Development of acquired resistance precipitating antibody in rabbits experimentally infested with females of Haemaphysalis longicornis (Ixodoidea: Ixodidae).

The resistance to the ixodid tick, Haemaphysalis longicornis (parthenogenetic Okayama strain), manifested as a reduction in engorged body weight, developed in rabbits subjected to a series of adult female infestations. A single infestation with females always produced resistance in hosts. This production appeared to depend little on the number of ticks per infestation. Unlike the previous papers, this study revealed that there was no reduction in the mean recovery rate of engorged females when ticks fed on a rabbit repeatedly infested with the ticks. A series of infestations were carried out comparatively to investigate the major biological characters of ticks, such as feeding, oviposition, and hatchability of eggs. As a result, there were no marked differences in these characters among the infestations. Especially, no differences were noticed in the concentration of ingested blood meal in detached females among the infestations. Precipitating antibodies were found in the sera of rabbits resistant to the tick-bite. They were subjected to fractionation by Sephadex G-200 chromatography and tested for sensitivity to 2-mercaptoethanol. As a result, they were proved to be of immunoglobulin of 7 S class.     

Development of Leucocytozoon caulleryi in chick embryos infected by biting of Culicoides arakawae through shell membrane.

Chick embryos were infected with Leucocytozoon caulleryi by biting of the midge, Culicoides arakawae, through the shell membrane. Schizonts of L. caulleryi were detected in the chorioallantoic membrane and most of the internal organs of embryos and of chicks after hatching. The development of schizonts was slower in embryos than in chickens. Soluble antigens of L. caulleryi were demonstrated by the precipitation test in the allantoic fluid and blood from the embryos and chicks. No erythrocytic stage of L. caulleryi, however, was observed in any embryo or chick.     

Changes in structural organization of surface membrane during erythrocyte maturation

The effect of concanavalin A and its succinylated derivative on cell agglutination and potassium compartmentation of mature and immature erythrocytes was observed. The binding of tetravalent concanavalin A to the surface glycoproteins of rabbit erythrocytes leads to a change in the properties of the surface membrane, which results in an induction of cell agglutination and concomitant release of potassium from the cells. Both of the phenomena induced by concanavalin A are temperature dependent, and observed at above 15°C.Divalent succinylated concanavalin A, lacking the inducing activity of surface glycoprotein cross-linking into patches and caps, caused neither cell agglutination nor change in the potassium compartmentation of erythrocytes and reticulocytes.In the case of immature reticulocytes, however, remarkable agglutination of the cells was induced without a change in the potassium compartmentation after treatment with tetravalent concanavalin A.It is suggested that changes in the molecular organization of the surface membrane occur in which potassium compartmentation of the reticulocytes becomes more susceptible to surface glycoprotein cross-linking during cellular maturation.     

Kinetcs and decay of fumarase activity of immobilized Brevibacterium ammoniagenes cells for continuous production of L-malic acid

The kinetics of the reversible fumarase reaction of immobilized Brevibacterium ammoniagenes cells and the decay behavior of enzyme activity were investigated in a plug flow system. The time course of the reaction in the immobilized cell column was well explained by the time-conversion equation including the apparent kinetic constants of the immobilized cell enzyme. The decay rate of fumarase activity was faster in the upper sections of the column (inlet side of the substrate solution) compared with the lower sections when 1M sodium fumarate (pH 7.0) was continuously passed through the column at 37°C. It was shown that the decay rate of the fumarase activity in the immobilized cell column depends on the flow rate of the substrate solution. The effect of flow rate on the decay rate of enzyme activity was considered to be related to the rate of contamination of enzyme with poisonous substances derived from the substrate solution or to the rate of leakage of enzyme stabilizers and/or enzyme itself from the immobilized cells.     

X-ray diffraction data for (1→3)-α-d-glucan triacetate

The crystal structures of (1→3)-α-d-glucan triacetates were studied by X-ray diffraction measurements on fibre diagrams. The oriented films annealed in water at high temperature were of higher crystallinity and occurred as two crystalline polymorphs (GTA I and GTA II) depending on the samples and also the annealing temperature. All reflections in GTA I were indexed with a pseudo-orthorhombic unit cell with a = 1·753, b = 3·018 and c(fibre axis) = 1·205 nm. From the fibre repeat data coupled with the density data and the presence of only the (003) reflection on the meridian, an extended three-fold helical structure was proposed. Although some reflections in GTA II split from the layer lines, the basic unit cell was a monoclinic system with a = 1·685, b = 3·878, c (fibre axis) = 1·210 nm and γ = 112·2°. A similar three-fold structure to GTA I was proposed from the almost identical fibre repeat and the conformational analysis on (1→3)-α-d-glucan. It was concluded that, on acetylation, the d-glucan structure changed from the fully extended two-fold helix to the extended three-fold accompanied by some extent of chain shrinking.     

Microbial endo-beta-N-acetylglucosaminidases acting on complex-type sugar chains of glycoproteins.

The activity and substrate specificity of endo-beta-N-acetylglucosaminidase [glycopeptide-D-mannosyl-N4-(N-acetyl-D-glucosaminyl)2-asparagine 1,4-N-acetyl-beta-glucosamino-hydrolase, EC 3.2.1.96] obtained from Mucor hiemalis (Endo-M) was compared with that of the enzyme obtained from Flavobacterium meningosepticum (Endo-F), which is the only enzyme available that acts on the complex oligosaccharides of asparagine-linked sugar chains in glycoproteins. They showed almost the same activities toward DNS-ovalbumin glycopeptide containing high-mannose and hybrid asparagine-linked oligosaccharides. However, Endo-M showed high activity towards DNS-asialotransferrin and DNS-transferrin glycopeptides, which contain complex biantennary oligosaccharides. Endo-M could weakly act even on DNS-asialofetuin glycopeptide containing complex triantennary oligosaccharides, while Endo-F could not. SDS-denatured asialotransferrin was deglycosylated by both enzymes in the presence of non-ionic detergent (NP-40) and EDTA, and the deglycosylated protein migrated to a lower molecular weight position than asialotransferrin on SDS-PAGE. However, even in the absence of detergent, Endo-M deglycosylated native asialotransferrin and transferrin. Deglycosylation of asialotransferrin was confirmed by means of Con A-Sepharose 4B column chromatography and SDS-PAGE.     

Effect of Ozone on the Activities of Reactive Oxygen Scavenging Enzymes in Rbc of Ozone Exposed Japanese Charr (Salvelinus Leucomaenis)

The effect of ozone exposure on the activities of reactive oxygen scavenging enzymes (SOD†, catalase, GSH-Px) in RBC of Japanese charr (Salvelinus leucomaenis) was examined. Ozone (0, 0.4 and 0.7 ppm as initial concentrations) was exposed to Japanese charr for 30 min, which definitely caused serious membrane damage to RBC of fish. Ozone exposure at 0.4 and 0.7 ppm decreased activities of both catalase and GSH-Px by 80 to 57+ of the control. On the other hand, the activities of SOD remained unaffected even by 0.7 ppm ozone exposure. A hypothesis on the RBC membrane damage and participation of SOD and heme-iron was proposed.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain

The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism.The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U₂ to U₁ was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U₁

U₂ reaction is proline isomerization. The rates of interconversion between N and U₂ changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.