An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.     

The role of the tryptophyl residue in the hypotensive peptide xenopsin

The role of the Trp⁶ residue in the biological activity of the hypotensive peptide xenopsin (<Glu-Gly-Lys-Arg-Pro-Trp-Ile-Leu-OH) was investigated. This residue was satisfactorily reduced to 2,3-dihydro-Trp on treatment with excess pyridine-borane in trifluoroacetic acid without any detectable change in other parts of the molecule. The analogous peptide, (Lys², Gly³) xenopsin, was also reduced in a similar manner. Both reduction products were purified by gel filtration and characterized by UV absorption, amino acid composition, and structural analysis.The reduced peptides were assayed on the fundus strip of isolated rat stomach and were found to possess less than 1 percent of the activity of the original peptides. Although each of the reduced analogs had an indoline substituted for an indole in the tryptophyl residue, their biological activity was virtually lost. This suggests that the tryptophyl residue of xenopsin is crucial for its biological activity.     

Gap Junctions Mediate Glucose Transport Between GLUT1-Positive and -Negative Cells in the Spiral Limbus of the Rat Cochlea

To elucidate the role of the spiral limbus in glucose transport in the cochlea, we analyzed the expression and localization of GLUT1, connexin26, connexin30, and occludin in the spiral limbus of the rat cochlea. GLUT1 and occludin were detected in blood vessels. GLUT1, connexin26, connexin30, and occludin were also expressed in fibrocytes just basal to the supralimbal lining cells. Connexin26 and connexin30 were present among not only these GLUT1-positive fibrocytes but also GLUT1-negative fibrocytes. In vivo glucose imaging using 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (6-NBDG, MW 342) together with Evans Blue Albumin (EBA, MW 68,000) showed that 6-NBDG was rapidly distributed throughout the spiral limbus, whereas EBA was localized only in the vessels. Moreover, the gap junctional uncoupler heptanol inhibited the distribution of 6-NBDG. These findings suggest that gap junctions play an important role in glucose transport in the spiral limbus, i.e., that gap junctions mediate glucose transport from GLUT1-positive fibrocytes to GLUT1-negative fibrocytes in the spiral limbus.     

Involvement of the Golgi region in the intracellular trafficking of cholera toxin

The intracellular pathway following receptor-mediated endocytosis of cholera toxin was studied using brefeldin A (BFA), which inhibited protein secretion and induced dramatic morphological changes in the Golgi region. In both mouse Y1 adrenal cells and CHO cells, BFA at 1 μg/ml caused a 80–90% inhibition of the cholera toxin (CT)-elevation of intracellular cAMP. The inhibition of the cytotoxicity of CT by BFA was also observed in a rounding assay of Y1 adrenal cells. The inhibition of CT cytotoxicity by BFA was dose dependent, with the ID₅₀ value similar to the LD₅₀ of BFA in Y1 adrenal cells. Binding and internalization of [¹²⁵I]-cholera toxin in Y1 adrenal cells was not affected by BFA. Unlike the BFA-sensitive cell lines such as Y1 adrenal and CHO cells, BFA at 1 μg/ml did not inhibit the cytotoxicity of CT in PtK₁ cells, of which the Golgi structure was BFA-resistant. These results strongly suggest that a BFA-sensitive Golgi is required for the protection of CT cytotoxicity by BFA. In contrast, elevation of the intracellular cAMP by forskolin, which acts directly on the plasma membrane adenylate cyclase, was not affected by BFA. These observations indicate that the intoxication of target cells by CT requires an intact Golgi region for its intracellular trafficking and/or processing. In this respect, CT shares a common intracellular pathway with ricin, Pseudomonas toxin, and modeccin, even though their structures and modes of action are very different. © 1993 Wiley-Liss, Inc.     

Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB ₁ -C-A-B ₂, from which two proteins, OdhAB₁C and OdhAB₂C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB₁ and OdhB₂ in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Twenty-four-hour variations in subcellular structures of rat type II alveolar epithelial cells

Summary Subcellular structures of type II alveolar epithelial cells in the rat lung were analyzed at six evenly spaced times over 24 h (light period: 06.00 h–18.00 h), using a morphometric technique. The cell volumes were maximal at 16.00 h and minimal at 08.00 h. The volume and surface densities of rough endoplasmic reticulum and mitochondria were low during the light period, and high during the dark period. Morphometric parameters of multivesicular bodies did not significantly fluctuate over 24 h, but they increased from 04.00 h to 08.00 h. The volume densities of lamellar bodies increased from 16.00 h to 20.00 h, and decreased from 00.00 h to 08.00 h. The change in numerical densities of lamellar bodies was inversely correlated to that in the volume densities. As shown by electron microscopy, small lamellar bodies predominated at 08.00 h, larger lamellar bodies increasing at 16.00h. Composite bodies often appeared at 08.00 h and 12.00 h. Type II cells thus appear to fluctuate, showing three phases over 24 h: formation, accumulation and secretion of lamellar bodies. In particular, it is noteworthy that the accumulation stage occurs during the resting phase of the rat, whereas the secretion stage occurs during its body-active phase.     

