Enzymatic Maceration of Plant Tissue

Tissue cells of Vicia faba were separated from the leaf pieces by the pectinase treatment after which the chlorophyll determination method was employed. The separation proceeded at a constant rate for 3 hours', the maximum rate of separation was at pH 5.3. The optimal pH shifted to 5.5 in the presence of 0.001 M 2Na-ethylenediaminetetraacetic acid (EDTA), which accelerated the separation at pH levels higher than 5.5. The separation was enhanced by NaCl and MgCL₂ and delayed by CaCl₂ and hypertonicity produced by sugars. Indoleacetic acid (IAA) and naphthaleneacetic acid (NAA) accelerated the separation at physiological concentrations. The rate of separation differed markedly by the age of the leaf and the species which offered the substrate material.     

Spatio-temporal distribution patterns of two tachinid flies,Epicampocera succincta andCompsilura concinnata parasitizingPieris

Larvae of genus Pieris in the northern part of Kyoto City are parasitized by two tachinid flies:Epicampocera succincta, a specialist on genus Pieris, and Compsilura concinnata, a generalist with very wide host-range. We surveyed the parasitism rates of Pieris by both flies for two years at six study areas. In these study areas, there lived three host species in the genus Pieris: P. rapae, P. melete, and P. napi, but neither tachinid parasitized P. napi to any significant extent. In the mountainous district, P. rapae and P. melete coexisted and their populations were relatively continuous, while in the lowland, only P. rapae larvae were abundant in spring and autumn, but even they disappeared in summer. Parasitisms by E. succincta occurred mainly in mountainous district and never in the lowland. C. concinnata parasitized Pieris in all the areas, but its parasitisms occurred mainly in autumn. We analyzed the factors affecting the spatial and temporal patterns of parasitism rates and presumed that the temporal discontinuity of host population restricted the distribution of the specialist parasitoid.     

Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Detection of multiple forms of human ceruloplasmin. A novel Mr 200,000 form

Three polypeptides with apparent Mr = 200,000, 135,000, and 115,000, reacting with antibody to human ceruloplasmin (Cp), were consistently found in sera of normal adult and newborn subjects, patients with Wilson's disease, as well as in the oxidase-active fraction of purified human Cp, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The concentrations of the three Cp polypeptides were proportional to the total Cp oxidase activity measured in whole serum. Peptide mapping revealed that the three Cp polypeptides were closely related. Cross-linking of Cp135 resulted in dimers with electrophoretic mobility similar to that of Cp200. A common shift in electrophoretic mobility following N-glycanase treatment indicated that all three polypeptides were N-glycosylated, and that the apparent differences in molecular mass could not be related to the carbohydrate moiety. Immunoprecipitates of cell lysates of [35S]cysteine labeled HepG2 cells revealed the presence of two species of newly synthesized Cp polypeptides, Mr 200,000 and 135,000, which were secreted into the media. Secretion of Cp200 by the human liver appears to be physiologic and may be the result of posttranslational modification of Cp135.     

Effective pretreatment by cysteine proteinase inhibitor for improved analysis of protein components of trematodes on SDS-PAGE

Since Fasciola sp. contained proteolytic enzyme(s), it was confirmed that degradation took place in protein components in extracts of the liver flukes, which resulted in lack of clarity of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Degradation was shown to occur mostly during a heating process of the extract samples. The proteolytic activity in the extracts was completely blocked and electrophoretic patterns were improved only by the use of cysteine proteinase inhibitor N-[N-(L-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine (E-64). Great improvement was also noted in electrophoretic patterns of the extracts of other trematodes, such as Paragonimus westermani, P. miyazakii and Clonorchis sinesis, when their extracts were treated with E-64.     

Praziquantel-induced secretion of proteolytic enzyme from Schistosoma mansoni cercariae

The effect of praziquantel (PZQ) on secretion of proteolytic enzyme by Schistosoma mansoni cercariae was examined using an azocoll assay. The cercariae secreted proteolytic enzyme in various concentrations of PZQ (0.1, 1, and 10 micrograms/ml), but secretion of enzyme was highest at the lowest concentration. PZQ-induced secretion of proteolytic enzyme was partially inhibited by treatment with verapamil and ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetra-acetic acid but not by calmodulin antagonist W-7 and protein kinase C inhibitor H-7.     

Synthesis and biological activity of (22E,25R)- and (22,25S)-22-dehydro- 1 alpha,25-dihydroxy-26-methylvitamin D3

Both 25-epimers of (22E)-22-dehydro-1 alpha,25-dihydroxy-26-methylvitamin D3 [22-dehydro-26-methyl-1,25-(OH)2D3] were synthesized. The biological activity of these compounds was tested in binding affinity to chick intestinal receptor protein of 1 alpha,25-dihydroxy-vitamin D3 [1,25-(OH)2D3] and in stimulating for intestinal calcium transport and bone calcium mobilization with vitamin D-deficient rats. The relative potency of (25R)- and (25S)-22-dehydro-26-homo-1,25-(OH)2D3 and 1,25-(OH)2D3 in competing for the intestinal cytosolic binding was 1.7:1.5:1. A similar order of activity was observed on intestinal calcium transport and bone calcium mobilization. In the ability for stimulation of intestinal calcium transport, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were about 3.6 and 2.1 times as active as 1,25-(OH)2D3, respectively. In bone calcium mobilization tests, (25R)- and (25S)-22-dehydro-26-methyl-1,25-(OH)2D3 were estimated to be 2.2 and 1.6 times as potent as 1,25-(OH)2D3, respectively.     

Monoclonal antibodies against alpha toxin of Clostridium perfringens

Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA). The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice. Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule. This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.