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.     

Chromatic Regulation of Photosystem Composition in the Cyanobacterial Photosynthetic System: Kinetic Relationship between Change of Photosystem Composition and Cell Proliferation

Kinetics of the change of photosystem (PS) composition in cyanobacteriainduced by chromatic light were studied in relation to cellproliferation. The study was made for two unicellular strains,Synechococcus NIBB 1059 and Synechocystis (Aphanocapsa) PCC6714. We found that (1) the change to a higher or lower PS I/IIratio was due to acceleration or suppression of apparent PSI formation, and (2) it progressed on a similar time scale tothat of the cell proliferation. The apparent rate constant ofthe change in the PS I/II ratio was proportional to that ofcell proliferation, µ, when this was low, but at highvalues of µ the increase in the rate constant of the changein the PS I/II ratio became smaller, causing a deviation fromthe linear relationship. Results indicate that under autotrophicconditions, the photoregulated composition change occurs asa result of thylakoid development, which accompanies cell proliferation. (Received June 23, 1986; Accepted December 5, 1986)     

Dynamics of appearance and disappearance of the ripple structure in multilamellar liposomes of dipalmitoylphosphatidylcholine

The physical properties of the pretransition (P beta'----L beta') of dipalmitoylphosphatidylcholine liposomes were investigated using freeze-fracture electron microscopy. The kinetics of pretransition examined in the previous paper using TEMPO spin probe (Tsuchida, K., et al. (1985) Biochim. Biophys. Acta 812, 249-254) was extensively studied by observing the ripple structures in the freeze-fractured surfaces at different time intervals. When the temperature is decreased from 38 degrees C to 30 degrees C, the ripple structure disappears in the following steps. The intervals between ripples begin to expand with the decrease of ripple density upon the temperature shift, and this process continues for several tens minutes. Then, each ripple disappears gradually and changes into a completely smooth surface at 3 h after the temperature shift. The comparison of relaxation times between the previous ESR measurement and the present experiment suggests that the fast relaxation observed in the previous study corresponds to the expansion of the intervals between ripples. On the other hand, the ripple structure of regular intervals appears rapidly in some places and then spreads over the whole area of fractured surface when the temperature is increased from 23 degrees C to 35 degrees C. The results obtained in this work and the previous ESR work strongly suggest that the formation and disappearance of ripple structure is closely related to the relaxation processes near the pretransition temperature.     

A complete deletion mutant of the Escherichia coli dnaKdnaJ operon

Southern hydridization analyses of genomic DNAs from various dnaJ mutants of Escherichia coli showed that mutant K7052, which has well characterized dnaK706 and dnaJ705 double mutantions, is a deletion mutant. The deletion is about 8.0 kb long and encompasses the whole of the dnaKdnaJ operon.     

A region-matched hydrophobic interaction between melittin and dimyristoylphosphatidylcholine in a ternary mixture of phosphatidylcholines

Interaction of melittin with phosphatidylcholine molecules in pure vesicles, binary mixtures and a ternary mixture of dimyristoylphosphatidylcholine IDMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC) was investigated by differential scanning calorimetry. Melittin binds preferentially with DMPC, and results in segregation of DMPC in binary mixtures of DMPC/DPPC and DMPC/DSPC and in a ternary mixture of DMPC/DPPC/DSPC. The results indicate that the hydrophobic part of peptide interacts preferentially with the phospholipid which has the same size of hydrophobic region or fatty acyl chains.     

Demonstration of a dawn phenomenon in normal adolescents

To ascertain whether the dawn phenomenon occurs in normal adolescents and, if so, to determine its mechanism, we measured nocturnal plasma glucose, insulin, glucagon, growth hormone, cortisol, and adrenocorticotropic hormone (ACTH) levels between 01.00 and 08.00 h in 10 healthy adolescents. The prehepatic insulin secretion rate was calculated based on C peptide levels. The metabolic clearance rate of insulin (MCRI) was calculated as the ratio of mean insulin secretion rate to mean insulin concentration. There was no change in plasma glucose, insulin, and glucagon between 01.00-04.00 and 05.00-08.00 h (paired t test). The MCRI was higher at 05.00-08.00 h compared to 01.00-04.00 h (9.30 +/- 1.50 vs. 4.87 +/- 1.11 ml.kg-1.min-1; p = 0.008). The prehepatic insulin secretion increased at 05.00-08.00 h relative to 01.00-04.00 h (1.1 +/- 0.2 vs. 0.6 +/- 0.1 pmol.kg-1.min-1; p = 0.013). Similarly, cortisol and ACTH levels were higher at 05.00-08.00 versus 01.00-04.00 h (323 +/- 33 vs. 102 +/- 22 nmol/l, p less than 0.001; 3.6 +/- 0.5 vs. 1.8 +/- 0.4 pmol/l, p = 0.006, respectively). Growth hormone was higher at 01.00-04.00 versus 05.00-08.00 h (7.6 +/- 1.2 and 3.0 +/- 0.9 microgram/l; p = 0.019). ACTH correlated with MCRI (r = 0.66; p = 0.002) and prehepatic insulin secretion (r = 0.75; p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)