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PubMed1993

The use of centrifugation to study early Drosophila embryogenesis.

By the end of 10th nuclear cycle, the somatic nuclei of the Drosophila embryo have migrated to the periphery of the egg. Centrifugation of embryos did not result in the displacement of these nuclei, since cytoskeletal elements anchor them to the cortex. But, mild centrifugal forces displace the centrally located, nascent yolk nuclei. If this increased sensitivity to hypergravity occurs before the beginning of nuclear differentiation during cycle 8, when the nascent yolk and somatic nuclei physically separate, then it would mark the earliest functional difference between these two lineages.

Animals↗
PubMed1995

Utilization of [14C]phenylalanine derived from arylphorin or free amino acid in Manduca sexta pharate adults.

The role of arylphorin as a storage protein was studied using 14C-arylphorin. 14C-arylphorin was produced optimally by incubating one-half fat body from Manduca sexta fifth instar larvae at 22 degrees C for 24 h, in 1 ml of medium containing amino acids at 25% of their physiological concentration with [U-14C]-phenylalanine (phe) provided initially without nonlabeled phenylalanine. Nonlabeled phe was provided after 1 h at 16% of its physiological concentration. The specific activity of 14C-arylphorin produced in vitro was 30 times greater than that generated in vivo. Injection of 14C-arylphorin into pharate adults was used to study the distribution of 14C-phe derived from this protein into 14CO2 and tissues for comparison with injection of free 14C-phe during the middle (days 6 to 12 pharate adult) and late (days 12 to 17 pharate adult) stages of adult development. Appearance of 14CO2 from 14C-arylphorin as compared to 14C-phenylalanine showed a slower time course during both the middle and late stages of development, in keeping with the time needed for degradation of the protein. In accord with faster phe turnover near the end of adult development, total 14CO2 production was greater and the retention of 14C in hemolymph and fat body was less compared to the middle stage of development regardless of whether 14C-arylphorin or 14C-phe was injected. In the middle stage of development, the appearance of 14C in the cuticle and head parts was greater, whereas incorporation into abdomen and thorax was less than during the late stage of development. Since the pattern of 14C distribution from 14C-arylphorin and 14C-phe was similar, one major function of arylphorin must be as a storage protein replenishing the supply of free amino acids used for synthesis of adult tissues. These results also suggest a limited contribution of M. sexta arylphorin to formation of the cuticle subsequent to day-6 pharate adult.

Amino Acids↗
PubMed1993

HSP86 and HSP84 exhibit cellular specificity of expression and co-precipitate with an HSP70 family member in the murine testis.

This study extends to the protein level our previous observations, which had established the stage and cellular specificity of expression of hsp86 and hsp84 in the murine testis in the absence of exogenous stress. Immunoblot analysis was used to demonstrate that HSP86 protein was present throughout testicular development and that its levels increased with the appearance of differentiating germ cells. HSP86 was most abundant in the germ cell population and was present at significantly lower levels in the somatic cells. By contrast, the HSP84 protein was detected in the somatic cells of the testis rather than in germ cells. The steady-state levels of HSP86 and HSP84 paralleled the pattern of the expression of their respective mRNAs, suggesting that regulation at the level of translation was not a major mechanism controlling hsp90 gene expression in testicular cells. Immunoprecipitation analysis revealed that a 70-kDa protein coprecipitated with the HSP86/HSP84 proteins in testicular homogenates. This protein was identified as an HSP70 family member by immunoblot analysis, suggesting that HSP70 and HSP90 family members interact in testicular cells.

Animals↗
PubMed1993

Cytoplasmic pH influences cytoplasmic calcium in MC3T3-E1 osteoblast cells.

