Expression and processing of human preprogastrin in murine medullary thyroid carcinoma cells.

Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.

Molecular pathogenesis of human immunodeficiency virus infection.

Molecular studies of the pathogenesis of human immunodeficiency virus (HIV) infections have proceeded rapidly following the molecular cloning and nucleotide sequence analysis of the HIV genome. Correlation of biochemical and functional studies of HIV-infected cells with the HIV nucleotide sequence has allowed the identification and preliminary functional characterization of many HIV proteins. These include structural proteins (gag), viral enzymes (pol), and viral regulatory proteins (tat, art). Cloned HIV DNA segments have been utilized as probes for in situ nucleic acid hybridization to study the distribution of HIV-infected cells in acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC) patients. These studies have demonstrated the infection of macrophages as an important component of HIV-induced neurologic disease. Only very low numbers of HIV-infected lymphocytes can be identified in the peripheral blood of infected individuals. Thus, the mechanism of CD4 cell depletion in the pathogenesis of AIDS remain obscure.

Transcription of novel open reading frames of AIDS retrovirus during infection of lymphocytes.

The retrovirus frequently isolated from patients with the acquired immune deficiency syndrome (AIDS) has two novel open reading frames previously designated "A" and "B." The "A" region was found to be specifically expressed as polyadenylated RNA's of 5.5 and 5.0 kilobases in infected cells. The "B" region was expressed as 1.8- to 2.0-kilobase RNA species. Additional full-length and spliced messenger RNA's of the env region were also identified.

Separation and characterization of the neoplastic and stromal elements of the R3230AC mammary adenocarcinoma.

The purification of the various types of cells in solid tumors is necessary for study of their biochemical and immunological functions. The R3230AC adenocarcinoma contains both a variety of stromal cells and well-differentiated neoplastic cells that possess many of the biosynthetic capabilities of normal rat mammary cells. Suspensions of cells from tumors weighing less than 1.2 g consisted of 70.4 +/- 7.2% (S.D.) malignant cells by morphology, 6.8 +/- 3.0% lymphocytes, 6.3 +/- 3.2% macrophages, 15.2 +/- 5.2% red blood cells, 0.8 +/- 0.5% granulocytes, and 0.5 +/- 0.6% unidentified cells. Sedimentation of the cells from the R3230AC tumor in a previously described isokinetic gradient resulted in a 5- to 6-fold purification of lymphocytes and macrophages. The modal fraction of malignant cells contained 95.3 +/- 2.9% malignant cells. Detection of alpha-lactalbumin by the direct peroxidase conjugate technique gave vacuolar staining of malignant cells, in contrast to the indirect and peroxidase-antiperoxidase techniques which stained ducts from normal lactating mammary gland and a wide variety of cells without vacuoles in the tumor. The best fixative for frozen sections, paraffin-embedded tissue, and cell suspensions was 50% ethanol-50% acetone. The suspensions of tumor cells contained 14.4 +/- 9.4% cells with histochemically demonstrable alpha-lactalbumin. Squamous metaplasia was commonly observed in tumors than lactating rats.

The separation of human palatine tonsillar cells in the reorienting gradient zonal rotor: a large-capacity enrichment of plasma cells.

Velocity sedimentation by centrifugation in a previously described isokinetic gradient, at unit gravity, or with an elutriator has proved to be highly effective as a method for the enrichment of many kinds of cells. For many biochemical and immunological purposes, the available techniques for the separation of cells have been incapable of separating a sufficient number of cells. With a newly designed rotor, we have separated more than twenty-fold more human tonsillar cells that can be separated in the previously described isokinetic gradient. Without exceeding the band capacity, we separated 692 million tonsillar cells. Plasma cells were more than seven-fold enriched from human tonsillar cells. When 692 million tonsillar cells were separated in the rotor, the five most enriched, contiguous fractions of plasma cells contained 44 million cells. The most enriched fraction contained 27% plasma cells. We are not aware of any method for velocity sedimentation which will separate such large numbers of tonsillar cells.