Analysis of birefringence decay profiles for nucleic acid helices possessing bends: the tau-ratio approach.

For nucleic acid helices in the 100-200-bp range, a central bend or point of flexibility increases the rate of rotational diffusion. In a transient electric birefringence (TEB) experiment, this increase is manifest as a reduction in the terminal (slowest) birefringence decay time. Previous experimental and theoretical work has demonstrated that the ratio of the decay times for a bent/flexible molecule and its fully duplex (linear) counterpart represents a sensitive, quantifiable measure of the apparent bend angle (tau-ratio approach). In the current work, we have examined the influence of helix parameters (e.g., persistence length, helix rise, diameter) on the tau-ratio for a given bend. The tau-ratio is found to be remarkably insensitive to variations and/or uncertainties in the helix parameters, provided that one employs bent and control molecules with the same sequence and length (apart from the bend itself). Although a single tau-ratio determination normally does not enable one to distinguish between fixed and flexible bends, such a distinction can be made from a set of tau-ratios for molecules possessing two variably phased bends. A number of additional uncertainties are examined, including errors in the estimation of the dimensions of nonhelix elements that are responsible for bends; such errors can, in principle, be estimated by performing a series of measurements for molecules of varying length.     

Influence of static and dynamic bends on the birefringence decay profile of RNA helices: Brownian dynamics simulations.

Bends in nucleic acid helices can be quantified in a transient electric birefringence (TEB) experiment from the ratio of the terminal decay times of the bent molecule and its fully duplex counterpart (tau-ratio method). The apparent bend angles can be extracted from the experimental tau-ratios through the application of static (equilibrium-ensemble) hydrodynamic models; however, such models do not properly address the faster component(s) of the birefringence decay profile, which can represent up to 80% of the total birefringence signal for large band angles. To address this latter issue, the relative amplitudes of the components in the birefringence decay profile have been analyzed through a series of Brownian dynamics (BD) simulations. Decay profiles have been simulated for three-, five-, and nine-bead models representing RNA molecules with central bends of 30 degrees, 60 degrees, and 90 degrees, and with various degrees of associated angle dispersion. The BD simulations are in close agreement with experimental results for the fractional amplitudes, suggesting that both amplitudes and terminal tau-ratios can be used as a measure of the magnitudes of bends in the helix axis. Although the current results indicate that it is generally not possible to distinguish between relatively fixed and highly flexible bends from single tau-ratio measurements, because they can lead to similar reductions in terminal decay time and amplitude, measurements of the dependence of the fractional amplitudes on helix length may afford such a distinction.     

Arterial and venous blood flows in cats in deep respiration following orthostatic and antiorthostatic tilts

In anesthetized cats, an increase in negative intrathoracic pressure following orthostatic and antiorthostatic tilts exerted no effects on the vena cava blood flow, whereas the carotid artery blood flow increased following the antiorthostatic tilt.     

Fluid flows and forces in development: functions, features and biophysical principles

Throughout morphogenesis, cells experience intracellular tensile and contractile forces on microscopic scales. Cells also experience extracellular forces, such as static forces mediated by the extracellular matrix and forces resulting from microscopic fluid flow. Although the biological ramifications of static forces have received much attention, little is known about the roles of fluid flows and forces during embryogenesis. Here, we focus on the microfluidic forces generated by cilia-driven fluid flow and heart-driven hemodynamics, as well as on the signaling pathways involved in flow sensing. We discuss recent studies that describe the functions and the biomechanical features of these fluid flows. These insights suggest that biological flow determines many aspects of cell behavior and identity through a specific set of physical stimuli and signaling pathways.     

Influence of temperature on the development and decay of thermotolerance and heat shock proteins

The development of thermotolerance and its decay in plateau-phase Chinese hamster cells are shown to be temperature-dependent phenomena. Development of tolerance, after an initial dose of 10 min at 45 degrees C, is appreciably slower between 20 and 28 degrees C than it is at 37 degrees C. Decay of tolerance is also slower in that temperature range; at 4-23 degrees C, it does not decay at all during the 96-h interval of the experiment. At 41 degrees C, thermotolerance decay, "step-down" cell killing, and thermotolerance induction apparently all occur and affect cell survival. The decay of HSP 70 mirrors that of thermotolerance, except at 41 degrees C. At that temperature very likely de novo synthesis of that protein becomes important in determining protein concentration. Our data show that care must be taken when extrapolating from kinetic data obtained with surface tissues in vivo to those in depth. The former are usually at a temperature between 25 and 32 degrees C; the latter are at 37 degrees C.     

