Gravitropic bending of cress roots without contact between amyloplasts and complexes of endoplasmic reticulum

The polar arrangement of cell organelles in Lepidium root statocytes is persistently converted to a physical stratification during lateral centrifugation (the centrifugal force acts perpendicular to the root long axis) or by apically directed centrifugation combined with cytochalasin-treatment. Lateral centrifugation (10 min, 60 min at 10\g or 50\g) causes displacement of amylplasts to the centrifugal anticlinal cell wall and shifting of the endoplasmic reticulum (ER) complex to the centripetal distal cell edge. After 60 min of lateral centrifugation at 10\g or 50\g all roots show a clear gravitropic curvature. The average angle of curvature is about 40° and corresponds to that of roots stimulated gravitropically in the horizontal position at 1\g in spite of the fact that the gravistimulus is 10-or 50-fold higher. Apically directed centrifugation combined with cytochalasin B (25 g\ml^-1) or cytochalasin D (2.5 g\ml^-1) incubation yields statocytes with the amyloplasts sedimented close to the centrifugal periclinal cell wall and ER cisternae accumulated at the proximal cell pole. Gravitropic stimulation for 30 min in the horizontal position at 1\g and additional 3 h rotation on a clinostat result in gravicurvature of cytochalasin B-treated centrifuged (1 h at 50\g) roots, but because of retarded root growth the angle of curvature is lower than in control roots. Cytochalasin D-treatment during centrifugation (20 min at 50\g) does not affect either root growth or gravicurvature during 3 h horizontal exposure to 1\g relative to untreated roots. As lateral centrifugation enables only short-term contact between the amyloplasts and the distal ER complex at the onset of centrifugation and apically directed centrifugation combined with cytochalasin-treatment even exclude any contact the integrity of the distal cell pole need not necessarily be a prerequisite for graviperception in Lepidium root statocytes.Abbreviations CB cytochalasin B - CD cytochalasin D - ER endoplasmic reticulum - g gravitational acceleration     

Cytoplasmic streaming in internodal cells ofNitella under centrifugal acceleration: a study done with a newly constructed centrifuge microscope

Summary We constructed a new centrifuge microscope of the stroboscopic type, with which the cytoplasmic streaming inNitella internodal cells under centrifugal acceleration was studied. Under moderate centrifugal acceleration (ca. 50–100×g), the direction of cytoplasmic streaming in an internodal cell ofNitella is parallel to the direction of the subcortical fibrils. The speed of endoplasm flowing contiguous to the subcortical fibrils is neither accelerated nor retarded by moderate centrifugal acceleration. The endoplasmic flow, however, stops suddenly following an electrical stimulus. The endoplasm contiguous to the subcortical fibrils is immobilized transiently at the time of streaming cessation induced by an electrical stimulus under centrifugal acceleration at 50–100×g, even at 900×g. It is suggested that transitory cross bridges between the immobilized endoplasm and the subcortical fibrils are formed at the time of streaming cessation. The bulk endoplasm flows as a whole in the direction parallel to that of the subcortical fibrils and stops promptly upon electrical stimulation. Soon after the stoppage the bulk endoplasm starts to flow passively in the direction parallel to that of the centrifugal acceleration as a result of the centrifugal force.Abbreviations APW artificial pond water - CMS centrifuge microscope     

Isolation of intact nuclei of high purity from mouse liver

Current methods of nuclear isolation from liver disrupt the plasmalemmae via homogenization and separation of the nuclei by high centrifugal force (HCF) through gradients of sucrose or other substances for up to 80 min. The use of HCF for such a long time increases the potential for nuclear damage and degradation by endogenous proteases. We compared four combinations of alterations to classical nuclear isolation methods as follows. Mouse liver was gently crushed through a fine mesh with and without in vivo perfusion with collagenase. The cell suspension was centrifuged at 600g to remove gross debris and then at moderate centrifugal force (MCF, 16,000g) or high centrifugal force (HCF, 70,000g) through sucrose gradients for 30 min. The purity of the isolated nuclei was assessed biologically and morphologically, including analyses of representative marker proteins for nuclei and cytoplasm. The results indicate that MCF and no collagenase provided the highest nuclear integrity and purity, whereas MCF with collagenase is a viable option if priority is given to yield. The method is especially suited for small samples and so should facilitate studies with human liver biopsies and livers from mice, the most widely used species for gene targeting.     

