Improved Performance for the Electrochemical Sensing of Acyclovir by Using the rGO-TiO2-Au Nanocomposite-Modified Electrode.

An electrochemical sensor for sensitive sensing of acyclovir (ACV) was designed by using the reduced graphene oxide-TiO2-Au nanocomposite-modified glassy carbon electrode (rGO-TiO2-Au/GCE). Transmission electron microscopy, X-ray diffractometer, and X-ray photoelectron spectroscopy were used to confirm morphology, structure, and composition properties of the rGO-TiO2-Au nanocomposites. Cyclic voltammetry and linear sweep voltammetry were used to demonstrate the analytical performance of the rGO-TiO2-Au/GCE for ACV. As a result, rGO-TiO2-Au/GCE exerted the best response for the oxidation of ACV under the pH of 6.0 PB solution, accumulation time of 80 s at open-circuit, and modifier amount of 7 µl. The oxidation peak currents of ACV increased linearly with its concentration in the range of 1-100 µM, and the detection limit was calculated to be 0.3 µM (S/N = 3). The determination of ACV concentrations in tablet samples also demonstrated satisfactory results.

One-pot green preparation of deep-ultraviolet and dual-emission carbon nanodots for dual-channel ratiometric determination of polyphenol in tea sample.

A novel deep-ultraviolet and dual-emission carbon nanodots (DUCDs)-based dual-channel ratiometric probe was prepared by a one-pot environmental-friendly hydrothermal process using guanidine as the only starting material for sensing polyphenol in tea sample (TPPs). Under the exposure to TPPs, the DUCDs not only provided a characteristic colorimetric response to TPPs, but also displayed TPPs-sensitive ratiometric fluorescence quenching. The detection mechanism was proved to be that enrichment-specific hydroxyl sites (e.g., -NH2 and -COOH) of DUCDs can specifically react with phenolic hydroxyl groups of TPPs to generate dynamic amide and carboxylate bonds by dehydration and/or condensation reaction. As a result, a new carbon nanomaterial with decrement of surface passivation groups, inherent light-absorbing, and invalid fluorescence emission was generated. The ratio (FL297nm/FL395nm) of fluorescence intensity at 297 nm and 395 nm of DUCDs excited at 275 nm decreased with increasing TPPs concentration. The linearity range was 5.0 ng/mL to 100 µg/mL with a detection limit (DL) of 3.5 ± 0.04 ng/mL for TPPs (n = 3, 3σ/k). Colorimetry of DUCDs, best measured as absorbance at 320 nm, was increased linearly in the TPP concentration range 200 ng/mL-200 µg/mL with a DL of 94.7 ± 0.04 ng/mL (n = 3, 3σ/k). The probe was successfully applied to the determination of TPPs in real tea samples, showing potential application prospects in food analysis.

UV-assisted one-pot synthesis of bimetallic Ag-Pt decorated reduced graphene oxide for colorimetric determination of hydrogen peroxide.

Bimetallic Ag-Pt nanoparticles decorated on the surface of reduced graphene oxide (Ag-Pt/rGO) were designed and selected as a nanozyme for the assay of hydrogen peroxide. The nanocomposites were prepared through a one-pot reduction of potassium chloroplatinate, silver nitrate, and graphene oxide under ultraviolet irradiation without using any extra chemical reducing agents or surfactants. The successful formation of Ag-Pt/rGO nanocomposites was confirmed by transmission electron microscopy, energy disperse spectroscopy mapping, X-ray photoelectron spectroscopy, and X-ray diffraction analysis. Significantly, Ag-Pt/rGO nanocomposites possessed excellent peroxidase-like activity toward the catalytic oxidation of 3,3',5,5'-tetramethylbenzidine to form a blue product in the presence of hydrogen peroxide. Steady-state kinetics studies suggested that Ag-Pt/rGO nanocomposites had high affinity to hydrogen peroxide. Based on these properties, a convenient and sensitive method for the colorimetric determination of hydrogen peroxide was developed. Under optimal conditions, the absorbance at 652 nm increases linearly in the 10-100 µM and 100 µM-1 mM ranges of hydrogen peroxide concentration, and the detection limit is 0.9 µM (S/N = 3). The method was successfully applied to the determination of hydrogen peroxide in real water samples. Graphical abstract Ag-Pt/rGO nanocomposites were prepared by a one-pot UV irradiation method and used as a novel nanozyme for colorimetric determination of H2O2.

