Cloning of genes for bacterial glycosyltransferases. I. Selection of hybrid plasmids carrying genes for two glucosyltransferases.

A method of identifying plasmids containing genes responsible for synthesis of nucleotide sugar:lipopolysaccharide glycosyltransferases is described. Hybrid ColE1 plasmids containing random fragments of the chromosome of Escherichia coli K12 were introduced into an indicator strain of Salmonella typhimurium which lacks UDP-glucose:lipopolysaccharide glucosyltransferase I due to an rfaG mutation. Plasmids capable of correcting the transferase defect were identified by their ability to convert the bacteriophage sensitivity pattern of the recipient strain from Ffm-sensitive to Ffm-resistant. Analysis of the lipopolysaccharide of the S. typhimurium/ColE1 hybrid strains and assay of cell extracts defined the new enzyme activities. Two plasmids were identified which carried the rfaG+ gene; one of these plasmids also contained genetic information for a second glucosyltransferase, the E. coli glucosyltransferase II, which normally is not present in S. typhimurium.     

Metabolic behavior of acetyl glyceryl ether phosphorylcholine on interaction with rabbit platelets

The metabolic fate of 1-O-[³H]alkyl-2-acetyl-sn-glycero-3-phosphorylcholine ([³H]-AGEPC) upon interaction with rabbit platelets was investigated. [³H]AGEPC was converted to a product identified as the long-chain fatty acyl analog. The reaction was unaffected by extracellular calcium. After a lag time of 30 to 60 s the kinetics of the conversion was linear. The rate of the reaction was found to be a function of platelet and AGEPC concentrations. Of the [³H]AGEPC (10^?9m) 85 ± 5% was processed into the-long chain fatty acyl analog within 1 h when incubated at 37 2C with a 1.25 × 10⁹ platelets per milliliter suspension. A maximal number of 1200 to 3600 [³H]AGEPC molecules were converted to the long-chain fatty acyl derivative per minute per platelet in the presence of 2 mm EDTA. Under similar conditions the 1-O-[³H]alkyl-2-(lyso)-sn-glycero-3-phosphorylcholine ([³H]lysoGEPC) also was transformed to a comparable long-chain fatty acyl derivative at a much slower rate and to a lower extent. No significant increase in lysoGEPC was noted in incubation mixtures containing [³H]AGEPC. The possible direct transacylation of AGEPC upon interaction with platelets is discussed as well as the possible involvement of this reaction in directly triggering the platelet response to AGEPC stimuli.     

Effect of primycin on some electric properties of the frog skeletal muscle

The effect of primycin, a guanidine-type antibiotic was studied on the electric properties and 42K+ uptake of the frog sartorius and semitendinosus muscle. Both in normal and choline chloride Ringer solution, primycin evoked a concentration and time dependent depolarization of the surface membrane of the muscle. This depolarization was significantly increased by Na ions. Primycin treatment was shown to evoke a dose-dependent decrease of the depolarization induced by 20 mM K+-Ringer. When the muscles were incubated in a Ringer solution containing choline chloride, during an incubation period of 30 min the uptake of 42K+ was decreased to 12% upon the exposure to 5 x 10(-6) mol primycin as compared to the control value. As the primycin-induced depolarization increased, the shape and amplitude of the action potentials elicited by square-wave electric impulses were altered and decreased, respectively. In sodium isaethionate Ringer 1--2 x 10(-6) M primycin induced a slow depolarization resulting in firing potentials. The results suggest that primycin depolarizes the surface membrane exclusively through the blockade of the resting K+ channels, the other phenomena being the results of this depolarizing effect.     

Monoclonal antibodies to an artificial immunogen, specifically demonstrating phosphotyrosine-containing proteins

The importance of protein phosphorylation at tyrosyl hydroxy groups in the control of cell proliferation has recently been established. For identification of tyrosine-phosphorylated proteins, monoclonal antibodies (Mabs) against artificial immunogens containing O-phosphotyrosine (pTyr) or tripeptide pTyr-Gly-Gly as haptens were generated; the haptens were coupled to carrier proteins (bovine serum albumin, human immunoglobulin, keyhole limpet hemocyanin). After immunization of mice with pTyr coupled to keyhole limpet hemocyanin, Mabs were generated which were highly specific for pTyr and did not cross-react with O-phosphoserine, O-phosphothreonine, tyrosine or nucleoside-5'-monophosphates. The Mabs specifically react with tyrosinephosphorylated proteins in the Rous sarcoma virus-transformed rat XC-cell.     

On the spontaneous adherence of myelin basic protein to T lymphocytes.

We have applied a double tagging system in order to study whether purified myelin basic protein is able to adhere to normal human peripheral T lymphocytes without the need to purify cells. Evaluation of myelin basic protein adherence to peripheral blood mononuclear cells was determined with biotinylated myelin basic protein and fluoresceinated avidin, and lymphocyte population was identified by the corresponding phycoerythrinated monoclonal antibody. The observed adherence of myelin basic protein to T lymphocytes was found to depend on protein conformation.     

Prolactin messenger ribonucleic acid concentrations throughout the ovine estrous cycle: assessment relative to prolactin serum and pituitary amounts

Prolactin (PRL) mRNA concentrations were assessed by nucleic acid hybridization assays in pituitaries of ewes representing the defined stages of the ovine estrous cycle. Concomitantly, pituitary and serum PRL concentrations were measured in these ewes using radioimmunoassays. It was observed that PRL serum, pituitary and mRNA concentrations tended to increase near the time of the gonadotropin preovulatory surge, particularly between 24 hrs before behavioral estrus to 5 hours after estrus. However, the changes in PRL mRNA, serum and pituitary concentrations were shown not to be statistically significant. These data suggest that PRL production during the sheep estrous cycle is maintained without dramatic changes in synthesis or secretion.