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Biomedical subjects

W C Hymer

Publications and source records attributed to W C Hymer.

At least 19 recordsLinked to original sources
PubMed1999

Electrophoresis in space.

Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed. Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground. Some studies have shown that resolution is also increased. However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation. Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity. If this result is confirmed, a further cellular mechanism of "sensing" the low gravity environment will have been found. Several free fluid electrophoresis devices are now available. Most have been tried at least once in microgravity. Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing. The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity. Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit. Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware. A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities--equipment like bioreactors and protein crystal growth chambers. The authors feel that such capabilities will lead to the production of commercially useful quantities of target products and to an accumulation of new knowledge relating to the complexities of electrostatic phenomena at the cell surface.

Animals↗
PubMed1997

Cellular electrophoretic mobility data: a first approach to a database.

Cellular electrophoretic mobility values of 288 types of eucaryotic cells were collected from literature published worldwide by a series of authors during the past forty years and arranged in a list. This list contains well-known recent electrophoretic results and also data that cannot be found anymore with modern literature retrieval systems. It will be a valuable help for scientists trying to purify cell populations. In addition, it confirms the observation that most eucaryotic cells have very similar electrophoretic mobilities, ranging from 40% above to 50% below the electrophoretic mobility of human erythrocytes, and thus reinforces the suggestion that electrophoretic mobilities of eucaryotic cells are subjected to strong biological controls.

Animals↗
PubMed1996

Feeding frequency affects cultured rat pituitary cells in low gravity.

In this report, we describe the results of a rat pituitary cell culture experiment done on STS-65 in which the effect of cell feeding on the release of the six anterior pituitary hormones was studied. We found complex microgravity-related interactions between the frequency of cell feeding and the quantity and quality (i.e. biological activity) of some of the six hormones released in flight. Analyses of growth hormone (GH) released from cells into culture media on different mission days using gel filtration and ion exchange chromatography yielded qualitatively similar results between ground and flight samples. Lack of cell feeding resulted in extensive cell clumping in flight (but not ground) cultures. Vigorous fibroblast growth occurred in both ground and flight cultures fed 4 times. These results are interpreted within the context of autocrine and/or paracrine feedback interactions. Finally, the payload specialist successfully prepared a fresh trypsin solution in microgravity, detached the cells from their surface and reinserted them back into the culture chamber. These cells reattached and continued to release hormone in microgravity. In summary, this experiment shows that pituitary cells are microgravity sensitive and that coupled operations routinely associated with laboratory cell culture can also be accomplished in low gravity.

Adrenocorticotropic Hormone↗
PubMed1996

Bioprocessing in microgravity: applications of continuous flow electrophoresis to rat anterior pituitary particles.

In this report we describe the results of a continuous flow electrophoresis (CFE) experiment done on STS-65 in which we tested the idea that intracellular growth hormone (GH) particles contained in a cell lysate prepared from cultured rat anterior pituitary cells in microgravity might have different electrophoretic mobilities from those in a synchronous ground control cell lysate. Collectively, the results suggested that CFE processing in microgravity was better than on earth; more sample could be processed/time (6 x) and more variant forms of GH molecules could be resolved as well. We had also hoped to carry out a pituitary cell CFE experiment, but failure of the hardware required that the actual cell electrophoresis trials be done on earth shortly after Shuttle landing. Data from these experiments showed that space-flown cells possessed a higher electrophoretic mobility than ground control cells, thereby offering evidence for the idea that exposure of cultured cells to microgravity can change their net surface charge-density especially when the cells are fed. Collectively, the results from this pituitary cell experiment document the advantage of using coupled cell culture and CFE techniques in the microgravity environment.

Animals↗
PubMed1996

Experimental modification of rat pituitary growth hormone cell function during and after spaceflight.