Au-Hg/rGO with enhanced peroxidase-like activity for sensitive colorimetric determination of H2O2.

The Au-Hg amalgam anchored on the surface of reduced graphene oxide nanosheets (Au-Hg/rGO) has been synthesized successfully and characterized by various techniques such as transmission electron microscopy, X-ray diffraction and X-ray photoelectron spectroscopy. The Au-Hg/rGO nanocomposites were found to possess excellent peroxidase-like catalytic activity and can quickly catalyze the oxidation of colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxTMB in the presence of H2O2. The obvious color change offered accurate determination of the H2O2 concentration by recording the absorbance at 652 nm using a UV-vis spectrophotometer. The linear response range for H2O2 was from 5 µM to 100 µM and the detection limit was 3.25 µM (S/N = 3). Furthermore, a kinetic study indicated that the catalytic behavior of Au-Hg/rGO nanocomposites followed the typical Michaelis-Menten theory and Au-Hg/rGO nanocomposites showed good affinity for H2O2. We envision that the simple and sensitive colorimetric detection system holds great promising applications in clinical diagnostics and food and environment monitoring.

A voltammetric sensor based on reduced graphene oxide-hemin-Ag nanocomposites for sensitive determination of tyrosine.

A novel voltammetric sensor was designed and used for the determination of l-tyrosine (l-Tyr) by surface modification of a glassy carbon electrode with reduced graphene oxide-hemin-Ag (rGO-H-Ag) nanocomposites. The nanocomposites were synthesized by a facile one-pot hydrothermal method and characterized by means of transmission electron microscopy and Raman spectroscopy. The determination of l-Tyr was investigated by cyclic voltammetry and further quantified using differential pulse voltammetry. The results revealed a significant enhanced electrochemical oxidation effect for l-Tyr at the nanocomposites modified electrode. Two linear ranges from 0.1 to 100 µM and 100 to 1000 µM as well as a low detection limit of 30 nM (S/N = 3) were obtained. In addition, the sensor also demonstrated good selectivity, reproducibility and stability.

Aggregation-Induced Emission Behavior of Dual-NIR-Emissive Zinc-Doped Carbon Nanosheets for Ratiometric Anthrax Biomarker Detection.

The development of near-infrared (NIR) emission nanoprobes for the ratiometric fluorescent determination of living cells in vitro/vivo is of great analytical importance. In this work, dual-NIR-emissive Zn-doped carbon-based nanosheets (Zn-CNSHs) were prepared with a beneficial and special donor-π-acceptor-conjugated (D-π-A-conjugated) spatial framework, which resulted in not only a much lower HOMO-LUMO energy level but also excellent biocompatibility and physicochemical properties. The Zn-CNSHs were prepared by simple one-pot solvothermal synthesis with zinc gluconate (ZGN) and a strong acid and exhibited two distinctive photoluminescence (PL) peaks at 620 and 720 nm with the 600 nm excitation. The 620 nm peak intensity was dependent on dipicolinic acid (DPA) owing to the aggregation-induced emission enhancement effect via the strong intersheet hydrogen bonds between Zn-CNSHs and DPA, enabling the ratiometric fluorescent determination of DPA, an important clinical anthrax biomarker. An excellent calibration curve showed linear regions over the range of 0.05-500 µM between the ratio of PL intensity (PL620 nm/PL720 nm) and the concentrations of DPA. The detection limit was down to 21.7 nM. Based on the high stability, low cytotoxicity, high selectivity, and outstanding PL-reliant sensitivity for the DPA assay, the nanoprobe has been successfully used to monitor DPA in serum, wastewater, and cells.