Evacuolation and enucleation of mesophyll protoplasts in self-generating percoll gradients

Mesophyll protoplasts from Brassica oleracea, B. napus, Nicotiana tobaccum and Solanum tuberosum were isolated and subjected to uttracentrifugation at 65000g for 30 min in percoll solutions containing various strengths of salt and osmotic stabilizing agents. After centrifugation, the self-generated percoll gradients were evaluated for their effectiveness in protoplast evacuolation and enucleation. The vacuoles, cell debris, evacuolated protoplasts and enucleated protoplasts were separated. Factors that affected evacuolation and enucleation in the percoll gradients were described. Mesophyll protoplasts produced by epidermis peeling and short enzyme incubation periods were more easily evacuolated and enucleated than those produced by leaf-slicing and long incubation periods. Lower centrifugal force at 25000g for 80 min was also successful in evacuolating and enucleating the mesophyll protoplasts. A green band that contained nearly pure evacuolated protoplasts, of which 45% were enucleated protoplasts, was obtained from the self-generated percoll gradient. Rhodamine 123 staining of mitochondria indicated that the evacuolated protoplasts were metabolically active and were capable of regenerating the vacuole and cell wall. Cell divisions were also observed when the evacuolated protoplasts were cultured.     

Effects of centrifugal force and centrifugation time on the sedimentation of plant organelles

The effect of centrifugal force and length of centrifugation time on the sedimentation of plant organelles was determined for corn (Zea mays L.) root homogenates. A centrifugal force of 6000g for at least 20 minutes was necessary to pellet 90% of the mitochondrial marker (cytochrome c oxidase). This initial centrifugation step is optimal for separating mitochondria from microsomes, since cross-contamination of endoplasmic reticulum and plasma membrane vesicles with mitochondria is minimized. Centrifugal forces of 8000g or 10,000g for 20 minutes and 13,000g for 15 minutes pellet 90% of the mitochondrial marker; however, these centrifugation conditions also sediment more plasma membrane and endoplasmic reticulum.     

Orientation of the photosynthetic flagellate,Peridinium gatunense,in hypergravity

The photosynthetic freshwater flagellate,Peridinium gatunense, uses both positive phototaxis and negative gravitaxis to move upwards in the water column. At higher fluence rates approaching those at the surface of their habitat, the cells tend to become unoriented and thus stop their upward movement. Orientation and motility ofPeridinium gatunense has been studied in the slow rotating centrifuge microscope (NIZEMI), which allows observation of swimming behavior during centrifugation acceleration between 1g and 5g. The movement vectors were analyzed by real time image analysis capable of tracking many cells simultaneously. At 1g the orientation was not very precise, but the degree of orientation increased significantly at higher acceleration forces up to about 3g. Most cells were capable of swimming even against an acceleration vector of 3.8g; at higher acceleration forces the cells were not able to cope with the centrifugal force. The linear velocity of cells swimming against 1g was about 20% lower than that of cells moving in other directions. The velocity decreased even more in cells swimming against higher acceleration forces.     

Standardization of Human Diploid Fibroblast Cultivation: Centrifugation Procedure

The effect of varying centrifugal forces on the growth rate, longevity, and adsorption on glass of human embryonic diploid lung fibroblasts was studied. Cells centrifuged at 120, 500, or 1,500 × g at each passage had similar growth rates but their longevity decreased slightly with increasing force. These forces had no influence on the proportion of cells attaching to the glass. When the material in the first supernatant was recentrifuged at 2,000 × g for 30 min and added to the cells precipitated in the first centrifugation, the longevity of these cells was increased by several cell divisions. Cells which were not centrifuged but added directly from the cell suspension in trypsin to the new culture grew at a slightly slower rate than the centrifuged cells and became senescent at an earlier time. However, the noncentrifuged cells adsorbed to glass better than those centrifuged.     

