Gliding mutants of Myxococcus xanthus with high reversal frequencies and small displacements

Myxococcus xanthus cells move on a solid surface by gliding motility. Several genes required for gliding motility have been identified, including those of the A- and S-motility systems as well as the mgl and frz genes. However, the cellular defects in gliding movement in many of these mutants were unknown. We conducted quantitative, high-resolution single-cell motility assays and found that mutants defective in mglAB or in cglB, an A-motility gene, reversed the direction of gliding at frequencies which were more than 1 order of magnitude higher than that of wild type cells (2.9 min-1 for DeltamglAB mutants and 2.7 min-1 for cglB mutants, compared to 0.17 min-1 for wild-type cells). The average gliding speed of DeltamglAB mutant cells was 40% of that of wild-type cells (on average 1.9 micrometers/min for DeltamglAB mutants, compared to 4.4 micrometers/min for wild-type cells). The mglA-dependent reversals and gliding speeds were dependent on the level of intracellular MglA protein: mglB mutant cells, which contain only 15 to 20% of the wild-type level of MglA protein, glided with an average reversal frequency of about 1.8 min-1 and an average speed of 2.6 micrometers/min. These values range between those exhibited by wild-type cells and by DeltamglAB mutant cells. Epistasis analysis of frz mutants, which are defective in aggregation and in single-cell reversals, showed that a frzD mutation, but not a frzE mutation, partially suppressed the mglA phenotype. In contrast to mgl mutants, cglB mutant cells were able to move with wild-type speeds only when in close proximity to each other. However, under those conditions, these mutant cells were found to glide less often with those speeds. By analyzing double mutants, the high reversing movements and gliding speeds of cglB cells were found to be strictly dependent on type IV pili, encoded by S-motility genes, whereas the high-reversal pattern of mglAB cells was only partially reduced by a pilR mutation. These results suggest that the MglA protein is required for both control of reversal frequency and gliding speed and that in the absence of A motility, type IV pilus-dependent cell movement includes reversals at high frequency. Furthermore, mglAB mutants behave as if they were severely defective in A motility but only partially defective in S motility.     

Increased oxygen consumption associated with breathing activity in fetal lambs

To examine the relationship between fetal O2 consumption and fetal breathing movements, we measured O2 consumption, umbilical blood flow, and cardiovascular and blood gas data before, during, and after fetal breathing movements in conscious chronically catheterized fetal lambs. During fetal breathing movements, O2 consumption increased by 30% from a control value of 7.7 +/- 0.7 (SE) ml X min-1 X kg-1. Umbilical blood flow was 210 +/- 21 ml X min-1 X kg-1 before fetal breathing movements; in 9 of 16 samples it increased by 52 +/- 12 ml X min-1 X kg-1, while in the other 7 it decreased by 23 +/- 9 ml X min-1 X kg-1. Umbilical arterial and venous O2 partial pressures and pH fell during fetal breathing movements, and the fall was greater when umbilical blood flow was decreased. Partial CO2 pressure rose in both vessels, and again the increase was greatest when umbilical blood flow fell during fetal breathing movements. Also associated with a fall in umbilical blood flow was the transition from low-amplitude irregular to large-amplitude regular fetal breathing movements. It is concluded that fetal breathing movements increase fetal O2 demands and are associated with a transient deterioration in fetal blood gas status, which is most severe during large-amplitude breathing movements.     

Extracellular matrix fluctuations during early embryogenesis

Extracellular matrix (ECM) movements and rearrangements were studied in avian embryos during early stages of development. We show that the ECM moves as a composite material, whereby distinct molecular components as well as spatially separated layers exhibit similar displacements. Using scanning wide field and confocal microscopy we show that the velocity field of ECM displacement is smooth in space and that ECM movements are correlated even at locations separated by several hundred micrometers. Velocity vectors, however, strongly fluctuate in time. The autocorrelation time of the velocity fluctuations is less than a minute. Suppression of the fluctuations yields a persistent movement pattern that is shared among embryos at equivalent stages of development. The high resolution of the velocity fields allows a detailed spatio-temporal characterization of important morphogenetic processes, especially tissue dynamics surrounding the embryonic organizer (Hensen's node).     

