The Role of Free Radicals in the Photodynamic Treatment of Fibrotic Skin Diseases.

The first derivatives of gelatin and type I collagen fluorescence spectra were characterized in order to describe the effect of free radicals on pyridinoline (PYD) cross-links. The different gas saturation conditions were used to investigate the effect of different free radicals. An analysis of first derivative fluorescence spectra suggests that PYD cross-link fluorescence emission is composed of three peaks in gelatin, but only two in type I collagen. The PYD cross-link was photo-degraded more than other gases in the presence of O2. This suggests that the singlet oxygen ((1)O2) plays a key role when using photodynamic therapy to treat skin fibrosis disease with Hypocrellin B (HB).

[Sample pretreatment methods of pesticide residues in Panax notoginseng of Chinese traditional medicine].

Two sample pretreatment methods of pesticide residues in Panax notoginseng of Chinese traditional medicine were developed. For Method I, the residues were extracted from homogenized tissue with n-hexane-dichloromethane (6:4) by means of ultrasonication, the crude extract was purified by an Envi-carb/NH2 solid-phase extraction (SPE) column. For Method II, matrix solid-phase dispersion (MSPD) technique was used for extracting and cleaning up. The eluates were concentrated by rotary evaporation, and then were redissolved in dichloromethane prior to GC-MS determination. The determination was performed in selected ion monitoring (SIM) mode with the external calibration for quantitative analysis. Under the optimal conditions, the results indicated that the methods are easier and faster, the recoveries of method I for the spiked standards at concentration of 0.01, 0.5, and 2.0 mg x kg(-1) were 81.90%-102.10% with the relative standard deviations (RSDs) of 3.60%-7.10%. The recoveries of method II were 96.26%-104.20% with the RSDs of 3.52%-7.94%. The detection limits (S/N) for residues of pesticides were in the range of 0.48-1.34 ng x g(-1). The results indicated that these multiresidue analysis methods can meet the requirements for determination of residue pesticides and can be appropriate for trace analysis of residue pesticides in Panax notoginseng.

Single chain fragment variable recombinant antibody as a template for Fc sensors.

A label free immunosensor for detection of Fc receptors expressed on cell surface was developed and characterized using a Quartz Crystal Microbalance (QCM) transducer. Taking advantage of the characteristics of single chain fragment variable (scFv) recombinant antibody and the multivalency of an antibody, the engineered recombinant scFv was immobilized onto preformed functionalized self-assembled monolayers (SAMs) template surface. The monomeric scFv can bind with the CH1 region of any rabbit IgG to form a highly oriented IgG layer with its Fc portion pointing toward a solution phase. This results in a highly oriented Fc sensor that can be used to study the thermodynamics and kinetics of binding between the Fc portion of immunoglobulin and the cell surface Fc receptor (FcR), an important area of the immune system. The Fc sensor was used to study the binding between Staphylococcus aureus and the Fc receptor on macrophage. Parallel characterization of cell surface Fc receptors in the same samples by ELISA was also performed.

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT(2) receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung.

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.

[Comparative study on trace elements in Elsholtzia bodineri Vaniot. from Honghe by FAAS].

In the present paper, the authors prepared all the samples by the same method, and optimized the work conditions for flame qtomic absorption spectrometry. FAAS was applied to determine the trace elements, such as Pb, Cr, Cd, Cu, Zn, Hg, Se etc, in Elsholtzia bodineri Vaniot. from Mengzi, Shiping, Mile and Jianshui located in Honghe state. The method is simple, rapid and accurate, and allows simultaneous determination of many elements. The recoveries were in the range of 98.7%-116.4%, and RSD was 0.07%-1.69%. The contents of Pb, Cd and Hg were 53.47, 6.03 and 7.68 microg x g(-1), respectively, in the sample from Jianshui, and were higher than those from other areas. The content of Mn was the highest, and was higher in Mengzi than in other areas, and the average content in the four samples was 913.48 microg x g(-1), which was higher than the content in the soil. Fe is the semi-microelement whose average content in Elsholtzia bodineri Vaniot. from Honghe was 688.46 microg x g(-1), the high-to-low content sequence of Fe in terms of location was Mengzi, Mile, Shiping and Jianshui. The average content of Zn was 116.64 microg x g(-1), and the highest one was from Shiping. But we did not obtain the data of Se in the Elsholtzia bodineri Vaniot. from Honghe state by AAS, and did not detect Hg in samples from Shiping and Mile. The content of the detected elements in Mengzi's sample had the sequence as follows; Mn > Fe > Zn > Pb > Cu > Cr > Cd > Hg>Se, while the sequence was Mn > Fe > Zn > Pb > Cu > Hg > Cd > Cr > Se in the sample of Jianshui, Mn > Fe > Z n >Pb > Cu > Cd > Cr > Hg > Se in Shiping, and Fe > Mn > Zn > Pb > Cu > Cd > Cr > Hg > Se in Mile. The results show that the content of the trace elements is high, especially, Mn, Fe and Zn, in Elsholtzia bodineri Vaniot. in Honghe. Therefore, Elsholtzia bodineri Vaniot., which can be used as drug or beverage, is worth exploitation.