We found that the cytoplasmic concentration of calcium (Cai) of MC3T3-E1 osteoblasts was influenced by the type of pH buffer we used in the perfusing medium, suggesting that intracellular pH (pHi) might influence Cai. To study this effect, the Cai and pHi were monitored as we applied various experimental conditions known to change pHi. Exposure to NH4Cl caused a transient increase in both pHi and Cai without a change in extracellular pH (pHo). Decreasing pHo and pHi by lowering the bicarbonate concentration of the medium decreased Cai, and increasing pHi by the removal of 5% CO2 increased Cai. Clamping pHi to known values with 10 microM nigericin, a potassium proton ionophore, also influenced Cai: acid pHi lowered Cai, whereas alkaline pHi increased it. The rise in Cai appears to be very sensitive to the extracellular concentration of calcium, suggesting the existence of a pH-sensitive calcium influx mechanism. We conclude that physiologic changes in pH could modulate Cai by controlling the influx of calcium ions and could change the time course of the Cai transient associated with hormonal activation.

3T3 Cells↗
PubMed1993

Studies of chondrogenesis in rotating systems.

A great deal of energy has been exerted over the years researching methods for regenerating and repairing bone and cartilage. Several techniques, especially bone implants and grafts, show great promise for providing a remedy for many skeletal disorders and chondrodystrophies. The bioreactor (rotating-wall vessel, RWV) is a cell culture system that creates a nurturing environment conducive to cell aggregation. Chondrocyte cultures have been studied as implants for repair and replacement of damaged and missing bone and cartilage since 1965 [Chesterman and Smith, J Bone Joint Surg 50B:184-197, 1965]. The ability to use large, tissue-like cartilage aggregates grown in the RWV would be of great clinical significance in treating skeletal disorders. In addition, the RWV may provide a superior method for studying chondrogenesis and chondrogenic mutations. Because the RWV is also reported to simulate many of the conditions of microgravity it is a very useful ground-based tool for studying how cell systems will react to microgravity.

Animals↗
PubMed1984

Delayed fertilization of anuran amphibian (Xenopus) eggs leads to reduced numbers of primordial germ cells.

Several media were tested for the extent to which they promoted high fertilization efficiencies in ovulated, stripped Xenopus eggs. One medium was selected for maintaining eggs in a 'delayed fertilization' (DelF) condition. DelF eggs displayed several unusual characteristics, including shift of the center of gravity, prominent sperm entrance site, and occasional polyspermy. The frequency of normal pattern formation varied according to the length of time eggs were maintained in the DelF condition. Various developmental abnormalities were observed during gastrulation, neurulation, and organogenesis. Most abnormalities appeared, however, to be related to morphogenesis of the endoderm. Primordial germ cell (PGC) development was examined in DelF eggs which displayed normal external morphological features at the swimming tadpole stage. PGC counts were usually normal in short-duration (eg, 5 hr) DelF eggs, but frequently substantially reduced or completely diminished in longer-duration (eg, 25h) tadpoles. Six spawnings were compared and shown to exhibit considerable variability in fertility, morphogenesis, and PGC development. Yolk platelet shifts and developmental parameters were examined in two additional spawnings. The subcortical cytoplasm in which the germ plasm is normally localized appeared to be disrupted in longer duration DelF eggs. That observation may account for low PGC counts in DelF tadpoles.

Animals↗
PubMed1993

Early amphibian (anuran) morphogenesis is sensitive to novel gravitational fields.

Anuran amphibian embryos (Xenopus laevis and Rana dybowskii) are sensitive to novel gravitational fields. Under simulated weightlessness, (i) the location of the first horizontal cleavage furrow was shifted toward the vegetal pole at the eight-cell stage; (ii) the position of the blastocoel was more centered, and the number of cell layers in the blastocoel roof was increased at the blastula stage; (iii) the dorsal lip appeared closer to the vegetal pole at the gastrula stage; and (iv) head and eye dimensions were enlarged at the hatching tadpole stage. Effects of simulated hypergravity were opposite to those of simulated weightlessness, except that hypergravity, unlike simulated weightlessness, reduced the number of primordial germ cells in feeding tadpoles. Despite those dramatic differences in the early embryogenesis, tadpoles at the feeding stage are largely indistinguishable from controls.

Animals↗
PubMed1991

Rhopalium development in Aurelia aurita ephyrae.

