Antitumor activity of a newly developed monoclonal antibody against ROR1 in ovarian cancer cells.

Receptor-tyrosine-kinase-like Orphan Receptor 1 (ROR1) is a tyrosine-protein kinase transmembrane receptor and ROR1 overexpression is associated with a poor prognosis in various cancers, including ovarian cancer. Targeting of ROR1 has been evaluated as a novel cancer therapy strategy. This study developed a novel chimeric anti-ROR1 Fab antibody (named ROR1-cFab) and then assessed the antitumor activity of this antibody in ovarian cancer cells, an in vitro model of preclinical cancer therapy. A ROR1-cFab prokaryotic expression vector was constructed from positive fusion cells (splenocytes from mice with high ROR1 immune titers were fused with myeloma cells) after three rounds of sub-clone affinity screening. Then, a variety of assays were employed to assess the binding selectivity and specificity of ROR1-cFab to ROR1 protein. Furthermore, CCK8, flow cytometric apoptosis, wound healing, and Transwell migration assays were used to assess antitumor activity of this newly developed anti-ROR1 antibody in ovarian cancer cells. We demonstrated that ROR1-cFab could specifically bind to ROR1 protein and ROR1-positive ovarian cancer A2780 cells. Functional assays revealed that ROR1-cFab inhibited tumor cell proliferation and migration, as well as inducing apoptosis of ROR1-positive A2780 cells in a dose dependent manner. These effects were not observed in ROR1-negative lose386 cells. In conclusion, ROR1-cFab is a novel anti-ROR1 antibody with a high affinity to ROR1 protein and inhibitory effects on ROR1-positive cells. Future studies will determine whether the ROR1-cFab might be a promising candidate for treatment of ROR1-positive ovarian cancer.

Study on the interaction of catalase with pesticides by flow injection chemiluminescence and molecular docking.

The interaction mechanisms of catalase (CAT) with pesticides (including organophosphates: disulfoton, isofenphos-methyl, malathion, isocarbophos, dimethoate, dipterex, methamidophos and acephate; carbamates: carbaryl and methomyl; pyrethroids: fenvalerate and deltamethrin) were first investigated by flow injection (FI) chemiluminescence (CL) analysis and molecular docking. By homemade FI-CL model of lg[(I0-I)/I]=lgK+nlg[D], it was found that the binding processes of pesticides to CAT were spontaneous with the apparent binding constants K of 10(3)-10(5) L mol(-1) and the numbers of binding sites about 1.0. The binding abilities of pesticides to CAT followed the order: fenvalerate>deltamethrin>disulfoton>isofenphos-methyl>carbaryl>malathion>isocarbophos>dimethoate>dipterex>acephate>methomyl>methamidophos, which was generally similar to the order of determination sensitivity of pesticides. The thermodynamic parameters revealed that CAT bound with hydrophobic pesticides by hydrophobic interaction force, and with hydrophilic pesticides by hydrogen bond and van der Waals force. The pesticides to CAT molecular docking study showed that pesticides could enter into the cavity locating among the four subdomains of CAT, giving the specific amino acid residues and hydrogen bonds involved in CAT-pesticides interaction. It was also found that the lgK values of pesticides to CAT increased regularly with increasing lgP, Mr, MR and MV, suggesting that the hydrophobicity and steric property of pesticide played essential roles in its binding to CAT.

Study on the chemiluminescence behavior of bovine serum albumin with luminol and its analytical application.

In this paper, the luminescence behavior of bovine serum albumin (BSA) and luminol was first studied by flow injection chemiluminescence (CL). It was found that the hyperchromic effect of luminol in the presence of BSA led to the acceleration of the electrons transferring rate of excited 3-aminophthalate, which greatly enhanced the CL intensity of luminol/dissolved oxygen reaction. The increments of CL intensity were proportional to the concentrations of BSA with a linear range from 0.01 to 7 nmol L(-1). It was also found that azithromycin could inhibit the CL intensity of luminol/BSA reaction. The decrements of CL intensity were logarithm over the concentrations of azithromycin ranging from 0.1 to 700 ng mL(-1). At a flow rate of 2.0 mL min(-1), a complete analytical process, which included sampling and washing, could be performed within 30s with relative standard deviations of less than 3.1%. This proposed method was successfully applied in assaying azithromycin in pharmaceutical and human serum samples with recoveries from 91.0 to 104.3%. The possible luminescence mechanism of luminol/BSA/azithromycin reaction was discussed in detail by CL, UV and fluorescence methods.

Study on the binding behavior of bovine serum albumin with cephalosporin analogues by chemiluminescence method.

