ATP induces a conformational change in lipid-bound cytochrome c.

Resonance energy transfer studies using a pyrene-labeled phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol (donor) and the heme (acceptor) of cytochrome c (cyt c) have indicated that ATP causes changes in the conformation of the lipid-bound protein (Rytömaa, M., Mustonen, P., and Kinnunen, P. K. J. (1992) J. Biol. Chem. 267, 22243-22248). Accordingly, after binding cyt c via its so called C-site to neat phosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, further quenching of donor fluorescence is caused by ATP, saturating at 2 mm nucleotide. ATP-induced conformational changes in liposome-associated cyt c could be directly demonstrated by CD in the Soret band region (380-460 nm). The latter data were further supported by time-resolved spectroscopy using the fluorescent cyt c analog with a Zn(2+)-substituted heme moiety. A high affinity ATP-binding site has been demonstrated in cyt c (Craig, D. B., and Wallace, C. J. A. (1993) Protein Sci. 2, 966-976) that is compromised by replacing the invariant Arg(91) to norleucine. Although no major effects on conformation and function of cyt c were concluded due to the modification, a significantly reduced effect by ATP on the lipid-bound [Nle(91)]cyt c was evident, implying that this modulation is mediated via the Arg(91)-containing binding site.

Escherichia coli beta-galactosidase is heterogeneous with respect to a requirement for magnesium.

Commercially obtained E. coli beta-galactosidase was stored at 25 degrees C in buffer containing 1 mM MgCl2 and in buffer containing no added MgCl2. Samples were removed at set times and the activity of individual enzyme molecules assayed. When stored in the presence of 1 mM magnesium, the number of active molecules did not change over a 2.5-h period. When stored in the absence of added MgCl2, over half the enzyme molecules became inactive within the first hour. However, those molecules which retained activity remained active for the duration of the experiment. This indicates that there may exist two populations of E. coli beta-galactosidase, one which requires storage in the presence of the higher concentration of Mg2+ in order to remain active. There was no observed correlation between this requirement for magnesium and reaction rate. Additionally, the presence of the 1 mM MgCl2 was found to decrease the average activity of the beta-galactosidase molecules under the conditions employed.

Laser-induced fluorescence detector for liquid chromatography: applications to protein analysis.

A laser-induced fluorescence detector for liquid chromatography was developed. This detector was assessed by utilizing it in conjunction with gel filtration chromatography. Using the 488 nm line of an argon ion laser for excitation and monitoring the emitted fluorescence centering at 535 nm, the limit of detection of fluorescein was 580 fM. Bovine serum albumin labeled with fluorescein was detected at a concentration of 500 fM.

Newly induced beta-galactosidase molecules have a higher activity than the basally expressed enzyme.

OBJECTIVE: The objective of this study was to compare the activities of individual molecules of induced and basally expressed Escherichia coli beta-galactosidase. BACKGROUND DATA: Single-molecule assays of enzymes have determined that individual molecules are not identical. They differ with respect to catalytic rate. The structural cause and cellular role of this microheterogeneity is as yet unknown. METHODS: E. coli were grown and induced to produce beta-galactosidase by treatment with isopropyl-beta-D-thiogalactopyranoside. Cells were lysed and the beta-galactosidase assayed with capillary electrophoresis instrumentation utilizing post-column, laser-induced fluorescence detection. The enzyme obtained from treated cells were compared to that from untreated cells. RESULTS: The activity of newly induced beta-galactosidase was found to be approximately 20% greater than that of the basally expressed enzyme. This measured difference is statistically significant. CONCLUSIONS: Production of beta-galactosidase in E. coli under differing conditions results in differences in the activities of the individual enzyme molecules.

beta-galactosidase assay using capillary electrophoresis laser-induced fluorescence detection and resorufin-beta-D-galactopyranoside as substrate.

beta-Galactosidase was incubated for 60 min with the fluorogenic substrate resorufin-beta-D-galactopyranoside, which is converted by the action of the enzyme into resorufin and galactose. A 160 pL aliquot of reaction mixture was analyzed by capillary electrophoresis utilizing laser-induced fluorescence detection. Based on the detection of the resorufin formed, the limit of detection of beta-galactosidase was 1.5 x 10(-15) M or 900 molecules of enzyme in a 1 microL sample.

Sodium dodecyl sulfate-capillary electrophoresis of proteins in a sieving matrix utilizing two-spectral channel laser-induced fluorescence detection.

