No abstract

Melanoma growth stimulatory activity (MGSA) is a mitogenic polypeptide secreted by Hs294T human melanoma cells. Comparison of the N‐terminal sequences of the 13 and 16 kd MGSA species with the cDNA sequence revealed that the mature form of human MGSA is maximally 73 amino acids long. Expression of the cDNA in mammalian cells results in the secretion of this peptide with mitogenic activity. MGSA is structurally related to the platelet‐derived beta‐thromboglobulin and to several other polypeptides. These factors may constitute a family of growth factors. MGSA mRNA was detected in a variety of cell types. The level of MGSA mRNA in melanoma cells is strongly elevated by treatment with MGSA. MGSA is the gene product of a recently detected gene gro. The gene was mapped to chromosome 4 (region q13––q21). This same region also contains genes for two of the structurally related factors, for c‐kit, a receptor for an as yet unidentified ligand, and for ‘piebald trait’, an inherited skin pigmentation disorder.

The Hs0294 human malignant melanoma cell line produces a monolayer mitogen that stimulates the serum free growth of low-density cultures of Hs0294 cells. This report describes the purification of that mitogen, termed MGSA for melanoma growth stimulatory activity, from serum-free conditioned medium from the Hs0294 cultures. MGSA has been purified from acetic acid extracts of lyophilized conditioned medium by gel filtration, reverse-phase high-pressure liquid chromatography (RP-HPLC), and preparative electrophoresis, resulting in a greater than 400,000-fold purification. MGSA bioactivity resides in acid- and heat-stable polypeptides of high and low molecular weight (24-28 kd and less than 14-16 kd). However, the majority of the activity is reproducibly associated with the approximately 16-kd moiety eluting from RP-HPLC at approximately 35% acetonitrile. Reduction with dithiothreitol or B-mercaptoethanol results in a loss of biological activity but does not convert the 24-28-kd moieties to the less than 14-16-kd forms of MGSA. 125I-MGSA that has been purified by preparative electrophoresis (16 kd) specifically binds to Hs0294 melanoma cells and retains 100% of the growth-stimulatory activity. The 16-kd MGSA stimulates the proliferation of Hs0294 cells at concentrations of 0.3-30 pM. The electrophoretic mobility of MGSA is also unaltered by the preparative electrophoresis procedure, further demonstrating that this procedure does not alter the biochemical integrity of the growth factor. Purified MGSA does not enable anchorage-independent growth of normal rat kidney (NRK) cells and is therefore different from the previously described transforming growth factors. The amino acid composition of MGSA differs from that of other previously described growth factors. These data demonstrate that MGSA represents a separate class of growth factors with biological and biochemical properties different from other growth factors.

The fine structure of the tegument of three trematode species, Schistosoma mansoni, Dicrocoelium dendriticum, and Fasciola hepatica, was studied by means of light scanning (SEM) and transmission electron microscopy (TEM) after in vitro exposure to 0, 1, 10, and 100 micrograms/ml of the anthelmintic praziquantel for 5, 15, 30, and 60 min. In S. mansoni and D. dendriticum the resulting vacuolization of the tegument was confined to numerous small areas scattered all over the surface of the parasites and this finally led to the disruption of the apical tegumental layer. No changes were found in the tegument of F. hepatica after treatment with praziquantel.

Praziquantel (EMBAY 8440, Droncit) is a new type of acylated isoquinoline-pyrazine. A single oral or subcutaneous dose of the compound is reliably effective against juvenile and adult cestodes in mice and rats. For the first time, with praziquantel a compound is available that is also effective on cestodes in the bile-duct. Using Hymenolepis nana in mice we were able to show that age, sex, strain and intensity of infestation of the host have no influence on the efficacy. The onset of the effect of praziquantel in vivo is rapid. Within 10 min the parasites were immobilized and contracted and excreted within a few hours with the faeces.

Adult Hymenolepis diminuta, H. microstoma, H. nana, Echinococcus multilocularis, and Taenia (Hydatigera) taeniaeformis have been exposed in vitro in media containing 0.1 to 100 micrograms praziquantel/ml. Already after 5 min characteristic tegumental lesions, that were restricted to the growth zone of the neck region, were recognized using both scanning and transmission electron microscopy. Within the tegument numerous vacuoles were formed that released their contents to the exterior and finally caused destruction of the tegument. Proglottides of the central or posterior strobilar portions were never damaged. Larvae of T. taeniaeformis (cysticerci) and E. multilocularis (alveolar cysts) were studied employing the same methods both after in vitro exposure to and after in vivo treatment of their hosts with praziquantel. Strobilocerci of T. taeniaeformis developed identical tegumental lesions after contact with praziquantel whether incubated in vitro or treated in vitro. The wall of the bladder containing the larva remained unaffected. Evaginated protoscolices of E. multilocularis were damaged by in vitro contact with praziquantel while invaginated protoscolices remained intact. After in vivo exposure there were some fully developed evaginated and damaged protoscolices whereas all invaginated protoscolices and the cyst wall with its germinative layer were unaffected.

No abstract

Autostimulatory growth factors may contribute to the ability of malignant cells to escape normal growth controls. We have previously shown that Hs0294 human malignant melanoma cells release into culture medium an acid-soluble, heat-stable, trypsin-sensitive, autostimulatory monolayer mitogen which can be purified from acetic acid extracts of conditioned medium by gel filtration, reverse-phase high-performance liquid chromatography, and preparative electrophoresis. The majority of this melanoma growth-stimulatory activity (MGSA) resides in a 16-Kd moiety, though bioactivity is also associated with 24-26 and less than 14-Kd forms of MGSA (Richmond and Thomas: J Cell Physiol 129:375, 1986). In order to further characterize this growth factor, monoclonal antibodies were prepared against a partially purified preparation of the autostimulatory melanoma mitogen. Monoclonal antibody clones were selected based on supernate inhibition of 3H-thymidine incorporation in serum-free Hs0294 melanoma cultures. One of these, termed FB2AH7, slows, but does not completely block, the growth of Hs0294 cells in serum-free medium in a dose-dependent manner. This antibody does not slow the growth of normal rat kidney fibroblasts, which neither produce nor require this mitogen, in either serum-free medium or medium containing 0.8% calf serum. This monoclonal antibody also blocks the mitogenic effects of partially purified preparations of this melanoma growth stimulatory activity (MGSA) on both Hs0294 cells and normal rat kidney fibroblasts. The FB2AH7 antibody has been demonstrated to bind MGSA by Western blot and by immunoprecipitation procedures. Western blot analysis of reverse-phase high-performance liquid chromatography purified growth factor demonstrated that FB2AH7 antibody binds to the 16-Kd and approximately 13-14-Kd forms of MGSA. FB2AH7 antibody can be used in immunoprecipitation experiments to bind the approximately 13-16-Kd forms of MGSA. The specificity of the binding of FB2AH7 antibody for MGSA but not other growth factors has been demonstrated in a modified dot blot assay. These data thus support the hypothesis that MGSA is an autostimulatory melanoma mitogen distinct from other growth factors.