Induction of reversible changes in cell-surface glycoconjugates and lung colonization potential by 13-cis retinoic acid

Murine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13-cis retinoic acid (RA), had dose-dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P ?.0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971 ?977, 1987). These findings suggest a possible relationship between the RA-induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 × 10^?6 M, 5 × 10^?7 M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor-cell subpopulations. Finally, in a scries of experiments designed to determine the reversibility of the RA treatment, the RA-induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA-induced changes in cell-surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA-induced modulation of tumor cell-surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in determining metastatic behavior.     

Meiofauna communities along a depth transect off Halley Bay (Weddell Sea-Antarctica)

Summary Meiofauna communities from 10 stations along a depth transect from approximately 500 to 2,000 m off the Halley Bay Station (Weddell Sea) are investigated. Representatives of about 30 smallsized taxa of higher category are found, most of them belonging to the meiofauna. Loricifera are recorded for the first time for the Southern Ocean. At one of the stations a maximum of 22 taxa occur, the mean number of taxa ranges from 7 to 16. Nematoda, Harpacticoida, Ostracoda, Polychaeta and Bivalvia are present at all sampling sites. Nematodes are always dominant representing more than 90% of the individuals per sample, followed by harpacticoids (3%) and kinorhynchs (1.2%). Important fractions of the meiofauna (an average of more than 50%) occur in strata below the top 0–1 cm layer. Maximal density is 3,800 individuals (10 cm^–2), the mean abundance per station ranges from 790 to 3,720 individuals (10 cm^–2) and the overall mean is 1,700 individuals (10 cm^–2). Multivariate analysis (TWINSPAN, Cluster analysis, DCA) discriminates between three communities which are correlated with depth and sediment characteristics: the near shelf-ice, the slope and the deep-sea communities.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

An immunofluorescence study of the effects of ultraviolet radiation on the organization of microfilaments, keratin intermediate filaments, and microtubules in human keratinocytes.

Indirect immunofluorescence microscopy has been used to investigate the ultraviolet (UV) radiation induced disruption of the organization of microfilaments, keratin intermediate filaments, and microtubules in cultured human epidermal keratinocytes. Following irradiation, concurrent changes in the organization of the three major cytoskeletal components were observed in cells incubated under low Ca2+ (0.15 mM) conditions. UV irradiation induced a dose-dependent condensation of keratin filaments into the perinuclear region. This collapse of the keratin network was accompanied by the reorganization of microfilaments into rings and a restricted distribution of microtubules, responses normally elicited by exposure to high Ca2+ (1.05 mM) medium. The UV induced alteration of the keratin network appears to disrupt the interactions between keratin and actin, permitting the reorganization of actin filaments in the absence of Ca2+ stimulation. In addition to the perinuclear condensation of keratin filaments, UV irradiation inhibits the Ca2+ induced formation of keratin alignments at the membrane of apposed cells if UV treatment precedes exposure to high Ca2+ medium. Incubation of keratinocytes in high Ca2+ medium for 24 hours prior to irradiation results in the stabilization of membrane associated keratin alignments and a reduced susceptibility of cytoplasmic keratin filaments to UV induced disruption. Unlike results from investigations with isogenic skin fibroblasts, no UV induced disassembly of microtubules was discernible in irradiated human keratinocytes.     

Micronuclei in human lymphocytes irradiated in vitro or in vivo

Venous blood from healthy donors or from patients with various lympho- and myeloproliferative diseases was incubated in vitro in the presence of cytochalasin B for the induction of binucleated lymphocytes. The time at which cytochalasin B was added depended on the proliferation rate of the lymphocytes. Proliferation was monitored using a semiautomatic microscope photometer/computer system. The background level of micronuclei in binucleated lymphocytes of the patients before radiotherapy was statistically indistinguishable from that of healthy persons. Blood from both groups was irradiated in vitro for the study of the dose-response relationship. The dose-response curves were very similar up to 3.75 Gy, and a somewhat lower micronucleus frequency was found in lymphocytes of patients after a 5-Gy exposure. These in vitro results were compared with in vivo exposure after total-body irradiation of leukemic patients. Due to heavy medication that accompanied radiation therapy, only two doses (1.25 and 2.5 Gy) could be checked after in vivo exposure. There was no statistically significant difference between in vitro and in vivo results after 1.25 Gy, but a slightly lower number of micronuclei was observed after in vivo exposure to 2.5 Gy.     

