Association of ABCA1 with syntaxin 13 and flotillin-1 and enhanced phagocytosis in tangier cells

The ATP-binding cassette transporter A1 (ABCA1) facilitates the cellular release of cholesterol and choline-phospholipids to apolipoprotein A-I (apoA-I) and several studies indicate that vesicular transport is associated with ABCA1 function. Syntaxins play a major role in vesicular fusion and have also been demonstrated to interact with members of the ABC-transporter family. Therefore, we focused on the identification of syntaxins that directly interact with ABCA1. The expression of syntaxins and ABCA1 in cultured human monocytes during M-CSF differentiation and cholesterol loading was investigated and syntaxins 3, 6, and 13 were found induced in foam cells together with ABCA1. Immunoprecipitation experiments revealed a direct association of syntaxin 13 and full-length ABCA1, whereas syntaxin 3 and 6 failed to interact with ABCA1. The colocalization of ABCA1 and syntaxin 13 was also shown by immunofluorescence microscopy. Silencing of syntaxin 13 by small interfering RNA (siRNA) led to reduced ABCA1 protein levels and hence to a significant decrease in apoA-I-dependent choline-phospholipid efflux. ABCA1 is localized in Lubrol WX-insoluble raft microdomains in macrophages and syntaxin 13 and flotillin-1 were also detected in these detergent resistant microdomains along with ABCA1. Syntaxin 13, flotillin-1, and ABCA1 were identified as phagosomal proteins, indicating the involvement of the phagosomal compartment in ABCA1-mediated lipid efflux. In addition, the uptake of latex phagobeads by fibroblasts with mutated ABCA1 was enhanced when compared with control cells and the recombinant expression of functional ABCA1 normalized the phagocytosis rate in Tangier fibroblasts. It is concluded that ABCA1 forms a complex with syntaxin 13 and flotillin-1, residing at the plasma membrane and in phagosomes that are partially located in raft microdomains.     

Delayed-relaxed response explained by hyperactivation of RelE

Escherichia coli encodes two rel loci, both of which contribute to the control of synthesis of macromolecules during amino acid starvation. The product of relA (ppGpp synthetase I) is responsible for the synthesis of guanosine tetraphosphate, ppGpp, the signal molecule that exerts stringent control of stable RNA synthesis. The second rel locus, relBE, was identified by mutations in relB that confer a so-called 'delayed-relaxed response' characterized by continued RNA synthesis after a lag period of approximately 10 min after the onset of amino acid starvation. We show here that the delayed-relaxed response is a consequence of hyperactivation of RelE. As in wild-type cells, [ppGpp] increased sharply in relB101 relE cells after the onset of starvation, but returned rapidly to the prestarvation level. RelE is a global inhibitor of translation that is neutralized by RelB by direct protein-protein interaction. Lon protease activates RelE during amino acid starvation by degradation of RelB. We found that mutations in relB that conferred the delayed-relaxed phenotype destabilized RelB. Such mutations confer severe RelE-dependent inhibition of translation during amino acid starvation, indicating hyperactivation of RelE. Hyperactivation of RelE during amino acid starvation was shown directly by measurement of RelE-mediated cleavage of tmRNA. The RelE-mediated shutdown of translation terminated amino acid consumption and explains the rapid restoration of the ppGpp level observed in relB mutant cells. Restoration of the prestarvation level of ppGpp, in turn, allows for the resumption of stable RNA synthesis seen during the delayed-relaxed response.     

Serum insulin-like growth factor I levels in growth hormone-deficient adults: influence of sex steroids

Measurement of serum insulin-like growth factor I (IGF-I) concentrations remains the single most important tool in the evaluation of growth hormone (GH) replacement in GH-deficient adults, and the therapeutic goal is to maintain the level within the age-adjusted normal range. In healthy adults, IGF-I levels do not differ between males and females, whereas spontaneous GH secretion is approximately twofold higher in females. Untreated GH-deficient women exhibit lower IGF-I levels compared with men, and the increase in serum IGF-I during GH replacement is also significantly less. Put together, these data suggest resistance to GH in women, which in healthy individuals is compensated for by increased GH secretion. Administration of oral oestrogen in healthy post-menopausal women suppresses hepatic IGF-I production and increases pituitary GH release, and oral oestrogen replacement in women with GH deficiency lowers IGF-I concentrations and increases the amount of GH necessary to obtain IGF-I target levels during treatment. These data clearly suggest that hepatic suppression of IGF-I production by oestrogen subserves the gender difference in GH sensitivity, but it is also likely that sex steroids may interact with the GH/IGF axis at further levels. There is also circumstantial evidence to indicate that testosterone stimulates IGF-I production, and it is speculated that a certain threshold level of androgens is essential to ensure hepatic IGF-I production. Whether these data should translate into earlier discontinuation of oestrogen replacement therapy in adult women with hypopituitarism merits consideration.     

