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.     

An improved method for staining cells of the endocrine polypeptide (APUD) series by masked metachromasia: application of the principle of ''fixation by excluded volume''.

Synopsis Masked metachromasia is demonstrated by staining with a metachromatic basic dye, after acid hydrolysis of suitably fixed tissue. We report that the addition of 20% Carbowax 20M (an inert polymer, mol. wt. about 20000) to the hydrolysis mixture improved the reaction. The improved method gives increased metachromasia, greater tolerance to variations in hydrolysis conditions, and demonstrates a greater proportion of cells — presumably due to a lower threshold of sensitivity. Lower molecular weight polymers (Carbowax 1000, Carbowax 6000) are less effective.     

The variable refractivity of the protein or polypeptide hormone-producing cells showing a unique luminescence in the dark-field microscope

Synopsis When cryostat sections of endocrine tissue were examined in a dark-field microscope, a brilliant granular luminescence was revealed in the endocrine cells thought to be concerned with protein or polypeptide hormone production. The sections were prepared from fresh materials either frozen in a cryostat chamber at –25°C, in dry ice-acetone, or fixed in formalin-calcium for 24 hr. The neurosecretory substance in the hypothalamus and the posterior lobe of the pituitary showed a blue luminescence; the acidophil cells of the anterior lobe of the pituitary, orange; basophil cells, green or blue; intermediate lobe cells, no luminescence; thyroid C cells, white-blue; pancreatic A cells, blue; B cells, orange; adrenomedullary cells, greenish blue; enterochromatin cells, green; and other endocrine cells in the gastrointestinal tract, blue or orange. After tearing and spreading the pituitary and hypothalamus with a pair of needles on a glass slide, and examining the teased specimen by dark-field microscopy, various cells of different luminescent colours became apparent in the anterior lobe of the pituitary, a blue fluorescent substance in the posterior lobe, and neurosecretory cell bodies in the hypothalamus. The different colours appear to be inherent in the granules of living tissues.     

Suppression of metachromasia of galactogen by organic solvents and its relation to their dielectric constants

Summary In order to elucidate the relationship between the dielectric constants of various organic solvents and their capacity to abolish the metachromasia of galactogen localised in the albumen gland of a land snail, effects of various alcohols, ketones, esters and ethers on the metachromasia were investigated. The metachromasia was labile to all these solvents and its suppression depended on their dielectric constants. The higher the dielectric constant of a solvent the lower was its capacity to suppress the metachromasia of galactogen. Such a relationship was not a generalised phenomenon but it was true within a group of solvents with a specific reactive group. The four groups of solvents can be arranged in a series: ethers, esters, alcohols, ketones, in order of their increasing efficiency to abolish the metachromasia. Saturated or unsaturated nature of the solvent also affected such a suppression.     

The structure of monellin and its relation to the sweetness of the protein.

The sweet protein monellin [1-3] has been shown to consist of two non-identical subunits of 50 and 42 amino acid residues, which were separated electrophoretically and chromatographically. Automatic sequential Edman degradation gave the complete sequence of the longer subunit, and a partial sequency of the shorter one. It was found that the sweetness of monellin requires the undissociated molecule. The individual subunits were not sweet, neither did they block the sweet sensation of sucrose or monellin. Blocking of the single SH of monellin abolished its sweetness as did reaction of the single methionyl residue with CNBr. Since the cysteinyl and methionyl residues appear to be adjacent, it is suggested that this part of the molecule is essential for its sweetness.     

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.     

Structure of an antifreeze polypeptide and its precursor from the ocean pout, Macrozoarces americanus

Serum antifreeze polypeptides (AFP) from Newfoundland ocean pout have been resolved by ion exchange chromatography and reverse phase high performance liquid chromatography into at least 12 components. The protein sequences of three of the AFP were determined using a combination of protein Edman degradation and cDNA sequencing. The AFP precursor protein encodes for a preprotein of 87 amino acids with no obvious prosequences. Two of the AFP (SP1-A and SP1-C) were separate gene products with minor amino acid sequence differences. The protein structure of SP1-C precursor is MKSVILTGLLFVLLCVDHMTASQSVVAT QLIPINTALTPAMMEGKVTNPIGIPFAEMSQIVGKQVNTPVAKGQTLMPNMVKTYVAGK. The third AFP (SP1-B) is a post-translation modification product of SP1-C. These experiments indicate that the ocean pout AFP are a multigene family with protein structure different from any other known polypeptide antifreezes.     

On the ultrastructure of polypeptide hormone-producing cells in the gut of the ascidian,Ciona intestinalis L. and the Bivalve,Mytilus edulis L.

Summary Ultrastructural evidence has been found for the presence of polypeptide hormone-producing cells in the gut of Ciona intestinalis L. and Mytilus edulis L. which do not appear to have been described before. Due to their localization and ultrastructural characteristics, it is suggested that the cells in Mytilus edulis probably produce an insulin-like substance and that some of these cells in Ciona intestinalis may produce 5-HT (5-Hydroxytryptamine). In each species only one granulated cell type can be observed. The granules, which are electron dense and membrane bound, also show a halo. The average diameter of the granules is 100–200 nm for Ciona and 200–400 nm for Mytilus.I thank Mr. G. Bargsten, M.A., Dept. of Marine Zoology, University of Kiel, for the supply of the animals     

Primary structure of fibrinogen, its relation to conformation and function of the molecule

Data available in literature are generalized for the amino acid sequence of three fibrinogen chains, their homology and a possibility for their common precursor to exist. The primary structure of resistant products of fibrinogen splitting by plasmin, compactness of their space organization and mutual arrangement in the intact molecule are described. The relation between the primary structure of fibrinogen and its high specificity as a thrombin substrate is traced. Evidence is considered concerning localization of active molecule centres to provide spontaneous polymerization of fibrin.     

Nonnative protein polymers: structure, morphology, and relation to nucleation and growth

Thermally induced aggregates of α-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (∼10³-10⁵ kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight (M_w). Aggregates in both systems are composed of nonnative monomers with elevated levels of β-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M_w with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For α-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation (τ_n) and growth (τ_g), as well as the stoichiometry of the nucleus (x). The analysis illustrates a general procedure to noninvasively and quantitatively determine τ_n, τ_g, and x for soluble (chain polymer) aggregates, as well as the relationship between τ_n/τ_g and aggregate M_w.