Essential Bacillus subtilis genes.

To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among approximately 4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

The effect of 1,25-dihydroxyvitamin D3 on the growth and differentiation of cultured human outer root sheath cells from normal subjects and patients with vitamin D-dependent rickets type II with alopecia.

We examined the effect of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on the growth and differentiation of cultured human hair outer root sheath cells (ORSC) from normal subjects and patients with vitamin D-dependent rickets type II (DDR-II) with alopecia. 1,25(OH)2D3 dose-dependently suppressed the plating efficiency, clonal growth, and DNA synthesis of normal ORSC. It enhanced the cornified envelope formation and caused morphological changes in the cells. All results indicated the existence of specific receptors for 1,25(OH)2D3 in the ORSC, and suggest that 1,25(OH)2D3 is a potent inhibitor of proliferation of ORSC as well as a stimulator of terminal differentiation. However, the cells from DDR-II patients with alopecia did not respond to 1,25(OH)2D3, suggesting a lack of the specific receptors in the cells. The differences in the cellular response to the hormone between the normal ORSC and those from the patients were apparent and easily distinguishable, therefore this experiment may be a rapid and simple diagnostic test for DDR-II patients with alopecia. Large number of hairs were difficult to obtain from patients with alopecia, and we developed a new culture method to accomplish these studies from a few plucked hair follicles. Our system may be useful in the culture of ORSC from limited number of follicles, and could be utilized to analyse the cellular characteristics of ORSC in patients with hair diseases.

[Difference between the hair follicle cell and epidermal keratinocyte in vitro].

We cultured human hair follicle cells (HFC) and epidermal keratinocytes (EK) using mixed collagen membranes as the substrate for the culture, and studied the degree of differentiation of these cells. Morphologically, polygonal epithelioid arrangements were seen in both cultures. However, in the culture of EK compared with HFC, large denucleated cells appeared within shorter periods of culture and stratification of cells were more prominent. In the culture of HFC, keratohyalin granules were not observed and cornified envelope formation was suppressed compared with that in EK. Then it is suggested that HFC and EK are different in the degree of keratinization and of other nature.

A new method for culturing human hair follicle cells on floating mixed collagen membranes.

A new method for culturing human hair follicle cells without a biological feeder layer has been developed. Dispersed hair follicle cells obtained from plucked hairs have been successfully grown on floating mixed collagen membranes (collagen types I and IV) and have reached a confluent state. They could be subcultured up to twice. Differentiation of the cells was maintained throughout a month in culture. Stratification of the cells and continuous epithelial pavement formation were seen, as in cultured epidermal keratinocytes.

Culture of dispersed hair follicle cells from plucked out hairs without a feeder layer.

Dispersed hair follicle cells from plucked out hairs have been successfully grown on collagen type IV without a biological feeder layer. The optimal calcium concentration for the culture of these cells was also studied and decided to be 0.3-0.5 mM. In this calcium range and on collagen type IV, colony formation and colony growth were best and big colonies with a paving stone-like cell arrangement were formed. The availability of such cultured cells for the treatment of missing skin is proposed instead of cultured epidermal keratinocytes which have been commonly used for wound dressing.