Actin filaments responsible for the location of the nucleus in the lentil statocyte are sensitive to gravity

The location of the nucleus in statocytes or lentil roots grown: 1), at 1 g on the ground, 2), on a 1 g centrifuge in space, 3), in simulated microgravity on a slowly rotating clinostat (0.9 rmp) 4), in microgravity in space was investigated and statistically evaluated. In cells differentiated at 1 g on the ground, the nuclear membrane was almost in contact with the plasmalemma lining the proximal cell wall, whereas in statocytes of roots crown on the clinostat there was a distance of 0.47 micrometers (horizontal clinorotation) and or 0.76 micrometers (vertical clinorotation) between these membranes. However, in microgravity the nucleus was the most displaced, 0.87 micrometers from the proximal cell wall. Centrifugation of vertically grown roots in the root-tip direction showed that the threshold of centrifugal force to detach all nuclei from the proximal cell wall was about 40 g. In statocytes developed in the presence of cytochalasin B at 1 g the nuclei were sedimented on the amyloplasts at the distal cell pole, demonstrating that the location of the nucleus depends on actin filaments. The results obtained are in agreement with the hypothesis that gravity causes a tension of actin filaments and that this part of the cytoskeleton undergoes a relaxation in microgravity.     

Cortical and cap sedimentation in gravitropic Equisetum roots

Although the rootcap is required for gravitropic sensing, various classical and contemporary data raise the question of whether additional sensing occurs away from the cap in roots. Roots of Equisetum hyemale L. (horsetail) were examined by light and electron microscopy to determine which cell components were distributed with respect to gravity both in and away from the rootcap. Adventitious roots from stem cuttings were gravitropic in a vertical orientation or if reoriented to the horizontal. Obvious amyloplast sedimentation was found in vertical and in reoriented roots 1) in cells in the center of the rootcap and 2) in young, elongating cortical cells located in two to three layers outside the endodermis. These cortical amyloplasts were smaller than cap amyloplasts and, unlike central cap amyloplasts, were occasionally found in the top of the cell. The nucleus was also sedimented on top of the amyloplasts in both cell types, both in vertical and in reoriented roots. Sedimentation of both organelles ceased as cortical cells elongated further or as cap cells became peripheral in location. In both cell types with sedimentation, endoplasmic reticulum was located in the cell periphery, but showed no obvious enrichment near the lower part of the cell in vertical roots. This is the first modern report of sedimentation away from the cap in roots, and it provides structural evidence that gravitropic sensing may not be confined to the cap in all roots.     

Investigation of sarcoplasmic reticulum SH-groups]

The amount and the reaction capacity of the thiol groups in the sarcoplasmic reticulum containing up to 86% of Ca-ATPase were determined using 7-chloro-4-nitrobenzo-2-hydroxo-1,3-diazole (NBD-chloride). The total amount of SH-groups interacting with NBD-chloride is about 9 moles/10(5) g of protein as determined in the excess of NBD-chloride (750 micrometers). With respect to their sensitivity to NBD-chloride the SH-groups may be divided into two classes: slow and fast ones (5,3 and 3,5 moles/10(5) g of protein, respectively). The modification constants for the fast and slow SH-groups are 0,16 and 0,015min-1. ATP (30 micrometers) decreases the number of fast groups by 1 mole/10(5) g of protein. At higher concentrations of ATP (1--3 mM) the amount of fast SH-groups is decreased by 3 moles/10(5) g of protein, their modification rate constant being decreased 2-fold. ATP at concentration of 1 mM, decreases the rate constant for the Ca-ATPase inactivation by NBD-chloride from 0.68 down to 0,073 min-1, which coincides with the modification rate constant for fast SH-groups (0,071 min-1) under the same conditions. Ca2+ at concentration of 10(-4) M increases the amount of fast thiol groups by 1 mole/10(5) g of protein, the rate constant of their modification by NBD-chloride being increased 2-fold. A half-maximal effect was observed in the presence of 5.10(-7) M Ca2+ . Mg2+ did not affect the total amount of fast thiol groups; however, it decreased their modification rate constant.     

