Abortive Infection of L Cells by Influenza B Virus: Defect in Bud Formation

Host-dependent restriction of influenza B virus replication in L cells was analysed in comparison with productive infection in MDCK or 1–5C-4 cells. The synthesis and intracellular distribution of virus-specific proteins and the production of cytoplasmic ribonucleoproteins in nonpermissive L cells were similar to those in permissive MDCK cells. However, an electron microscopic study of infected L cells showed neither extracellular virions nor budding virus particles on the cell surface, in contrast to MDCK cells which produced numerous virus particles. PAGE analysis of the plasma membrane isolated from the cells demonstrated no significant difference in the composition of viral polypeptides between permissive 1-5C-4 and nonpermissive L cells. It was noted that the abortiveness of influenza B virus infection in L cells may be due to a defect in host cell function involved in the initiation of virus budding.     

Characterization by [3H]Dihydroergocryptine Binding of Alpha-Adrenergic Receptors in Neuroblastoma × Glioma Hybrid Cells

[3H]Dihydroergocryptine ([3H]DHE) was shown to bind to sites in membranes from neuroblastoma X glioma hybrid cells (NG 108-15) that had the characteristics expected of alpha-adrenergic receptors. The binding was saturable with 0.3 pmol [3H]DHE bound per mg of protein and of high affinity, with an apparent dissociation constant (KD) of 1.8 nM. The specificity of the binding site for various ligands was more similar to that of alpha 2 receptors than to that of alpha 1. No specific binding of [3H]WB-4101 was found in the membranes derived from NG 108 cells. This finding also indicated that the [3H]DHE binding site in the cell is the alpha 2 receptor. GTP lowered the affinity of agonists for the [3H]DHE binding site, although the nucleotide hardly affected the affinity of antagonists including [3H]DHE.     

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.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

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.     

A Comparative Study of the Structures of {alpha}-Glucans from Suspension-Cultured Nonglutinous and Glutinous Rice Cells

-Glucans (average mol wt, 1.3 ? 10⁴) extracted with perchloricacid from 8-day-old suspension-cultured nonglutinous (var. Sasanishiki)and glutinous rice (var. Miyakogane) cells were compared. Theresults of hydrolysis by alpha;-, ß- and iso-amylasesand methylation analysis of the -glucans suggested that theirbasic structures are almost the same. These -glucans are highly-branchedpolysaccharides with an average chain length of about 9–10,with exterior and interior chain lengths of about 6–7and 2–3, respectively. ¹Current address: Laboratory of Food Science, Faculty of Education,Hirosaki University, Hirosaki, Aomori 036, Japan. (Received April 27, 1987; Accepted March 2, 1988)     

Common antigenic determinant in the receptor binding sites of Escherichia coli enterotoxin and cholera toxin

Abstract A mutant (TUH No. 9) of a porcine strain of enterotoxigenic Escherichia coli (ETEC) produces as abnormal B subunit (B') of heat-labile enterotoxin (LT), which has aspartate instead of glycine at residue 33 from the N-terminus and does not bind to the receptor, GM1 ganglioside. The antigenicities of the receptor-binding site of LT were analyzed.
The antibody, which could not bind to the B' subunit in the anti-B subunit of porcine LT(LTp)-serum, could bind to cholera toxin (CT), LTp and LT produced by a human ETEC strain (LTh), suggesting that it recognizes a common epitope of LTp, LTh and CT. Thus glycine at residue 33 from the N-terminus in the B subunit of CT, LTh and LTp may be related to the common epitope of these three toxins. The bindings of CT, LTh and LTp to the antibody were inhibited by the GM1 ganglioside.
These data indicate that the antibody recognizes a common epitope in the receptor (GM1 ganglioside)-binding site of CT, LTh and LTp.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

The chloroplastchIL gene of the green algaChlorella vulgaris C-27 contains a self-splicing group I intron

ThechiL gene product is involved in the light-independent synthesis of chlorophyll in photosynthetic bacteria, green algae and non-flowering plants. The chloroplast genome ofChlorella vulgaris strain C-27 contains the first example of a splitchiL gene, which is interrupted by a 951 bp group I intron in the coding region. In vitro synthesized pre-mRNA containing the entire intron and parts of the flanking exon sequences is able to efficiently self-splice in vitro in the presence of a divalent and a monovalent cation and GTP, to yield the ligated exons and other splicing intermediates characteristic of self-splicing group I introns. The 5 and 3 splice sites were confirmed by cDNA sequencing and the products of the splicing reaction were characterized by primer extension analysis. The absence of a significant ORF in the long P9 region (522 nt), separating the catalytic core from the 3 splice site, makes this intron different from the other known examples of group I introns. Guanosine-mediated attack at the 3 splice site and the presence of G-exchange reaction sites internal to the intron are some other properties demonstrated for the first time by an intron of a protein-coding plastid gene.     

Dibromothymoquinone (DBMIB) Replaces the Function of QA at 77 K in the Isolated Photosystem II Reaction Center (D1-D2-Cytochrome b559) Complex: Difference Spectrum of the P680+(DBMIB-) State

Transient absorbance changes of the primary electron donor chlorophylla (P680) and acceptor pheophytin a (H) were measured at 77 Kby nanosecond laser spectroscopy in the D1-D2-cytochrome b₅₅₉photosystem II reaction center complex containing dibromomethylisopropylbenzoquinone (DBMIB). After the laser excitation of the reactioncenter in the presence of DBMIB, only the P680⁺-(DBMIB^-) statewas detected. P680⁺ mainly decayed with a t_1/e of 11 ms. Inthe absence of DBMIB, the excitation produced the P680⁺H^- radicalpair. The radical pair produced the triplet state (P680^T) witha t_1/e of 50 ns, and P680^T then decayed with a t_1/e of 2.1 ms.It was concluded that H_- was oxidized by DBMIB in a time rangefaster than the detecting time resolution (3.5 ns) even at 77K. The rapid oxidation of H^- by DBMIB was also confirmed bythe suppression of delayed fluorescence with a decay t_1/e of50 ns. The P680⁺(DBMIB^-)/P680(DBMIB) difference spectrum exhibiteda Q_y, band with a peak at 682 nm with a shoulder at 673 nm.The spectral shape was almost temperature insensitive between77 and 265 K. The feature of this spectrum in the wavelengthrange between 330 and 720 nm was compared with that of P680_T/P680or H^-/H at 77 K. (Received May 8, 1996; Accepted June 24, 1996)