Calcimedins: purification and characterization from chicken gizzard and rat and bovine livers

A procedure for the simultaneous extraction and purification of four calcimedins from chicken gizzard, rat liver, and bovine liver is described. These proteins bind to hydrophobic resins in a calcium-dependent manner similar to calmodulin and troponin C. The four calcimedins purified had molecular weights 67,000 (67K), 35,000 (35K), 33,000 (33K), and 30,000 (30K) as determined by SDS polyacrylamide gel electrophoresis. Their ability to bind calcium was demonstrated using the Hummel-Dreyer method. Their tissue concentration ranged between 1-4 mg/100 g wet weight in the three tissues studied. During gel filtration, calcimedins 67K and 35K, had Rf (Ve-Vo/Vt-Vo) values of 0.46 and 0.74, respectively, indicating monomeric structure. However, the 33K and 30K calcimedins had Rf values of 0.26 (molecular weights greater than 90,000) suggesting that they occur as subunit complexes in their native state. Antibodies raised against the 67K and 35K calcimedins showed cross reactivity suggesting possible common origin. However, peptide mapping studies showed that they are independent proteins with considerable peptide homology. Antibodies to 30/33K calcimedins did not cross-react with either 67K or 35K calcimedins. Moreover, their peptide maps were strikingly different from those of 67K and 35K calcimedins indicating that they are unique. At present, the regulatory function of this group of proteins is not clear. Indirect evidences support the possibility that they are involved in membrane associated events, such as endocytosis and secretion.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.     

Immunological Studies on Dermatophytes IV. Chemical Structures and Serological Reactivities of Polysaccharides from Microsporum praecox, Trichophyton ferrugineum, Trichophyton sabouraudii, and Trichophyton tonsurans

The chemical structures and serological specificities of polysaccharides isolated from four species of dermatophytes, Microsporum praecox, Trichophyton ferrugineum, T. sabouraudii, and T. tonsurans, were investigated. Each of these species yielded a mixture of crude polysaccharides which could be separated into three water-soluble, neutral polysaccharides free of nitrogen. These were grouped as galactomannan I, galactomannan II, and glucan. The galactomannans I were quite similar in chemical structure. When measured by complement fixation, their serological cross-reactivities were similar with rabbit antisera to each of these species except T. sabouraudii. The differences in their relative reactivities with this antiserum could be correlated with differences in structure and specificity of this antiserum for galactofuranose end groups. The galactomannans II differed both in chemical structure and in their serological reactivities with antisera to each of these species. The galactomannan II from T. ferrugineum differed most in chemical structure and was the least reactive serologically. The glucans also differed in both structure and serological reactivities.     

CHLOROPLAST DEVELOPMENT IN OCHROMONAS DANICA

When dark-grown cells of Ochromonas danica are placed in the light, the amount of chlorophyll a per cell increases 82-fold; the content of carotenoid pigment, 24-fold. Concomitantly with this increase in chlorophyll and carotenoid pigment, the small proplastid of dark-grown cells develops into a large lamellate chloroplast. During the first 12 hours in the light, vesicles appear within the loose clusters of dense chloroplast granules, enlarge, align themselves into rows (plates in three dimensions), and fuse into discs. Double discs may form from the more or less simultaneous fusion of two adjacent plates of vesicles or by the addition of vesicles to an already formed single disc. Three-disc bands arise by the addition of a disc to an already formed two-disc band through the approach and fusion of more vesicles. After 24 hours in the light, most of the chloroplast bands contain three discs, but the chloroplasts are still small. After 48 hours in the light, almost all the cells contain full-sized chloroplasts with a full complement of three-disc bands. However, at this time the amount of chlorophyll a and carotenoid pigment is only one-half of maximum. During the next 3 days in the light, as the number of chlorophyll and carotenoid molecules per chloroplast approximately doubles, there is a compression of the discs in each band (from 180 to 130 A) and a precise alignment of their membranes. Changes also occur in the nucleus when dark-grown cells are placed in the light. There is an increase in the number of small nucleolar bodies, many of which lie directly against the nuclear envelope, and in a few cells a dense mass of granules is seen between the two membranes of the nuclear envelope.     

