Reexamination of the effect of endotoxin on cell proliferation and transfection efficiency.

Plasmid DNA purified from bacterial cells can be contaminated with endotoxin to different extents, depending on the purification method. Earlier reports indicate that endotoxin can decrease transfection efficiency in many eukaryotic cell lines; however, the amount of endotoxin required for inhibition is unclear. We determined endotoxin effects in several cell lines and observed that endotoxin levels greater than or equal to 10,000 endotoxin units (EU) were needed to significantly affect cell proliferation and viability; levels greater than 2000 EU/mu g DNA were required to significantly inhibit transfection for all but one (Huh-7) of the cell lines tested. These endotoxin levels are significantly higher than endotoxin contamination in plasmid DNA purified by anion exchange, CsCl2 gradient and endotoxin-free purification technology, but not as high as a crude alkaline lysis preparatory method. Plasmid DNA prepared using anion exchange technology was comparable to endotoxin-free technology in terms of transfection efficiency. Even Huh-7 cells, which are markedly more sensitive to endotoxins, have comparable transfection efficiencies using plasmid DNA purified by either of these two methods. We conclude that for those cell lines commonly used for transfection studies, endotoxin-free, quality DNA is not necessary because significantly higher levels of bacterial endotoxins are required to inhibit either cell proliferation or transfection.

Paper-based archiving of mammalian and plant samples for RNA analysis.

The ability to archive biological samples for subsequent nucleic acid analysis is essential for tissue specimens and forensic samples. FTA Card is a chemically treated filter paper designed for the collection and room temperature storage of biological samples for subsequent DNA analysis. Its usefulness for the preservation of biological samples for subsequent RNA analysis was tested. Here, we demonstrate that RNA in biological samples stored on FTA Cards is stable and can be used successfully for RT-PCR and northern blot analysis. RNA stability depends on the storage temperature and the type of biological specimen. RNA in mammalian cells stored on FTA Cards is stable for over one year at temperatures at or below -20 degrees C and for two to three months in samples stored at room temperature. For plant leaf, longer storage times (> 5 days) require temperatures at or below -70 degrees C following sample application. FTA Cards may constitute a method not only for convenient collection and storage of biological samples but also for rapid RT-PCR analysis of tissue and cell samples.

Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA.

Reverse transcriptase (RT) is the key enzyme required for conversion of RNA to DNA. Cloning of Moloney murine leukemia virus (MMLV) RT has enable engineering an RT that lacks endogenous RNase H activity. RT catalyzes cDNA synthesis more efficiently in the absence of RNase H. We describe here a number of properties of MMLV RT and RNase H-minus MMLV RT not summarized in a single location elsewhere, providing a basis for best use of these enzymes in cDNA synthesis. In addition, general guidelines and detailed protocols are provided for use of MMLV RTs in one tube double-stranded cDNA synthesis, in [32P]cDNA synthesis, and in RT-PCR and long RT-PCR.

Comparison of intravenous nitroprusside, nitroprusside-captopril, and nitroglycerin for deliberate hypotension during posterior spine fusion in adults.

Three techniques for deliberate hypotension (mean arterial pressure, 60 to 70 mmHg) were prospectively compared in adults undergoing posterior spine fusion. Patients received either IV sodium nitroprusside, sodium nitroprusside with oral captopril pretreatment, or IV nitroglycerin. Patient groups were comparable in age, sex, weight, baseline hemodynamic and laboratory parameters, duration of surgery, and duration of hypotension. Absolute blood loss was significantly less in the group receiving nitroglycerin; however, there were no differences between groups when corrected for operative exposure (milliliter per spine segment exposed). Nitroprusside was effective in producing target blood pressure in all patients. Nitroglycerin was ineffective in two patients and two other patients required greater than 20 micrograms/kg/min. Both groups receiving nitroprusside developed significant postinfusion increases in arterial pressure. Blood pressure fell significantly after induction of anesthesia in patients receiving captopril. Cardiac index, heart rate, pulmonary capillary wedge pressure, intrapulmonary shunt, and arterial blood gases were comparable and did not change significantly in any group. Systemic vascular resistance fell during infusion in all groups and remained depressed after infusion in patients receiving nitroglycerin. Plasma renin activity was significantly increased in the group receiving captopril due to loss of feedback inhibition of renin release and rose significantly during infusion in those patients receiving nitroprusside alone. There were no complications. Nitroprusside with and without captopril pretreatment was associated with postoperative increases in arterial pressure, although not to hypertensive levels, probably due to loss of captopril activity after single-dose administration. The use of nitroglycerin was limited by lack of potency. There was no demonstrable clinical advantage for any of the three techniques.

