PVX-tolerant potato development using a nucleic acid-hydrolyzing recombinant antibody.

3D8 scFv, a catalytic recombinant antibody developed in the MRL mouse, exhibits nucleic acid-hydrolyzing activity. Previous studies have demonstrated that tobacco plants harboring 3D8 scFv antibodies showed broad-spectrum resistance to infection by both DNA and RNA viruses. In this study, potatoes were transformed with the 3D8 scFv gene and screened by potato virus X (PVX) challenge. Starting with the T0 and T1 potato lines, PVX-tolerant T1 potatoes were identified in the field and characterized by ELISA and RT-PCR analysis. T2 potatoes were propagated for T3 generation and additional virus challenges in the field, and 44% of the 3D8 scFv T3 transgenic potatoes grown in GMO fields were found to be tolerant to PVX infection. Tubers from PVX-tolerant T3 lines were 60% bigger and 24% heavier, compared with tubers from PVX-susceptible transgenic lines and wild-type potatoes. Three-step virus challenge experiments and molecular characterization techniques were used for plants grown in growth chambers or fields to identify 3D8 scFv-transgenic, PVX-tolerant potatoes. These studies also revealed that the viral tolerance enabled by 3D8 scFv persisted during asexual propagation.

Biological Behavior of Osteoblast Cell and Apatite Forming Ability of the Surface Modified Ti Alloys.

Titanium as one kind of biomaterials comes in direct contact with the body, making evaluation of biocompatibility an important aspect to biomaterials development. Surface chemistry of titanium plays an important role in osseointegration. Different surface modification alters the surface chemistry and result in different biological response. In this study, three kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coating successfully formed on the Ti surfaces in simulated body fluid. Strong mixed acid increased the roughness of the metal surface, because the porous and rough surface allows better adhesion between Ca-P coatings and substrates. After modification of titanium surface by mixed acidic solution and subsequently H2O2/HCL treatment evaluation of biocompatibility was conducted from hydroxyapatite formation by biomimetic process and cell viability on modified titanium surface. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Results from this study indicated that surface treatment methods affect the surface morphology, type of TiO2 layer formed and subsequent apatite deposition and biological responses. The thermo scientific alamarblue cell viability assay reagent is used to quantitatively measure the viability of mammalian cell lines, bacteria and fungi by incorporating a rapid, sensitive and reliable fluorometric/colorimetric growth indicator, without any toxic and side effect to cell line. In addition, mixed acid treatment uses a lower temperature and shorter time period than widely used alkali treatment.

Biomimetic Deposition of Hydroxyapatite by Mixed Acid Treatment of Titanium Surfaces.

A simple chemical method was established for inducing bioactivity of Ti metal. In the present study, two kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coatings successfully formed on the Ti surfaces in the simulated body fluid. Strong mixed acid etching was used to increase the roughness of the metal surface, because the porous and rough surfaces allow better adhesion between Ca-P coatings and substrate. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Some specimens were treated with a 5 M NaOH aqueous solution, and then heat treated at 600 °C in order to form an amorphous sodium titanate layer on their surface. This treated titanium metal is believed to form a dense and uniform bone-like apatite layer on its surface in a simulated body fluid (SBF). This study proved that mixed acid treatment is not only important for surface passivation but is also another bioactive treatment for titanium surfaces, an alternative to alkali treatment. In addition, mixed acid treatment uses a lower temperature and shorter time period than alkali treatment.

Exoplanet detection. A terrestrial planet in a ~1-AU orbit around one member of a ~15-AU binary.

Using gravitational microlensing, we detected a cold terrestrial planet orbiting one member of a binary star system. The planet has low mass (twice Earth's) and lies projected at ~0.8 astronomical units (AU) from its host star, about the distance between Earth and the Sun. However, the planet's temperature is much lower, <60 Kelvin, because the host star is only 0.10 to 0.15 solar masses and therefore more than 400 times less luminous than the Sun. The host itself orbits a slightly more massive companion with projected separation of 10 to 15 AU. This detection is consistent with such systems being very common. Straightforward modification of current microlensing search strategies could increase sensitivity to planets in binary systems. With more detections, such binary-star planetary systems could constrain models of planet formation and evolution.

