A novel Vero cell line for use as a mammalian host-vector system in serum-free medium

We have established a novel cell line from a Vero cell derivative that is useful for expression of exogenous genes and protein production. Parental Vero-317 cells can grow in biotin-containing Eagle's MEM without supplements. By transforming this cell line with replication origin-defective SV40 DNA, which contains a temperature-sensitive tsA58 large T antigen gene, we established the Verots S3 cell line that amplified a SV40-origin containing plasmid. The cell line expressed a human growth hormone (hGH) gene insert with higher efficiency than COS-7 cells in 5% serum-containing MEM and could grow and continue hGH expression in protein-free MEM. However, temperature-sensitive shut down of hGH production was observed not immediately but 3 days after the temperature shift from 33°C to 39.5°C.     

Inhibition of cofactor activity of protein S by a complex of protein S and C4b-binding protein. Evidence for inactive ternary complex formation between protein S, C4b-binding protein, and activated protein C

To elucidate the mechanism by which C4b-binding protein inhibits the cofactor activity of protein S for anticoagulant-activated protein C, the interactions between protein S, activated protein C, and C4b-binding protein were studied using solid-phase enzyme immunoassays. Both activated protein C and C4b-binding protein bound to protein S fixed to microplate wells. C4b-binding protein did not inhibit the binding of activated protein C to protein S, nor did activated protein C inhibit the binding of C4b-binding protein to protein S. Activated protein C bound to a protein S-C4b-binding protein complex which was cross-linked with a chemical reagent as well as it bound to free protein S. Protein S-C4b-binding protein complex competitively inhibited activated protein C-binding to free protein S and also the cofactor activity of free protein S. Immunoblotting analysis showed ternary complex formation with protein S, C4b-binding protein, and activated protein C in the liquid phase by treatment with the cross-linking reagent. These findings suggest that the protein S-C4b-binding protein complex inhibits the cofactor activity of free protein S probably by inhibition of functionally active protein S-activated protein C complex formation by the apparent competitive formation of an inactive ternary complex with protein S, C4b-binding protein, and activated protein C.     

Localization of thrombomodulin-binding site within human thrombin

A binding site for thrombomodulin on human thrombin (alpha-thrombin) was elucidated by identifying an epitope for a monoclonal antibody for thrombin (MT-6) which inhibited the activation of protein C by the thrombin-thrombomodulin complex by directly inhibiting the binding of thrombin to thrombomodulin. An 8.5-kDa fragment isolated by digestion of thrombin with Staphylococcus aureus V8 protease followed by reversed-phase high performance liquid chromatography (HPLC) and a peptide isolated by reversed-phase HPLC after reduction of the 8.5-kDa fragment, which was composed of three peptides linked by disulfide-bonds, bound directly to MT-6 and thrombomodulin. The amino acid sequence of the peptide coincided with the sequence of residues Thr-147 to Asp-175 of the B-chain of thrombin. A synthetic peptide corresponding to Thr-147 to Ser-158 of the B-chain inhibited the binding of thrombin to thrombomodulin. Elastase-digested thrombin, which was cleaved between Ala-150 and Asn-151, lost its binding affinity for both MT-6 and thrombomodulin. These findings indicate that the binding site for thrombomodulin is located within the sequence between Thr-147 and Ser-158 of the B-chain.     

Exocytotic release of secretory granules from endocrine cells in the midgut of insects

Summary Exocytotic release of the secretory granules of the endocrine cells in the midgut of a cockroach, Periplaneta americana, was studied by means of fixation with tannic acid in combination with glutaraldehyde and osmium tetroxide. A sequence of images indicative of exocytosis suggests the following steps in this process: (1) A delicate connection appears between the granule-limiting membrane and the plasma membrane. (2) The plasma membrane approaches the granule, forming a concave indentation. (3) The granule-limiting membrane fuses with the plasma membrane and opens to give rise to an omega profile. (4) The granule content is voided into extracellular space. Exocytosis occurs not only at the base of the cell but occasionally at its side facing adjacent cells. (5) The exocytotic invagination after release becomes smaller and narrower; sometimes a coated pit with bristles appears. Multiple exocytosis, and exocytosis in the endocrine cells of the nidus, i.e., the regenerative cell mass, are also described.     

Tuftsin: a naturally occurring immunopotentiating factor. I. In vitro enhancement of murine natural cell-mediated cytotoxicity

Tuftsin is a physiologic tetrapeptide, which has recently been shown to possess immunoadjuvant properties including the stimulation of macrophage and granulocyte phagocytosis, migration, bactericidal, and tumoricidal activities. Tuftsin has also been reported to possess in vivo immunologically mediated anti-tumor potential. To determine the potential role of tuftsin as an antineoplastic immunoadjuvant, the in vitro effects of tuftsin on murine natural cell-mediated cytotoxicity were studied. We observed that in vitro treatment of mouse splenic effector cells with synthetic tuftsin induced a pronounced enhancement of natural killer cell (NKC) cytotoxicity against the T cell lymphoma Yac-1. The magnitude of NKC enhancement was directly dependent upon the concentration of tuftsin employed, with maximum NKC stimulation observed at tuftsin concentrations of 50 to 100 microgram/ml. The tuftsin induced enhancement of NKC activity was not strain specific, since equivalent stimulation was seen in CBA/J, C56BL/10, and DBA/2 mice. Elimination of macrophages, monocytes, T cells, and immunoglobulin-bearing cells had no effect on the dose-dependent tuftsin stimulation of natural cell-mediated cytotoxicity; thus the characteristics of the effector cells activated by tuftsin were consistent with those reported for NKC. We also observed that treatment of splenic effector cells with tuftsin prolonged the cytotoxic capabilities of these cells beyond 18 hr.     

