Increase in the activity of Rhizopus delemar lipase on water-soluble esters by its binding with phosphatidylcholine

Rhizopus (Rh.) delemar (ATCC 34612) C-lipase was found to exhibit a slight activity towards water-soluble esters. The hydrolytic reaction of this lipase on alpha-naphthyl acetate was competitively inhibited by the presence of olive oil or Tween 80. This finding showed that both substrates, insoluble triglyceride and water-soluble ester, were hydrolyzed at the same site on the enzyme. The activities on water-soluble esters (alpha-naphthyl acetate, beta-naphthyl acetate, methyl acetylsalicylate and Tween 80) increased on binding of lipase with phosphatidylcholine (PC), although the activity on olive oil did not change. The increase in activity on water-soluble esters was due to the increase in the Vmax for its hydrolysis. It appears that local structural change of the catalytic site on lipase occurred on binding of PC to the lipase molecule and resulted in an increase in the activity on water-soluble esters. The temperature dependence of the hydrolysis of water-soluble esters demonstrated that the activation energy was lowered on binding of PC to the lipase molecule, and this resulted in an increase in the activity.     

Physiological Effects of a and {alpha} Sexual Agglutination Substances on Mating Reaction in the Yeast Saccharomyces cerevisiae

The sex-specific glycoprotein agglutination substance, responsiblefor sexual agglutination, solubilized from the surface of haploidcells of a or a mating type by the autoclave method had thefollowing effects on mating reaction in Saccharomyces cerevisiae.Sexual agglutination was inhibited by the agglutination substanceof the opposite mating type in living cells as well as in heat-killedcells. Formation of zygotes was completely inhibited, when botha and a cells were treated with the agglutination substanceof the opposite mating type. The a and a agglutination substanceswere inactivated by cells of the opposite mating type, withthe degree of inactivation being greater for the former. Theenzyme responsible for the inactivation of a agglutination substanceseems to be carboxypeptidase Y. ¹ This paper is dedicated to the late Professor J. Ashida, KyotoUniversity. ² Present address: Department of Plant Pathology, Universityof California, Davis, CA. 95616, U.S.A. (Received November 1, 1982; Accepted January 19, 1983)     

Cytogenetics of ticks (Acari: Ixodoidea)

Chromosomes and sex determination of 9 species of Haemaphysalis assigned to 4 subgenera are described. H. (tAlloceraea) kitaokai possesses an XX∶XO sex chromosome system with 18 autosomes plus XX in females; 18 plus X in males. H. (Kaiseriana) hystricis has 18 +XX and 18 + XY in females and males, respectively, in most specimens, but a supernumerary chromosome is present in some individuals. A supernumerary chromosome was also observed in 1 male H. (Aborphysalis) formosensis. These two species are the second and third species of ticks reported to have supernumerary chromosomes. H. formosensis, H. (Kaiseriana) bispinosa, H. (Haemaphysalis) campanulata, H. (H.) flava, H. (H.) megaspinosa, H. (H.) japonica, and H. (H.) pentalagi possess 20 autosomes plus 2 sex chromosomes in females and 20+1 sex chromosomes in males. Phylogenetic relationships within the genus Haemaphysalis are briefly discussed.     

Peroxidatic activity of catalase in the cortical granules of sea urchin eggs

The presence of peroxidatic activity of catalase in eggs of the sea urchins Hemicentrotus pulcherrimus and Temnopleurus toreumaticus was investigated by the ultrastructural cytochemical techniue and by biochemical assay on homogenates of eggs from before fertilization to the 2-cell stage. Biochemical assays showed that the unfertilized eggs had strong catalase activity whereas fertilized eggs had weak activity owing to the rapid decrease of activity after fertilization. The activity did not change from immediately after fertilization to the 2-cell stage. Cytochemical examination showed that the peroxidatic activity of catalase was mainly localized in the lamellae in the cortical granules. Disintegrated cortical granules with no lamellae and substances in the perivitelline space derived from breakdown of the cortical granules had no peroxidatic activity of catalase.     

