Flufenoxuron, an insect growth regulator, promotes peroral infection by nucleopolyhedrovirus (BmNPV) budded particles in the silkworm, Bombyx mori L.

A novel method was developed to infect perorally the silkworm Bombyx mori L. with budded particles of nucleopolyhedrovirus (BmNPV) using flufenoxuron, an insect growth regulator. NPV vectors are used to obtain proteins that occur naturally in minute amounts. NPV vectors are constructed conventionally by replacing the polyhedrin gene with the foreign gene of interest. These vectors thus do not produce polyhedra. The budded virus particle suspension of these vectors is produced in a cell culture and used as the stock inoculum. Budded NPV particles do not infect their host perorally. The inoculum is injected manually into the individual host using a syringe. It was found that B. mori L. fed on the insect growth regulator flufenoxuron were sensitive to BmNPV budded particles given perorally. Over 90% of B. mori L. ingesting BmNPV budded particles (1.3×10⁶ TCID₅₀ units per larva) after consuming an artificial diet for 24 h, containing 0.1% (w/w) flufenoxuron died of the viral infection. The peroral inoculation of BmNPV budded particles by flufenoxuron may thus lead to industrial pharmaceutical production using a baculovirus vector for large numbers of insect hosts.     

Detection and quantitation of Solenopsis invicta virus in fire ants by real-time PCR

A quantitative real-time PCR (QPCR) method was developed to detect and quantify the amount of Solenopsis invicta virus (SINV) infecting individual ants of S. invicta. The two-step method utilized a gene-specific oligonucleotide primer targeting the SINV RNA-dependent RNA polymerase (RdRp) for cDNA synthesis. Dithiothreitol used in the cDNA synthesis step was found to significantly decrease the detection sensitivity for SINV RdRp and was therefore omitted. SINV RdRp cDNA was then quantified by QPCR using SYBR Green dye and a standard curve generated from SINV RdRp plasmid clones. A standard curve was successfully constructed from clones of the SINV RdRp region. A strong linear relationship [r² = 0.998; y = (−3.63 ± 0.37)x + (39.19 ± 1.33)] between C_T and starting SINV RdRp copy number was observed within a dynamic range of 5–5 × 10⁶ copies. SINV RdRp copy number was determined with the optimized QPCR method in individual S. invicta ants taken from an infected field colony. Worker ants exhibited the highest RdRp copy number (2.1 × 10⁹ copies/worker ant) and pupae exhibited the lowest (4.2 × 10² copies/pupa). Mean RdRp copy number was lowest in early larvae and pupae. Overall, SINV RdRp copy number increased through larval development, sharply declined during pupation, then sharply increased in adults.     

New modulated metallic macrocycles: Electrochemistry and their interaction with calf thymus DNA

New modulated pentacoordinate complexes [C₁₇H₃₄N₇O₂Cu]ClO₄ (5), [C₁₇H₃₄N₇O₂Co]ClO₄ (6), [C₁₇H₃₄N₇O₂Ni] ClO₄ (7) have been synthesized by the interaction of 1,8-dihydro-1,3,6,8,10,13 Cu(II) (2), Co(II) (3), Ni(II) (4) hexaazacyclotetradecane complexes and Hsalea N-(2-hydroxy benzyl)-2-amino-1-ethanol ligand (1). All the complexes have been characterized by infrared, electron paramagnetic resonance, ¹H and ¹³C nuclear magnetic resonance (NMR), 2D correlation spectroscopy NMR and ultraviolet–visible (UV–vis) spectroscopy. In all the complexes, the metal center is encapsulated by the Hsalea ligand in a pentacoordinate environment. Conductance measurements show that the complexes are ionic in nature. UV–vis absorption titration and viscometric studies have been carried out to ascertain the interaction of complexes 5 and 6 with calf thymus DNA (CT DNA). The experimental results suggest that complex 5 binds to CT DNA through partial intercalation of the aromatic ring into the base pair of DNA while complex 6 binds to CT DNA by electrostatic mode. The intrinsic binding constants K_b of complex 5 and 6 were found to be 6.8 × 10⁻⁵ M⁻¹ and 1.8 × 10⁻⁴ M⁻¹, respectively. The binding of complexes 5 and 6 with CT DNA has also been investigated by cyclic voltammetry.     

