Laparoscopic ovum pick-up followed by in vitro embryo production for the reproductive rescue of aged goats of high genetic value.

The efficiency of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) in the propagation of aged goats with poor reproductive performance was evaluated in the present study. Follicular development was stimulated in donor goats with 80 mg follicle-stimulating hormone and 300 IU equine chorionic gonadotrophin administered 36 h before LOPU. In addition, goats were heat synchronised with intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days and a luteolytic injection of 125 microg cloprostenol 36 h before sponge removal and LOPU. Following in vitro maturation (IVM), oocytes were fertilised in vitro with frozen-thawed semen produced using the egg yolk-free Bioxcell extender (IVM, L'Aigle, France). The average number of follicles aspirated (17.9 +/- 8.0 per goat), oocytes recovered (15.7 +/- 8.4 per goat) and cleavage after IVM/in vitro fertilisation followed by a short 24-h in vitro culture in modified synthetic oviduct fluid medium (72 +/- 7%) were similar to results reported previously by our group and others in younger goats. A total of 296 embryos was transferred into 50 heat-synchronised recipients, of which 40 became pregnant (80%) and 38 progressed all the way to term, delivering 86 live kids. The present study indicates that LOPU-IVEP can be used successfully to extend the reproductive life of valuable goats that have acquired difficulties becoming pregnant by artificial insemination after multiple kiddings.

Assessment of mumps virus growth on various continuous cell lines by virological, immunological, molecular and morphological investigations.

Vero cells have been used as a convenient laboratory substrate for the isolation of mumps virus but may not be very sensitive and may select for particular adapted variants from clinical specimens. Continuous cell lines were evaluated for their ability to support the replication of mumps virus. Criteria included the production of infectious virus, detection of intracellular mumps proteins by immunofluorescence and electron microscopy and detection of specific nucleic acid by RT-PCR. Of the cells tested, CaCo-2, PLC/PRF/5, and Vero cells produced infectious virus, with Vero and CaCo-2 being the most permissive. The other substrates tested included cells of murine, canine and human origin showed signs of intracellular proteins and RNA but the amounts produced were much lower, and no infectious virus was detected in some cases. The virus use was a low passage of a Vero derived wild type strain, and it will ultimately be necessary to continue the studies with an unpassaged clinical specimen to identify a cell line able to isolate mumps virus at high efficiency and in unmodified form.

Multiphase transfer processes in waste rock piles producing acid mine drainage 1: Conceptual model and system characterization.

Acid mine drainage (AMD) results from the oxidation of sulfides, mainly pyrite, present in mine wastes, either mill tailings or waste rock. This is the first of two papers describing the coupled physical processes taking place in waste rock piles undergoing AMD production. Since the oxidation of pyrite involves the consumption of oxygen and the production of heat, the oxidation process initiates coupled processes of gas transfer by diffusion and convection as well as heat transfer. These processes influence the supply of oxygen that is required to sustain the oxidation process. This first paper describes a general conceptual model of the interaction of these coupled transfer processes. This general conceptual model is illustrated by the physicochemical conditions observed at two large sites where extensive characterization programs revealed widely different properties. The South Dump of the Doyon mine in Canada is permeable and has a high pyrite oxidation rate leading to high temperatures (over 65 degrees C), thus making temperature-driven air convection the main oxygen supply mechanism. The Nordhalde of the Ronnenberg mining district in Germany contains lower permeability material which is less reactive, thus leading to a more balanced contribution of gaseous diffusion and convection as oxygen supply mechanisms. The field characterization and monitoring data at these sites were thoroughly analyzed to yield two coherent sets of representative physical properties. These properties are used in the second paper as a basis for applications of numerical simulation in AMD-producing waste rock piles.

Multiphase transfer processes in waste rock piles producing acid mine drainage 2. Applications of numerical simulation.