CELLULAR DISTRIBUTION OF 16S ACETYLCHOLINESTERASE

Multiple molecular forms of acetylcholinesterase (AChE; EC 3.1.1.7), in crude extracts of various tissues from the rat, were distinguished by velocity sedimentation analysis on linear sucrose gradients. Skeletal muscle samples containing end-plate regions showed three different forms of AChE with apparent sedimentation coefficients of 16, 10 and 4s. The 16s form was not detected in non-innervated regions of skeletal muscle, large intestine smooth muscle, whole brain tissue, red blood cells or plasma. Spinal cord, a predominantly motor cranial nerve and mixed (sensory and motor) peripheral nerves contained 16, 10, 6.5 and 4S AChE. Ventral motor roots, supplying the sciatic nerve, contained these four forms of the enzyme, while corresponding dorsal sensory roots were devoid of the 16S form. The 16s-AChE confined to ventral roots can be attributed totally to motor neurons and not to Schwann cells composing these roots. Whether the 16s-AChE presently found in motor nerves has chemical identity with that found at motor end-plates is the basis of future experiments.     

Amyloplast sedimentation kinetics in gravistimulated maize roots

Amyloplast sedimentation in gravistimulated maize (Zea mays L.) roots was measured using the change in angle from the center of the cell to each amyloplast as an index of sedimentation. Using tissue fixed after gravistimulation, the relationship between mean amyloplast angle and the duration of gravistimulation was found to be linear when plotted on a logarithmic time scale. Extrapolated values for the onset of angular change are 5.9 s after the start of gravistimulation for the entire population of amyloplasts and 11.8 s for lead amyloplasts. By multiplying the instantaneous angular velocity (in radians) by the cell center to amyloplast radius, it is possible to calculate the initial sedimentation velocity to be 19.1 m min^-1 at 5.9 s. During sedimentation, the mean amyloplast angles surpass the calculated cell corner angle of 123° at 2.2 min for all amyloplasts and at 19 s for lead amyloplasts near the new lower wall. Thus, substantial sedimentation occurs within the presentation time, calculated to be 4.1 min. These kinetics are consistent with several hypotheses of graviperception.Symbol t_p presentation time     

Light-difference detector for reading Rayleigh fringe patterns from the ultracentrifuge

We have developed a dual-photocell, light-difference detector, easily attached to a comparator screen, which provides rapid and direct location of fringe centers from Rayleigh interferograms without the need for digital micrometers or measurement of optical densities. The device provides a pulse for digital micrometers which triggers the printing of fringe centers during manual movement of the stage, providing a two-thirds saving in time. From evaluation of sedimentation equilibrium patterns it was found that the precision of (1) concentration measurement is 0.003 fringes and (2) molecular weight determinations is several parts per thousand.     

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.     

Substrate-concentration of dependences of tension, shortening velocity and ATPase activity of glycerinated single muscle fibers