Molecular beacon real-time PCR detection of swine viruses

Rapid and reliable detection of viral pathogens is critical for the management of the diseases threatening the economic competitiveness of the swine farming industry worldwide. Molecular beacon assays are one type of real-time polymerase chain reaction (PCR) technology capable of fast, specific, sensitive, and reliable viral detection. In this paper, the development of molecular beacon assays as novel tools for the rapid detection of Aujeszky's disease virus, African swine fever virus, porcine circovirus type 2 and porcine parvovirus is described. The assays are capable of rapidly detecting 2 × 10¹ copies of target and are linear between 2 × 10⁹ and 2 × 10² copies. They can detect virus specifically in clinical samples such as whole blood, serum and tissue. In comparison to conventional PCR they are either as sensitive or more sensitive. As such these molecular beacon assays represent a powerful tool for the detection of these viruses in swine.     

Inhibitoty Effects of Local Anesthetics on Migration, Extracellular Release of Lysosomal Enzyme, and Superoxide Anion Production in Human Polymorphonuclear Leukocytes

This study examined the effects of four typical local anesthetics, lidocaine, prilocaine, procaine and tetracaine, on the functioning of human polymorphonuclear leukocytes (PMN). PMN were stimulated by fMet-Leu-Phe (FMLP) or phorbol myristate acetate (PMA) to elicit chemotaxis, extracellular release of beta-glucuronidase (BGL) and superoxide anion (SOA) production. the four agents inhibited chemotaxis efficiently and in a concentration-dependent manner but had only weak effects on the release of BGL. the effect of tetracaine was strongest, followed by lidocaine, then prilocaine, whereas the effect of procaine was blunt. the 50% inhibitory concentrations (IC₅₀ in molarity) of the four local aesthetics for chemetaxis were as follows: tetracaine=4.1×10^-4, lidocaine=3.2×10^-3, prilocaine=3.6×10, procaine=4.9×10^-3, those for SOA production induced by FMLP were : tetraaine=3.1×10^-4, lidocine=5.9×10^-3, prilocaine=1.9×10^-2, procaine=1.2×10^-2, those for SOA production indced by PMA were : tetracaine=1.1×10^-3, lidocine=1.2×10^-2, prilocaine=1.5×10-2, procaine=2.5×10^-2, and those for rlease of BGL were : tetracaine=1.6×10^-, lidocaine=5.3×10^-3, prilocaine=2.8×10^-2, procaine=1.2×10^-1. the IC₅₀ seemed to relate to the anesthetic's chemical structures and their inhibitory properties on PMN functions, as lidocaine and prilocaine, which are aminoamide type anesthetics, preferentially inhibited chemotaxis, whereas tetracaine and procaine, aminoester type anesthetics, inhibited SOA production induced by FMLP. the results suggest that the inhibitory effects of local anesthetics on human PMN functions are also correlated with local anesthetic potency and vary according to differences in their chemical structures.     

Acquisition of lipoproteins in the procyclic form of Trypanosoma brucei

The procyclic form of Trypanosoma brucei binds and internalizes bovine high density lipoprotein (HDL) particles in a saturable process; the binding and uptake of ¹²⁵I-labeled HDL are inhibited by excess unlabeled HDL. We calculated that each procyclic trypanosome exposes ≈1.0×10⁶ binding sites for bovine HDL, with an equilibrium dissociation constant (K_d) of ≈1.26×10⁻⁷ M. Uptake of HDL particles does not occur at 4°C. At 28°C, a significant amount of the internalized HDL particles were efficiently degraded through a process that is sensitive to the presence of 50 μM chloroquine. These results suggested that the uptake of HDL particles in procyclic T. brucei may occur via receptor mediated endocytosis, leading to proteolytic degradation of the particles in an acidic and endocytic compartment.     