Acid mine drainage (AMD) results from the oxidation of sulfides, mainly pyrite, present in mine wastes, either mill tailings or waste rock. This is the second of two papers describing the coupled physical processes taking place in waste rock piles undergoing AMD production. Since the oxidation of pyrite involves the consumption of oxygen and the production of heat, the oxidation process initiates coupled processes of gas transfer by diffusion and convection as well as heat transfer. These processes influence the supply of oxygen that is required to sustain the oxidation process. This second paper describes a numerical simulator used to represent the interaction of these coupled transfer processes. Numerical simulations are applied to two large sites with extensive characterization programs and widely different properties and behavior that were described in the first paper. The South Dump of the Doyon mine in Canada is permeable and has a high pyrite oxidation rate, thus making temperature-driven air convection the main oxygen supply mechanism. The Nordhalde of the Ronnenberg mining district in Germany contains lower permeability material which is less reactive, thus leading to a more balanced contribution of gaseous diffusion and convection as oxygen supply mechanisms. Overall, simulations allow a coherent representation of the conditions monitored within the waste rock piles and the confirmation of their physical properties. Conceptual simulations are also carried out to illustrate the potential effect of border membranes and layered co-mingling as mitigation methods used to control AMD production in either active or future waste rock piles.

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes.

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP. Positive strand probes were used as a negative control. Cells were fixed with a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol with progressive lowering of temperature and embedded in Lowicryl K4M or HM20 at -35 degrees C. Permeabilization or pre-treatment of sections with proteinase K was not essential. The hybridization mixture was applied for 3-4h at 37 degrees C and probe was visualized by direct immuno-staining with sheep anti-digoxigenin antibodies conjugated to 10nm gold. This method would be suitable for future studies of the pathogenesis of retroviral infections and as a basis for further development of the EM ISH technique. EM ISH of in vitro infections of immunodeficiency viruses has shown the location of viral RNA in immature and mature viruses and its relationship to multimerized Gag protein during viral budding. The label for RNA has also been found in the cytoplasm of infected cells; it was mainly located adjacent to the plasma membrane and unassociated with visible Gag proteins. This may indicate that viral RNA migrates to the plasma membrane independently of the Gag protein and may, in some instances, arrive at the plasma membrane prior to the Gag protein. Viral RNA has also been found in the nucleus of peripheral blood mononuclear cells (PBMC) that were showing no morphological evidence of infection. The RNA was typically located in the nucleolus and in peripheral dense chromatin. These cells, which displayed morphological features of macrophage lineage, may have been the initial cell type to be infected in the PBMC.

Comparison of in vitro and in vivo methods to study stability of PLGA microencapsulated tetanus toxoid vaccines.

The purpose of this study was to investigate the utility of various in vitro and in vivo methods to assess the stability of experimental vaccines containing tetanus toxoid (TT) within PLGA microspheres. In vitro, the breakdown of the encapsulating polymers into their acid components led to changes in the structure of TT, as determined by the physico-chemical methods, rendering it undetectable by capture ELISA and altering its structural integrity. The changes in TT were directly related to increasing acidity of the vaccine supernate. Purified toxoid (not encapsulated) exposed to low pH (2.5) underwent similar changes but re-neutralisation of buffer containing free toxoid, even after one week at pH 2.5 led to some re-folding of protein as determined by fluorescence spectroscopy and gel filtration chromatography. The microencapsulated vaccines were still able to generate an antibody response in mice even after prolonged pre-incubation at 37 degrees C and the apparent absence of detectable toxoid in the vaccine supernate. Electron microscopy demonstrated differences in the amount of degradation between different formulations of microspheres. Vaccines that had retained their spherical morphology after incubation in vitro for up to 28 days were able to induce protective antibodies response equal to that of freshly prepared vaccines, which indicates that the toxoid within intact microspheres remained immunogenic. Immunochemical and physico-chemical detection methods, performed on antigen released from PLGA vaccines in vitro, are valuable in providing information on product characteristics but may not be able to predict effectiveness and should be used with in vivo methods to evaluate the stability of such formulations.

Development of a continuous IL-7-dependent murine pre-B cell line PB-1 suitable for the biological characterisation and assay of human IL-7.

Human interleukin-7 (IL-7) is a cytokine that appears to be critical for early T- and B-cell development and although IL-7 is currently under investigation as a therapeutic agent in a variety of hematolymphopoietic disorders, there have been few instances of the detection or investigation of this cytokine using a biological assay. This has been due, in the main, to the lack of a widely available, stable, easy to maintain and use, IL-7 responsive cell line. We have developed a pre-B-cell line, PB-1, from murine bone marrow, that is dependent on IL-7 for growth and has been maintained continually for up to 1 year without loss of responsiveness. The cells survive freezing and reviving, having been stored for periods of up to 4 years. The IL-7 bioassay is reproducible and sensitive, able to reliably detect 50 pg/ml IL-7. The assay is completely unresponsive to any other stimulatory cytokines tested and is not affected by a wide variety of inhibitory cytokines, with the exception of high levels of interferon alpha. The assay can be made completely specific for human IL-7 by including specific neutralizing antibodies for IL-7 and has been shown to be suitable for the estimation of IL-7 in both plasma and serum samples.

