Sch 20569 is a semisynthetic derivative of gentamicin with activity against many gentamicin-resistant gram-negative bacilli. We compared its bactericidal action with that of gentamicin and amikacin against 171 clinical isolates of Enterobacteriaceae, Staphylococcus aureus, and Pseudomonas aeruginosa. Sch 20569 and amikacin showed markedly greater activity than gentamicin against Escherichia coli, Klebsiella, Enterobacter, Citrobacter, and indole-positive Proteus, primarily by virtue of their lethal effect on gentamicin-resistant strains (minimal bactericidal concentration -12.5 ,g/ml). Indole-negative Proteus isolates were uniformly sensitive to Sch 20569, whereas several were resistant to both gentamicin and amikacin. Amikacin was most active against Providencia, as was gentamicin against Serratia. All three agents exhibited similar activity against Pseudomonas. Staphylococcus aureus was more sensitive to gentamicin and Sch 20569 than to amikacin.Sch 20569 is a new semisynthetic aminoglycoside derived from sisomycin, an antibiotic produced by the growth of Micromonospora inyoensis. In molecular structure, both Sch 20569 and sisomycin most closely resemble gentamicin C0a, a component of the gentamicin complex ( Fig. 1). Because we have encountered an increasing number of gentamicin-resistant gramnegative infections at the Manhattan V.A. Hospital (5), we studied the bactericidal efficacy of this new aminoglycoside and amikacin against both gentamicin-sensitive and -resistant clinical isolates. MATERIALS AND METHODSGroups of twenty isolates of Escherichia coli, Klebsiella, Enterobacter, Serratia, Proteus, Pseudomonas, and Staphylococcus aureus; and 11 strains of Citrobacter were chosen for comparative tube dilution sensitivities against gentamicin, amikacin, and Sch 20569. Minimal inhibitory and bactericidal concentrations (MIC and MBC, respectively) were assayed by tube dilution in 1-ml volumes of MuellerHinton broth, using each antibiotic in simultaneous tests against a single isolate. Based upon prior studies with aminoglycoside antibiotics (4), it was assumed that the constant cation content of a single lot of Mueller-Hinton broth would affect the activity of each antibiotic equally. The MBC was defined as that concentration which killed >99% of 105 organisms in 18 h as demonstrated by subculture onto drug-free solid media. The difference between MIC and MBC values was one or two tube dilutions in the majority of instances; thus, only the MBC values will be presented. Resistance to gentamicin or Sch 20569 is defined as an MBC > 12.5 ,ug/ml; amikacin resistance is defined as an MBC 2 25 ,ug/ml. RESULTS E. coli. Figure 2 shows that Sch 20569 had fourfold greater activity than amikacin against many strains ofE. coli and that both were quite effective against all 13 gentamicin-resistant strains. Against sensitive strains, the activity of gentamicin and Sch 20569 was almost identical.Klebsiella. Similar results were obtained with Klebsiella (Fig. 3). Again, all gentamicinresistant strains were sensitive to both Sch...

Phosphatidylinositol transfer proteins (PITPs) and their yeast counterpart (SEC14p) possess the ability to bind phosphatidylinositol (PtdIns) and transfer it between membranes in vitro. However, the biochemical function of these proteins in vivo is unclear. In the present study, the physiological role of PITP was investigated by determining the biochemical consequences of lowering the cellular content of this protein. WRK-1 rat mammary tumour cells were transfected with a plasmid containing a full-length rat PITPalpha cDNA inserted in the antisense orientation and the resultant cell clones were analysed. Three clones expressing antisense mRNA for PITPalpha were compared with three clones transfected with the expression vector lacking the insert. The three antisense clones had an average of 25% less PITPalpha protein than control clones. Two of the three antisense clones also exhibited a decreased rate of growth. All three antisense clones exhibited a significant decrease in the incorporation of labelled precursors into PtdCho during a 90-min incubation period. Under the same conditions, however, there was no change in precursor incorporation into PtdIns. Further experimentation indicated that the decrease in precursor incorporation seen in antisense clones was not due to an increased rate of turnover. When choline metabolism was analysed more extensively in one control (2-5) and one antisense (4-B) clone using equilibrium-labelling conditions (48 h of incubation), the following were observed: (1) the decrease in radioactive labelling of PtdCho seen in short-term experiments was also observed in long-term experiments, suggesting that the total amount of PtdCho was lower in antisense-transfected clones (this was confirmed by mass measurements); (2) a similar decrease was seen in cellular sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an average two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, decreased; and (5) cellular CDPcholine was not significantly altered.

Agonist-stimulated phosphoinositide turnover is accompanied by compensatory resynthesis of these lipids. Several lines of evidence suggest that resynthesis of phosphatidylinositol (PtdIns) involves phosphorylation of diacylglycerol (DG) (salvage pathway) rather than acylation of glycerol phosphate (de novo pathway), although a contribution from the de novo pathway has not been ruled out. To determine the relative contribution of the de novo and salvage pathways in stimulated PtdIns resynthesis, an inhibitor of de novo synthesis (Triacsin C) was incubated simultaneously with the hormone agonist. Results indicate that at early times (90 min), hormone-stimulated PtdIns synthesis proceeds predominantly via the salvage pathway, although some de novo synthesis is also taking place. At later times (24 h), stimulated synthesis is solely via the de novo pathway. Increasing cellular DG content by either adding exogenous DG or treating cells with bacterial phospholipase C (bPLC) results in deacylation of the DG rather than phosphorylation; however, inhibition of this deacylation fails to stimulate phosphorylation by DG kinase (DGK), suggesting channeling of the DG substrate between PLC and DG kinase. Receptor activation is not required for activation of DGK, since treatment with a calcium ionophore induces the same Triacsin C-insensitive PtdIns synthesis. Depletion of the polyphosphoinositide pools by treatment with wortmannin prevents both hormone and A23187-induced polyphosphoinositide hydrolysis; however, A23187 is still able to induce hydrolysis of PtdIns and subsequent compensatory resynthesis. The inability of R59949 to inhibit either hormone-induced or ionophore-induced PtdIns resynthesis suggests that the alpha isoform is not involved; however, its possible that the channeling phenomenon prevents the inhibitor from gaining access to the diacylglycerol kinase enzyme. Further study will be required to determine which isoform catalyzes hormone-induced resynthesis of PtdIns.

Previous reports have suggested the existence of at least two pools of cellular myo-inositol (Ins); it has been further hypothesized that only one of these pools is utilized during hormone-activated, cyclic phosphatidylinositol (PtdIns) resynthesis. In an effort to investigate this possibility, we have undertaken kinetic studies of Ins metabolism in WRK-1 cells. Our results indicate that a single pool of Ins is involved in both basal and activated PtdIns synthesis. Ins generated by the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) mixes with the existing pool of free Ins and is not used exclusively for resynthesis of PtdIns.