Method of analysis of non-competitive enzyme immunoassays for antibody quantification

A computerized analysis of a quantitative enzyme-linked immunoadsorbent assay (EIA) using a non-specific immunoglobulin (IgG) of known concentration as the standard has been developed for measuring specific antibody levels in serum without the need for affinity purification of the positive control antibody. The computer program utilized logit-log linear regression analysis of sigmoid serial dilution curves plus a weighted least-squares best curve fit analysis and an iterative manipulation to eliminate errant data points. The EIA was performed using serial dilutions of standard and unknown antibodies, and a double sandwich technique. A comparison of antibody levels determined by EIA using non-specific IgG as a standard relative to antibody levels determined using affinity-purified specific antibody as a standard were 1.04, 0.53, 0.48, and 0.97 for four different polyclonal antibody systems. Five monoclonal antibodies to carcinoembryonic antigen gave ratios as described above of 1.07, 1.59, 1.73, 2.32, and 2.42. The corresponding antibody affinity constants (1/mol) were 1.0 × 10⁸, 3.8 × 10⁸, 5.5 × 10⁹, 1.8 × 10¹⁰, and 2.6 × 10¹⁰ respectively. This method permits accurate quantification of serum antibody levels when affinity-purified antibodies are not readily available and avoids errors due to loss of antibody activity during affinity purification.     

A method for the determination of the adherence of granulocytes to microtitre plates

We report a new partially automated method for the measurement of the adherence of PMN in vitro. Adherence to a plastic surface was detected by measuring leukocyte alkaline phosphatase activity of the adherent cells, with a Titertek Multiscan system.

Using three different cellular concentrations (1 × 10⁶, 5 × 10⁵, 2.5 × 10⁵ PMN/ml) the response curve was linear to 45 min and adhesion was maximal by 30 min. The specificity of the reaction was acceptable as was the assay reproducibility (intra-assay coefficient of variation <8%; inter-assay coefficient of variation <11%).     

Mitogenic Factor from Chronically Infected Guinea Pigs

A lymphokine produced by antigen stimulated lymphocytes, induces blastogenesis in cultures of lymphocytes which are not sensitive to the specific antigen. The in vitro production of this factor (MF) was accomplished utilizing Peritonea exudate (PE) cells from Coccidioides immitis infected guinea pigs. Production of MF by lymphoid cultures paralleled skin test reactivity of the donor animal. Removal of adherent cells from the PE population did not decrease the production of MF; conversely, a more significant production of MF was effected by the adherent cell depleted populations. Maximal production of MF was achieved at non-adherent cell concentrations from 4 × 10⁶ to 8 × 10⁶ cells/ml. Cell concentrations below 4 × 10⁶/ml produced material which inhibited DM synthesis in test cultures. MF was separated from the inhibitory substance(s) by column chromatography of the crude preparations on Sephadex G-75. Inhibitor(s) eluted in the void volume (Vo), and the MF eluted in an effluent volume (Ve) which was greater than the total bed volume (Vt) suggesting that MF is adsorbed by Sephadex beads.     

Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system

Isolated hepatocytes in spheroid configuration exhibit a high degree of cell–cell contacts, which are important in the maintenance of viability and liver specific functions. In the absence of a vascular network, the cells in a large spheroid size experience mass transfer limitations of metabolites and oxygen in the core of aggregates. In this paper transport phenomena related to the diffusion and reaction of oxygen, glucose and lactate are mathematically described and experimentally verified for hepatocyte spheroids cultured in a rotating-wall polystyrene system (RWPS) not permeable for gases and in a rotating-wall membrane system (RWMS) with oxygen-permeable membrane. The concentration profiles of glucose, oxygen and lactate in the hepatocyte spheroids were estimated for different diameters of aggregates by solving the mass transfer equations for simultaneous diffusion and reaction, by finite element method. Simulation results evidenced that, for aggregates with size lower than 300 μm cultured in both RWPS and RWMS systems, the concentration profiles of glucose and lactate towards the core of spheroids (effective diffusion coefficients in the order of 10⁻¹¹ m²/s) are not significantly affected by the metabolic rate (c.a 10⁻⁶ μg/mm³/s for glucose and about one order of magnitude less for lactate). On the contrary, the transport of oxygen (diffusion coefficient: 3.4×10⁻¹⁰ m²/s, reaction rate: 1.5×10⁻⁵ μg/mm³/s) is critically affected by the size of the multicellular spheroids and significant gradients in oxygen concentration may develop in spheroids. Aggregates with a size greater than 200 μm suffer severe oxygen limitation in the most part of its size attaining the lowest partial pressure in the centre. The improved viability predicted by the model culturing hepatocyte spheroids in the RWMS, characterized by a higher O₂ permeability with respect to RWPS, was experimentally confirmed. The results demonstrated that the mathematical model used in this study represents a useful support to experimental procedures in order to obtain hepatocyte spheroids with optimal size.     

Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors

Gene therapy aims to complement or, ideally, correct defective genes. The broad clinical application of this emerging technology requires the development of safe high-capacity gene delivery vehicles that combine efficient transduction of dividing as well as quiescent cells with sustained transgene expression. Here we present a new hybrid vector system that unites favorable attributes of adenoassociated virus (AAV) and adenovirus (Ad) vectors in a single particle. This was achieved by inclusion of Ad packaging elements in different sized recombinant AAV genomes. In the presence of AAV replicative functions and a recombinant helper Ad, AAV/Ad hybrid particles were generated via encapsidation of AAV-dependent replicative intermediates into Ad capsids. In stringent in vitro models based on transduction of proliferating cells we show that AAV/Ad hybrid vectors are superior to Ad vectors in establishing prolonged transgene expression and can be used to deliver DNA fragments of at least 27 kb.     

Simultaneous quantitation and genotyping of hepatitis B virus by real-time PCR and melting curve analysis

Hepatitis B virus (HBV) genotype and HBV DNA levels have been implicated in clinical evaluation and prognosis of patients with chronic HBV infection. The aim of the present study was to develop a rapid and sensitive method for simultaneous HBV DNA quantitation and differentiation between HBV genotypes B and C in a single-step reaction by real-time PCR and melting curve analysis using SYBR Green I fluorescent dye. The genotypes obtained by this method were compared with those examined by PCR-RFLP and direct sequencing on 52 serum samples of patients with chronic HBV infection. Using the results obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of HBV genotyping by PCR-RFLP and melting curve analysis was 90.38 and 92.31%, respectively. The geometric mean of HBV DNA levels was 3.42×10⁶, 2.10×10⁶, 1.19×10⁵ and 3.10×10⁴ copies/μl in asymptomatic carriers, patients with chronic hepatitis, cirrhosis and hepatocellular carcinoma, respectively. It is concluded that this method has the advantages of rapidity, reproducibility and accuracy, which would be feasible and attractive for large-scale analysis, particularly in regions where HBV genotypes B and C are prevalent.     

Slow isomerization step in the interaction between mouse dopamine transporter and dopamine re-uptake inhibitor N-(3-iodoprop-2E-enyl)-2beta-carbo-[3H]methoxy-3beta-(4'-methylphenyl)nortropane

The kinetics of the association and dissociation of the tritium-labeled selective and potent dopamine transporter inhibitor N-(3-iodoprop-2E-enyl)-2β-carbo-[³H]methoxy-3β-(4′-methylphenyl)nortropane ([³H]PE2I) with the transporter of mouse striatal membranes was studied. The analysis revealed that the specific binding of [³H]PE2I occurs within a homogeneous population of binding sites in these membranes. The relatively slow binding process was characterized by the pseudo-first-order rate constant k_obs. The plot of these rate constants versus free radioligand concentration was hyperbolic, demonstrating that at least two kinetically distinguishable steps can be identified in the interaction of dopamine transporter with this inhibitor. The fast and reversible binding step, characterized by dissociation constant K_A = 51 ± 23 nM, is followed by a slow but also reversible isomerization step of the complex, characterized by the isomerization rate constant k_i = (7 ± 2)10⁻² s⁻¹ and by the rate constant k_−i = (3.9 ± 0.5)10⁻³ s⁻¹ for the reverse process. This isomerization step increases the apparent affinity of the ligand and probably consists of a conformational transition of the transporter protein, induced by the inhibitor molecule.     

