Effects of viral transformation on synthesis and secretion of collagen and fibronectin-like molecules by embryonic chick chondrocytes in culture

Monolayer cultures of chondrocytes isolated from 11-day-old chick embryo vertebral cartilage were transformed by Rous sarcoma virus, and the effects of transformation on synthesis and secretion of extracellular proteins by these cells were studied. Transformation resulted in decreased synthesis of type II collagen which did not appear to be due to underhydroxylation of collagenous protein but to a decrease in the total amount synthesized. Carboxymethyl-cellulose chromatography and polyacrylamide disc gel electrophoresis failed to demonstrate any alpha 2 chains as a result of the transformation, suggesting that conversion of type II to type I collagen did not occur. In contrast to the decrease in collagen synthesis, synthesis of a molecule with biochemical characteristics similar to fibronectin increased markedly in virally transformed cultures. Although there were no significant differences in the amount of fibronectin-like molecules in the cell layers of normal and transformed chondrocytes, a marked increase of these molecules in the culture media of the transformed cells was demonstrated. These findings were confirmed by experiments with temperature-sensitive mutants of the virus.     

Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.     

Toxins in Blast-diseased Rice Plants

The occurence of tenuazonic acid (T.A.), which had been isolated from the culture broth of blast fungus, in blast-diseased rice plants was surveyed to ascertain whether or not this substance is one of the vivotoxins. T.A. was detected in four of six samples of blast-diseased rice plants, two of which had relatively high T.A. contents; 379 and 91 mg per kg of the samples (dry weight).

Besides T.A., coumarin, o-coumaric acid and piricularin were also isolated from blast-diseased rice plants. The molecular formula of the last substance, which was tentatively presented in a previous paper, was corrected to C₁₈H₃₀N₂O₅ from the results of high resolution mass spectrometry.     

Photoswelling and light-inactivation of isolated chloroplasts I. Change in lipid content in light-aged chloroplasts

The formation of lipid peroxide and changes in the lipid compositionof isolated chloroplasts aged in the light or dark, were investigatedin more detail. Lipid peroxide formation was observed in thethylakoid membrane as well as in the supernatant from dark-agedchloroplasts. Light was necessary for its formation in bothsystems. We confirmed that the peroxidation of lipids formedduring aging did not induce the inhibition of photochemicalactivities in chloroplasts. Aged chloroplasts underwent decompositionof their endogeneous monogalactolipid and phosphatidylcholine(lecithin) resulting in free fatty acids and lysophosphatidylcholine(lysolecithin). Decomposition of monogalactolipid occurred inboth the light- and dark-aged chloroplasts. The change of lecithinto lysolecithin was stimulated by illumination. This suggeststhat the peroxidation of lipids occurs as a result of the illuminationof free fatty acids released from monogalactolipid and lecithinin the thylakoid membranes, and that the change of lecithinto lysolecithin is related to the inactivation of photochemicalactivities and to swelling in light-aged chloroplasts. ¹ Present address: Department of Microbiology, Ishikawa ResearchLaboratory for Public Health and Environment, Minma, Kanazawa,Japan. (Received August 15, 1974; )     

Effect of ricin on biosynthesis of myeloma protein (IgA) and general cellular proteins in MOPC-315 cells.

Ricin, a phyto-toxin from the seeds of Ricinus communis, preferentially inhibits synthesis of myeloma protein by MOPC-315 cells as compared to the synthesis of general proteins. In contrast, cycloheximide inhibited equally both myeloma protein synthesis as well as general protein synthesis. These results suggest that proteins synthesized near or on cell membranes are more sensitive to Ricin which binds to the acceptor molecule on the plasma membrane.     

Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains: IDENTIFICATION OF THE RNA RECOGNITION LOOP IN RIG-I-LIKE RECEPTORS*

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5′-triphosphated single-stranded RNA (5′ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5′ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5′ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.