A fluorescence depolarization study of the orientational distribution of crossbridges in muscle fibres

A fluorescence depolarization study of the orientational distribution of crossbridges in dye-labelled muscle fibres is presented. The characterization of this distribution is important since the rotation of crossbridges is a key element in the theory of muscle contraction. In this study we exploited the advantages of angle-resolved experiments to characterize the principal features of the orientational distribution of the crossbridges in the muscle fibre. The directions of the transition dipole moments in the frame of the dye and the orientation and motion of the dye relative to the crossbridge determined previously were explicitly incorporated into the analysis of the experimental data. This afforded the unequivocal determination of all the second and fourth rank order parameters. Moreover, this additional information provided discrimination between different models for the orientational behaviour of the crossbridges. Our results indicate that no change of orientation takes place upon a transition from rigor to relaxation. The experiments, however, do no rule out a conformational change of the myosin S 1 during the transition. Correspondence to: Y. K. Levine     

Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa-Effekt

Zusammenfassung Es wurden analysenreine Proben der Romanowsky-Farbstoffe Eosin Y, Erythrosin B und Tetrachlorfluoreszein hergestellt.Im DC der Farbstoffproben konnten keine Verunreinigungen nachgewiesen werden. Die Absorptionsspektren der Farbstoffdianionen in wäßriger alkalischer Lösung und der Farbstoffsäuren in 95%igem Ethanol wurden bei sehr kleinen Farbstoffkonzentrationen gemessen und der molare Extinktionskoeffizient der längstwelligen Absorptionsbande der monomeren Farbstoffspezies bestimmt (Tabelle 1). Die Extinktionskoeffizienten können zur Standardisierung von Farbstoffproben verwendet werden. Die Absorptionsspektren von Eosin Y hängen in wäßriger Lösung von der Farbstoffkonzentration ab. Aus der Konzentrationsabhängigkeit wurden mit einem neuen, sehr empfindlichen Verfahren zwei Assoziationsgleichgewichte ermittelt. Bereits in sehr verdünnter Lösung bilden sich Dimere, bei erhöhter Konzentration Tetramere, Die Dissoziationskonstante der DimerenD in MonomereM beträgt bei pH=12, 293K:K ₂₁=2,9 × 10^–5 M; der TetramerenQ in DimereD:K ₄₂=2,4 × 10^–3 M. Aus den gemessenen Spektren von Eosinlösungen verschiedener Konzentration, pH=12, und den GleichgewichtskonstantenK ₂₁,K ₄₂ haben wir die Spektren der reinen Monomeren, Dimeren und Tetrameren bestimmt.M hat eine langwellige Absorptionsbande: , _M =1,03 x 10₅ M_-1 cm_-1;D eine Bande: , _D =1,74 x 10₅ M_-1 cm_-1;Q zwei Banden: , , _Q1=1,65 x 10⁵, _Q2=1,96 x 10⁵ M^-1 cm^-1. Das Absorptionsspektrum der Dimeren wird quantenmechanisch interpretiert.

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Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, Erythrosin B, tetrachlorofluorescein, Spectroscopic characterization of pure dyes, association of Eosin Y

Summary Analytically pure smaples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependend on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimersD in monomersM at 293 K, pH=12, isK ₂₁=2,9×10^–5 M; of the tetramersQ in dimersDK ₄₂=2,4×10^–3 M. From the experimental spectra of eosin solutions at various concentrations, pH=12, and the equilibrium constantsK ₂₁,K ₄₂ the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, , _M =1,03 x 10₅ M_-1 cm_-1;D also one absorption band, , _D =1,74 x 10₅ M_-1 cm_-1;Q two absorption bands, , , _Q1=1,65 x 10⁵, _Q2=1,96 x 10⁵ M^-1 cm^-1. The absorption spectrum of the dimers is discussed by quantum mechanics.

     

Identification of post-translational modified amino acids

Summary Methylated lysines (N -mono-methylated, N -di-methylated and N -tri-methylated) have been identified after derivatization with orthophthaldialdehyde (OPA) by using pre-column and post-column derivatization techniques.Also the N -acetylated lysine and N -formylated lysine have been identified by OPA post-column derivatization techniques but only in free form due to their instability under acidic conditions which are used for protein hydrolysis.Additionally, all the modified amino acids mentioned above have been derivatized with DABITC/PITC, an Edman reagent, and identified as DABTH-derivatives on thin-layer polyamide sheets.     