Carcinogen-induced histone alteration in normal human mammary epithelial cells.

Little is known about early carcinogen-induced protein alterations in mammary epithelium. Detection of early alterations would enhance our understanding of early-stage carcinogenesis. Here, normal human mammary epithelial cells (HMECs) were exposed to dietary and environmental carcinogens [2-amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP), 4-aminobiphenyl (ABP), benzo[a]pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin] individually or in combination. A phage display library of single-chain variable fragment antibodies was used to screen protein targets altered by the treatment. In combination with matrix-assisted laser desorption time of flight, we identified histone H3 as a target antigen. Although histone H3 total protein remained unchanged in control and treated HMEC, the methylation of lysine 4 was altered. A reduction in mono-methyl histone H3 (Lys 4) was observed in treated HMEC compared with control HMEC. This alteration was shown to be dependent on carcinogen concentration and specific for PhIP and ABP. To characterize potential histone demethylation mechanisms, localization and protein expression patterns of lysine-specific demethylase 1 (LSD1) were analyzed. In control HMEC, LSD1 was present at the nuclear periphery. However, following 72 h carcinogen treatment, LSD1 localized within the nucleus. Within 48 h after treatment, mono-methyl histone H3 (Lys 4) was restored and LSD1 localization was reversed. Protein expression levels of LSD1 were also increased in treated HMEC compared with control HMEC. Our data suggest that the induction of a single enzyme, LSD1, represents an early response to carcinogen exposure, which leads to the demethylation of histone H3 (Lys 4), which, in turn, may influence the expression of multiple genes critical in early-stage mammary carcinogenesis.

Efficient Degradation of Aqueous Rhodamine B Irradiation Under Indoor Light Using a SiO2 -Hypocrellin B Complex.

An efficient method for the degradation of the dye Rhodamine B (RhB) is reported. A SiO2 -hypocrellin B (SiO2 -HB) complex was found to work as a catalyst to degrade RhB in aqueous solution at room temperature and atmospheric pressure, over the pH range 1-10, under indoor light conditions. This method is capable of removing 82.68% of RhB within 20 min at pH 9. The SiO2 -HB complex was characterized by both scanning electron microscopy and surface area analysis. The reaction progress was examined using ultrahigh pressure liquid chromatography/mass spectrometry and UV-visible spectroscopy. This process represents an efficient means of decontaminating dye-containing wastewaters in either highly acidic or weakly alkaline environments.

Anti-tax interacting protein-1 (TIP-1) monoclonal antibody targets human cancers.

Radiation-inducible neo-antigens are proteins expressed on cancer cell surface after exposure to ionizing radiation (IR). These neo-antigens provide opportunities to specifically target cancers while sparing normal tissues. Tax interacting protein-1 (TIP-1) is induced by irradiation and is translocated to the surface of cancer cells. We have developed a monoclonal antibody, 2C6F3, against TIP-1.Epitope mapping revealed that 2C6F3 binds to the QPVTAVVQRV epitope of the TIP-1 protein. 2C6F3 binds to the surface of lung cancer (A549, LLC) and glioma (D54, GL261) cell lines. 2C6F3 binds specifically to TIP-1 and ELISA analysis showed that unconjugated 2C6F3 efficiently blocked binding of radiolabeled 2C6F3 to purified TIP-1 protein. To study in vivo tumor binding, we injected near infrared (NIR) fluorochrome-conjugated 2C6F3 via tail vein in mice bearing subcutaneous LLC and GL261 heterotopic tumors. The NIR images indicated that 2C6F3 bound specifically to irradiated LLC and GL261 tumors, with little or no binding in un-irradiated tumors.We also determined the specificity of 2C6F3 to bind tumors in vivo using SPECT/CT imaging. 2C6F3 was conjugated with diethylene triamine penta acetic acid (DTPA) chelator and radiolabeled with 111Indium (111In). SPECT/CT imaging revealed that 111In-2C6F3 bound more to the irradiated LLC tumors compared to un-irradiated tumors. Furthermore, injection of DTPA-2C6F3 labeled with the therapeutic radioisotope, 90Y, (90Y-DTPA-2C6F3) significantly delayed LLC tumor growth. 2C6F3 mediated antibody dependent cell-mediated cytotoxicity (ADCC) and antibody dependent cell-mediated phagocytosis (ADCP) in vitro.In conclusion, the monoclonal antibody 2C6F3 binds specifically to TIP-1 on cancer and radio-immunoconjugated 2C6F3 improves tumor control.

