The measurement of bioreductive capacity of tumor cells using methylene blue

PURPOSE: Methylene blue (MB) can be used as an intracellular electron acceptor. The purpose of this study was to demonstrate the usefulness of MB for the determination of total bioreductive capacity of cell suspensions. METHODS AND MATERIALS: We measured oxygen consumption by Clark electrode and pentose cycle activity by release of 14CO2 from 1-14C-glucose. RESULTS: Methylene blue catalyzes the reaction of intracellular reductants NADPH, NADH, and reduced glutathione (GSH) with oxygen, causing the production of hydrogen peroxide. The reaction rate correlates with the negative charge of molecule (NADPH(-4) > NADH(-2) > GSH(-1)), suggesting that reaction with positively charged oxidized MB is the limiting step of the reaction. In a cellular system MB causes the electron flow from cellular endogenous substrates to oxygen. It is activated by the disruption of the NADP+/NADPH ratio due to several processes. These are direct oxidation of NADPH and GSH, the GSH peroxidase catalyzed reaction of GSH with H2O2, followed by NADPH oxidation by oxidized glutathione (GSSG). This results in increased cellular oxygen consumption and stimulation of the oxidative limb of pentose cycle (PC) in the presence of MB. The cellular effect of MB differs from other electron accepting drugs. Diamide and tert-butylhydroperoxide act as direct oxidants, while MB is an electron carrier to oxygen. Accordingly, MB shows the highest effect on PC activation and oxygen consumption. CONCLUSIONS: Our results indicate that MB may be used for the determination of the total bioreductive capacity of the cells, measured by oxygen consumption and PC activation.     

Three-dimensional solution structure of alpha-conotoxin MII by NMR spectroscopy: effects of solution environment on helicity

alpha-Conotoxin MII, a 16-residue polypeptide from the venom of the piscivorous cone snail Conus magus, is a potent and highly specific blocker of mammalian neuronal nicotinic acetylcholine receptors composed of alpha3 beta2 subunits. The role of this receptor type in the modulation of neurotransmitter release and its relevance to the problems of addiction and psychosis emphasize the importance of a structural understanding of the mode of interaction of MII with the alpha3 beta2 interface. Here we describe the three-dimensional solution structure of MII determined using 2D 1H NMR spectroscopy. Structural restraints consisting of 376 interproton distances inferred from NOEs and 12 dihedral restraints derived from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. The final set of 20 structures is exceptionally well-defined with mean pairwise rms differences over the whole molecule of 0.07 A for the backbone atoms and 0.34 A for all heavy atoms. MII adopts a compact structure incorporating a central segment of alpha-helix and beta-turns at the N- and C-termini. The molecule is stabilized by two disulfide bonds, which provide cross-links between the N-terminus and both the middle and C-terminus of the structure. The susceptibility of the structure to conformational change was examined using several different solvent conditions. While the global fold of MII remains the same, the structure is stabilized in a more hydrophobic environment provided by the addition of acetonitrile or trifluoroethanol to the aqueous solution. The distribution of amino acid side chains in MII creates distinct hydrophobic and polar patches on its surface that may be important for the specific interaction with the alpha3beta2 neuronal nAChR. A comparison of the structure of MII with other neuronal-specific alpha-conotoxins provides insights into their mode of interaction with these receptors.     

Leukemia inhibitory factor modulates interleukin-1beta-induced activation of the hypothalamo-pituitary-adrenal axis

We have shown that leukemia inhibitory factor (LIF) is expressed in corticotroph cells and stimulates POMC gene expression and ACTH secretion in vivo and in vitro. We therefore examined the regulation of in vitro and in vivo pituitary LIF expression by cytokines known to stimulate the hypothalamo-pituitary-adrenal axis. In the corticotroph cell line AtT-20/D16v-F2, recombinant murine interleukin-1beta (IL-1beta; 0.1-10.0 ng/ml) caused a 5- to 10-fold increase in LIF messenger RNA (mRNA) levels. LIF mRNA expression was induced as early as 1 h, peaked at 2 h, and still persistently elevated above the baseline after 8 h. This effect of IL-1beta on LIF mRNA expression was abolished by preincubation with human IL-1 receptor antagonist (100 ng/ml) or antimurine IL-1beta antibody (10 microg/ml). Tumor necrosis factor-alpha (20 ng/ml) only modestly increased LIF mRNA, but was synergistic with IL-1beta (up to 2.5-fold). In contrast, IL-2 and IL-6 did not alter LIF mRNA. In C57BL/6 mice, i.p. injection of 100 ng IL-1beta increased plasma ACTH and corticosterone levels after 1 h (P < 0.02). In addition, pituitary LIF mRNA content was increased for up to 2 h in response to IL-1beta. In comparison to wild-type (+/+) B6D2F1 mice, LIF knockout mice with a deleted LIF gene (-/-) exhibited decreased plasma ACTH (631 +/- 61 vs. 376 +/- 50 pg/ml; P < 0.01) and corticosterone (783 +/- 85 vs. 433 +/- 51 ng/ml; P < 0.01) levels 1 h after i.p. IL-1beta administration. In conclusion, corticotroph LIF mRNA expression is specifically stimulated by IL-1beta and tumor necrosis factor-alpha. The attenuated hypothalamo-pituitary-adrenal response to IL-1beta in LIF knockout mice indicates that the effect of IL-1beta on ACTH secretion is modulated by LIF. Thus, LIF appears to function as an immune-neuroendocrine modulator signaling the hypothalamo-pituitary-adrenal axis.     

Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7

Granzyme B (GrB) is predicted to trigger apoptosis by activating preferred caspases, but the zymogens that are directly processed by the granzyme and the requirements for these interactions remain unclarified. We examined this dilemma by comparing the kinetics and pattern of GrB-mediated activation of the executioner caspase-7 in vitro and in vivo. GrB rapidly activates procaspase-7 in vitro by cleaving between the large and small subunits leaving the propeptide intact. During GrB-mediated apoptosis, the caspase-7 propeptide is removed and cleavage occurs between the subunits. Strikingly, caspase-7 is unprocessed in caspase-3-deficient MCF-7 cells exposed to GrB but is rapidly activated when the cells are solubilized. Transfection with caspase-3 restores the removal of the caspase-7 propeptide and the capacity of GrB to subsequently activate the caspase. The data suggest that GrB activates caspase-3, which then removes the propeptide of caspase-7 allowing activation by GrB. Thus GrB initiates the death pathway by processing the accessible caspase-3, and the caspase-7 propeptide regulates trans-activation of the zymogen by granzyme. As a consequence, two proteases, caspase-3 and GrB, are required to activate procaspase-7.     

Impact of chronic cough on quality of life

STUDY DESIGN: The biomechanical influence of in situ setting hydroxyapatite cement was examined for use in pedicle screw revision surgery. Pull-out testing of control and pedicle screws augmented with hydroxyapatite cement was performed in human cadaver vertebrae. OBJECTIVES: To determine the immediate effect of using hydroxyapatite cement to augment revision pedicle screws after failure of the primary pedicle screw fixation. SUMMARY OF BACKGROUND DATA: The potential problems associated with using polymethylmethacrylate to augment revision pedicular instrumentation have prompted the search for other solutions. The introduction of resorbable hydroxyapatite pastes may have provided new biocompatible solutions for pedicle screw revision. METHODS: Ten human cadaver vertebrae were instrumented with 6.0-mm pedicle screws in each pedicle. The screws were loaded to failure in axial tension (pull-out). The failed pedicles then were instrumented with 7.0-mm pedicle screws, either augmented with hydroxyapatite cement or nonaugmented, which also were loaded to failure. Finally, the nonaugmented 7.0-mm screw hole was reinstrumented with a hydroxyapatite cement-augmented, 7.0-mm pedicle screw and loaded to failure. RESULTS: The pull-out strength of the 7.0-mm, hydroxyapatite cement-augmented screws was 325% (P = 2.9 x 10(-5)) of that of the 6.0-mm control screws, whereas the strength of the 7.0-mm nonaugmented screws was only 73% (P = 2.0 x 10(-2)) of that of the 6.0-mm control screws. The 7.0-mm screws augmented with hydroxyapatite cement also were able to salvage 7.0-mm pull-out sites to 384% (P = 6.9E-5) of the pull-out strength of the 7.0-mm nonaugmented screws. CONCLUSIONS: Hydroxyapatite cement may be a mechanically viable alternative to polymethyl methacrylate for augmenting revision pedicular instrumentation and should be considered for future experimental, animal, and clinical testing.     

Effects of drug solubility, drug loading, and polymer molecular weight on drug release from Polyox tablets

