A microfluorometric study of masked metachromasia in cells of the endocrine polypeptide (APUD) series

Synopsis Masked metachromasia can be demonstrated by staining with a metachromatic basis dye after a Feulgen-type hydrolysis of suitably fixed tissue, and is believed to be indicative of polypeptides with a high concentration of side-chain acidic groups and a random-coil conformation. In this investigation, the metachromatic fluorochrome Coriphosphine O was used. After staining, the degree of metachromasia under various conditions and in several tissues was assessed by microspectrofluorometric measurements of the ratio of metachromatic fluorescence (red) to orthochromatic fluorescence (green). This technique was employed, in the first instance, to determine the optimum staining conditions; details of the final staining method are presented. Measurements of metachromasia in different tissues under standardized conditions showed that the degree of metachromasia varied between different cell types in the APUD series.     

Fluorescence metachromasia in polypeptide hormone-producing cells of the APUD series, and its significance in relation to the structure of the precursor protein

Synopsis A fluorescence metachromatic modification of the masked basophilia method is described. It is based on the acridine dye Coriphosphine O. Excitation and emission spectra of green (orthochromatic) and red (metachromatic) fluorescent tissue components are presented.When the method is applied to suitably fixed sections, metachromasia is demonstrable in cells of the polypeptide hormone-secreting APUD series, and in a few other situations.The view that side-chain carboxyl groups are demonstrated by the masked basophilia technique is considered to be accurate but inadequate. It is proposed that the technique, and its fluorescence modification, are influenced more by secondary than by primary protein structure. In particular, it is suggested that the conformation of the protein precursors of polypeptide hormones, in the storage granules of endocrine cells, is predominantly random-coil.     

An improved polychrome staining method for thick epoxy sections.

Improved polychrome staining of 1-1.5 mum epoxy sections is achieved with sequential applications of a single basic fuchsin-methylene blue mixture at two different pH values. The dye solution is applied for 2-3 min at 50-52 C first at pH 7.9, then at pH 6.7. In sections of mouse mammary tissue, epithelial cells are stained deep blue, connective tissue pink, and fat cells bright olive-green. This simple technique consistently yields uniform, vivid, contrasting colors that sharply delineate the elements of the complex glandular architecture of the mammary gland. Similar polychromatic effects are obtained in applications to other tissues, such as stomach, adrenal gland, mammary tumor and artery.     

An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4)

A simple method for the lectin histochemical visualization of rat microglial cells is described. Advantages include ease of fixation of brain tissue using paraformaldehyde, and rapidity of tissue processing by vibratome sectioning. Furthermore, in addition to providing good structural preservation, the method achieves improved lectin binding, resulting in complete labeling of all microglial cells and in superior visualization of cellular processes. The lectin histochemical technique for rat microglia has the potential to be adapted to any mammalian species, and should prove valuable for neuroscientists interested in studying this glial cell type.     

An improved method for the separation of cells by sedimentation at unit gravity.

An apparatus is described for the separation of cells by sedimentation velocity at lg. The viscosity of the sample was raised by the addition of polyethylene oxide and subsequently the sample was layered on top of a density gradient via a sieve. With the aid of this procedure the various ploidy classes of rat-liver cells were enriched and murine leukemia cells were separated according to the phases of their life cycle.     

Use of the excluded protecting group (EPG) method for peptide synthesis.

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.     

An improved diffusion chamber (DC) for culture of hematopoietic cells.

A DC with Nuclepore filter wall has been described. Recovery of in vitro incubated human nucleated peripheral blood cells and in vivo growth of mouse bone marrow cells in intraperitoneally implanted DCs were improved when compared to the growth obtained in the more commonly used Millipore filter chambers.     

The capping of lymphocytes and other cells, studied by an improved method for immunofluorescence staining of frozen sections.

Experiments have been carried out on the capping by lectins and antibodies of surface receptors of mouse splenic T and B lymphocytes and other cells, in which the surface distribution of the lectin or antibody, and the intracellular distribution of myosin or actin, were determined on the same cells by a double fluorescence technique. For this purpose, a general method for intracellular staining was developed which is intended to preserve sensitive antigens and fragile ultrastructural elements. The method involves mild formaldehyde fixation of the cells or tissues, infusion with concentrated sucrose, rapid freezing, and the preparation of frozen sections thinner than 1 micrometer thickness. The immunofluorescent or other appropriate fluorescent reagents are then applied to the thawed section. In the present experiments, intracellular actin was detected using a fluorescent staining method based on the interaction of F-actin with heavy meromyosin, while intracellular myosin was detected by an indirect immunofluorescence procedure. Our findings were that the formation of a cap by each of the lectins or antibody reagents was always accompanied by a concentration of myosin and actin directly under the cap. These and other results suggest that capping is an active process in which actin and myosin participate directly in the formation of all caps. This proposal carries important new implications for the molecular mechanism of capping.     

