Abstract. Lectin binding, endo-chitinase binding and enzymatic degradation studies show that the epidermal cuticle of the bony fish Paralipophrys trigloides (Blenniidae) is chitinous. This is the first evidence that a vertebrate species possesses a chitinous tissue. Recently a Xenopus gene has been identified which has significant sequence similarity to the catalytic domain of yeast chitin synthase III, a chitin producing enzyme ~,2. Taken together these two findings imply that chitin synthesis capability may be a basic vertebrate feature.

The tyramide amplification technique has recently been developed for signal enhancement in enzyme-linked immunosorbent assays and western blots. This method relies on using labelled tyramides as substrates for peroxidase, resulting in an immobilization of the labelled tyramide residues (tyramide reaction). We succeeded in establishing reliable protocols for the use of the tyramide reaction at the electron microscopic (EM) level. As model systems we chose the visualization of DNA in late spermatocytes, of actin in skeletal muscle, and the visualization of an rDNA probe after DNA-DNA in situ hybridization. We observed a significant increase in signal density after performing the tyramide reaction at the EM level. The tyramide amplification technique at the ultrastructural level therefore appears to be a useful tool to detect even a few epitopes present at the surface of a section as shown after in situ hybridization. It offers advantages over other amplification systems, such as the peroxidase-mediated deposition of diaminobenzidine, because of an increased spatial resolution, whereas specificity and sensitivity are comparable to the conventional immunogold detection method.

We report on the effect of different doses and times of incubation of the cytostatic drug actinomycin D (AMD) on nucleolar morphology, rRNA gene transcription and rDNA gene localization using in situ hybridization and the immunocytochemical detection of the human upstream binding factor (UBF) at the electron microscopic level in HeLa cells. Low doses of AMD (0.001 micrograms/ml, 30 min) selectively block rRNA gene transcription but alter neither nucleolar morphology nor the localization of rDNA with respect to the nucleolar components. Treatment with high doses of AMD (0.05 micrograms/ml, 1 h) resulted in a retraction of the rDNA out of the nucleolus in addition to the well-known blocking of rDNA transcription, total nuclear transcription and nucleolar segregation. Under these conditions accumulations of rDNA were found in patches of chromatin at the nucleolar periphery. We conclude that the blocking of rRNA gene transcription and the changes in nucleolar morphology, both induced by AMD at different doses, are independent phenomena.

Immunoelectron microscopy using ultrasmall gold markers is a very sensitive method to detect molecules at high resolution. In order to discriminate the gold particles in the electron microscope, enlargement of gold particles is necessary. So far, mostly silver ions were used for deposition onto the surface of gold grains. In our study, we tested the selective deposition of gold instead of silver ions to enlarge gold particles. This was performed following immunogold detection of DNA at the surface of ultrathin sections embedded in the acrylic resin LR White (postembedding approach). Morphometric analysis of the distribution of DNA in human spermatocytes revealed that the method offers very good specificity and sensitivity and therefore is a good alternative to the use of silver for signal enhancement. This technique was also applied to the detection of ribosomal genes in human testis at the electron microscopic level by in situ hybridization. Ribosomal genes were localized in peri-and intranucleolar chromatin as well as in the dense fibrillar component of nucleoli.

The Grey horse phenotype, caused by a 4.6 kb duplication in Syntaxin 17, is strongly associated with high incidence of melanoma. In contrast to most human melanomas with an early onset of metastasis, the Grey horse melanomas have an extended period of benign growth, after which 50% or more eventually undergo progression and may metastasize. In efforts to define changes occurring during Grey horse melanoma progression, we established an in vitro model comprised of two cell lines, HoMel-L1 and HoMel-A1, representing a primary and a metastatic stage of the melanoma, respectively. The cell lines were examined for their growth and morphological characteristics, in vitro and in vivo oncogenic potential, chromosome numbers, and expression of melanocytic antigens and tumor suppressors. Both cell lines exhibited malignant characteristics; however, the metastatic HoMel-A1 showed a more aggressive phenotype characterized by higher proliferation rates, invasiveness, and a stronger tumorigenic potential both in vitro and in vivo. HoMel-A1 displayed a near-haploid karyotype, whereas HoMel-L1 was near-diploid. The cell lines expressed melanocytic lineage markers such as TYR, TRP1, MITF, PMEL, ASIP, MC1R, POMC, and KIT. The tumor suppressor p53 was strongly expressed in both cell lines, while the tumor suppressors p16 and PTEN were absent in HoMel-A1, potentially implicating significance of these pathways in the melanoma progression. This in vitro model system will not only aid in understanding of the Grey horse melanoma pathogenesis, but also in unraveling the steps during melanoma progression in general as well as being an invaluable tool for development of new therapeutic strategies.