Production and characterization of mouse ureteric bud cell-specific rat hybridoma antibodies utilizing subtractive immunization and high-throughput screening.

The highly branched collecting system of the kidney arises developmentally from the ureteric bud (UB) by a process of branching morphogenesis. This process is critical for the normal development of the collecting ducts and pelvis of the kidney, and is tightly controlled by the spatial and temporal expression of numerous proteins. To identify cell proteins that are differentially expressed by the UB relative to those expressed by the highly differentiated collecting ducts of the adult kidney, two mouse cell populations derived from either the early UB or the adult inner medullary collecting duct (IMCD) were used. A subtractive immunization strategy was performed in rats to generate monoclonal antibodies that preferentially reacted with antigens on UB, but not IMCD cells. In addition, the technique of antibody printing, a novel high-throughput antibody screening method for determining the specificities of a large number of monoclonal antibodies, is described. The methodologies outlined in this manuscript have broad applicability as they demonstrate that subtractive immunization can be performed in rats with cells derived from mice. Additionally, the high-throughput screening methods should facilitate the use of subtractive immunization for identifying antibodies that can distinguish differences in proteins expressed in closely related cell types.

Targeted multiplex imaging mass spectrometry with single chain fragment variable (scfv) recombinant antibodies.

Recombinant scfv antibodies specific for CYP1A1 and CYP1B1 P450 enzymes were combined with targeted imaging mass spectrometry to simultaneously detect the P450 enzymes present in archived, paraffin-embedded, human breast cancer tissue sections. By using CYP1A1 and CYP1B1 specific scfv, each coupled to a unique reporter molecule (i.e., a mass tag) it was possible to simultaneously detect multiple antigens within a single tissue sample with high sensitivity and specificity using mass spectrometry. The capability of imaging multiple antigens at the same time is a significant advance that overcomes technical barriers encountered when using present day approaches to develop assays that can simultaneously detect more than a single antigen in the same tissue sample.

Radiation-guided drug delivery to mouse models of lung cancer.

PURPOSE: The purpose of this study was to achieve improved cancer-specific delivery and bioavailability of radiation-sensitizing chemotherapy using radiation-guided drug delivery. EXPERIMENTAL DESIGN: Phage display technology was used to isolate a recombinant peptide (HVGGSSV) that binds to a radiation-inducible receptor in irradiated tumors. This peptide was used to target nab-paclitaxel to irradiated tumors, achieving tumor-specificity and enhanced bioavailability of paclitaxel. RESULTS: Optical imaging studies showed that HVGGSSV-guided nab-paclitaxel selectively targeted irradiated tumors and showed 1.48 ± 1.66 photons/s/cm(2)/sr greater radiance compared with SGVSGHV-nab-paclitaxel, and 1.49 ± 1.36 photons/s/cm(2)/sr greater than nab-paclitaxel alone (P < 0.05). Biodistribution studies showed >5-fold increase in paclitaxel levels within irradiated tumors in HVGGSSV-nab-paclitaxel-treated groups as compared with either nab-paclitaxel or SGVSGHV-nab-paclitaxel at 72 hours. Both Lewis lung carcinoma and H460 lung carcinoma murine models showed significant tumor growth delay for HVGGSSV-nab-paclitaxel as compared with nab-paclitaxel, SGVSGHV-nab-paclitaxel,and saline controls. HVGGSSV-nab-paclitaxel treatment induced a significantly greater loss in vasculature in irradiated tumors compared with unirradiated tumors, nab-paclitaxel, SGVSGHV-nab-paclitaxel, and untreated controls. CONCLUSIONS: HVGGSSV-nab-paclitaxel was found to bind specifically to the tax-interacting protein-1 (TIP-1) receptor expressed in irradiated tumors, enhance bioavailability of paclitaxel, and significantly increase tumor growth delay as compared with controls in mouse models of lung cancer. Here we show that targeting nab-paclitaxel to radiation-inducible TIP-1 results in increased tumor-specific drug delivery and enhanced biological efficacy in the treatment of cancer.

