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.     

Matrix Gla protein C-terminal region binds to vitronectin. Co-localization suggests binding occurs during tissue development.

Matrix Gla protein (MGP) regulates calcification in cartilage and arteries. MGP synthesis during embryonic development and its binding and regulation of growth factors and morphogens of the TGF-beta/BMP superfamily suggests that it has additional functions. Assay by far-western gel overlays and gel filtration shift shows MGP binds vitronectin. Binding is saturable and consistent with a single class of binding sites. MGP binds to vitronectin but not collagen, fibromodulin, heparin, osteocalcin, chondroitin sulfate, laminin, ovalbumin or albumin. We have identified a vitronectin binding site within a 17-amino acid peptide 61-77 near the carboxyl-terminus that corresponds to a naturally occurring MGP C-terminus. MGP and the 61-77 MGP peptide also binds to fibronectin. MGP and vitronectin are focally co-localized in embryonic tissues. Co-localization in vivo suggests that the MGP and vitronectin interactions may modify cell-matrix interactions. Alternatively, vitronectin-bound MGP may have altered function for modulating BMP2 or TGF-beta activity. The current study demonstrates that MGP has a novel binding activity for vitronectin, an extracellular protein that promotes cell-matrix interactions and regulates coagulation.     

Rate of DNA Synthesis during Meiotic Prophase in Lily Microsporocytes in the Presence of Deoxyadenosine and Nalidixic Acid

The rate and period of DNA synthesis during meiotic prophasewere examined using lily microsporocytes. Meiocytes at the earlyleptotene stage were cultured for discrete periods in the presenceof inhibitors of DNA synthesis, deoxyadenosine and nalidixicacid. Deoxyadenosine, which arrests meiotic development at theearly zygotene stage, markedly suppressed DNA synthesis to 35%of control at 2 mM. Nalidixic acid simply reduced the rate ofDNA synthesis, resulting in prolongation of the synthetic period.The relevance of DNA synthesis to meiotic development is discussed. (Received January 12, 1987; Accepted May 7, 1987)     

Identification of jasmonic acid in Mimosa pudica and its inhibitory effect on auxin- and light-induced opening of the pulvinules

Jasmonic acid was identified from Mimosa pudica L. plants by mass spectrometry, high performance liquid chromatography and thin layer chromatography. Effects of authentic jasmonic acid on pulvinule movement and transpiration of the pinnae were compared with those of abscisic acid. Jasmonic acid and abscisic acid each at 10⁻⁵ M inhibited both auxin- and light-induced opening of the pulvinules. A closure-inducing activity of jasmonic acid at 10⁻⁴ M was greater than that of abscisic acid at 10⁻⁴ M. Pinnae transpiration was reduced by 10⁻⁵ M abscisic acid but not by 10⁻⁴ M jasmonic acid.     

Reversion of the apical hook of pea in the dark and its promotion by a red light pulse.

At the developmental stage at which the apical hook passed the 3rd and 4th nodes, dark-grown seedlings of pea ( Pisum sativum L. cv. Progress No.9) opened the hook upright and then formed a new hook above the node nearly in the opposite direction to the previous one. In cv. Alaska, in contrast, many (about 84%) seedlings closed the hook in the original direction after they partially (up to about 110°) opened it at the 3rd node, thus doing a wagging movement, while a small percentage (about 16%) of the seedlings reversed the hook direction. Exposure to red light of cv. Alaska seedlings for 10 min increased the percentage of the hook reversion up to 71% or more. The hook reversion was never observed except when the hook part passed the nodes, suggesting the involvement of the nodes in the phenomenon.     

Purification and characterization of the DNA-binding activity of the Epstein-Barr virus DNA polymerase accessory protein BMRF1 gene products, as expressed in insect cells by using the baculovirus system.

A recombinant baculovirus containing the complete sequence for the Epstein-Barr virus (EBV) BMRF1 gene product, the EBV DNA polymerase accessory protein, under the control of the polyhedrin promoter was constructed. Insect cells infected with the recombinant virus produced two phosphoproteins of 52 and 50 kDa and one unphosphorylated protein of 48 kDa, recognized by anti-BMRF1 protein-specific monoclonal antibody. The major protein bands were 50 and 48 kDa. The expressed BMRF1 gene products were purified to near homogeneity from the nuclear extract of the recombinant baculovirus-infected insect cells by double-stranded DNA-cellulose column chromatography followed by heparin-agarose column chromatography. The purified BMRF1 gene products exhibited higher binding affinity for double-stranded DNA than for single-stranded DNA without ATP hydrolysis. The protein-DNA interaction did not necessarily require a primer terminus. The present system will open the way for the biochemical characterization of the EBV DNA polymerase accessory protein.     

Structure and assembly of the cortical microtubule cytoskeleton in the green alga,Boodlea coacta

Summary Examination was made of the structure and assembly of the cortical microtubule (MT) cytoskeleton in the coenocytic green algaBoodlea coacta (Dickie) Murray et De Toni by immunofluorescence microscopy. Cortical MTs inBoodlea protoplasts are arranged randomly but some show a meridional arrangement within 6 h after protoplast formation. At 6–9 h such MTs become highly concentrated and parallel to each other in certain areas. At 12 h the concentration is uniformly high throughout the cell, indicating the completion of high density meridional arrangement of cortical MTs. Cortical MTs exhibiting a high density, meridional arrangement show characteristic disassembly by treatment with 10 M amiprophos-methyl (APM) or cold treatment (0 °C). Disassembly occurs by each MT unit at positions skipping 30–40 m in the transverse direction, and neighboring MTs subsequently disassemble to form MT groups. Each group becomes slender and then disappears completely within the following 24 h. The meridional arrangement of cortical MTs is disrupted by N-ethylmaleimide (NEM) accompanied by a remarkable reduction in density. The remaining MTs form groups at 30–40 m intervals from each other, as also occurs with drug or cold treatment, but disruption and density return to normal levels following removal of NEM. It appears that there are meridionally oriented channels, anchor-rich and anchor-poor, in the plasma membrane. The channels could be distributed alternately and anchors could be deposited in a cross-linking manner with cortical MTs to form a stable cortical MT-cytoskeleton. MTs comprising the cortical MT cytoskeleton could be oriented by meridionally oriented channels of anchors which are distributed following establishment of cell polarity.Abbreviations APM amiprophos-methyl - MT microtubule - MTOC microtubule organizing center - NEM N-ethylrnaleimide     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.