Toward Practical Biological Control of Parasitic Nematodes in Domestic Animals

In a series of laboratory and field experiments where the nematophagous fungus Arthrobotrys oligospora was mixed directly with feces it has been demonstrated that it is possible to use nematophagous fungi for biological control of animal parasitic nematodes. A procedure used for selection of nematophagous fungi that can pass the digestive tract of ruminants, horses, and pigs is described. The selected fungus, Duddingtonia flagrans, has been used in further field experiments, and the results have confirmed that by the addition of D. flagrans to feed supplement it is possible to reduce the parasitic burden significantly.     

Substance P in the suprachiasmatic nucleus of the rat: an immunohistochemical and in situ hybridization study

Using a biotin-streptavidin-horseradish peroxidase (HRP) immunohistochemical technique the distribution of substance P-immunoreactive neuronal elements was investigated in the rat suprachiasmatic nucleus (SCN). Substance P-immunoreactive nerve fibres and varicosities were distributed throughout the suprachiasmatic nucleus, with the largest accumulation in its ventral part. Because this location overlaps with the innervation of retinal afferents, the distribution and density of substance P-immunoreactive fibres in bilaterally enucleated rats were compared to normal rats. The density of substance P-immunoreactive fibres and nerve terminals in the ventral part of the suprachiasmatic nuclei was reduced in the rats with bilateral destruction of the optic nerves, whereas the density of fibres and nerve terminals in the dorsal part as well as other retinal target areas in the thalamus and mesencephalon was unaffected. In rats pretreated with an intraventricular injection of colchicine several substance P-immunoreactive perikarya were identified in the suprachiasmatic nucleus. The immunoreactive neurons, measuring 9.7 m±1.1 m in diameter, were frequently observed in the central core of the nucleus and to a lesser extent in the dorsomedial and ventrolateral subparts. Using in situ hybridization histochemistry pre-protachykinin-A mRNA was found in the same part of the SCN indicating that synthesis of substance P takes place in SCN neurons. Using a double immunohistochemical approach applying diaminobenzidine and benzidinedihydrochloride as chromagens substance P-, vasoactive intestinal peptide (VIP)-, and vasopressin/neurophysin-immunoreactivities were identified in the same brain section. The substance P-immunoreactive perikarya constituted a separate population of SCN neurons, which were not vasopressin-, neurophysin- or VIP-immunoreactive. Taken together, these observations show that substance P is contained in the retinohypothalamic pathway and within a group of SCN cell bodies, indiating that substance P may play a role in the generation and entrainment of circadian rhythmicity.     

Atypical Aeromonas salmonicida Isolated from Diseased Turbot (Scophtalmus maximus L.)

Atypical Aeromonas salmonicida were isolated from 3 outbreaks of disease among farmed turbot (Scophthalmus maximus L.) in 3 different farms, 1 from Norway (Nl) and 2 from Denmark (DK1 and DK2). In all 3 cases, the incidence of disease and mortality was high and the main characteristic pathological finding was skin ulcers and septicaemia. The isolated bacteria were subjected to a thorough phenotypic and genotypic examination and comparison in the laboratory. All 3 isolates belonged to A. salmonicida but dis-played some very different biochemical properties. However, the 2 Danish strains, DK1 and DK2 had identical ribotypes but different from that of Nl, whereas the plasmid pro-files of DK1 and Nl were identical but different from that of DK2. These observations emphasize the need for an improvement of our understanding of the taxonomy and epi-demiology of atypical A. salmonicida.     

RNA-DNA hybridization analyses of tRNA-Val-3b in Drosophila melanogaster

Summary Transfer RNA was extracted from 50–300 mg of adult flies and specifically labeled in vitro. The level of individual isoacceptors was quantitated by efficient annealing to Drosphila tRNA genes carried on recombinant DNA plasmids immobilized on nitrocellulose filters. The level of tRNA _3b ^Val in the tRNA isolated from flies deficient in the major tRNA _3b ^Val loci has been examined. The results show that deletion of the major tRNA _3b ^Val loci resulted in a reduction of approximately 50% in the level of tRNA _3b ^Val but did not produce the Minute phenotype; furthermore the effects of deficiencies at two loci were approximately additive.     

Bf polymorphism. A very fast variant from Nigeria

Summary In a collection of Nigerian serum samples typed for alleles of factor B of the alternative complement pathway, a very high frequency of Bf^F was found (0.69). In addition, a new variant was found in two samples. This variant (F^1.29) moved faster than BfF₁. It was hemolytically active.Supported in part by The Medical Research Council of Canada     

Quantitative immunoassay of total cellular GAP junction protein connexin32 during liver regeneration using antibodies specific to the COOH-terminus.

A radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were used to determine relative concentrations of liver connexin32 (CX32) in rats. The RIA and ELISA utilize synthetic peptides corresponding to regions of the carboxyl-terminus and antibodies raised in rabbits against these peptides. Assuming that affinities of antisera are similar for peptide and native CX32, total cellular CX32 was found to exceed the amount of gap junction protein at the cell surface calculated from morphometric analyses by 1.5-2.0 fold. This finding raises the possibility that some of the protein is present in cytoplasmic compartments or as occult precursors in the plasma membrane. Studies of CX32 content in regenerating rat liver support this conclusion and show a time course of loss and recovery of CX32 that agrees with those reported in studies using other techniques.     