We report a method for protein labeling, separation by capillary electrophoresis in a polymer sieving matrix, and detection by laser-induced fluorescence. Different dyes are used to label standard and sample proteins. A two-spectral channel detector resolves fluorescence from the sample and standards. Comparison of the migration time of the sample and standards permits the precise determination of molecular weight, irrespective of variations in run-to-run migration times.

General protease assay method coupling solid-phase substrate extraction and capillary electrophoresis.

Capillary electrophoresis with laser-induced fluorescence detection was used to develop a universal, highly specific protease assay. In this method, a peptide, biotinylated at the N-terminus, is labeled with fluorescein at a lysine residue near the C-terminus. Impurities are removed from the fluorescence labeling mixture by solid-phase extraction of the substrate on immobilized streptavidin, followed by extensive washing. The purified fluorescent substrate is dissociated from the streptavidin and incubated with the protease. The peptide sequence between the biotin and fluorescent label contains the cleavage sequence of the protease of interest. After cleavage, the fluorescent product does not contain a biotin group. A second solid-phase extraction is used to remove unreacted substrate to dramatically lower the background signal. The product is detected by capillary electrophoresis, which provides powerful discrimination against products generated by nonspecific proteases. With chymotrypsin as a test protease, product was detected with as little as 10 pg/mL (4.6 x 10(-13) M) chymotrypsin, or 5 amol of enzyme in the 10-microL sample volume.

Thrombotic thrombocytopenic purpura associated with HIV and visceral Kaposi's sarcoma treated with plasmapheresis and chemotherapy.

We present a case of a patient who is HIV positive and developed both thrombotic thrombocytopenia purpura and visceral Kaposi's sarcoma (KS) with hemorrhage. This case presents a difficult management problem in that the patient's bleeding originated from KS lesions and did not quickly abate with plasmapheresis therapy despite both clinical and laboratory improvement after 2-4 days. Chemotherapy was initiated on day 13 and the patient's condition improved markedly afterward. We believe the addition of chemotherapy to plasmapheresis hastened the improvement of our patient's thrombotic thrombocytopenic purpura (TTP) and KS-related bleeding. Therefore, under similar conditions, we recommend combining plasmapheresis and chemotherapy at the onset of therapy.

Do not resuscitate orders--managing the dilemma.

The status of DNR orders (or equivalent declarations) in patients undergoing surgery will continue to present considerable challenges for both healthcare providers and patients, or their alternate decision makers. It is essential that all parties understand the specifics of each DNR order, focusing not only on the actual content of the order or declaration but also on the context in terms of location, timing and circumstance. The principle of "respect for persons" should guide, inform and shape the approach followed with each patient. Meaningful dialogue and "negotiation" will be required. Make no assumptions! The "required reconsideration" of pre-existing DNR orders should be the basic approach followed. There is no single "solution" for all DNR-related issues in the peri-operative period. What may appear obvious to the anaesthetist may be viewed entirely differently by the patient, or even by other members of the care giving team. There is no justification for either the automatic suspension or the automatic continuation of DNR orders in patients undergoing surgery. A patient-specific and situation-specific approach and "solution" is required. Similar principles will apply in acute care settings other than the operating room. Full engagement by health care workers in the processes addressing these issues should be a personally enriching experience.

Once weekly azithromycin therapy for prevention of Mycobacterium avium complex infection in patients with AIDS: a randomized, double-blind, placebo-controlled multicenter trial.

We conducted a randomized, double-blind, placebo-controlled multicenter trial of azithromycin (1,200 mg once weekly) for the prevention of Mycobacterium avium complex (MAC) infection in patients with AIDS and a CD4 cell count of < 100/mm3. In an intent-to-treat analysis through the end of therapy plus 30 days, nine (10.6%) of 85 azithromycin recipients and 22 (24.7%) of 89 placebo recipients developed MAC infection (hazard ratio, 0.34; P = .004). There was no difference in the ranges of minimal inhibitory concentrations of either clarithromycin or azithromycin for the five breakthrough (first) MAC isolates from the azithromycin group and the 18 breakthrough MAC isolates from the placebo group. Of the 76 patients who died during the study, four (10.5%) of 38 azithromycin recipients and 12 (31.6%) of 38 placebo recipients had a MAC infection followed by death (P = .025). For deaths due to all causes, there was no difference in time to death or number of deaths between the two groups. Episodes of non-MAC bacterial infection per 100 patient years occurred in 43 azithromycin recipients and 88 placebo recipients (relative risk, 0.49; 95% confidence interval, 0.33-0.73). The most common toxic effect noted during the study was gastrointestinal, reported by 78.9% of azithromycin recipients and 27.5% of placebo recipients. Azithromycin given once weekly is safe and effective in preventing disseminated MAC infection, death due to MAC infection, and respiratory tract infections in patients with AIDS and CD4 cell counts of < 100/mm3.

