Perspectives for subunit vaccines for the control of ticks

For the first time, successful vaccination against a tick has been carried out using a single defined antigen. Further, it has been shown that it is feasible to produce active antigenic material by recombinant DNA technology. This represents a significant advance towards the development of an alternative means of tick control. Nevertheless, as with any new product and new technology, much developmental work still has to be done before one can be confident that a practical means of tick control will result. From the published information, it does not seem that research on vaccines against other tick species is as advanced as that on Boophilus microplus. The work on B. microplus may, however, provide a short cut to the development of further tick vaccines.     

Assessment of Needs for Plasma for Fractionation in Europe

In the early 1990s, a series of outbreaks of hepatitis C (HCV) infections clustering among recipients of certain lots of plasma-derived medicinal products (PDMP) alarmed regulatory authorities, manufacturers and the public alike. Also, a few episodes of Hepatitis A (HAV) infections occurred in haemophiliacs receiving solvent-detergent-treated factor VIII concentrates. Thus, several measures were brought into effect to reestablish the safety of the incriminated products and to further increase the margin of safety of PDMP in general. Therefore, intramuscular immunoglobulins had to be free of HCV RNA as shown by nucleic acid amplification technology (NAT) in the final products. Furthermore, the manufacturing process of PDMP had to be validated for both viral inactivation and elimination. Finally, HCV-NAT was to be standardised and implemented as a validated test of plasma pool samples.In 1994, a joint meeting of EPFA, EAPPI and Regulatory Authorities was held in Brussels to outline the state of the art and to delineate the actions to be taken. Five years later, in 1999, the incidence rates of HIV, HBV and HCV in unpaid blood donors have been minimized, especially in European countries. With probabilities for window period donations as low as 0.6 in 1 million for both HIV and HCV and 2.1 in 1 million for HBV in Switzerland, labile blood products have reached extreme, but not absolute safety. The introduction of HCV-NAT roughly doubles this safety resulting in a 1 in 3 million probability of a window donation.Concomittantly, extensive viral validation studies document effective inactivation and removal of viruses in PDMP. The demonstrated margins of safety, expressed as logarithmical reduction factors (LRF), range from 4 to over 20 log(10), depending on product, virus, and inactivation procedure used. Further progress to even safer PDMP shall be acomplished by consolidating the GMP processes, abandoning of obsolete requirements and harmonising national regulations within Europe. Before introducing new measures for additional agents such as HAV or Parvovirus B 19, gains and risks and even potential new threats have to be carefully assessed. Alternative efforts for the safeguard of patients, e.g. vaccination for HAV, need to be balanced against the risks of changing established and validated manufacturing procedures of PDMP with long-lasting safety records.     

A Modification of Stone's Test for Trend for Binary Outcome

Stone (1988) suggested the “first isotonic regression estimator” as a tool for drawing inferences on possibly increased cancer case counts among several subregions around a putative source. He assumed the case counts to be Poisson distributed and therefore introduced a rare disease assumption into his approach. However, when analyzing cross sectional data one would rather refer to prevalence estimates among these subregions around a point risk source (for example the origin of chemical fallout). Therefore we applied antitonic regression estimation in Binomial distributions to derive a test statistic and a p value to test for a possible trend in the observed prevalence data around the putative source. The computation of this p value will be illustrated as well as severe difficulties concerning its interpretation. Further the Maximum Likelihood Ratio approach will be used to derive an alternative test statistic.     

Evidence for a close spatial location of the binding sites for CO2 and for photosystem II inhibitors

1. CO2-depletion of thylakoid membranes results in a decrease of binding affinity of the Photosystem II (PS II) inhibitor atrazine. The inhibitory efficiency of atrazine, expressed as I50-concentration (50% inhibition) of 2,6-dichlorophenolindophenol reduction, is the same in CO2-depleted as well as in control thylakoids. This shows that CO2-depletion results in a complete inactivation of a part of the total number of electron transport chains. 2. A major site of action of CO2, which had previously been located between the two electron acceptor quinone molecule B (or R) and Photosystem II inhibitor atrazine as suggested by the following observations: (a) CO2-depletion results in a shift of the binding constant (kappa b) of [14C]atrazine to thylakoid membranes indicating a decreased affinity of atrazine to membrane; (b) trypsin treatment, which is known to modify the Photosystem II complex at the level of B, strongly diminishes CO2 stimulation of electron transport reactions in CO2-depleted membranes; and (c) thylakoids from atrazine-resistant plants, which contain a Photosystem II complex modified at the inhibitor binding site, show an altered CO2-stimulation of electron flow. 3. CO2-depletion does not produce structural changes in enzyme complexes involved in Photosystem II function of thylakoid membranes, as shown by freeze-fracture studies using electron microscopy.     

