Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Periodic appearance and disappearance of microvilli associated with cleavage cycles in the egg of the ascidian, Halocynthia roretzi

The surface of eggs of the ascidian Halocynthia roretzi, observed with SEM, is essentially smooth until immediately before cell division when numerous microvilli appear and remain during cytokinesis. As the dividing blastomeres become closely adherent, however, the microvilli disappear and the eggs recover their smooth surface. This periodic appearance-disappearance of microvilli is repeated at each cleavage cycle up to at least the 32-cell stage. During blastomere adhesion, microvilli that have appeared near the plane of the first cleavage or of the bilateral symmetry seem to fuse together across the plane to form a zipper-like complex of cytoplasmic processes, which might be responsible for attachment of the two halves of these bilaterally symmetrical embryos via the blastomeres bordering the plane of symmetry.     

Photosynthetic activities of a thermophilic blue-green alga

Photosynthetic activities of a thermophilic blue-green alga,a species of Synechococcus, were studied with special referenceto its growth at high temperatures. A rapid algal growth occurredin the temperature range between 50 and 60?C, showing the maximumrate, six doublings per day, at about 57?C. Photosynthetic oxygenevolution and methyl viologen photoreduction in the cells werealso active at high temperatures and the optimum temperaturesfor these activities agreed with that of the algal growth. Thegrowth and photosynthetic activities were very low at room temperatureand irreversibly inactivated at temperatures above 60?C. The thylakoid membranes isolated from the alga were also photochemicallyactive at high temperatures. The membranes mediated ferricyanidephotoreduction coupled with a stoichiometric oxygen evolutionat a rate comparable to that of photosynthetic oxygen evolutionin the cells. The optimum temperature for the reaction was ashigh as 50?C. The membranes also showed a photosystem I-mediatedreaction at high temperatures. These observations indicate thatthe thylakoid membranes are intrinsically thermophilic in thisorganism. Thus the growth of the alga at high temperatures canbe well correlated to thermophilic properties of the photosyntheticapparatus. (Received February 20, 1978; )     

Cytochemical and electron microscopic demonstration of organelle translocation and the inhibition of such translocation by colchicine during the mitosis of rat hepatocytes

Hepatocyte lysosomes, mitochondria, and peroxisomes show a dramatic translocation during mitosis induced by partial hepatectomy. During prophase, all three organelles move to the perinuclear cytoplasm. In metaphase, they become concentrated in the polar regions. During telophase, these organelles form clusters in the juxtanuclear regions. This organelle translocation is inhibited by the administration of a low concentration of colchicine, suggesting an involvement of microtubules in their movement.     

Expression of cDNA for batroxobin, a thrombin-like snake venom enzyme

The cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the fusion protein with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batroxobin with enzymatic activity was obtained by the refolding of recombinant batroxobin.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

Formation of functionally active chloroplasts is determined at a limited stage of leaf development in virescent mutants of rice

The ability to form functionally active chloroplasts is determined at a certain early stage of leaf development in three non-allelic temperature-sensitive virescent mutants of rice. Temperature-shift analysis, together with anatomical observations, indicates that the intrinsic developmental signals of the virescent genes are expressed at the stage immediately following the formation of basic leaf structure, but just before the onset of leaf elongation. These signals control the expression of chloroplast-encoded genes but do not affect the subsequent morphological development of the leaf or the photo-regulation of the expression of nuclear genes encoding chloroplast proteins.     

Molecular modelling of the structures of endothelin antagonists. Identification of a possible structural determinant for ET-A receptor binding.

Computer-aided molecular modelling of the endothelin (ET-A) receptor antagonists, BQ-123 and BE-18257B, shows that they have very similar 3D structures. Parts of their 3D structures are also shown to match closely with that reported for residues 6-8 in endothelin-1. On the basis of these similarities (and with supporting evidence from literature data on endothelin structure-activity relationships) a structural determinant is proposed for ET-A receptor binding, and novel designs of peptide are suggested for providing more potent and selective ET-A receptor antagonists.     

cDNA cloning of an extracellular dermal glycoprotein of carrot and its expression in response to wounding

Suspension-cultured cells of carrot (Daucus carota L.) synthesize and secrete a glycoprotein that is normally found only in dermal tissues (epidermis, endodermis and periderm). This protein, previously called GP57, is now referred to as EDGP (E xtracellular D ermal G lyco P rotein). We purified sufficient quantities of EDGP to obtain amino-acid sequences on two internal tryptic peptides and screened a cDNA library of young carrot roots with antiserum to EDGP and with oligonucleotides corresponding to the peptides. Here we report the derived amino-acid sequence of EDGP. Sequence comparisons show that it has 40% amino-acid sequence identity with 7S basic globulin, a protein that is released when soybean seeds are soaked in hot water for a few hours. We suggest that these two proteins belong to a new family of dermal proteins. As far as we know, this is the first reported derived amino-acid sequence for protein that is specific to the epidermis and other dermal tissues. The level of EDGP mRNA is low in dry seeds, but increases rapidly in growing seedlings as they develop dermal tissues. The level of mRNA is low in storage roots, but increases rapidly in response to wounding. The presence of EDGP in dermal tissues and its up-regulation in response to wounding indicate a role in the response of plants to biotic and-or abiotic stresses. An unusual feature of the amino-acid sequence of EDGP is that it contains a short motif, which is present at the active site of aspartyl proteases such as pepsin and chymosin.Abbreviations cDNA copy DNA - 2,4-D 2,4-dichlorophen-oxyacetic acid - EDGP extracellular dermal glycoprotein - 7SBG 7S basic globulin Supported by a contract from the United States Department of Energy (Energy Biosciences) (to M.J.C.) and a Grant-in-Aid for Special Research on Priority Areas (01660002, Cellular and Molecular Basis for Reproductive Processes in Plants) from the Ministry of Education, Science and Culture, and by the Fund from Basic Research Core System of Science and Technology Agency, Japan (to S.S.).