Phase diagrams in the binary systems of 2,4,7-trinitrofluoren-9-one with aromatic and heteroaromatic compounds

Phase diagrams of the binary systems formed by 2,4,7-trinitrofluoren-9-one (TNF) and naphthalene (N), anthracene (AN), pyrene (P), fluorene (F), dibenzothiophene (DBT) or carbazole (C) have been elaborated by differential scanning calorimetry and hot-stage microscopy. Apart from known 1 : 1 charge transfer complexes, the existence of incongruently melting DA₂ compounds has been found in ANTNF, PTNF and CTNF systems Evidence of a solid—solid transition in the C · TNF complex is also given.The literature data for the eutectic temperature and composition in ANTNF and CTNF systems have been verified and completed and the melting enthalpies of DBT,C, N · TNF, AN · (TNF)₂, P · (TNF)₂, F · TNF, DBT · TNF, C · TNF and C · (TNF)₂ are given.     

Post-translational processing of tumor necrosis factor production

To elucidate the mechanism of tumor necrosis factor (TNF) production, we analyzed proteins produced in macrophages sharing the epitope of TNF according to the priming and triggering of TNF production. Rabbit alveolar macrophages primed with Bacillus Calmette-Guerin (BCG) were isolated and cultured in vitro with 35S-methionine, and the proteins produced were analyzed using anti-rabbit TNF monoclonal antibody. Primed with BCG, alveolar macrophages synthesized two proteins with molecular sizes of 50 and 17 kilodaltons (kDa) (p50 and p17) sharing the same epitope with mature TNF within the cells. These two proteins were released into the medium where other proteins were detected without TNF-activity. Cultured with lipopolysaccharide (LPS triggering), the primed alveolar macrophages released TNF-activity into the medium where p17 together with many larger proteins was detected by immunoprecipitation. In vitro translation of messenger ribonucleic acid (mRNA) from BCG-primed macrophages showed that primary TNF has a molecular size of 28 kDa (p28). These results suggest that active TNF of p17 is secreted when triggered via post-translational processing of the precursor molecules synthesized through priming with BCG.     

Ultrasensitive Electrochemical Immunosensor for Detection of Tumor Necrosis Factor‐α Based on Functionalized MWCNT‐Gold Nanoparticle/Ionic Liquid Nanocomposite

A novel and highly sensitive electrochemical immunosensor was developed for the detection of protein biomarker tumor necrosis factor‐alpha (TNF‐α) based on immobilization of TNF‐α‐antibody (anti‐TNF‐α) onto robust nanocomposite containing gold nanoparticles (AuNP), multiwalled carbon nanotubes (MWCNTs) and ionic liquid (1‐buthyl‐3‐methylimidazolium bis (trifluoromethyl sulfonyl)imide). Functionalized MWCNT‐gold nanoparticle was produced by one‐step synthesis based on the direct redox reaction. The electrochemical properties of nanocomposite were characterized by electrochemical impedance spectroscopy and cyclic voltammetry. The anti‐TNF‐α was immobilized or entrapped in the nanocomposite and used in a sandwich type complex immunoassay with anti‐TNF‐α labeled with horseradish peroxidase as secondary antibody. Under optimum conditions, the immunosensor could detect TNF‐α in a linear range from 6.0 to 100 pg mL^?1 with a low detection limit of 2.0 pg mL^?1. The simple fabrication method, high sensitivity, good reproducibility, stability, as well as acceptable accuracy for TNF‐α detection in human serum samples are the main advantages of this immunosensor, which might have broad applications in protein diagnostics and bioassay.     

Immunomagnetic separation of tumor necrosis factor alpha. I. Batch procedure for human temporomandibular fluid.

A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.     