Space-flown rats show a number of flight-induced changes in the structure and function of pituitary growth hormone (GH) cells after in vitro postflight testing (W. C. Hymen, R. E. Grindeland, I. Krasnov, I, Victorov, K. Motter, P. Mukherjee, K. Shellenberger, and M. Vasques. J. Appl. Physiol. 73, Suppl.: 151S-157S, 1992). To evaluate the possible effects of microgravity on growth hormone (GH) cells themselves, freshly dispersed rat anterior pituitary gland cells were seeded into vials containing serum +/- microM hydrocortisone (HC) before flight. Five different cell preparations were used: the entire mixed-cell population of various hormone-producing cell types, cells of density < 1.071 g/cm3 (band 1), cells of density > 1.071 g/cm3 (band 2), and cells prepared from either the dorsal or ventral part of the gland. Relative to ground control samples, bioactive GH released from dense cells during flight was reduced in HC-free medium but was increased in HC-containing medium. Band 1 and mixed cells usually showed opposite HC-dependent responses. Release of bioactive GH from ventral flight cells was lower; postflight responses to GH-releasing hormone challenge were reduced, and the cytoplasmic area occupied by GH in the dense cells was greater. Collectively, the data show that the chemistry and cellular makeup of the culture system modifies the response of GH cells to microgravity. As such, these cells offer a system to identify gravisensing mechanisms in secretory cells in future microgravity research.

Animals↗
PubMed1996

Experimental modification of rat pituitary prolactin cell function during and after spaceflight.

This study was done to evaluate the effects of microgravity on prolactin (PRL) cells of the male rat pituitary gland. We used the identical passive closed-vial cell culture system that was described for the culture of growth hormone cells (W. C. Hymen, R. E. Grindeland, T. Salada, P. Nye, E. Grossman, and P. Lane. J. Appl. Physiol. 80:955-970, 1996). After an 8-day spaceflight, all flight media (containing released PRL), as well as extracts (containing intracellular PRL), contained significantly lower amounts of immunoreactive PRL than their corresponding ground control samples. On the other hand, these same samples, when assessed for their biological activities by two different in vitro lymphocyte assays, yielded disparate results that may reflect posttranslational modifications to the hormone molecule. Other data showed that 1) the apparent molecular weights of released PRL molecules were not altered by microgravity, but 2) the region from which the PRL cells came (dorsal or ventral) made a significant difference in the amount and activity of PRL released from the flight cells. Because there is much current interest in the role that PRL may play in the regulation of the immune system and because changes in both cellular and humoral immunity accompany spaceflight, this study could help define future microgravity research in this area.

Animals↗
PubMed1994

Prolactin-immune interactions in carcinogen-induced rat mammary tumors.

Because many mammary tumors are prolactin (PRL) dependent, tumor-bearing animals are immunocompromised, and PRL directly affects the immune system, we examined the endocrine and immune systems of rats initiated with nitrosomethylurea (NMU) to cause mammary tumors. We tested: a) PRL cells in the pituitary; b) pituitary PRL as detected by radioimmunoassay (RIA), Nb2 bioassay, and induction of interleukin-2 receptors on splenocytes; c) induction of IL-2R on lymphocytes in response to a standard PRL; d) CD phenotype of the splenocytes and tumor infiltrating lymphocytes. We found that 80% of all NMU-treated animals developed mammary tumors 10 to 13 weeks post-injection. PRL cell number, size, and granule content were unaffected. When tested by RIA or by the Nb2 bioassay, there appeared to be approximately 50% less PRL secreted (2 weeks post-injection) by cells of the NMU-treated than the vehicle-treated animals. However, when tested by IL-2R assay, PRL cells of NMU-treated animals secreted 50% more activity. Splenocytes from the treated animals, 2-6 weeks post-injection, expressed fewer IL-2R in response to standard PRL. NMU treatment (12 wks post-injection) increased the numbers of T-cytotoxic cells by 49%, had no effect on T-helpers, and increased the number of IL-2R positive splenocytes by 81%. Our interpretation is that NMU treatment interferes with the feedback of lymphokines on the pituitary with a decrease in the form of PRL detected by the RIA and Nb2 assays and an increase in the form which activates splenocytes, and thus changes the composition and function of the immune system.