Functionalization of poly(methyl methacrylate) (PMMA) as a substrate for DNA microarrays

A chemical procedure was developed to functionalize poly(methyl methacrylate) (PMMA) substrates. PMMA is reacted with hexamethylene diamine to yield an aminated surface for immobilizing DNA in microarrays. The density of primary NH₂ groups was 0.29 nmol/cm². The availability of these primary amines was confirmed by the immobilization of DNA probes and hybridization with a complementary DNA strand. The hybridization signal and the hybridization efficiency of the chemically aminated PMMA slides were comparable to the hybridization signal and the hybridization efficiency obtained from differently chemically modified PMMA slides, silanized glass, commercial silylated glass and commercial plastic Euray™ slides. Immobilized and hybridized densities of 10 and 0.75 pmol/cm², respectively, were observed for microarrays on chemically aminated PMMA. The immobilized probes were heat stable since the hybridization performance of microarrays subjected to 20 PCR heat cycles was only reduced by 4%. In conclusion, this new strategy to modify PMMA provides a robust procedure to immobilize DNA, which is a very useful substrate for fabricating single use diagnostics devices with integrated functions, like sample preparation, treatment and detection using microfabrication and microelectronic techniques.     

Acid-induced unfolding of the amino-terminal domains of the lethal and edema factors of anthrax toxin

The two enzymatic components of anthrax toxin, lethal factor (LF) and edema factor (EF), are transported to the cytosol of mammalian cells by the third component, protective antigen (PA). A heptameric form of PA binds LF and/or EF and, under the acidic conditions encountered in endosomes, generates a membrane-spanning pore that is thought to serve as a passageway for these enzymes to enter the cytosol. The pore contains a 14-stranded transmembrane beta-barrel that is too narrow to accommodate a fully folded protein, necessitating that LF and EF unfold, at least partly, in order to pass. Here, we describe the pH-dependence of the unfolding of LF(N) and EF(N), the 30kDa N-terminal PA-binding domains, and minimal translocatable units, of LF and EF. Equilibrium chemical denaturation studies using fluorescence and circular dichroism spectroscopy show that each protein unfolds via a four-state mechanism: N<-->I<-->J<-->U. The acid-induced N-->I transition occurs within the pH range of the endosome (pH 5-6). The I state predominates at lower pH values, and the J and U states are populated significantly only in the presence of denaturant. The I state is compact and has characteristics of a molten globule, as shown by its retention of significant secondary structure and its ability to bind an apolar fluorophore. The N-->I transition leads to an overall 60% increase in buried surface area exposure. The J state is expanded significantly and has diminished secondary structure content. We analyze the different protonation states of LF(N) and EF(N) in terms of a linked equilibrium proton binding model and discuss the implications of our findings for the mechanism of acidic pH-induced translocation of anthrax toxin. Finally, analysis of the structure of the transmembrane beta-barrel of PA shows that it can accommodate alpha-helix, and we suggest that the steric constraints and composition of the lumen may promote alpha-helix formation.     

The structure of endo-beta-1,4-galactanase from Bacillus licheniformis in complex with two oligosaccharide products

The beta-1,4-galactanase from Bacillus licheniformis (BLGAL) is a plant cell-wall-degrading enzyme involved in the hydrolysis of beta-1,4-galactan in the hairy regions of pectin. The crystal structure of BLGAL was determined by molecular replacement both alone and in complex with the products galactobiose and galactotriose, catching a first crystallographic glimpse of fragments of beta-1,4-galactan. As expected for an enzyme belonging to GH-53, the BLGAL structure reveals a (betaalpha)(8)-barrel architecture. However, BLGAL betaalpha-loops 2, 7 and 8 are long in contrast to the corresponding loops in structures of fungal galactanases determined previously. The structure of BLGAL additionally shows a calcium ion linking the long betaalpha-loops 7 and 8, which replaces a disulphide bridge in the fungal galactanases. Compared to the substrate-binding subsites predicted for Aspergillus aculeatus galactanase (AAGAL), two additional subsites for substrate binding are found in BLGAL, -3 and -4. A comparison of the pattern of galactan and galactooligosaccharides degradation by AAGAL and BLGAL shows that, although both are most active on substrates with a high degree of polymerization, AAGAL can degrade galactotriose and galactotetraose efficiently, whereas BLGAL prefers longer oligosaccharides and cannot hydrolyze galactotriose to any appreciable extent. This difference in substrate preference can be explained structurally by the presence of the extra subsites -3 and -4 in BLGAL.     