Development of 120 microsatellite markers for Primula sieboldii E. Morren for linkage mapping

Primula sieblodii E. Morren is a perennial herb, classified as ‘Near Threatened’ in the Japanese Red List. Using the magnetic enrichment method, we have developed 120 microsatellite markers and screened polymorphisms within the species as a whole as well as within a single population. At the species level, the average number of alleles per locus, and the average observed and expected heterozygosities were 7.06, 0.48, and 0.74, respectively; at the level of the single population these values were 5.53, 0.52, and 0.64, respectively. The microsatellite markers presented here will be valuable research tools in forthcoming projects, such as QTL mapping as well as for association studies of P. sieboldii.     

The outlook for the development of live vaccines for the prevention of melioidosis

The effectiveness of immunization with Burkholderia pseudomallei attenuated strains (Pur and Ts), heterologous vaccines and the recombinant culture of Francisella tularensis RM2 carrying a plasmid with fragments of B. pseudomallei chromosome was studied in four species of experimental animals, essentially differing in their sensitivity to melioidosis. The most immunogenic B. pseudomallei mutants, introduced subcutaneously, created a statistically significant level of protection in animals, moderately sensitive to melioidosis, but proved to be ineffective in highly sensitive animal models when tested under the same conditions. In aerogenic infection the effectiveness of the experimental vaccines under study in all species of the animals was on the same level. The study showed good prospects of using tularemia vaccine for inducing heterologous immunity to melioidosis, as well as the possibility of its use as the basis of a bivalent gene-engineering vaccine.     

A Proposed Framework for Investigation of Cause for Environmental Effects Monitoring

Environmental Effects Monitoring (EEM) programs in Canada have been developed for the pulp and paper and metal mining industries, and require a cyclical evaluation of the receiving environment to determine whether effects exist when the facilities are in compliance with existing regulations. Identifying the cause of environmental effects is a specific, identified stage in this monitoring program, but as yet there has not been a synthesis of what is meant by “identification of cause”. We propose a multitiered guidance framework for the identification of the cause of environmental effects after they have been detected, confirmed, and their extent and magnitude documented. As part of point source confirmation, the framework includes levels to define whether there is an effect, whether it is related to the effluent discharge facility, and whether response patterns in the receiver are characteristic of a particular stressor type. The next tier involves investigating individual process wastes within the facility to determine the components that are contributing to effects caused by exposure to the final effluent. The last three tiers of the framework relate to characterizing the chemical classes involved in the effect and, ultimately, to identifying the specific chemicals associated with the responses. Although there is increasing knowledge of specific causes of environmental effects gained as one progresses through the levels of investigation, there is a concomitant increase in effort and costs required. Stakeholder input is critical in determining the depth of the investigation as well as how to proceed once the environmental effects information is available.     

Strategy for selection of methods for separation of bioparticles from particle mixtures

The desired product of bioprocesses is often produced in particulate form, either as an inclusion body (IB) or as a crystal. Particle harvesting is then a crucial and attractive form of product recovery. Because the liquid phase often contains other bioparticles, such as cell debris, whole cells, particulate biocatalysts or particulate by-products, the recovery of product particles is a complex process. In most cases, the particulate product is purified using selective solubilization or extraction. However, if selective particle recovery is possible, the already high purity of the particles makes this downstream process more favorable. This work gives an overview of typical bioparticle mixtures that are encountered in industrial biotechnology and the various driving forces that may be used for particle-particle separation, such as the centrifugal force, the magnetic force, the electric force, and forces related to interfaces. By coupling these driving forces to the resisting forces, the limitations of using these driving forces with respect to particle size are calculated. It shows that centrifugation is not a general solution for particle-particle separation in biotechnology because the particle sizes of product and contaminating particles are often very small, thus, causing their settling velocities to be too low for efficient separation by centrifugation. Examples of such separation problems are the recovery of IBs or virus-like particles (VLPs) from (microbial) cell debris. In these cases, separation processes that use electrical forces or fluid-fluid interfaces show to have a large potential for particle-particle separation. These methods are not yet commonly applied for large-scale particle-particle separation in biotechnology and more research is required on the separation techniques and on particle characterization to facilitate successful application of these methods in industry.     

Semiautomatic regulator for the preparation of microelectrodes for electrophysiology

This paper describes an electronic device for making metallic microelectrodes to be used for electrophysiological experiments. These microelectrodes are endowed with a small tissue-electrode capacity and a high mechanical strength. The same microelectrodes are applicable for derivation of spikes as in the case of microstimulation of nervous structures during repeated penetrations.     

Orientation of Small Specimens for Cryosectioning

A simple technique is introduced to achieve symmetrically oriented frozen sections of small specimens such as young fish or frog larvae. Small samples are especially difficult to orient if they are already frozen to the chuck in a freezing microtome. Orientation of the sample in a mold filled with embedding medium prior to freezing permits sectioning as well as easy labeling and storage of the specimens. The use of a stereo microscope during orientation is optional.     

An improved embalming procedure for long-lasting preservation of the cadaver for anatomical study

A successful embalming procedure necessary for long-lasting preservation of the cadaver and its subsequent anatomical dissection has been undertaken in our laboratory. In short, the procedure consists of a preembalming treatment with blood clot disperser, removal of blood clots, drainage of blood, and arterial embalming with an embalming machine via both carotid and femoral triangles of the body. The embalming fluid is prepared from methyl alcohol and a small amount of formalin as the fixatives, ethylene glycol as a preservative, and liquefied phenol as a mould preventive. Coloring of the blood vessels is also useful in their identification. Other matters relevant to embalming problems are also discussed.     

Technique for interpretation of electromyography for concentric and eccentric contractions in gait

We present a technique to combine muscle shortening and lengthening velocity information with electromyographic (EMG) profiles during gait. A biomechanical model was developed so that each muscle's length could be readily calculated over time as a function of angles of the joints it crossed. The velocity of shortening and lengthening of the muscle fiber was then calculated, and with computer graphics this information was overlaid on the EMG profiles. Thus, researchers and clinicians were not only able to interpret the processed EMG signal as level of activity (tension) but also to gain insight as to the muscles' role as generators (muscle shortening) or absorbers (muscle lengthening) of energy. Six common muscles are documented, using database profiles; soleus (SOL), medial gastrocnemius (MG), tibialis anterior (TA), vastus lateralis (VL), rectus femoris (RF), and semitendinosus (ST). The protocol thus demonstrates a relatively simple technique for calculating muscle fiber velocity and for combining that velocity information with EMG activity profiles.     

A testing sequence for reducing rejection of potential biological control agents for weeds

Many insect agents, selected for the biological control of weeds and tested under restricted cage conditions, have been rejected as unsafe for introduction because of an apparent increase in host range compared with that observed under natural conditions. These aberrant results appear to be due to the insects' inability to follow the normal behavioural sequence based on appropriate cues which lead to correct host selection. It is suggested here that a reverse sequence of testing which progressively reduces the degree of restriction and deletes unattacked plants at each stage, until only a few remain to be tested under conditions as near natural as possible, would allow previously rejected insects to be used as biological control agents.     

Lack of evidence for an immunosuppressive role for MUC1

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells. Received: 20 August 1998 / Accepted: 3 December 1998     

APL program for evaluation of heat processes for spherical foods

The present program solves the heat transfer equations for sphericalfoods by a numerical procedure that uses the Crank–Nicolsonmethod. The program is implemented for conditions with as fewrestrictions as possible to enhance versatility. The resultsagree with the analytical solutions for two particular casesof boundary conditions. The program also computes the thermaleffect in the foodstuff from the temperature data obtained bythe numerical program.     

Diagnostic Criteria for Differentiation of Pseudomonads Pathogenic for Man

To determine the most useful diagnostic characters for the differentiation of pseudomonads pathogenic for man, including Pseudomonas maltophilia (Alcaligenes bookeri), P. stutzeri (Bacillus denitrificans), P. pseudomallei (Malleomyces pseudomallei), and apyocyanogenic strains of P. aeruginosa, a comparative examination was made of 58 strains of these bacilli for their morphological and biochemical characteristics. It was concluded that the criteria for differentiating these bacteria are type of flagellation, growth on S S Agar, fluorescein production, oxidation of carbohydrates in the medium of Hugh and Leifson, nitrogen gas production, gluconate oxidation, gelatinase activity, urease activity, lysine decarboxylase activity, arginine dihydrolase activity, oxidase reaction, sensitivity to polymyxin, requirement for methionine as a growth factor, and assimilation of organic compounds as the sole source of carbon and energy.     

