Characterization of messenger RNA from epimastigotes and metacyclic trypomastigotes of Trypanosoma cruzi

The cell-free translation products of polyribosomal and post-polyribosomal mRNAs from the non-infective epimastigotes and the infective metacyclic trypomastigotes of the parasitic protozoan Trypanosoma cruzi were compared by two-dimensional polyacrylamide gel electrophoresis. The result show that although many polypeptides are conserved, quantitative and qualitative differences are observed between both differentiation stages. The results also indicate the existence of post-polyribosomal mRNAs in equilibrium with polyribosomal counterparts. The immunoprecipitation of the in vitro synthesized polypeptides with chagasic human serum and the serum raised against an 85-kDa glycoprotein (P2-WGA), potentially involved in the process of T. cruzi penetration into mammalian cells, shows that while the chagasic serum recognizes the same 72-kDa, 68-kDa and 46-kDa polypeptides in both differentiation stages, the anti-P2-WGA serum immunoprecipitates a single 48-kDa polypeptide from in vitro translation products of metacyclic trypomastigotes.     

Measurements of the specific activity of the nucleoside triphosphate pool of sea-urchin embryos following 8-3H-guanosine administration

The nucleoside triphosphate (NTP) pool during the early development of sea-urchin, Paracentrotus lividus, was measured using high-performance liquid chromatography (HPLC). Each NTP pool was measured at four stages between the eight-blastomere stage and the early blastula stage, as was the specific activity of each NTP following an initial administration of 8-3H-guanosine. Unexpectedly high UTP (uridine-5'-triphosphate) concentrations and radioactivity levels in UTP were observed. Studies of the sea-urchin, Strongilocentrotus purpuratus, also revealed elevated levels of 8-3H-guanosine incorporation into UTP. The present procedure was found to be suitable for the unequivocal identification of UTP.     

GRF-induced feeding: Evidence for protein selectivity and opiate involvement

Growth hormone-releasing factor (GRF) is a hypothalamic peptide named for its ability to induce release of growth hormone from the anterior pituitary. GRF also acts as a neurotransmitter in the suprachiasmatic nucleus/medial preoptic area (SCN/MPOA) to stimulate food intake. The purpose of this series of experiments was to explore the nature of GRF-induced feeding, with a particular emphasis on macronutrient selectivity, and to examine the role of opiate activity in the paraventricular nucleus of the hypothalamus (PVN). Chow intake stimulated by GRF microinjection (1 pmol/0.5 μl) into the SCN/MPOA was blocked by injection of methyl-naltrexone (3 μg/0.5 μl) into the PVN. In animals habituated to macronutrient diets (Teklad, WI), GRF preferentially stimulated intake of protein at 2 and 4 h postinjection, whereas it had no effect on carbohydrate intake. Further, this effect was blocked by injection of naloxone (40 nmol/0.5 μl) into the PVN. Microinjection of morphine (0, 1, 10, and 17 μg/0.5 μl) into the PVN also specifically stimulated protein intake at 2 and 4 h postinjection. These results suggest that feeding derived from GRF actions in the SCN/MPOA is macronutrient selective, and is dependent on PVN opiate activity for expression.     

Human carcinoma cell lines xenografted in athymic mice: biological and antigenic characteristics of an intraabdominal model

Summary In order to investigate in vivo clinical applications of murine monoclonal antibodies directed against human ovarian carcinoma a preclinical in vivo model was developed using BALB/c athymic mice. Three human carcinoma cell lines (MCF7, HT29, and SW626) were injected into the peritoneal cavity of pristane-primed animals and the biological and antigenic characteristics of the i.p. grown tumors were studied. The animals were killed when moribund or 6–8 weeks after tumor injection. At autopsy tumor take was observed in 85% of the injected animals, whereas palpable nodules were evident in only 83%. Examination of the peritoneal cavity revealed intraabdominal carcinomatosis with tumor masses varying in size between 0.2 and 0.5 cm in diameter and tumor sheets. The most frequently affected organs were the diaphragm, the liver, and the reproductive system. Ascitic fluid formation was rare and no animal developed tumors outside the peritoneal cavity. To determine whether the in vivo tumors retained the same antigenic characteristics as the in vitro cell lines, four monoclonal antibodies (MBrl, MOv2, MOv8, and MOv15) directed against ovarian carcinoma-associated antigens and two different experimental approaches (immunofluorescence and immunoblotting) were used. Variations at either a quantitative or a qualitative level were observed for some antigens, whereas no evident changes were apparent for others. In particular, the antigens detected by MBr1 and MOv15 on the MCF7 line both maintained high levels of expression and immunoblotting staining pattern, whereas the antigens detected by MOv2 on the HT29 and SW626 lines, although present at a high level, clearly changed their staining pattern. As regards the antigens recognized by MOv8 and MOv15 on the HT29 and SW626 lines, we observed a drastic decrease in the level of their expression and in many cases a drop below the threshold of detectability of the test. The intraabdominal carcinomatosis described partially mimics the growth characteristics of human ovarian cancer and maintains the expression of some antigenic markers associated with epithelial tumors of the ovary and may therefore be useful in devising immunodiagnostic and/or immunotherapeutic strategies for ovarian carcinoma.     