An electron microscopic study of the cerebral blood vessels of the opossum

Summary Blood vessels of the opossum brain are paired, artery and vein, and end in a closed loop. Both anatomically and physiologically they are true end-arteries. The two limbs are enclosed within a single basement membrane and are separated from each other by an intercapillary cell thought to be analogous to the usual capillary pericytes. The similarity of this vascular arrangement to that in the rabbit placenta and in the medulla of the kidney is discussed in relation to the counter-current multiplier system.This work was supported in part by the Beaumont-May Institute of Neurology and by a grant, B-425, from the United States Public Health Service.Research Fellow of the National Multiple Sclerosis Society.     

FINE STRUCTURE OF THE NEUROHYPOPHYSIS OF THE OPOSSUM (DIDELPHIS VIRGINIANA)

The neurohypophysis of the opossum (Didelphis virginiana) was studied by electron microscopy in order to amplify Bodian''s classic light microscopic observations in which he demonstrated a definite lobular pattern. The lobule of the opossum neurohypophysis is divided into three regions: a hilar, a palisade, and a septal zone. The hilar portion contains bundles of nerve fibers, the extensions of the hypothalamo-hypophyseal tract containing neurofilaments but few neurosecretory granules. In the opossum, pituicytes have a densely fibrillar cytoplasm. Herring bodies are prominent in the hilar region. They are large bodies packed with neurosecretory granules that have been described as end bulb formations of axons. From the hilar region, axons fan out into a palisade zone where the nerve terminals packed with neurosecretory granules, mitochondria, and microvesicles abut upon basement membranes. The neurosecretory granules are similar to those present in the neurohypophysis of other mammals, except for an occasional huge granule of distinctive type. Material morphologically and histochemically resembling glycogen occurs as scattered particles and as aggregates within nerve fibers. The septal zone, containing collagen, fibroblasts, and numerous small capillaries, is separated from the adjacent glandular tissue by a basement membrane.     

Survey for mitochondrial-desmosome complexes in differentiating epithelia

Summary Mitochondria are frequently found to be closely associated with the plaques of desmosomes in a variety of columnar or cuboidal epithelia of fetal or early postnatal mammals (mouse, rat, human being). The organs in which mitochondrial-desmosome complexes were found include stomach, small intestine, pancreas, kidney, epididymis, seminal vesicle, coagulating gland, thyroid gland. The association has not been observed in simple squamous epithelium (vascular endothelium). Mitochondria lie quite close to desmosomes in the stratum spinosum of stratified squamous mucous epithelium of fetal animals and also to axo-dendritic synapses in still poorly differentiated central nervous system. Mitochondria have also been detected close to attachment sites in ectoderm of the early frog gastrulae. Here there is as yet no visible plaque material.We suggest that the mitochondria may provide energy or some chemical for the formation of the plaque. This hypothesis does not explain why the complexes are not found in poorly differentiated epithelia from older animals.Dedicated to Professor Berta V. Scharrer on her 60th birthday, with affection and admiration. — This study was supported by U.S.P.H.S. research grants NB-05219 and GM-10757 from the National Institutes of Health.     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.     

Morphology and distribution of tanycytes in the third ventricle of the adult rat. A study using semithin methacrylate sections

Light microscopy and semithin methacrylate sections were used to study the tanycytic projections and morphology in the floor of the third ventricle of the rat. The tanycytic cell soma was located in the ependyma. The luminal surface showed minute protrusions into the ventricular space and their basal processes projected across the width of the parenchyma of the infundibular region. During their course, tanycytic processes made contact with capillaries in the parenchyma and pial surface, suggesting that they might be involved in uptake and/or delivery mechanisms between the cerebrospinal fluid, hypothalamic cells and blood vessels.