Dose-dependent nonlinear response of the main phase-transition temperature of phospholipid membranes to alcohols

Summary The effect of 1-alkanols upon the main phase-transition temperature of phospholipid vesicle membranes between gel and liquid-crystalline phases was not a simple monotonic function of alkanol concentration. For instance, 1-decanol decreased the transition temperature at low concentrations, but increased it at high concentrations, displaying a minimal temperature. This concentration-induced biphasic effect cannot be explained by the van't Hoff model on the effect of impurities upon the freezing point. To explain this nonlinear response, a theory is presented which treats the effect of 1-alkanols (or any additives) on the transition temperature of phospholipid membranes in a three-component mixture. By fitting the experimental data to the theory, the enthalpy of the phase transition H ^* and the interaction energy, _AB ^* between the additive and phospholipid molecules may be estimated. The theory predicts that when _AB ^* >2 (where _AB ^* = _AB,/RT _o,T _o being the transition temperature of phospholipid), both maximum and maximum transition temperatures should exist. When _AB ^* = 2, only one inflection point exists. When _AB ^* < 2, neither maximum nor minimum exists. The alkanol concentration at which the transition temperature is minimum (X _min) depends on the _AB ^* value: the larger the _AB ^* values, the smaller theX _min. When _AB ^* is large enough,X _min values become so small that the plot T vs.X shows positive T in almost all alkanol concentrations. The interaction energy between 1-alkanols and phospholipid molecules increased with the increase in the carbon chain-length of 1-alkanols. In the case of the dipalmitcylphosphatidylcholine vesicle membrane, the carbon chain-length of 1-alkanols that caused predominantly positive T was about 12.     

Electron tomography of fast frozen, stretched rigor fibers reveals elastic distortions in the myosin crossbridges

As a first step toward freeze-trapping and 3-D modeling of the very rapid load-induced structural responses of active myosin heads, we explored the conformational range of longer lasting force-dependent changes in rigor crossbridges of insect flight muscle (IFM). Rigor IFM fibers were slam-frozen after ramp stretch (1000 ms) of 1-2% and freeze-substituted. Tomograms were calculated from tilt series of 30 nm longitudinal sections of Araldite-embedded fibers. Modified procedures of alignment and correspondence analysis grouped self-similar crossbridge forms into 16 class averages with 4.5 nm resolution, revealing actin protomers and myosin S2 segments of some crossbridges for the first time in muscle thin sections. Acto-S1 atomic models manually fitted to crossbridge density required a range of lever arm adjustments to match variably distorted rigor crossbridges. Some lever arms were unchanged compared with low tension rigor, while others were bent and displaced M-ward by up to 4.5 nm. The average displacement was 1.6 +/- 1.0 nm. "Map back" images that replaced each unaveraged 39 nm crossbridge motif by its class average showed an ordered mix of distorted and unaltered crossbridges distributed along the 116 nm repeat that reflects differences in rigor myosin head loading even before stretch.     

Tomographic Three-dimensional Reconstruction of Insect Flight Muscle Partially Relaxed by AMPPNP and Ethylene Glycol