Rhopalia of developing ephyrae were examined using the SEM and TEM at 24 h intervals following strobilation induction. Kinocilia are shorter in the ephyra stage than in polyps. A few ephyra-type kinocilia are found in rhopalia as early as 24 h after induction, before a distinct rhopalium is seen. By 72 h, the shorter kinocilia predominate and are almost as numerous as in ephyrae (120 h). Many of the kinocilia are associated with mechanoreceptor cells (MR) found in the rhopalia. These MR cells are compared to those reported for medusae. Although newly released ephyrae lack a touch plate, the MR cells in their rhopalia along with the statocyst and neuromuscular system apparently enable these organisms to detect and respond to gravity.

Animals↗
PubMed2003

Vergence-dependent adaptation of the vestibulo-ocular reflex.

The gain of the vestibulo-ocular reflex (VOR) normally depends on the distance between the subject and the visual target, but it remains uncertain whether vergence angle can be linked to changes in VOR gain through a process of context-dependent adaptation. In this study, we examined this question with an adaptation paradigm that modified the normal relationship between vergence angle and retinal image motion. Subjects were rotated sinusoidally while they viewed an optokinetic (OKN) stimulus through either diverging or converging prisms. In three subjects the diverging prisms were worn while the OKN stimulus moved out of phase with the head, and the converging prisms were worn when the OKN stimulus moved in-phase with the head. The relationship between the vergence angle and OKN stimulus was reversed in the fourth subject. After 2 h of training, the VOR gain at the two vergence angles changed significantly in all of the subjects, evidenced by the two different VOR gains that could be immediately accessed by switching between the diverged and converged conditions. The results demonstrate that subjects can learn to use vergence angle as the contextual cue that retrieves adaptive changes in the angular VOR.

Adaptation, Physiological↗
PubMed1990

Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm (Manduca sexta).

Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both "flooding-dose" and "non-carrier" techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 micromoles/insect) together with the [3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24-53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76-86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 micrograms/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.

Animals↗
PubMed1984

Properties of electrophoretic fractions of human embryonic kidney cells separated on Space Shuttle flight STS-8.

Suspensions of cultured primary human embryonic kidney cells were subjected to continuous flow electrophoresis on Space Shuttle flight STS-8. The objectives of the experiments were to obtain electrophoretically separated fractions of the original cell populations and to test these fractions for the amount and kind of urokinase (a kidney plasminogen activator that is used medically for digesting blood clots), the morphologies of cells in the individual fractions, and their cellular electrophoretic mobilities after separation and subsequent proliferation. Individual fractions were successfully cultured after return from orbit, and they were found to differ substantially from one another and from the starting sample with respect to all of these properties.

Cell Line↗
PubMed1986

Amphibian egg cytoplasm response to altered g-forces and gravity orientation.

Elucidation of dorsal/ventral polarity and primary embryonic axis development in amphibian embryos requires an understanding of cytoplasmic rearrangements in fertile eggs at the biophysical, physiological, and biochemical levels. Evidence is presented that amphibian egg cytoplasmic components are compartmentalized. The effects of altered orientation to the gravitational vector (i.e., egg inversion) and alterations in gravity force ranging from hypergravity (centrifugation) to simulated microgravity (i.e., horizontal clinostat rotation) on cytoplasmic compartment rearrangements are reviewed. The behavior of yolk compartments as well as a newly defined (with monoclonal antibody) non-yolk cytoplasmic compartment, in inverted eggs and in eggs rotated on horizontal clinostats at their buoyant density, is discussed.

Animals↗
PubMed1989

A step in embryonic axis specification in Xenopus laevis is simulated by cytoplasmic displacements elicited by gravity and centrifugal force.

Determination of the body pattern in Xenopus embryos is known to involve at least six steps. One of these steps can be experimentally simulated by inclining the fertilized egg with respect to gravity or centrifugal force (10-30 g x 4 min, directed 90 degrees to the animal-vegetal axis). In these eggs, the dorsal structures of the body axis form from the side of the egg that was uppermost in the gravitational or centrifugal field. This topography is seen even if the sperm entry point side (the prospective ventral side in control eggs) was uppermost. In addition, conjoined twin embryos form at very high frequencies in response to certain conditions of single or double centrifugation. Cytological analysis shows that the dorsal structures invariably form from the side(s) of the egg away from which vegetal cytoplasm was displaced. This is similar to the situation in the unperturbed egg, where the subcortical cytoplasm of the vegetal hemisphere rotates some 30 degrees relative to the surface, and the dorsal structures form from the side of the egg away from which the subcortical cytoplasm moved. The displacements elicited by centrifugation probably substitute for the normal displacements brought about by the subcortical rotation. These and other data suggest that the subcortical rotation is a crucial step in the process of axis determination. The subcortical rotation is an autonomous activity of the activated egg, and can displace cytoplasm against gravity. I believe that the subcortical rotation will function normally at microgravity, and I expect that overall development and axis polarity at microgravity will be normal. This will be tested in spaceflight.

