Effects of Choline and Quiescence on Drosophila Choline Acetyltransferase Expression and Acetylcholine Production by Transduced Rat Fibroblasts

Rat-1 fibroblasts were transduced to express Drosophila choline acetyltransferase. The presence of an active enzyme in these cells (Rat-1/dChAT) was confirmed using various methods. Rat-1/dChAT fibroblasts released acetylcholine (ACh) into the culture medium. Moreover, intra- and extracellular levels of ACh could be increased by adding exogenous choline chloride. In addition, serum starvation or confluence-induced quiescence caused an 80% decrease in recombinant choline acetyltransferase activity (compared with actively growing cells). ACh release was also repressed in quiescent fibroblast cultures. Exogenous choline could mitigate the decrease in ACh release. These results indicate that Rat-1 fibroblasts can be genetically modified to produce ACh and that ACh release can be controlled by introducing choline into the culture medium. Furthermore, these data demonstrate that the expression of the retroviral promoter used in this study decreases with the onset of quiescence; however, exogenous choline can increase the amount of ACh released by quiescent fibroblasts.     

Inducible gene expression by retrovirus-mediated transfer of a modified tetracycline-regulated system.

The ability to regulate gene expression via exogenous stimuli will facilitate the study of gene functions in mammalian cells. In the present study, we modified the tetracycline-controlled inducible system by the addition of the ligand-binding domain of the estrogen receptor to the carboxy terminus of the tTA transactivator. A single retroviral vector can transduce both the transactivator gene and the gene of interest controlled by the tTA-inducible promoter into mammalian cells. We show that cell lines expressing the transactivator can readily be established and that expression of the gene of interest depends on the removal of tetracycline and the addition of estrogen. By using this system, cell lines with inducible expression of the G protein of vesicular stomatitis virus, a potentially toxic gene product, were established. The combination of a powerful inducible system and retrovirus-mediated gene transfer can not only be used to study gene function but may also be applied in the future to clinical trials in human gene therapy.     

Practical Procedures for the Purification of Bacterial Viruses

The efficiencies of the various methods used for phage concentration have been compared. The two-phase concentration method (with polyethylene glycol and dextran sulfate) gave maximal recoveries of infectivity for coliphages of the T-even and T-odd series and for ribonucleic acid phages and single-stranded deoxyribonucleic acid phages. Precipitation of phages by acid gave high yields when applied to T2 and T4 phages but not with T3 and T7 coliphages. Differential centrifugation was efficient when sedimented phages were gently dispersed before repeating the centrifugation cycle. The efficiencies of the various methods have also been confirmed by electron microscope studies, which also show that the two-phase concentration method gave rise to intact phages. Zone centrifugations in sucrose gradients (12.5 to 52.5%) indicated that coliphages of the T-even series sediment faster than T-odd coliphages; they may thus be separated from each other and from empty ghosts by centrifugation at 100,000 x g for 40 min. Equilibrium centrifugation in preformed cesium chloride gradients was also useful for phage concentration and purification. This study also deals with some optical properties of purified phages; optical cross sections and absorbance ratios (at 260 and 280 nm) of the various preparations are given.     

Polymorphism of the HLA DR1 haplotype in the Israeli population investigated at the serological,cellular, and genomic levels

In the present report, we used serological, cellular, and restriction fragment length polymorphism (RFLP) to investigate the DR1 haplotype in the Israeli population. We describe an Israeli homozygous typing cell (HTC), HLA-DwLVA, which defines a new lymphocyte-activating determinant associated with Bw65, DR1 and distinct from Dwl. The parents of this donor, non-Ashkenazi Algerian Jews, are first cousins and share HLA-Cw8, Bw65, BfS, DR1, DQw1, DPw4. No specificity could be assigned to HLA-DwLVA using the 91 Ninth Workshop HTCs. Two families and forty unrelated DR1 individuals were studied with DwLVA and a panel of DR1/Dw1 HTCs. HLA-DwLVA showed segregation as a single determinant within families. This new specificity was present in 24 out of 40 (60%) unrelated DR1 individuals, indicating that in the Israeli population DwLVA is the main lymphocyte-defined determinant associated with the serologically defined DRI specificity, in contrast to non-Jewish Caucasoids where DR1 is significantly associated with Dw1. The vast majority of DwLVA-positive carriers were also Bw65 carriers, indicating that Bw65, DR1, DwLVA may represent a typical allele combination in the Israeli population. The RFLP analysis established the correlation of certain RFLPs with Dw1 and DwLVA. In addition, we describe a cluster of RFLPs that may correspond to a new Dw subtype associated with DR1, for which no serological and cellular reagents have been described so far.     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

Characterization of the hormone-sensitive Ca2+ uptake activity of the hepatic endoplasmic reticulum

The characteristics and kinetics of calcium uptake activity were studied in isolated hepatic microsomes. The sustained accumulation of calcium was ATP- and oxalate-dependent. Glucagon increased microsomal Ca2+ uptake upon either in vivo injection, or in vitro perfusion of the hormone in the liver. In contrast, the effect of insulin depended on the route of administration. Calcium accumulation by subsequently isolated hepatic microsomes increased when insulin was injected intraperitoneally whereas it decreased when the hormone was perfused directly into the liver. These effects of glucagon and insulin were dose dependent. When insulin was added to the perfusate prior to the addition of glucagon, insulin blocked the glucagon-stimulated increase in microsomal Ca2+ uptake. Cyclic AMP mimicked the effect of glucagon on microsomal Ca2+ accumulation when the cyclic nucleotide was perfused into the liver. The effects of glucagon and insulin on the kinetics of hepatic microsomal Ca2+ uptake were investigated. In microsomes isolated from perfused rat livers treated with glucagon the V of the uptake was significantly increased over the control values (12.2 vs. 8.6 nmol Ca2+ per min per mg protein, P less than 0.02). In contrast, the addition of insulin to the perfusate significantly decreased the V of Ca2+ uptake by subsequently isolated microsomes (6.8 vs. 8.3 nmol Ca2+ per min per mg protein, P less than 0.05). However, neither hormone had an effect on the apparent Km for Ca2+ (4.1 +/- 0.5 microM) of the reaction. The effect of these hormones on the activity of Ca2+-stimulated ATPase was also studied. No significant changes in either V or Km for Ca2+ of the enzymatic reaction were detected.