Adoptive immunity in mice challenged with L1210/DTIC clones

Summary New antigenic specificities, not detectable on parental cells, have been induced by many investigators in mouse lymphomas by treatment with the antitumor agent 5(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC). The antigens are transmissible, after withdrawal of the drug treatment, as an inheritable character. The mechanism of induction, the molecular nature, and the number of the new antigenic specificities have not been completely elucidated. Four clones from murine leukemia L1210 isolated and expanded in vitro were treated in vivo with DTIC and the new sublines were studied in detail. The four drug-treated sublines studied exhibited strong immunogenicity since they were rejected by syngeneic animals. Immunosuppressed animals challenged with 10⁷ A/DTIC or P/DTIC cells were reciprocally protected by the adoptive transfer of spleen cells from donors that had rejected a lethal challenge of A/DTIC or P/DTIC clones. In a similar fashion, the adoptive transfer of spleen cells obtained from animals that had rejected the Q/DTIC or the R/DTIC clones protected immunosuppressed mice challenged with Q/DTIC or R/DTIC cells. No antitumor activity was observed in cross-protective schedules other than those indicated. It was been concluded that (a) the L1210 leukemia line does not have antigenic cells, (b) four DTIC-treated clone sublines were rejected by compatible hosts, and (c) two mutually exclusive sets of antigens were expressed in four antigenic clone sublines.Research supported in part by P.F.O. Contract Grant from C. N. R., Rome, Italy     

Neutral gene flow in the presence of a selected gene with random or assortative mating

Neutral gene flow in an island model in the presence of a gene subject to selection has been analysed. The selection regime on the gene is such that the maintenance of a stable interpopulation differentiation at this locus is allowed for very low values of the migration rate. Mating at this locus may be random or assortative. Many models of zygotic or prezygotic isolating mechanisms with unifactorial inheritance are represented by this genetic model. Two general solutions for migration rates tending toward zero have been obtained for the case of migration preceding selection and vice versa. It is found that the zygotic isolating mechanisms and one type of prezygotic isolating mechanisms have the same qualitative and quantitative effects for the same values of selection coefficients and of recombination frequencies between the selected gene and the neutral gene. A second type of prezygotic mechanism behaves in a different way: the reduction of gene flow caused by such mechanisms is also a function of the “mating coefficients” of the various genotypes.     

Biotransformation of mercury by bacteria isolated from a river collecting cinnabar mine waters

One hundred six strains of aerobic bacteria were isolated from the Fiora River which drains an area of cinnabar deposits in southern Tuscany, Italy. Thirty-seven of the strains grew on an agar medium containing 10g/ml Hg (as HgCl₂) with all of these strains producing elemental mercury. Seven of the 37 strains also degraded methylmercury. None of 106 sensitive and resistant strains produced detectable monomethylmercury although 15 strains produced a benzene-soluble mercury species. Two strains of alkylmercury (methyl-, ethyl- and phenylmercury) degrading bacteria were tested for the ability to degrade several other analogous organometals and organic compounds, but no activity was detected toward these compounds. Mercury methylation is not a mechanism of Hg resistance in aerobic bacteria from this environment. Growth of bacteria on the agar medium containing 10g/ml HgCl₂ was diagnostic for Hg detoxification based on reduction.     

In vivo assembly of chromatin on pBR322 sequences cloned into yeast plasmids

In order to study the in vivo assembly of chromatin on prokaryotic DNA templates, we have transformed yeast cells with plasmids pAJ50 and pRB58, which contain pBR322 sequences. In both cases nucleosomes are assembled in vivo on pBR322 DNA, although the nucleosomes are not homogeneous in size. To explore whether there is any preference for nucleosome assembly along pBR322 sequences, we have used an indirect end labeling method. The results indicate that most nucleosomes are placed at random on pBR322, although the probability for histone octamers to interact with some short regions is somewhat reduced. These regions coincide with sequences in which the frequency distribution of nucleosomes reconstituted in vitro (E. Caffarelli et al. (1988) Eur. J. Biochem. 171, 497-501) is low. Sequence determinants that direct chromatin assembly in vitro seem thereby to act to some extent in vivo.     

Genomic distribution of copia-like transposable elements in somatic tissues and during development of Drosophila melanogaster

The genomic distribution of elements of the copia, 412, B 104, mdg 1, mdg 4 and 1731 transposon families was compared by the Southern technique in DNA preparations extracted from brains, salivary glands and adult flies of two related Drosophila lines. The copia, 412 and mdg 1 sequences were also probed in DNA from sperm, embryos, and 1st and 2nd instar larvae. The homogeneity of the patterns observed shows that somatic transposition is unlikely to occur frequently. A correlation between mobility and the euchromatic or heterochromatic location of transposable elements is discussed. In addition, an explanation of the variable band intensities of transposable elements in Southern autoradiographs is proposed.