Rigor insect flight muscle (IFM) can be relaxed without ATP by increasing ethylene glycol concentration in the presence of adenosine 5′-[β′γ- imido]triphosphate (AMPPNP). Fibers poised at a critical glycol concentration retain rigor stiffness but support no sustained tension (“glycol-stiff state”). This suggests that many crossbridges are weakly attached to actin, possibly at the beginning of the power stroke. Unaveraged three-dimensional tomograms of “glycol-stiff” sarcomeres show crossbridges large enough to contain only a single myosin head, originating from dense collars every 14.5 nm. Crossbridges with an average 90° axial angle contact actin midway between troponin subunits, which identifies the actin azimuth in each 38.7-nm period, in the same region as the actin target zone of the 45° angled rigor lead bridges. These 90° “target zone” bridges originate from the thick filament and approach actin at azimuthal angles similar to rigor lead bridges. Another class of glycol-PNP crossbridge binds outside the rigor actin target zone. These “nontarget zone” bridges display irregular forms and vary widely in axial and azimuthal attachment angles. Fitting the acto-myosin subfragment 1 atomic structure into the tomogram reveals that 90° target zone bridges share with rigor a similar contact interface with actin, while nontarget crossbridges have variable contact interfaces. This suggests that target zone bridges interact specifically with actin, while nontarget zone bridges may not. Target zone bridges constitute only ∼25% of the myosin heads, implying that both specific and nonspecific attachments contribute to the high stiffness. The 90° target zone bridges may represent a preforce attachment that produces force by rotation of the motor domain over actin, possibly independent of the regulatory domain movements. Force production by myosin heads during muscle contraction has long been modeled as a transition of attached crossbridges from a 90° to a 45° axial angle. Efforts to image crossbridge forms and angles intermediate between 90° heads in ATP-relaxed insect flight muscle (IFM)¹ and the 45° angled bridges in rigor have used nucleotide analogs such as adenosine 5′-[β′γ-imido] triphosphate (AMPPNP) in stable equilibrium states to drive the crossbridges backwards from the 45° angle in rigor to an attached 90° preforce form, otherwise similar to myosin heads in ATP-relaxed fibers (Reedy et al., 1988; Tregear et al., 1990). However, AMPPNP alone will not fully relax IFM, and crossbridges binding AMPPNP retain many rigor-like features (Schmitz et al., 1996; Winkler et al., 1996). On the other hand, AMPPNP in combination with ethylene glycol will relax IFM. When poised at a critical glycol concentration, muscle stiffness is as high as rigor, suggesting crossbridge attachment, but fibers will not bear sustained tension (Clarke et al., 1984; Tregear et al., 1984). Two-dimensional (2-D) analysis of electron micrographs showed that this stiff glycol-PNP state resembled ATP-relaxed fibers in having dense collars every 14.5 nm along the thick filament and thin crossbridges originating from these collars at various axial angles around 90°. However, unlike relaxed muscle, stiff glycol-PNP fibers showed both 90° angled bridges that were regularly spaced every 38.7 nm and more intensity on the 19.3-nm layer line in optical and x-ray diffraction patterns (Reedy et al., 1988; Tregear et al., 1990). Crossbridges in this partially relaxed, glycol-PNP state are important because they may represent the form of the initial attachment of myosin with bound nucleotide preceding force generation (Marston and Tregear, 1984; Tregear et al., 1984; Reedy et al., 1988). This putative preforce 90° crossbridge could not be characterized in 3-D because its variable form and lattice arrangement precluded imaging by averaging methods of 3-D reconstruction. Recently, nonaveraging tomographic methods have been developed and successfully applied to rigor and aqueous-PNP, facilitating characterization of variable crossbridge forms that occur in situ (Taylor and Winkler, 1995, 1996; Schmitz et al., 1996; Winkler and Taylor, 1996). IFM is superb for structural study because the symmetry and spatial arrangement of filaments results in paired crossbridges on opposite sides of the actin filament. This in turn has given rise to a unique shorthand terminology. The individual crossbridge forms are not unique to IFM, only their symmetrical placement about the thin filament. The filament arrangement also facilitates the microtomy of a type of thin section with coplanar filaments that provide views of the entire crossbridge. The best of these, the myac layer, is a 25-nm-thick longitudinal section containing alternating myosin and actin filaments. In rigor, the maximum number of myosin heads attach to actin, forming doublet pairs every 38.7 nm, the “double chevrons” (Reedy, 1968). “Lead bridges,” which form the pair proximal to the M-band, consist of both heads of a myosin molecule and show an overall axial angle of 45° (Taylor et al., 1984). “Rear bridges,” which form the pair proximal to the Z-disk, consist of a single myosin head angled closer to 90°. Crossbridges originate from the thick filament along helical tracks so the azimuths of their origins follow a regular pattern. Relative to the thin filament in the myac layer, the lead bridges originate from the left-front and back-right of the adjacent thick filaments, while