The long-term effects of a token economy on safety performance in open-pit mining.

A token economy that used trading stamps as tokens was instituted at two dangerous open-pit mines. Employees earned stamps for working without lost-time injuries, for being in work groups in which all other workers had no lost-time injuries, for not being involved in equipment-damaging accidents, for making adopted safety suggestions, and for unusual behavior which prevented an injury or accident. They lost stamp awards if they or other workers in their group were injured, caused equipment damage, or failed to report accidents or injuries. The stamps could be exchanged for a selection of thousands of items at redemption stores. Implementation of the token economy was followed by large reductions in the number of days lost from work because of injuries, the number of lost-time injuries, and the costs of accidents and injuries. The reductions in costs far exceeded the costs of operating the token economy. All improvements were maintained over several years.

Isolation and characterization of homogeneous acetate kinase from Salmonella typhimurium and Escherichia coli.

Acetate kinase from Salmonella typhimurium and Escherichia coli was purified to electrophoretic homogeneity. The amino acid compositions of both proteins were similar, and the apparent molecular weights were the same, about 40,000 for the putative monomers. The native proteins gave higher molecular weights, suggesting that the enzymes may be oligomers, perhaps with two polypeptide subunits. Steady-state kinetic studies were performed with the enzymes isolated from both organisms and the kinetic constants were determined. The Km values were 0.07 and 7 mM for ATP and acetate, respectively. In contrast to earlier studies using less pure preparations, the homogeneous enzymes from both strains were active only with acetate but not with propionate or butyrate. The enzyme activity was cold-labile, and the length of reactivation time in the presence of Mg X ATP and acetate was dependent on protein concentration, suggesting that the monomer may not be catalytically active. The enzyme was phosphorylated with [gamma-32P]ATP and the phosphoprotein was isolated. Phosphoacetate kinase was capable of transferring the phosphate group to either ADP or acetate. The accompanying paper (Fox, D. K., Meadow, N. D., and Roseman, S. (1986) J. Biol. Chem. 261, 13498-13503) shows that the phosphoryl group of phosphoacetate kinase can also be reversibly transferred to Enzyme I of the phosphoenolpyruvate:glycose phosphotransferase system.

Phosphate transfer between acetate kinase and enzyme I of the bacterial phosphotransferase system.

Interactions between homogeneous acetate kinase and proteins of the phosphoenolpyruvate:glucose phosphotransferase system (PTS) were studied. The phosphorylation of D-glucose was followed spectrophotometrically using a coupled assay system, and acetate kinase and GTP were found to substitute for phosphoenolpyruvate provided that each of the PTS proteins was present in the mixture. To further define the phosphoryl transfer reaction pathway, the system was simplified to include only the homogeneous, soluble PTS proteins. 32P was transferred from [gamma-32P]ATP to the protein IIIGlc, but this transfer reaction required acetate kinase, and the PTS proteins Enzyme I and HPr. These results suggested that acetate kinase interacts with the first protein in the PTS sequence, Enzyme I. Acetate kinase was therefore incubated with [32P] phospho-Enzyme I, and a direct transfer of the phosphoryl group was observed without the addition of any other protein. These results show that there is a reversible transfer of the phosphoryl group between Enzyme I and acetate kinase. The possible role of this interaction in regulating sugar uptake by the Krebs cycle is discussed.