The luminol-bovine serum albumin chemiluminescence system was proposed for the first time. It was found that the hydrophilic luminol bound to the hydrophilic domain at Trp(134) of BSA with accelerating the electrons transferring rate of excited 3-aminophthalate, which led to the enhancement CL intensity of luminol at 425 nm. The increment of chemiluminescence intensity was proportional to the concentrations of bovine serum albumin from 5.0 × 10(-11) to 1.0 × 10(-8)mol L(-1) with the linear equation of ΔI=7.47 C(BSA)+4.89 (R(2)=0.9950). Based on the remarkable quenching effect of cephalosporin on the luminol-bovine serum albumin chemiluminescence system, the interaction of bovine serum albumin-cephalosporin was studied by flow injection-chemiluminescence method. A valuable model for studying the interaction of bovine serum albumin-cephalosporin was constructed and the formula lg[(I(0)-I)/I]=lg K(D)+n lg[D] was obtained. The binding parameters calculated by the model did agree very well with the results obtained by fluorescence quenching method. The major binding force of bovine serum albumin with cephalosporins was the hydrophobic effect. The binding ability of cephalosporin analogues to bovine serum albumin followed the pattern: cefoperazone, ceftriaxone and cefotaxime>cefuroxime and cefaclor>cefadroxil, cefradine and cefazolin, which was close to the order of their antibacterial ability. Using flow injection chemiluminescence method also obtained the stoichiometric ratio, the average of association constant K(P) and dissociation degree α of luminol-bovine serum albumin were 1:1, 1.12 × 10(7)L mol(-1) and 0.086, respectively.

A valuable way for understanding the relationships between lysozyme and cephalosporin analogues by flow injection chemiluminescence.

The photochemical reaction mechanism of lysozyme with cephalosporin analogues was investigated with luminol used as a luminescence probe by flow injection chemiluminescence. It was found that Glu35 and Asp52 of lysozyme accelerated the rate of excited 3-aminophthalate electrons transferring and enhanced the chemiluminescence signal of luminol, producing steady-state chemiluminescence in the flow injection system with relative standard deviations less than 3.0%. It was also found that cephalosporin analogues could enter into the site of Trp62 in lysozyme forming 1 : 1 complex which leads to a conformational change of lysozyme, giving the effect of chemiluminescence quenching from luminol-lysozyme. Based on the photochemical behavior of luminol/lysozyme and cephalosporin, a model of lysozyme-cephalosporin interaction, lg[(I(0)-I)/I]=lgK(D) + nlg[D], was established. Using the proposed model, the interaction parameters and the binding ability of lysozyme with cephalosporin were successfully obtained, and the results agreed very well with the results obtained by fluorescence.

DNA ploidy cytometry testing for cervical cancer screening in China (DNACIC Trial): a prospective randomized, controlled trial.

PURPOSE: This randomized, controlled trial was designed to determine whether the DNA cytometry testing is superior to the conventional cytologic testing for mass cervical cancer screening. EXPERIMENTAL DESIGN: After approval by the institutional ethics review boards from three separate screening centers, a total of 23,993 Chinese women ages 20 to 65 years were randomly assigned into one of the two groups: a DNA cytometry testing group (11,999 women) and a cytologic testing group (11,994 women). Each woman underwent the other testing after first attending the assigned screening test. Women with positive results after assigned testing additionally underwent colposcopy and human papillomaviruses detections, and those with cervical precancerous or cancerous lesions received appropriate treatment. Sensitivity and specificity estimates were adjusted for verification bias. Analyses were by intention to treat and per protocol ways. RESULTS: In the cytometric DNA testing group, cervical cancer was diagnosed in 40 subjects, compared with 24 subjects in the cytologic testing group [hazard ratio for the detection of advanced cancer in the DNA cytometry testing group, 0.42; 95% confidence interval (CI), 0.27-0.60]. The sensitivity of the DNA cytometry testing for cervical cancer was 91.7% (95% CI, 64.3-95.8), whereas the sensitivity of cytologic testing was 44.5% (95% CI, 25.2-61.3; P = 0.008). The specificity was 54.1% (95% CI, 31.6-69.0) for DNA cytometry testing and 70.6% (95% CI, 46.8-82.5; P = 0.003) for cytologic testing. The sensitivity of both tests used together was 100%, and the specificity was 91.8%. A total of 187 subjects reported mild to severe adverse events after treatment with positive results in 319 women. CONCLUSIONS: Our results highlight the benefit of the DNA cytometry testing strategy in mass cervical cancer screening with greater sensitivity and positive predicted value than the conventional cytologic testing in developing settings.

Subpicogram determination of melamine in milk products using a luminol-myoglobin chemiluminescence system.

A sensitive chemiluminescence method based on a luminol-myoglobin system is proposed for the determination of melamine in milk products. It was found that the mixed solutions of melamine and myoglobin could react to form a complex on line, which could greatly inhibit the chemiluminescence intensity generated from the reaction between luminol and myoglobin. The decrease in chemiluminescence intensity was proportional to the concentration of melamine, giving a calibration graph linear over the concentration from 10 pg mL(-1) to 50 ng mL(-1) (R(2) = 0.9988) with a detection limit of 3 pg mL(-1) (3sigma). At a flow rate of 2.0 mL min(-1), one analysis cycle, including sampling and washing, could be accomplished in 20 s with a relative standard deviation of <4.0%. The proposed method was applied successfully to the determination of melamine in milk products, and the recovery was from 93.4 to 106.5%. The possible mechanism of luminol-myoglobin-melamine reaction is given.

