Dynamics of the in-run in ski jumping: a simulation study

A ski jumper tries to maintain an aerodynamic position in the in-run during changing environmental forces. The purpose of this study was to analyze the mechanical demands on a ski jumper taking the in-run in a static position. We simulated the in-run in ski jumping with a 4-segment forward dynamic model (foot, leg, thigh, and upper body). The curved path of the in-run was used as kinematic constraint, and drag, lift, and snow friction were incorporated. Drag and snow friction created a forward rotating moment that had to be counteracted by a plantar flexion moment and caused the line of action of the normal force to pass anteriorly to the center of mass continuously. The normal force increased from 0.88 G on the first straight to 1.65 G in the curve. The required knee joint moment increased more because of an altered center of pressure. During the transition from the straight to the curve there was a rapid forward shift of the center of pressure under the foot, reflecting a short but high angular acceleration. Because unrealistically high rates of change of moment are required, an athlete cannot do this without changing body configuration which reduces the required rate of moment changes.     

Defective CD4<Superscript>+</Superscript>CD25<Superscript>+</Superscript>regulatory T cell functioning in collagen-induced arthritis: an important factor in pathogenesis,counter-regulated by endogenous IFN-γ

Mice with a deficiency in IFN-γ or IFN-γ receptor (IFN-γR) are more susceptible to collagen-induced arthritis (CIA), an experimental autoimmune disease that relies on the use of complete Freund's adjuvant (CFA). Here we report that the heightened susceptibility of IFN-γR knock-out (KO) mice is associated with a functional impairment of CD4⁺CD25⁺ T_reg cells. Treatment of wild-type mice with depleting anti-CD25 antibody after CFA-assisted immunisation with collagen type II (CII) significantly accelerated the onset of arthritis and increased the severity of CIA. This is an indication of a role of T_reg cells in the effector phase of CIA. IFN-γR deficiency did not affect the number of CD4⁺CD25⁺ T cells in the central and peripheral lymphoid tissues. In addition, CD4⁺CD25⁺ T cells isolated from naive IFN-γR KO mice had a normal potential to suppress T cell proliferation in vitro. However, after immunisation with CII in CFA, the suppressive activity of CD4⁺CD25⁺ T cells became significantly more impaired in IFN-γR-deficient mice. Moreover, expression of the mRNA for Foxp3, a highly specific marker for T_reg cells, was lower. We further demonstrated that the effect of endogenous IFN-γ, which accounts for more suppressive activity in wild-type mice, concerns both T_reg cells and accessory cells. Our results demonstrate that the decrease in T_reg cell activity in CIA is counter-regulated by endogenous IFN-γ.     

Experimental evaluation of the power balance model of speed skating.

Prediction of speed skating performance with a power balance model requires assumptions about the kinetics of energy production, skating efficiency, and skating technique. The purpose of this study was to evaluate these parameters during competitive imitations for the purpose of improving model predictions. Elite speed skaters (n = 8) performed races and submaximal efficiency tests. External power output (P(o)) was calculated from movement analysis and aerodynamic models and ice friction measurements. Aerobic kinetics was calculated from breath-by-breath oxygen uptake (Vo(2)). Aerobic power (P(aer)) was calculated from measured skating efficiency. Anaerobic power (P(an)) kinetics was determined by subtracting P(aer) from P(o). We found gross skating efficiency to be 15.8% (1.8%). In the 1,500-m event, the kinetics of P(an) was characterized by a first-order system as P(an) = 88 + 556e(-0.0494t) (in W, where t is time). The rate constant for the increase in P(aer) was -0.153 s(-1), the time delay was 8.7 s, and the peak P(aer) was 234 W; P(aer) was equal to 234[1 - e(-0.153(t-8.7))] (in W). Skating position changed with preextension knee angle increasing and trunk angle decreasing throughout the event. We concluded the pattern of P(aer) to be quite similar to that reported during other competitive imitations, with the exception that the increase in P(aer) was more rapid. The pattern of P(an) does not appear to fit an "all-out" pattern, with near zero values during the last portion of the event, as assumed in our previous model (De Koning JJ, de Groot G, and van Ingen Schenau GJ. J Biomech 25: 573-580, 1992). Skating position changed in ways different from those assumed in our previous model. In addition to allowing improved predictions, the results demonstrate the importance of observations in unique subjects to the process of model construction.     