Determination of the average shape of flagellar bends: a gradient curvature model

Data obtained by manual digitization of photographs of flagellar bending waves have been analyzed by determining size parameters for the bends by least-squares fitting of a model waveform. These parameters were then used to normalize the data so that the average shape of the bends could be determined. Best fits were obtained with a model waveform derived from the constant curvature waveforms used previously but with provision for a linear change in curvature across the central region of the bend-the gradient curvature model (GCM). The central regions of the GCM bending waves are separated by transition regions with length determined by a parameter called the truncation factor (FT). Fitting the GCM to sine-generated bending waves give optimal fit when FT = 0.34. Fitting the GCM to four different samples of flagellar bending waves gave best fits with values of FT ranging from 0.17 for ATP-reactivated Lytechinus spermatozoa beating at approximately 10 Hz to 0.32 for live spermatozoa of Arbacia. The difference between the Arbacia waveforms and a sine-generated waveform is therefore very small, but a sine-generated waveform lacks the degree of freedom represented by FT that is required to fit other waveforms optimally. The residual differences between the waveform data and optimal GCM waveforms were averaged and found to be small. In most cases, the curvature in the central region of the optimal GCM decreased in magnitude towards the tip of the flagellum; however, this slope was highly variable and sometimes positive. Significant variations in both this slope and FT were found in individual bends as they propagated along a flagellum.     

Effect of pH and cell cycle progression on development and decay of thermotolerance

Synchronous Chinese hamster ovary cells were heated in G1 and incubated at 37 degrees C at pH 6.75 or pH 7.4 before they were heated a second time. The magnitude and rate of development and decay of thermotolerance were greatly reduced at pH 6.75. This was also observed for asynchronous cells. Furthermore, the heat-induced delay in cell cycle progression was greatly enhanced at low pH and correlated with the reduced rate for development and decay of thermotolerance. However, studies with [3H]TdR to kill cells entering S phase showed that the decay of thermotolerance is relatively independent of the cell cycle. Therefore, low pH apparently slows many cell processes, including those associated with heat-induced cell cycle delay and the rate of development and decay of thermotolerance.     

Movement of turritella spermatozoa: direction of propagation and chirality of flagellar bends.

The marine snail, Turritella communis, produces two types of spermatozoa, named apyrene and eupyrene. Eupyrene spermatozoa are usually paired, but unpaired ones are involved in fertilization. Movements of these spermatozoa were analyzed using a video camera with a high-speed shutter. The eupyrene spermatozoa usually swim with the head foremost but are able to swim flagellum foremost. A reversal of the direction of their swimming was found to be the result of a change in the direction of flagellar bend propagation, which changed with calcium concentration. Reversal of the direction of bend propagation was accompanied by a reversal of direction of the rotational movement of the spermatozoa around their long axis, suggesting that the bending waves keep the sense of their three-dimensional form. The swimming speed of apyrene spermatozoa in natural seawater was about one-eighth of that of the eupyrene ones and remained almost constant in highly viscous medium.The swimming speed of conjugated eupyrene spermatozoa was the same as that of unpaired spermatozoa over a wide viscosity range (<3,000 cP). No advantage of swimming by two spermatozoa could be detected in Turritella spermatozoa.     

Helical phasing between DNA bends and the determination of bend direction.

The presence and location of bends in DNA can be inferred from the anomalous mobility of DNA fragments or protein-DNA complexes during electrophoresis in polyacrylamide gels. Direction of bending is not so easily determined. We show here that a protein-induced bend, when linked to a protein-independent DNA bend by a segment of variable length, exhibits an electrophoretic mobility that varies in a sinusoidal manner with the length of the linker. Mobility minima occur once for each addition to the linker of one helical turn of DNA. Since minima should occur when two bends reinforce one another, the direction of one bend relative to the other can be determined from the distances between the two centers of bending at which minima occur. Our results strongly support the idea that the A5-6 tracts in kinetoplast DNA bend towards the minor groove while the bend at the recombination site of the gamma delta resolvase (binding site I of the gamma delta res site) bends towards the major groove.     

Utilization of glucose and amino acids in insect cell cultures: Quantifying the metabolic flows within the primary pathways and medium development

The current understanding of insect cell metabolism is very limited. In order to gain some insight into the growth and metabolism of insect cells Spodoptera frugiperda (Sf9), a comprehensive characterization of culture conditions for cells grown in the IPL-41 medium was made by measuring the amino acid composition of the growth medium and the cell extract, the macromolecular composition of the cells (DNA, RNA, and protein), medium concentrations of various metabolites and sugars, and the evolved CO(2). Since in the IPL-41-based serum-free medium all of the amino acids except cysteine are in great excess of what is needed by the cells for energy and protein production, a medium formulation with an osmolarity similar to the IPL-41 but with a lower amino acid content than IPL-41 was also developed. The new medium also lacks maltose and sucrose (contains only glucose), supported cell growth to a high cell density of 8 x 10(6) cells/mL. The cellular and energetic yields indicated that a tight coupling between the biosynthetic and energetic reactions was attained for cells grown in the new medium. Moreover, it was found that the intermittent feeding of glucose may not be required as the cell yield and growth rate were comparable whether the same total amount of glucose was provided intermittently or was included initially in the medium. The eventual cessation of growth in the new medium is believed to be due to the amino acid limitation because concentrations of both glutamine and glutamate were very low at the end of the growth phase. Thus, further optimization, which may include higher initial glutamine in the medium or its intermittent feeding, could lead to a further increase in the cell density. Finally, a stoichiometrically based analysis of metabolic reactions confirmed the operation of the key pathways and was used to quantify the distribution of metabolites among primary metabolic reactions. The quantitative flow values were used to highlight some key aspects of insect cell metabolism. (c) 1993 John Wiley & Sons, Inc.     