Effect of cryogenic preservation (−80 °C) on polymorphonuclear neutrophil integrity as determined by biochemical analysis

The effect of cryopreservation on plasma membrane and granule associated enzymes of polymorphonuclear neutrophils (PMNs) was studied. The activity of PMNs to generate superoxide anions during phagocytosis was very sensitive to cryopreservation and exhibited approximately 60% inhibition in 24 hr. The total enzyme activity was not as affected during 1-month cryopreservation as that observed with the extracellular release of enzymes. Acid p-nitrophenyl phosphatase and peroxidase were released slightly from frozen and thawed PMNs. However, the extracellular release of LDH, a cytosol marker, and β-glucuronidase and lysozyme, granuleassociated enzymes, increased with cryopreservation time. The degree of release of these enzymes was LDH > β-glucuronidase > lysozyme. A considerable amount of LDH was extracellularly released after 1-month storage. Frozen and thawed PMNs became sensitive to hypotonic solutions, although fresh, nonfrozen PMNs were very resistant to hypotonic lysis. The hypotonic fragility increased even after 1 hr of cryopreservation.Addition of ATP to the preservation medium did not improve enzyme activity, enzyme release, or stimulated superoxide anion generation but increased the hypotonic fragility of PMNs. However, albumin showed protective effects against cryopreservation injury to the O₂^?-generating system, the extracellular enzyme release, and osmotic fragility.     

Mode of action of Bacillus thuringiensis δ-endotoxin: General characteristics of intoxicated Bombyx larvae

The general pathology induced by δ-endotoxin in terms of larval behavior and hemolymph chemistry has been widely studied in the so-called Type I insect, Bombyx mori. The succession of symptoms is divided into four arbitrary stages: Stage 0, appearance and locomotion normal, no feeding; Stage 1, slightly sluggish; Stage 2, extremely sluggish; and Stage 3, complete paralysis. The action of δ-endotoxin is highly specific to the midgut since contractile movement of both foregut and hindgut continues long after all locomotor activity and heartbeat have stopped. Immediately after the silkworm stops feeding and blood pH sharply rises, there is an associated abrupt rise in the K⁺ concentration of hemolymph. Thereafter, the rise in K⁺ is linear while the rise in pH is not. In vivo measurements have not yielded the same simple linear dependence of pH on K⁺ concentrations that is found in in vitro mixtures of hemolymph and midgut juice. Ligation experiments showed that the same pathological sequence (rise of pH and K⁺ concentration, and general paralysis) follows whether the toxin has unrestricted access to the entire midgut or only part of it (anterior or posterior). From the results of injections of midgut juice or various salt solutions into hemocoel, we came to the conclusions that the blood pH and the symptoms are not necessarily parallel and the intact midgut and Malpighian tubules have strong functions for ion regulation.     

Test reactions for a stopped-flow apparatus: Reduction of 2,6-dichlorophenolindophenol and potassium ferricyanide by l-ascorbic acid

A reaction system suitable for testing the function of a stopped-flow apparatus of high performance was searched for. The reduction of 2,6-dichlorophenol-indophenol by l-ascorbic acid at pH 2.0 was recommended as the most practical test reaction. In due course of the study the reaction mechanism of the reduction was discussed.     

Induction of body distension by operations on the nervous system in larvae of the swallowtall, Papilio xuthus

Removal of the ganglion or severance of the nerve cords at the thorax in mature larvae of the swallowtail, Papilio xuthus, induced systemic distension of the body by swallowing excess air. Such a distension, however, was never induced by simultaneous extirpation of the brain, suboesophageal ganglion, or frontal ganglion, or by severance of the recurrent nerve. Removal of an abdominal ganglion induced distension of the posterior part of the body accompanied by shrinkage of the anterior part. The latter phenomenon appears to be induced by a different mechanism from that of systemic distension.     

Purification and Some Properties of Nucleases from Shii-take,Lentinus edodes

Three kinds of nuclease preparations, each of which having both endonuclease activity that formed 5′-mononucleotides and 3′-nucleotidase activity, were separated and partially purified from Shii-take, Lentinus edodes. Both enzyme activities of each preparation showed a similar thermostability and electrophoretic mobility on Polyacrylamide gel, and a competitive relationship was observed between RNA and 3′-AMP in their enzyme reactions. From these results, it is concluded that both enzyme activities of these three preparations reside in a single protein, respectively. They resemble one another in substrate specificity, cleavage pattern of RNA and thermostability, but are distinguishable from one another by molecular weight, electrophoretic mobility and optimum pH for degradation of RNA.