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.     

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.     

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.     

Isolation and Identification of α-(γ-Aminobutyryl)-Hypusine

A new dipeptide, alpha-(gamma-aminobutyryl)-hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid-soluble fraction of bovine brain with five steps of ion-exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.     

General recombination mechanisms in extracts of meiotic cells

RecA-like proteins have been purified from somatic and meiotic cells of mouse and lily. The rec proteins have been designated s-rec and m-rec to indicate their respective tissues of origin. The two proteins differ in molecular weight and in their response to temperature, the latter being consistent with the optimal temperature for physiological function of their tissues of origin. There is a major increase in m-rec protein with the entry of cells into meiosis, the peak of activity being early pachytene. Extracts of the cells and also those of yeast (Saccharomyces cerevisiae) have been prepared that have the capacity to catalyze homologous recombination. These extracts behave similarly to the m-rec proteins upon entry of cells into meiosis. Yeast transferred to sporulation medium displays a 100-fold increase in the recombination activity of the extract at about the time of entry into meiosis. The occurrence of peak levels of m-rec and recombination activity in extracts from cells in early pachytene points strongly to that stage as the time at which the enzymatic phase of recombination occurs.     

Preliminary crystallographic data for cross-linked (lysine7-lysine41)-ribonuclease A

A cross-linked derivative of ribonuclease A, N^ε,N^ε′-(2,4-dinitrophenylene-1,5)-(lysine⁷-lysine⁴¹)-RNase A, has been crystallized by dialysis against 30% ($\text{v}\text{v}$) ethanol/water mixtures buffered at high pH. Single crystals belong to the orthorhombic space group P2₁2₁2₁, $\text{a = 37.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}$, with one molecule in the Crystallographic asymmetric unit.