Tension P0 and ATPase activity J0 of glycerinated single muscle fibers under isometric concentration as well as the velocity V0 of unloaded shortening were measured as a function of substrate concentration [S]. The stiffness of fibers with sinusoidal length changes at 1 kHz was used as a qualitative measure of the amount of rigor complex. P0 is an increasing function of [S] at low substrate concentrations and has a broad maximum at about 10-40 micrometers MgATP. In this concentration range, 10-40 micrometers, V0 still has a very small value. Then it increases and finally reaches at a plateau at about 1 mM MgATP. J0 increases as P0 does. However, it reaches at a saturated level at about the same concentration as V0. Either 0.5 mM 8-BrATP or 1 mM PPi was added to the substrate solutions to reduce the amount of rigor complex at low substrate concentrations. The addition of PPi of 8-BrATP decreases P0 dominantly at low concentrations of substrate and shifts the maximum to about 100 micrometers MgATP. 8-BrATP considerably increases V0 at low substrate concentrations while V0 is decreased by added PPi. The temperature coefficients, Q10 values were obtained for P0, J0, and V0. The values are essentially constant, 2.1-2.4, in the cases of P0 and J0, and about the same values were found for V0 at very low substrate concentrations. However, they become about 3.3 in the concentration range from 34 micrometers and 2.3 mM. The P-V relation was obtained at 11 micrometers and 2.3 micrometers MgATP. The normalized P-V relation at 11 micrometers was unchanged when 8-BrATP was added. The results are discussed in connection with the mechanism of actomyosin-ATPase activity as well as that of the elementary cycle of the motive force generation.     

Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation

Fluorescence optical detection in sedimentation velocity analytical ultracentrifugation allows the study of macromolecules at nanomolar concentrations and below. This has significant promise, for example, for the study of systems of high-affinity protein interactions. Here we describe adaptations of the direct boundary modeling analysis approach implemented in the software SEDFIT that were developed to accommodate unique characteristics of the confocal fluorescence detection system. These include spatial gradients of signal intensity due to scanner movements out of the plane of rotation, temporal intensity drifts due to instability of the laser and fluorophores, and masking of the finite excitation and detection cone by the sample holder. In an extensive series of experiments with enhanced green fluorescent protein ranging from low nanomolar to low micromolar concentrations, we show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of the impact of these effects on the recorded data. Systematic deviations of fluorescence optical sedimentation velocity data analyzed using conventional sedimentation models developed for absorbance and interference optics are largely removed after these adaptations, resulting in excellent fits that highlight the high precision of fluorescence sedimentation velocity data, thus allowing a more detailed quantitative interpretation of the signal boundaries that is otherwise not possible for this system.     

Microtubule dynamics in a cytosolic extract of fetal rat brain

Brain microtubule dynamics were studied by video-enhanced differential interference contrast microscopy in a cytosolic extract from fetal rat brain, prepared under conditions designed to produce minimal alterations in microtubule stability. With urchin sperm axoneme fragments as nucleation seeds, the extract was shown to support cellular-like microtubule dynamics. Microtubules elongated from one end of the axonemes, and did not spontaneously self-assemble in the absence of axonemes. The following microtubule kinetic parameters were measured in the extract: velocity of elongation (8.1 mm/min), velocity of rapid shortening (5.8 mm/min), catastrophe frequency (0.17 min-1), and rescue frequency (1 min-1). These parameters were in close agreement with reported values for growth cones of living neurons. Microtubule properties in the fetal brain extract were shown to be affected by agents with known effects on the cytoskeleton. pp60c-src, a tyrosine kinase important in cell adhesion molecule-dependent axon growth, caused small increases in the frequency of microtubule catastrophe (0.31 min-1) and rescue (2 min-1) without changing the velocities of elongation or rapid-shortening. Although pp60c-src phosphorylated purified porcine brain tubulin in vitro, it did not elicit significant changes in its polymerization properties, suggesting that other cytoskeletal proteins in the brain extract are involved in modulating microtubule dynamics. The cytosolic extract of fetal rat brain provides a useful system for studying regulation of microtubule assembly in neuronal growth cones.     

