Pre-Golgi degradation of yeast prepro-alpha-factor expressed in a mammalian cell. Influence of cell type-specific oligosaccharide processing on intracellular fate.

We demonstrate that expression of yeast prepro-alpha-factor in GH3 rat pituitary cells results in its degradation in an endoplasmic reticulum (ER) or early Golgi compartment, suggesting that this wild-type prohormone is recognized as abnormal or misfolded in the context of a mammalian ER. In GH3 cells, as in yeast, the nascent polypeptide is efficiently targeted to the ER, where it undergoes cleavage of its amino-terminal signal peptide and core glycosylation to form glycosylated pro-alpha-factor (gp alpha f). Subsequently, however, this species disappears from cells with a half-time of 25-30 min (including a 10-20-min lag), and no alpha-factor- or proregion-derived products can be detected.

Purification of recombinant human secretory phospholipase A2 (group II) produced in long-term immobilized cell culture.

Recombinant human secretory phospholipase A2 (Group II) was expressed in long-term culture of immobilized Chinese hamster ovary cells utilizing a continuous-perfusion airlift bioreactor. The bioreactor was continuously perfused with cell-culture medium supplemented with 5% fetal calf serum at an average flow rate of 5 liters/day for 30 days. Recombinant phospholipase A2, at concentrations ranging from 100 to 500 micrograms/liter, was purified to apparent homogeneity by an efficient two-step procedure involving a silica-based cation-exchange resin and hydrophobic interaction chromatography (greater than 65% recovery of phospholipase A2).

Induction of phospholipase A2 gene expression in human hepatoma cells by mediators of the acute phase response.

Serum levels of phospholipase A2 (PLA2) activity have been shown to be elevated in cases of septic shock and rheumatoid arthritis. The cellular origin of serum PLA2, however, is not known. In this report, we demonstrate that human group II PLA2 expression and secretion are induced in hepatoma cells (HepG2) following treatment with interleukin-6 (IL-6), tumor necrosis factor (TNF), and interleukin-1 (IL-1).

Human immunodeficiency virus type 1 protease cleaves the intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein.

The intermediate filament proteins vimentin, desmin, and glial fibrillary acidic protein are cleaved in vitro by human immunodeficiency virus type 1 protease (HIV-1 PR). Microsequencing showed that HIV-1 PR cleaved both human and murine vimentin between leucine-422 and arginine-423 within the sequence between positions 418 and 427, Ser-Ser-Leu-Asn-Leu/Arg-Glu-Thr-Asn-Leu (SSLNL/RETNL). Minor cleavages at other sites were also observed.

Retrovirus-mediated expression of preprosomatostatin in rat pituitary GH3 cells: targeting of somatostatin to the regulated secretory pathway.

Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, prepro-SRIF, consisting of a signal peptide and a proregion of 80-90 amino acids; mature SRIF is located at the carboxyl-terminus of the precursor. We have used a recombinant retroviral expression vector encoding anglerfish prepor-SRIF-I to infect rat pituitary GH3 cells. The aim of these studies was to investigate the intracellular storage and secretion of the total pool of endogenous GH compared to that of SRIF.

The propeptide of preprosomatostatin mediates intracellular transport and secretion of alpha-globin from mammalian cells.

We have investigated the role of the somatostatin propeptide in mediating intracellular transport and sorting to the regulated secretory pathway. Using a retroviral expression vector, two fusion proteins were expressed in rat pituitary (GH3) cells: a control protein consisting of the beta-lactamase signal peptide fused to chimpanzee alpha-globin (142 amino acids); and a chimera of the somatostatin signal peptide and proregion (82 amino acids) fused to alpha-globin. Control globin was translocated into the endoplasmic reticulum as determined by accurate cleavage of its signal peptide; however, alpha-globin was not secreted but was rapidly and quantitatively degraded intracellularly with a t 1/2 of 4-5 min.

The role of paired basic amino acids in mediating proteolytic cleavage of prosomatostatin. Analysis using site-directed mutagenesis.

Many small peptide hormones are synthesized as larger precursors in which the mature hormone sequence is flanked by pairs of basic amino acids. These precursors often undergo extensive post-translational modifications; a critical step in this process is proteolytic excision of the hormone at the paired basic residues. To determine the role of paired basic amino acids as recognition signals for cleavage by processing enzymes, we investigated the heterologous expression of prosomatostatin (the pro-somatotropin release inhibiting factor (pro-SRIF).

Retrovirus-mediated expression of preprosomatostatin: posttranslational processing, intracellular storage, and secretion in GH3 pituitary cells.

Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, preproSRIF, consisting of a signal peptide and a proregion of 80-90 amino acids. The mature hormone, which is located at the carboxyl terminus of the precursor, is preceded by a single pair of basic amino acids. We are studying preproSRIF to investigate intracellular sorting, proteolytic processing, and storage of peptide hormone precursors in the secretory pathway.

DNA base composition of filarial nematodes.

We have determined the molar content of guanine + cytosine (GC content) of DNA of the filarial nematode (Brugia malayi, Brugia pahangi and Dirofilaria imitis) and of the free-living soil nematodes Caenorhabditis elegans and have analysed the DNA for the presence of methylcytosine. Two independent methods, thermal denaturation and direct analysis of base content by HPLC following enzymatic hydrolysis, reveal that the GC content of filarial nematodes is 26-28%. We have been unable to find methylcytosine in the DNA of B.