Effect of salt and pH on the activation of photoactive yellow protein and gateway mutants Y98Q and Y98F

We investigated the photocycle of mutants Y98Q and Y98F of the photoactive yellow protein (PYP) from Halorhodospira halophila. Y98 is located in the beta4-beta5 loop and is thought to interact with R52 in the alpha3-alpha4 loop thereby stabilizing this region. Y98 is conserved in all known PYP species, except in Ppr and Ppd where it is replaced by F. We find that replacement of Y98 by F has no significant effect on the photocycle kinetics. However, major changes were observed with the Y98Q mutant. Our results indicate a requirement for an aromatic ring at position 98, especially for recovery and a normal I1/I2 equilibrium. The ring of Y98 could stabilize the beta4-beta5 loop. Alternatively, the Y98 ring could transiently interact with the isomerized chromophore ring, thereby stabilizing the I2 intermediate in the I1/I2 equilibrium. For Y98Q, the decay of the signaling state I2' was slowed by a factor of approximately 40, and the rise of the I2 and I2' intermediates was slowed by a factor of 2-3. Moreover, the I1 intermediate is in a pH-dependent equilibrium with I2/I2' with the ratio of the I1 and I2 populations close to one at pH 7 and 50 mM KCl. From pH 5.5 to 8, the equilibrium shifts toward I1, with a pKa of approximately 6.3. Above pH 8, the populations of I1 and I2/I2' decrease due to an equilibrium between I1 and an additional species I1' which absorbs at approximately 425 nm (pKa approximately 9.8) and which we believe to be an I2-like form with a surface-exposed deprotonated chromophore. The I1/I2/I2' equilibrium was found to be strongly dependent on the KCl concentration, with salt stabilizing the signaling state I2' up to 600 mM KCl. This salt-induced transition to I2' was analyzed and interpreted as ion binding to a specific site. Moreover, from analysis of the amplitude spectra, we conclude that KCl exerts its major effect on the I2 to I2' transition, i.e., the global conformational change leading to the signaling state I2' and the exposure of a hydrophobic surface patch. In wild type and Y98F, the I1/I2 equilibrium is more on the side of I2/I2' as compared to Y98Q but is also salt-dependent at pH 7. The I2 to I2' transition appears to be controlled by an ionic lock, possibly involving the salt bridge between K110 on the beta-scaffold and E12 on the N-terminal cap. Salt binding would break the salt bridge and weaken the interaction between the two domains, facilitating the release of the N-terminal domain from the beta-scaffold in the formation of I2'.     

Photoreversal kinetics of the I1 and I2 intermediates in the photocycle of photoactive yellow protein by double flash experiments with variable time delay

We investigated the kinetics of photoreversal from the I(1) and I(2) intermediates of photoactive yellow protein (PYP) by time-resolved optical absorption spectroscopy with double flash excitation. A first flash, at 430 nm, initiated the photocycle. After a variable time delay, the I(1) intermediate was photoreversed by a second flash, at 500 nm, or a mixture of I(2) and I(2)' intermediates was photoreversed by a second flash, at 355 nm. By varying the delay from 1 micros to 3 s, we were able to selectively excite the intermediates I(1), I(2), and I(2)'. The photoreversal kinetics of I(2) and I(2)' at 21 different delays and two wavelengths (340 and 450 nm) required two exponentials for a global fit with time constants of tau(1) = 57 +/- 5 micros and tau(2) = 380 +/- 40 micros (pH 6, 20 degrees C). These were assigned to photoreversal from sequential I(2) and I(2)' intermediates, respectively. The good agreement of the delay dependence of the two amplitudes, A(1) and A(2), with the time dependence of the I(2) and I(2)' populations provided strong evidence for the sequential model. The persistence of A(1) beyond delay times of 5 ms and its decay, together with A(2) around 500 ms, suggest moreover that I(2) and I(2)' are in thermal equilibrium. The wavelength dependence of the photoreversal kinetics was measured at 26 wavelengths from 510 to 330 nm at the two fixed delays of 1 and 10 ms. These data also required two exponentials for a global fit with tau(1) = 59 +/- 5 micros and tau(2) = 400 +/- 40 micros, in good agreement with the delay results. Photoreversal from I(2)' is slower than from I(2), since, in addition to chromophore protonation, the global conformational change has to be reversed. Our data thus provide a first estimate of about 59 micros for deprotonation and 400 micros for the structural change, which also occurs in the thermal decay of the signaling state but is obscured there since reisomerization is rate-limiting. The first step in photoreversal is rapid cis-trans isomerization of the chromophore, which we could not resolve, but which was detected by the instantaneous increase in absorbance between 330 and 380 nm. In agreement with this observation, the spectrum of the I(2)'(trans) intermediate, derived from the A(2) amplitude spectrum, has a much larger extinction coefficient than the spectrum of the I(2)'(cis) intermediate. With a first flash, at 430 nm, and a second flash, at 500 nm, we observed efficient photoreversal of the I(1) intermediate at a delay of 20 micros when most molecules in the cycle are in I(1). We conclude that each of the three intermediates studied can be reversed by a laser flash. Depending on the progression of the photocycle, reversal becomes slower with the time delay, thus mirroring the individual steps of the forward photocycle.     

