Improved clonal and nonclonal growth of human,rat and bovine adrenocortical cells in culture

Summary This report describes the development of a culture system for long-term growth and cloning of human fetal adrenocortical cells. Optimal conditions for stimulating clonal growth were determned by testing the efficacy of horse serum (HS), fetal bovine serum (FBS), fibroblast growth factor (FGF), epidermal growth factor (EGF), fibronectin, and a combination of growth factors, UltroSer G, in stimulating growth from low density. Optimal conditions for clonal growth were achieved using fibronectin-coated dishes and DME/F12 medium with 10% FEBS, 10% HS, 2% UltroSer G, and 100 ng/ml FGF or 100 pM EGF. Conditions for growth at clonal density were found to be optimal for growth of early passage, nonclonal cultures at higher densities. The improved growth conditions used for cloning were shown to allow continued long-term growth of nonclonal human adrenocortical cells without fibroblasts overgrowth. All cells in cultures grown in HS, FBS, and UltroSer G had morphologic characteristics of adrenocortical cells, whereas cells grown in FBS only rapidly became overgrown with fibroblasts. Clonal and nonclonal early passage human adrenocortical cells had smilar mitogenic responses to FGF and EGF. Whereas FGF, EGF, and UltroSer G showed similar stimulation of DNA synthesis and clonal growth in human adrenocortical cells and human adrenal gland fibroblasts, the tumor promoter 12-O-teradecanoylphorbol-13-acetate stimulated growth only in adrenocortical cells and was strongly inhibitory to growth in fibroblasts. In both cell types, forskolin inhibited DNA synthesis. Human adrenocortical cell cultures were functional and synthesized cortisol, dehydroepiandrosterone, and dehydroepiandrosterone sulfate. The improved growth conditions for clonal growth of human adrenocortial cells also provided optimal conditions for long-term growth of cultured rat adrenocortical cells and ncreased the cloning efficiency of cultured bovine adrenocortical cells. This work was supported by Research grants AG-00936 and AG-06108 from the National Institute on Aging, Bethesda, MD.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B₄ and C₄. After 3–4 min, the levels of LTB₄ and LTC₄ were 93 and 35 pmol/310⁷ cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC₄ were 8 and 20 times lower than those of LTB₄ after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

The Influence of Feeding and Oral Rehydration on the Bioavailability of Oxytetracycline in Calves

The influence of feeding on the bioavailability of oxytetracycline was studied in preruminant calves. Oxytetracycline was given in water as a drench to fasting calves or was mixed in the milk replacer. Compared to water the bioavailability was significantly reduced (53.5%) when oxytetracycline was mixed in the milk re-placer. A further reduction, 83.3 %, occurred when the calves were treated one hour post milk feeding. Also concentrate was found to reduce the bioavailability. Very high serum levels were recorded when the drug was given in an oral rehydration solution, pH 4.9, containing glycine. The values obtained when an alkaline (pH 8.3) solution without glycine was used did not differ from the levels recorded when oxytetracycline was given in water. It was suggested that the use of oxytetra-cyclines in feeds may be questioned because of their well-known complex forming ability.     

Suproxide dismutase in human testis preparations

A protein fraction from human testis was structurally investigated. The main component of the fraction reported to contain inhibin-like activity was purified and analyzed by tryptic digestion. The peptides obtained identified the protein as an enzyme, superoxide dismutase, previously known to be present in seminal plasma. The results show that superoxide dismutase is a major enzyme, also of testicular material. They further demonstrate the importance of using pure fractions, and controls such as checks with structural analysis or synthetic peptides, in the work of elucidating the nature of inhibin and other hormonal peptides.     

Electrophoretic behavior of the H+-ATPase proteolipid from bovine heart mitochondria

The proteolipid subunit of H⁺-ATPase was labeled by [¹⁴C]N,N-dicyclohexylcarbodiimide in bovine heart mitochondria. The radioactive labeling was followed using various systems of sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). When using discontinuous SDS-PAGE (Laemmli, U.K., 1970,Nature (London)227, 680–685) a monomeric (Mr 7600±1500) and a dimeric form (Mr 17,800±1200) of the proteolipid were detected, while only the monomeric form was found on urea (8 M) containing gels (SDS-PAGE according to Laemmli; or Swank, R. T., and Munkers, K. D., 1971,Anal. Biochem. 39, 462–477). When using SDS-PAGE with Na-P_i buffer (Weber, K., and Osborn, M., 1969,J. Biol. Chem. 244, 4406–4442), only a dimeric form of the proteolipid (Mr 15,000±1000) was detected. Experimental data indicate that the different patterns of proteolipid separation are related to the presence of the two distinct proteolipid conformations in the SDS solution.     

