Dynein arms are oscillating force generators

Eukaryotic flagella beat rhythmically. Dynein is a protein that powers flagellar motion, and oscillation may be inherent to this protein. Here we determine whether oscillation is a property of dynein arms themselves or whether oscillation requires an intact axoneme, which is the central core of the flagellum and consists of a regular array of microtubules. Using optical trapping nanometry, we measured the force generated by a few dynein arms on an isolated doublet microtubule. When the dynein arms on the doublet microtubule contact a singlet microtubule and are activated by photolysis of caged ATP8, they generate a peak force of approximately 6pN and move the singlet microtubule over the doublet microtubule in a processive manner. The force and displacement oscillate with a peak-to-peak force and amplitude of approximately 2 pN and approximately 30 nm, respectively. The geometry of the interaction indicates that very few (possibly one) dynein arms are needed to generate the oscillation. The maximum frequency of the oscillation at 0.75 mM ATP is approximately 70 Hz; this frequency decreases as the ATP concentration decreases. A similar oscillatory force is also generated by inner dynein arms alone on doublet microtubules that are depleted of outer dynein arms. The oscillation of the dynein arm may be a basic mechanism underlying flagellar beating.     

Calcium ion as a second messenger for o-nitrophenylsulfenyl-adrenocorticotropin (NPS-ACTH) and ACTH in bovine adrenal steroidogenesis

o-Nitrophenyl sulfenyl-modified ACTH (NPS-ACTH) stimulated steroidogenesis acutely in bovine fasciculata-reticularis cells without increase in cellular cAMP synthesis. Application of NPS-ACTH to the cultured cells induced Ca2+ signals in individual cells as detected by video-enhanced microscopic fluorescence measurements. The percentage of Ca2+ signaling cells corresponded well with the increase of steroidogenesis induced by NPS-ACTH below 1 nM. Treatment of the cells with nicardipine, a Ca2+ channel blocker, suppressed the Ca2+ signals except for the transient increase just after the addition of NPS-ACTH and also blocked completely the stimulative effect on the steroidogenesis of NPS-ACTH below 1 nM. At a dosage of NPS-ACTH higher than 10 nM, the stimulative effect of steroidogenesis was partly suppressed by nicardipine and also by AA-861, a lipoxygenase inhibitor. The action of NPS-ACTH might be mediated by both Ca2+ and lipoxygenase metabolite(s) of arachidonic acid as dual second messengers. The effect of ACTH in pM range on the steroidogenesis was suppressed completely by the treatment with nicardipine and AA-861 at the same time, indicating that the action was mediated by both Ca2+ and the lipoxygenase metabolite(s) but not by cAMP. cAMP plays a significant role as a second messenger for ACTH action only at ACTH concentrations greater than 10 pM.     

Three-dimensional measurement of ice crystals in frozen beef with a micro-slicer image processing system

A novel technique has been developed for measuring the three-dimensional (3-D) structure and distribution of ice crystals formed in frozen beef by using a micro-slicer image processing system (MSIPS). The system has functions to reconstruct the 3-D image based on the image data of exposed cross-sections obtained by multi-slicing of a frozen sample with the minimum thickness of 1 μm and to display the internal structure as well as an arbitrary cross-section of the sample choosing observation angles. The size and distribution of ice crystals can be determined from the 2-D quantitative information, such as the periphery and area of the crystals. The effects of freezing conditions on the morphology and distribution of the ice crystals were demonstrated quantitatively from the observations of raw beef stained by fluorescent indicator. For the samples frozen at −15 °C, the network structure of ice crystals were observed mainly at intercellular space, having approximately 100 μm in cross-sectional size, while that prepared at −120 °C showed the spherical crystals of 10–20 μm in diameter within the cells. The 3-D image of the sample demonstrated that the growth of ice columns was restricted by the intrinsic structure of muscle fibers. The proposed method provided a new tool to investigate the effects of freezing conditions on the size, morphology and distribution of ice crystals.     

Design of oral drug delivery systems - Past, present and future

This paper reviews the past and the present thinking about peroral delivery research and development and how future approaches may be significantly different, both qualitatively and quantitatively. The future should involve the use of comprehensive models capable of incorporating physico-chemical data and biological information such as gastrointestinal flow, how and where drug absorption occurs, and whether and where metabolism of the drug occurs during gastrointestinal transit. Special challenges would involve the use of such models in research protocols in the optimization of drug delivery systems.     

