Enhanced retroviral transduction of 5-fluorouracil-resistant human bone marrow (stem) cells using a genetically modified packaging cell line.

Pluripotent hematopoietic stem cells (PHSC) are rare cells capable of multilineage differentiation, long-term reconstituting activity and extensive self-renewal. Such cells are the logical targets for many forms of corrective gene therapy, but are poor targets for retroviral mediated gene transfer owing to their quiescence, as retroviral transduction requires that the target cells be cycling. To try and surmount this problem we have constructed a retroviral producer line that expresses the membrane-bound form of human stem cell factor (SCF) on its cell surface. These cells are capable, therefore, of delivering a growth signal concomitant with recombinant retroviral vector particles. In this report we describe the use of this cell line to transduce a highly quiescent population of cells isolated from adult human bone marrow using the 5-fluorouracil (FU) resistance technique of Berardi et al. Quiescent cells selected using this technique were transduced by cocultivation with retroviral producers expressing surface bound SCF or with the parent cell line that does not. Following coculture, the cells were plated in long-term bone marrow culture for a further 5 weeks, before plating the nonadherent cells in semisolid media. Colonies forming in the semisolid media over the next 14 days were analyzed by polymerase chain reaction for the presence of the retroviral vector genome. Over six experiments, the transduction frequency of the quiescent 5-FU resistant cells using the SCF-expressing producer line averaged about 20%, whereas those transduced using the parent producer line showed evidence of reduced levels or no transduction.

Analysis of connections between nitric oxide synthase neurons in the myenteric plexus of the guinea-pig small intestine.

In the myenteric plexus of the guinea-pig ileum, a sub-population of descending interneurons contains nitric oxide synthase. Final neurons in descending motility pathways, inhibitory circular muscle motor neurons, also contain nitric oxide synthase. In this study we used ultrastructural immunocytochemistry to determine whether nitric oxide synthase descending interneurons provide inputs to all nitric oxide synthase neurons. The presence of nitric oxide synthase inputs to 35 nitric oxide synthase nerve cells from three animals was examined. Nine nerve cells from one ganglion were studied in serial section. Every nerve cell received inputs (close contacts and synapses) from nitric oxide synthase terminals. The number of inputs to the nine serially sectioned neurons ranged from 13 to 45. The inputs were found in about equal numbers on the cell bodies and the dendrites. There was no significant correlation between the size of nitric oxide synthase neurons and the number of nitric oxide synthase inputs they received. There was also no correlation between the number of nitric oxide synthase inputs and the number of 5-hydroxytryptamine inputs (determined in a previous study) received by nitric oxide synthase neurons. Random sections through an additional 26 nitric oxide synthase neurons (seven in the same ganglion and 19 from another two myenteric ganglia from different animals) were examined and nitric oxide synthase synapses and close contacts were observed on each neuron. Nitric oxide synthase interneurons and motor neurons are morphologically indistinguishable.(ABSTRACT TRUNCATED AT 250 WORDS)

Nitric oxide synthase is localized predominantly in the Golgi apparatus and cytoplasmic vesicles of vascular endothelial cells.

The localization of nitric oxide synthase (NOS) in vascular endothelial cells of submucosal blood vessels from the guinea-pig ileum was examined using NADPH diaphorase histochemistry at the light microscopic level, and endothelial NOS immunohistochemistry at the light and electron microscopic level. The pattern of staining observed following NADPH diaphorase histochemistry and endothelial NOS immunohistochemistry was identical. Endothelial cells of the arterioles, capillaries and venules showed small patches of intense, perinuclear staining. Under the electron microscope, endothelial NOS immunoreactivity was found predominantly in association with the Golgi apparatus and with the membranes of some vesicles. Small regions of the plasma membrane and the rough endoplasmic reticulum also showed some immunoreactivity. The presence of NOS in the Golgi apparatus and in vesicles raises the possibility that NOS may be exteriorized by endothelial cells, and hence that nitric oxide is synthesized extracellularly.

Polypeptide-metal cluster connectivities in Cd(II) GAL4.

Two-dimensional 1H-113Cd correlation NMR spectra have been used to determine the polypeptide/metal cluster connectivities in Cd(II) GAL4. The results show that the protein contains a two metal ion cluster where Cys-11 and Cys-28 are the bridging ligands.

Metal ion co-ordination in the DNA binding domain of the yeast transcriptional activator GAL4.

The structure of the DNA binding domain of the yeast transcriptional activator GAL4 was investigated by extended X-ray fine structure (e.x.a.f.s.). Two samples of GAL4 were studied, one containing cadmium as a structural probe (Cd(II)GAL4) and the other containing the 'native' zinc (Zn(II)-GAL4). The results suggest that the structure of the DNA binding domain of GAL4 contains a two metal ion cluster distinguishing it from the 'zinc finger' proteins typified by the Xenopus laevis transcription factor TFIIIA.

Fluid flow studies on vascular endothelium cultured on microporous membranes.

We have designed a two compartment system in which upper and lower compartments are separated by a monolayer of vascular endothelial cells grown on a porous membrane. The culture of pig aortic endothelial cells and their growth to confluence on porous PTFE membranes is described, and the principal characteristics of membrane-cultured cells are compared with those of similar cells cultured on solid surfaces. A decrease in fluid flow across the membrane-cultured cells was associated with their growth to apparent confluence: from low levels, fluid flow could be increased markedly by altering the environmental temperature of the cells. The possible uses and limitations of such a system in the interpretation of the role of endothelial cells in selective transport and compartmentation of fluids and cellular components of the blood are discussed.