Tumor Inhibition by Effector Cells Cultured from Progressing Sarcomas

The effect of cytotoxic lymphoid cells emerging in primary cultures of an antigenic sarcoma of strain 13 guinea pigs was investigated on tumor growth in vivo. The growth of lethal tumor inocula was inhibited or the tumor cells were completely rejected in normal syngeneic recipients treated s.c. with low doses (5×10⁵) of effector cells which were mixed with the tumor cells. A higher dose (2.5×10⁶) of killer cells did not affect significantly tumor growth in normal recipients. Significant tumor inhibition was observed in X-irradiated recipients given high doses (2.2×10⁶) of effector cells locally although rejection did not occur with high frequency. Local treatment of X-irradiated recipients with low doses (5×10⁵) of effector cells did not influence tumor growth. Systemic treatment of normal or X-irradiated recipients with effector cells had little effect at any dose, although tumor inocula were occasionally rejected. Sonication of the cytotoxic effector cells prior to administration inhibited their tumor suppressing effect. Animals that rejected the first tumor inoculum were immune to a second lethal dose of sarcoma cells. It is indicated by the results that tumor rejection in vivo is not simply a matter of killer-target cell interaction, but rather a complex and poorly understood phenomenon.     

The Rna Binding Subset of Adenosine Antibodies

Hapten specific antibodies were elicited against adenosine-BSA conjugate (Ado*-BSA) in rats. By radiohapten assay, two major populations of hapten specific antibodies were identified. They were directed against either adenosine trialcohol(A^ox-red) or morpholino-adenosine(Morph-Ade). The association constant (Ka) for the ³H-A^ox-red binding population was determined to be 1.02 × 10⁷ M^-1. Majority of the A^ox-red specific antibodies were crossreactive with Morph-Ade, but they did not cross-react at all with adenosine(A) or deoxyadenosine(dA). ³H-A binding subsets were actually Morph-Ade specific and constitute less than 10% of the hapten specific antibodies. The Ka value for the ³H-A binding was determined to be 4.5 × 10⁶ M^-1. A very minor and highly cross-reactive subset of Morph-Ade specific antibodies participate in RNA binding. Though a larger proportion of Morph-Ade specific antibodies interact with ³H-A, only a fraction of them bind to RNA. The extreme crossreactivity and hence, the highly adaptive nature of the binding sites of the antibodies interacting with RNA, might be a stringent requirement for recognising adenine residues of nucleic acids in solution.     

A quantitative analysis of rat adrenal chromaffin tissue: Morphometric analysis at tissue and cellular level correlated with catecholamine content

A morphometric analysis of normal Wistar rat adrenal medulla following perfusion fixation and Araldite embedding, was correlated with catecholamine levels on fresh tissue, measured by high-performance liquid chromatography. The mean volume of whole adrenal is 13.2 mm³ and the mean medullary volume 1.3mm³. Volume density estimates showed that the medulla is composed of 63% chromaffin tissue with an adrenaline to noradrenaline storing cell ratio of 4.4:1. The vasculature occupies 20%, neuronal tissue 5% and interstitial tissues 12% of the medulla. A comparison was made of cell volumes, cell numbers and volume and surface density estimates of cytoplasmic organdies in adrenaline and noradrenaline storing cells. The mean cell volume of adrenaline storing cells at 1300 μm³ is larger than that of noradrenaline storing cells at 980 μm³. A single adrenal medulla contains4.4−5.7 × 10⁵ adrenaline cells and1.5−1.9 × 10⁵ noradrenaline cells. Chromaffin granules account for approximately 30% of the volume of the cytoplasm; the numerical density of granules at different sites in the cell was calculated for adrenaline cells. The volume density of mitochondria (4%) and the surface density of mitochondrial membranes (the ratio of outer to inner membrane being approximately 1:2.3) were similar in both cell types. Rough endoplasmic reticulum was the only organelle to show a significant difference in volume and surface density between the two cell types. Adrenaline storing cells have stacks of rough endoplasmic reticulum which have two to three times the surface and volume densities of that found diffusely scattered throughout noradrenaline cells. The adrenaline content of an adrenaline storing cell is0.14 × 10⁻⁶ μM and that of a granule 3.0 × 10⁻¹² or3.8 × 10⁻¹² μ moles depending on the method of calculation. The noradrenaline content of noradrenaline storing cells can only be calculated on the assumption that all noradrenaline is stored in this cell type though it is likely that some is contained within adrenaline cells. Based on this assumption the noradrenaline content is0.17 × 10⁻⁶μ moles per cell and5 × 10⁻¹² μ moles per granule. The present study provides baseline morphometric data on the rat adrenal medulla at tissue and cellular level correlated with amine levels in adrenaline and noradrenaline storing cells and granules.     