TIP-1 translocation onto the cell plasma membrane is a molecular biomarker of tumor response to ionizing radiation.

BACKGROUND: Tumor response to treatment has been generally assessed with anatomic and functional imaging. Recent development of in vivo molecular and cellular imaging showed promise in time-efficient assessment of the therapeutic efficacy of a prescribed regimen. Currently, the in vivo molecular imaging is limited with shortage of biomarkers and probes with sound biological relevance. We have previously shown in tumor-bearing mice that a hexapeptide (HVGGSSV) demonstrated potentials as a molecular imaging probe to distinguish the tumors responding to ionizing radiation (IR) and/or tyrosine kinase inhibitor treatment from those of non-responding tumors. METHODOLOGY/PRINCIPAL FINDINGS: In this study we have studied biological basis of the HVGGSSV peptide binding within the irradiated tumors by use of tumor-bearing mice and cultured cancer cells. The results indicated that Tax interacting protein 1 (TIP-1, also known as Tax1BP3) is a molecular target that enables the selective binding of the HVGGSSV peptide within irradiated xenograft tumors. Optical imaging and immunohistochemical staining indicated that a TIP-1 specific antibody demonstrated similar biodistribution as the peptide in tumor-bearing mice. The TIP-1 antibody blocked the peptide from binding within irradiated tumors. Studies on both of human and mouse lung cancer cells showed that the intracellular TIP-1 relocated to the plasma membrane surface within the first few hours after exposure to IR and before the onset of treatment associated apoptosis and cell death. TIP-1 relocation onto the cell surface is associated with the reduced proliferation and the enhanced susceptibility to the subsequent IR treatment. CONCLUSIONS/SIGNIFICANCE: This study by use of tumor-bearing mice and cultured cancer cells suggested that imaging of the radiation-inducible TIP-1 translocation onto the cancer cell surface may predict the tumor responsiveness to radiation in a time-efficient manner and thus tailor radiotherapy of cancer.

Intramolecular regulation of phosphorylation status of the circadian clock protein KaiC.

BACKGROUND: KaiC, a central clock protein in cyanobacteria, undergoes circadian oscillations between hypophosphorylated and hyperphosphorylated forms in vivo and in vitro. Structural analyses of KaiC crystals have identified threonine and serine residues in KaiC at three residues (T426, S431, and T432) as potential sites at which KaiC is phosphorylated; mutation of any of these three sites to alanine abolishes rhythmicity, revealing an essential clock role for each residue separately and for KaiC phosphorylation in general. Mass spectrometry studies confirmed that the S431 and T432 residues are key phosphorylation sites, however, the role of the threonine residue at position 426 was not clear from the mass spectrometry measurements. METHODOLOGY AND PRINCIPAL FINDINGS: Mutational approaches and biochemical analyses of KaiC support a key role for T426 in control of the KaiC phosphorylation status in vivo and in vitro and demonstrates that alternative amino acids at residue 426 dramatically affect KaiC's properties in vivo and in vitro, especially genetic dominance/recessive relationships, KaiC dephosphorylation, and the formation of complexes of KaiC with KaiA and KaiB. These mutations alter key circadian properties, including period, amplitude, robustness, and temperature compensation. Crystallographic analyses indicate that the T426 site is phosphorylatible under some conditions, and in vitro phosphorylation assays of KaiC demonstrate labile phosphorylation of KaiC when the primary S431 and T432 sites are blocked. CONCLUSIONS AND SIGNIFICANCE: T426 is a crucial site that regulates KaiC phosphorylation status in vivo and in vitro and these studies underscore the importance of KaiC phosphorylation status in the essential cyanobacterial circadian functions. The regulatory roles of these phosphorylation sites--including T426--within KaiC enhance our understanding of the molecular mechanism underlying circadian rhythm generation in cyanobacteria.

Engineering peptide linkers for scFv immunosensors.

Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors.

Recombinant antibody piezoimmunosensors for the detection of cytochrome P450 1B1.