Storage granules of thyroid C cells in the dog: a cytochemical and ultrastructural study, in relation to the masked metachromasia reaction.

Masked metachromasia can be demonstrated in thyroid C cells, and other cells of the APUD series, by staining with a metachromatic basic dye after hydrolysis of suitably fixed tissue. The reaction is thought to be due to the presence of polypeptides with a high concentration of side-chain acidic groups. Since most APUD cells possess storage granules, presumed to contain a polypeptide hormone, it has been assumed that the masked metachromasia reaction gives information concerning the contents of these granules. However, there has been an increasing suspicion that the reaction might actually be due to the membrane bounding these granules, rather than to the contents. We have examined, cytochemically and ultrastructurally, dog thyroid tissue which has been subjected to fixation and hydrolysis as in the usual method for masked metachromasia. We found that the membrane surrounding the C cell granules is removed by hydrolysis, confirming the hypothesis that the reaction is due to the contents (hormone and/or matrix)rather than to the membrane. Tissues were fixed in an aqueous mixture containing glutaraldehyde (6 25% v/v), picric acid (three-quarters saturation) and sodium acetate (I% W/V)adjusted to PH 7 with sodium hydroxide. This was found to be a very satisfactory fixative for electron microscopy Some morphological details of C cells were noted, such as the richness of desmosomes between C cells in this species, and frequent direct contact with the colloid.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP- or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas. Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide- and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide- or glucagon-immunoreactivity were also found. Glucagon- and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

Coexistence of pancreatic polypeptide (PP)-and glucagon-immunoreactivity in pancreatic endocrine cells of mouse

Summary Immunocytochemical double staining techniques were used to study PP- and glucagon-like-immunoreactivity in pancreatic endocrine cells of mouse. An antiserum against FMRFamide appeared to react with all PP-immunoreactive endocrine cells. With fluorescence microscopy most PP/FMRFamide-immunoreactive cells also showed glucagon-immunoreactivity, but cells containing only PP-or glucagon-like substances were found as well. The proportion of cells containing PP-, glucagon, and both immunoreactivities varied strongly from islet to islet in all parts of the pancreas.Using an electron microscopical immunogold double staining procedure on Lowicryl-embedded pancreas, PP/FMRFamide-and glucagon-immunoreactivity appeared to be present in the majority of endocrine A cells; both immunoreactivities were randomly distributed within the granules of these cells. Cells containing only PP/FMRFamide-or glucagon-immunoreactivity were also found. Glucagon-and a faint FMRFamide-immunoreactivity was also observed in osmicated epon-embedded tissue. Independent of their immunoreactivity all positive cells showed the same round electron dense secretory granules.     

An improved method for rearing Halotydeus destructor (Acari: Penthaleidae) in the laboratory

We developed an improved method for rearing Halotydeus destructor in the laboratory. Mite numbers increased rapidly over the summer of 1992–1993 when fed on vetch, Vicia sativa cv Blanchefleur; in contrast mite numbers were low and fell over the second half of the summer of 1990–91 when fed on subterranean clover, Trifolium subterraneum cv Junee. On V. sativa at a fluctuating temperature from 11 to 18°C 200 mites produced a mean of 1308 and 1455 progeny per transfer in 2 years, a 6–7-fold rate of increase per transfer. The mean transfer time was 35 days and using the interval between transfers as a measure of generation time, the mites completed six to seven generations with no evidence for development of diapause eggs from August until April. There was considerable variability in the numbers of mites produced per pot, associated in part with the presence of Verticillium sp. fungus in 35% of the pots during transfers. Improvement in H. destructor rearing resulted from the use of V. sativa cv Blanchefleur as a food source, maintenance of high humidity and adequate ventilation within cages, and the transfer of mites to fresh food sources at early nymphal stages, which reduced the spread of fungal infections.