Study on the binding behavior of lysozyme with cephalosporin analogues by fluorescence spectroscopy.

It was first found that the intrinsic fluorescence of lysozyme at 340 nm can be quenched by cephalosporin analogues through the static quenching and non-radiative energy transferring procedure. In the acetate buffer solution with pH 7.0 and 298 K, the quenching fluorescence intensity was in a good linearity over the concentration of drugs in the range of 1-100 micromol L(-1), 0.1-100 micromol L(-1), 0.5-100 micromol L(-1) and 0.05-100 micromol L(-1) for cefradine, cefuroxime, cefotaxime and ceftriaxone, respectively. The quenching ability or the binding ability of the studied drugs followed the pattern: ceftriaxone > cefotaxime > cefuroxime > cefradine, which was close to the order of their antibacterial ability. The binding parameters including the association constant and the number of binding potential point were calculated at different temperatures (288, 298 and 308 K), and thermodynamic parameters DeltaH degrees, DeltaS degrees and DeltaG degrees were given. The binding mode of lysozyme with cephalosporins showed that the hydrophobic effect might play a major role. The binding distance between cephalosporin and tryptophan residue in lysozyme was obtained. The results provided the quantitative information for the binding of cephalosporin to lysozyme, and it was suggested that the drugs probably bound to the active site near Trp62 in lysozyme.

Nanoparticles as a vaccine adjuvant of anti-idiotypic antibody against schistosomiasis.

BACKGROUND: The development of new adjuvants for human use has been the focus of attention. This study's aim is to explore the possibility of using nanoparticle Ca nanoparticles (CA) as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis and its protective mechanisms. METHODS: Nanoparticle CA-NP30 conjugate (CA-NP30) was fabricated. BALB/c mice were immunized actively with CA-NP30 to evaluate its effects of protective immunity on mice. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 and the concentrations of IFN-gamma and IL-4 in supernatant of splenocytes were determined via ELISA. RESULTS: Nanoparticle CA could enhance significantly the protective immunity of NP30 against infection of Schistosoma japonicum and the worm reduction rose from 36.0% (NP30 alone) to 52.6%. The serum levels of specific IgG, IgG1 and IgG2a antibodies against NP30 increased remarkably, as compared with those of the group immunized with NP30 alone. The concentration of IFN-gamma in supernatant of splenocyte was drastically elevated [the groups immunized with CA-NP30 and NP30 alone were (493.80 +/- 400.74) pg/ml and (39.03 +/- 39.58) pg/ml, respectively], but the concentration of IL-4 showed no significant difference from that of NP30 alone [(27.94 +/- 9.84) pg/ml vs (27.28 +/- 14.44) pg/ml]. CONCLUSIONS: Nanoparticle CA could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. The mechanism could be that CA-NP30 enhances humoral and cellular immune responses in mice.

[Construction and expression of single chain Fv gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum].

OBJECTIVE: To construct single chain Fv (scFv) gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum. METHODS: The heavy and light chain variable region genes of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum were inserted into two corresponding sites of expression vector pTHA90, and a scFv gene was constructed with a short peptide (Gly4Ser)3 linker gene. The recombinants were determined by digesting with XhoI/SpeI, XbaI/EcoRI and XhoI/EcoRI, and then were introduced into E. coli Top10. The antigen binding activity of expressed product was detected with ELISA. RESULTS: The recombinants were determined by digesting with endonucleases and expected bands were identified. The value of expressed scFv was 3 times higher than negative control by ELISA(OD492 = 1.06). CONCLUSION: The scFv gene of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum was successfully cloned, and the expressed scFv fragment could interact specifically with antigen NP48.

[Study of the acupuncture effect on monoamine transmitters in rabbit plasma and brain tissue by high performance liquid chromatography with electrochemical detection].

A rapid and simple method for the study of the acupuncture effect on monoamine transmitters and related compounds in rabbit plasma and brain tissue by high performance liquid chromatography with electrochemical detection was developed. An ODS column was selected as the separation column at 25 degrees C, and pH 4.50, 0.02 mol/L of trisodium citrate-0.05 mol/L sodium phosphate dibasic to methanol (95:5, volume ratio) without ion-pair at a flow rate of 1.0 mL/min. Four compounds, epinephrine (E), norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT), were simultaneously separated and determined under the above conditions. Twenty rabbits were investigated after the acupuncture action upon the central neurotransmitters. The sufficient data showed that acupuncture could significantly affect the activities of the neurotransmitters including E, NE, DA and 5-HT, and the changed functions of the neurotransmitter systems induced by acupuncture not only lead to the neurotransmitter content increase both in brain and plasma but also cause the increase of rabbit breed ability. The results show that the method is very simple and fast. The method is valuable not only for clinical diagnosis but also for research work.