Reduced graphene oxide-Hemin-Au nanohybrids: Facile one-pot synthesis and enhanced electrocatalytic activity towards the reduction of hydrogen peroxide.

A facile and effective strategy is demonstrated for the synthesis of ternary reduced graphene oxide-Hemin-Au (rGO-H-Au) nanohybrids. The nanohybrids were synthesized through a one-pot in situ reduction of GO and HAuCl4 under alkaline conditions using GO, Hemin and HAuCl4 as the starting materials. The synthesis process can be finished within 1h in a solution phase, without adding any additional surfactant, stabilizing agent and toxic or harsh chemical reducing agents. The resulting nanohybrids were characterized by UV-vis spectroscopy, Raman spectroscopy, transmission electron microscopy (TEM), and so on. Electrochemical measurements showed that the rGO-H-Au nanohybrids exhibited good electrocatalytic activity for the reduction of hydrogen peroxide (H2O2). Based on this property, a simple and highly sensitive amperometric biosensor for H2O2 had been developed. The linear relationships were obtained from 0.1 µM to 40 µM and the detection limit was estimated to be 30 nM. The simple and sensitive sensing platform showed great promising applications in the pharmaceutical, clinical and industrial detection of H2O2.

Direct electrolytic exfoliation of graphite with hemin and single-walled carbon nanotube: Creating functional hybrid nanomaterial for hydrogen peroxide detection.

We present a new, facile and efficient method to prepare functional graphene (GN) hybrid nanomaterials using direct electrolytic exfoliation of graphite robs in hemin (HN) and single-walled carbon nanotube (SWCNT) solution. During the exfoliation process, HN and SWCNT were simultaneously adsorbed on the surface of GN nanosheets through noncovalent π-π interaction, and then 3D GN-HN-SWCNT hybrid nanomaterials were formed. Due to the synergic effect among GN, HN, and SWCNT, these hybrid nanomaterials possessed excellent electrocatalysis properties and were used to construct novel electrochemical biosensor for H2O2 determination. The results displayed a wide linear range of 0.2 µM-0.4 mM and a low detection limit of 0.05 µM. Moreover, the developed sensor was successfully applied for real samples, such as beverages, and showed great promise in routine sensing applications.

[Open reduction and mini-plate internal fixation for the treatment of fracture and dislocation of coccyx].

OBJECTIVE: To explore the therapeutic methods of fracture and dislocation of coccyx and evaluate its curative effects. METHODS: From May 2002 to March 2010,56 patients with fracture and dislocation of coccyx were divided into surgical treatment group and non-surgical treatment group. There were 7 males and 20 females in surgical treatment group with an average age of (48.1 +/- 0.6) years (ranged, 29 to 62 years), treated with open reduction and mini-plate internal fixation. There were 8 males and 21 females in non-surgical treatment group with an average age of (47.5 +/- 0.9) years (ranged, 19 to 54 years),treated with manipulative reduction. All patients were underwent X-ray examination and were finally diagnosed before treatment. Clinical symptoms and Visual Analogue Scales (VAS) of all patients were statistically analyzed before and after treatment. RESULTS: There was no significant difference between two groups in gender, age, BMI index and VAS evaluation. All patients were followed up from 12 to 25 months with an average of 17.2 months. In surgical treatment group,there were 26 cases with I/a incision and 1 case with II/a incision; the excellent rate of clinical symptom was respectively 92.6% and 100% at leaving hospital and final follow-up; the improvement rate of VAS was 97.6% and was excellent result;internal fixtures were removed at the 1 to 2 years after treatment and no unwell symptoms occurred; VAS of all patients in the group was 0 point. In non-surgical treatment group,the excellent rate of clinical symptom was respectively 72.4% and 82.8% at leaving hospital and final follow-up; the improvement rate of VAS was 72.1% and was good result. There was significant difference in clinical results between two groups (P<0.05). CONCLUSION: The results indicated that fracture and dislocation of coccyx should be treated in time. For the treatment of patients with manipulative reduction failures, instability reduction by X-ray examination and serious rectal irritation, open reduction and mini-plate internal fixation can obtain satisfactory results.

[Effect of electroacupuncture combined with auricular point tapping and pressing on serum insulin and testosterone in the patients of obese women with polycystic ovary syndrome].

OBJECTIVE: To assess the therapeutic effect and mechanism of electroacupuncture combined with auricular point tapping and pressing on the obese women with polycystic ovary syndrome. METHODS: Thirty-nine cases of obese women with polycystic ovary syndrome were treated with electroacupuncture combined with auricular point tapping and pressing, body points as Tianshu (ST 25), Fenglong (ST 40), Guanyuan (CV 4) and Siman (KI 14) etc. were selected, and ear points as Kou (mouth), Wei (stomach) and Pi (spleen) etc. were selected. After 3 courses, the therapeutic effect, the body mass index (BMI), the waist circumference (WC) and the changes of the serum insulin (Ins) and testosterone (T) were compared before and after treatment. RESULTS: Of the 39 cases, 10 cases were cured, 25 cases were effective, 4 cases were ineffective, with a total effective rate of 89.7%; there were significant differences in BMI, WC, Ins and T of the patients compared with that before treatment (all P < 0.01). CONCLUSION: Electroacupuncture combined with auricular point tapping and pressing has a good clinical effect on obese women with polycystic ovary syndrome, the treatment mechanism may realized by regulating the serum insulin and the testosterone of the patients.

Human pregnane X receptor activation and CYP3A4/CYP2B6 induction by 2,3-oxidosqualene:lanosterol cyclase inhibition.

The effects of [4'-(6-allyl-methyl-amino-hexyloxy)-2'-fluoro-phenyl]-(4-bromophenyl)-methanone fumarate (Ro 48-8071), an inhibitor of 2,3-oxidosqualene:lanosterol cyclase (cyclase), were evaluated on CYP3A4 and CYP2B6 mRNA content in primary cultured human hepatocytes. In seven hepatocyte culture preparations, 24-h treatment with 3, 10, or 30 microM Ro 48-8071 produced median increases in CYP3A4 mRNA content that were 2.2-, 7.1-, and 8.5-fold greater than untreated control, respectively, and produced increases in CYP2B6 mRNA content that were 3.0-, 4.6-, and 3.4-fold greater than control, respectively. Increases in CYP3A4 immunoreactive protein content were also measured in Ro 48-8071-treated hepatocytes. To evaluate the effects of cyclase inhibitor treatments further, a pregnane X receptor (PXR)-responsive transactivation assay in HepG2 cells was used. Ro 48-8071, trans-N-(4-chlorobenzoyl)-N-methyl-(4-dimethylaminomethylphenyl)-cyclohexylamine (BIBX 79), and 3beta-(2-diethylaminoethoxy)androst-5-en-17-one HCl (U18666A) induced luciferase expression from a PXR-responsive reporter with EC(50)s of 0.113, 0.916, and 0.294 microM, respectively. Treatment of the HepG2 system with (E)N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[(3,3'-bithiophen-5-yl)methoxy]benzenemethanamine (NB-598), an inhibitor of squalene monooxygenase, at concentrations sufficient to achieve cholesterol biosynthesis inhibition significantly inhibited cyclase inhibitor-mediated, but not rifampicin-mediated, reporter induction. Direct treatment of the HepG2 system with 1 to 10 microM squalene 2,3:22,23-dioxide, but not squalene 2,3-oxide, significantly activated PXR-responsive reporter expression. Also, squalene 2,3:22,23-dioxide bound to human PXR in vitro with an IC(50) of 3.35 microM. These data indicate that cyclase inhibitors are capable of producing CYP3A4 and CYP2B6 induction in primary cultured human hepatocytes, and that an endogenous squalene metabolite is a conserved intracrine activator of PXR.

