Two-dimensional motion of DNA bands during 120° pulsed-field gel electrophoresis. I. Effect of molecular weight

The instantaneous position and velocity of bands of linear, double-stranded DNA were measured during 120° pulsed-field electrophoresis in 1% agarose gels, using a video micrometer capable of simultaneous measurements in two dimensions. When the direction of the field was switched, the band initially retraced the last portion of its path during the preceding pulse. The distance the band moved backward increased with DNA length: 48.5 kb (kilobase pair) DNA moved backward only 0.2 μm, but 1110 kb DNA moved backward 24 μm, before setting off in a positive direction. The velocity of the DNA band was particularly rapid during the backward movement: the magnitude of the velocity spike increased with M, reaching 2.4 μm/s for 1110 kb DNN, which was about 5 times the steady-state velocity. The velocity in the y direction, perpendicular to the mean drift direction, allowed an even larger transient spike, which also increased with M. Simulation of the dynamics of long DNA chins undergoing gel electrophoresis by a dynamic Monte Carlo method gave instantaneous xy position and velocity in excellent agreement with experiment. The simulation included extensional motions of the DNA within the tube of interconnected agarose pores as well as the possibility of loops (hernias) that escape laterally from the tube. © 1995 John Wiley & Sons, Inc.     

An analysis of the association of collagen based on structural models.

The existence and distribution of vertical, interacting edges on cylindrical surfaces, which were derived from the basic structural features of the collagen molecule, have been examined as a function of the angle θ of the superhelix. Over the range θ = 26°–33° and near θ = 36°, the strongest intermolecular association was detected at 29.95°. This model exhibited three similar, self-interacting edges, separated by approximately 120°. The potential for three similar but nonidentical edges lies in the geometry of the collagen molecule, but the existence of such edges and their ability to self-associate is dependent upon the unique amino acid sequence. The relative strengths of association of each edge with itself and with the other edges are not identical, but the present analysis does not permit a judgment as to whether this variation could result in functional specificity in such interactions. All three chains participate in each edge though their contribution to the relative interaction strength is not identical. These edges showed distinct maxima when displaced past one another by integral intervals of D, where D = 233 residue positions (quarter-stagger). The strongest relative interaction occurs at a displacement of 1D.     

Crystallization of the purine salvage enzyme adenine phosphoribosyltransferase

Adenine phosphoribosyltransferase from the protozoan parasite Leishmania donovani has been crystallized in the presence of the substrate Mg²⁺-α-D -5-phosphoribosyl-1-pyrophosphate (PRPP) or the product adenosine-5-monophosphate, as well as in the absence of ligand. These crystals belong to the space group P6₁22 or its enantiomorph P6₅22, with unit cell dimensions of a = b = 64.0 Å, c = 240.5 Å, α = β = 90°, and γ = 120°. The crystals diffract to 1.9 Å. © 1996 Wiley-Liss, Inc.     

Germination and emergence of Sorghum bicolor. genotypic and environmentally induced variation in the response to temperature and depth of sowing

Abstract The germination of Sorghum bicolor seeds of 9 genotypes was tested at temperatures between 8°C and 48°C on a thermal gradient plate. Samples were tested from three regions of the panicle expected to differ in temperature during grain filling. Seeds of a tenth genotype, SPV 354, produced in controlled-environment glasshouses at different panicle temperatures, were tested similarly. In addition, the emergence of SPV 354 was measured from planting depths of 2 and 5 cm at mean soil temperatures of 15, 20 and 25°C. Four methods of calculating mean germination rate for the nine genotypes were compared. Germination characters like base, optimum and maximum temperature (T_b, T_o, T_m), thermal time (θ)and the germination rate at T_o(R_max showed only small differences between methods. There was a range of genotypic variation in all characters: T_b 8.5–11.9°C; T_o, 33.2–37.5°C; T_m, 46.8–49.2°C; θ, 23.4–38.0°Cd; R_max, 0.69–1.14-d_-1. In contrast, mean germinability (G) was between 90% and 100% over the temperature range 13–40°C. Panicle temperature had no effect on any germination character in SPV 354. However, deeper burial increased θ for emergence and decreased G, irrespective of soil temperature except at 5 cm. Increasing panicle temperature, by reducing seed size, reduced G and increased θ by about 10% only at 15°C and 5 cm depth.     