Osmotic response of individual cells during freezing. II. Membrane permeability analysis

An analytical model is presented to simulate the freezing of individual yeast cells. In addition the model is solved numerically on a digital computer to obtain values for cell volume as a function of temperature, based on the thermal protocol during freezing, and the transport parameters of the cell membrane. The numerical procedure was modified to enable values for the membrane hydraulic permeability reference coefficient, Lpg, and activation energy, ELp, to be deduced by nonlinear analysis of complementary experimental data (10). It was observed that the apparent values of both Lpg and ELp increase with cooling rate, from Lpg = 0.0116 micrometer 3 micrometers-2 atm-1 min-1 and ELp = 19.4 kJ mol-1 for 9 degrees K/min to Lpg = 2.11 micrometers 3 micrometer-2 atm-1 min-1 and ELp = 101 kJ mol-1 for 35 degrees K/min. The deduced permeabilities fall within the range of values determined in a prior study by Levin (6). Analysis with the model also indicates that the turgor pressure exerts a negligible effect on yeast exposed to freezing stress.     

Induction by an hepatic carcinogen, 1-nitroso-5,6-dihydrouracil, of single and double strand breaks of liver DNA with rapid repair

The nitrosoureas derived from 3 naturally occurring ureides were administered to rats and the velocity sedimentation of hepatic DNA in alkaline and neutral sucrose gradients determined. The potent hepatocarcinogen 1-nitroso-5,6-dihydrouracil induced apparent double strand as well as single strand breaks in liver DNA within 30 minutes. This damage seemed to be repaired within 4 hours. In contrast, 1-nitrosohydantoin and δ-nitroso-L-citrulline, neither of which are known hepatocarcinogens, did not modify the velocity sedimentation of hepatic DNA.     

Interactions of photo- and geotropism with periodical oscillations of growing pea root (Pisum sativum L.)

Oscillation movements of primary roots of pea seedlings were investigated after low- and high-energy irradiation (10^?2 and 10⁶ W m^?2) which was applied continuously and in pulses at intervals of 3 and 30 min. Oscillation amplitudes of control roots grown in darkness were lower (1 mm) than those of irradiated ones recorded in our previous experiments. The elongation rate of roots grown under scattered and low-energy irradiation as well as their mean oscillation amplitude (2.6 mm) and frequency (8 h) correspond to the standard values recorded in previous experiments. The same effects on root growth and oscillations had scattered electronic flash irradiation. Side-irradiation did not influence the oscillation movements of growing roots. Their frequency and oscillation amplitudes were not changed. Their longitudal axis was, however, deflected 2.75° from the vertical one due to the negative phototropic response of the root. The growth oscillations may be, according to present results, interpreted as consequences of tropic growth controlling system. External factors, like irradiation, may influence oscillation parameters,i.e. their amplitudes and after application of side-irradiation also the direction of longitudinal axis of oscillation spirals. The resultant trajectory is composed from movements which resulted from geotropic and phototropic responses of the roots.     

A competitive labeling method for the determination of the chemical properties of solitary functional groups in proteins.

The properties of the functional groups in a protein can be used as built-in-probes of the structure of the protein. We have developed a general procedure whereby the ionization constant and chemical reactivity of solitary functional groups in proteins may be determined. The method may be applied to the side chain of histidine, tyrosine, lysine, and cysteine, as well as to the amino terminus of the protein. The method, which is an extension of the competitive labeling technique using [3H]- and [14C]1-fluoro-2,4-dinitrobenzene (N2ph-F) in a double-labeling procedure, is rapid and sensitive. Advantage is taken of the fact that after acid hydrolysis of a dinitrophenylated protein, a derivative is obtained which must be derived from a unique position in the protein. The method has been applied to the solitary histidine residue of lysozyme, alpha-lytic protease, and Streptomyces griseus (S.G.) trypsin, as well as to the amino terminus of the latter protein. The following parameters were obtained for reaction with N2ph-F at 20 degrees C in 0.1 N KCl: the histidine of hen egg-white lysozyme, pKa of 6.4 and second-order velocity constant of 0.188 M-1 min-1; the histidine of alpha-lytic protease, pKa of 6.5 and second-order velocity constant of 0.0235 M-1 min-1; the histidine of S.G. trypsin, pKa of 6.5 and second-order velocity constant of 0.0328 M-1 min-1; the valyl amino terminus of S.G. trypsin, pKa of 8.1 and second-order velocity constant of 0.403 M-1 min-1. In addition, the results obtained provide clues as to the microenvironments of these functional groups, and indicate that the proteins studied undergo pH-dependent conformational changes which affect the microenvironment, and hence the chemical reactivity of these groups.     