Age- and sex-dependent expression of multiple murine hepatic hydroxysteroid sulfotransferase (SULT2A) genes.

Hydroxysteroid sulfotransferase (SULT2A) enzymes play important roles in hepatic steroid and xenobiotic metabolism. Unlike humans, which express one SULT2A, inspection of mouse genome information indicated the presence of seven SULT2A genes within a cluster on chromosome 7. The age- and sex-dependent expressions of the seven murine SULT2A family members were characterized in the livers of C57BL/6 mice using real-time RT-PCR. The transcripts for three of the SULT2A forms (NCBI reference/model sequences XM_001471624, NM_009286 and NM_001111296) were abundant in pre-pubertal male and female mouse liver but were essentially silenced in the livers of adult male mice. The mRNAs of three other SULT2A forms (NM_001101534, XM_894052 and NM_001081325) were also expressed in pre-pubertal male and female mouse liver, but at markedly reduced levels relative to those of the abundant forms. The mRNA levels of these lower-abundance forms were further suppressed in adult animals. A seventh SULT2A mRNA (XM_983034) was expressed in adult male and female mouse liver, but was not detected in pre-pubertal mouse liver of either sex. Full-length amplifications with primers targeting untranslated regions confirmed that all SULT2A forms were expressed. However, while the XM_001471624, NM_001111296, NM_001101534, XM_894052 and NM_001081325 transcripts were detected at their predicted sizes, the NM_009286 and XM_983034 transcripts each lacked two predicted exons. These results demonstrate that seven murine SULT2As display different profiles of age- and sex-dependent hepatic expression.

Positive and negative regulation of human hepatic hydroxysteroid sulfotransferase (SULT2A1) gene transcription by rifampicin: roles of hepatocyte nuclear factor 4alpha and pregnane X receptor.

The effects of rifampicin treatment on SULT2A1 mRNA expression were evaluated in 23 preparations of primary cultured human hepatocytes. In contrast to the consistently occurring induction of CYP3A4, a prototypical pregnane X receptor (PXR) target gene, rifampicin treatment increased SULT2A1 mRNA levels in 12 of the hepatocyte preparations, but it produced little change or even suppression in the others. Transient transfection of HepG2 cells with a series of reporter constructs implicated two SULT2A1 5'-flanking regions as containing rifampicin-responsive information. Each of these regions contained a hepatocyte nuclear factor 4 (HNF4) binding site (at nucleotide [nt] -6160 and -54), as demonstrated by in vitro binding and site-directed mutagenesis. HNF4alpha bound to the HNF4-54 region of the endogenous SULT2A1 gene, as indicated by chromatin immunoprecipitation. Cotransfection of HepG2 cells with pregnane X receptor (PXR) dose-dependently suppressed reporter expression from SULT2A1 constructs containing the HNF4 sites, and rifampicin treatment augmented the suppression. Rifampicin treatment concentration-dependently suppressed SULT2A1 reporter expression at the same concentrations that progressively induced expression from a PXR-responsive CYP3A4 reporter, whereas higher rifampicin concentrations reversed the SULT2A1 suppression. The suppressive effect of rifampicin was diminished, whereas the activating effect was augmented, in HepG2 cells with RNA interference-mediated PXR knockdown. These results suggest that HNF4alpha plays a central role in the control of SULT2A1 transcription and that rifampicin-liganded PXR suppresses SULT2A1 expression by interfering with HNF4alpha activity. By contrast, the rifampicin-inducible SULT2A1 expression that occurs in many human hepatocyte preparations seems to be mediated through a PXR-independent mechanism.

Regulation of human hepatic hydroxysteroid sulfotransferase gene expression by the peroxisome proliferator-activated receptor alpha transcription factor.