Islet amyloid polypeptide-induced membrane leakage involves uptake of lipids by forming amyloid fibers

Fibril formation of islet amyloid polypeptide (IAPP) is associated with cell death of the insulin-producing pancreatic beta-cells in patients with Type 2 Diabetes Mellitus. A likely cause for the cytotoxicity of human IAPP is that it destroys the barrier properties of the cell membrane. Here, we show by fluorescence confocal microscopy on lipid vesicles that the process of hIAPP amyloid formation is accompanied by a loss of barrier function, whereby lipids are extracted from the membrane and taken up in the forming amyloid deposits. No membrane interaction was observed when preformed fibrils were used. It is proposed that lipid uptake from the cell membrane is responsible for amyloid-induced membrane damage and that this represents a general mechanism underlying the cytotoxicity of amyloid forming proteins.     

Interaction between dopamine and its transporter: role of intracellular sodium ions and membrane potential

The present study addresses the effect of intracellular Na(+) and membrane potential on the binding of dopamine (DA) to the dopamine transporter (DAT). Perforation of plasma membranes of DAT-expressing cells with gramicidin diminished DA uptake and decreased the potency (increases K(i)) of DA in inhibiting the binding of cocaine analog [(3)H]2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT). It also compromised the ability of external Na(+) to reduce DA K(i). No substantial effect on DA K(i) was observed upon gramicidin treatment in Na(+)-free buffer, membrane depolarization with high [K(+)](o), or elevation of [Na(+)](i) with monensin under non-depolarizing conditions. Elevation of DA K(i) was greater at more positive potentials when [Na(+)](i) was raised to a similar level, or at higher [Na(+)](i) when the membrane was depolarized to a similar level. In cells expressing D313N DAT, DA K(i) was significantly higher but less sensitive to gramicidin than that in wild-type (WT) cells. In contrast, DA K(i) in cell-free membranes was insensitive to Na(+), gramicidin, and D313N mutation. The data suggest that (i) intracellular Na(+) plays a role in affecting the external access to DA binding sites at DAT on depolarized plasma membranes of cells, and (ii) access to DA binding sites in cell-free membranes may occur from the intracellular side of the membrane. Unlike DA binding, CFT binding to both cells and membranes was sensitive to Na(+) and D313N mutation but insensitive to gramicidin, consistent with exclusively external access to sites that are different from but conformationally linked to those for DA.     

Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine.     

Mucosal vaccine delivery of antigens tightly bound to an adjuvant particle made from food-grade bacteria

Mucosal immunization with subunit vaccines requires new types of antigen delivery vehicles and adjuvants for optimal immune responses. We have developed a non-living and non-genetically modified gram-positive bacterial delivery particle (GEM) that has built-in adjuvant activity and a high loading capacity for externally added heterologous antigens that are fused to a high affinity binding domain. This binding domain, the protein anchor (PA), is derived from the Lactococcus lactis AcmA cell-wall hydrolase, and contains three repeats of a LysM-type cell-wall binding motif. Antigens are produced as antigen-PA fusions by recombinant expression systems that secrete the hybrid proteins into the culture growth medium. GEM particles are then used as affinity beads to isolate the antigen-PA fusions from the complex growth media in a one step procedure after removal of the recombinant producer cells. This procedure is also highly suitable for making multivalent vaccines. The resulting vaccines are stable at room temperature, lack recombinant DNA, and mimic pathogens by their bacterial size, surface display of antigens and adjuvant activity of the bacterial components in the GEM particles. The GEM-based vaccines do not require additional adjuvant for eliciting high levels of specific antibodies in mucosal and systemic compartments.