Longitudinal tracking of muscular power changes of NCAA Division I collegiate women gymnasts

Gymnastics relies upon power as a critical component of sports-specific fitness. The purpose of this study was to monitor long-term training adaptations in the power of National Collegiate Athletics Association Division I women gymnasts. Twenty members of a women's gymnastic team (aged 18-22) were tracked over 3 years with the first year a baseline year of testing. Whole body power for the counter-movement (CMJ) and squat (SJ) vertical jump was obtained via force plate analyses at 2 assessment time points during each year (February and November). Results showed significant (p < or = 0.05) and continued increases in peak power output in the CMJ and SJ at each biannual assessment. Improvements of 46% (+1010 W) for the CMJ and 43% (+900 W) for the SJ were observed at the end of the tracking period. Peak power for the CMJ and SJ were recorded at 3210 W (+/-350 W) and 3000 W (+/-152 W), respectively. Associated improvements in the time to peak power of 36% (-0.239 second) and 38% (-0.151 second) were also found for the CMJ and SJ. There were no significant changes in body mass or total skinfold thickness, however, a shift toward improved fat free mass (i.e., lean muscle mass) was apparent. These data underscore the importance that specificity, and more importantly power development, should play in the conditioning of collegiate women gymnasts' training programs.     

Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy

To study the orientation and dynamics of myosin, we measured fluorescence polarization of single molecules and ensembles of myosin decorating actin filaments. Engineered chicken gizzard regulatory light chain (RLC), labeled with bisiodoacetamidorhodamine at cysteine residues 100 and 108 or 104 and 115, was exchanged for endogenous RLC in rabbit skeletal muscle HMM or S1. AEDANS-labeled actin, fully decorated with labeled myosin fragment or a ratio of approximately 1:1000 labeled:unlabeled myosin fragment, was adhered to a quartz slide. Eight polarized fluorescence intensities were combined with the actin orientation from the AEDANS fluorescence to determine the axial angle (relative to actin), the azimuthal angle (around actin), and RLC mobility on the <10 ms timescale. Order parameters of the orientation distributions from heavily labeled filaments agree well with comparable measurements in muscle fibers, verifying the technique. Experiments with HMM provide sufficient angular resolution to detect two orientations corresponding to the two heads in rigor. Experiments with S1 show a single orientation intermediate to the two seen for HMM. The angles measured for HMM are consistent with heads bound on adjacent actin monomers of a filament, under strain, similar to predictions based on ensemble measurements made on muscle fibers with electron microscopy and spectroscopic experiments.     

Periodate oxidation of chitosans with different chemical compositions

Periodate oxidation of chitosans with different chemical compositions were investigated by determining the consumption of periodate consumed, and the amount of ammonia and formaldehyde liberated during the reaction. Oxidised chitosans were further characterised by size-exclusion chromatography with online multi-angle light scattering (SEC-MALLS) to obtain the molecular weight distributions, and by elemental analysis to obtain the N/C ratio. Chitosans became only partially oxidised by periodate, reaching degrees of oxidation around 0.5, when oxidising with excess periodate. Overconsumption of periodate is attributed to the extensive depolymerisation, which occurs concomitantly with the oxidation, thereby exposing novel reducing and non-reducing ends which consume additional periodate. Both the rate and extent of overoxidation, and the rate of depolymerisation decreased with increasing F(A). A chitosan-specific degradation mechanism is probably involved in the depolymerisation in addition to the general free-radical-mediated degradation.     

Evidence of piezoelectric resonance in isolated outer hair cells

Our results demonstrate high-frequency electrical resonances in outer hair cells (OHCs) exhibiting features analogous to classical piezoelectric transducers. The fundamental (first) resonance frequency averaged f(n) approximately 13 kHz (Q approximately 1.7). Higher-order resonances were also observed. To obtain these results, OHCs were positioned in a custom microchamber and subjected to stimulating electric fields along the axis of the cell (1-100 kHz, 4-16 mV/80 microm). Electrodes embedded in the side walls of the microchamber were used in a voltage-divider configuration to estimate the electrical admittance of the top portion of the cell-loaded chamber (containing the electromotile lateral wall) relative to the lower portion (containing the basal plasma membrane). This ratio exhibited resonance-like electrical tuning. Resonance was also detected independently using a secondary 1-MHz radio-frequency interrogation signal applied transversely across the cell diameter. The radio-frequency interrogation revealed changes in the transverse electric impedance modulated by the axial stimulus. Modulation of the transverse electric impedance was particularly pronounced near the resonant frequencies. OHCs used in our study were isolated from the apical region of the guinea pig cochlea, a region that responds exclusively to low-frequency acoustic stimuli. In this sense, electrical resonances we observed in vitro were at least an order of magnitude higher (ultrasonic) than the best physiological frequency of the same OHCs under acoustic stimuli in vivo. These resonance data further support the piezoelectric theory of OHC function, and implicate piezoelectricity in the broad-band electromechanical behavior of OHCs underlying mammalian cochlear function.