An economical adaptation of the RosetteSep procedure for NK cell enrichment from whole blood,and its use with liquid nitrogen stored peripheral blood mononuclear cells

This study reports an economical adaptation of the RosetteSep™ procedure for enrichment of NK cells designed for whole blood and its use with liquid nitrogen stored peripheral blood mononuclear cells (PBMC). Combining 45×10⁶ PBMC with 45×10⁸ Alsevers' stored red blood cells (RBC) in 1 ml requires 50 μl of RosetteSep™ bifunctional antibody cocktail and provides NK cells of 99% purity with average yields of 43%.     

Patients with chronic pancreatitis have an impaired oxidative burst ability of blood monocytes

The destruction by phagocytic cells of ingested microorganisms is best ensured by an intact respiratory burst with production of superoxide anion and other metabolites. The aim of the present study was to investigate the status of the reticulo-endothelial system, as assessed by superoxide anion generation of blood monocytes, in 18 patients with chronic pancreatitis (CP) as compared to 15 with chronic renal failure (CRF), 14 with diabetes mellitus (DM) and to 20 healthy volunteers. Macrophage suspensions (1 × 10⁶) were prepared from blood samples withrawn after an overnight fast were tested for conventional phagocytosis function (carbon partical ingestion: percent and intensity) and for superoxide anion generation (chemiluminescent activation: cpm of light integrated intensity). Conventional phagocytosis function tests did not show any differemce among the groups examined. However, the peak value of generated susperoxide anion (× 10⁴ cpm) was significantly lower in CP patients as compared to either healthy controls (14.9 ± 4.3 vs. 28.3 ± 4.7, P < 0.001) and CRF (24.8 ± 3.9, P < 0.01) and DN patients (22.8 ±_4.9, P < 0.05). Further, the time elapsed to generate such a peak was significantly longer in CP patients (374 ± 106 s) than in controls (208 ± 35 s, P < 0.01). This study suggests that patients with CP present a defective monocytes burst ability which is unrelated to flare-up of the disease and to the underlying malnutrition. Further, this phenomenon appears to occure to a far greater extent as compared to other long-standing chronic illnesses.     

A novel hyaluronan-based biomaterial (Hyaff-11) as a scaffold for endothelial cells in tissue engineered vascular grafts

Current prosthetic small diameter vascular grafts show poor long-term patency rates, leading to the pursuit of a biological alternative. Hyaff-11 is a hyaluronan-based biodegradable polymer developed for tissue-engineering applications. This study aimed to determine whether human vascular endothelial cells attach to Hyaff-11 scaffolds and produce a subendothelial matrix. Two forms of fibrous, non-woven Hyaff-11 scaffolds: unpressed and pressed felts, were analysed. Attachment of human venous endothelial cells was investigated after 1, 5, 10 and 20 days in culture using SEM and confocal microscopy. The deposition of subendothelial matrix components was investigated by immunofluorescent staining.

We demonstrate that endothelial cells adhere to the individual fibres of both unpressed and pressed scaffolds: with a seeding density of 1×10⁶ cells/cm², 94% of the cells attached to Hyaff-11 fibres after 24 h. The pressed material provided the best environment for cell growth, allowing the formation of a complete endothelial monolayer after 20 days. Furthermore, endothelial cells on Hyaff-11 pressed felts deposited an organised subendothelial matrix containing laminin, fibronectin, type IV and type VIII collagen. This work indicates Hyaff-11 based biopolymers as suitable scaffolds to promote endothelialisation within the next generation of vascular grafts.