The human "peripheral-type" benzodiazepine receptor: regional mapping of the gene and characterization of the receptor expressed from cDNA.

A cDNA for the human "peripheral-type" benzodiazepine receptor (PBR) was isolated from a liver cDNA library. The 851-nucleotide probe hybridized with a approximately 1 kb mRNA in Northern blots of RNA extracted from various human tissues and cell lines. The human PBR probe was hybridized to DNA from a somatic cell hybrid mapping panel to determine that the gene maps to chromosome 22. With a regional mapping panel for chromosome 22, we localized the gene within band 22q13.31. The ligand-binding properties of the receptor expressed from the cDNA were examined in transient expression experiments and compared to the endogenous human PBR. The PBR ligand [3H]PK 11195 had high affinity for the expressed receptor in COS-1 cells, but the affinities of a pair of isoquinoline propanamide enantiomers differed remarkably in expressed and endogenous human PBR. These findings reveal that the host cell and/or post-translational modification may have an important influence on PBR function.     

Mutagenesis of the conserved 51-nucleotide region of Sindbis virus.

We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication.     

The control of O6-methylguanine-DNA methyltransferase (MGMT) activity in mammalian cells: a pre-molecular view

Human peripheral blood lymphocytes (PBLs) can have a range of O⁶-methylguanine-DNA methyltransferase (MGMT) activities. PBLs from some individuals may have almost no MGMT activity. Such individuals have most often been subject to malignancy or to immunodeficiency disease. Long-term lymphoblastoid lines (LCLs) prepared from PBLs of normal subjects by Epstein-Barr virus (EBV) transformation have MGMT activities which are in general somewhat higher than the PBLs from which they derive. Such cultures are therefore generally MGMT-positive. Only in rare cases, and generally from patients with low MGMT activity, are freshly obtained lines with very low activity obtained. There is however a 4-fold range of MGMT activity over which multiple lines derived from the same PBL sample can be found. Long-term cultivation can lead to LCLs with low activity as well as to lines of high activity. On rare occasions an MGMT-positive line may, within a few divisions, give a negative line. Some (but not all) MGMT-negative (or very low) lines have been known to gain (some) activity. Chinese hamster ovary (CHO) cell lines are in general very low in MGMT activity. Lines of higher activity can be selected by treatment with mutagenic crosslinking alkylating agents. Chinese hamster lines with high MGMT activity can be obtained by transfection with human DNA from MGMT-positive cells. Lines with significant activity can also be obtained by transfection of CHO cells with human DNA from MGMT-negative (or very low) cells. Resistance to MNNG treatment can be acquired without the acquisition of significant MGMT activity. Crosses of lines of high and low MGMT activity give equivocal results. Hybrids of low × low activity have no activity. Crosses of positive × positive strains give varied results. It has not been possible to identify MGMT-positive hybrids as including one particular chromosome by this type of experiment. There is no evidence for a general adaptive effect on MGMT synthesis greater than the variation within the cell cycle.     

The development of the U.S. EPA health effects research laboratory frozen blood cell repository program

We have suggested that proper blood-cell freezing and storage is necessary in longitudinal studies with reduced between tests error, for specimen sharing between laboratories and for convenient scheduling of assays. Our present purpose is to describe the design and use of a prototype computer program, Cryovial, for data-based management of frozen cell repositories. We are pleased to provide, free of charge upon request, a 5.25" (specify 0.36 or 1.2 MB) disk containing all files required to run Cryovial.     

