Monoclonal antibodies against the mercaptoethanol-sensitive structure of a cell-cell adhesion protein of Polysphondylium pallidum

Monoclonal antibodies were prepared against a putative cell-cell adhesion glycoprotein, with an apparent molecular mass of 64,000 (gp64), of the cellular slime mold, Polysphondylium pallidum. Five monoclonal antibodies obtained by means of an enzyme-linked immunoadsorbent assay did not bind to the antigens which were subjected to gel electrophoresis and blotting method in the presence of a reducing agent, but they did bind specifically to the antigens prepared in unreducing conditions of samples and then processed by the same blotting method. To solubilize gp64 in a sodium dodecyl sulfate (SDS)-sample buffer without mercaptoethanol (heated) or SDS-sample buffer with 2-mercaptoethanol (nonheated) was critical for the antibody binding onto gp64 on a membrane. Hence the antibodies seem to bind to a surface portion(s) of the localized protein structure folded up by disulfide cross-linkages. One of the antibodies obtained blocked cell-cell adhesion by about 20%.     

In vitro production of Bombyx mori silk fibroin by organ culture of the posterior silk glands; isotope labeling and fluorination of the silk fibroin

An in vitro silk fibroin production system has been developed by culture of posterior silk glands from Bombyx mori. A large amount of the silk fibroin was produced continuously and effectively with a rotation culture procedure. Modified Grace's insect medium was used, and oxygen bubbling in the medium was performed. In addition, half of the medium was replaced with fresh medium every 6 h. The production yield of silk fibroin produced after 100 h culture was 81 mg/g wet weight of posterior silk gland. This culture system was used successfully for efficient (15)N isotope labeling of silk fibroin, which is required for (15)N solid state nuclear magnetic resonance (NMR) analysis of silk fibroin. Moreover, the introduction of fluorinated amino acids into silk fibroin was also carried out using this culture system. (c) 1993 John Wiley & Sons, Inc.     

Interferon modulates the messenger RNA of G1-controlling genes to suppress the G1-to-S transition in Daudi cells

Interferon (IFN) is one of the potent antiproliferative cytokines and is used to treat some selected cancers. IFN arrests the growth of Burkitt Iymphoma derived cell line Daudi cells in the G1 phase. G1-to-S progression is controlled by positive and negative regulatory genes. Therefore, we investigated the effects of IFN on G1-controlling genes. Expression of cyclin-dependent kinases (Cdks 2, 3, 4, 5, 6), MO 15/Cdk7, and cyclins E and H was studied to assess positive regulators, while p15^Ink4B, p16^Ink4, p18, p21^CipI, and p27^Kip1 were assessed as negative regulators. Cdks 2, 4, 6 and cyclin E were markedly down-regulated. MO15/Cdk7 expression showed little change, but its regulatory subunit (cyclin H) was down-regulated like cyclin E. Expression of p15^Ink4B and p16^Ink4 was not observed. p18 was induced until 48 h and its expression returned to the initial level at 72 h. In contrast, p21^Cip1 mRNA expression remained at the baseline level throughout IFN treatment, while the expression of p27^Kip1 increased at 48 and 72 h. Taken together, these data indicate that IFN changes the messenger RNA of G1-controlling genes towards the suppression of G1-to-S transition.     

Murine monoclonal antibody recognizing human α(1,3/1,4)fucosyltransferase

We prepared a mouse monoclonal antibody, FTA1-16, that specifically recognizes human (1,3/1,4)fucosyltransferase without crossreactivity to any other members of the (1,3)fucosyltransferase family. The specificity was confirmed by both immunofluorescense staining of native antigens in the Golgi apparatus and Western blotting analysis, using stable transformant cells transfected with each gene of the (1,3)fucosyltransferase family. Western blotting analysis on a series of human tumour cell lines from various tissues revealed that some epithelial cancer cell lines from digestive organs expressed an amount of (1,3/1,4)fucosyltransferase in good correlation with expression of sialyl Lewis a antigen. Immunohistochemical staining by FTA1-16 on colon cancer tissues revealed enhanced expression of the enzyme in cancer cells in comparison to normal cells. Finally, the antigenic epitope recognized by FTA1-16 was determined using truncated recombinant peptides which were expressed inE. coli. A minimal length determined was a fragment, amino acid positions 132–153, of the (1,3/1,4)fucosyltransferase.     