A new assay based on solid-phase extraction procedure with LC-MS to measure plasmatic concentrations of tenofovir and emtricitabine in HIV infected patients

A new solid-phase extraction (SPE) method has been developed and validated on a liquid chromatography (LC) coupled with a mass spectrometer for the determination of plasma concentrations of tenofovir (TNF) and emtricitabine (FTC) in HIV infected patients. Chromatographic separation was achieved with a gradient (acetonitrile and water with formic acid 0.05%) on an Atlantis 4.6 mm x 150 mm, reversed phase analytical column. Detection of TNF, FTC, and internal standard (IS) was achieved by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. Calibration ranged from 15.6 to 4000 ng/mL for TNF and 11.7 to 3000 ng/mL for FTC. Plasma was analyzed, and the limit of quantitation was 15.6 ng/mL for TNF and 11.7 ng/mL for FTC; limit of detection was 2 ng/mL for TNF and 1.5 ng/mL for FTC. Mean recovery of TNF, FTC, and IS were 46.5% [relative standard deviation (RSD): 8.8%] and 88.8% (RSD: 1.0%), and 81.7% (RSD: 3.1%), respectively. The method did not show any significant interference with antiretrovirals or other concomitant drugs administered to patients, and no significant "matrix effects" were observed. The method was applied for the determination of antiretroviral plasma concentration of HIV-positive patients treated with FTC and/or TNF, in combination with various other antiretrovirals.     

Possible involvement of a tumor necrosis factor (TNF)-like mediator as an endogenous pyrogen in fever induction by Nocardia rubra cell wall skeleton (N-CWS)

Tumor necrosis factor (TNF), a cytokine produced in macrophages, also acts as an endogenous pyrogen (EP). To investigate whether TNF has a role in the fever induced by Nocardia rubra cell wall skeleton (N-CWS), the relationship between fever and TNF production was studied in guinea pigs. N-CWS injected i.v. to guinea pigs caused biphasic fever and had L-929 cell-killing activity which resembled that of TNF in the sera 30 min before the first phase of fever appeared. In vitro, L-929 cell-killing activity was demonstrated in the culture supernatant of guinea pig peritoneal macrophages pretreated with N-CWS, and the activity increased dependently on N-CWS concentration or culture duration. When the supernatant of the macrophages was fractionated by gel filtration and each fraction was assayed for fever-inducing and L-929 cell-killing activities, the fraction with the cell-killing activity also induced fever with characteristics similar to that by i.v. injection of N-CWS in guinea pigs. These results suggest that TNF acts as an EP on the fever induced by N-CWS in guinea pigs.     

Induction of smectic A phases with calamitic molecules and 2,4,7-trinitrofluorenone: the influence of branching and chain length

Binary systems consisting of 2,5-diphenyl-1,3,4-thiadiazole derivatives incorporating an allene unit in one of the terminal chains and the electron acceptor 2,4,7-trinitrofluorenone (TNF) have been investigated. Though the diphenylthiadiazole cores do not represent typical electron donor units, the nematic and smectic C phases observed for the pure compounds were suppressed and replaced by smectic A phases which in most cases have a higher stability than the nematic phases of the pure compounds. The substitution pattern around the allene moiety allowed a systematic study of the influence of steric effects on the mesophase induction by TNF. Compounds with long and especially those with branched terminal chains can take up a larger number of TNF molecules and can reach a higher stability of the induced SmA phase than those with shorter and unbranched chains. The induction of SmA phases is explained as the result of attractive intermolecular interaction between the diphenylthiadiazole rigid cores and TNF molecules provided by donor-acceptor interactions and quadrupole interactions, as well as a consequence of microsegregation and space filling effects.     

Microwaves and cellular immunity. I. Effect of whole body microwave irradiation on tumor necrosis factor production in mouse cells

Whole body microwave sinusoidal irradiation of male NMRI mice with 8.15-18 GHz (1 Hz within) at a power density of 1 microW/cm2 caused a significant enhancement of TNF production in peritoneal macrophages and splenic T lymphocytes. Microwave radiation affected T cells, facilitating their capacity to proliferate in response to mitogenic stimulation. The exposure duration necessary for the stimulation of cellular immunity ranged from 5 h to 3 days. Chronic irradiation of mice for 7 days produced the decreasing of TNF production in peritoneal macrophages. The exposure of mice for 24 h increased the TNF production and immune proliferative response, and these stimulatory effects persisted over 3 days after the termination of exposure. Microwave treatment increased the endogenously produced TNF more effectively than did lipopolysaccharide, one of the most potential stimuli of synthesis of this cytokine. The role of microwaves as a factor interfering with the process of cell immunity is discussed.     