Novel method for in vitro O-glycosylation of proteins: application for bioconjugation

Here, we describe a new method for the bioconjugation of a nonglycoprotein with biomolecules. Using polypeptide-alpha- N-acetylgalactosaminyltransferase II (ppGalNAc-T2), we transfer a C2-modified galactose that has a chemical handle, such as ketone or azide, from its respective UDP-sugars to the Ser/Thr residue(s) of an acceptor polypeptide fused to the nonglycoprotein. The protein with the modified galactose is then coupled to a biomolecule that carries an orthogonal reactive group. As a model system for the nonglycoprotein, we engineered glutathione- S-transferase (GST) protein with a 17-amino-acid-long fusion peptide at the C-terminal end that was expressed as a soluble protein in E. coli. The ppGalNAc-T2 protein, the catalytic domain with the C-terminal lectin domain, was expressed as inclusion bodies in E. coli, and an in vitro folding method was developed to produce milligram quantities of the active enzyme from a liter of bacterial culture. This ppGalNAc-T2 enzyme transfers from the UDP-sugars not only GalNAc but also C2-modified galactose with a chemical handle to the Ser/Thr residue(s) in the fusion peptide. The chemical handle at the C2 of galactose is used for conjugation and assembly of bionanoparticles and preparation of immuno-liposomes for a targeted drug delivery system. This novel method enables one to glycosylate, using ppGalNAc-T2, the important biological nonglycoproteins, such as single-chain antibodies, growth factors, or bacterial toxins, with an engineered 17-residue peptide sequence at the C-terminus of the molecule, for conjugation and coupling.     

A convenient method for the synthesis of phosphoramidite non-nucleotide inserts for preparation of functionalized oligonucleotides

A simple approach to the synthesis of amidophosphite synthons of achiral non-nucleotide inserts using 4-(2-(4,4??-dimethoxytrityloxy)ethyl)morpholine-2,3-dione as a backbone of key precursor was suggested. Non-nucleotide synthons were synthesized using this approach that were suitable for synthesis of acridine-containing oligonucleotide derivatives, as well as oligonucleotides with branched carbohydrate-phosphate backbone.     

Development of a 16S rDNA-targeted PCR assay for monitoring of Lactobacillus plantarum and Lact. rhamnosus during co-cultivation for production of inoculants for silages

AIMS: This paper reports a simple, rapid approach for the parallel detection of Lactobacillus plantarum and Lact. rhamnosus in co-culture in order to produce an inoculant mixture for silage purposes. METHODS AND RESULTS: The 16S rDNA-targeted PCR primers were established for parallel detection of Lact. plantarum and Lact. rhamnosus in a single multiplex PCR. A protocol for application of these primers in direct PCR as well as colony-direct (CD) PCR was developed. These primers were also applicable for the estimation of the relative amount of each DNA type in mixed probes (semi-quantitative PCR). CONCLUSIONS: The PCR assay presented in this study is a robust, fast and semi-quantitative approach for detection of Lact. plantarum and Lact. rhamnosus in liquid cultures as well as on agar plates. SIGNIFICANCE AND IMPACT OF THE STUDY: This method provides an effective tool for the establishment of a regime for co-cultivation of Lact. plantarum and Lact. rhamnosus. This would enable faster and thus cost-reduced production of ensiling inoculants.     

Methodology for an assessment of derived radiation levels for agrocenoses

Radiation protection of agrarian ecosystems should be considered as an integral part of a system for radiation protection of environment, with a special concern to agroecosystems' features. A methodology is proposed for an assessment of maximum permissible doses of radiation impact for agrocenoses based on an unified analysis of available data about effects of radiation in cultivated plants. It is considered as a component of radiation protection system for agricultural ecosystems. Critical doses and dose rates are estimated for crops under different exposure situations. It is shown that doses that could result in decreasing indexes of productivity and survival for main crops below 50% are unlikely up to 170-200 Gy and 15-17 Gy at an acute exposure of dormant seeds and vegetative plants, correspondingly. At chronic exposure, above 10% loss of productivity in crops is not expected at dose rates below 3-10 mGy/h.