rear bridges originate from the left-back and right-front. At their actin ends, the crossbridge attachments follow the changing rotation of the actin protomers along the actin helix. The combination of the azimuth of the origin and the azimuth of the crossbridge contact to actin define the azimuthal angle of the crossbridge.Target zone is the name given to the region of the thin filament where crossbridges bind (Reedy, 1968); by implication this is the region of the thin filament where actin monomers are most favorably placed for actomyosin interaction. In our previous 3-D reconstructions of rigor and aqueous-PNP (Schmitz et al., 1996; Winkler et al., 1996), it was recognized that troponin maintained a constant position with respect to the most regularly positioned crossbridges, the lead bridges, and could thus be used as a landmark to determine the actin dyad orientation in the lead bridge target zone. The most sterically favorable actin position for crossbridge binding in the IFM lattice is midway between troponin densities, where lead bridges bind. The strained structure of the rigor rear bridges suggests that they bind at the very edge of the target zone (Schmitz et al., 1996; Winkler et al., 1996). The target zone defined by lead bridges alone is narrower than target zones previously considered for rigor muscle (Reedy, 1968) because it does not include rear bridge targets. When aqueous AMPPNP was added to rigor IFM, the tension dropped by two thirds, but the stiffness remained as high as rigor. This initially suggested a reversal of the power stroke, but 3-D reconstructions revealed that the lead bridges remained attached, midway between troponin densities, at axial and azimuthal angles close to rigor. The drop in tension without a large change in axial angle seemed to contradict the lever arm hypothesis for motion producing force. However, a cause for the loss of tension was found in tomograms, which showed that rear bridges detached and were replaced by nonrigor bridges bound to actins outside of the rigor target zone, to sites not selected by crossbridges even under the high-affinity conditions of rigor. These nontarget bridges in aqueous-PNP had variable axial and azimuthal angles and appeared to bind actin with variable contact interfaces. This suggested that they were nonspecifically bound to actin. Moreover, their variable structure did not suggest how a simple axial angle change could convert them to a familiar form, such as an angled rigor bridge. However, an intriguing doublet crossbridge group with a consistent structure was recognized in aqueous-PNP. Immediately M-ward of the “lead” rigor-like bridge was a “nonrigor” bridge bound at a 90° or antirigor angle. In this doublet, called a mask motif, both lead and M-ward nonrigor bridge pairs had similar azimuths and contact interfaces with actin and bound within the lead bridge target zone. A simple angle change could convert the M-ward, nonrigor bridge in a mask motif to a single headed lead bridge. Thus, in the mask motif, the lead bridge could be at the end of the power stroke, with the M-ward, nonrigor bridge near the beginning. The pairing of rigor and antirigor angled crossbridges bound to the same target zone suggests that crossbridges might act as a relay during muscle contraction (Schmitz et al., 1996). The affinity of myosin for actin in aqueous-PNP is high compared with weak binding intermediates thought to represent the beginning of the power stroke (Green and Eisenberg, 1980; Biosca et al., 1990). Therefore, the M-ward crossbridge in the mask motif may not represent the best candidate for a preforce crossbridge. Thus, it is important to characterize crossbridge structure in a state with lower actomyosin affinity, such as the stiff glycol-PNP state, where earlier 2-D analysis indicated that weakly attached 90° bridges are prevalent (Reedy et al., 1988). In this work, we have used two spatially invariant features, troponin position and lead crossbridge origins, to identify distinct classes of crossbridges. The invariant position of troponin recognized in 3-D reconstructions allows us to identify the lead bridge target zone and the actin dyad orientation relative to the bound crossbridges. In addition, the “front-back” rule for the azimuth of the origins of the lead target zone bridges distinguishes crossbridges that bind actin with the correct azimuth for specific binding from those that bind nonspecifically. By fitting the myosin subfragment 1 (S1) atomic structure to the in situ bridges, we can compare the positions of the motor and regulatory domains. Previous results and models have introduced the idea that during a power stroke, the crossbridge rotates over the actin binding site while acting as a long, relatively rigid lever arm (Huxley and Simmons, 1971), while others propose that the motor domain position remains constant and light chain domain movements provide a shorter lever arm (Rayment et al., 1993b ; Whittaker et al., 1995). Our previous results (Reedy et al., 1987, 1988; Schmitz et al., 1996; Winkler et al., 1996) and the present work show (a) that regulatory domain position can vary significantly while motor domain position remains constant and (b) that the motor domain can bind actin with varying orientations. This work supports the possibility that both rotation of the motor domain on actin and movements of the regulatory domain could contribute to the power stroke.     

Plant chaperonins: a role in microtubule-dependent wall formation?

Summary The cytosolic chaperonin containing t-complex peptide-1 (CCT) is involved in the correct folding of newly synthetized actin and tubulin molecules. To get insight into potential additional functions of plant CCT, the localization of the subunit CCT was followed throughout cell cycle, cell elongation, and cell differentiation in the tobacco cell culture VBI-O with relation to the microtubular cytoskeleton by double-immunofluorescence and confocal microscopy. The CCT subunit was found to colocalize with sites of microtubule nucleation such as nuclear envelope and preprophase band. In addition, CCT was associated with tubulin in sites of elevated wall synthesis such as phragmoplast or along secondary-wall thickenings. CCT and its substrate tubulin were found to be soluble during periods of cytoskeletal dynamics, whereas sedimentable, vesicle-bound forms of CCT and tubulin prevailed during cell differentiation. The sedimentability of CCT was increased by calcium, whereas it was detached from microsomes by ATP. CCT can bind to both polymerized microtubules and tubulin dimers. These data suggest an additional function of plant CCT in microtubule-driven transport of vesicles that contain cell-wall material.Abbreviations CCT cytosolic chaperonin containing t-complex polypeptide 1     

The effect of different growing conditions on water relations parameters of leaf epidermal cells of Tradescantia virginiana L.

Summary Tradescantia virginiana L. plants were cultivated under contrasting conditions of temperature, humidity, light quality and intensity, and nutrient status in order to investigate the effect of growth conditions on the water relations parameters of the leaf epidermal cells. Turgor pressure (P), volumetric elastic modulus (), half-time of water potential equilibration (T _1/2), hydraulic conductivity (L _p ) were measured with the miniaturized pressure probe in single cells of the upper and lower epidermis of leaves. Turgor differed (range: 0.1 bar to 7.2 bar) between treatments with lowest values under warm and humid conditions and additional supply of fertilizer, and highest values under conditions of low air humidity and low nutrient supply. The volumetric elastic modulus changed by 2 orders of magnitude (range: 3.0 bar to 350 bar, 158 cells), but was only affected by the treatments, in as much as it was dependent on turgor. The turgor dependence of , measured on intact leaves of T. virginiana, was similar to that for cells of the isolated (peeled) lower epidermis, where as a function of turgor was linear over the whole range of turgors. This result has implications for the discussion of pressure/volume curves as measured by the pressure bomb where changes in bulk leaf are frequently discussed as adaptations to certain treatments. The measurements of the hydraulic conductivity indicate that this parameter varies between treatments (range of means: 2.4×10^-6 cm s^-1 bar^-1 to 13.4×10^-6 cm s^-1 bar^-1). There was a negative correlation for L _p in cells of intact leaves as a function of turgor which was altered by the growing conditions. However, a correlation with turgor could not be found for cells from isolated epidermis or cells from a uniform population of plants. The large variation in L _p from cell to cell observed in the present and in previous studies was accounted for in a study of 100 cells from a uniform population of plants by the propagation of measurement errors in calculating L _p . The results suggest that in T. virginiana cellular water relations are changed mainly by the turgor dependence of .     

Linear dichroism studies of binding site structures in solution: Complexes between DNA and basic arylmethane dyes

The interaction between B-form DNA and twelve cationic triaryl-methane dyes was studied with respect lo optical properties and stabilities, using linear dichroism (LD) and aqueous two-phase partition techniques. Monovalent dyes derived from crystal violet as a rule form a single strong complex (K₁ ca 10⁵ M^?1; site density per nucleotide base n₁ ca 0.1 at 0.1M ionic strength) in which the plane of the dye is at an angle of less than 50° to the local DNA helix axis. The complex with fuchsin is weaker (10⁴M^?1) but can be explained by a similar orientation. For some of the dyes (those with pseudo-C_2v symmetry) XXXre angular orientations of two molecule-fixed axes can be obtained. For the divalent methyl green a second complex appears to be formed at low ionic strength. Methyl green (and to some extent 2-thiophene green and malachite green) show exciton splitting in the LD spectrum and circular dichroism assignable to exciton coupling between transition dipoles roughly parallel to the helical strands, indicating a dye-dye interaction. Tne optical data, supported by fitting experiments with space-filling models, suggests a general structure for the binding site. The dye is not intercalated but is bound to exposed hydrophobic regions in the major groove. The ligand is in part (the charged amino groups) in contact with the phosphoribose chain but its main surface lies against the hydrophobic base-pair stack. For a diphenylmethane dye, Michler's hydrol blue, a perpendicular orientation was observed, possibly due to intercaiation.     