Isolation, characterization and expression of the complementary DNA for human tumor necrosis factor (TNF-alpha)

A cDNA for human TNF-alpha (615bp) was isolated by means of polymerase chain reaction (PCR) using first strand cDNA from PMA-induced HL-60 cells as template. The result from sequencing the 615 bp cDNA fragment indicated that it corresponded to the entire sequence of mature human TNF coding region. Direct expression of mature human TNF was achieved using a plasmid pHT-1 constructed by ligation of the cDNA and a synthetic DNA. The IPTG-induced bacterial product (hTNF) showed cytotoxicity to mouse L-929 cells. The TNF activity was further identified by neutralization of a specific monoclonal antibody against human TNF-alpha. Approximately 80,000 units of activity were detected per ml of culture at A600 = 2.     

Synergistic induction of polypeptides by tumor necrosis factor and interferon-gamma in cells sensitive or resistant to tumor necrosis factor: assessment by computer based analysis of two-dimensional gels using the PDQUEST system

Tumor necrosis factor (TNF) synergistically enhanced the antiproliferative activity of interferon-gamma (IFN-gamma) in both TNF-sensitive and TNF-resistant variants of the cervical carcinoma line, ME-180. TNF alone had no apparent effect on the levels of synthesis of individual proteins in either of these variant cell lines as assessed by computerized two-dimensional gel analysis of cell lysates using the PDQUEST system. However, IFN-gamma enhanced the levels of 18 polypeptides and suppressed the levels of 10 polypeptides in both cell lines. When used in combination in both cell lines, TNF and IFN-gamma induced the synthesis of 10 polypeptides that were not induced by either agent alone. These synergistically induced polypeptides may be crucial to the mechanism of the synergistic antiproliferative action of TNF and IFN-gamma in ME-180 cells.     

Expression of a partial synthetic human TNF cDNA in E. coli.

A recombinant human tumour necrosis factor (rhTNF) cDNA was constructed. The TNF gene was isolated from a human genomic gene library. There are four exons in the TNF gene. The fourth exon codes for 140 amino acids of the TNF matured protein which is composed of 157 amino acids. A major portion of the fourth exon was isolated and then ligated to a synthesized DNA fragment coding for the remaining amino acids. The partial synthetic hTNF (rhTNF) cDNA thus generated was subcloned into a vector and successfully expressed in E. coli. 5-1 fermentator was used to produce rhTNF. About 20 g (wet weight) of bacterial pellet per liter medium and 10(6)-10(7) units of cytotoxicity to L929 cells per milliliter medium were obtained. rhTNF was purified by HPLC and dried with a freeze dryer. rhTNF with a purity of about 95% in the form of white powder was obtained. The sequence of ten amino acids at the amino terminus of the rhTNF was determined. The result showed that it was identical with that of the natural human TNF.     

Immunomagnetic separation of tumor necrosis factor alpha. II. In situ procedure for the human gingival space.

An in situ procedure has been developed for the separation of tumor necrosis factor (TNF) alpha directly from the fluid in the human gingival space. Paramagnetic beads coated with anti-TNF monoclonal antibodies were introduced into the gingival space of the subject with a polypropylene-tipped calibrated delivery system and retrieved using a permanent magnet designed to fit into the space. After retrieval, the amount of immunoadsorbed TNF was quantified using an immunochemical assay called the "cluster assay". The results indicate that following the appropriate preparation of the site, over 95% of the beads could be recovered. With this method we found that 62% of those cavities sampled contained TNF and that the values ranged from 0.10 to 13.0 ng/ml with a mean value of 1.7 ng/ml. A comparison of these values with those obtained from the same space using other methods suggests that the immunomagnetic method was more effective in retrieval of TNF. Because the separation is performed in situ we have named the procedure "chromatobiosis".     

