions, the stability constant in D₂O increased threefold. The isotope effect depends strongly on magnesium ion concentration and is possibly due to some specific mechanism of magnesium ion complexing involving water molecules.     

Involvement of adenosine A2B receptor in radiation-induced translocation of epidermal growth factor receptor and DNA damage response leading to radioresistance in human lung cancer cells

BackgroundAdenosine receptors are involved in tumor growth, progression, and response to therapy. Among them, A2B receptor is highly expressed in various tumors. Furthermore, ionizing radiation induces translocation of epidermal growth factor receptor (EGFR), which promotes DNA repair and contributes to radioresistance. We hypothesized that A2B receptor might be involved in the translocation of EGFR.MethodsWe investigated whether A2B receptor is involved in EGFR translocation and DNA damage response (γH2AX/53BP1 focus formation) of lung cancer cells by means of immunofluorescence studies. Radiosensitivity was evaluated by colony formation assay after γ-irradiation.ResultsA2B receptor was expressed at higher levels in cancer cells than in normal cells. A2B receptor antagonist treatment or A2B receptor knockdown suppressed EGFR translocation, γH2AX/53BP1 focus formation, and colony formation of lung cancer cell lines A549, calu-6 and NCI-H446, compared with a normal cell line (beas-2b). γ-Irradiation-induced phosphorylation of src and EGFR was also attenuated by suppression of A2B receptor expression.ConclusionActivation of A2B receptor mediates γ-radiation-induced translocation of EGFR and phosphorylation of src and EGFR, thereby promoting recovery of irradiated lung cancer cells from DNA damage.General significanceOur results indicate that A2B receptors contribute to radiation resistance in a cancer-cell-specific manner, and may be a promising target for radiosensitizers in cancer radiotherapy.     

Neonicotinoid resistance and cDNA sequences of nicotinic acetylcholine receptor subunits of the western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae)

The western flower thrips, Frankliniella occidentalis (Pergande), is difficult to control because of high insecticide resistance. In this study, susceptibility to major insecticides was examined in two Japanese strains (H-1 and KC) and a Chinese strain (BJ) using a leaf-dipping method. All three strains were resistant to permethrin and acetamiprid at agriculturally recommended doses. The median lethal concentration (LC₅₀) for acetamiprid was 1720 ppm in strain H-1, 4780 ppm in strain KC and >6680 ppm in strain BJ. In the presence of piperonyl butoxide, an inhibitor of cytochrome P450 monooxygenases, the LC₅₀ for acetamiprid was 312 ppm in strain H-1, 837 ppm in strain KC and 1250 ppm in strain BJ. These results suggested that metabolism by cytochrome P450 monooxygenases is involved in acetamiprid resistance in these strains, though other factors also seem to play a role. Furthermore, cDNA cloning of the nicotinic acetylcholine receptor (nAChR) subunits was performed using degenerate primers, and the presence or absence of a point mutation in nAChR β1 was confirmed. The R81T mutation that had been reported in Myzus persicae (Sulzer) nAChR β1 was not found in F. occidentalis strains tested in this study.     

The influence of heavy water of low concentration on Spirogyra,Planaria and on enzyme action

Further experiments support the original suggestion of one of the authors (T. C. B.) that low concentrations of the heavy isotope of hydrogen in water will yield more significant physiological results than pure heavy water. The water employed had been slightly concentrated by commercial electrolysis and had a specific gravity of 1.00061 or 1 part in 2,000 of deuterium. Short lengths (5 to 50 cells) ofSpirogyra filaments lived longer in the isotope water (average 6.3 days for 355 cells) as compared to their longevity in ordinary distilled water (average 3.3 days for 322 cells). The experiments indicate that this effect was not due to differences in salt, CO₂, or O₂ content of the water samples.Planaria maintained their body size for longer periods in the isotope water. Fermentation by suspensions of commercial yeast, and digestion of starch by pancreatic amylase were inhibited only after a period of incubation of the enzyme in the heavy water; the degree of inhibition of the former reaction was, however, very nearly indentical for incubation periods of 16 and 166 hours indicating a stoichiometrical relationship between the isotope and the enzyme.     