Reversing-pulse electric birefringence of poly(γ-benzyl-L-glutamate). III. The temperature dependence of the transient and steady-state behavior in cyclohexanone

The reversing-pulse electric birefringence (RPEB) technique was applied to the study of the temperature effect on the electrooptical and hydrodynamic properties of a fractionated [Glu(OBzl)]_n sample, which is molecularly dissolved in cyclohexanone. The aim was to develop a standard analytical method for thermal denaturation and temperature-induced conformational transitions. The field-on reverse and steady-state signal, and the field-off decay signal, were measured at 535 nm and at a constant low field strength (ca. 3 kV/cm) over a wide temperature range (5–90°C). The steady-state birefringence and the relaxation time in the decay process were also measured at two constant temperatures (5 and 70°C) over a wide field strength range (E ≤ 20 kV/cm). By the combination of these two different sets of RPEB measurements, the unwanted effect of the high pulse field on polymer conformation at elevated temperatures could be minimized. Together with the density and viscosity of cyclohexanone measured between 5 and 95°C, the following quantities could be evaluated: the weight-average permanent dipole moment and polarizability anisotropy, the reduced optical anisotropy factor (Δg/n), the weight-average length, and the degree of polydispersity. All these quantities, except for Δg/n, were found to be almost independent of temperature (5–90°C) and concentration (1.54–4.27 mM).     

The crystal structure of 2-deoxy-β-d-arabino-hexopyranose at −150°

2-Deoxy-β-d-arabino-hexopyranose, C₆H₁₂O₅, is orthorhombic, P2₁2₁2₁, with cell dimensions at ?150° [20°], a = 6.484(2) [6.510(3)], b = 10.364(2) [10.427(4)], c = 11.134(3) [11.153(5)] Å, V = 748.2 [757.1] ų, Z = 4, D_x = 1.457 [1.440], and D_m = [1.455] g.cm^?3. The intensities of 1269 reflections were measured by using MoKα radiation. The structure was solved by direct methods, and refined by full-matrix least-squares, with anisotropic, thermal parameters for the carbon and oxygen atoms, and isotropic parameters for the hydrogen atoms. The pyranose has the ⁴C₁(d) conformation, with puckering parameters Q = 0.563 Å, θ = 3.9°, and ? = 350.3°. The departure from ideality is very small, and less than that in β-d-glucopyranose, Q = 0.584 Å and θ = 6.9°. The β-glycosidic, CO bond is short, 1.383(4) Å, and the OCOH torsion angle is ?87°, consistent with the anomeric effect. The hydrogen-bonding scheme consists of infinite chains, with side chains terminating at a ring-oxygen atom.     

Acute Changes In Knee‐Extensors Torque,Fiber Pennation,and Tendon Characteristics

The aim of the current study was to examine the relationships between quadriceps torque, vastus lateralis pennation angle (θ), and patella tendon stiffness (K) at 07:45 and 17:45 h. Using short‐duration static contractions, simultaneous recordings were made of vastus lateralis (VL) electromyograph (EMG), θ and patella tendon K. Peak isometric extension torque (Peak torque Ext_corr) increased by 29.4±6.5% at a knee angle of 70° (p=0.03) in the evening compared to the morning. In the contracted muscle, a 35.0±11.0% (p=0.02) time‐of‐day (TOD)‐related change in θ (to a greater evening compared to morning θ) was observed. Morning and evening measures of θ were also made, both at rest and at a standardized force level (250 N), to separate architecture change effects from increased torque capacity effects. Significant increments in θ in both the resting muscle (13.0±5.1%, p=0.046) and during the standardized exertions (8.0±3.1%, p=0.04) were observed in the evening versus the morning. Increases in θ with TOD were significantly correlated with the 40% (p=0.018) decrease in K both during the standardized contractions (r=0.788, p<0.001) and at rest (r=0.77, p=0.026). These data show that TOD affects K and θ and that these two important factors involved in in‐vivo muscle torque generation capacity are associated. The data also show that despite the potentially deleterious effects of the direction of the changes in both K and θ with TOD, peak torque Ext_corr still shows a significant upward shift in the evening relative to the morning.     