Random root movements in weightlessness

The dynamics of root growth was studied in weightlessness. In the absence of the gravitropic reference direction during weightlessness, root movements could be controlled by spontaneous growth processes, without any corrective growth induced by the gravitropic system. If truly random of nature, the bending behavior should follow socalled 'random walk' mathematics during weightlessness. Predictions from this hypothesis were critically tested.
In a Spacelab ESA-experiment, denoted RANDOM and carried out during the IML-2 Shuttle flight in July 1994, the growth of garden cress ( Lepidium sativum ) roots was followed by time lapse photography at 1-h intervals.
The growth pattern was recorded for about 20 h. Root growth was significantly smaller in weightlessness as compared to gravity (control) conditions.
It was found that the roots performed spontaneous movements in weightlessness. The average direction of deviation of the plants consistently stayed equal to zero, despite these spontaneous movements. The average squared deviation increased linearly with time as predicted theoretically (but only for 8–10 h).
Autocorrelation calculations showed that bendings of the roots, as determined from the 1-h photographs, were uncorrelated after about a 2-h interval.
It is concluded that random processes play an important role in root growth. Predictions from a random walk hypothesis as to the growth dynamics could explain parts of the growth patterns recorded. This test of the hypothesis required microgravity conditions as provided for in a space experiment.     

Preparation of mammalian cell-enclosing subsieve-sized capsules (<100 microm) in a coflowing stream

The droplet breakup technique with an immiscible liquid coflowing stream was investigated for the preparation of mammalian cell-enclosing subsieve-sized capsules of less than 100 microm in diameter. The major parts of the droplet generation device were a needle of several hundred micrometers in diameter for extruding the cell-suspending sodium alginate aqueous solution and a tubule of 2.5 mm in diameter through which the extruded alginate solution flowed into ambient immiscible liquid paraffin. The needle was positioned upstream in the vicinity of the coaxial tubule. The droplet diameter of the viscous sodium alginate aqueous solution could be controlled from several dozen to several hundred micrometers by changing the velocities of the inner and ambient fluids and the diameter of the needle. By utilizing a 300-microm diameter needle, CHO-K1 cell-enclosing droplets of 48 +/- 8 microm in diameter were obtained by extruding a cell-suspending sodium alginate solution at a velocity of 1.2 cm/sec into the ambient liquid paraffin flowing at a velocity of 23.5 cm/sec. The breakup process did not influence the viability of the enclosed cells, since more than 95% of the CHO-K1 cells remained alive after the enclosing process.     

Characterization of the 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol.

The 1 alpha,25-dihydroxyvitamin D3 receptor from rat intestinal cytosol has been partially characterized. Sucrose density gradient sedimentation and analytical gel filtration analyses of this receptor yielded values of 3.1 S, 80,000, and 36 A for the sedimentation coefficient, molecular weight (Mr), and Stokes molecular radius (Rs), respectively. The receptor was found to be a protein by its susceptibility to protease but not nuclease digestion, and studies with N-ethylmaleimide and iodoacetamide revealed the presence of a reduced cysteine residue near the ligand binding site of the receptor. Kinetic and equilibrium binding studies showed an equilibrium dissociation constant of 7.4 x 10(-10) M (4 degrees C), an association rate constant of 1.7 x 10(7) M-1 min-1 (0 degrees C) and a dissociation rate constant of 7.2 x 10(-4) min-1 (4 degrees C, t1/2 = 16 h).