Roles of matrix, p2, and N-terminal myristoylation in human immunodeficiency virus type 1 Gag assembly.

Human immunodeficiency virus type 1 Gag protein is cotranslationally myristoylated at the N terminus and targeted to the plasma membrane, where virus particle assembly occurs. Particle assembly requires the ordered multimerization of Gag proteins, yet there is little direct evidence of intermediates of the reaction or of the domains that lead to each stage of the oligomerization process. In this study, following the expression in insect cells of C-terminally truncated Gag proteins and their purification, both the multimeric nature of each Gag protein and the ability to form Gag virus-like particles (VLP) were analyzed. Our results show that (i) the matrix (MA) domain forms a trimer and contributes to a similar level of oligomerization of the assembly-competent Gag; (ii) the p2 domain, located at the capsid/nucleocapsid junction, is essential for a higher order of multimerization (>1,000 kDa); (iii) the latter multimerization is accompanied by a change in Gag assembly morphology from tubes to spheres and results in VLP production; and (iv) N-terminal myristoylation is not required for either of the multimerization stages but plays a key role in conversion of these multimers to Gag VLP. We suggest that the Gag trimer and the > 1,000-kDa multimer are intermediates in the assembly reaction and form before Gag targeting to the plasma membrane. Our data identify a minimum of three stages for VLP development and suggest that each stage involves a separate domain, MA, p2, or N-terminal myristoylation, each of which contributes to HIV particle assembly.

Approaches to the control of acellular pertussis vaccines.

The quality control of acellular pertussis vaccines presents particular problems related to the differences in composition and method of detoxification used in the various type of preparation. These vaccines are not amenable to potency assay by the active mouse protection test used for whole-cell pertussis vaccines and assurance of protective activity is problematic. In contrast, monitoring of these vaccines for safety is relatively straightforward and is centred on assays for the lipooligosaccharide endotoxin, active pertussis toxin and absence of reversion to toxicity of detoxified product. The absence of heat-labile toxin, tracheal cytotoxin and adenyl cyclase toxin is assumed provided that adequate validation of the process has been performed. Confirmation of the antigenic content of the detoxified bulk components is difficult to achieve by conventional binding assays based on monoclonal antibodies because of changes in accessibility of reactive sites post-toxoiding. However, single radial diffusion assay using polyclonal antisera permits estimation of pertussis toxoid (PT), filamentous haemagglutinin (FHA) and pertactin (P69). Dot blot immunoassay can be used for the fimbrial agglutinogens 2 and 3 (Fim 2 and 3) and potentially could also be used to check the composition of final filling lots for PT, FHA, P69 and Fim 2 and 3. Gel electrophoresis and immunoblotting can be applied to monitor purity of purified bulk components and the characteristics of these change after chemical detoxification. Electron microscopy provides a useful semi-quantitative supporting method for checking purity of bulk components. Physico-chemical examination, particularly CD and fluorescence spectroscopy, offer a means of monitoring the consistency of detoxified bulk components. No completely satisfactory method is available for monitoring potency. Immunogenicity assays may be useful for checking consistency but do not necessarily correlate with protection. At present, active protection against aerosol challenge offers the best prospect of a functional assay.

Evaluation of transmission electron microscopy for examination of components of acellular pertussis and combination vaccines.

New acellular pertussis and combination vaccines vary in the concentration and presence or absence of specific components and in extent of adsorption to adjuvants. There is a pressing need to develop new control methods for acellular pertussis vaccines. Negative staining electron microscopy has been evaluated as a method for assessing the purity of individual vaccine components and the amount of adsorption to aluminium gels. Negative staining showed the characteristic morphology of vaccine components and permitted detection of contaminants and morphological changes. Reproducible results were obtained by use of a standardized negative staining technique and confidence in the technique was increased by comparison of previously unexamined specimens with a specimen that had been characterized by repeated observations. The degree of adsorption to aluminium adjuvants could only be assessed by observation of the amount of non-adsorbed material in the specimen. Negative staining electron microscopy can be used as one of a number of techniques for control of acellular pertussis combination vaccines.