Human hydroxysteroid sulfotransferase or (HUMAN)SULT2A1 catalyzes the sulfonation of procarcinogen xenobiotics, hydroxysteroids, and bile acids and plays a dynamic role in hepatic cholesterol homeostasis. The treatment of primary cultured human hepatocytes with a peroxisome proliferator-activated receptor alpha (PPARalpha)-activating concentration of ciprofibrate (10(-) (4) M) increased (HUMAN)SULT2A1 mRNA, immunoreactive protein, and enzymatic activity levels by approximately 2-fold. By contrast, expression of (RAT)SULT2A3, the rat counterpart to (HUMAN)SULT2A1, was induced by treatment of primary hepatocyte cultures with an activator of the pregnane X receptor, but not PPARalpha. In HepG2 cells, transient transfection analyses of luciferase reporter constructs containing upstream regions of the (HUMAN)SULT2A1 gene implicated a candidate peroxisome proliferator response element (PPRE) at nucleotides (nt) -5949 to -5929 relative to the transcription start site. Site-directed mutagenesis and electrophoretic mobility shift assay studies confirmed that this distal PPRE (dPPRE), a direct repeat nuclear receptor motif containing one intervening nt, represented a functional PPRE. Chromatin immunoprecipitation analysis indicated that the (HUMAN)SULT2A1 dPPRE was also a functional element in the context of the human genome. These data support a major role for the PPARalpha transcription factor in the regulation of hepatic (HUMAN)SULT2A1. Results also indicate that important species differences govern the transactivation of SULT2A gene transcription by nuclear receptors.

Regulation of glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription in primary cultured rat hepatocytes: role of CCAAT/enhancer-binding protein liver-enriched transcription factors.

The mechanism responsible for glucocorticoid receptor (GR)-mediated induction of rat hepatic hydroxysteroid sulfotransferase (SULT2A-40/41) gene transcription was investigated. We previously reported that the region of the SULT2A-40/41 5'-flanking region delimited by -158 to -77 nucleotides relative to the transcription start site was sufficient to support GR-inducible expression. This region of the SULT2A-40/41 gene does not contain a consensus glucocorticoid receptor-responsive element, but does contain two consensus sites for liver-enriched CCAAT/enhancer-binding protein (C/EBP) transcription factors. In the present study, incubation of primary cultured rat hepatocytes with a GR-activating concentration (10(-7) M) of a potent glucocorticoid, dexamethasone or triamcinolone acetonide (TA), rapidly produced increases in C/EBPalpha and C/EBPbeta nuclear protein contents, as measured by Western blot or in vitro DNA-binding activity analysis, that preceded increases in SULT2A-40/41 mRNA and protein levels. Transient cotransfection of SULT2A-40/41 reporter plasmids with a dominant negative C/EBP expression plasmid completely blocked TA-inducible SULT2A-40/41 reporter gene expression. Linker scanning and site-directed mutagenesis of the proximal SULT2A-40/41 5'-flanking region, complemented by in vitro DNA-binding analyses, indicated that the more distal C/EBP site was important for controlling SULT2A-40/41 promoter activity. These data support a role for GR-inducible C/EBPalpha and C/EBPbeta expression in the transactivation of hepatic SULT2A-40/41 expression.

Transactivation of glucocorticoid-inducible rat aryl sulfotransferase (SULT1A1) gene transcription.