Animals↗
PubMed1993

Age-related changes in the secretion of growth hormone in vivo and in vitro in infantile and prepubertal Holstein bull calves.

The average concentration of GH in blood is high at birth and declines during the period of sexual maturation in bulls. The objectives of these studies were (1) to define age-related changes in vivo in the pulsatile secretion of GH from birth to puberty, (2) to determine whether pituitary cell content of GH and characteristics of the secretion of GH in vitro reflect age-related changes in vivo, and (3) to examine whether responsiveness to GH-releasing hormone (GHRH) and somatostatin (SRIF) in vitro changed with age in Holstein bull calves. In experiment 1, calves were bled every 15 min for 12 h at < 1, 12 and 42 weeks of age (n = 5/group), these being representative of infantile, juvenile and pubertal stages of development. Calves were killed 3 to 5 days later and the pars distalis of the anterior pituitary gland was enzymatically dispersed into a suspension of single cells. Aliquots of cells were extracted with 0.01 mol NaHCO3/l to determine the content of GH and cultured for 18 and 72 h. As expected, the average concentration of GH in plasma decreased with age (P < 0.001). The initial decrease in GH was caused by a reduction in the baseline concentration between birth and 12 weeks of age. There was a marked decrease in GH pulse amplitude between 12 and 42 weeks of age and a further reduction in the baseline concentration. In contrast, the pulse frequency of GH increased (P < 0.05) from < 1 week to 12-weeks of age and remained constant thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1992

Effects of spaceflight on rat pituitary cell function.

The secretory capacity of growth hormone (GH) and prolactin (PRL) cells prepared from rats flown in space on the 12.5-day mission of COSMOS 1887 and the 14-day mission of COSMOS 2044 was evaluated in several postflight tests on Earth. The results showed statistically significant and repeatable decrements in hormone release, especially when biologic (rather than immunologic) assays were used in the tests. Significant and repeatable intracellular changes in GH cells from the flight animals were also found; most important were increases in the GH-specific cytoplasmic staining intensities and cytoplasmic areas occupied by hormone. Tail suspension of rats for 14 days, an established model for mimicking musculoskeletal changes in rats flown in space, resulted in some changes in GH and PRL cell function that were similar to those from animals flown in space. Our results add to a growing body of data that describe deconditioning of physiological systems in spaceflight and provide insights into the time frame that might be required for readaptation of the GH/PRL cell system on return to Earth.

Animals↗
PubMed1992

Prolactin variants in ram adenohypophyses vary with season.

Secretion of PRL in sheep is affected by photoperiod being highest during the spring and summer, lowest in fall and winter. The objectives of this study were to determine if 1) the production of variant forms of PRL, and 2) immuno- and bioactivities of PRL (iPRL and bPRL) differ during times of the year selected to represent periods of low, transitional and high PRL secretion. Twelve mature rams were maintained on pasture and killed in October, December, and April (n = 4/month). Individual pituitary glands were dispersed, cells obtained, and fixed for immunocytochemical flow cytometry, extracted with 0.01 N NaHCO3 or cultured in serum-free, defined media. The Mr of PRL extracted from cells immediately following dispersion ranged from 14-140K, with significantly more bands greater than 40K being detected from rams sacrificed in December than from those killed in October and April (P less than 0.01). No bands of PRL greater than 25K were observed when samples were reduced with beta-mercaptoethanol prior to electrophoresis, indicating that the high Mr forms were disulfide-linked aggregates. Culture media from October and April contained variants of PRL that ranged from 22-40K but those greater than 25K were generally not observed from cells harvested during December. Extracts of cells after 24 h in culture contained fewer high Mr species during December than had been present in initial extracts from that month. In contrast, during April more high Mr intracellular forms were present after culture than had been detected prior to culture during that month. The percentage of lactotrophs averaged 50.0 +/- 2.5, 47.4 +/- 5.7, and 59.4 +/- 5.5 for October, December, and April, respectively. Initial lactotroph content (pg/lactotroph) of iPRL was higher (P = 0.06) in April (46.0 +/- 17.0) when compared to October and December (8.0 +/- 2.0 and 20.0 +/- 10.0, respectively). In contrast, the bPRL content of initial extracts was higher (P = 0.05) in December (267.0 +/- 68.0) than in October (101.0 +/- 35.0), but not than in April (190.0 +/- 70.0). Although iPRL and bPRL concentrations in culture media were similar for the 3 months, the intracellular iPRL (P less than 0.001) and bPRL (P less than 0.0001) content after culture was greatest during April. In summary, in addition to the well-documented seasonal changes in blood concentrations of PRL, different molecular forms of PRL were found within the pituitary at different times of the year and seasonal variations in iPRL and bPRL did not occur in parallel.