Animals↗
PubMed1989

The amphibian egg as a model system for analyzing gravity effects.

Amphibian eggs provide several advantageous features as a model system for analyzing the effects of gravity on single cells. Those features include large size, readily tracked intracellular inclusions, and ease of experimental manipulation. Employing novel gravity orientation as a tool, a substantial data base is being developed. That information is being used to construct a 3-D model of the frog (Xenopus laevis) egg. Internal cytoplasmic organization (rather than surface features) are being emphasized. Several cytoplasmic compartments (domains) have been elucidated, and their behavior in inverted eggs monitored. They have been incorporated into the model, and serve as a point of departure for further inquiry and speculation.

Animals↗
PubMed1989

Subcellular components of the amphibian egg: insights provided by gravitational studies.

Most cytoplasmic regions of fertilized amphibian eggs move with respect to the gravity vector in experimentally gravity oriented eggs. The pattern and extent of this movement varies among different batches of eggs. This variation in apparent cytoplasmic viscosity (or, conversely, cytoplasmic mobility) can be correlated with variations in subsequent morphogenesis of experimental, gravitationally manipulated eggs. Therefore, the proper interpretation of gravity experiments with amphibian eggs requires that one understand the subcellular basis for this variation on cytoplasmic mobility. Variation in the packing of the major cytoplasmic organelle, the yolk platelets, or the organization and amount of cytoskeletal components may explain the variation in cytoplasmic mobility. Evidence is presented that the variation in yolk volume density (fraction of total cytoplasmic volume occupied by yolk platelets) does not account for the variation in cytoplasmic mobility in Xenopus laevis eggs. Experimental evidence from cold-shocked inverted eggs indicates that microtubules may be involved in determining cytoplasmic mobility. However, quantitative evidence that the microtubule levels and state of the microtubules (polymerized vs. non-polymerized) in the whole Xenopus laevis egg does not correlate directly with cytoplasmic mobility is presented. The apparent conflict these data represent regarding the role of the cytoskeleton in determining cytoplasmic mobility is discussed.

Animals↗
PubMed1992

Microgravity effects on sea urchin fertilization and development.

Gravity has been a pervasive influence on all living systems and there is convincing evidence to suggest that it alters fertilization and embryogenesis in several developmental systems. Notwithstanding the global importance of gravity on development, it has only been recently possible to begin to design experiments which might directly investigate the specific effects of this vector. The goal of this research program is to explore and understand the effects of gravity on fertilization and early development using sea urchins as a model system. Sea urchin development has several advantages for this project including the feasibility of maintaining and manipulating these cells during spaceflight, the high percentage of normal fertilization and early development, and the abundant knowledge about molecular, biochemical, and cellular events during embryogenesis which permits detailed insights into the mechanism by which gravity might interfere with development. Furthermore, skeletal calcium is deposited into the embryonic spicules within a day of fertilization permitting studies of the effects of gravity on bone calcium deposition.

Animals↗
PubMed1992

Understanding the organization of the amphibian egg cytoplasm: gravitational force as a probe.

A combination of hypergravity (centrifugation) and hypogravity (clinostat) studies have been carried out on amphibian (frog, Xenopus) eggs. The results reveal that the twinning caused by centrifugation exhibits substantial spawning to spawning variation. That variation can be attributed to the apparent viscosity of the egg's internal cytoplasm. Simulated hypogravity results in a relocation of the egg's third (horizontal) cleavage furrow, towards the equator. Substantial egg-to-egg variation is also observed in this "cleavage effect". For interpreting spaceflight data and for using G-forces as probes for understanding the egg's architecture the egg variation documented herein should be considered.

Animals↗