Pel, the protein that permits lambda DNA penetration of Escherichia coli, is encoded by a gene in ptsM and is required for mannose utilization by the phosphotransferase system.

Mannose uptake and phosphorylation in Escherichia coli is catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS). The mannose-specific complex of the PTS, designated IIMan, comprises lipid and two membrane proteins, II-AMan and II-BMan. The proteins are encoded by ptsM, located at approximately equal to 40 minutes on the E. coli chromosome. A different genetic marker, pel, maps with ptsM, and is required for lambda DNA penetration of the cytoplasmic membrane. Earlier studies suggested that both pel function and II-BMan are encoded by the same gene, while a different gene (also in ptsM) encodes II-AMan. In the present studies, a ptsM clone, pCS13, was isolated from an E. coli HindIII gene bank in pBR322 and restored both mannose termentation and pel+ function to ptsM mutants defective in II-BMan. Subclones of pCS13 show that two distinct genes, manY and manZ, encode the pel+ function and the II-BMan protein, respectively; each gene may have its own promoter; whereas the protein encoded by manY (Pel) alone seems sufficient for lambda sensitivity, all three gene products are required for mannose fermentation, transport of the mannose analogue 2-deoxyglucose, and phosphorylation of the latter by cytoplasmic membranes. Thus, Pel is required for function of the IIMan complex. The efficiency of the complex may depend on the ratio of Pel to IIMan.

Heparin, protamine, and ionized calcium in vitro and in vivo.

The possibility that hypotension associated with protamine or heparin might be related to changes in levels of serum ionized calcium values was determined by in vitro and in vivo studies in dogs. In vitro protamine did not decrease serum calcium levels, but heparin did in a dose-dependent fashion. The reduction ranged from 7% with 10 units/ml of heparin to 20% with 100 units/ml of heparin. Ionized calcium concentrations initially decreased by heparin were restored toward control levels by our increasing the dose of protamine, indicating that the electrostatic attraction between protamine and heparin molecules is stronger than that between heparin and ionized calcium. Despite significant reductions in blood pressure and heart rate, clinical doses of protamine did not decrease ionized calcium in vivo. Although the results of the in vitro study suggested that heparin-induced hypocalcemia might occur in vivo, in vivo heparin caused neither a decrease in ionized calcium nor hypotension. The reduction of ionized calcium by heparin might have been rapidly compensated for in vivo. The results indicate that hypotension due to protamine or heparin is unlikely to be related to changes in serum ionized calcium levels.

Pulse oximetry and circulatory kinetics associated with pulse volume amplitude measured by photoelectric plethysmography.

Through a catheter placed in a superficial vein on the finger, we observed a pulsatile venous pressure. To delineate the relationship between the pulsatile venous pressure and the pulse volume amplitude (PVA) recorded by photoelectric plethysmography (PEPG), both tracings were simultaneously recorded. When the PVA changed acutely or gradually, the venous pulse pressure and mean venous pressure simultaneously followed the same trend. We also found that mean PVO2 (135 mm Hg) was greater when the PVA and venous pulse pressure increased above the level (50 mm Hg) observed when they decreased. These findings suggested that the finger pulse detected by PEPG, as well as by pulse oximetry, is caused by pulsations in veins rather than by pulsations in arterial beds. In experiments to evaluate the validity of this hypothesis, we found that the average value of hemoglobin saturation (%SaO2) measured by the pulse oximeter of the dependent fingertip and finger base when dependent was 1.5% and 7.8% lower than when the fingertip and finger base were elevated. Also, the PVA detected by the pulse oximeter followed the same trend as %SaO2. This finding was explained by venous congestion in the dependent finger. On the basis of the high venous pressure, the behavioral trends between the PVA and venous pressure, the high PVO2, and the low %SaO2 and PVA in the dependent finger, we conclude that the PVA of the PEPG is determined mainly by venous pulse volume generated by shunting of arterial pulse via open arteriovenous (AV) anastomoses in the cutaneous circulation.

Evidence for two promoters upstream of the pts operon: regulation by the cAMP receptor protein regulatory complex.

Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH. The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP.