Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (delta psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial delta psi and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 micrograms ml-1 for 40 min for Rhodamine, and 1 microM for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. 'Round-shaped' osteoclasts had significantly higher mitochondrial delta psi and volume in the apical regions than in the basolateral portions (p < 0.00001). In contrast, mitochondrial delta psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.     

Human immunodeficiency virus replication in AIDS patients with Mycobacterium avium complex bacteremia: a case control study. California Collaborative Treatment Group

The development of opportunistic infections and the administration of vaccines have been associated with transient increases of human immunodeficiency virus (HIV) RNA plasma levels in HIV-infected patients. To determine the relationship between Mycobacterium avium complex (MAC) bacteremia and HIV RNA levels, HIV RNA levels in patients who developed MAC bacteremia (cases) were compared with levels in patients who remained free of MAC disease (controls). Cases and controls were matched for CD4 cell count, prophylaxis against MAC disease, antiretroviral therapy, and duration of follow-up. Mean baseline HIV RNA levels were 4.8 log10 copies/mL in cases and 4.6 log10 copies/mL in controls (P = 0.22). HIV RNA levels increased by a median of 0.4 log in cases but not controls at the time of MAC bacteremia (P = 0.01). In AIDS patients, the onset of MAC bacteremia is associated with a modest but significant increase in serum HIV RNA levels. Increased HIV replication may contribute to the higher mortality associated with MAC bacteremia.     

Analysis of gradient algorithms for TLS-based adaptive IIR filters

Steepest descent gradient algorithms for unbiased equation error adaptive infinite impulse response (IIR) filtering are analyzed collectively for both the total least squares and mixed least squares-total least squares framework. These algorithms have a monic normalization that allows for a direct filtering implementation. We show that the algorithms converge to the desired filter coefficient vector. We achieve the convergence result by analyzing the stability of the equilibrium points and demonstrate that only the desired solution is locally stable. Additionally, we describe a region of initialization under which the algorithm converges to the desired solution. We derive the results using interlacing relationships between the eigenvalues of the data correlation matrices and their respective Schur complements. Finally, we illustrate the performance of these new approaches through simulation.     

Production of monoclonal antibodies using a secretion capture report web

We describe a novel method for the production of monoclonal antibodies using a secretion capture report web (SCRW). Following HAT selection in bulk culture, individual hybridomas are encapsulated in biotinylated agarose drops. Antibody secreted by the hybridoma is captured within the agarose drop using an avidin bridge and biotinylated anti-mouse immunoglobulin. The secreted antibody is detected by a fluorescent reporter which can be either a second anti-mouse antibody or an antigen. The binding of the reporter can be quantitated and the desired hybridoma directly cloned by flow cytometry. Multiparameter (i.e., two-color) reporter analysis can also be used to selectively enrich and clone rare hybridomas secreting antibodies directed to unique epitopes. The method allows the characterization of thousands of clones per second and the isolation of hundreds of clones per day.     

Detection of amphetamine following administration of fenproporex

Drugs that are metabolized to amphetamine or methamphetamine are potentially significant concerns in the interpretation of amphetamine-positive drug testing results. A number of different compounds have been reported to produce amphetamine in the urine of users. One of these compounds, fenproporex, has been shown to produce amphetamine. Previous reports indicate that the parent compound can be detected only for a few hours following administration, whereas the amphetamine can be detected for several days. Administration of fenproporex to five healthy volunteers resulted in amphetamine being detected in the urine of all subjects. Peak concentrations of amphetamine were detected at approximately 6-20 h postdose and ranged from approximately 1200 to 2100 ng/mL amphetamine. Amphetamine could be detected (> 5 ng/mL) in the urine for up to 119 h. Analysis of the metabolically produced amphetamine showed the presence of both enantiomers, which can be helpful in the differentiation of some illicit amphetamine use from the use of this precursor drug. More significantly, all samples that contained amphetamine at a concentration of at least 500 ng/mL were shown to also contain measurable amounts of the parent compound.     

Modelling central bursting in the extrusion of particulate reinforced metal matrix composite materials

Elastic-viscoplastic damage constitutive equations for particulate reinforced metal matrix composite (MMC) materials have been used to investigate central burst formation in extrusion. The equations have been implemented into finite-element software with which extrusion processes for a range of area reductions and volume fractions of particulate reinforcement have been simulated.The analyses carried out have enabled an extrusion limit diagram to be established for a particle reinforced aluminium matrix composite. The results show that for volume fraction of reinforcement up to approximately 20%, the influence of the volume fraction of particle on central bursting is small compared with the process parameters area reduction and semi-cone die angle. Hence, the method of selection of process parameters to avoid central bursting in MMC materials is likely to be the same as that for monolithic materials.The results show that with increasing volume fraction of reinforcement, significant changes in the patterns of damage evolution occur, with increasing volume fraction leading to the development of damage in the central region of the extrudate that does not exist for lower volume fractions. However, increasing the volume fraction of reinforcement tends to decrease the level of surface damage developing within the extrudate.