Evaluation of the bonding strength of dental zirconia with veneering porcelains.

The effect of thermo-cycling treatment on the bond strength and flexural strength of porcelain veneered zirconia was evaluated. After thermo-cycling treatment between 5 degrees C to 55 degrees C, porcelain-zirconia bond strength and zirconia flexural strength was not significantly affected. In the phase analyses using XRD after thermo-cycling treatment, both the experimental group and the control group showed only tetragonal phases. That is, the porcelain-zirconia bond strength and zirconia flexural strength were not affected by low temperature degradation. So low temperature aging treatment did not reduce the flexural strength and the effect of temperature applied to the aging treatment could beignorable.

Peripheral venous pressure as an alternative to central venous pressure in patients undergoing laparoscopic colorectal surgery.

BACKGROUND: Peripheral venous pressure (PVP) is strongly correlated with central venous pressure (CVP) during various surgeries. Laparoscopic surgery in the Trendelenburg position with pneumoperitoneum typically increases CVP. To determine whether PVP convincingly reflects changes in CVP, we evaluated the correlation between PVP and CVP in patients undergoing laparoscopic colorectal surgery. METHODS: Both CVP and PVP were measured simultaneously at predetermined time intervals during elective laparoscopic colorectal surgery in 42 patients without cardiac disease. The pairs of venous pressure measurements were analysed for correlation, and the Bland-Altman plots of repeated measures were used to evaluate the agreement between CVP and PVP. RESULTS: A total of 420 data pairs were obtained. The overall mean CVP was 11.3 (sd 4.5) mm Hg, which was significantly lower than the measured PVP of mean 12.1 (4.5) mm Hg (P=0.005). There was a strong positive correlation between overall CVP and PVP (correlation coefficient=0.96, P<0.0001). The mean bias (PVP-CVP) corrected for repeated measurements using random-effects modelling was 0.9 mm Hg [95% confidence interval (CI) 0.54-1.19 mm Hg] with 95% limits of agreement of -1.2 mm Hg (95% CI -1.75 to -0.62 mm Hg) to 2.9 mm Hg (95% CI 2.35-3.48 mm Hg). CONCLUSIONS: PVP displays a strong correlation and agreement with CVP under the increased intrathoracic pressure of pneumoperitoneum in the Trendelenburg position and may be used as an alternative to CVP in patients without cardiac disease undergoing laparoscopic colorectal surgery.

Effect of glass phase on the dissolution of hydroxyapatite.

Nano size defect formation at grain boundary during the dissolution of hydroxyapatite in water was evaluated by adding several sintering additives for sinterability enhancement. In the case of sintered pure hydroxyapatite, significant dissolution occurred after immersion in distilled water or in simulated body fluid. The dissolution initiated at the grain boundaries creating nano-size defects like small pores that afterwards grew up to micro scale by increasing immersion time. This dissolution resulted in grain separation at the surfaces and finally in fracture. The dissolution concentrated on the grains adjacent to pores rather than those in the dense region. So hydroxyapatite ceramics containing glass powders were prepared to prevent the dissolution by strengthening grain boundary. Calcium silicate and phosphate glasses were added at 0 to 10 mass% and sintered at 1200 degrees C for 2 h in air with moisture protection. Glass phase was incorporated into hydroxyapatite to act as the sintering aid followed by crystallization in order to improve the mechanical properties without reducing biocompatibility. Dissolution tests, as well as X-ray diffraction and SEM showed little decomposition of hydroxyapatite to secondary phases and the fracture toughness increased compared to pure hydroxyapatite.