The effects of beta-mercaptoethanol and sodium dodecyl sulfate on the Humicola insolens beta-glucosidase

Hydrolysis of p-nitrophenyl-beta-D-glucoside by the beta-glucosidase of a thermophilic and cellulolytic fungus, Humicola insolens was stimulated by two-fold in the presence of high concentrations of beta-mercaptoethanol. This enzyme did not have any free sulfhydryl groups and high concentrations of beta-mercaptoethanol (5% v/v) reduced all of the three disulfide bonds present in the enzyme. In contrast, the hydrolysis of cellobiose and cellulose polymers was inhibited by 50% under the same conditions. Sodium dodecyl sulfate (1% w/v) even in combination with beta-mercaptoethanol did not show any significant effects on this enzyme. These unusual properties suggest that this enzyme may be of significant importance for understanding the structure of the enzyme.     

Viral proteases as targets for chemotherapeutic intervention

Many viruses encode proteinases that are essential for infectivity, and are consequently attractive chemotherapeutic targets. The biochemistry and structure of the human immunodeficiency virus proteinase have been characterized extensively, and potent peptide-mimetic inhibitors have been developed. Techniques and strategies used to improve the efficiency of these compounds are likely to be applicable to other viral proteinases.     

Monoclonal antipeptide antibodies to the glucocorticoid receptor.

The generation of monoclonal antibodies to synthetic peptides of the glucocorticoid receptor is described. Two antibodies to sequences from the DNA binding region are IgMs. Two other antibodies to sequences in the steroid binding region and the C-terminus belong to the IgG class. The specificity of the IgG binding to the receptor in an ELISA assay is demonstrated by competition with the relevant peptides. Both IgGs are able to recognize the receptor in Western blots, but do not form stable complexes in sucrose gradients. Steroid binding to the receptor is not influenced by preincubation with antibodies. This indicates that denaturation or distortion of the receptor is necessary for the accessibility of these antibodies to their epitopes. Both antibodies can be used to stain the glucocorticoid receptor in neoplastic cells of patients suffering from chronic lymphatic leukemia.     

The N-Myc oncoprotein is associated in vivo with the phosphoprotein Max(p20/22) in human neuroblastoma cells.

Proteins encoded by the proto-oncogenes c-myc, L-myc, and N-myc contain at their carboxy-terminus a tripartite segment comprising a basic DNA binding region (BR), a helix-loop-helix (HLH) and a leucine zipper motif (Zip), that are believed to be involved in DNA binding and protein-protein interaction. The N-Myc oncoprotein is overexpressed in certain human tumors that share neuroectodermal features due to amplification of the N-myc gene. Using a monoclonal antibody directed against an N-terminal epitope of the N-Myc protein in immunoprecipitations performed with extracts of neuroblastoma cells, two nuclear phosphoprotein, p20/22, forming a hetero-oligomeric complex with N-Myc are identified. Both proteins are phosphorylated by casein kinase II in vitro. By partial proteolytic maps we show that p20 and p22 are structurally related to each other and that p20 is identical with Max, a recently described in vitro binding partner of myc proteins. Time course experiments show the presence of the complex in cellular extracts immunoprecipitated within a 5 min interval after the preparation of the cell extract. While the expression of N-myc is restricted, expression of both Max(p20/22) and the murine homolog Myn(p20/22) was observed in cells of diverse human and murine embryonal lineages as detected by heterologous complex formation. By introduction of expression vectors containing the wild type N-myc gene or N-myc genes with in frame deletions or point mutations into recipient cells and subsequent immunoprecipitation of the resulting N-Myc proteins we show that the HLH-Zip region is essential to the formation of the N-Myc-p20/22 complex.