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.     

Immunocytochemical study of the intracellular localization of M protein of vesicular stomatitis virus

Summary The purpose of this paper is to describe the immunocytochemical localization of M protein of vesicular stomatitis virus (VSV) in infected cells. Vero cells, MDBK cells, Swiss 3T3 cells, and BHK cells were examined at various times after infection. For immunofluorescent staining, the cells were fixed with PLP fixative and then treated with 0.05% Triton X-100 before incubation with antibodies. Three hours after infection, M protein exhibited diffuse immunostaining throughout the cytoplasm and later accumulated along the cell membrane. The localization of M protein differed from the granular localization of the nucleocapsid N protein of VSV in the cytoplasm. For electron microscopy, the cells were fixed first in a mixture of 2% paraformaldehyde and 0.05% glutaraldehyde and then with PLP fixative, this being followed by treatment with 0.05% saponin. They were then immunostained using the immunoperoxidase method. The M protein was found to be distributed throughout the cytoplasm and later under the cell membrane, especially at virus budding sites. We also used postembedding immunostaining and freeze-fracture immunostaining to avoid the translocation of M protein caused by the detergent treatment. These techniques confirmed our previous results. Our findings are consistent with the view that the M protein of VSV is synthesized on free ribosomes and is then associated with the cell membrane where viral assembly may occur.S. Ohno was a visiting fellow from the Fogarty International Center at the National Institutes of Health, USA, from September 1981 to August 1983, while some parts of this work were in progress.     

Cell type-specific expression of the genes for the protein kinase C family: down regulation of mRNAs for PKC alpha and nPKC epsilon upon in vitro differentiation of a mouse neuroblastoma cell line neuro 2a

By the use of cloned cDNAs for protein kinase C isozymes alpha, beta I, beta II, gamma, and those for novel protein kinase C, epsilon and zeta, the expression of the corresponding mRNA species was examined in various mouse tissues, human lymphoid cell lines, and mouse cell lines of neuronal origin. In adult brain, mRNAs for all the isozymes of PKC family are expressed. However, the expression of these mRNA species in brain is low at birth. A similar pattern of expression was also observed for beta I/beta II mRNAs in spleen. These expression patterns are in clear contrast to that for beta I/beta II mRNAs in thymus where the mRNAs are expressed at birth and the levels of expression decrease with age. Human lymphoid cell lines express large amounts of PKC beta mRNAs in addition to PKC alpha. Further, nPKC epsilon mRNA is expressed in some of these cell lines. On the other hand, all the mouse cell lines of neuronal origin tested express nPKC epsilon and zeta in addition to PKC alpha. In a mouse neuroblast cell line, Neuro 2a, down modulation of mRNAs for both PKC alpha and nPKC epsilon was observed in association with in vitro differentiation.     

Alkaline Bismuth Solution as an En Bloc Stain for Formaldehyde-Glutaraldehyde Potassium Permanganate Fixed Fungal Spores

An alkaline solution of bismuth subnitrate reacted well with the cell membranes and cell walls of formaldehyde-glutaraldehyde potassium permanganate fixed Alternaria spores, demonstrating them with greater contrast than in sections stained with uranyl acetate and lead citrate. Optimal fine structure of fungal spores was obtained by en bloc staining with alkaline bismuth solution after aldehyde and permanganate fixation. The contrast of the cell organelles and cell walls was high enough in sections cut after the alkaline bismuth en bloc stain for direct ultrastructural observation. Our results indicate that the alkaline bismuth stain is useful either as an en bloc or section stain for aldehyde and permanganate fixed fungal spores.     

A new member of the protein kinase C family, nPKC theta, predominantly expressed in skeletal muscle.

A new protein kinase C (PKC)-related cDNA with unique tissue distribution has been isolated and characterized. This cDNA encodes a protein, nPKC theta, which consists of 707 amino acid residues and showed the highest sequence similarity to nPKC delta (67.0% in total). nPKC theta has a zinc-finger-like cysteine-rich sequence (C1 region) and a protein kinase domain sequence (C3 region), both of which are common in all PKC family members. However, nPKC theta lacks a putative Ca2+ binding region (C2 region) that is seen only in the conventional PKC subfamily (cPKC alpha, -beta I, -beta II, and -gamma) but not in the novel PKC subfamily (nPKC delta, -epsilon, -zeta, and -eta). Northern (RNA) blot analyses revealed that the mRNA for nPKC theta is expressed predominantly in skeletal muscle. Furthermore, nPKC theta mRNA is the most abundantly expressed PKC isoform in skeletal muscle among the nine PKC family members. nPKC theta expressed in COS1 cells serves as a phorbol ester receptor. By the use of an antipeptide antibody specific to the D2-D3 region of the nPKC theta sequence, nPKC theta was recognized as a 79-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in mouse skeletal muscle extract and also in an extract from COS1 cells transfected with an nPKC theta cDNA expression plasmid. Autophosphorylation of immunoprecipitated nPKC theta was observed; it was enhanced by phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate but attenuated by the addition of Ca2+. These results clearly demonstrate that nPKC theta should be considered a member of the PKC family of proteins that play crucial roles in the signal transduction pathway.