Characterization, stability and in-vivo distribution of asialofetuin glycopeptide incorporating DSPC/CHOL liposomes prepared by mild cholate incubation.

In this study, a small triantennary asialoglycopeptide of fetuin (A-F2) was used as a ligand to direct liposomes to hepatocytes. A-F2 was cleaved from asialofetuin, purified, conjugated with fatty acids and incorporated into pre-formed sonicated DSPC/Chol (2:1) liposomes. A mild cholate incubation method for incorporating the A-F2 ligand on pre-formed vesicles was used. In preliminary in vivo experiments 111In3+ encapsulated in A-F2/palmityl liposomes was seen to accumulate in the liver of mice significantly faster than when encapsulated in non-ligand bearing liposomes of the same lipid composition (studied before), justifying further investigation of this system. The presence of the A-F2/fatty acid conjugate in a functional form on the vesicle surface was confirmed by their reversible agglutination in the presence of Ricinus communis agglutinin (RCA120). Effects of ligand incorporation on the vesicle size distribution, z-potential, membrane integrity and stability were monitored. The results demonstrate that highest ligand incorporation was achieved when liposomes and ligand were co-incubated in the presence of 1 mM sodium cholate. Incorporation increased with the length of the fatty acid used for A-F2 conjugation. Ligand-bearing liposomes were demonstrated to be smaller in diameter (about 30%) with a more positive z-potential in comparison to control vesicles while ligand incorporation did not influence the liposome membrane integrity. The size of the ligand-incorporating vesicles was maintained after 24 hours of incubation in isotonic buffer, proving that the vesicles do not aggregate. Although the preliminary biodistribution results may suggest that ligand bearing liposomes are accumulating in the liver, further cell culture, in vivo distribution and especially liver fractionation studies are required in order to clarify the intrahepatic localization of these liposomes and the ability to target liver hepatocytes in vivo.

Determination of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical formulation by high performance liquid chromatography with electrochemical detection.

A novel HPLC method with electrochemical detection has been developed for the determination of recombinant human epidermal growth factor (rhEGF) in pharmaceutical products. rhEGF was separated from other components in formulation on a reversed-phase C18 column with 24% acetonitrile in 0.1 M phosphate buffer (pH 4.75). The optimum electrochemical oxidation of EGF was obtained at 0.85 V vs. Ag/AgCl in a glassy carbon working electrode due to electroactive tyrosine, tryptophan, methionine, and arginine residues. The quantitation range was from 1.0 to 200 ng of rhEGF with the linear correlation coefficient greater than 0.999. The method was successfully applied for the quantitation of rhEGF in a pharmaceutical preparation.

Inhibition of calmodulin-dependent calcium-ATPase and phosphodiesterase by various cyclopeptides and peptide alkaloids from the Zizyphus species.

The effects of various sedative cyclopeptides and peptide alkaloids from the Zizyphus species on calmodulin-dependent Ca2+-ATPase and phosphodiesterase were investigated. Calmodulin-induced activation of Ca2+-ATPase was strongly inhibited by sanjoinine-A dialdehyde (IC0O, 2.3 microM), -Ah1 (IC50, 4.0 microM), -A (IC50, 4.6 microM), and -G2 (IC50, 7.2 microM), while calmodulin-induced activation of phosphodiesterase was strongly inhibited by both deachuine-S10 (IC30, 4.9 microM) and sanjoinine-D (IC50, 9.0 microM). The inhibitory activity of the various cyclopeptides and peptide alkaloids on Ca2+-ATPase was found to correlate well with their sedative activity.

Determination of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation by capillary electrophoresis.

