Instability of Rts1 (drug-resistant factor) replicon: stabilization by DNA fragments derived from Rts1.

Rts1 is a large naturally occurring plasmid which has a kanamycin resistance gene and exhibits various temperature-sensitive phenotypes. A smaller derivative of plasmid, pOK, contains the Rts1 replicon and the kanamycin resistance gene of Rts1. This plasmid, pOK, is much more unstable than Rts1 at 42.5 degrees C. A DNA fragment, G3, 1590 nucleotides long from Rts1 DNA, stabilized pOK completely at 42.5 degrees C but only in the cis configuration. G3 did not change the copy number of pOK. The pOK derivative containing G3 was destabilized by the presence of a compatible plasmid containing G3. G3 has four inverted repeats, two 14-base direct repeats, and three ORFs. Smaller fragment of G3 also had a stabilization effect and these studies showed that the ORF does not play any role in stabilization.

Instability of standard calibrators may be involved in overestimating vancomycin concentrations determined by fluorescence polarization immunoassay.

Fluorescence polarization immunoassay (FPIA) is widely used to determine serum vancomycin concentrations, and it has been shown to over-estimate vancomycin concentrations in sera from renally impaired patients. This phenomenon has generally been thought to result from interference by vancomycin crystalline degradation products (CDP-1). In this study, we confirmed that serum vancomycin concentrations in various patients determined by FPIA were higher than those determined by high-performance liquid chromatography (HPLC) or enzyme multiplied immunoassay (EMIT). However, the quantitative differences in the serum vancomycin concentrations determined by FPIA versus HPLC were higher than the CDP-1 concentrations, even when the cross-reactivity of FPIA to CDP-1 is assumed to be 100%. When the vancomycin calibrators for FPIA were stored at 4 degrees C for 30 days, their concentrations determined by FPIA and HPLC decreased by 14 and 20%, respectively, and CDP-1 corresponding to 20% of primary vancomycin was formed. When stored at 25 degrees C, the degradation of vancomycin was more marked. We concluded that not only the cross-reactivity of FPIA to CDP-1 but also the instability of calibrators may cause the overestimation of serum vancomycin concentrations determined by FPIA.

In vitro cytostatic effect of TNF (tumor necrosis factor) entrapped in immunoliposomes on cells normally insensitive to TNF.

The cytostatic activity of TNF entrapped in novel immunoliposomes with a specific antibody against target cells is described. A two step conjugation method was used for the preparation of these targeted immunoliposomes. In the first step, liposomes containing N-4-(p-maleimidophenyl)butyryl phosphatidylethanolamine (MPB-PE) were conjugated with a goat anti-mouse IgG Fab' fragment which recognizes the Fc portion of a mouse antibody against the target cell markers. In the second step, the mouse antibody against human tumor cells was conjugated to the liposomes. Using these targeted immunoliposomes, we demonstrated that cells usually insensitive to TNF such as Daudi cells, MT-2 cells and T-24 cells could become sensitive to TNF in vitro. The cytostatic activity of these immunoliposomes was blocked by the addition of a lysosomotropic agent such as NH4Cl or chloroquine. Significant uptake of 125I-TNF into T-24 cells was observed when these immunoliposomes were used, and this uptake of TNF was inhibited by cytochalasin B or chloroquine. Free 125I-TNF was not taken up by these cells.

[Partitioning and measurement of respiratory impedance into central and peripheral airways with catheter tip type transducer].

In this study, we have examined the availability of catheter tip type transducer for the partitioning of respiratory impedance. Total respiratory resistance and reactance from 1 to 20 Hz determined by forced oscillation method with random noise input in anesthetized dogs were partitioned by catheter tip type transducer into central airway resistance (Rc) and reactance (Xc) as well as peripheral resistance (Rp) and reactance (Xp). Intratracheal acetylcholine (0.1g) increased Rp at the wide frequency range (from about 1 to 20 Hz), although Rc did not change with it. The decreases of Rc at higher frequencies (over 12Hz) were produced by intratracheal salbutamol (10mg), whereas Rp did not change significantly. These results suggest that the predominant site of action of intratracheal acetylcholine and salbutamol is peripheral airway and central airway respectively. We have concluded that the respiratory impedance can be partitioned noninvasively with catheter tip type transducer without pneumothorax and hemorrhage caused with the retrograde catheter. This method can be applied clinically during general anesthesia or in intensive care.

[Effect of sevoflurane on respiratory impedance measured by the forced oscillation method with random noise input].

This study was performed to investigate the effect of sevoflurane, a new inhalation anesthetic, on the respiratory impedance measured by forced oscillation method with random noise input. Random noise (1 to approximately 25Hz) oscillated by a loudspeaker was imposed on the respiratory system of dogs through a tracheostomy and the oscillatory pressure wave and flow wave were monitored at the same point with a pressure transducer and a pneumotachograph. This procedure was repeated during inhalation of 100%-oxygen, 2.0%-then 4.0%-sevoflurane-oxygen mixture, respectively. Impedance was calculated with fast Fourier transform by spectrum analyzer and a personal computer. During inhalation of 4.0%-sevoflurane-oxygen mixture, respiratory impedance especially respiratory resistance decreased at the high frequency area (6to approximately 20Hz), but 2.0%-sevoflurane-oxygen mixture did not cause a significant change of respiratory impedance. This study suggests that inhalation of an anesthetic concentration of sevoflurane produces weak bronchodilation compared with halothane or enflurane.