A new technique is described for the study of mammalian pituitary cells in vivo and in vitro. The method involves encapsulation of freshly trypsinized rat, sheep or human pituitary cells in XM-50 Amicon hollow fibers followed by their intracranial implantation into hypophysectomized rats. These pituitary fiber units promoted recipient growth for ∼3 weeks before weight gains plateaued. Body composition analyses showed that significant quantities of protein, fat and ash accounted for the weight gain. Morphological study of the capsule contents 7–39 days postimplantation revealed the presence of intact somatotrophs and corticotrophs. The hollow fibers may provide an immunologically privileged site by virtue of the fact that the 50,000 dalton pores making up the lumen surface permit pituitary hormones to diffuse from the capsule, but theoretically do not permit immunoglobulin molecules to penetrate the capsule. Growth of hypox rats receiving capsules containing allogeneic rat pituitary cells, sheep cells or pieces of human postmortem pituitary support this concept. Furthermore, rats implanted with human PRL adenoma cells had detectable quantities of circulating hPRL 100 days post-implantation. It is suggested that the pituitary hollow fiber units function when they come in contact with a ventricular surface of a hypox animal. With these units, it will be possible to study function of the same group of pituitary cells in vitro and in vivo.

A new continuous flow electrophoretic separator for cells and macromolecules was built and tested in laboratory experiments and in the microgravity environment of space flight. Buffer flows upward in a 120-cm long flow chamber, which is 6 cm wide X 1.5 mm thick in the laboratory version and 16 cm wide X 3.0 mm thick in the microgravity version. Electrophoretic subpopulations are collected in 197 fractions spanning 16 cm at the upper end of the chamber. The electrode buffer is recirculated through front and back cooling chambers, which are also electrode chambers. Ovalbumin and rat serum albumin were used as test proteins in resolution and throughout tests; resolution of these two proteins at 25% total w/v concentration in microgravity was the same as that found at 0.2% w/v concentration in the laboratory. Band spreading caused by Poiseuille flow and conductance gaps was evaluated using polystyrene microspheres in microgravity, and these phenomena were quantitatively the same in microgravity as in the laboratory. Rat anterior pituitary cells were separated into subpopulations enriched with cells that secrete specific hormones; growth-hormone-secreting cells were found to have high electrophoretic mobility, whereas prolactin-secreting cells were found to have low electrophoretic mobility. Cultured human embryonic kidney cells were separated into several electrophoretic subfractions that produced different plasminogen activators; a medium-high-mobility subpopulation and a medium-low-mobility subpopulation each produced a different molecular form of urokinase, whereas a high- and an intermediate-mobility subpopulation produced tissue plasminogen activator. Canine pancreatic islets of Langerhans cells were separated into subpopulations, which, after reaggregation into pseudoislets, were found to be enriched with cells that secrete specific hormones; insulin-secreting beta cells were found in lowest mobility fractions, whereas glucagon-secreting alpha cells were found in the highest mobility fractions. Results of particle electrophoresis experiments were comparable in microgravity and in the laboratory, since cell densities that overloaded the carrier buffer (resulting in zone sedimentation) were avoided, and a 500-fold increase in protein throughput was achieved without compromising resolution in microgravity.

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Previous studies in Leishmania mexicana have identified the cytoskeletal protein KHARON as being important for both flagellar trafficking of the glucose transporter GT1 and for successful cytokinesis and survival of infectious amastigote forms inside mammalian macrophages. KHARON is located in three distinct regions of the cytoskeleton: the base of the flagellum, the subpellicular microtubules, and the mitotic spindle. To deconvolve the different functions for KHARON, we have identified two partner proteins, KHAP1 and KHAP2, that associate with KHARON. KHAP1 is located only in the subpellicular microtubules, while KHAP2 is located at the subpellicular microtubules and the base of the flagellum. Both the KHAP1 and KHAP2 null mutants are unable to execute cytokinesis but are able to traffic GT1 to the flagellum. These results confirm that KHARON assembles into distinct functional complexes and that the subpellicular complex is essential for cytokinesis and viability of disease-causing amastigotes but not for flagellar membrane trafficking.

Light scatter patterns produced by living cells in the flow cytometer are known to provide useful information with regard to their size and internal structure. The purpose of this study was to determine if light scatter signals produced by live male rat anterior pituitary cells could be used as markers to aid in the identification and separation of different hormone-containing cell types. The typical light scatter pattern (4 X 10(5) cells/sample X 15 min) had three ridges in the forward angle light scatter (FALS) perpendicular light scatter (PLS) bivariate cell distribution. FALS signals could be correlated with the size of different cell types and PLS signals with their content of cytoplasmic secretory granules. Agranular cells dominated the low PLS ridge while moderately granulated PRL cells and heavily granulated GH cells dominated the medium and high PLS ridges, respectively. These light scatter patterns were reproducible both within and between different cell suspensions. Inclusion of dopamine in the pituitary gland dissociation medium, a treatment known to increase intracellular PRL contents of mammotrophs, increased the intensity of the PLS signals of a large population of cells, presumably PRL cells. Pituitary cells prepared from different aged male rats also showed changes in light scatter. Cell sorting on the basis of FALS-PLS signals established the relationship between cell type and light scatter pattern.