Expression and Transmission of Wild-Type Pigmentation in the Skin of Transgenic Orange-Colored Variants of Medaka (Oryzias latipes) Bearing the Gene for Mouse Tyrosinase

Transgenic fish carrying a reconstructed mouse tyrosinase gene, mg-Tyrs-J, were produced by microinjecting the gene into the oocyte nucleus of an orange-colored variant of medaka (Oryzias latipes). Of 64 oocytes microinjected and subsequently inseminated, 13 embryos developed normally beyond hatching and three of them exhibited brown skin pigmentation in the adult as was commonly observed in the wild type of this species. Light and electron microscopic examination disclosed a ubiquitous distribution of typical melanophores in the skin of these transgenic fish. Judging from their population density and distribution pattern, it was presumed that melanogenesis in these fish was elicited in amelanotic melanophores that resided in the skin of the orange-colored fish of this variant. Immunofluorescence with use of the anti-mouse tyrosinase antiserum lacking reactivity to medaka tyrosinase clearly disclosed that the gene introduced was expressed in the melanophores of transgenic fish. Crosses of female transgenic fish and males from an orange-colored variant yielded offspring exhibiting wild-type or orange-colored pigmentation in a ratio of 1:1, thus implying that mg-Tyrs-J integrated into the medaka genome behaves like a dominant gene. Little melanogenesis was observed in xanthophores, leucophores and iridophores in transgenic fish, suggesting possible specificity in recognition of teleostean cell types (i.e., melanophores) by the regulatory region of the mouse tyrosinase gene.     

Ten microsatellite loci from Zamia integrifolia (Zamiaceae)

Ten microsatellite loci isolated from Zamia integrifolia are described. All 10 are polymorphic, with three to 10 alleles across 36 members of a single population from South Florida. Heterozygosities ranged from 0.139 to 0.889. Two loci depart significantly from Hardy–Weinberg equilibrium, and exhibit heterozygote deficiency. One locus pair exhibits significant linkage disequilibrium. The primers have also successfully amplified loci from Zamia portoricensis and Zamia ambliphyllidia. These loci will be utilized for population studies in the Caribbean Zamia pumila complex.     

The accuracy of endometrial cytology in the diagnosis of endometrial adenocarcinoma

tajima m., inamura m., nakamura m., sudo y. and yamagishi k. (1998) Cytopathology 9 , 369–380
The accuracy of endometrial cytology in the diagnosis of endometrial adenocarcinoma
We have examined 62 234 cytological samples of endometrium to establish the accuracy and false-positive rate for the diagnosis of endometrial adenocarcinoma. The patients were either attending the gynaecological out-patients clinic with symptoms or were asymptomatic women attending for routine population screening as part of our cancer detection programme, the numbers from these two sources being equal. Out of 138 cases identified as endometrial adenocarcinoma by cytology 126 (91.3%) were confirmed histologically in our hospital. Twelve cases (8.7%) were shown to be false-positives. Re-examination of these led to the same false-positive diagnosis in all 12 cases. This was attributable to similarities of nucleo–cytoplasmic ratio, irregular arrangement of nuclei, variation in nuclear shape and in the numbers of nucleoli in repair cells and hyperplastic cells compared with the carcinoma cases. Most of the false-negative reports were due to insufficient material, pale staining in malignant cells or diagnostic error. Refraction measurement of the density of nuclei of cancer cells using equipment for which the patent is pending enabled objective measurement of nuclear density which indicated that the nuclei were not stained darkly enough in false-negative cases.     

The Melanotropic Peptide, [Nle4, d-Phe7]α-MSH,Stimulates Human Melanoma Tyrosinase Activity and Inhibits Cell Proliferation

Seventeen human melanoma cell (HMC) lines, both melanotic and amelanotic, were incubated in the continuous presence of a potent melanotropic peptide hormone analog, [Nle⁴,d -Phe⁷]α-MSH, for 72 hr with daily changes of medium. Only one cell line (HD, melanotic) consistently responded to the hormone analog by increased tyrosinase activity. Three (one melanotic, two amelanotic) of the HMC lines also failed to respond to the peptide by either increased or decreased enzyme activity when incubated continuously in the presence of the peptide for longer periods of time (6,15,27,43 days). The HD cell line, however, again responded with increasingly enhanced basal enzyme activity the longer the cells were incubated in the presence of the melanotropin. One amelanotic cell line (C8161) responded with enhanced enzyme activity when grown to confluency in the continuous presence of the peptide. Basal tyrosinase activity of the C8161 cell line may have increased as cell density in the flasks increased. These results suggest that under conditions of increased cell number, phenotypic expression of tyrosinase activity in so called “amelanotic” (tyrosinase-negative) cells is increased and can be enhanced further by stimulation with a melanotropic peptide. Under conditions of increased cell number, the presence of [Nle⁴,d -Phe⁷]α-MSH caused morphological differentiation (shape change); the cells became enlarged and very dendritic. The number of cells in monolayer (surface of the flask) and in the medium were drastically reduced in both melanotic and “amelanotic” cell lines incubated with [Nle⁴,d -Phe⁷]α-MSH. The data support other published reports that melanotropic peptides inhibit human melanoma cell growth (proliferation) in vitro, most likely through a cytostatic mechanism. [Nle⁴,d -Phe⁷]α-MSH also exhibited a prolonged (residual) inhibitory action on HD cell proliferation. In other words, inhibition of cell growth (proliferation) of the HMCs was evident even several days after removal of the melanotropic peptide from the incubation medium.     

Selective proliferation of human γδ T cells in vitro

The effect of monoethylphosphate (MEP,commercial available or synthesized) together with IL-2 on the selective proliferation of human γδT cells in vitro from peripheral blood mononuclear cells (PBMC) of healthy donors and of cancer patients was investigated.The γδT cells were stimulated by MEP to proliferate in a dose-dependent manner.The effect of synthesized MEP was 10 times greater than that of commercial MEP.When the PBMCs of healthy donors were cultured for 25 d in the medium containing different concentrations of MEP,the total cell number increased about 1000-3000 fold;and the ratio of γδT cells reached to 70-80%.The selective expansion of γδT cells depended on the synergic action of MEP and IL-2.The bulk cultured γδT cells exhibited obvious cytotoxic activities against allogenic tumor cell lines (SQ-5,K562 and Daudi) and autologous tumor cells.The culture system described here not only offers a simple method for obtaining a large number of γδT cells which may become a new effector in the adoptive immunotherapy,but also provides a useful model for the further studies of the structure and function of γδT cells in vitro.