Water relations of growing pea epicotyl segments

The water relations of growing epicotyl segments of pea (Pisum sativum L.) were studied using the miniaturized pressure probe. For epidermal cells stationary turgor pressures of P=5 to 9 bar and half-times of water exchange of individual cells T _1/2=1 to 27 s were found. The volumetric clastic modulus () of epidermal cells varied from 12 to 200 bar and the hydraulic conductivity, Lp=0.2 to 2·10^-6 cm s^-1 bar^-1. For cortical cells P=5 to 11 bar, T _1/2=0.3 to 1 s, Lp=0.4 to 9·10^-5 cm s^-1 bar^-1 and =6 to 215 bar. The T _1/2 of cortical cells was extremely low and the Lp rather high as compared to other higher plant cells. The T _1/2-values of cortical cells were sometimes observed to change from short to substantially longer values (T _1/2=3 to 20 s). Both short and long pressure relaxations showed all the characteristics of non-artifactual curves. The change is apparently due to an increase in Lp and not , but the reason for the change in cell permeability to water is not known.In osmotic exchange experiments on peeled segments using solutions of different solutes, the half-time of osmotic water exchange for the whole segment was approximately 60 s. Water exchange occurred too quickly to be rate controlled by solute diffusion in the wall space. The data suggest that the short T _1/2-values in the cortical cells are the physiologically relevant ones for the intact tissue and that a considerable component of water transport occurs in the cell-to-cell pathway, although unstirred layer effects at the boundary between the segment and solution may influence the measured half-time. Using the theory of Molz and Boyer (1978, Plant Physiol. 62, 423–429), the gradient in water potential necessary to maintain the uptake of water for cell enlargement can be calculated from the measured diffusivities to be approximately 0.2 and 1 bar for growth rates of 1% h^-1 and 5% h^-1, respectively. Thus, although the T _1/2-values are short and Lp rather high, there may be a significant osmotic disequilibrium in the most rapidly growing tissue and as a consequence the influence of water transport on the growth rate cannot be excluded.Abbreviations P turgor pressure - T _1/2 half-time of water exchange of individual cell - Lp hydraulic conductivity - volumetric elastic modulus - t _1/2 average half-time of water exchange of tissue     

Characterization and expression analysis of CD3ɛ and CD3γ/δ in fugu, Takifugu rubripes

CD3 is an essential component of the CD3-TCR complex. In this report, we describe the cloning, characterization, and expression analysis of the CD3 and CD3/ chain genes from fugu, Takifugu rubripes. Two distinct CD3 homologue cDNAs, designated as CD3-1 and CD3-2, and a CD3/ homologue cDNA were isolated from the fugu thymus. The deduced amino acid sequences of these cDNAs exhibit conserved essential CD3 chain motifs and overall structures. RT-PCR analysis demonstrated that the CD3 and CD3/ genes were expressed in lymphoid organs (e.g. thymus, head kidney, trunk kidney and spleen), mucosal tissues (gill, skin, and intestine), and peripheral blood leucocytes (PBL). The CD3 and TCR genes were expressed only in the surface IgM^– population, which were separated from PBL using an anti-fugu IgM monoclonal antibody. In addition, in situ hybridization confirmed that CD3-expressing cells were distributed randomly in the head kidney, trunk kidney, and spleen, but in the thymus were restricted to the lymphoid outer zone and epithelioid inner zone only. Collectively, these results suggest that CD3 molecules are useful markers for the identification of T cells in teleost fish. The present study thus provides a critical step in identifying T cells in this model organism.Nucleotide sequence data reported in this paper are available in the DBJ/EMBL/GenBank databases and have been assigned the accession numbers AB166798 (CD3-1), AB166799 (CD3-2), and AB166800 (CD3/).     

Asparagine and glutamine side-chain conformation in solution and crystal: A comparison for hen egg-white lysozyme using residual dipolar ouplings

Experimental ¹⁵N–¹H and ¹H–¹H residual dipolar couplings (RDCs) for the asparagine (Asn) and glutamine (Gln) side chains of hen egg-white lysozyme are measured and analysed in conjunction with ¹N relaxation data, information about ¹ torsion angles in solution and molecular dynamics simulations. The RDCs are compared to values predicted from 16 high-resolution crystal structures. Two distinct groups of Asn and Gln side chains are identified. The first contains residues whose side chains show a fixed, relatively rigid, conformation in solution. For these residues there is good agreement between the experimental and predicted RDCs. This agreement improves when the experimental order parameter, S, is included in the calculation of the RDCs from the crystal structures. The comparison of the experimental RDCs with values calculated from the X-ray structures shows that the similarity between the oxygen and nitrogen electron densities is a limitation to the correct assignment of the Asn and Gln side-chain orientation in X-ray structures. In the majority of X-ray structures a 180° rotation about ² or ³, leading to the swapping of N ² and O ¹, is necessary for at least one Asn or Gln residue in order to achieve good agreement between experimental and predicted RDCs. The second group contains residues whose side chains do not adopt a single, well-defined, conformation in solution. These residues do not show a correlation between the experimental and predicted RDCs. In many cases the family of crystal structures shows a range of orientations for these side chains, but in others the crystal structures show a well-defined side-chain position. In the latter case, this is found to arise from crystallographic contacts and does not represent the behaviour of the side chain in solution.     