The structure of native cellulose

Native cellulose has been shown to consist of a crystalline array of parallel chains, based on the X-ray diffraction data for specimens from the sea alga Valonia ventricosa. The unit cell is monoclinic with dimensions a = 16.34 Å, b = 15.72 Å, c = 10.38 Å (fiber axis), and β = 97.0°. The space group is P2₁ and the cell contains disaccharide segments of eight chains. Models containing chains with the same sense (parallel) or alternating sense (antiparallel) were refined against the intensity data using rigidbody least squares procedures. The results show a preference for a parallel chain structure with specific chain polarity with respect to the c axis. The refinement places the ? CH₂OH side chains approximately 20′ from the so-called tg conformation, with a result that an 02′? H…06 intramolecular bond is formed. The structure also contains an 03? H…05′ intramolecular bond and an 06? H…03 intermolecular bond along the a axis. All these bonds lie in the 020 planes, and the structure is an array of hydrogen-bonded sheets. A major consequence of this work is that regular chain folding can be ruled out and cellulose is seen as extended chain polymer single crystals.     

Behavioural evidence that magnetic field effects in the land snail,Cepaea nemoralis,might not depend on magnetite or induced electric currents

Although extremely low frequency (ELF) magnetic fields (<300 Hz) appear to exert a variety of biological effects, the magnetic field sensing/transduction mechanism(s) remains to be established. Here, using the inhibitory effects of magnetic fields on endogenous opioid peptide-mediated “analgaesic” response of the land snail. Cepaea nemoralis, we addressed the mechanism(s) of action of ELF magnetic fields. Indirect mechanisms involving both induced electric fields and direct magnetic field detection mechanisms (e.g., magnetite, parametric resonance) were evaluated. Snails were exposed to a static magnetic field (B_DC=78±1 μT) and to a 60 Hz magnetic field (B_AC=299±1 μT peak) with the angle between the static and 60 Hz magnetic fields varied in eight steps between 0° and 90°. At 0° and 90°, the magnetic field reduced opioid-induced analgaesia by approximately 20%, and this inhibition was increased to a maximum of 50% when the angle was between 50° and 70°. Because B_AC was fixed in amplitude, direction, and frequency, any induced electric currents would be constant independent of the B_AC/B_DC angle. Also, an energy transduction mechanism involving magnetite should show greatest sensitivity at 90°. Therefore, the energy transduction mechanism probably does not involve induced electric currents or magnetite. Rather, our results suggest a direct magnetic field detection mechanism consistent with the parametric resonance model proposed by Lednev. © 1996 Wiley-Liss, Inc.     

Anomalies in sedimentation. IV. Decrease in sedimentation coefficients of chains at high fields

A theory is presented for the decrease of sedimentation coefficient at high centrifugal fields recently reported for samples of DNA by Rubenstein and Leighton and others. The theory uses the model of a chain of beads and springs to represent the molecule. Kirkwood's approximation is used for the sedimentation coefficient. The decrease in sedimentation coefficient with field comes about as a result of the uneven frictional forces in the chain, which on the average are less on segments near the center of the chain than on those near the ends. As a result the ends of the chain tend to drag behind the center, and the average intersegment distances are increased; consequently the hydrodynamic shielding of one segment by another is reduced, and the average friction is increased. The effect is thus characteristic of single molecules; intermolecular interaction is not involved. The sedimentation coefficient, S, varies as a power series in a parameter y that measures the distortion produced by the uneven friction: S = S⁰(1?D₂y² + D₄y⁴ ? ·). where S⁰ is the limiting value of S at zero centrifugal field and D₂ and D₄ are constants; y is proportional to the cen speed squared tunes the molecular weight squared divided by S⁰. It has been observed that the effects of centrifuge speed on S are negligible below certain critical values of the speed and molecular weight, but increase dramatically immediately above these values; this follows naturally from the high powers of the speed and molecular weight that appear in the above equation.     