Regulation of the gene for estrogenic 17-ketosteroid reductase lying on chromosome 17cen----q25

17-Ketosteroid reductase (17KSR), also known as 17 beta-hydroxysteroid dehydrogenase, catalyzes the reversible interconversion of estradiol to estrone and of androstenedione to testosterone. Using a recently cloned human placental 17KSR cDNA, we show that the 1.4-kilobase mRNA for this enzyme is detected only in tissues producing estrogens, and a 2.4-kilobase mRNA is detected in some estrogenic tissues and some androgenic tissues. This tissue distribution suggests that the interconversion of androstenedione and testosterone may be mediated by a different enzyme. Southern blotting studies show that the mRNA for this estrogenic 17KSR is encoded by two very similar genes localized to chromosome 17cen----q25 by analysis of DNA from mouse/human somatic hybrid cell lines. 8-Br-cAMP increases the abundance of estrogenic 17KSR mRNA as well as mRNAs for other steroidogenic enzymes in JEG-3 choriocarcinoma cells. By contrast, cAMP decreases estrogenic 17KSR mRNA in primary cultures of human cytotrophoblasts and human granulosa cells, a pattern of tropic regulation that differs from other steroidogenic enzyme mRNAs.     

DNA topoisomerase inhibitors block erythropoiesis and delay hemoglobinization in vitro

To examine the importance of topological constraints on DNA during erythroid development, we measured the effects of camptothecin and teniposide, two tumoricidal agents which are also specific inhibitors of type I and type II topoisomerases respectively, on the formation of hematopoietic colonies by cultured human bone marrow cells. When added to bone marrow culture, each inhibitor alone impairs the formation of early BFU-E-derived colonies, late CFU-E-derived colonies and mixed hematopoietic (CFU-GEMM-derived) colonies by up to 100%. Inhibition of colony formation is directly related to the time of inhibitor addition and the inhibitor concentration tested. Although either inhibitor alone reduces colony formation by 90%, when added together at a submaximal concentration, camptothecin and teniposide exert a synergistic suppressive effect. Furthermore, addition of topoisomerase inhibitors to culture impairs hemoglobinization of colony erythroblasts in a time-dependent fashion. In contrast to the effects of topoisomerase inhibitors, the antiproliferative agent aphidicolin reduces erythroid colony number and size without altering hemoglobinization of colony erythroblasts. Since neither topoisomerase inhibitor alters the morphology of cultured cells, the capacity of cells to exclude trypan blue or the potential to form erythroid colonies through the interval required for the first progenitor cell division, it is unlikely that camptothecin or teniposide are cytotoxic to hematopoietic cells. Human mononuclear cells enriched in bone marrow lymphocytes and nucleated erythroblasts from both human and mouse sources release DNA into the detergent soluble fraction. Release requires functional topoisomerases and is altered by acute exposure to topoisomerase inhibitors. Our results suggest that topoisomerases are critical not only to proliferation but also to differentiation of human marrow erythroid progenitor cells and stem cells in culture.     

Biosynthesis of human preproapolipoprotein A-II

The primary translation product of human apolipoprotein A-II was purified from wheat germ and ascites cell-free lysates programmed with RNA isolated from either a hepatocellular carcinoma cell line (HepG2) or intestinal epithelium. A-II mRNA represents 0.2% of the translatable RNA in these hepatocytes and in jejunal epithelium. Plasma high density lipoprotein-associated A-II is a 77-amino acid polypeptide. The primary translation product is 100 amino acids long and contains a 23-amino acid NH2-terminal extension. Cotranslational cleavage of the cell-free product indicated that this NH2-terminal sequence consists of an 18-amino acid long signal peptide, Met-Lys-Leu-Leu-Ala-Ala-X-Val-Leu-Leu-Leu-X-X-Cys-X-Leu-X-X-, and a 5-amino acid long propeptide, Ala-Leu-Val-Arg-Arg. This functional division was confirmed by sequencing the stable intracellular form of apolipoprotein A-II isolated from HepG2 cells. Approximately 45% of the proapo-A-II is cleaved to the mature form during export from HepG2 cells. The COOH-terminal dipeptide conforms to the rule that prosegments are cleaved after paired basic residues. We have previously shown (Gordon, J. I., Sims, H. F., Lentz, S. R., Edelstein, C., Scanu, A. M., and Strauss, A. W. (1983) J. Biol. Chem. 258, 4037-4044) that proapolipoprotein A-I is not cleaved during export from these cells and contains a prosegment with a COOH-terminal Gln-Gln dipeptide. Therefore, proteolytic processing of the two principal high density lipoprotein-associated apolipoproteins proceeds along different pathways.     