BACKGROUND: This study augments a randomized controlled trial to analyze the cost-effectiveness of 2 standardized treatments for major depression relative to each other and to the "usual care" provided by primary care physicians. METHODS: A randomized controlled trial was conducted in which primary care patients meeting DSM-III-R criteria for current major depression were assigned to pharmacotherapy (where nortriptyline hydrochloride was given) or interpersonal psychotherapy provided in a standardized framework or a primary physician's usual care. Two outcome measures, depression-free days and quality-adjusted days, were developed using information on depressive symptoms over time. The costs of care were calculated. Cost-effectiveness ratios comparing the incremental outcomes with the incremental costs for the different treatments were estimated. Sensitivity analyses were performed. RESULTS: In terms of both economic costs and quality-of-life outcomes, patients assigned to the pharmacotherapy group did slightly better than those assigned to interpersonal psychotherapy. Both standardized therapies provided better outcomes than primary physician's usual care, but each consumed more resources. No meaningful cost-offsets were found. The incremental direct cost per additional depression-free day for pharmacotherapy relative to usual care ranges from $12.66 to $16.87 which translates to direct cost per quality-adjusted year gained from $11270 to $19510. CONCLUSIONS: Standardized treatments for depression lead to better outcomes than usual care but also lead to higher costs. However, the estimates of the cost per quality-of-life year gained for standardized pharmacotherapy are comparable with those found for other treatments provided in routine practice.     

Screening calcaneal ultrasound and risk factors for osteoporosis among lesbians and heterosexual women

We compared calcaneal ultrasound measurements (speed of sound [SOS], broadband ultrasound attenuation [BUA], and stiffness index [SI]) of lesbian and heterosexual women to examine the medical and lifestyle risk factors for osteoporosis in each group. This was an exploratory, community-based, cross-sectional study. Subjects were mailed food frequency, health, and physical activity questionnaires. Weight, height, and calcaneal ultrasound measurements were taken at one office visit. In communities in southern and eastern Maine, 71 lesbians and 77 heterosexual women between the ages of 30 and 50 with regular menses and in good general health were the subjects. Statistical analysis used t-tests and chi-square tests to evaluate differences between study groups. Linear regression models were used to evaluate risk factors for low calcaneal ultrasound measurements. There were no differences between the lesbian and heterosexual women in age, body mass index (BMI), exercise, calcium intake, alcohol use, or calcaneal ultrasound measurements. There was a positive association between BUA and both BMI and alcohol consumption (p < 0.01). Antidepressant use significantly reduced SOS and SI (p < 0.05). There were no differences in calcaneal ultrasound measurements between lesbian and heterosexual women. BMI was strongly and positively associated with BUA. Antidepressant use in both populations was associated with a significant reduction in calcaneal bone mass. Studies are needed to define the relationship of depression and its treatment to bone mineral density and the future risk of osteoporosis.     

A mutation in the Escherichia coli F0F1-ATP synthase rotor, gammaE208K, perturbs conformational coupling between transport and catalysis

Cross-linking studies on the Escherichia coli F0F1-ATP synthase indicated a site of interaction involving gamma and epsilon subunits in F1 and subunit c in F0 (Watts, S. D., Tang, C., and Capaldi, R. A. (1996) J. Biol. Chem. 271, 28341-28347). To assess the function of these interactions, we introduced random mutations in this region of the gamma subunit (gamma194-213). One mutation, gammaGlu-208 to Lys (gammaE208K), caused a temperature-sensitive defect in oxidative phosphorylation-dependent growth. ATP hydrolytic rates of the gammaE208K F0F1 enzyme became increasingly uncoupled from H+ pumping above 28 degreesC. In contrast, Arrhenius plot of steady-state ATP hydrolysis of the mutant enzyme was linear from 20 to 50 degreesC. Analysis of this plot revealed a significant increase in the activation energy of the catalytic transition state to a value very similar to soluble, epsilon subunit-inhibited F1 and suggested that the mutation blocked normal release of epsilon inhibition of ATP hydrolytic activity upon binding of F1 to F0. The difference in temperature dependence suggested that the gammaE208K mutation perturbed release of inhibition via a different mechanism than it did energy coupling. Suppressor mutations in the polar loop of subunit c restored ATP-dependent H+ pumping and transition state thermodynamic parameters close to wild-type values indicating that interactions between gamma and c subunits mediate release of epsilon inhibition and communication of coupling information.     

A study of chest radiography with mobile X-ray units

A survey of radiographic technique and estimated entrance surface dose has been carried out for 364 chest radiographs performed with mobile X-ray equipment in the Intensive Therapy Unit (ITU) and 30 wards at Aberdeen Royal Infirmary. Data for these two types of location were compared, as were those for two film/screen systems used on the wards. Image quality assessments were made on sets of radiographs for two patients. Entrance skin doses for chest radiographs performed in the ITU were 50% greater than on the wards with the same film/screen system. The main technique difference was the use of shorter focus-to-skin distances (FSDs) in ITU. Doses with the Kodak Insight system were 20% higher than those using Du Pont Quanta III in similar locations. No correlation was found between image quality and entrance surface dose (ESD). Results from the survey were used to recommend exposure factors for shorter FSDs. A follow-up study revealed a 35-45% reduction in ESD for Kodak Insight and a 20% reduction for Quanta III.