Solution properties of synthetic polypeptides. V. Helix–coil transition in poly(β-benzyl L-aspartate)

Simple approximate expressions have been derived from the theory of Zimm and Bragg for use in the analysis of experimental data on the helix-coil transition in polypeptide. On the basis of the resulting expressions practical procedures are proposed to determine two basic parameters characterizing a thermally induced transition, i.e., helix initiation parameter σ and enthalpy change for helix formation, ΔH. They have been applied to the data for poly(β-benzyl L -aspartate) (PBLA) with the result: σ = 1.6 × 10^?4 and ΔH = ?450 cal/mole for PBLA in m-cresol; σ = 0.6 × 10^?4 and ΔH = 260 cal/mole for PBLA in chloroform containing 5.7 vol-% of dichloroacetic acid. This result gives evidence that σ may change not only from one polypeptide to another but also for a given polypeptide in different solvents. The change in limiting viscosity number [η] accompanying the transition was measured in the same solvents. The curve of [η] versus helical content had a relatively monotonic shape for the chloroformdichloroacetic acid solutions as compared with that for the m-cresol solutions, indicating that [η] depended largely on σ. Provided that [η] is a direct measure of the mean-square radius of gyration, 〈S²〉, the results are consistent with the theoretical predictions of Nagai and of Miller and Flory for 〈S²〉.     

Fine Structure of Healthy and TMV-Infected Tobacco Cells Grown in Tissue Culture

Three types of tobacco (Nicotiana tabacum cv. Havana 38) callus: 1) healthy stem callus, 2) TMV-infected stem callus, 3) TMV-infected leaf callus; and leaves differentiated from healthy stem callus, and from TMV-infected leaf callus were compared for fine structure. In addition, the fine structure was observed of plastids in cells of leaves differentiated from callus isolated from stem sections of TMV-infected hybrid tobacco plants (N. tabacum cv. Havana 38 ×N. glutinosa) grown under high temperature. The cytoplasmic organelles in tissue cultured cells were similar to those in cells of greenhouse-grown tobacco plants. Except for plastids, TMV infection did not noticeably affect morphologically other cellular organelles in tissue culture cells. In TMV-infected leaf callus, numerous small bodies were seen in plastid-like bodies, while vesicle-like structures were observed in the stroma of plastids in leaves differentiated from callus of hybrid tobacco inoculated with TMV. Morphological variations of mitochondria, such as swelling and vacuolization of the inner matrix, occurred frequently in TMV-infected leaf callus. Needle-like crystalline inclusions or looped inclusions composed of many fine, long filaments were considered TMV particles orientated parallel to each other. The TMV particles were detected in the cytoplasm of tissue culture cells.     

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.     

Intravesical treatment of bladder cancer with recombinant human interferon-β

Summary In order to examine its clinical efficacy, recombinant human interferon- (rIFN-) was instilled intravesically into 51 patients with superficial bladder cancer. Ten patients, who received intermittent intravesical instillation at a dose of (3–36) × 10⁶ U rIFN- on days 1–3 every week, showed no response. Thirty-two patients received intravesical instillation at a dose of (3–36) × 10⁶ U every day for 10–20 days. Eight patients showed partial response, indicating an efficacy rate of 25%. Nine patients received divided doses of 18 × 10⁶ U twice a day every day for 10–20 days. Six patients showed partial response, indicating an efficacy rate of 67%. This value was significantly higher than that obtained by administering divided doses. The response to intravesical instillation therapy with rIFN- varies with treatment protocol. Frequent and longer exposure to rIFN- may induce better regression of superficial bladder cancer. Six incidences of side-effects were found in five cases (9.8%): pollakiuria in one, pain on micturition in two, fever in two, and eruption in one case. All of these side-effects were slight and reversible after drug withdrawal. Laboratory tests showed only a few changes with low severity. Thus, rIFN- is potentially a new drug for instillation therapy of superficial bladder cancer, in view of the absence of adverse effects.     

Cloning of two protease genes fromRhodocyclus gelatinosa APR 3-2 and their expression inEscherichia coli

Two different protease genes were cloned fromRhodocyclus gelatinosa APR 3-2 inEscherichia coli HB 101/ with pBR329 or its derivatives. The recombinant plasmids designated as pRP100 and pRP300 contained 11.2 and 10.6 kb DNA fragments, respectively. The differences of both plasmids in restriction enzyme maps indicate that these plasmids contained different protease genes. DNA fragments coding for protease, 6.4 kb and 4.5 kb from pRP100 and pRP300, were subcloned into pRP329 and designated as pRP101 and pRP301, respectively. The two cloned proteases were excreted in culture medium ofE. coli, and ß-lactamase ofE. coli, which was originally localized in periplasmic space, was also excreted in the medium.