The purpose of the current study was to establish the role of the glucocorticoid receptor (GR) and androgen receptor (AR) transcription factors in the transactivation of rat aryl sulfotransferase (SULT1A1) gene transcription and to identify the functional hormone-responsive element(s) in the SULT1A1 gene. A cis-acting inverted repeat with three intervening bases (IR3) was identified in the 5'-flanking of the SULT1A1 gene that mediates the transactivation of SULT1A1 gene transcription by both the GR and AR. CV-1 cells were cotransfected with SULT1A1-luciferase reporter plasmids and either wild-type or mutant GR or AR expression constructs. In cotransfectants expressing the wild-type GR, treatment with triamcinolone acetonide produced an approximately 4- to 6-fold induction of luciferase activity in IR3-containing SULT1A1 reporter plasmids. IR3-containing SULT1A1 reporter constructs were also activated by treatment with the synthetic androgen R1881 in cells cotransfected with wild-type but not mutant AR. In primary cultured rat hepatocytes, androgen-inducible expression of IR3-containing SULT1A1 reporter plasmids required cotransfection with AR expression plasmid. Targeted disruption of the SULT1A1 IR3 by mutation of a conserved GT sequence in the 3' half-site of the element ablated GR and AR responsiveness. These results indicate that a proximal IR3 element in the 5'-flanking region of the rat SULT1A1 gene is sufficient for the transactivation of SULT1A1 gene transcription by the GR and AR, and that relative to the GR, functional AR activity is reduced in primary cultured rat hepatocytes.

Identification and cloning of a new protein that binds the 3(') untranslated region of alpha-striated tropomyosin.

The 3' untranslated region of muscle tropomyosin (TM UTR) induces muscle differentiation when transcribed in primary fibroblasts. This sequence binds protein in extracts from cell types that differentiate upon TM UTR transcription. To identify the protein(s) bound by the TM UTR, an avian embryo fibroblast library was induced to express protein in solution and extracts from these pools were screened with electromobility shift assays using a TM UTR RNA probe. Positive pools were progressively fractionated until a pool containing a single positive clone was obtained. The TM UTR-binding protein (UBP) clone thus isolated contains 751 nt, 618 of which represent a single open reading frame. UBP is related to a human autoantigen, Sjogren's syndrome antigen B (SSB) beginning with the start of the UBP open reading frame. This homology is to the 5' end of SSB in a region containing an RNA-binding motif of 70 amino acids. The deduced amino acid sequence of UBP predicts phosphorylation sites for protein kinase C, casein kinase 2, and cAMP-dependent protein kinase and asparginine glycosylation sites. The observed size of UBP by UV cross-linking with a TM UTR probe is of the same size as the protein bound in fibroblast extract. UBP is expressed in primary fibroblasts, but not in fibroblast or myogenic cell lines, suggesting that its expression is restricted. The full-length UBP mRNA is approximately 3 kB, suggesting a long 5' untranslated region. Transient transfection of cultured cells with UBP directs production of a protein that binds the TM UTR, confirming that these sequences interact in vivo. These observations suggest that we have identified a novel protein that binds to the TM UTR in vitro and in vivo. Determining the function of this protein will facilitate determining the mechanism by which the TM UTR induces differentiation.

DNA immune responses induced by codelivery of IL-12 expression vectors with hepatitis C structural antigens.

OBJECTIVE: To demonstrate the utility of DNA vaccines for the tailored methods, the efficacy of enhanced immune responses, and the types of increased immune responses. METHODS: Four recombinant plasmids constructed included the coding regions for the core protein (pC) and for the core, E1 and E2 together (pCE1E2), IL-12 p35 and p40. These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/C mice for measurement of specific antibodies and cytotoxic T-lymphocyte (CTL) responses. RESULTS: All the recombinant plasmids were shown to express specific antigens stably in mammalian cells. Codelivery of pIL-12 expression cassettes with pC and pCE1E2 in mice resulted in the enhancement of Ag-dependent CTL responses and the reduction of specific Ab response. The CTL activity was: pC=18.65%+/-5.71%, pCE1E2=20.07%+/-11.11%, pC+pIL-12=60.11%+/-17.37%, pCE1E2+pIL-12=67.48%+/-15.57%, respectively. The average A values of anti-HCV were pC=0.415+/-0.127, pCE1E2=0.358+/-0.096, pC+pIL-12=0.210+/-0.086, pCE1E2+pIL-12=0.258+/-0.125. CONCLUSION: Codelivery of pIL-12 with plasmid DNA can enhance the efficacy of immune responses and shift the type of immune responses.