-2, or -3. The increase in the number of melanoblasts or melanocytes was comparable with that ofmelanoblasts or melanocytes cocultured with secondary keratinocytes in MDMDF or MDMD. Also,pure cultured primary melanoblasts in MDM were further cultured with MDMM supplemented withET-1, -2, or -3 from 14 d. A dramatic increase in the percentage of melanocytes in the melanoblast-melanocyte population was observed after 7 d; however, no increase in the percentage of melanocytes wasobserved in the absence of ET-1, -2, or -3. The increase was comparable with that of melanocytescocultured with secondary keratinocytes in MDMM. Moreover, anti-ET-1, -2, and -3 antibodies inhibitedboth the proliferation of melanoblasts or melanocytes in MDMDF or MDMD and the differentiationof melanocytes in MDMM in primary culture. These results suggest that ET-1, -2, and -3 are one memberof the keratinocyte-derived factors that are involved in regulating the proliferation and differentiation ofmouse epidermal melanocytes in primary culture. First Paragraph]
Growth pattern, morphological characters and molecular characters were investigated in carbon ion beam-irradiated orchid rhizomes. The ion beam irradiation significantly inhibited growth of the rhizomes as the beam dose increased. Even the frequency showed low, plants with abnormal chlorophyll containing leaves were induced at only 10Gy among the shoot induced plantlets. Some of the abnormal rhizomes were propagated and regenerated. Scorable products from 20 primers were obtained by RAPD analysis and most of the plants showed the similar DNA band patterns between control and the ion beam treated rhizomes. While the ion beam-treatment specific DNA bands were appeared in AFLP analysis of the ion beam-treated rhizomes.
Human hair follicles, which are distributed in various and specific sites of the body, appear to have an inherited susceptibility for androgen-dependent growth. Beard, axillary, and frontal scalp dermal papilla cells (DPC) were recently shown to possess the characteristics of androgen target cells. These DPC show strong expression of androgen receptors, and the expression of type II 5α reductase is restricted to beard and frontal scalp DPC. These findings suggest that DPC mediate the signals of androgen to follicular epithelial cells in a paracrine fashion. We developed an in vitro co-culture system using DPC and keratinocytes (KC) to characterize the mode of androgen action in human hair follicles. Androgen significantly stimulated the proliferation of KC co-cultured with beard DPC, indicating that beard DPC produce androgen-dependent diffusible growth factors. Insulin-like growth factor-I was identified as one of the androgen-dependent paracrine growth factors produced by beard DPC. We also identified the inhibitory role of androgen on the growth of KC co-cultured with DPC from androgenetic alopecia (AGA) when the DPC were transfected with an expression vector encoding the androgen receptor. This growth suppression of KC wasmediated by transforming growth factor-β1 (TGF-β1) derived from DPC of AGA, suggesting that TGF-β1 is a paracrine mediator for AGA.
Ultraviolet (UV) irradiation is a major source of environmental damage to skin. Melanin pigmentation protectsagainst this damage by absorbing UV photons and UVgenerated free radicals before they can react with DNA and other critical cellular components; and UV-induced melanogenesis or tanning is widely recognized as exposed skin’s major defense against further UV damage. This article reviews extensive data suggesting DNA damage or DNA repair intermediates directly triggers tanning and other photoprotective responses. Evidence includes the observations that tanning is enhanced in cultured pigment cells by accelerating repair of UV-induced cyclobutane pyrimidine dimers or by treating the cells withUV-mimetic DNA-damaging chemicals. Moreover, small single stranded DNA fragments such as thymidinedinucleotides (pTpT), the substrate for almost all DNA photoproducts, also stimulates tanning when added to cultured pigment cells or applied topically to intact skin.In bacteria, single stranded DNA generated by DNA damage or its repair activates a protease that in turn derepresses over 20 genes whose protein products enhance DNA repair and otherwise promote cell survival, a phenomenon termed the SOS response. Interestingly, pTpT also enhances repair of UV-induced DNA damage in human cells and animal skin, at least in part by activating the tumor suppressor protein and transcription factor p53 and thus upregulating a variety of gene products involved inDNArepair and cell cycle regulation. Together, these data suggest that human cells have an evolutionarily conserved SOS-like response in which UV-induced DNA damage serves as signal to induce photoprotective responses such as tanning and increased DNA repair capacity. The responses can also be triggered in the absence of DNA damage by addition of smallsingle-stranded DNA fragments such as pTpT. First Paragraph]
It was performed the evaluation of the effectiveness of the 3B® product in the inhibition of bacterial and antiviral growth. The 3B® is an equilibrated mixture of controlled strong and weak acids, of pH <1 and innocuous to the human tissues and mucous. The study determined in vitro its inhibitory effectiveness on the growth of microorganisms in water of Maracaibo Lake, served waters taken from the outlet of the clarifier of water treatment plant located in Maracaibo, Venezuela, The same evaluation using 3B in cattle fecal feces in three concentrations High (Non diluted) Medium (diluted 1:50 ml of Water) and Low (diluted in 1:100 ml of water). The results of the in vitro tests with all cultured water and after 24 hours of contact express the total inhibition (0nmp/100 ml) of microorganisms. The results of the culture with cattle fecal feces and after 72 hours of direct contact resulted on the total inhibition of the microorganisms Proteus sp., Escherichia coli, Klebsiella pneumoniae, Bacillus Gram negative, Enterobacter aerogenes, Enterobacter cloacae, Filamentous Fungus and the absolute and fast disappearance of feces odor.
Tyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and ¯anking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5¢ ¯anking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5¢ ¯anking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling speci®c gene expression in many other types of cells. We also provide evidence that the 5¢ ¯anking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte- speci®c expression of tyrosinase are discussed. First Paragraph]
In the last decade, several new aspects of glucocorticoid (GC)-actions on immune cells have been recognized. This recognition has been largely obtained through clinical observations of stress-induced exacerbations of certain dermatologic diseases. To clarify whether GC modulates cutaneous inflammatory reactions besides its known anti-inflammatory effect, ®rst we examined the effect of long-term application of topical GC on several kinds of inflammatory responses induced in the murine model and demonstrated that these regimens signi®cantly augmented the classical contact sensitivity reaction, the croton oil-induced irritant reaction, and the IgEmediated biphasic cutaneous reaction. In addition, large dose topical steroid and its withdrawal enhanced scratching behavior in hapten-challenged mice. This augmented scratching behavior correlated with the induction of preprotachykinin mRNA expression in the challenged skin. In an in vitro experiment, a low-dose, stress-induced level of glucocorticoid signi®cantly upregulated hapten-induced proinflammatory cytokine (IL1a) production by murine keratinocyte cell line Pam 212 and induced substance P peptide production from cultured human keratinocytes. Our results suggest that unsuitable use of GC in addition to stress-induced GC may modulate immune function in the skin through aberrant production of tachykinin, such as substance P or other epidermal cell derived cytokines. First Paragraph]
In this study, it was investigated how estrogens (17-β-estradiol, E2) affect the estrogen receptor (ER) expression and gene regulation of male versus female human scalp hair follicles in vitro. Anagen VI follicles from frontotemporal scalp skin were microdissected and organ-cultured for up to 9 d in the presence of E2 (1–100 nm). Immunohistochemistry was performed for ERβ-expression, known to be predominant in human scalp hair follicles, and for TGF-β2-expression (as negative key hair growth modulator), and E2-responsive genes in organcultured human scalp hair follicles (48 h, 10 nM) were explored by cDNA microarray, using a commercial skin focus chip (Memorec, Cologne, Germany). The distribution pattern of ERβ and TGF-β2-immunoreactivity differed between male and female hair follicles after 48 h culture. Of 1300 genes tested, several genes were regulated sex-dependent differently. The study reveals substantial sex-dependent differences in the response of frontotemporal human scalp hair follicles to E2. Recognition and systematic dissection of the E2-dependent gene regulation will be crucial for the development of more effective, gender-tailored management strategies for female versus male pattern balding.
Recently, we demonstrated the expression of rhodopsin in the tail ®n of the Xenopus tadpole, in which photosensitive melanophores exist (Miyashita et al, The photoreceptor molecules in Xenopus tadpole tail ®n, in which melanophores exist. Zool Sci 18:671± 674, 2001). The presence of opsin molecules in pigment cells of lower vertebrates raises the possibility that pigment cells in animal skin function as photosensors generally. To explore this possibility in higher vertebrates, we tried to detect photoreception molecules in mammalian melanocytes. We extracted total RNA from Melan a2, a cell line of immortal murine melanocyte, which is derived from C57BL mice. The DNA sequence obtained by reverse transcriptase- polymerase chain reaction (RT-PCR) ampli ®cation was homologous to the corresponding portion of the sequence of ocular rhodopsin of mice. Western blotting and uorescent immunocytochemistry showed the existence of the opsin protein in the melanocytes. Another cell line, EL4, which is derived from lymphoma of C57BL/6N, scarcely expresses opsin mRNA, as judged by RT-PCR. Thus expression of the opsin gene is not ubiquitous among immortal cell lines. Detection of rhodopsin mRNA in murine tissues of C57BL/6N by RT-PCR showed its presence in the eye and skin but not in the liver. The role of the opsin molecule in melanocyte is not known at present, but this will provide additional insight into photoreception systems in animal skin. First Paragraph]
Ultraviolet B (UVB) irradiation induces apoptosis of keratinocytes, where p53 has been suggested to playan important role. Recently we have shown that UVB irradiation induces apoptosis of SV40-transformedhuman keratinocytes (SVHK cells). Because p53 function is impaired in SVHK cells by large Tantigen, a UVB-induced p53-independent apoptotic pathway was suggested. We investigated the UVBinduced apoptotic pathway using various keratinocytes. Cultured mouse keratinocytes of homozygous p53 de®cient mice (p53(±/±)) were markedly resistant to UVB-induced apoptosis compared with keratinocytes from wild or heterozygous p53 de®cient mice (p53(±/+)). Twenty per cent of keratinocytes derived from p53 (±/±) mice, however, induced apoptosis following UVB irradiation. Analysis using caspase inhibitors disclosed activation of caspase 8 and 3 in UVB-irradiated SVHK cells. Keratinocytes derived from MRL/lpr mice, which have mutated Fas antigen, showed diminished UVB-induced apoptosis suggesting that Fas antigen is signi®cantly involved in UVB-induced apoptosis. Immunohistochemical analysis revealed that UVB irradiation induces aggregation of Fas antigen showing a dense dot-like staining, which was also observed in SVHK cells treated with agonistic anti-Fas antibody, CH11. Pretreatment of antagonistic anti-Fas antibody, ZB4, inhibited CH11-induced but not UVB-induced multimerization of Fas antigen. Furthemore, UVB irradiation did not affect the basal expression of Fas ligand mRNA, protein and soluble Fas ligand. These results indicate that UVB irradiation induces multimerization of Fas antigen that results in apoptosis without the Fas ligand. First Paragraph]