Isolation and characterization of stable nanofiber from turmeric spent using chemical treatment by acid hydrolysis and its potential as antimicrobial and antioxidant activities

Turmeric spent, a by-product of turmeric processing industries, was used as a source to prepare nanofibers (NF). The chemical treatments methods followed by acid hydrolysis accompanied with high pressure homogenization were used to prepare NF. The resulting turmeric nanofibers (TNF) were characterized by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and thermogravimetric analysis (TGA/DTA). The TNF presented needle like structure, high thermal stability, an average width of 38.5?nm, average length of 245.7?nm, and giving an aspect ratio (L/D) of 23.15. The prepared TNF showed pronounced antimicrobial activity against Bacillus cereus, Escherichia Coli, Salmonella typhimurium and Staphylococcus aureus and also registered good antioxidant activity. The results showed that TNF were successfully obtained from turmeric spent and might be potentially applied in different fields, such as pharmaceutical, biological active species, nutraceuticals, components for food industries and bionanocomposites.     

2,4,5,7-Tetranitrofluorescein in solutions: novel type of tautomerism in hydroxyxanthene series as detected by various spectral methods

Stepwise dissociation and tautomerism of 2,4,5,7-tetranitrofluorescein (TNF) were studied by using vis-spectroscopy in dimethylsulfoxide (DMSO), in aqueous acetone, and in cetyl-trimethylammonium chloride (CTAC) micellar solutions at ionic strength of the bulk phase 4.00M KCl. The pK(a) values in DMSO and 90 mass% (CH3)2CO as well as the 'apparent'pK(a)(a) values of the substance in micellar media were determined spectrophotometrically. The neutral (molecular) form H2R is found to be completely converted into the colorless lactone. Moreover, the lactonic structure, yellow due to 'nitrophenolate' absorption band, predominates also in the case of TNF dianion R2-. Contrary to the unsubstituted fluorescein, and like 2,4,5,7-tetrabromofluorescein (eosin), the monoanion HR- of TNF with lambda(max) 522-525 nm and E(max) approximately (60-62)x10(3) dm(3)mol(-1)cm(-1) exists mainly as a deeply and intensively colored structure with non-ionized carboxylic and ionized hydroxylic group; its fluorescence spectra in various media are registered. In 90% acetone, the Stokes shift is 1.17x10(3)cm(-1), fluorescence lifetime equals 2.3 ns. An extremely expressed trend to dianion-lactone formation of R2- ion of TNF is confirmed in the systems studied. For TNF in DMSO, in aqueous acetone, in surfactant micelles, and in trichloromethane extracts of ionic associatiates with N(n-Bu)4+ and N(n-Hept)4+, the deeply colored 'quinon-phenolate' dianion, typical for all hydroxyxanthenes, is not registered at all. The sequence of dissociation of functional groups in solution is confirmed using IR spectroscopy in DMSO.     

TNFa在血管内皮细胞ECV304中的靶向表达研究

在缺失了3'LTR U3区内病毒的启动子／增强子序列的逆转录病毒载体pLXSNd中,用血管内皮生长因子受体KDR的特异性启动子调控了TNFa在血管内细胞ECV304中的靶向表达。将构建的载体pLXSN-TNFa,pLXSNd-KDRp-TNFa和空载体pLXSN用PA317细胞包装后获得重组病毒,并用重组病毒分别感染NIH3T3细胞和ECV304细胞,培养物上清的ELISA结果证明,KDR启动子指导的TNFa在KDR阳性细胞ECV304中的表达量为在KDR阴性细胞NIH3T3中的表达量的8倍;而TR指导的TNFa在这两种细胞中的表达无明显差异,实现了TNFa在血管内皮细胞中的靶向表达,这可能为肿瘤基因治疗提供新途径。     

A simple and fast LC–MS/MS method for the simultaneous determination of tenofovir alafenamide and tenofovir in human plasma

A simple and fast liquid chromatography–tandem mass spectrometry method was established and validated for the simultaneous determination of tenofovir alafenamide (TAF) and tenofovir (TNF) in human plasma. A simple protein precipitation procedure was employed to extract analytes from plasma. Chromatographic separation was performed on an Eclipse Plus C₁₈ column utilizing a fast gradient elution starting with 2% of 2 mM ammonium acetate–formic acid (100/0.1, v/v) followed by increasing the percentage of acetonitrile. Detection was performed on a tandem mass spectrometer equipped with an electrospray ionization source operated in the positive ionization mode, using the transitions m/z 477.2 → m/z 346.1 for TAF and m/z 288.1 → m/z 176.1 for TNF. TAF-d₅ and TNF-d₇ were used as the internal standard of TAF and TNF, respectively. The method was validated in the concentration ranges 1.25–500 ng/mlfor TAF and 0.300–15.0 ng/ml for TNF with acceptable accuracy and precision.     