Behavioral responses to short periods of lowered gravitational force in blind goldfish

1. The movements of blind goldfish flown in an aircraft through vertical flight patterns show a consistent correlation with the varyingg loads as recorded by ag meter.
2. Decreasing theg load in all tested cases caused the fish to rapidly dive downwards after an approximate delay of 0.5–1.5 sec. The opposite reaction, a tilt and movement upwards, was observed in transitions from a lower to a higherg load, though the latter reaction was not as pronounced.
3. Because any potential influence of visual cues, swimming bladder reflexes, and changing barometric pressure was excluded by experimental precautions, we concluded that the response of the fish to varyingg loads were vestibular reflexes probably resulting from displacements of the otoliths during vertical accelerations.
4. It is concluded from data in the literature and from the present results, that the behavioral responses resulting from static or dynamic forces are counterdirected toward the otolith displacements caused by these forces. Consequently, the behavioral, responses are counterdirected toward the gravitational pull on the animal, but must have the same direction as mechanical forces, which accelerate the whole organism and displace the otolith by inertia. It is concluded therefore, that the otolith reflexes have adaptive value only in a gravitational or centrifugal field, but are unsuitable to subserve any stabilizing function in the case of inertial forces during weightlessness.

     

Effect of centrifugation on the viability of Burkitt lymphoma cells

This study reports some findings on the effects of centrifugation on the viability of mammalian cells. The authors used Burkitt lymphoma cells cultivated in a synthetic medium containing 10% fetal calf serum for all experiments. Batch centrifugations were conducted in a RC2-B centrifuge (Ivan Sorvall, Incorporated, Norwalk, Connecticut USA) operated at 0 and 25°C. During centrifugation we exposed the cells to gravitational fields ranging from 24,800 to 42.200g. The results showed that at, 0°C and 25,800 or 42,000g no loss in cell viability occurred for up to 90 min exposures in the centrifugal field. However, at 25°C and for gravitational fields of 24,800 and 42,000g, there were appreciable losses in cell viability. Continuous centrifugation studies in the Sharples supercentrifuge (Division of Penn Salt Corporation, Warminister, Pennsylvania USA) were also conducted with bowl speeds up to 28,000 rpm (19,000g) and flow rates ranging from 1.4 to 20 1, hr. Slight, losses in cell viability were noted and postulated as caused by the shear stresses encountered by the cells. Some pumping studies using the lymphoma cells substantiate this conclusion.     

Spermatozoa recovery and post-thawing quality of brown bear ejaculates is affected for centrifugation regimes

Sperm cryopreservation protocols for brown bear (Ursus arctos) require the centrifugation of semen samples to increase sperm concentration and to clean urine in contaminated samples. We evaluated the effect of centrifugation regimes (time and relative centrifugal force—RCF) on the quantity of sperm recovered and the quality of post-thawed sperm. Thirteen brown bears were electroejaculated. The ejaculates were diluted 1:1 in Tris–citric acid–glucose (TCG) extender and centrifuged with different RCF/time combinations: 600×g, 1,200×g and 2,400×g, for 3, 6 or 12 min. After centrifugation, spermatozoa were diluted in TES–Tris–fructose extender with egg yolk and glycerol (final glycerol concentration of 8%) and frozen in 0.25-mL straws. In the post-thawed semen, motility was assessed by CASA, and acrosomal status (PNA-FITC), viability (SYBR-14 with propidium iodide) and chromatin status (SCSA) were determined by flow cytometry. The longest centrifugation time (12 min) significantly decreased some motility parameters. Sperm recovery significantly decreased in brown bear at 600×g. Our results suggest that brown bear spermatozoa are more sensitive to long centrifugation times than to high RCF. Centrifugation regimes showed no effects on the post-thawing chromatin status. We recommend preparing the brown bear semen for freezing by centrifugation 1,200×g or 2,400×g for 6 min, after electroejaculation and dilution 1:1 in TCG extender, since these procedures increase the spermatozoa recovery without harmful effects on the post-thawed quality of brown bear spermatozoa.     