Molecular configuration of amylose and its complexes in aqueous solutions. Part II. Relation between the DP of helical segments of the amylose–iodine complex and the equilibrium concentration of free iodine

The iodine which is added to an aqueous amylose solution is bound only partly by the amylose while forming the blue complex and partly remains free. The equilibrium normality of the free and the bound iodine at half-saturation of amylose by iodine is designated as [I_f]_v and [I_b]_w, respectively. The stability of the poly iodine chain formed within the axis of amylose helices depends on its length, i.e., indirectly on the DP of the amylose helices: the greater this stability, the lower the [I_f]_v value. The amylose molecule consists of helical segments. Such a molecule may behave as a random coil. The average length of the helical segments in freshly prepared amylose-iodine complexes depends on temperature, pH, iodide concentration, the presence of other complex-forming agents, and the DP of the amylose. This latter factor is investigated in the present paper. By the aid of an automatically recording photometrictitrating device the coherent values of [I_b] and [I_f] were determined. Plotting these values against DP _n for mechanochemically degraded as well as for periodateo-xidized amyloses resulted in curves consisting of two linear sections. The break of the curves occurred between DP _n 110 and 130. It was concluded that below DP _n = 100 the DP of helical segments (= sDP _n) is identical to the DP _n of the total molecule, i.e., the molecule consists of only a single, relatively stiff helix. Above this limit the molecule contains several helical segments. The DP of these helical segments can be calculated as follows: sDP _n = 141.1 ? 10.2 × 10⁵[I_f]_v. This equation is considered to be valid for 0.5–0.6 mg. amylose in 100 ml. 0.1N HCl at 20°C., λ = 650 mμ, euuvet diameter 3.4 cm., the feed rate of the iodate-iodide titrating solution (in acid medium resulting in a 5 × 10^?3N I₂ solution with a molar iodide to iodine ratio of 1.5) is 0.4ml./min. Amylose molecules of, e.g., DP ⁿ = 1380 consist of an average of 11.4 segments having a DP of about 120 and consisting of an average of 15–18 helical turns.     

Solution conformations of B. subtilis ribosomal 5S RNA: a calorimetric study

The unfolding of B. subtilis 5S RNA is examined by direct calorimetric measurement in the presence of various concentrations of Na⁺ and Mg²⁺. The composite differential scanning calorimetry (DSC) curve is analyzed into 3–5 individual two-state melting transitions. In the absence of added Na⁺ or Mg²⁺, the 5S RNA segments melt together at T_m = 40°C. Addition of Na⁺ stabilizes the molecular structure (T_m = 56°C) and widens the melting temperature range, so that up to five component transitions are observed. Addition of Mg²⁺ alone produces a very stable structure (T_m = 75°C) with highly cooperative melting. Finally, addition of both Na⁺ and Mg²⁺ produces the highest stability (T_m = 76°C). The results are interpreted according to hypothetical secondary and tertiary base-pairing schemes. The conformational changes demonstrated here may facilitate the movement of the protein synthesis machinery during RNA translation.     

Conformational analysis of polytripeptides (Gly-Pro-Ala)n, (Gly-Ala-Hyp)n,and (Gly-Ala-Ala)n in connection with the problem of collagen structure

Conformational analysis of triple helics of a type of collagen was performed with typical collagen tripeptide sequences based on Gly-Pro-Ala, Gly-Ala-Hyp, and Gly-Ala-Ala. During energy minimization, the possibility of continual deformation of the pyrrolidine cycle was taken into account in order to achieve better accuracy in the resulting structure. The (Gly-Pro-Ala)_n structure is almost isomorphic to the (Gly-Pro-Hyp)_n structure obtained in the previous work [Tumanyan, V. G. & Esipova, N.G. (1982) Biopolymers 21 , 475–497]. For a collagen-type structure, the optimal conformation of (Gly-Ala-Hyp)_n tends to have a decreased unit twist (t = 15°), although the energy advantage with respect to the conformation with t = 45° is not so significant. A similar situation is observed for (Gly-Ala-Ala)_n. In this case, the energy decrease during unwinding to t = 15° from t = 45° is quite small. The conformations of (Gly-Ala-Hyp)_n and (Gly-Ala-Ala)_n with t = 15° exhibit a similarity with a triple complex of polyproline II helices—a noncoiled coil such as (Gly-Pro-Hyp)_n and (Gly-Pro-Ala)_n. A similar structure may be postulated for subcomponent cq1 of the first component of a human complement containing substantial Gly-X-Pro and Gly-X-Y tripeptide derivatives in the primary structure (X, Y = any amino acid). The results suggest that the observed helical symmetry of collagen (t = 36°) is a consequence of superposition of diffraction patterns (for sufficiently long segments) from various helices (t varies from ～15° for Gly-X-Hyp and Gly-X-Y to ～56° for Gly-Pro-Ala). For short alternating segments, some unification of different helical structures is possible.     

Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl^? buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 Å. The crystals belong to the trigonal lattice type with the spacegroup P3₁21 or P3₂21, respectively, have the cell dimensions a = b = 96.8 Å, c = 196.2 Å, α = β = 90°,γ = 120°. The crystals have a theoretical packing density of 2.7 ų/Da, assuming one molecule with a molecular weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset. © 1995 Wiley-Liss, Inc.     

Crystallization of Thermus thermophilus histidyl-tRNA synthetase and Its complex with tRNAHis

Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus. The protein has been crystallized separately with histidine and with its cognate tRNA^His. Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant. The crystals of HisRS with histidine belong to the spacegroup P2₁2₁2 with cell parameters a = 171.3 Å, b = 214.7 Å, c = 49.3 Å, α = β = γ = 90°. A complete data set to a resolution of 2.7Å with an R_merge on intensities of 4.1% has been collected on a single frozen crystal. A partial data set collected on a crystal of HisRS in complex with tRNA_His shows that the crystals are tetragonal with cell parameters a = b = 232 Å, c = 559 Å, α = β = γ = 90° and diffract to about 4.5 Å resolution. © 1995 Wiley-Liss, Inc.     

Vibrational analysis of peptides,polypeptides, and proteins. XVIII. Conformational sensitivity of the α-helix spectrum: αI- and αII-Poly(L-alanine)

The α_II-helix (? = ?70.47°, ψ = ?35.75°) is a structure having the same n and h as the (standard) α_I-helix (? = ?57.37°, ψ = ?47.49°). Its conformational angles are commonly found in proteins. Using an improved α-helix force field, we have compared the vibrational frequencies of these two structures. Despite the small conformational differences, there are significant predicted differences in frequencies, particularly in the amide A, amide I, and amide II bands, and in the conformation-sensitive region below 900 cm^?1. This analysis indicates that α_II-helices are likely to be present in bacteriorhodopsin [Krimm, S. & Dwivedi, A. M. (1982) Science 216 , 407–408].     

X-Ray and stereochemical studies on xylan diacetate

By means of x-ray fiber diffraction, it has been found that xylan diacetate crystallizes with two chains or four residues in a monoclinic cell (space group P2₁): a = 7.64, b = 12.44, c (fiber axis) = 10.31 Å, and γ = 85°. Pairs of residues are related by a twofold screw axis in the c direction. Based on the observed fiber repeat and chain symmetry, the probable conformation of a pair of xylose diacetate residues joined via a β-1,4′ linkage has been obtained by energy minimization methods. The conformations corresponding to a threefold screw axis and a twofold screw axis along the chain have been compared and the reason why xylan diacetate assumes a twofold screw axis seems to be due to intermolecular packing effects rather than intramolecular non-bonded interactions.     