Animals↗
PubMed1991

Function of prolactin cells in the individual rat pituitary gland is location dependent.

Anterior pituitary glands from individual ovariectomized (ovx) or ovx-estrogen (E2) treated rats were sectioned into 1/8 cubes. Each section was incubated for four consecutive 15 min periods in order to measure the release of immunoreactive and bioactive prolactin (PRL); each individual section was then trypsinized into a single cell suspension for determination of PRL cell numbers in that section. Hormone release (ng PRL/1000 PRL cells) was not uniform throughout the gland; the consistency of the secretory patterns demonstrated that the amount of PRL release from the gland was location-dependent. Statistical analysis of the data showed that the most active cells were in the gland's left lobe, while the least active were in the right lobe. Within these lobes, the dorsal-caudal and ventral-rostral left lobe areas released the most hormone in vitro while those in the dorsal-rostral, dorsal-caudal and ventral-rostral right lobe areas were least active. Injection of ovx rats with E2 for 2 days altered these secretory patterns. This sectioning procedure should prove useful in future studies addressing issues of cell-cell interaction and geographic location as they relate to pituitary cell function.

Analysis of Variance↗
PubMed1991

The pituitary: aging and spaceflown rats.

Decrements in growth hormone (GH) release we observed in two spaceflight experiments and four tail-suspended rat studies mimic age-associated changes in the mammalian pituitary GH system seen by Meites and others. The spaceflight data suggest that formation of high molecular weight bioactive disulfide-linked aggregates of the 20 and 22K monomeric GH forms may be reduced in microgravity, thereby, reducing target tissue activity. Correlative studies to confirm spaceflight as a model for pituitary GH system aging should include: 1) investigation of mechanisms of intracellular hormone packaging, 2) consequences to biological activity of the hormone molecule, and 3) study of intracellular microtubule dynamics.

Aging↗
PubMed1991

Prolactin-induced mitogenesis of lymphocytes from ovariectomized rats.

PRL can induce interleukin-2 (IL-2) receptor expression in splenocytes from ovariectomized (OVX) female rats. In this further study of the effects of PRL on lymphocytes in vitro we found that PRL induced IL-2 receptor expression, IL-2 production, and proliferation of splenocytes and thymocytes from OVX rats. Cells from male rats were not affected. The proliferative response, as measured by [3H]thymidine incorporation, depended on the concentration of PRL and the presence of adherent cells in the culture. After a 48-h incubation with PRL (1 microgram/ml), splenocytes from OVX rats incorporated essentially the same amount of [3H]thymidine as cells incubated with the polyclonal T-cell mitogen Concanavalin-A (ConA). As determined by autoradiography, approximately 40% of the splenocytes responded to PRL or to ConA. After incubation of splenocytes and thymocytes with PRL, bioactive IL-2 was detected in culture medium only from cells of OVX female rats, while incubation with ConA caused IL-2 production by lymphocytes from both male and OVX rats. However, ConA induced IL-2 activity sooner than PRL. Immunofluorescent-flow cytometric analysis revealed time-dependent increases in percentages of IL-2 receptor-positive splenocytes as well as increases in percentages of total T-cells and cells of the CD8 and, to a lesser extent, the CD4 subclass after PRL stimulation.

