Development of a simple and rapid immunochromatographic strip test for diarrhea-causative porcine rotavirus in swine stool.

A rapid and simple immunochromatography (IC) strip test, for specific detection of porcine rotavirus (PRV) in stool specimen, was developed. Monoclonal antibodies (mAbs) to the OSU strain of PRV have been produced in mice. Among them, two hybridoma clones that generate mAb-1 and mAb-2, respectively, specific for VP6 protein of PRV, have been selected. In the IC configuration, mAb-1, one of the selected mAbs was used to the designed coat microparticles (MP), while another mAb-2 was used to fix it on the nitrocellulose membrane strip to form a result line. The control line was formed on the same membrane strip past the result line by fixing anti-mouse IgG antibody. The IC test was capable of detecting 1000 plaque-forming units of PRV/ml in less than 5min, and the binding capacity was demonstrated by specific recognition of PRV only, but not other porcine diarrhea viruses, transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). The IC test produced positive results with all the nine PRV-positive stool specimens and negative results with five different non-PRV specimens, which were identified previously by the polymerase chain reaction (PCR) test, respectively. The results indicate an excellent concordance between the two methods, suggesting a potential application of the three combinated IC tests (PRV, TGEV and PEDV) for the on-site, rapid screening of porcine diarrhea cases.

Antibacterial activity of agricultural waste derived wollastonite doped with copper for bone tissue engineering.

Bioactive ceramic materials with metal ions generation brought great attention in the class of biomaterials development and widely employed as a filler material for bone tissue regeneration. The present study aimed to fabricate calcium silicate based ceramic material doped with copper metal particles by sol-gel method. Rice straw of agricultural waste was utilized as a source material to synthesize wollastonite, then wollastonite was doped with copper to fabricate copper doped wollastonite (Cu-Ws) particles. The synthesized materials were subjected to physio-chemical characterization by TEM, DLS, FTIR, XRD and DSC analysis. It was found that the sizes of the WS particles was around 900nm, while adding copper the size was increased upto 1184nm and the addition of copper to the material sharpening the peak. The release of Cu ions was estimated by ICP analysis. The anti-bacterial potentiality of the particles suggested that better microbial growth inhibition against E. coli (Gram negative) and S. aureus (Gram positive) strains from ATCC, in which the growth inhibition was more significant against S. aureus. The biocompatibility in mouse Mesenchymal Stem cells (mMSC) showed the non-toxic effect up to 0.05mg/ml concentration while the increase in concentration was found to be toxic to the cells. So the particles may have better potential application with the challenging prevention of post implantation infection in the field of bone tissue engineering (BTE).

Sequence analysis of the 5'-flanking region of the gene encoding human N-acetylglucosaminyltransferase III.

We have isolated and characterized the immediate (1651 bP) 5'-flanking region of the gene (GnT-III) encoding N-acetylglucosaminyltransferase III (GnT-III) from a human placental genomic library. Analysis of promoter elements shows a similarity to the 5'-flanking region of murine 1,4-galactosyltransferase. The sequence lacks obvious TATA elements and CCAAT boxes; however, putative regulatory sites, including 2 potential cAMP-response regulatory elements (CRE), 11 insulin-response element consensus sequences (IRE), 7 potential AP-2 binding sites, 2 SP1 consensus sequences (GC boxes) and 2 sequences similar to the half-palindromic glucocorticoid-responsive element (GRE), are present.

Peptide-specific CTL induction in HBV-seropositive PBMC by stimulation with peptides in vitro: novel epitopes identified from chronic carriers.

Cytotoxic T lymphocytes (CTL) recognize and destroy virus-infected cells, and it has been established that epitope-based peptides could induce such CTL in vivo as well as in vitro. In this study attempts were made to define the epitopes that are recognized by the CTL, and thus a series of 9- to 10-mer peptides derived from the amino acid sequences of hepatitis B virus (HBV) proteins were synthesized on the basis of the previously described HLA-A2 peptide binding motif. The binding assay of the synthetic peptides using transporter-associated with antigen processing (TAP)-deficient human cell line, T2, showed that eight out of 11 peptides tested enhanced the expression of HLA-A2 molecules on the T2 cell surface. Some of these peptides triggered activation of CTL in peripheral blood mononuclear cells of HBV-seropositive chronic carriers. The activated CTL in turn recognized and killed the T2 cells pulsed with the same peptides. This study shows that novel HLA-A2-restricted epitopes exist in the natural repertoire of immunity against HBV. These findings can be useful in developing peptide-based therapeutics against viral infections.

Solid-phase immunoassay using a flow cytometer: quantitative and qualitative determination of protein antigens and a hapten.

