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.     

Enzymatic sulfation of galactosyl- and lactosylceramides in cell lines derived from renal tubules.

1. The renal cell lines, JTC-12 and MDCK, not only synthesize galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate in vivo, but also contain enzymes that catalyze the transfer of sulfate to galactosylceramide and lactosylceramide in vitro. 2. Concentration of cations necessary for maximum sulfotransferase activity occurred at 40 mM Ca2+ with galactosylceramide and 15 mM Ca2+ with lactosylceramide as the substrate. Na+ was also found to stimulate the sulfation of galactosylceramide, but was slightly inhibitory for the sulfation of lactosylceramide. 3. The products of the in vitro assay mixture were characterized as galactosylceramide 3-sulfate and lactosylceramide 3'-sulfate by a variety of TLC separations. 4. The apparent Km of JTC-12 cells for galactosylceramide was 17 microM, while that for lactosylceramide was 82 microM. The Km values of MDCK cells were comparable to those of JTC-12 cells. Competition studies suggested that galactosylceramide and lactosylceramide were sulfated by a single enzyme in both cell lines.     

Biosynthesis of monosulfogangliotriaosylceramide and GM2 by N-acetylgalactosaminyltransferase from rat brain

Some properties of the enzyme activity that catalyzes the transfer of N-acetylgalactosamine from UDP-N-acetylgalactosamine to exogenous lactosylceramide-II3-sulfate (SM3) and N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100,000 X g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II3-sulfate (SM2) and N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The Km values for UDP-N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 microM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme.     

Metabolic pathways from 1 alpha,25-dihydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3-26,23-lactone. Stereo-retained and stereo-selective lactonization

The present study was carried out in order to elucidate the metabolic pathway from 1 alpha,25-(OH)2D3 to 1 alpha,25-(OH)2D3-26,23-lactone. For that purpose, we stereospecifically synthesized the vitamin D3 derivatives 1 alpha,23(S),25-(OH)3D3, 1 alpha,23(S),25(R),26-tetrahydroxyvitamin D3, and 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-lactol. The in vitro metabolism of these compounds was examined in kidney homogenates and intestinal mucosa homogenates from 1 alpha,25-(OH)2D3-supplemented chicks. The naturally occurring 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone was produced (in increasing amounts) from 1 alpha,25-(OH)2D3, 1 alpha,25(R),26-(OH)3D3, 1 alpha,23(S),25-(OH),D3, 1 alpha,23(S),25(R),26-(OH)4D3, and 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol. These results indicated that there are two possible metabolic pathways from 1 alpha,25-(OH)2D3 to 1 alpha,23(S),25(R),26-(OH)4D3: the major one is by way of 1 alpha,23(S),25-(OH)3D3 and the minor one is by way of 1 alpha,25(R),26-(OH)3D3. 1 alpha,23(S),25(R),26-Tetrahydroxyvitamin D3 is further metabolized to 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactone via 23(S),25(R)-1 alpha,25-dihydroxyvitamin D3-26,23-lactol. In the course of our studies, a new biosynthetic vitamin D3 metabolite was isolated in pure form. This metabolite was identified as 23(S),25(R)-1 alpha,25-(OH)2D3-26,23-lactol by UV spectrophotometry and mass spectrometry. Furthermore, we establish in this report that the lactonization of 1 alpha,23,25,26-(OH)4D3 and 1 alpha,25-(OH)2D3-26,23-lactol occurs in a stereo-retained and stereo-selective fashion.     

Diagnosis of a case of pulmonary carcinosarcoma by detection of rhabdomyosarcoma cells in sputum

Cytologic examination of sputum samples from an elderly patient revealed the presence of two cell populations: squamous cell carcinoma cells and rhabdomyosarcoma cells. The abnormal squamous cells showed both keratinizing and nonkeratinizing forms while some of the rhabdomyosarcoma cells showed cross striations. Sputum cytology was thus able to suggest a diagnosis of pulmonary carcinosarcoma. Histologically, the tumor was composed mainly of sarcomatous tissue showing various kinds of cells: fusiform or fibrous cells, round anaplastic cells, spindled cells with typical cross striations and myoblastic cells. A partially myxomatous degeneration was present. In addition, squamous cell carcinoma proliferated along the bronchi and formed small invasive cell nests in the sarcomatous tissue. No transition between the two components was noted. Both cellular constituents had metastasized to an interlobar lymph node.