Use of dual wavelength spectrophotometry and continuous enzymatic depletion of oxygen for determination of the oxygen binding constants of hemoglobin

A small stopped-flow cuvette was built into a computer-controlled Cary 210 spectrophotometer. The enzymatic depletion of oxygen in solutions of hemoglobin and myoglobin was initiated by flowing the hemeproteins with the enzyme against a solution of the hemeproteins containing the appropriate substrate. The deoxygenation was homogeneous throughout the solution. Oxygen activity was calculated at each instant of time from the fractional saturation of Mb, determined from observations at the Hb/HbO2 isosbestic wavelength. Fractional saturation of Hb was determined from absorbances at the Mb/MbO2 isosbestic wavelength. The spectrophotometer cycled between these two wavelengths during the deoxygenation. The deoxygenation of HbO2 was largely complete in 20-25 min, whereas the deoxygenation of MbO2 was allowed to proceed for about 1 h. This procedure eliminates equilibration of Hb solutions with a gas phase and replaces oxygen electrode readings with spectrophotometric sensing by Mb, providing essentially instantaneous determinations of oxygen activity and hence 250-500 or more independent data points per run. The Mb and Hb data vectors require several manipulations to correct for small relative displacements in time and for small non-isosbestic effects. Detailed consideration of the enzyme kinetics allowed oxygen activities to be determined in regions where Mb is a poor sensor. Studies of HbO2 deoxygenation as a function of wavelength show that the determination of the four Adair constants requires in addition the determination of three spectroscopic parameters. Values of the apparent Adair constants, determined without these spectroscopic parameters, depend strongly on the monitoring wavelength.     

Resonance Raman investigation of a soluble cytochrome c552 from alkaliphilic Bacillus firmus RAB

The environment of the heme site of a low-potential soluble cytochrome (c552) from alkaliphilic Bacillus firmus RAB has been characterized with resonance Raman scattering and compared to that of horse heart cytochrome c. The Raman data indicate that vibrational bands sensitive to the axial ligation of the heme, as well as modes sensitive to the heme peripheral environment in cytochrome c552, are distinct from those of horse heart cytochrome c. The spectra of cytochrome c552 display resonance Raman modes indicative of a methionine as the sixth ligand in the oxidized form, while the reduced form appears to contain a nitrogenous-based sixth ligand. In addition, Q-band excitation reveals differences among vibrational modes in cytochrome c552 that are sensitive to the amino acid environment surrounding the heme.     

Kinetics and specificity of alginate lyases: Part I, A case study

Purified preparations of alginate lyase from Klebsiella aerogenes and Haliotis sp. were investigated for activity and degradation patterns with alginate and alginate fragments having different compositions and sequences. With fragments approaching homopolymers of guluronate and mannuronate, Michaelis-Menten kinetics were obeyed and kinetic parameters could be obtained. Degradation of alginates containing all four possible linkages in various proportions, followed by isolation of the fragments and identification of the end groups by n.m.r. spectroscopy, indicated that the enzyme preparations can attack more than one type of linkage. The results are discussed with reference to the concept of specificity for enzymes with copolymeric substrates having non-regular distributions of units.     

Preparation and application of a photoreactive thrombin analogue: binding to human platelets

alpha-Thrombin has previously been shown to bind to specific, saturable glycoproteins on the platelet surface. Modification of the thrombin active site with tosyllysyl chloromethyl ketone (TosLysCH2Cl) does not alter thrombin's binding characteristics. Interaction of alpha-thrombin with high-affinity binding sites (KD = 10(-9) M) initiates the platelet response which involves proteolytic hydrolysis of this glycoprotein. Although TosLysCH2Cl--thrombin binds to and competes for the same sites as alpha-thrombin, it cannot induce platelet stimulation because it is enzymatically inactive. In this study, we describe the preparation and application of photoreactive tritium-labeled thrombin analogues. The alpha-thrombin derivative retains its platelet-stimulating and enzymatic activities and, upon photoactivation, covalently binds to specific platelet membrane components. When freshly washed human platelets are exposed to less than saturation doses (less than or equal to 2 nM) of the thrombin derivatives in the dark and photoactivated, a single labeled complex is detected. The same experiment with greater than saturating doses (greater than or equal to 20 nM) of the thrombin derivative yields a similar complex as well as two additional ones. Molecular weight estimates of these thrombin-bound complexes were obtained by gel filtration and NaDodSO4--polyacrylamide gel electrophoresis. The low dose (high affinity) complex with TosLysCH2Cl--thrombin has an approximate molecular weight of 200 000, while that with active alpha-thrombin is smaller, approximately 120 000, due to enzymatic cleavage. The additional complexes detected with the high thrombin dose had estimated molecular weights of 400 000 and 46 000, respectively, and appeared to be the same for TosLysCH2Cl--thrombin and for the alpha-thrombin coupled platelets. These isolated complexes appear to correspond to the two previously detected populations of thrombin binding sites on the platelet.