A simple assay method of recombinant human epidermal growth factor (rhEGF) in a pharmaceutical preparation was studied and validated by capillary electrophoresis (CE) using micellar electrokinetic chromatography (MEKC) techniques. Factors affecting the migration behavior and separation performances of the peptide; type of buffer, pH, buffer concentration, and concentration of sodium dodecyl sulfates (SDS) were investigated to optimize the analytical performance. CE was performed using running buffer, 50.0 mM borate (pH 8.5) containing 12.5 mM SDS at 20 kV of the applied voltage. Calibration curves for the rhEGF showed good linearity (r>0.999) over the wide dynamic range from 1.25 to 100 microg/ml. Sample analysis was performed by using standard addition method to eliminate the matrix effects of dosage vehicle. This method is assumed to be useful for quality control (QC) of various forms of pharmaceutical products of the peptide.

Lipopolysaccharide enhances substance P-mediated neutrophil adherence to epithelial cells and cytokine release.

Lipopolysaccharide (LPS) is implicated in many respiratory tract inflammatory diseases. Tachykinins, especially substance P (SP) through the NK-1 receptor, mediate leukocyte adhesion to the endothelial or airway epithelial cells. Here we assessed the enhancement by LPS of tachykinin-mediated neutrophil adherence to alveolar epithelial cells, and associated interleukin-1 beta (IL-1beta) and tumor necrosis factor (TNF-alpha) release. Neutrophil adherence to A549 epithelial cell was not increased by LPS (100 ng/ml), or SP (10(-)(12)-10(-)(8) M) alone, but was significantly enhanced by their combination (LPS + SP). Neutrophil adherence to epithelial cells induced IL-1beta and TNF-alpha release from A549 cells either spontaneously or stimulated by SP or LPS. LPS + SP significantly enhanced IL-1beta and TNF-alpha release. The NK-1 receptor antagonist L-732,138 inhibited this enhancement response. Prevention of neutrophil adherence by CD11b/CD18 blocking antibody or by placing a filter on the epithelial monolayer diminished spontaneous or LPS + SP-enhanced IL-1beta and TNF-alpha release. Pretreatment with the serine protease inhibitor cocktail also inhibited LPS + SP-enhanced neutrophil adherence-dependent IL-1beta and TNF-alpha release as well as their mRNA expression. In conclusion, we have demonstrated LPS enhanced SP-mediated neutrophil adherence and associated IL-1beta and TNF-alpha release from the A549 epithelial monolayer, partly through NK-1 receptors. Neutrophil adherence to epithelial cells may release serine protease to induce IL-1beta and TNF-alpha release and their synthesis.

Overexpression of the transforming growth factor-beta-inducible gene betaig-h3 in anterior polar cataracts.

PURPOSE: In anterior polar cataracts and the fibrosis that can occur after cataract surgery, lens epithelial cells synthesize abundant extracellular matrix molecules and transdifferentiate into myofibroblast-like cells. Transforming growth factor (TGF)-beta has been implicated as a key player in these cataractous changes. The purpose of this study was to determine whether the TGF-beta-inducible gene h3 (betaig-h3) is expressed in lens epithelial cells from patients with anterior polar cataracts and to test whether betaig-h3 is induced by TGF-beta in cultured lens epithelial cells. METHODS: Lens epithelial cells attached to the anterior capsules of human cataractous lenses and noncataractous lenses were examined for the expression of betaig-h3 mRNA and protein using reverse transcription-polymerase chain reaction and immunohistochemical analyses. The effect of TGF-beta on betaig-h3 gene expression was also tested in human lens epithelial B-3 cells using Northern and Western blot analyses. RESULTS: betaig-h3 mRNA was not detected in lens epithelial cells from patients with clear lenses or patients with nuclear cataracts. Significant expression of mRNA for betaig-h3 was observed in lens epithelial cells from patients with anterior polar cataracts. Immunohistochemical analysis using anti-betaig-h3 antiserum indicated that betaig-h3 protein was present within the subcapsular plaques of anterior polar cataracts. Treatment of human lens epithelial B-3 cells with TGF-beta1 led to an increase in betaig-h3 mRNA and the secretion of betaig-h3 protein into the culture medium. CONCLUSIONS: betaig-h3 may serve as a marker for anterior polar cataracts in addition to previously known proteins, fibronectin, type I collagen, and alpha-smooth muscle actin. The functions of this protein in lens pathology need to be further investigated.