A fluoroimmunoassay employing a flow cytometer as the fluorescence-detecting device is described. Three kinds of antigens, murine immunoglobulin (Ig), human chorionic gonadotropin (hCG) and progesterone were chosen as examples of the assay using fluorescein-labeled antibodies. Cyanogen bromide-activated agarose beads were used as solid-phase supporters. The flow cytometric immunoassay was applied to both qualitative and quantitative analyses; determination of murine Ig isotypes, quantitative determination of Ig, hCG and a hapten, progesterone. This assay produced very reproducible and less-fluctuating data since thousands of particles in the assay were collected and processed to produce a single value for fluorescence intensities. Furthermore, the working range of the assay in terms of antigen concentration was much broader than that of enzyme immunoassay. Therefore, we believe that microparticles like agarose beads could be useful solid-phase supporters in immunoassay, and the flow cytometer could provide a reliable alternative to the fluorescence-detecting device in immunoassay.

Molecular cloning and characterization of Mycobacterium bovis BCG pcp gene encoding pyrrolidone carboxyl peptidase.

The Mycobacterium bovis bacilli Calmette-Guerin (BCG) pcp gene that encodes the pyrrolidone carboxyl peptidase (Pcp) was cloned from a lambdagtll genomic library and sequenced. The nucleotide sequence contains a 669 bp open reading frame coding for a protein of 222 amino acid residues with a calculated molecular mass of 23,209 Da. The deduced amino acid sequence is highly homologous to the Pcps from Bacillus amyloliquefaciens, Pseudomonas fluorescens, Bacillus subtilis, Streptococcus pyogenes, and Staphylococcus aureus. A multiple sequence alignment revealed highly conserved domains. The BCG pcp gene was overexpressed in Escherichia coli. The Pcp was purified to homogeneity. The recombinant protein was further confirmed by an enzymatic assay.

Effects of the rate of solvent evaporation on the characteristics of drug loaded PLLA and PDLLA microspheres.

We investigated the effects of the rate of solvent removal by varying ambient pressure at a fixed temperature on the morphology, particle sizes, drug encapsulation efficiency and releases pattern of lidocaine loaded poly-L-lactatide (PLLA) and poly-D,L-lactatide (PDLLA) microspheres, prepared with O/W emulsion-solvent evaporation process. Prepared in the fast rate of solvent evaporation (FRSE) process by reducing ambient pressure, smoothly morphological surface of drug loaded PLLA and PDLLA microspheres was observed. While in the normal rate of solvent evaporation (NRSE) process, roughness or pinhole surface was only found at drug loaded PLLA microspheres. Fabricated in the FRSE process, both PLLA and PDLLA microspheres showed smaller particle sizes and lower drug encapsulation efficiencies than those prepared in NRSE process. In regard to two materials, PLLA microspheres had higher drug encapsulation efficiencies than PDLLA ones for both processes. Although initial burst releases of drug were observed for both PLLA and PDLLA microspheres prepared in whatever solvent removal process, drug release for PLLA microspheres was slightly less than that for PDLLA ones in the earlier stage of drug release. However, in the subsequent stage of drug release, there was no difference between two materials. In corporation with different crystalline characteristics of PLA polymer and its derivatives, FRSE process by reducing ambient pressure could be further applied to produce different characteristics of microspheres for drug delivery.

A method using biodegradable polylactides/polyethylene glycol for drug release with reduced initial burst.

Post-coating of biodegradable polylactides (PLA)/polyethylene glycol (PEG) microspheres with a gelatin film produced by dipping the microspheres into a gelatin solution to reduce the initial drug release burst was investigated. Biodegradable block PLA/PEG microspheres, prepared by w/o emulsion/solvent evaporation, showed that the hydrophilic segment PEG protruded to the sphere surface. However, these microspheres also showed a large burst release in the initial period. Post-coating of the PLA/PEG microspheres with a gelatin film by dipping the microspheres into a dilute gelatin solution effectively inhibited the initial burst release rate in the drug release tests. Post-coating of gelatin reduced 98% of the burst release. With thicker coatings, there were slower releasing rates, and the release rate can be simply related to the coating thickness.

Release of ciprofloxacin from poloxamer-graft-hyaluronic acid hydrogels in vitro.

Recently, in situ gel formation has extensively been studied to enhance ocular bioavailability and duration of the drug activity. In this study, we report grafting of poloxamer onto the hyaluronic acid for application of tissue engineering oriented ophthalmic drug delivery system. Graft copolymers were prepared by coupling mono amine-terminated poloxamer (MATP) with hyaluronic acid (HA) backbone using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxylsuccinimide (NHS) as coupling agents. The coupling of MATP with HA was clarified by 1H NMR and FT-IR spectroscopy. The gelation temperature of graft copolymers was dependent on the content of HA and the concentration of poloxamer. From drug release studies in vitro, ciprofloxacin was sustainedly released from the poloxamer-g-hyaluronic acid hydrogel due to the in situ gel formation of the copolymer and viscous properties of HA.