Mechanism of function of dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter in Halobacterium halobium: pH effect

The regulatory roles of medium pH, a transmembrane pH gradient (delta pH), and an electrical potential (delta phi) on the activation of the N,N'-dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter were studied in the membrane vesicle of Halobacterium halobium in the dark. Neither delta pH nor delta phi independently activated the antiporter but a combination could. The initial rate of Na+ extrusion did not proportionally relate to the size of delta microH+ imposed. The delta microH+-coupled Na+ efflux in the presence of delta phi (-140 mV) increased as external pH decreased, regardless of the size of delta pH, suggesting the existence of one external H+-binding site (apparent pKa 4.6) whose protonation determines primarily the Na+/H+-exchange activity. On the other hand, the dependence of the Na+ efflux on cytoplasmic pH varied with the size of delta pH imposed and the apparent pKa for the cytoplasmic H+ increased with elevating delta pH. The resulting pKa difference across the membrane seems to be the key mechanism for the facilitation of Na+-coupled H+ influx. In other words, delta pH modulates Na+/H+-exchange activity through manipulating the H+ affinity on the cytoplasmic regulatory site. The Na+ extrusion was gated by the threshold delta phi of -100 mV regardless of the size of existing delta pH. delta phi acts on the protonated antiporter and converts it into an active state which becomes delta pH reactive.     

Holocene sedimentary history of some coastal plains in Hokkaido,Japan IV. Diatom assemblages in the sediments from Kushiro moor (2)

Diatom assemblages of sediments obtained from three sites on Kushiro Moor were analyzed to investigate the Holocene sedimentary history. The results showed that: 1) The Takkobu site was originally at the bottom of the paleo-Kushiro Bay, and after-wards the paleo-Takkobu Lagoon developed, became sealed off, and changed to a freshwater lake. The succession to peat moor probably began about 2000 yr B.P. at the Takkobu site. 2) The Tsurui site was originally at the bottom of the paleo-Kushiro Bay, then changed to the paleo-Kushiro Lagoon and became peat moor as a result of the first Holocene regression, which finished about 3600 yr B.P. The site then returned to a brackish lake again, probably due to the second Holocene transgression between 3600 and 3000 yr B.P., thereafter passing through brackish lake and freshwater lake stages, and eventually becaming peat moor at about 2000 yr B.P., 3) At the Chuo site, the second paleo-Kushiro Bay developed again as a result of the second Holocene transgression, which finished about 3000 yr B.P. Thereafter, brackish or freshwater lakes, rivers, and then peat moor developed in the central area of Kushiro Moor. 4) The second marine diatom zone (MD₂ Zone), which indicates the second Holocene transgression, complete by about 3000 yr B.P., is detected only at the Chuo site in the central area of Kushiro Moor.     

A re-evaluation of the cytology of cat Pacinian corpuscles

Summary The ultrastructure of cat mesenteric Pacinian corpuscles in cross and longitudinal sections has been examined. The terminal ends of lamellar cells of the inner core have been identified in longitudinal sections through the proximal portion of the inner core. These terminal bulbous expansions contain characteristic concentric membranes of rough endoplasmic reticulum and in some cases masses of oval membranous inclusions. The central axon as seen in cross section is oval in profile, having X-(short) and Y-(long) axes, and each axonal face is characterized by specializations of the axolemma. At the X-axis, the inner lamellae of the inner core tightly abut a smooth axolemma, with no intervening connective tissue matrix, in a manner reminiscent of a neuroepithelium. The axolemma of the Y-axis has numerous axonal spines (microspikes) that project into the cleft in the inner core. The extent of the axolemma having axonal spines can only be appreciated in longitudinal sections. The clefts contain a specialized connective tissue with elastic and collagen fibrils. The connective tissue compartment of fibers and matrix separating individual inner core lamellae is unique, in that it contains extremely thin collagen fibrils measuring approximately 15 nm in diameter. The diameter of collagen fibrils increases as the cleft is approached. Here the fibrils resemble typical endoneural collagen.