Phosphorylation of HSP27 during development and decay of thermotolerance in Chinese hamster cells

The small molecular weight heat shock protein HSP27 was recently shown to confer a stable thermoresistant phenotype when expressed constitutively in mammalian cells after structural gene transfection. These results suggested that HSP27 may also play an important role in the development of thermotolerance, the transient ability to survive otherwise lethal heat exposure after a mild heat shock. In Chinese hamster O23 cells increased thermoresistance is first detected at 2 h after a triggering treatment of 20 min at 44 degrees C, attains a maximum at 5 hours, and decays thereafter with a half-life of 10 h. We found that the development and decay of transient thermotolerance cannot be solely explained on the basis of changes in the cellular concentration of HSP27. The cellular HSP27 concentration is not increased appreciably at 2 h after heat shock and attains a maximum at 14 h. Similar results were obtained in the case of another heat shock protein, HSP70. HSP70 follows slightly faster kinetics of accumulation (peaks at 10 h) and decays much more rapidly (ti/2 = 4h) than HSP27 (t1/2 = 13h). HSP27 has 3 isoelectric variants A, B, and C of which B and C are phosphorylated. In cells maintained at normal temperature, HSP27A represents more than 90% of all HSP27. Shifting the cell culture temperature from 37 to 44 degrees C induces the incorporation of 32P into the more acidic B and C forms, a process that occurs very rapidly since the reduction in the concentration of the A form and a corresponding increase in the level of B and C is detectable by immunoblot analysis within 2.5 min at 44 degrees C. Analyses performed at various times during development and decay of transient thermotolerance revealed a close relationship between the effect of heat shock on HSP27 phosphorylation and cell ability to survive. For example, fully thermotolerant cells (5 h post-induction) are refractory to induction of HSP27 phosphorylation by a 20-min heat shock. The induction of HSP27 phosphorylation was also studied in a family of clonal cell lines of O23 cells that are thermoresistant as a result of the constitutive expression of a transfected human HSP27 gene. In these thermoresistant cells, phosphorylation of the endogenous hamster HSP27 is induced to a level comparable to that found in the thermosensitive parental cells. However, phosphorylation of the exogenous human protein, which represents more than 80% of total HSP27 in these cells, was much less induced.(ABSTRACT TRUNCATED AT 250 WORDS)     

New insights into the structure of An tracts and B'-B' bends in DNA

Energy calculations suggest that the currently available NOE distance constraints for An tracts in DNA are incapable of distinguishing between structures with a narrowed minor groove arising from a large propeller twist with a small inclination or from a small propeller twist with a large negative inclination. Furthermore, analysis of published data, together with energy estimations, strongly argue against bifurcated hydrogen bonding between A and T residues being the cause of the anomalous structural properties of An tracts. A conformational analysis of the B'-B' junction has been performed in which a single variable base pair has been inserted between two regions of B' structure. We have calculated low-energy structures for AnGAn,AnCAn,AnTAn,AnCTn, and TnCAn duplexes, where the An and Tn tracts were fixed in the anomalous B' conformation. Upon optimization, all these structures were found to contain a pronounced roll-like bending into the major groove at the site of the insertion. The important factors in the formation of these B'-B' bends are the destruction of the B' conformation and the concomitant widening of the minor groove at the junction region in order to reduce minor groove interstrand base clashes and improve interstrand stacking energy. If the B' conformation has strong negative inclination, the improved intrastrand stacking energy also contributes to the bending. In calculations of duplexes with An and Tn tracts in the B conformation instead of B', the bending disappears.     

Riding the wave: structural and energetic principles of helical membrane proteins

Genome sequencing efforts have revealed that perhaps as many as 20-40% of open reading frames in complex organisms may encode proteins containing at least one helical transmembrane segment. Contrasting with this approaching tidal wave of helical membrane proteins is the fact that our understanding of the sequence-structure-function relationships for membrane proteins lags far behind that of soluble proteins. This looming reality emphasizes the tremendous biochemical and structural work that remains to be done on helical membrane proteins in order to elucidate the structural and energetic principles that specify and stabilize their folds, which define their functions. These facts are not lost on the pharmaceutical industry, where successful therapeutics and major discovery efforts are targeting membrane proteins.     

Sedimentation coefficients of DNA topoisomers: the helical wormlike chain

The sedimentation coefficient s_N of the DNA topoisomer with the linking number N is evaluated as a function of N and chain length on the basis of a (circular) twisted wormlike chain, i.e., a special case of the helical wormlike chain. Evaluation is carried out by an application of the Oseen–Burgers procedure of hydrodynamics to the cylinder model with the preaveraged Oseen tensor. The necessary mean reciprocal distance between two contour points is obtained by a Monte Carlo method. It is shown that s_N increases as |ΔN| is increased from 0 in the range of small |ΔN|, where ΔN = N ? N , with N the number of helix turns in the linear DNA chain in the undeformed state. It is found that there is semiquantitative agreement between the Monte Carlo values and the experimental data obtained by Wang for s_N.