A biotin-avidin labeled enzyme immunoassay for the quantitation of serum TSH using protein-layered solid phase.

A sensitive enzyme immunoassay for serum TSH has been developed utilizing the tight binding between biotin and avidin, and three layered protein polystyrene beads as solid phase. To increase binding capacity of TSH and sensitivity of the assay, the polystyrene beads were coated sequentially with mouse immunoglobulin as first layer, rabbit antimouse immunoglobulin as second layer and monoclonal anti-TSH as third layer. A serum sample was incubated simultaneously with a monoclonal anti-TSH immobilized polystyrene beads and a second monoclonal anti-TSH covalently attached to biotin. After washing, the antibody bound serum TSH-anti-TSH-biotin complex is reacted with horseradish peroxidase (HRP)-labeled avidin. Following a second wash, the bound HRP activity was measured colorimetrically. Reproducible results were obtained within 4 hours for serum TSH in the range between 0 microIU/ml and 50 microIU/ml with detection limit of 0.1 microIU per test.

Changes in viscosity of low shear rates and viscoelastic properties of oxidative erythrocyte suspensions.

We exposed erythrocytes in soluble hemoglobin and Fe+2, in which hydroxyl radical (OH*) might be generated, and measured low shear rate viscosity and viscoelasticity of erythrocyte suspensions at Hct of 40%. The quantities of the lipid peroxidation product, malonyldialdehyde (MDA), for oxidized samples were higher than that for control (e.g., 2.20 +/- 0.46 nmol and 1.70 +/- 0.42 nmol, n = 6, p = 0.01, respectively). The viscosity values of oxidized erythrocyte suspensions for all tested shear rates were higher than those for the control samples (p < 0.05 or better, n = 6). Dynamic viscosity (eta') of oxidized erythrocyte samples was higher than that of control samples at the tested shear stress of 30 mPa whereas it was not observed in elasticity values (eta"). We tentatively concluded from the study, that oxidized erythrocytes would be more prone to form aggregates and increase viscosity of blood at low shear rates. Therefore, they might impair blood flow in the microcirculation.

A model for analyzing the formation of thrombin in vessels.

A pseudo steady-state model is proposed to analyze the effects of a vessel-length, blood-flow velocities, and the concentration levels of Va/Xa/phospholipid complex on the formation of thrombin in an arterial vessel. The outlet concentrations of thrombin are calculated in either physiological or pathological coagulation process. Among those factors, blood-flow velocity is the most important factor which affects the amount of thrombin formation. The model and its results might be used as a reference for those who clinically manage coagulation problems of patients with severe sepsis.

Mechanical properties of microwave hydrothermally synthesized titanate nanowires.

In this investigation titanate nanowires were synthesized by a microwave hydrothermal process and their nanomechanical characterization was carried out by a compression experiment via buckling instability using a nanomanipulator inside a scanning electron microscope. Nanowires of diameters 120-150 nm and length tens of microns can be synthesized by keeping a commercial nanoparticle inside a microwave oven at 350 W and 210 °C for 5 h. The nanowire was clamped between two cantilevered AFM tips attached to two opposing stages of the manipulator for nanomechanical characterization. The elasticity coefficients of the titanate nanowires were measured by applying a continuously increasing load and observing the buckling instability of the nanowires. The buckling behavior of a nanowire was analyzed from the series of SEM images of displacement of the cantilever attached to the nanowire due to application of load. The critical loads for different sized titanate nanowires were determined and their corresponding Young's modulus was computed with the Euler pinned-fixed end model. The Young's modulus of these microwave hydrothermal process synthesized titanate nanowires were determined to be approximately in the range 14-17 GPa. This investigation confirms the capability of the nanomanipulator via the buckling technique as a constructive device for measuring the mechanical properties of nanoscale materials.

Effect of lactate dehydrogenase isoenzymes on the coupled enzymatic assay for alanine aminotransferase activity.

We show an example of the importance of specifying the form of isoenzyme and source of indicator enzymes to be used in coupled enzymatic assays. When we compared H-4 (pig heart) and M-4 (rabbit muscle) isoenzymes of lactate dehydrogenase for their suitability as indicator enzymes in the assay for alanine aminotransferase activity, we found that about fourfold as much M-4 as H-4 was required in terms of lactate dehydrogenase activity to reflect accurately equivalent amounts of alanine aminotransferase activity. Moreover, the substrate specificities of the two isoenzymes differed quantitatively.

Viscosity measurements for LEH suspended in different plasma expanders.