The phase I enzyme known as cytochrome P450 1B1 (CYP1B1) is involved in the metabolism of many endogenous and exogenous compounds, including carcinogens. CYP1B1 is overexpressed in a wide variety of human diseases ranging from diabetes to malignancies, such as invasive breast cancer. Because of its microsomal location in the cell, CYP1B1 could not be measured directly by existing methods but only assessed indirectly via the determination of the catalytic products. We report here a rapid, sensitive piezoimmunosensor for detection of CYP1B1 using single-chain fragment variable antibodies (scFv) as recognition elements and a quartz crystal microbalance (QCM) as the transducer. Three anti-CYP1B1 scFvs (designated B-66, D-23, and L-21) were biotinylated and used to capture and specifically detect CYP1B1 from samples in solution. ScFvs are smaller than most commonly used antibodies and can be coated onto QCM surfaces at much higher density to improve sensor sensitivity and specificity. The scFv-QCM biosensors showed excellent sensitivity (detection limit, 2.2 +/- 0.9 nM) and specificity with a dissociation constant K(d) = (1.54 +/- 0.59) x 10(-7) M. CYP1B1 were quantitatively detected in normal and malignant cell lysates (e.g., human T47D breast cancer cell microsomes). Results demonstrate that an anti-CYP1B1 scFv-QCM immunosensor could be used to detect P450 enzymes in biological samples.

Engineered recombinant single-chain fragment variable antibody for immunosensors.

A recombinant single-chain fragment variable (scFv) antibody (designated A10B) was engineered to contain two histidines within the linker peptide used to join the scFv heavy and light chains. A piezoimmunosensor using the scFv was successfully developed. A10B scFv bound to the gold piezoimmunosensor surface were correctly oriented, retained antigen-binding activity, and coupled at high surface concentration. These results, and results obtained from an earlier study using an scFv containing a linker cysteine, suggest that the location on the linker sequence in which the amino acids were incorporated was well tolerated by the scFv and did not interfere with scFv antigen-binding activity. The scFv-modified QCM sensor was thoroughly characterized and used to specifically detect antigen in crude serum sample and had a sensitivity of 2.3 +/- 0.15 nM (n = 4) with a linear range over 2.3 x 10(-9) - 3.3 x 10(-(8) M. The piezoimmunosensor was also used to study the kinetics and thermodynamics of antigen/scFv antibody binding.

Single-chain fragment variable antibody piezoimmunosensors.

In this paper, we describe a novel nonlabeled biosensor with high diagnostic potential for rapid and sensitive detection of antigens in complex biological samples. The biosensor comprises a piezoimmunosensor (PZ) displaying a specially constructed recombinant antibody on its surface. The recombinant single-chain fragment variable (scFv) antibody contained a cysteine within the linker amino acid sequence used to join the scFv variable heavy and light chains. The presence of cysteine induced the scFv construct to self-assemble as a densely packed rigid monolayer on the gold surface of a quartz crystal microbalance. scFv molecules in this self-assembled monolayer (SAM) exhibited a defined orientation and high areal densities, with scFv-modified microbalance surfaces displaying 35 times as many variable antigen-binding sites per square centimeter as surfaces modified with whole antibody. Experimental data show that the scFv SAM PZ is superior to Fab fragment, Fab fragment containing a free sulfhydryl group (i.e., Fab-SH), and whole antibody PZs regarding sensitivity and specificity. Because of their small uniform size (MW approximately 27000) and the ease with which they can be modified using genetic engineering, scFv's have significant advantages over whole antibodies in microbalance biosensor systems. We demonstrate here that the use of scFv containing a cysteine within the scFv linker sequence (i.e., scFv-cys) for preparation of biosensor surfaces markedly increases the density of available antigen-binding sites, yielding a system that is highly selective, rapid, and capable of detecting low concentrations of antigens in complex samples.

Selection of active ScFv to G-protein-coupled receptor CCR5 using surface antigen-mimicking peptides.

This study describes the use of cyclic peptides for use in the selection of single-chain (ScFv) antibodies specific for the HIV-1 coreceptor CCR5, a representative G-protein-coupled receptor (GPCR). A tandem ligation strategy was developed for preparing biotinylated cyclic peptides, first through an orthogonal end-to-end ligation and then a chemoselective ligation with functionalized biotin. Cyclic peptides mimicking the extracellular loops of CCR5 and their unconstrained counterparts were then used for solution-phase selection of ScFv antibodies from a phage display antibody library. Antibodies reactive with CCR5 on cells were detected using a homogeneous high throughput assay. Of 19 isolated ScFv antibodies that bound to CCR5+ cells, three inhibited CCR5-mediated but not CXCR4-mediated HIV infection. Only ScFvs selected by binding to cyclic constrained peptides exhibited inhibitory activity. Our results demonstrate that surface-antigen mimetics of a GPCR are effective tools for selecting active, site-specific ScFv antibodies that hold promise as immunological reagents and therapeutics.