Lentil root statoliths reach a stable state in microgravity

 The kinetics of the movement of statoliths in gravity-perceiving root cap cells of Lens culinaris L. and the force responsible for it have been analysed under 1 g and under microgravity conditions (S/MM-03 mission of Spacehab 1996). At the beginning of the experiment in space, the amyloplasts were grouped at the distal pole of the statocytes by a root-tip-directed 1-g centrifugal acceleration. The seedlings were then placed in microgravity for increasing periods of time (13, 29, 46 or 122 min) and chemically fixed. During the first 29 min of microgravity there were local displacements (mean velocity: 0.154 μm min⁻¹) of some amyloplasts (first at the front of the group and then at the rear). Nevertheless, the group of amyloplasts tended to reconstitute. After 122 min in microgravity the bulk of amyloplasts had almost reached the proximal pole where further movement was blocked by the nucleus. After a longer period in microgravity (4 h; experiment carried out 1994 during the IML 2 mission) the statoliths reached a stable position due to the fact that they were stopped by the nucleus. The position was similar to that observed in roots grown continuously in microgravity. Treatment with cytochalasin D (CD) did not stop the movement of the amyloplasts but slowed down the velocity of their displacement (0.019 μm min⁻¹). Initial movement patterns were the same as in control roots in water. Comparisons of mean velocities of amyloplast movements in roots in space and in inverted roots on earth showed that the force responsible for the movement in microgravity (F_c) was about 86% less (F_c = 0.016 pN) than the gravity force (F_g = 0.11 pN). Treatment with CD reduced F_c by two-thirds. The apparent viscosity of the statocyte cytoplasm was found to be 1 Pa s or 3.3 Pa s for control roots or CD treated roots, respectively. Brownian motion or elastic forces due to endoplasmic reticulum membranes do not cause the movement of the amyloplasts in microgravity. It is concluded that the force transporting the statoliths is caused by the actomyosin system. Received: 22 March 1999 / Accepted: 18 December 1999     

Effect of gravity and centrifugal acceleration on auxin transport in corn coleoptiles

Summary Inversion of corn coleoptile sections resulted in a 10–20% inhibition of basipetal transport of 3-indoleacetic acid (IAA) and a more pronounced inhibition (20–50%) of the transport of 1-naphthaleneacetic acid (NAA).The effect of inversion on basipetal NAA transport was compared in wild-type corn and in the amylomaize mutant which contains smaller and slower sedimenting amyloplasts: the gravity induced inhibition was higher in the wild type coleoptiles (27% versus 9%).In wild type the inhibitory effect on basipetal NAA transport appeared within less than 30 min after inversion; then the effect remained relatively constant over at least 2 hr of transport. When the sections were returned to the upright position the transport rate increased, reaching the level of upright controls within 30 min.An effect of gravity on lateral transport of NAA was also demonstrated and shown to be expressed within 10 min after placing the tissue horizontally.When basipetal transport was tested in the direction of gravity and/or centrifugal acceleration, auxin movement incrased with increasing acceleration. Transport against centrifugal acceleration (10 x g) was less than transport of control sections (inverted at 1 x g).The results agree with the hypothesis that starch statoliths act by a pressure mechanism on the membrane transport system of auxin.     

Gravitational force modulates muscle activity during mechanical oscillation of the tibia in humans

Mechanical oscillation (vibration) is an osteogenic stimulus for bone in animal models and may hold promise as an anti-osteoporosis measure in humans with spinal cord injury (SCI). However, the level of reflex induced muscle contractions associated with various loads (g force) during limb segment oscillation is uncertain. The purpose of this study was to determine whether certain gravitational loads (g forces) at a fixed oscillation frequency (30 Hz) increases muscle reflex activity in individuals with and without SCI. Nine healthy subjects and two individuals with SCI sat with their hip and knee joints at 90° and the foot secured on an oscillation platform. Vertical mechanical oscillations were introduced at 0.3, 0.6, 1.2, 3 and 5g force for 20 s at 30 Hz. Non-SCI subjects received the oscillation with and without a 5% MVC background contraction. Peak soleus and tibialis anterior (TA) EMG were normalized to M-max. Soleus and TA EMG were <2.5% of M-max in both SCI and non-SCI subjects. The greatest EMG occurred at the highest acceleration (5g). Low magnitude mechanical oscillation, shown to enhance bone anabolism in animal models, did not elicit high levels of reflex muscle activity in individuals with and without SCI. These findings support the g force modulated background muscle activity during fixed frequency vibration. The magnitude of muscle activity was low and likely does not influence the load during fixed frequency oscillation of the tibia.     

Measurement of the motive force of the migrating slug ofDictyostelium discoideum by a centrifuge method

Summary The motive force of the migrating slug of the cellular slime mouldDictyostelium discoideum was measured by the use of centrifugal force. Changes in shape of the slugs due to the use of centrifugal force were prevented by letting them migrate in an agar capillary. The motive force thus obtained was proportional to the slug volume, the value per unit volume being 5.8×10⁶ dyne/cm³ (58 N/cm³). This is in good agreement with the value measured by the use of hydrostatic pressure.     

