Tatsuya Tamaoki

Inhibition of protein kinase C by calphostin C is light-dependent

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, inhibits protein kinase C by competing at the binding site for diacylglycerol and phorbol esters. Calphostin C is a polycyclic hydrocarbon with strong absorbance in the visible and ultraviolet ranges. In characterizing the activity of this compound, we unexpectedly found that the inhibition of [3H]phorbol dibutyrate binding was dependent on exposure to light. Ordinary fluorescent light was sufficient for full activation. The inhibition of protein kinase C activity in cell-free systems and intact cells also required light. Light-dependent cytotoxicity was seen at concentrations about 5-fold higher than those inhibiting protein kinase C.

Topical application of cyclosporin A induces rapid-remodeling of damaged anagen hair follicles produced in cyclophosphamide administered mice

Adult C3H mice which had either anagen IV or anagen VI hair follicles were given the anti-tumor drug cyclophosphamide, and cyclosporin A or minoxidil were topically applied to the mice daily from the 4th day after cyclophosphamide administration. In the mice that had anagen IV-hair follicles, 0.5% cyclosporin A induced very thick and long hairs after 21 days of cyclophosphamide administration, while vehicle and 1% minoxidil induced sparsely visible, short hairs. In the mice which received cyclosporin A, the injured hair follicles seemed to remodel themselves into intact anagen hair follicles and restart the production of hairs, instead of shifting to telogen. In the mice that had anagen VI-hair follicles at the time of cyclophosphamide administration, complete alopecia occurred within the first 7 days in all groups. After 14 days of cyclophosphamide administration, hair regrowth was observed in both the 0.5% cyclosporin A-group and the 1% minoxidil- group with the predominant effect over the vehicle. This study shows that anagen hair follicles respond to cyclophosphamide in different ways depending on their stages (IV and VI), and that the damaged anagen IV hair follicles have the potential of remodeling themselves, which is promoted by topical cyclosporin A administration.

HAIR FOLLICLE ELONGATION IN ORGAN CULTURE OF SKIN FROM NEWBORN AND ADULT MICE

By means of skin organ culture, some biological characteristics of hair follicle elongation were examined. When skin sections from 6-day-old C3H mice were cultured, spontaneous elongation of hair follicles was maintained. Without insulin, hair follicle elongation was poorly maintained irrespective of the presence of serum at 20%. Insulin could be replaced by IGF-I at 100 ng/ml. During in vitro elongation of hair follicles, bromodeoxyuridine was incorporated into germinal epithelial cells around dermal papillae. Skin sections from 4-week-old C3H mice did not show hair follicle elongation in complete medium. However, when 0.5 mM minoxidil was added to the medium, concentration dependent thickening and elongation of hair follicles was observed. In contrast, in vitro elongation of newborn pelage hair follicles was not enhanced by minoxidil. These results suggest that this system with the use of skin sections from 4-week-old C3H mice would be a potential in vitro model of human androgenic alopecia in which the anagen phase is suppressed but the suppression is partially released by minoxidil.

Optimization of cell culture conditions for G-CSF (granulocyte colony-stimulating factor) production by genetically engineered Namalwa KJM-1 cells

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 μg/ml/day using a 25 cm2 tissue culture flask, even though the cell number was above 7×105 cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, BiofermenterTM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×107 cells/ml), and the amount of G-CSF reached 41 μg/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.

KF19418, a new compound for hair growth promotion in vitro and in vivo mouse models

KF19418, a newly synthesized compound, stimulated proliferation of cultured hair bulb cells from new born mice in concentration-dependent manner in the range under 10 microM. In the culture system of whole skin pieces from 4-week-old mice which we earlier established, KF19418 promoted hair follicle elongation as in the case of minoxidil. After topical application for 2 weeks of KF19418 or minoxidil to dorsal skin of hair-clipped mouse alopecia model, KF19418 at 1% suspension accelerated hair regrowth at a rate comparable to 1% minoxidil solution. Thus, it was shown that KF19418 directly stimulated hair follicle in vitro and had hair growth promoting activities in vivo.

Expression of intact Ki-ras p21 protein in Escherichiacoli

We have constructed recombinant plasmids capable of expressing in Escherichia coli the intact ras p21 protein encoded by Kirsten murine sarcoma virus. The Ki-ras gene was inserted into an expression vector carrying the E. coli tryptophan promoter and E. coli lipoprotein transcriptional terminator. The resulting plasmids direct the synthesis of large quantities of p21 protein, which represented 20% of the total cellular protein. The Ki-ras p21 protein is immunoprecipitated with monoclonal antibody to p21, and exhibits guanine nucleotide binding activity and autophosphorylation activity. The purified Ki-ras p21 expressed in E. coli has shown to have intact N-terminal and C-terminal amino acid sequences predicted by the nucleotide sequences and migrate as -23K in SDS/polyacrylamide gels.

Modulation of oligosaccharide structure of a pro-urokinase derivative (pro-UKΔGS1) by changing culture conditions of a lymphoblastoid cell line Namalwa KJM-1 adapted to serum-free medium

Pro-UKΔGS1 was designed as a long-life and thrombin-resistant derivative of pro-urokinase (pro-UK) by deleting the growth factor domain of pro-UK and introducing a glycosylation site near the thrombin cleaving site for thrombin-resistance using site-directed mutagenesis. An expression plasmid for pro-UKDGS1, pIH1UKΔGS1SEd1–5 was constructed and introduced into Namalwa KJM-1, a lymphoblastoid cell line adapted to serum-free medium, and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKΔGS1 producer (resistant to 200 nM of MTX), clone 2–9, was selected and used for further studies.Under the conventional conditions, i.e. at 37°C in serum-free ITPSGF medium (based on RPMI-1640 medium), the oligosaccharide structure of pro-UKΔGS1 produced by clone 2–9 mainly consisted of fucose (Fuc)-containing biantennary complex-type oligosaccharide. Addition of dexamethasone (Dex), changed the carbohydrate contents in the media, and a shift down of incubation temperature caused a change in oligosaccharide structure of pro-UKΔGS1 from mainly Fuc-containing biantennary to mainly Fuc-containing tri-and tetraantennary complex-type oligosaccharide. The modulated pro-UKΔGS1 showed superiorin vivo activity for a canine femoral thrombosis formed by inserting a copper-coil.

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 μg ml−1. Addition of glucose and tri-iodothyronine (T3) improved productivity, and the maximal productivity of pro-UKS1 was 67 μg ml−1 day−1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.

Stable Production of Pro-urokinase and its Derivative by Namalwa KJM-1 Cells Adapted to a Serum-free Medium

We have previously reported that Namalwa KJM-1 cells adapted to a serum-free medium and derived from Namalwa (B lymphoblastoid) cells were more useful than CHO (Chinese hamster ovary) cells as the host cell line for producing various recombinant proteins with a dhfr gene coamplification method, because CHO cells secrete a cysteine endopeptidase (Satoh, 1990).