Reproductive strategies of the orang-utan: New data and a reconsideration of existing sociosexual models

Recent field data indicate that MacKinnon’s model of the orang-utan’s sexual and agonistic activity needs to be revised. In this model, male reproductive activity is concentrated in an extended phase of subadulthood and in early adulthood. According to this model, the role of older adult males is primarily that of range guardian, and in that role they would ensure that the offspring they had generated earlier would have safe access to food resources. This study presents cases suggesting that subadult males, even though sexually active, may have low reproductive success. In previous studies adult males were shown to display less sexual initiative than subadult males. In this study an adult male was at times involved infrequent mating activity in response to proceptive activity of females in the course of consortship. This adult male proved to be a successful breeder, thus refuting the hypothesis of adult male sterility. The female is most likely to conceive through cooperative mating in lengthy consortships with the dominant resident adult male. We hypothesize that the extended subadult phase represents a submissive strategy, allowing subadult males to remain in the home range of adult males but with minimal reproductive success.     

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.     

Protein composition of the carboxysomes of Thiobacillus neapolitanus

For purifying carboxysomes of Thiobacillus neapolitanus an isolation procedure was developed which resulted in carboxysomes free from whole cells, protoplasts and cell fragments. These purified carboxysomes are composed of 8 proteins and at the most of 13 polypeptides. The two most abundant proteins which make up more than 60% of the carboxysomes, are ribulose-1,5-bisphosphate carboxylase and a glycoprotein with a molecular weight of 54,000. The shell of the carboxysomes consists of four glycoproteins, one also with a molecular weight of 54,000. The other proteins are present in minor quantities. Ribulose-1,5-bisphosphate carboxylase is the only enzyme which could be detected in the carboxysomes and 3-phosphoglycerate was the only product formed during incubation with ribulose-1,5-diphosphate and bicarbonate. The supernatant of a broken and centrifuged carboxysome suspension contained the large subunit of ribulose-1,5-bisphosphate carboxylase. The small subunit of ribulose-1,5-bisphosphate carboxylase was found in the pellet together with the shell proteins which indicates that the small subunit of ribulose-1,5-bisphosphate carboxylase is connected to the shell.Abbreviations RuBisCO ribulose-1,5-bisphosphate carboxylase - PMSF phenylmethylsulfonyl fluoride - PAA gelectrophoresis, polyacrylamide gelelectrophoresis - SDS sodium dodecyl sulphate - CIE crossed immunoelectrophoresis - IEF isoelectric focusing     

Monoclonal antibody-defined antigens of human prostate cancer cell line PC3

Summary Over 600 hybridomas were derived from the immunization of mice with live cells and aqueous extracts of the human prostatic carcinoma cell line PC3. A total of 26 hybridomas with restricted reactivities were selected, subcloned and antibodies tested on a variety of tumor and normal cells. Seven monoclonal antibodies showed reactivity for prostate cancer and other tumor cell lines, including breast carcinomas. Three of the antibodies obtained after immunization with live cells reacted with live cells only and three of the four antibodies obtained after immunization with cell extract reacted with cell extracts and spent culture media. The fourth antibody in the latter group was reactive only in the immunoperoxidase staining assay. Antibody PrS5 recognized a 90,000 molecular weight molecule from ¹²⁵I-surface-labeled cells in immunoprecipitation analysis. Antibodies PrE3 and PrD8 detected a nonacid glycolipid pentasaccharide from PC3 cells and meconium, and a glycoprotein of 115,000 molecular weight from ¹²⁵I-surface-labeled red blood cells. The similar patterns of reactivity in RIAs and antigen analysis suggest that antibodies PrE3 and PrD8 recognize the same molecule. The results emphasize the usefulness of immunohistochemistry in the testing of monoclonal antibodies and the impact of the form in which the antigen is presented on the resultant antibody specificity