Uptake of lipoproteins by in situ perfused rat ovaries: identification of binding sites for high density lipoproteins

We have examined the uptake and distribution of 125I-labeled human high density lipoprotein, apolipoprotein E-free (hHDL3), 125I-rat high density lipoprotein (HDL), and human HDL (hHDL) reconstituted with [3H]cholesteryl linoleate after their in situ vascular perfusion to ovaries of gonadotropin-primed immature rats on days 6-9 post human chorionic gonadotropin (hCG)-injection. Some rats were treated with 4-aminopyrazolopyrimidine to reduce plasma lipoproteins and ovarian cholesteryl ester stores. Perfused ovaries were analyzed biochemically and autoradiographically, and progestin content of the ovarian effluent was quantified. Infusion of ovine luteinizing hormone and hHDL increased ovarian progestin secretion severalfold, indicating that the perfused ovary was functional. After perfusion with HDL reconstituted with [3H]cholesteryl linoleate, radioactive progestin appeared in the effluent; thus, sterol carried by exogenous HDL was converted to steroid. At 37 degrees C, uptake of 125I-hHDL3 was greatest after 15 min of perfusion with label. This was decreased by 80% when the perfusion was carried out at 4 degrees C and by 70-95% when excess unlabeled hHDL, but not human low density lipoprotein (hLDL), was included in the perfusate with 125I-hHDL. Aminopyrazolopyrimidine treatment enhanced 125I-hHDL uptake twofold. After perfusion for 15 min with 125I-hHDL3, radioactivity in the ovary was high for 3-30 min of HDL-free wash, then declined 75% by 30-60 min. With light and electron microscope autoradiography, 125I-hHDL3 was localized to corpora lutea, both along luteal cell surfaces and over their cytoplasm. The plasma membrane grains appeared to be associated with segments that lacked bristle coats. Perfusion with 125I-rat HDL produced a similar pattern of labeling. In ovaries perfused with 125I-BSA, silver grains were concentrated over macrophage-like cells but were sparse over luteal cells. We conclude that the in situ perfused rat ovary takes up 125I-hHDL3 by a temperature-dependent, lipoprotein-specific process, and that this lipoprotein is accumulated by luteal cells.     

Propagation of Autographa californica nuclear polyhedrosis virus in cell culture: Methods for infecting cells

Autographa californica nuclear polyhedrosis virus (AcNPV) produced in Trichoplusia ni (TN-368) cells was used to infect other cell cultures. Methods were developed to recover and obtain high titers of virus from infected cells for subsequent use as inocula. To release cell-associated nucleocapsids, the cells were lysed by sonication and freeze-thawing. The infectivity of enveloped nucleocapsids was greatly reduced by freeze-thawing, while sonication was not as detrimental. The titer of plaque-forming units (pfu) was reduced about 12-fold when passed through 0.45-μm filters. The virus and cells were manipulated to determine the most efficient methods for inoculating cells while yielding the highest numbers of polyhedra. The viral inocula may be left on cells during virus replication, and cells may be centrifuged at 380 g prior to exposure to virus without affecting the yield of polyhedra. The production of polyhedra is affected by cell density, and, of the densities tested, 7.65 × 10⁵ cells/ml yielded the maximum number of polyhedra per cell (142). However, the highest number of polyhedra per milliliter of culture (2.2 × 10⁸) was obtained with 3.8 × 10⁶ cells/ml. The numbers of polyhedra per cell did not vary when cells were taken from fermentor cultures at 0–144 hr and were infected with virus.