Equilibrium muscle crossbridge behavior: The interaction of myosin crossbridges with actin

The interaction of myosin crossbridges with actin under equilibrium conditions is reviewed. Similarities and differences between the weakly- and strongly-binding interactions of myosin crossbridges with actin filaments are discussed. A precise, narrow definition of weakly- binding crossbridges is given. It is postulated that the fundamental interaction of crossbridges with actin is that the crossbridge heads are mobile after attachment in the first case but not in the second. It is argued that because the weakly-binding crossbridge heads are mobile after attachment, the heads appear to function independently of each other. The lack of head mobility in attached strongly-binding crossbridges makes the strongly-binding crossbridge heads appear to act cooperatively. This model of the strongly-binding crossbridge gives an explanation for two important and otherwise unexplained observations. It explains why the rate constant of force decay after a small stretch is a sigmoidal function of nucleotide analogue concentration, and why, in the presence of analogues or in rigor, the rate constant of force decay after a small stretch is often significantly slower than the rate constant for myosin subfragment-1 detachment from actin in solution. The model of the weakly-binding crossbridge accurately describes the behavior of the myosin·ATP crossbridge.     

Green-synthesis, characterization, photostability and polarity studies of novel schiff base dyes using spectroscopic methods

Preparation, characterization, photostability and polarity studies of novel Schiff base dyes using spectroscopic methods were achieved. The Schiff base dyes were prepared by the reaction of salicylalde-hyde/2-hydroxy-l-naphthaldehyde with aminophenazone under microwave irradiation. The spectroscopic (FT-IR, ¹H NMR, ¹³C NMR, Mass) studies and elemental analyses were in good agreement with chemical structure of synthesized compounds. In addition, UV-Vis and fluorescence spectroscopic experiments showed that these dyes are good absorbent and fluorescent. Based on the photostability study of these dyes, minimal to no loss in fluorescence intensities of 4-[(2-hydroxy-benzylidene)-amino]1,5-dimemyl-2-phe-nyl-1,2-dihydro-pyrazol-3-one (D1) (6.14%) and 4-[(2-hydroxy-naphthalen-l-ylmethylene)-amino]-1,5-dimethyl-2-phenyl-1,2-dihydro-pyrazol-3-one (D2) (2.95%) was observed with an increase in the exposure time using time-based fluorescence steady-state experiments. These studies also inferred that these Schiff base dyes have a high photostability against photobleaching. In addition, Dye 2 is found to be more sensitive than Dye 1 to the polarity of the microenvironment provided by different solvents based on the results of fluorescence polarity studies.     

Comparison of three granular support materials for anaerobic fluidized bed systems

Summary Three different materials, kaolin, pozzolana and biolite (a material used in a commercial anaerobic fluidized bed treatment process) when tested as supports for an anaerobic fluidized bed system had similar physical and fluidization properties but behaved differently towards the biomass hold-up. However, all three systems attained similar removal efficiency rates.Nomenclature U Fluidization velocity (m/s) - U₁ Terminal fluidization velocity (m/s) - g Local acceleration due to gravity (m/s²) - _s Solid density (kg/m³) - _f Fluid density (kg/m³) - P Pressure drop (Pa) - HRT Hydraulic retention time (days) - H_mf Height of bed at minimum fluidization (m) - H Height of bed (m) - C_d Drag coefficient (dimensionless) - W Mass of solids in bed (kg) - dp Particle diameter (m) - A Cross-sectional area of column (m²) - h column height (m) - Rc_t Terminal Reynolds no. - Voidagc (fractional free volume, dimensionless) - _mf Voidage (fractional free volume) at minimum of fluidization (dimensionless)     

Apolipoprotein E (apoE) polymorphism and its influence on ApoE concentrations in the cerebrospinal fluid in Finnish patients with Alzheimer's disease