Further evidence for hexagonal organization of HIV gag protein in prebudding assemblies and immature virus-like particles.

The fullerene-like model for the organization of HIV gag encoded precursor pr55gag was based on the study of prebudding assemblies at the plasma membrane of cells infected with a recombinant baculovirus expressing HIV-1 gag protein. The objective of the present study was to support the model by image processing of virus-like particles (VLP). In this work we used VLP purified by density gradient centrifugation, which caused partial or occasionally complete loss of the lipid bilayer in some VLP without the use of detergent. In addition more plasma membrane-associated pr55gag protein assemblies were processed. Image processing of negatively stained specimens revealed the presence of threefold symmetry and a hexagonal network of rings with a resolution of 29 A in VLP and better than 25 A in membrane associated assemblies. The center-to-center spacing of the rings was 67 A in VLP and 70 A in membrane assemblies. Patches of gag protein oligomers at the plasma membrane were usually round and varying in size, but some of them were triangular. Indication of triangular-shaped gag protein assemblies was also seen in partly dissociated VLP. Since the hexagonal network is formed by the uncleaved gag polyprotein, we conclude that the threefold symmetry applies to all domains including p24gag. The presence of threefold symmetry and the hexagonal network in VLP are consistent with the hypothesis that immature HIV particles possess icosahedral symmetry.

Detection of a trimeric human immunodeficiency virus type 1 Gag intermediate is dependent on sequences in the matrix protein, p17.

Previous studies have shown that single amino acid changes in the amino-terminal matrix (MA) domain, p17, of the human immunodeficiency virus type 1 Gag precursor Pr55, can abrogate virion particle assembly. In the three-dimensional structure of MA such mutations lie in a single helix spanning residues 54 to 68, suggesting a key role for this helix in the assembly process. The fundamental nature of this involvement, however, remains poorly understood. In the present study, the essential features of the MA helix required for virus assembly have been investigated through the analysis of a further 15 site-directed mutants. With previous mutants that failed to assemble, residues mapped as critical for assembly were all located on the hydrophobic face of the helix and had a key role in stabilizing the trimeric interface. This implies a role for the MA trimer in virus assembly. We support this interpretation by showing that purified MA is trimeric in solution and that mutations that prevent virus assembly also prevent trimerization. Trimerization in solution was also a property of a larger MA-capsid (CA) Gag molecule, while under the same conditions CA only was a monomer. These data suggest that Gag trimerization driven by the MA domain is an intermediate stage in normal virion assembly and that it relies, in turn, on an MA conformation dependent on the hydrophobic core of the molecule.

Resorbable lamellar particles of polylactide as adjuvants for influenza virus vaccines.

Lamellar particles and microspheres were produced by precipitation from solutions of resorbable, biocompatible, semi-crystalline poly(L-lactide)[PLA] and amorphous poly(DL lactide co-glycolide)[PLG] copolymer, respectively, to investigate their adjuvanticity towards adsorbed influenza virus. Both types of substrate were capable of adsorbing large quantities of virus (> 15% w/w) and retaining virus (> 60% of the initial load) over an 8 week time scale in-vitro. Potent immune responses were obtained in mice after the intra-muscular injection of adsorbed vaccine systems. The response to virus adsorbed on PLA lamellar particles was almost five times that obtained using PLG microspheres and fourteen times that using aqueous vaccine. The lamellar forms of PLA may function as an immunomodulator enhancing phagocytic activity due to their irregular shape and may be useful in improving the immune response to a variety of protein and viral antigens.

Structure-function studies of the human immunodeficiency virus type 1 matrix protein, p17.

The human immunodeficiency virus type 1 (HIV-1) matrix protein, p17, plays important roles in both the early and late stages of the viral life cycle. Using our previously determined solution structure of p17, we have undertaken a rational mutagenesis program aimed at mapping structure-function relationships within the molecule. Amino acids hypothesized to be important for p17 function were mutated and examined for effect in an infectious proviral clone of HIV-1. In parallel, we analyzed by nuclear magnetic resonance spectroscopy the structure of recombinant p17 protein containing such substitutions. These analyses identified three classes of mutants that were defective in viral replication: (i) proteins containing substitutions at internal residues that grossly distorted the structure of recombinant p17 and prevented viral particle formation, (ii) mutations at putative p17 trimer interfaces that allowed correct folding of recombinant protein but produced virus that was defective in particle assembly, and (iii) substitution of basic residues in helix A that caused some relocation of virus assembly to intracellular locations and produced normally budded virions that were completely noninfectious.

Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly.

Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag particle assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble particles. Mutations that abolished both in vitro p24 binding and particle assembly mapped to the C terminus of p24 confirming this region as critical for virion assembly. We suggest the division of virion assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information.

A molecular determinant of human immunodeficiency virus particle assembly located in matrix antigen p17.

We report single-point mutations that are located in the matrix protein domain of the gag gene of human immunodeficiency virus type 1 and that prevent Gag particle formation. We show that mutations of p17 that abolish human immunodeficiency virus particle assembly also prevent the dimerization of p17 protein, as measured directly by a protein-protein binding assay. In the three-dimensional structure of p17, mutations that abolish dimerization are located in a single alpha helix that forms part of a fingerlike projection from one side of the molecule. Peptides derived from this region of p17 also reduce the level of p17 dimer when they are added to p17-expressing cells and compete for p17 self-association when present in protein-protein binding assays. We propose that the dimerization of the Gag precursor that occurs by the interdigitation of alpha helices on adjacent matrix molecules is a key stage in virion assembly and that the prevention of such an interaction is the molecular basis of particle misassembly.

An electrophoretic method to detect cold-induced dissociation of proteins in crude extracts of higher plants.

The phenomenon of cold denaturation has been firmly established recently in several proteins. Some multimeric enzymes from plant origins are believed to dissociate under some circumstances in the cold. To determine the presence and number of soluble, non-membrane-bound protein that undergoes cold-induced dissociation in plants, we have devised a special two-dimensional polyacrylamide gel electrophoretic method using two native gradient gels. Examination of the gel run at 0 degrees C in the second dimension showed the presence of four cold-dissociated proteins running below the diagonal and staining intensely with silver, in the extract of maize leaves. The electrophoretic method described here is expected to be a convenient way to detect cold-induced dissociation of soluble proteins in crude extracts of various tissues. It is also possible to estimate roughly the molecular weights of both the cold-dissociated subunit and the native protein from which it is derived.

Superovulatory response to a single subcutaneous injection of Folltropin-V in beef cattle.

A series of 4 experiments were designed to evaluate the feasibility of superstimulation in beef cattle with a single sc injection of the porcine pituitary extract, Folltropin-V. In the preliminary study (Experiment 1), superovulatory response of cows (n=7) treated with a single sc injection of 400 mg NIH-FSH-P1 Folltropin-V was not different than that of cows (n=8) superstimulated with twice daily im injections over 4 d, or a single sc injection plus an injection of eCG (n=12). Experiments 2 and 3 were designed to determine the optimal site of a single sc injection. In Experiment 2, cows (n=25) with body condition scores (BCS) of 1 to 2 were used. The mean number of CL counted and ova/embryos collected was lower (P<0.05) in cows treated with the single sc injection in the neck region than in cows treated with a single sc injection behind the shoulder, or with the twice daily im injection treatment. In Experiment 3, cows (n=49) with BCS of 3 to 5 were used. There were no differences in the number of CL, total ova/embrvos collected, fertilized ova and transferable embryos whether treatments were given in the neck region or behind the shoulder, or whether the cows were implanted or not implanted with Syncro-Mate-B. Experiment 4 was designed to determine the optimal superstimulatory dosage of Folltropin-V administered by a single sc injection. Superovulatory response of cows treated with the higher doses (400 mg, 600 mg or 800 mg NIH-FSH-P1) was higher (P<0.05) than those treated with 200 mg NIH-FSH-P1. The number of unovulated (>or=10 mm) follicles at the time of ova/embryo collection was higher (P<0.05) in the 600 and 800 mg groups, and progesterone concentration at estrus was higher (P<0.05) in cows treated with 800 mg than with 400 or 200 mg. It was concluded that a single, bolus sc injection of 400 mg NIH-FSH-P1 of Folltropin-V is as efficacious as the 4-d, twice daily im treatment protocol for inducing superovulation in beef cows. The amount of subcutaneous fat and site of injection appeared to affect the efficacy of a single sc injection; a single bolus sc injection of Folltropin-V behind the shoulder resulted in the most predictable superovulatory response.