Endotoxemia augments neurogenic plasma exudation in guinea pig lungs.

BACKGROUND: Lipopolysaccharide (LPS) is closely associated with the development of infection-induced deleterious pulmonary reactions. In this study, we investigated the enhancement effects of LPS on tachykinin-mediated plasma exudation in the lungs of guinea pigs. The role of oxidants was also explored. METHODS: Intravenous LPS (100 mu kg-1) or its vehicle was administered 0 to 3 hours prior to bilateral electrical or sham stimulation of the cervical vagus nerves in animals anesthetized with urethane and artificially ventilated. Plasma exudation into the lungs was assessed by measurement of extravasated 125I-albumin which had been intravenously administered before stimulation. RESULTS: The plasma exudation in the lungs increased after bilateral cervical vagal stimulation. LPS alone did not induce significant plasma exudation. The vagally-mediated plasma exudation was enhanced by LPS with the peak effect 1 hour after LPS administration. LPS also enhanced exogenous substance P (10(-8) mol kg-1, i.v.)-induced plasma exudation. The vagally-induced plasma exudation was abolished by a specific neurokinin-1 (NK-1) receptor antagonist, L-732,138. The LPS-induced enhancement response was also attenuated by L-732,138. The vagally-induced plasma exudation was not affected by superoxide dismutase (SOD, 5000 U kg-1, i.p.) pretreatment. However, SOD significantly inhibited the LPS-enhanced neurogenic plasma leakage. The LPS-induced enhancement was not completely abolished by either L-732,138 or SOD pretreatment alone, but by a combination of both. CONCLUSION: LPS augments neurogenic plasma exudation partly through NK-1 receptors to increase vascular permeability and partly via the generation of oxidative metabolites. Tachykinins released from nerve endings may contribute to endotoxin-related pulmonary inflammatory responses.

Lipopolysaccharide enhances neurogenic plasma exudation in guinea-pig airways.

1. Lipopolysaccharide (LPS) is implicated in many pulmonary and airway inflammatory diseases. Tachykinins released from nerve endings increase vascular permeability. In this study, we have assessed the enhancement by LPS of tachykinin-mediated plasma exudation in guinea-pig airways, and examined the role of oxidants as well as leukocyte adherence. 2. LPS (100 microg kg(-1), i.v.) was administered 0-3 h before bilateral electrical stimulation of the cervical vagus nerves in animals anaesthetized with urethane and ventilated. Vagal stimulation increased vascular permeability in the airways. LPS enhanced the vagally-mediated plasma exudation with the peak effect at 1 h after LPS administration. LPS alone induced no significant plasma exudation. LPS also enhanced exogenous substance P (10(-8) mol kg(-1), i.v.)-induced plasma exudation. 3. The NK-1 receptor antagonist L-732,138 abolished vagally-induced plasma exudation and significantly inhibited the enhancement by LPS. Pretreatment with superoxide dismutase (SOD, 5000 U kg(-1), i.p.) did not affect the vagally-induced plasma exudation, but inhibited the LPS-enhanced neurogenic plasma leakage. The LPS-enhanced vagally-induced plasma exudation was not completely inhibited by either L-732,138 or SOD pretreatment alone, but was blocked by the combination of both pretreatments. 4. Neutrophil depletion by cyclophosphamide alone did not influence vagally-induced plasma exudation, but significantly inhibited the LPS-enhanced response. 5. In conclusion, we have demonstrated LPS enhanced neurogenic plasma exudation by augmenting the response to tachykinins, partly through NK-1 receptors, to directly increase vascular permeability or to enhance leukocyte adhesion-mediated endothelial cell injury. Tachykinins released from nerve endings may contribute to endotoxin-related airway inflammatory responses.