Prevention of TNF-induced necrotic cell death by rottlerin through a Nox1 NADPH oxidase

Previous studies have demonstrated that rottlerin, a specific PKCdelta inhibitor, potentiates death receptor- mediated apoptosis through a cytochrome c-dependent or -independent pathway. However, its ability to regulate necrotic cell death, as well as the underlying mechanism, remains unknown. We found that in murine fibrosarcoma L929 cells, treatment with rottlerin protected the cells against TNF-induced necrosis, whereas it sensitized the cells to apoptosis induced by co-treatment with Hsp90 inhibitor geldanamycin and TNF, in a manner independent of its ability to inhibit PKC-delta. TNF treatment induced rapid accumulation of mitochondrial superoxide (O2-) through the Nox1 NADPH oxidase when cells undergo necrosis. Moreover, pretreatment with rottlerin failed to induce the GTP-bound form of small GTPase Rac1 by TNF treatment, and subsequently suppressed mitochondrial O2- production and poly(ADP-ribose) polymerase activation, thus inhibiting necrotic cell death. Therefore, our study suggests that Nox1 NADPH oxidase is a new molecular target for anti-necrotic activity of rottlerin upon death-receptor ligation.     

Structure and dynamics of a discotic liquid-crystalline charge-transfer complex.

The structure of the charge-transfer complex hexakis(n-hexyloxy)triphenylene-2,4,7-trinitro-9-fluorenone (HAT6-TNF) has been characterized by neutron scattering, X-ray diffraction (XRD), optical microscopy, and dielectric relaxation spectroscopy (DRS). On the basis of these data and the 1:1 stoichiometry, a consistent structure for the complex is proposed. This structure differs markedly from structures previously proposed for similar materials, because the TNF molecules are found to be situated between the discotic columns rather than sandwiched between the discotic molecules of a given column. The addition of TNF to HAT6 is found to stiffen the structure, and quasi-elastic neutron scattering shows that the local dynamics of the discotic molecules in HAT6-TNF is slowed by the presence of the TNF molecules. This scenario is consistent with the observation of two VFT-type (VFT=Vogel-Fulcher-Tamman) dielectric relaxation processes that relate to the columnar glass transition and a polyethylene-like hindered glass transition originating from the nano-phase-separated fraction of the aliphatic tails.     

The stereochemistry of natural thionuphlutine sulfoxides isolated from nuphar luteum

The combined application of the three research techniques (ASIS, LIS and the S-O bond anisotropy-induced PMR shift analysis) helped in the assignment of the sulfinyl bond configuration in natural isomeric thionuphlutine (TNF) sulfoxides. As in the NTBN S-oxides the more polar TNF S-oxide, 2, was assigned the beta configuration, and the less polar, 3, the alpha configuration.     

Murine pro-tumor necrosis factor expressed in Saccharomyces cerevisiae HF7c localizes to membrane/particulate

Tumor necrosis factor (TNF) is a cytokine that is produced by immune cells in response to bacterial and viral stimuli and plays important roles in various inflammatory diseases. TNF is produced as a membrane-bound precursor, which is then cleaved to release soluble mature protein. We expressed murine pro-TNF in Saccharomyces cerevisiae and examined processing and cellular localization of the recombinant protein. Yeast cells were transformed with an expression construct carrying the pro-TNF gene under the control of alcohol dehydrogenase promoter. Immunoblotting analysis of cell homogenate revealed expression of 26 kD pro-TNF in transformed cells. Upon centrifugation, pro-TNF transformed cells fractionated into the membrane/particulate. In a clone that expresses a high level of pro-TNF, mature 17 kD TNF was detected in the culture medium, although the amount was far smaller than that of cell-associated pro-TNF. Flow cytometric analysis of yeast spheroplasts demonstrated the presence of TNF on the cell surface. Our results show that pro-TNF expressed in yeast mainly resides in the cellular membrane with an orientation similar to that of pro-TNF produced in mammalian cells. Our data suggest that the transformed yeast cells can be used for the genetic analysis of pro-TNF processing machinery in immune cells.