IMPORTANCE OF PHYSICAL VARIABLES ON THE SEASONAL DYNAMICS OF EPILITHIC ALGAE IN A HIGHLY SHADED CANYON STREAM1

The seasonal abundance of epilithic algae was correlated with major physico-chemical parameters in a first-order, heavily shaded stream in northern Arizona. Diatoms made up over 85%, by numerical abundance, of the epilithon community Light energy, water temperature, and stream discharge were most highly correlated with seasonal abundance of epilithic diatom taxa when analyzed with stepwise multiple regression. None of the chemical variables measured in the study (NO₃-N, O-PO₄, SiO₂, including PH) was found to be significantly correlated with the seasonal community structure of epilithic diatoms. Total diatom cell densities showed a significant negative correlation to stream bed light energy. Likewise, total diatom cell densities along a transect in the stream bed showed a negative correlation to current velocity during those months when base flow was low and stable, and current velocity was ≤25 cm·sec-¹. Most diatom taxa had highest cell densities at temperatures < 16°C and at daily mean stream bed light levels < 400 μE·m^?2·s^?1. Highest cell densities of green algae occurred at temperatures between 6–16°C and at daily mean stream bed light levels of > 400 μE·m^?2·s^?1. Blue-green algae (cyanobacteria) grew best at the highest recorded water temperatures and daily mean stream bed light energy (16–20°C and 900–1200 μE·m^?2·s^?1). Abrupt increases in NO₃-N coincided with a brief pulse of Nostoc pruniforme colonies during June, and leaf drop from Alnus oblongifolia during October.     

The ontogeny of the locomotor wave in sevruga fish (Acipenser stellatus Pall.)

Continuous filming of the swimming movements of sevruga prelarvae (Acipenser stellatus Pall.) was performed over the first to the fifteenth post-hatching days. These data were used to determine three locomotor characteristics: {f(v, θ), A(v, θ), w(v, θ)} where f is the frequency of the transverse oscillations; A is the amplitude of the tail fin movements; w is the speed of the locomotor wave; v is the speed of the locomotion; and θ is the number of days during which the filming was carried out. In addition, the comparison of two characteristics was performed. These are the step length L(v, θ) and the wavelength λ(v, θ), where L ≡ vT is the distance that a fish covers in time T ≡ 1/f and λ is λ ≡ wT. The results led to the conclusion that the prelarva swimming movements achieve the carangiform mode by the end of the second week. This is characteristic of adults and coincides with the beginning of external feeding. The mode is represented by an equation that follows from the generalized Bainbridge’s law: f = f ₀ + f ₁ v/H. The equation includes one anatomical parameter H of total length and two kinematic parameters f ₀ and f ₁, which can be calculated by the linear approximation method. These are the kinematic parameters that are changed in the early ontogenesis when 1 ≤ θ ≤ 15. However, the parameters become stable when θ ≥ 15. This means that the parameter values are constant and similar to those of adult fish. The similar properties of stabilization and variability have the kinematic parameters of other locomotor characteristics.

     

Active knee joint velocity replication measures are stable and accurate in healthy individuals

The purpose of this study was to determine the stability and accuracy of active knee joint velocity replication methods in healthy subjects. We used a repeated measures design with 14 healthy volunteers. Measures of velocity replication were performed in two ranges of knee joint flexion (0°–30° and 60°–90°), across four testing velocities (5, 10, 15, and 30°/s) in two movement directions (flexion and extension). Statistical analysis included intraclass correlation coefficients (ICCs; 2, k) and associated standard error of the measures calculated between day 1 and 2. We performed z-tests between all possible combinations of ICC pairs using Fisher’s Z transformations to determine if any significant differences existed between observed ICCs. We also calculated correlation ratios (η²) to explain the source of variability in the calculated ICCs. To assess measurement accuracy, we calculated constant error and absolute error between criterion and replication velocities. Results on ICCs and standard error of the measurements (SEMs) ranged from r = ?0.44 ± 7.00 to 0.88 ± 0.72°/s. Calculated z-tests indicated six paired ICCs were significantly different (?p < 0.1). In all six pairs, the faster test velocity had a lower ICC magnitude. The η² calculations demonstrated that inconsistent performance between day 1 and 2 caused the low ICC magnitudes observed with faster testing velocities. Significantly more absolute error occurred at 30 and 15°/s compared with 5°/s. Significantly less constant error was observed for 30°/s compared with 15°/s. A significant direction by range of motion interaction indicated less constant error for flexion movements in the 60°–90° range of motion (ROM) as compared with extension movements in either ROM. Healthy individuals could actively replicate slower criterion velocities in the mid and end ranges of knee joint motion in both movement directions with an acceptable amount of consistency and accuracy. The data support the use of velocity replication in future investigations on proprioceptive function.