Establishment of rat dermal papilla cell lines that sustain the potency to induce hair follicles from afollicular skin.

Dermal papilla cells in culture show a lower proliferative capacity compared with dermal fibroblasts, and lose their in situ potency to induce hair follicles in the epidermis at more than 10 passage numbers. This study overcomes these limitations of cultured papilla cells and for the first time demonstrates that papilla cells can be serially cultured for a long period without losing their hair-inductive potency. Outgrowth and the ensuing proliferation of papilla cells were markedly stimulated when explants of rat vibrissa papillae were cultured with rat sole-derived keratinocytes. Such feeder effects of the keratinocytes could be replaced to some extent with conditioned medium of the cells. Serial cultivation of papilla cells was established by maintaining them in the conditioned medium in which they were subcultured for more than 90 passages with an approximate population doubling time of 30 h, a value similar to that of rat dermal fibroblasts. During the subculture, they showed morphologic characteristics and phenotypic expressions of original papilla cells. Even after at least 70 passages, papilla cells sustained the innate hair follicle inductive ability at a level comparable with that of intact dermal papillae. The established cell lines did not show tumorigenicity when they were subcutaneously implanted into nude mice. The culture method developed in this study should facilitate the search for a biochemical entity of dermal papilla cells.

Kdap, a novel gene associated with the stratification of the epithelium.

The skin develops and differentiates during embryogenesis, which is concertedly regulated by a variety of genes. The present study isolated from the rat embryonic skin a novel differentiation-associated gene named Kdap (keratinocyte differentiation-associated protein) by suppression subtractive hybridization between the skin of 14day postcoitus (dpc) embryo (the prehair-germ stage) and that of 17dpc embryo (the hair-germ stage). Its mRNA contained four spliced forms in these tissues. The gene encoded a protein of total 98 amino acids with a calculated molecular mass of 11kDa and an isoelectric point of 6.1 as an unspliced form. The two splicing zones were well conserved among rat, mouse, and human. This protein had a high hydrophobic N-terminal region, a possible signal sequence, and contained two putative N-myristoylation sites and two casein kinase II phosphorylation sites. In situ hybridization experiments detected Kdap transcripts exclusively in the suprabasal cell layers of the embryonic epidermis. Intense expression was also seen in suprabasal cells in regions of infundibulum of the hair follicle. These results indicated that Kdap provides a new insight into the mechanism of differentiation and the maintenance of stratified epithelia.

The upper dermal sheath has a potential to regenerate the hair in the rat follicular epidermis.

It has been established that the dermal papilla and the lower dermal sheath of a hair follicle can induce a new hair bulb in follicular epidermis. However, the upper dermal sheath has been believed not to have such an inductive capacity. In contrast to this generally accepted notion, the present study claims the inductive capacity of the upper dermal sheath of a rat vibrissa follicle, as pelage-type hairs were autonomously produced in the amputated upper halves of the follicles when implanted under the kidney capsule for 8 weeks. The study with monoclonal antibodies specific to different follicular tissues clearly revealed the regeneration of small hair bulbs in these implants and suggested that the fine hairs were formed as a result of the interaction between the upper dermal sheath cells and the follicular basal cells. Newly formed bulbs located at two separate sites, one at the amputated end and the other in the upper region of the follicles. No bulb was formed near the bulge area where follicular stem cells had been reported to locate. However, the possibility of involvement of the stem cells in the bulb formation remains, as migration and rearrangement of epidermal cells had occurred in the implants. Together with the comparative experiments on the inductive potential of various dermal compartments, the present study showed that the upper dermal sheath possesses a hair-inducing ability which is weak, as compared to the lower dermal compartments, but sufficient to induce the follicular epidermis to differentiate into the pelage-like hair-producing bulb, and would be activated only under this specific condition.

Elution concentration of proteins cut from two-dimensional polyacrylamide gels using Pasteur pipettes.

The present study devised a new procedure for concentrating proteins cut from primary two-dimensional polyacrylamide gels prior to their structural characterization. Gel spots containing a protein were cut from several identical two-dimensional gels and loaded on the top of a high tensile strength stacking gel in a Pasteur pipette. The protein was concentrated electrophoretically into a small volume in the narrow tip of the pipette. The concentrated protein was then blotted from the stacking gel onto a polyvinylidene difluoride membrane, where the protein was subjected to tryptic on-membrane digestion. Utilizing this system, we identified 22 proteins chosen randomly from two-dimensional gels of proteins from rat dermal papilla cells by internal microsequencing. As much as 1.5 mL volume (cut gel spots in protein sample buffer) could be loaded onto the Pasteur pipettes, generally yielding a final on-membrane area of approximately 2 mm2 after elution concentration and electroblotting onto polyvinylidene difluoride membranes. We concluded that this newly devised system is effective and useful for concentrating proteins prior to structural characterization, and that larger volumes than previously recommended can be effectively concentrated, with little or no effect on the final on-membrane area occupied by the concentrated proteins.

A quantitative structure-activity relationship for antitumor activity of long-chain phenols from Ginkgo biloba L.

With the aim of obtaining compounds with strong antitumor activity, a quantitative structure-activity relationship (QSAR) of antitumor phenolic compounds (long-chain phenols) was derived using the Hansch-Fujita equation. The ED50 values against Chinese hamster V-79 cells were analyzed in terms of log P as the hydrophobic parameter and the energy of the lowest unoccupied molecular orbital (ELUMO) calculated by using the modified neglect of differential overlap (MNDO) method as the electronic parameter, by means of multiple regression analysis. It was found that the activities mainly depended on log P (an optimum log P of 8.3) and a low-lying ELUMO value. 4-Undecylcatechol, selected on the basis of the above results, exhibited strong antitumor activity against Sarcoma 180 ascites and P-388 lymphocytic leukemia.

A cytotoxic substance from Sangre de Grado.

Taspine has been isolated as a cytotoxic substance from Sangre de Grado, sap of Croton palanostigma (Euphorbiaceae), by bioassay guided fractionation. The cytotoxicity (IC50) of taspine was found to be 0.39 microgram/ml against KB cells and 0.17 microgram/ml against V-79 cells.