The apoE phenotype of 83 patients with probable Alzheimer's disease (AD) and of 164 non-demented controls was determined by isoelectric focusing and Western blotting. The proportion of the e4 allele was 0.548 in AD and 0.202 in controls (P<0.0001). The effect was seen in both early-onset and late-onset AD patients. The risk of AD in 4 homozygotes was 18-fold greater than in individuals without the 4 allele. ApoE concentrations were measured in serum and cerebrospinal fluid (CSF) from a subgroup of patients with AD (n=72) and controls (n=84) by a sandwich enzyme-linked immunosorbent assay. Although serum apoE concentrations were lower in individuals with the 4 allele than in those without the e4 allele, CSF apoE concentrations did not vary in different phenotype groups. However, CSF apoE levels were lower in AD patients than in controls. We conclude that the inheritance of the 4 allele of apoE is a risk factor for AD in the Finnish population.     

Determination of the backbone torsion angle ɛ in nucleic acids

Summary The multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2 and H3 in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle (C4-C3-O3-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around , without the knowledge of ³J_cp coupling constants.     

Steady-state fluorescence polarization studies of the orientation of myosin regulatory light chains in single skeletal muscle fibers using pure isomers of iodoacetamidotetramethylrhodamine.

The regulatory light chain (RLC) from chicken gizzard myosin was covalently modified on cysteine 108 with either the 5- or 6-isomer of iodoacetamidotetramethylrhodamine (IATR). Labeled RLCs were purified by fast protein liquid chromatography and characterized by reverse-phase high-performance liquid chromatography (HPLC), tryptic digestion, and electrospray mass spectrometry. Labeled RLCs were exchanged into the native myosin heads of single skinned fibers from rabbit psoas muscle, and the ATR dipole orientations were determined by fluorescence polarization. The 5- and 6-ATR dipoles had distinct orientations, and model orientational distributions suggest that they are more than 20 degrees apart in rigor. In the rigor-to-relaxed transition (sarcomere length 2.4 microm, 10 degrees C), the 5-ATR dipole became more perpendicular to the fiber axis, but the 6-ATR dipole became more parallel. This orientation change was absent at sarcomere length 4.0 microm, where overlap between myosin and actin filaments is abolished. When the temperature of relaxed fibers was raised to 30 degrees C, the 6-ATR dipoles became more parallel to the fiber axis and less ordered; when ionic strength was lowered from 160 mM to 20 mM (5 degrees C), the 6-ATR dipoles became more perpendicular to the fiber axis and more ordered. In active contraction (10 degrees C), the orientational distribution of the probe dipoles was similar but not identical to that in relaxation, and was not a linear combination of the orientational distributions in relaxation and rigor.     

Regulation of Fibre Contraction in a Rat Model of Myocardial Ischemia

Background

The changes in the actomyosin crossbridge cycle underlying altered contractility of the heart are not well described, despite their importance to devising rational treatment approaches.

Methodology/Principal Findings

A rat ischemia–reperfusion model was used to determine the transitions of the crossbridge cycle impacted during ischemia. Compared to perfused hearts, the maximum force per cross-sectional area and Ca²⁺ sensitivity of fibers from ischemic hearts were both reduced. Muscle activation by photolytic release of Ca²⁺ and ATP suggested that the altered contractility was best described as a reduction in the rate of activation of noncycling actomyosin crossbridges to activated, cycling states. More specifically, the apparent forward rate constant of the transition between the nonforce bearing A-M.ADP.Pi state and the bound, force bearing AM*.ADP.Pi state was reduced in ischemic fibers, suggesting that this transition is commensurate with initial crossbridge activation. These results suggested an alteration in the relationship between the activation of thin filament regulatory units and initial crossbridge attachment, prompting an examination of the post-translational state of troponin (Tn) T and I. These analyses indicated a reduction in the diphosphorylated form of TnT during ischemia, along with lower Ser23/24 phosphorylation of TnI. Treatment of perfused fibers by 8-Br-cAMP increased Ser23/24 phosphorylation of TnI, altering the reverse rate constant of the Pi isomerization in a manner consistent with the lusitropic effect of β-adrenergic stimulation. However, similar treatment of ischemic fibers did not change TnI phosphorylation or the kinetics of the Pi isomerization.

Conclusions

Ischemia reduces the isomerization from A-M.ADP.Pi to AM*.ADP.Pi, altering the kinetics of crossbridge activation through a mechanism that may be mediated by altered TnT and TnI phosphorylation.