Animals↗
PubMed1990

Separation of rat pituitary secretory granules by continuous flow electrophoresis.

The separation of growth hormone-containing cytoplasmic secretory granules from the rat pituitary gland by continuous flow electrophoresis is described. The results are consistent with the hypothesis that granule subpopulations can be separated due to differences in surface charge; these, in turn, may be related to the oligomeric state of the hormone.

Animals↗
PubMed1990

Prolactin induction of interleukin-2 receptors on rat splenic lymphocytes.

A case for the involvement of PRL in the regulation of the immune system is strong. However, no mechanism by which PRL exerts this regulation has yet been identified. We studied the in vitro effects of PRL on splenocytes from ovariectomized (OVX) rats and discovered that PRL induced the formation of interleukin-2 (IL-2) cell surface receptors. However, PRL did not induce IL-2 secretion. This response, which was dependent on the concentration of PRL, also depended upon the estrogen status of the splenocyte donor; thus, splenocytes from OVX rats or rats in diestrus responded to PRL, whereas those from estrogen-treated OVX rats or rats in estrus did not. We propose that in vivo exposure of PRL, under certain physiological conditions, may prime a pool of splenocytes to express IL-2 cell surface receptors, allowing these cells to be responsive to variations in local concentrations of IL-2.

Animals↗
PubMed1990

Growth hormone aggregates in the rat adenohypophysis.

Although it has been known for some time that GH aggregates are contained within the rat anterior pituitary gland, the role that they might play in pituitary function is unknown. The present study examines this issue using the technique of Western blotting, which permitted visualization of 11 GH variants with apparent mol wt ranging from 14-88K. Electroelution of the higher mol wt variants from gels followed by their chemical reduction with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. With the blot procedure we found 1) that GH aggregates greater than 44K were associated with a 40,000 x g sedimentable fraction; 2) that GH aggregates were not present in glands from thyroidectomized rats, but were in glands from the thyroidectomized rats injected with T4; 3) that GH aggregates were uniquely associated with a heavily granulated somatotroph subpopulation isolated by density gradient centrifugation; and 4) that high mol wt GH forms were released from the dense somatotrophs in culture, since treatment of the culture medium with beta-mercaptoethanol increased GH immunoassayability by about 5-fold. Taken together, the results show that high mol wt GH aggregates are contained in secretory granules of certain somatotrophs and are also released in aggregate form from these cells in vitro.

Animals↗
PubMed1990

A new tissue-slicing method for the study of function and position of somatotrophs contained within the male rat pituitary gland.

A tissue-slicing method has been developed for obtaining quantitative information relative to the function and distribution of somatotrophs in the individual pituitary of the male rat. The adenohypophysis was cut along midtransverse, coronal, and sagittal planes, dividing the gland into eight distinct slices. Basal (unstimulated) GH release was measured from sections placed in gyrotory incubation for three consecutive periods (20 min each). Response to rat hypothalamic GH-releasing hormone (rhGHRH) was studied in slices placed in perifusion and in dispersed cells placed in static incubation. Tissues and cells were analyzed by light and electron microscopy and flow cytometry. Cells retained good morphological integrity and viability (greater than 85%) after incubation (1 h) or perifusion (5 h). Somatotrophs were evenly distributed sagittally and rostrocaudally, but were unevenly distributed dorsoventrally; mean values for dorsal and ventral sections were 45.3 +/- 2.5% and 38.9 +/- 1.9%, respectively. Differential basal GH release was observed after the first gyrotory incubation period; values differed by as much as 3-fold. These differences became less apparent with subsequent incubations. Pituitary slices in perifusion showed a differential response to rhGHRH. For some sections GH release increased up to 2-fold; in other sections the response was either negligible or absent. Dissociated cells from sections also showed differential response to rhGHRH; increases ranged from 160-250% above unstimulated values. These studies demonstrate that tissue slicing is a useful method for investigating pituitary cell function in vitro. The correlation between function and position suggests that heterogeneity of somatotrophs is related in part to their location in the gland.

Animals↗