A protein in the oxygen-evolving complex in the chloroplast is associated with symptom expression on tobacco leaves infected with cucumber mosaic virus strain Y

To elucidate the molecular basis of symptom expression in virus-infected plants, the changes in proteins between tobacco, Nicotiana tabacum cv. Ky57, leaves inoculated with cucumber mosaic virus strain Y [CMV(Y)] and strain O [CMV(O)], were compared by 2-dimensional (2-D) gel electrophoresis. The appearance of chlorotic spots in CMV(Y)-inoculated tobacco leaves accompanied an increase of 3 polypeptides and a decrease in 6 polypeptides, as compared with those in the CMV(O)-inoculated tobacco which showed no clear symptoms. The decrease in the amounts of two polypeptides of 22 and 23 kDa was particularly significant: these two polypeptides were compared with a 24 kDa polypeptide, which co-migrated with them in 2-D gel electrophoresis but did not clearly decrease at an early stage of infection, as well as major other proteins of CMV(Y)-inoculated tobacco leaves. However, the 22, 23 and 24 kDa polypeptides showed the same peptide mapping pattern. Furthermore, the 12 amino acid residues at N-termini of the three polypeptides match those of the extrinsic 23 kDa polypeptide of an oxygen-evolving complex from spinach. A comparative analysis of the 22, 23 and 24 kDa polypeptides in N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, revealed that the 22 kDa polypeptide derives from N. sylvestris and the 23 kDa polypeptide from N. tomentosiformis; the 24 kDa polypeptide derives from both ancestral Nicotiana species. The results indicate that the polypeptides whose amounts differentially decrease with the progress of symptom expression in N. tabacum inoculated with CMV(Y) are one component of the oxygen-evolving complex in photosystem II.     

Auxin Changes Both the Extensibility and the Yield Threshold of the Cell Wall of Vigna Hypocotyls

Elongation of plant stem is governed by two simultaneous processes:irreversible yielding of the cell wall and uptake of water.Among many candidates for the parameters that regulate and/or restrict growth, we focused on the mechanical propertiesof the cell wall and determined those parameters that governthe process of IAA-induced growth by means of the pressure-jumpmethod combined with the pressure-probe technique. The elongation growth of segments excised from the elongationzone of Vigna hypocotyls was accelerated by xylem perfusionwith 10^–4 M IAA. During the promotion of growth, boththe extensibility () of the cell wall and the effective turgor(Pⁱ–Y) increased while only a little or no change in theintracellular pressure (Pⁱ) occurred. These results indicate that IAA increases not only the extensibilityof the cell wall but also the effective turgor, i.e., the drivingforce for yielding of the cell wall. However, the driving forceis not increased by the increase in Pⁱ but by the decrease inthe yield threshold (Y). These results suggest that Y is adjustableduring the regulation of growth. ¹Present address: Department of Biology, Faculty of Science,Okayama University, Okayama, 700 Japan (Received September 20, 1990; Accepted November 27, 1990)     

Metabolic Flux Analysis of the Phenylpropanoid Pathway in Wound-Healing Potato Tuber Tissue using Stable Isotope-Labeled Tracer and LC-MS Spectroscopy

In the May issue of Plant and Cell Physiology     

Orofacial pain increases mRNA level for galanin in the trigeminal nucleus caudalis of the rat

The changes of preprogalanin mRNA levels in the superficial dorsal horn neurons (laminae I and II) of the trigeminal nucleus caudalis in response to orofacial pain induced by the injection of 5% formalin into the lips of rats was investigated and compared to those of preproenkephalin A mRNA and preprodynorphin mRNA in the same region by means of in situ hybridization histochemistry. Rapid and marked increases of preprogalanin and preprodynorphin mRNA were observed on the side of the injection, but the increase of preproenkephalin A mRNA level was less pronounced than that of the other two mRNAs, indicating that these peptides have different roles in the dorsal horn analgesic mechanism and that galanin, in addition to opioid peptides, may have a highly specific role in this mechanism.