Outline of a mathematical approach to the cancer problem

The fact that the growth of every organism, as it develops through cell multiplication from the ovum, eventually ceases, makes plausible the hypothesis that each cell of an organism exerts some kind of inhibitory influence upon the growth and multiplication of every other cell through the medium of some factorj. Each cell, however, is considered to possess an amount of a “growth factor”g, the activity of which is inhibited byj. In the course of development, most cells differentiate. A number of them remains relatively undifferentiated, and those are used for repairs of natural tear and wear as well as for healing of wounds. With age either the number of those cells or their “potency” decreases, which accounts for a general decrease in the speed of wound healing. Due to statistical fluctuations, the amount of theg is not uniformly distributed amongst the cells. Some of them have accidentally a greater amount than others. As the number or potency of the undifferentiated cells decreases with age, so does their inhibitory influence upon other cells. Therefore, those cells which do have an accidental dental excess ofg-factor will begin to grow and multiply, producing neoplasms. Putting the above assumptions into mathematical form, it is possible to derive a relation which gives theprobability of cancer incidence as a function of age. The theoretical relation is compared to the rather inadequate empirical data and found in agreement with them. A relation for the speed of wound healing as a function of age is also derived, and also found in general agreement with the rather scant data. Several other conclusions are discussed and possibilities of their practical applications pointed out.     

Influence of the rate and the share of water freezing on hydrogen and oxygen isotope separation

The influence of the rate v and the share of freezing water g on the separation of deuterium D and oxygen ¹⁸O was studied by mass spectrometry. Evidence was obtained supporting the well known facts that upon freezing of water, (1) the concentration of D in ice is higher than in water; (2) the degree of separation for D is higher than for ¹⁸O; (3) an increase in the concentration of D and ¹⁸O in ice takes place as the v value decreases. It was shown for the first time that, at g < 0.05, the concentrations of D at high v values are higher than at g > 0.05, and at low v values, they are less than at g > 0.05.     

Observations and measurements of sea urchin eggs with a centrifuge microscope

Eggs of the sea urchins, Arbacia punctulata and Lytechinus variegatus were observed with a centrifuge microscope. The protoplasmic viscosity calculated from the displacement velocity of the nucleus in a centrifugal field is about 30 poises. The surface forces of the unfertilized egg, which were determined from the relationship between the deformations of the egg in centrifugal fields and the magnitudes of the centrifugal forces, are 0.046 dyne/cm in Arbacia and 0.087 dyne/cm in Lytechinus (mean values). Both of these values increase as the deformation of the egg increases. The cleavage plane of the first cleavage of the egg in the centrifugal field (35–140 × g) is parallel to the direction of the centrifugal force. The flattening of the fertilized egg in a constant centrifugal field changes during development, probably owing to the change in the surface force. The flattening attains a minimum shortly before the onset of cleavage and another minimum during the cleavage, corresponding to a peak of the surface before the cleavage and another peak during the cleavage.     

Comparative studies on synaptosomes: Applicability of the rapid method for preparing synaptosomes to elasmobranch brain

A flotation technique for the rapid preparation of synaptosomes, originally developed for invertebrate nervous tissue, has now been successfully applied to that of an elasmobranch fish (Mustelis canis, dogfish). The technique involves submitting the supernatant, obtained after a homogenate has been centrifuged at low speed to remove nuclei and tissue debris to centrifugal fields of intermediate intensity (10⁶ g/min), appears to separate well-sealed synaptosomes from those less well sealed as judged by the criteria of osmotic shrinkage, enzyme occlusion, and choline uptake. The sealed synaptosomes do not equilibrate with the 0.8 M sucrose used as the homogenization medium and rise to form a coherent pellicle at the top of the tube. Due to the short (1–1/1/2 hr) preparation time, such synaptosomes may well prove useful in further metabolic studies.     

Morphology of Arabidopsis Grown under Chronic Centrifugation and on the Clinostat

Morphological measurements were made on populations of Arabidopsis thaliana grown from seed for 21 days under essentially constant environmental conditions except for the influence of gravitational or centrifugal accelerations. Growth conditions were what had been proposed for experiments in an artificial satellite. Observations are reported for plants grown at normal 1-g upright or on horizontal clinostats and for plants grown on a centrifuge. Increased g-force, up to 15 times normal, was found to have significant but small effects on some morphological end points. The plants' sensitivity to the magnitude of the g-force was much less than to its vector direction.