Multiple labeling of proteins.

Fluorescent dyes are often used to label proteins before analysis by capillary electrophoresis. Fluorescent labeling produces spectacular improvements in sensitivity compared with UV absorbance detection of the native protein. However, labeling of the protein can lead to significant band broadening. This band broadening is interpreted as a result of multiple labeling of the protein, wherein one or more fluorescent molecules are bound to the protein. The heterogeneous reaction products, which are presumed to have different mobilities, generate a broad peak during electrophoresis. There has been little direct evidence for multiple labeling as the cause of band broadening of proteins. In this paper, we perform electrophoresis on native green fluorescence protein, along with the reaction products produced by fluorescence labeling. For short incubations, a series of regularly spaced components are resolved by free-zone electrophoresis; upon longer incubation, the product peaks merge together, forming a broad envelope.

Detection of Aequorea victoria green fluorescent protein by capillary electrophoresis laser induced fluorescence detection.

Aequorea victoria green fluorescent protein was assayed by capillary electrophoresis using post-capillary laser-induced fluorescence detection in a sheath flow cuvette. The limit of detection was 3.0 x 10(-12) M protein in an injection volume of 17 nL, corresponding to a mass of 3100 molecules.

Do not resuscitate orders in the operating room.

PURPOSE: The background to a current analysis of the management of "do not resuscitate" (DNR) orders in the operating room is reviewed, with an emphasis on the current status of resuscitation/DNR issues in Canada. SOURCE: The Joint Statement on Resuscitative Interventions published by the Canadian Medical Association and cooperating organizations and the report of the Senate of Canada Special Committee on Euthanasia and Assisted Suicide are examined for information relevant to the DNR issue. Guidelines on the management of DNR orders in the operating room, published by the American Society of Anesthesiologists and the American College of Surgeons are used to provide a perioperative DNR order management approach consistent with the Joint Statement on Resuscitative Interventions. PRINCIPAL FINDINGS AND CONCLUSIONS: The dominant principle is that of the patient's right to self determination. This right can be exercised either directly by the patient, or through an appropriate alternate, or in the form of an advance directive. DNR orders are not incompatible with subsequent surgical care in an operating room. It is wrong to suspend automatically DNR orders in the perioperative period. It is wrong to continue DNR orders automatically in the perioperative period. It is wrong to make assumptions about the meaning of an individual DNR order. An appropriate approach to the perioperative management of pre-existing DNR orders is one based on "required reconsideration." All anaesthetists must be aware of their responsibilities in managing patients with DNR orders in place.

Studies of 8-azido-ATP adducts reveal two mechanisms by which ATP binding to cytochrome c could inhibit respiration.

We have proposed that the binding of ATP at a site of substantial affinity and specificity could regulate the activity of cytochrome c with its physiological partners and thus the overall efficiency of mitochondrial electron transport. We now describe the use of ATP affinity-labeled protein to test the effect of occupancy of that site, which includes the invariant arginine 91, on the activity of cytochrome c with purified cytochrome c reductase and oxidase and its association with the mitochondrial inner membrane. Electron-transfer activities with the reductase and oxidase were inhibited by site occupancy to 41% and 11-15% of native values, respectively. The marked difference in the degree of inhibition of activity that distinguishes the reactions with the two major physiological partners was sufficient to cause, in whole mitochondria, a demonstrable shift from a situation in which there is a rate-limiting transfer from the reductase to cytochrome c, to a state where rates are more evenly matched for transfers between cytochrome c and the two redox partners. Site occupancy also substantially reduces the ionic strength necessary for half-maximal dissociation of cytochrome c from the membrane. These data imply that the decreased efficiency of electron transfer caused by ATP attachment can be attributed to a decrease in the protein's activity with individual physiological partners, possibly compounded with a decrease in its affinity for the inner mitochondrial membrane, and suggest that feedback regulation by ATP of cellular respiration operates in like manner.