Cytosol Preparations are Inadequate for Quantitating Unoccupied Receptors for 1,25-Dihydroxyvitamin D3

Abstract

Recent studies in this laboratory have indicated that 90% of the unoccupied receptors for 1,25-dihydroxyvitamin D₃ [1,25(OH)₂-D₃] are associated with nuclear components when chick intestinal mucosa is homogenized in low salt buffer (TED: 10 mM Tris, 1.5 mM EDTA, 1.0 mM dithiothreitol, pH 7.4). This observation suggested that previously reported cytosol 1,25(OH)₂D₃ receptors could result instead from salt extraction of nuclear receptors. The studies herein indicate that tissue 1,25(OH)₂D₃ receptor recovery is 30–50% lower in cytosol prepared from KTED (0.3 M KC1 + TED) or STKM (0.25 M sucrose, 50 mM Tris, 25 mM KC1, 5 mM MgCl₂, pH 7.4) than in TED-prepared chromatin. Thus tissue concentrations of unoccupied 1,25(OH)₂D₃ receptors can be closely estimated in TED-chromatin; full quantitation can be achieved by summing the number of receptors in TED-chromatin plus TED-cytosol. Incubation at different temperatures for varying times yielded maximal receptor recovery (6.1 pmol/g mucosal wet weight) at 4°C for 4–24 h or at 23° for 30 min. Scatchard analyses confirmed that only a single class of high affinity (K_d 0.4 nM) binding sites was present under all incubation conditions. Dithiothreitol significantly improved receptor recovery both in cytosol and in chromatin preparations. Conversely, inclusion of 20% glycerol caused an artificial increase in specific H-1,25(OH)₂D₃ binding due to a second class of chromatin binding sites with ten-fold higher K_d (8.1 nM) and a greater number of binding sites than the 1,25(OF)₂D₃ receptor. In conclusion, the TED-chromatin assay procedure provides better quantitation of the tissue content of unoccupied 1,25(OH)₂D₃ receptors than do previously described techniques. The presence of unoccupied nuclear-associated 1,25(OH)₂D₃ receptors in other target tissues emphasizes the potential for erroneous physiological conclusions if these chromatin-associated receptors are overlooked.     

A synthetic BMP-2 mimicking peptide induces glioblastoma stem cell differentiation

BackgroundGlioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. In this context, Bone Morphogenetic Proteins (BMPs) have been demonstrated to induce CSC differentiation and to sensitize GBM cells to treatments.MethodsThe BMP-2 mimicking peptide, named GBMP1a, was synthesized on solid-phase by Fmoc chemistry. Structural characterization and prediction of receptor binding were obtained by Circular Dicroism (CD) and NRM analyses. Activation of BMP signalling was evaluated by a luciferase reporter assay and western blot. Pro-differentiating effects of GBMP1a were verified by immunostaining and neurosphere assay in primary glioblastoma cultures.ResultsCD and NMR showed that GBMP1a correctly folds into expected tridimensional structures and predicted its binding to BMPR-IA to the same epitope as in the native complex. Reporter analysis disclosed that GBMP1a is able to activate BMP signalling in GBM cells. Moreover, BMP-signalling activation was specifically dependent on smad1/5/8 phosphorylation. Finally, we confirmed that GBMP1a treatment is sufficient to enhance osteogenic differentiation of Mesenchymal Stem Cells and to induce astroglial differentiation of glioma stem cells (GSCs) in vitro.ConclusionsGBMP1a was demonstrated to be a good inducer of GSC differentiation, thus being considered a potential anti-cancer tool to be further developed for GBM treatment.General significanceThese data highlight the role of BMP-mimicking peptides as potential anti-cancer agents against GBM and stimulate the further development of GBMP1a-based structures in order to enhance its stability and activity.     

内源微生物驱油及其对油藏微生物活动的影响

【目的】新疆油田六中区为典型水驱普通稠油油藏，水驱效果较差，油藏具有丰富的内源微生物，本研究通过分析内源微生物驱油对油藏微生物活动的影响，确定内源微生物驱油技术在该类油藏的应用潜力。【方法】采用高通量测序及分析化学技术，系统研究实施内源微生物驱油技术后油藏细菌群落结构组成、细菌总数和功能菌群的浓度以及采出液的流体性质，总结内源微生物驱油对油藏微生物活动的影响。【结果】现场试验注入激活剂和空气后，内源微生物被显著激活，细菌群落结构发生明显变化，细菌总数及功能菌群浓度普遍提高了2–3个数量级；各种内源微生物代谢活动显著增强，与地层流体相互作用后，原油明显被乳化，最终石油采收率提高5.2%。【结论】对于内源微生物较为丰富的水驱普通稠油油藏，内源微生物驱油技术对油藏微生物活动的影响显著，具有显著的技术优势和较大的应用潜力，微生物群落结构、功能菌群浓度及其相关代谢产物可以作为评价内源微生物驱油现场激活效果的重要指标，为其他内源微生物驱油现场试验提供技术参考。     

Functional relevance of the cannabinoid receptor 2 — heme oxygenase pathway: A novel target for the attenuation of portal hypertension

Aims

In liver cirrhosis, inflammation triggers portal hypertension. Kupffer cells (KC) produce vasoconstrictors upon activation by bacterial constituents. Here, we hypothesize that the anti-inflammatory action of the cannabinoid receptor 2 (CB₂) agonists JWH-133 and GP 1a attenuate portal hypertension.

Main methods

In vivo measurements of portal pressures and non-recirculating liver perfusions were performed in rats 4 weeks after bile duct ligation (BDL). Zymosan (150 μg/ml, isolated liver perfusion) or LPS (4 mg/kg b.w., in vivo) was infused to activate the KC in the absence or presence of JWH-133 (10 mg/kg b.w.), GP 1a (2.5 mg/kg b.w.) or ZnPP IX (1 μM). Isolated KC were treated with Zymosan (0.5 mg/ml) in addition to JWH-133 (5 μM). The thromboxane (TX) B₂ levels in the perfusate and KC media were determined by ELISA. Heme oxygenase-1 (HO-1) and CB₂ were analyzed by Western blot or confocal microscopy.

Key findings

JWH-133 or GP 1a pre-treatment attenuated portal pressures following KC activation in all experimental settings. In parallel, HO-1 expression increased with JWH-133 pre-treatment. However, the inhibition of HO-1 enhanced portal hypertension, indicating the functional role of this novel pathway. In isolated KC, the expression of CB₂ and HO-1 increased with Zymosan, LPS and JWH-133 treatment while TXB₂ production following KC activation was attenuated by JWH-133 pre-treatment.

Significance

JWH-133 or GP 1a treatment attenuates portal hypertension. HO-1 induction by JWH-133 plays a functional role. Therefore, the administration of JWH-133 or GP 1a represents a promising new treatment option for portal hypertension triggered by microbiological products.     

7‐Ketocholesterol impairs phagocytosis and efferocytosis via dysregulation of phosphatidylinositol 4,5‐bisphosphate

The plasma membrane is inhomogeneously organized containing both highly ordered and disordered nanodomains. 7‐Ketocholesterol (7KC), an oxysterol formed from the nonenzymatic oxidation of cholesterol, is a potent disruptor of membrane order. Importantly, 7KC is a component of oxidized low‐density lipoprotein and accumulates in macrophage and foam cells found in arterial plaques. Using a murine macrophage cell line, J774, we report that both IgG‐mediated and phosphatidylserine‐mediated phagocytic pathways are inhibited by the accumulation of 7KC. Examination of the well‐studied Fcγ receptor pathway revealed that the cell surface receptor abundance and ligand binding are unaltered while downstream signaling and activation of spleen tyrosine kinase is not affected. However, while the recruitment of phospholipase Cγ1 was unaffected its apparent enzymatic activity was compromised resulting in sustained phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P₂] levels and polymerized actin at the base of the phagocytic cup. Additionally, we found that 7KC prevented the activation of PLCβ downstream of the P2Y₆ G‐protein coupled receptor and that 7KC impaired PLCγ activity in response to a direct elevation of cytosolic calcium induced by ionomycin. Finally, we demonstrate that 7KC partly attenuates the activity of rapamycin recruitable constitutively active PLCβ3. Together, our results demonstrate that the accumulation of 7KC impairs macrophage function by altering PtdIns(4,5)P₂ catabolism and, thus, impairing actin depolymerization required for the completion of phagocytosis.      

Metabolism of Adrenergic Receptors and Adenylate Cyclase

Abstract

The alpha₁ and beta-adrenergic receptor metabolism was studied at cell confluency in BC₃H₁ and C₆ glioma cells. After their irreversible blockade with phenoxybenzamine and a bromoacetyl derivative of pindolol (Br-AAM-pindolol) respectively the receptor reappearance allows to determine a half life of 23 hours for the alpha₁-adrenergic receptor in BC₃H₁ and a quasi absence of beta-adrenergic receptor metabolism in C₆ glioma cells at confluency. In contrast, beta-adrenergic receptor is rapidly synthesized during cell division. This metabolic stability of beta-adrenergic receptor at confluency was also observed in BC₃H₁ cells using the heavy isotope labeling of the beta-adrenergci receptor (half life of 8 days). This stability was also confirmed by the observation that at confluency in C6 glioma cells, beta adrenergic receptors reappeared at the cell surface after a complete down-regulation. In parallel with the study of the half life of adrenergic receptors, we determined in BC3H1 the halflife of the forskolin stimulated catalytic unit of the adenylate cyclase using heavy isotope tabling method.     

Difference in extractability of estradiol- and tamoxifen-receptor complex in the nuclei from MCF-7 cells with nonidet P-40

The extraction of [³H]estradiol- and [³H]tamoxifen-receptor complex in the nuclei from MCF-7 cells with the nonionic detergent Nonidet P-40 has been studied. We found that there is a striking difference in the extractability of estradiol- and tamoxifen-receptor complex from nuclei with 0.5% Nonidet P-40. The nuclear bound estradiol-receptor complex is scarcely extractable with Nonidet P-40. In contrast, almost all of the nuclear bound tamoxifen-receptor complex is extractable. The nuclear [³H]tamoxifen-receptor complex extracted in the presence of Nonidet P-40 sediments in two peaks at 7 S and 5 S. The latter sedimentation rate is the same with that of the nuclear [³H]tamoxifen-receptor complex extracted with 0.4 M KC1. The nuclear [³H]estradiol-receptor complex extracted with 0.4 M KC1 sediments at 4 S.The results suggest that interaction of tamoxifen-receptor complex with chromatin is different from that of estradiol-receptor complex.     

NOD1配体抑制人早孕期滋养细胞的侵袭功能

目的:明确固有免疫受体NOD1对人早孕期滋养细胞侵袭功能的调控及对侵袭相关因子分泌的影响。方法:采用免疫细胞化学法鉴定原代滋养细胞NOD1的表达,用transwell侵袭实验检测激活NOD1后滋养细胞侵袭功能的改变,ELISA检测配体刺激后滋养细胞MMP2和MMP9的分泌情况。结果:免疫细胞化学结果显示滋养细胞分离鉴定成功,且原代滋养细胞可以表达固有免疫受体NOD1。使用NOD1的特异性配体及非特异性配体,发现激活NOD1可以抑制滋养细胞的侵袭,且非特异性配体LPS可以下调侵袭相关金属基质蛋白酶分子MMP2和MMP9的分泌,特异性配体i E-DAP仅下调MMP9的分泌而对MMP2的分泌无影响。结论:固有免疫模式识别受体NOD1可以在早孕期滋养细胞表达,可调控滋养细胞的侵袭功能,其激活会导致侵袭相关分子MMP2和MMP9的分泌下降。     

Temperature-dependent change in rate-limiting step of the magnesium-stimulated ITPase of myosin.

The effects of temperature on Mg-ITPase activity of heavy meromyosin and myosin subfragment 1 were measured in 0.1 M KC1. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.e. one mol per two mol of myosin active sites, at 20 degrees C. However, it was almost zero mol below 8degrees C. Effects of KC1 concentration and pH on ITPase activity of heavy meromyosin at 20 degrees C were different from those below 8 degrees C, suggesting that the rate-limiting step in the Mg-ITP hydrolysis of myosin depends on temperature. The effect of temperature on the actin activation of heavy meromyosin Mg-ITPase was analyzed by measuring the temperature dependence of double-reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at low temperture. The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule.     

The Effects of Temperature and Heavy Water on Cell Division in Heat-Synchronized Cells of Tetrahymena*

SYNOPSIS. A study was undertaken of the rates of cell division of heat-synchronized cells of Tetrahymena pyriformis GL at various temperatures in water and 20, 30 and 40% heavy water. The results suggest that division rate is limited by a protein which undergoes both high and low temperature denaturation and that this protein is partially stabilized against heat-denaturation while becoming more susceptible to cold denaturation in the presence of heavy water. Thus, the optimum temperature for division shifts upward as the heavy water concentration is increased, with a maximum shift of 1 C occurring in 40% heavy water. In addition, division activity occurs in heavy water at 34 C, a temperature at which cells kept in water are blocked. Furthermore, the sharp increase in slope seen in the low temperature portion of Arrhenius plots of the data, occurs at higher temperatures when heavy water is present. Finally, at virtually all temperatures, except the highest, heavy water has a depressive effect on division rate indicating a general inhibitory influence of deuterium-substituted water on rate processes within the cell.     

Insulin activation of NADH ferricyanide reductase in human erythrocytes is mediated by the insulin receptor tyrosine kinase: a comparative study in normal and diabetic states

Summary

We have previously reported that NADH ferricyanide reductase in human erythrocytes is stimulated by insulin. Hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors like vanadate, indicating the involvement of glycolysis in the mechanism by which insulin stimulates ferricyanide reduction. Activation of erythrocyte metabolism in response to insulin could be a result of hormone binding to its receptor, inducing phosphorylation of band 3 (at a site for reversible association of glycolytic enzymes) and/or other membrane proteins like the Na⁺/H⁺ antiport. Activation of the antiporter protein by insulin can stimulate glycolysis by an increase in intracellular pH, an effect which is prevented by amiloride. Evidence for a role for tyrosine phosphorylation in triggering the reductase activation came from studies with protein kinase inhibitors. Genistein, sphingosine and acridine orange have been shown to prevent insulin-stimulated ferricyanide reduction, implicating tyrosine phosphorylation as an important signal for activation of the enzyme by insulin. To evaluate activation of the enzyme by insulin stimulated phosphorylation, a comparative study was done using erythrocytes from healthy and diabetic humans. We measured ferricyanide reductase activities in basal and insulin stimulated states. Basal activities were lower in diabetics than in normal humans. Nevertheless, hormone stimulated activities were similar, despite earlier reports of decreased receptor phosphorylation of exogenous substrates in type 2 diabetics. These observations, together with previous ones, suggest that insulin-receptor kinase interaction may mediate the action of insulin on human erythrocytes by phosphorylation of cellular proteins like band 3 and/or the Na⁺/H⁺ antiport.     

Antiestrogen action: Differential nuclear retention and extractability of the estrogen receptor

Since there is a much longer uterine nuclear retention of the U-11,100A (antiestrogen) receptor complex (UARC) than of the estradiol receptor complex (ERC) at 4–12 hrs after injection, experiments were designed to determine if there is a difference between the relative nuclear affinities for the two RCs as determined by extraction with various ionic strength mediums. Although the UARC was retained longer in the nuclear fraction $\text{in}$$\text{vivo}$, the UARC was completely extractable with 0.3M KC1 or 50mM spermine, whereas the ERC demonstrates a saltresistant form. This suggests that the ERC is more tightly bound to nuclear components through this salt-resistant form of the receptor. In addition, various intercalating agents were used to distinguish the different nuclear chromatin DNA sites where the UARC and ERC may be binding. With actinomycin D (50 uM) more ERC than UARC was retained in the nuclear fraction. However, with ethidium bromide (100uM) less ERC than UARC was retained. Also, the ERC selectively released by ethidium bromide is precisely that fraction not released by salt. These results indicate that the UARC and ERC bind to different chromatin loci.     

In vitro translation studies of the cytoplasmic nonpolysomal particles containing messenger RNA.

We analyzed the translational capacity of different kinds of free cytoplasmic messenger ribonucleoprotein complexes (free mRNP) in a Hela cell cell free system. Native free mRNP are not translated although free mRNP washed with 0.5 M KC1 can direct polypeptide synthesis. Furthermore, the 0.5 M KC1 wash possesses a factor which inhibits the translation of 0.5 M KC1 washed free mRNP as well as globin mRNA naked mRNA from plasmocytoma, or Hela cells. We also demonstrated that native free mRNP are able to form a complex with ribosomal subunits in the presence of initiation factors. This indicates that inhibition of translation by the 0.5 M KC1 wash occurs either at some point after initiation complex formation or at the elongation step.     

Modulation of the chemokines KC and MCP-1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice

Activation of the aryl hydrocarbon receptor (AhR) by TCDD may lead to the induction of proinflammatory cytokines in various cell types and organs such as liver leading to active chronic inflammation. Here we studied the expression of the chemokines keratinocyte chemoattractant (KC) and monocyte chemoattractant protein 1 (MCP-1) in different organs of mice after exposure to TCDD. TCDD exposure led to an early and clear induction of KC in liver and spleen on day 1 which was sustained over a period of 10 days. The level of MCP-1 mRNA was induced by TCDD on day 1 in spleen, lung, kidney, and liver, which was further increased at day 7. Increase of KC and MCP-1 at day 7 in liver, thymus, kidney, adipose, and heart was associated with elevated levels of the macrophage marker F4/80, indicating the infiltration of macrophages in these organs. Induction of KC requires a functional AhR since mice with a mutation in the AhR nuclear localization domain (AhR(nls)) were found to be resistant to TCDD-induced expression of KC. These results are the first showing the induction of the chemokines KC and MCP-1 in multiple organs of mice associated with an increase of the macrophage marker F4/80 indicating the involvement in TCDD's inflammatory response like infiltration of macrophages.