Rheological interpretation of heat generation associated with fatigue of polycarbonate

Temperature change was measured of polycarbonate under monotonically increasing tensile and pulsating tensile loads. In the former case, the specimen temperature began to rise when an appreciable amount of viscoelastic strain was noticed on the stress—strain diagram. The rise, θ_v, could be formulated as a function of the viscoelastic strain, εv: In fatigue tests, the average temperature began to rise immediately after the decrease due to the thermoelastic effect. The amount of the heat generation, σ, was nearly constant for each cycle throughout the fatigue process and has a relation to the fatigue life, N_f, (α? a)·N_f = constant, where a is another adjustable constant. From a rheological aspect of dissipation energy, the equation is transformed to a relation between the viscoelastic strain and the fatigue life as εV^1/2 · N_f = constant, which is similar to the one for metals given by Manson and Coffin.⁶ The temperature rise in the fatigue was also related to the viscoelastic strain. The relation is of the same form as for static tension but less by a factor of one order.     

Simple determination of residual anticoccidial drugs (diclazuril and nicarbazin) in chicken tissues by HPLC

A simple and rapid determination of anticoccidial drug residues, diclazuril (DCZ) and nicarbazin (NCZ), in chicken tissues has been developed. DCZ and NCZ were extracted with acetonitrile from chicken liver, muscle, and fat. The extract was rinsed with n-hexane saturated with acetonitrile and then evaporated. The residue was dissolved in 1.4 mL of acetonitrile-methanol (1:1), then 1.0 mL of n-hexane saturated with acetonitrile-methanol (1:1) was added, and the mixture was partitioned by the addition of 0.6 mL of water. DCZ and NCZ in the aqueous layers were determined by HPLC on an Xterra RP-18 column with acetonitrile-0.5% ammonium acetate containing 0.01 mol/L tetra-n-butylammonium hydrogen sulfate (43:57) as the mobile phase. The mean recoveries (n = 5) of DCZ and NCZ spiked in chicken tissues at the maximum residue levels were 92.0-95.6% (CV 2.4-3.0%) and 87.3-89.4% (CV 1.7-2.8%), respectively. The detection limits of DCZ and NCZ were 0.01 and 0.004 microgram/g, respectively.     

Analysis of protein adsorption and binding at biosensor polymer interfaces using X-ray photon spectroscopy and scanning electrochemical microscopy

We describe a method, based on X-ray photoelectron spectroscopy (XPS) measurements, to assess the extent of protein adsorption or binding on a variety of different muTAS and biosensor interfaces. Underpinning this method is the labeling of protein molecules with either iodine- or bromine-containing motifs by using protocols previously developed for radiotracer studies. Using this method, we have examined the adsorption and binding properties of a variety of modified electrodeposited polymer interfaces as well as other materials used in muTAS device fabrication. Using polymer interfaces modified with poly(propylene glycol) (PPG) chains, our results indicate that a chain of at least approximately 30 monomer units is required to inhibit nonspecific adsorption from concentrated protein solutions. The XPS methodology was also used to probe specific binding of avidins and enzyme conjugates thereof to biotinylated and mixed biotin/PPG-modified polymer interfaces. In one example, using competitive binding, it was established that the mode of binding of a peroxidase-streptavidin conjugate to a biotinylated modified polymer interface was primarily via the streptavidin moiety (as opposed to nonspecific binding via the enzyme conjugate). XPS evaluation of nonspecific and specific peroxidase-streptavidin immobilization on various functionalized polymers has guided the design and fabrication of functionalized interdigitated electrodes in a biosensing muTAS device. Subsequent characterization of this device using scanning electrochemical microscopy (SECM) corroborated the adsorption and binding previously inferred from XPS measurements on macroscale electrodes.     

Study on chemical structures of poly (tetrafluoroethylene-co-perfluoroalkylvinylether) by soft-EB irradiation in solid and molten state

Poly (tetrafluoroethylene-co-perfluoroalkylvinylether) (PFA) was irradiated by soft electron beam (soft-EB) under nitrogen gas atmosphere in solid-state and its molten state, respectively. The changes of thermal property and chemical structures of irradiated PFA in solid-state and molten state were studied by differential scanning calorimetric analysis (DSC) and solid-state ¹⁹F magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. By DSC analysis, the melting temperature shifted to lower temperatures, and crystallinity decreased with increasing soft-EB dose. By solid-state ¹⁹F MAS NMR spectroscopy, the new signals was observed and the detected new signals in irradiated PFA at 315 °C and at 30 °C were due to the tertiary carbon group with branching site (Y-type crosslinking site), perfluoro-propylene site and chain end methylene groups, respectively.Moreover, the molar ratio of perfluoroalkylvinylether (FAVE) structure to -CF₂- units decreased with increasing dose.