Effect of endogenous nitric oxide on tumour necrosis factor-alpha-induced leukosequestration and IL-8 release in guinea-pigs airways in vivo.

1. Tumour necrosis factor-alpha (TNF-alpha) is implicated in the pathogenesis of many pulmonary and airway diseases. TNF-alpha stimulation may release interleukin-8 (IL-8) in airways mediated via an increase in intracellular oxidant stress. In the present study, we have assessed leukosequestration and IL-8 release in the airways in response to intratracheal administration of human recombinant TNF-alpha, and examined the modulatory role of endogenous NO by pretreatment with a NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). 2. TNF-alpha (10(2)-10(-4) u) was administered intratracheally in male guinea-pigs which were anaesthetized with urethane and were ventilated artificially. TNF-alpha induced a time- and dose-related increase in neutrophil numbers and a concomitant increase in human IL-8 equivalent level retrieved from bronchoalveolar lavage (BAL) with the peak effect at 10(3) u at 6 h of TNF-alpha injection (late phase). Intratracheal administration of recombinant human (rh)IL-8 (0.025, 0.25, 2.5 ng) producing a similar range of human IL-8 equivalent levels in BAL as measured in our results induced neutrophil recovery in BAL fluid to a similar extent. Administration of anti-IL-8 antibody prevented the late phase of neutrophil recruitment induced by TNF-alpha or rhIL-8. 3. Pretreatment with L-NAME significantly enhanced the TNF-alpha (10(3) u)-induced neutrophil recruitment and human IL-8 equivalents production at 6 h, but not at 1 h of TNF-alpha administration (early phase). L-Arginine reversed the responses to L-NAME. Pretreatment with 0.2% DMSO (i.v.) significantly inhibited TNF-alpha-induced neutrophil recruitment and human IL-8 equivalents release both in the early and late phase of the responses. Pretreatment with DMSO also inhibited the enhancement effect of L-NAME on the late phase of TNF-alpha-induced responses. DMSO failed to modify exogenous rhIL-8-induced neutrophil recruitment. Neither L-NAME nor DMSO alone induced any significant change in neutrophil numbers or human IL-8 equivalent level in BAL fluid. 4. Neutrophil depletion by cyclophosphamide pretreatment failed to modify TNF-alpha-induced human IL-8 equivalent release. 5. The expression of beta 2-integrin, CD11b/CD18 on neutrophils was increased only in the late but not early phase of TNF-alpha stimulation. L-NAME failed to modify these responses. 6. In conclusion, we demonstrated that NO may be an important endogenous inhibitor of TNF-alpha-induced leukocyte chemotaxis via inhibition of IL-8 production. Thus, the production of NO in airway inflammatory diseases may play a negative feedback role in self-limiting the magnitude of inflammatory responses.

Discrepancy of recovery times related to potency between atracurium and mivacurium simultaneously administered in isolated forearms.

Recovery from potent non-depolarising muscle relaxants is slower than from the less potent agents. However, recovery from mivacurium, which is more potent than atracurium, is faster than from atracurium following systemic administration. In an attempt to confirm this discrepancy we compared recovery times following simultaneous administration of equipotent doses of atracurium and mivacurium into the isolated forearms of human volunteers (n = 10). This method enabled us to study the interaction of muscle relaxants with receptors at the neuromuscular junction separated from the effects of plasma drug concentration. In these experiments, the recovery times from maximum block to 50% recovery of control twitch height were significantly longer with mivacurium than with atracurium (mean 25.2(SD 4.7) versus 22.6(3.1) min, p < 0.01). We found that the evidence that mivacurium has a slower recovery than the less potent atracurium may be true using the bilateral, isolated forearm technique and that the discrepancy might be due to a difference in the pharmacokinetic variables of the two drugs.