Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow

Summary The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Histochemical studies of mucosubstances in metaplastic epithelium of the stomach,with special reference to the development of intestinal metaplasia

Summary Processes in the development of intestinal metaplasia of the stomach were investigated from the morphological and histochemical approaches using light and electron microscopic techniques. The specimens taken from 38 gastric carcinomas and 15 gastric and/or duodenal ulcers were subjected to this study. Morphological appearances of the intestinal metaplasia observed in routine examination with hematoxylin and eosin staining was able to be divided into complete and incomplete metaplasia by the light and electron microscopic histochemical stainings of the mucosubstances. The columnar cells at the area of the incomplete metaplasia had both the properties of the intestinal epithelia and the gastric foveolar epithelia. The incomplete as well as the complete metaplasia arose from the generative cells at the isthmus of the gland. The generative cells, however, sometimes gradually transformed to produce the complete metaplastic cells. The two processes of the development of the intestinal metaplasia were proposed and discussed.     

Chemical ionization and electron impact mass spectra of oligosaccharides derived from sphingoglycolipids

Chemical ionization (CI) mass spectra with isobutane and ammonia for the oligosaccharides obtained from sphingoglycolipids were compared with their electron impact (EI) mass spectra. The oligosaccahride moieties were liberated from the parent glycolipids and were further reduced with sodium borohydride. They were analyzed as their permethyl peracetyl and pertrimethylsilyl derivatives. In the CI spectra, peaks corresponding to QM+ and/or [M-59]+ were observed in all of the peracetylated oligosaccharides examined. In CI with ammonia as the reagent, H+ was transferred to nitrogen-containing saccharides to produce [MH]+ and NH4 was transferred to nitrogen-free saccharides to yield [M+NH4]+ as QM+. Non-reducing ends yielded very intense peaks in CI spectra. On the other hand, the reduced end, glucitol, produced rather prominent peaks in EI spectra. Fragment ions due to cleavage of glycosidic bonds were major ones under the CI conditions, and they could be used for elucidating the sugar sequence in the oligosaccharides. An additional characteristic feature in the CI spectra was that ions due to scission of hexosaminyl glycosidic linkages were observed with very high intensities.     

The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. I. Tryptic peptides

A total of 25 tryptic peptides was isolated from the S-beta-carboxymethyl derivative of Clostridium pasteurianum iron protein (N2). In order to obtain the various peptides in pure state, a combination of gel permeation, cation and anion exchange column chromatographic methods, as well as various ascending paper chromatographic methods were adopted. Sequence studies of the tryptic peptides were carried out mainly by a modified manual Edman degradation procedure and also by automated analysis, carboxypeptidase digestion, and by hydrazinolysis. Thus, 242 residues (88.6%) out of a total of 273 amino acid residues were sequenced in the present study. The sum of the amino acid residues in the tryptic peptides isolated from iron protein (N2) accounted for the 273 amino acid residues present in the iron protein.     

Electrical properties and active solute transport in rat small intestine

Summary The transepithelial resistance, the cell membrane resistance and the ratio of resistances of the serosal (baso-lateral) to the mucosal (brush border) cell membrane were measured in rat duodenum, jejunum and ileum by means of microelectrode techniques. These measured values were not affected in the presence of actively transported solutes in the mucosal bathing fluid.Contribution of an electrical conductance through the extracellular shunt pathway to the total transepithelial conductance was quantitatively estimated using an electrically equivalent circuit analysis. These values estimated in respective tissues of small intestine were approx. 95% of the total transepithelial conductance, remaining unaffected by an active solute transport.From these data, the changes in emf's of the mucosal and serosal membrane induced byd-glucose or glycine were separately evaluated.     

Oscillations of membrane potential in L cells

Summary Effects of divalent cations on oscillations of membrane potentials (i.e., spontaneous repetitive hyperpolarizing responses) and on hyperpolarizing responses induced by electrical stimuli as well as on resting potentials were studied in large nondividing L cells. Deprivation of Ca²⁺ from the external medium inhibited these hyperpolarizing responses accompanying slight depolarization of the resting potential. Sr²⁺ or Mn²⁺ applied to the external medium in place of Ca²⁺ was able to substitute for Ca²⁺ in the generation of hyperpolarizing responses, while Mg²⁺, Ba²⁺ or La³⁺ suppressed hyperpolarizing responses. The addition of A23187 to the bathing medium or intracellular injection of Ca²⁺, Sr²⁺, Mn²⁺ or La³⁺ induced membrane hyperpolarization. When the external Ca²⁺, Sr²⁺ or Mn²⁺ concentration was increased, the resting potential also hyperpolarized, in a saturating manner. The amplitude of maximum hyperpolarization produced by high external Ca²⁺ was of the same order of magnitude as those of hyperpolarizing responses and was dependent on the external K⁺ concentration. In the light of these experimental observations, it was deduced that the K⁺ conductance increase associated with the hyperpolarizing excitation is the result of an increase in the intracellular concentration of free Ca²⁺ mainly derived from the external solution.     

Glycinebetaine enhances and stabilizes the evolution of oxygen and the synthesis of ATP by cyanobacterial thylakoid membranes.

Glycinebetaine (betaine), an osmoregulant in halophilic plants, stabilized the evolution of oxygen and the synthesis of ATP by thylakoid membranes from the cyanobacterium Synechocystis PCC6803 when it was present during the preparation and incubation of the thylakoid membranes. Moreover, betaine enhanced the evolution of oxygen and the synthesis of ATP when present during assays. When betaine at 1.0 M was present during the preparation of thylakoid membranes and during the measurement of activity, the rate of evolution of oxygen was equivalent to that of intact cells.     

A simple method for the release of asparagine-linked oligosaccharides from a glycoprotein purified by SDS-polyacrylamide gel electrophoresis.

A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.     

Crystallization and preliminary X-ray study of the cathepsin B complexed with CA074, a selective inhibitor.

Cathepsin B from bovine spleen has been purified and crystallized as a complex with a specific inhibitor CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- isoleucyl-L-proline], using the hanging-drop method. The complex crystals obtained from 50 mM-citrate buffer (pH 3.5) belong to the tetragonal space group P4(1) (or P4(3)) with a = 73.06 A and c = 141.59 A, and diffract beyond 2.2 A resolution. There are two complex molecules per asymmetric unit giving a packing density of 3.37 A3/Da and indicating a high solvent content of 63.5%.