To investigate the possibility of hemodilution with oxygen carrying fluid, we measured the viscosity effects of LEH which was suspended in different weight percentage of plasma expanders for different shear rates. All of LEH/Plasma expander suspensions show shear thinning flow behavior, and the viscosity of those suspensions increase with increased the weight percentage of each expander in suspensions for a fixed shear rate. Compared the viscosity data to those of human blood at 40% of hematocrit, LEH/albumin suspensions contained with 4% to 8% of albumin are the most suitable, whereas LEH/dextran (Dex) suspensions contained with 6% Dex70c is the least favorable suspension media for LEH. In point of viscosity effects, hemodilution with LEH/albumin or LEH/0.9% oxypolygelatin (Gel) may be valuable for further testing their efficacy in animal model.

An engineering model to characterize oxygen transfer rates for liposome encapsulated hemoglobin (LEH).

An engineering model was designed to evaluate oxygen transfer rates for LEH and other oxygen carriers under wall shear rates from 150 sec-1 to 450 sec-1. The results showed that increasing the shear rates (or flow rates) of oxygen carriers flowed inside of hollow fiber tubes would increase oxygen transfer rates to outside media. The values of overall oxygen transfer rate coefficients for LEH, based on 1 g/dl of hemoglobin contents, were about 2 to 2.5 times higher than those values for human blood at all of tested shear rates (e.g., 5.1 x 10(-5) cm/sec and 2.1 x 10(-5) cm/sec for LEH and blood at wall shear rates of 450 sec-1, respectively). Moreover, the results of oxygen transfer efficiency for LEH calculated by this model were consistent with the similar results reported by Usuba et al.[3] obtained by animal study. With an engineering model, we possibly estimate the effects of other factors such as viscosity on the oxygen transfer rates for LEH in microcirculation.

Microencapsulation of gentamicin in biodegradable PLA and/or PLA/PEG copolymer.

Biodegradable carriers containing gentamicin for local treatment of bone infection were developed. This paper describes the preparation and in vitro evaluation of these biodegradable implants. Poly-L-lactic acid (PLA) and poly-L-lactic acid:polyethylene glycol (PLA/PEG) disk implants containing gentamicin sulphate were obtained by compression of microspheres prepared by a double emulsion process. The mean particle size distribution of the microspheres, based on volume, ranged from 95-270 microm. The gentamicin sulphate loading of the microspheres, after a methylene chloride-water extraction procedure, exceeded 90% of the theoretical value. In vitro dissolution studies on the microspheres and implants with drug loadings 10-40% w/w indicated that the rate of drug release from both PLA and PLA/PEG implants increased, with an increase in drug loading. The release of gentamicin from microspheres was dependent on the properties of PLA and/or PLA/PEG. The PLA/PEG copolymer was more hydrophilic than the PLA homopolymer, and with a smaller pH change in the microenvironment with polymer being degraded. In comparison, the PLA/PEG implant released antibiotic faster and had a larger inhibitory zone based on the Bauer-Kirby experiments used to test the inhibitory activity of antimicrobial devices. Experimental results showed that the biodegradable PLA/PEG gentamicin delivery system had a potential for prophylaxis of post-operative infection.

Structural role of amino acids 99-110 in recombinant human erythropoietin.

Erythropoietin is the prime regulator of red blood cell production. Previous studies demonstrated that antipeptide antibodies to amino acids 99-119 and 111-129 bind to two non-overlapping domains and inhibit the hormone's action (Sytkowski, A.J. & Donahue, K. A. (1987) J. Biol. Chem. 262, 1161-1165). Oligonucleotide-directed mutagenesis now shows that amino acids 99-110 (domain 1) but not 119-129 (domain 2) are important to erythropoietin's structure and function. Mutagenesis of wild-type human erythropoietin cDNA was used to produce a series of mutant proteins with sequential deletion of three adjacent amino acids and insertion of the sequence Glu-Phe across the two domains. Transient expression in COS-7 cells revealed 2.0-kb transcripts encoded by all of the cDNAs. Domain 2 mutants exhibited specific biological activities similar to that of the wild type. In contrast, domain 1 mutants were not secreted. In vitro transcription and translation of the domain 1, domain 2 and wild-type cDNAs resulted in the isolation of 23.5-kDa and 32-kDa proteins in the absence or presence of pancreatic microsomes, respectively, consistent with efficient translation of all of the mutants and equivalent post-translational processing of each protein. The data suggest that mutation within domain 1 results in the intracellular biosynthesis of erythropoietins with altered structure, rendering them subject to rapid degradation. The bioassay of erythropoietins synthesized entirely in vitro demonstrated that domain 1 mutants were inactive, whereas both wild type and domain 2 mutant hormones exhibited biologic activity. The results are consistent with a critical role for amino acids 99-110 in the structure of human erythropoietin.