Interleukin 1 alpha mRNA in virus-transformed T and B cells

IL-1 alpha cDNA clone was isolated from a T cell line infected by the human T lymphotropic retrovirus type-I (HTLV-I/ATLV). We found significant amounts of mRNA hybridizing to IL-1 alpha cDNA not only in HTLV-I-transformed T cells but also in Epstein-Barr Virus-transformed B cells. A part of IL-2 receptor inducing activity in Adult T cell leukemia (ATL) cell line seems to be due to IL-1 alpha.     

Isolation and characterization of a monoclonal nonspecific suppressor factor (MNSF) produced by a T cell hybridoma

By fusing Con A-activated BALB/c mice spleen cells with AKR thymoma BW5147 cells, we prepared a hybridoma producing a monoclonal nonspecific suppressor factor (MNSF). This factor inhibits a generation of LPS-induced immunoglobulin-secreting cells. We used ELISA for the bioassay of MNSF activity. With this method, a stable E17 hybridoma clone was selected, and its product in culture medium was isolated and characterized. MNSF fractionated on Sephadex G-100 in saline buffer shows a form with multiple m.w., but fractionated in 0.4 M pyridine-acetic buffer, it is limited to two species of approximately 24Kd and 16Kd. The MNSF was purified by hydroxyapatite chromatography, with marked effectiveness. MNSF activity was found exclusively in the 0.35 M sodium phosphate elution, and the content was further fractionated on subsequent gel filtration in the high ionic strength buffer described above. The purified factor exhibited two forms, of 24Kd and 16Kd, and showed peaks of pI 5.3 and 5.7, respectively, on isoelectric focusing. The MNSF preparation described here is stable at 56 degrees C and unaffected by 2-mercaptoethanol, but is unstable at pH 2.0 and is sensitive to tryptic proteolysis. We injected the hybridoma cells into the peritoneal cavity of pristane-primed F1 (AKR/J X BALB/c) mice, and a large amount of pure MNSF was obtained from the ascites, the characteristics of which were similar to those in the culture supernatant. Thus, the MNSF obtained from the E17 hybridoma consists of functionally identical but physicochemically different discrete proteins. This simple method of purification can serve as a probe for further characterization of MNSF and its application in in vivo experiments.     

Cytolytic activity of antigen-specific T cells with helper phenotype

We have investigated an unusual cytolytic activity displayed in vitro by cloned T cells which have the cell surface phenotype of helper T cells. When the cloned T cells are cultured with the appropriate antigen and antigen-presenting cells (APC), the T cells become activated in that they produce lymphokines and proliferate in an antigen-specific and major histocompatibility complex-restricted manner. At the same time, these T cells cause lysis of the APC. In addition, innocent non-histocompatible bystander cells present in the cultures can also be killed. The cytolytic activity may be involved in a mechanism of immune regulation.     

Dissociation between plasma and atrial content of atrial natriuretic polypeptide (ANP) following sodium load in rats

To investigate the time course effect of sodium intake on release and synthesis of atrial natriuretic polypeptide (ANP), plasma and atrial content of ANP were measured in rats which had been fed either a high or a low salt diet for 1, 3, 7, 14 and 35 days. Plasma ANP in rats fed the high salt diet for one day was significantly higher than in those fed the low salt diet. However, there were no significant differences between the groups fed either the high or the low salt diet for 3 days or more. In contrast to the direction of change in plasma ANP, atrial content of ANP in rats fed the high salt diet for one day tended to be lower and was significantly lower in those fed for 3 and 7 days than in the low salt diet group, while there were no significant differences between both groups that were fed for 14 and 35 days. These results suggest that ANP is rapidly released into the circulation when sodium is loaded, however, the atrial storage of ANP remains depleted for about one week.     

The pyrogallol related compounds reduce UV-induced mutations in Escherichia coli B/r WP2

Plant components with bio-antimutagenic activity were screened on UVC (254 nm)-induced mutagenesis using E. coli B/r WP2. The components with a pyrogallol moiety including gallic acid, (-)-epicatechin gallate (ECG), (-)-epigallocatechin (EGC) and (-)-epigallocatechin gallate (EGCG) reduced the mutation induction, but other components such as caffeic acid, chlorogenic acid and quercetin did not. The above compounds with a pyrogallol moiety were also effective on UVAB (295-400 nm)-induced mutagenesis, while they showed little effect on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutagenesis. As this bio-antimutagenic effect was not seen in the DNA excision-repair-deficient strains WP2s and ZA159, the activity by the above plant components might be based on the promotion of the excision-repair system in E. coli B/r WP2.     

Nalidixic acid-resistant mutations of the gyrB gene of Escherichia coli

Summary DNA fragments of 3.4 kb containing the gyrB gene were cloned from Escherichia coli KL-16 and from spontaneous nalidixic acid-resistant mutants. The mutations (nal-24 and nal-31) had been determined to be in the gyrB gene by transduction analysis. Nucleotide sequence analysis of the cloned DNA fragments revealed that nal-24 was a G to A transition at the first base of the 426th codon of the gyrB gene, resulting in an amino acid change from aspartic acid to asparagine, and nal-31 was an A to G transition at the first base of the 447th codon, resulting in an amino acid change from lysine to glutamic acid. This indicates that mutations in the gyrB gene are responsible for nalidixic acid resistance.     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)     

Structure of the trypsin-binding domain of Bowman-Birk type protease inhibitor and its interaction with trypsin

The crystal structure of the complex formed by bovine trypsin and Bowman-Birk type protease inhibitor AB-I extracted from azuki beans (Vigna angularis) 'Takara' has been analyzed. The structure was solved by the application of the phase combination of single isomorphous phases and trypsin model phases, followed by phase improvement using the iterative Fourier technique. From the resulting electron density map, a three-dimensional atomic model of the trypsin binding domain of AB-I has been built. The peptide chain at the trypsin reactive site turns back sharply at Pro29 and forms a 9-residue ring (Cys24-Cys32). The 'front side' of this ring, consisting of the reactive site (Cys24-Met28), interacts with trypsin in a similar manner to other families of inhibitors and forms a stable complex, which seems to be maintained by the interactions with the 'back side' of this ring (Pro29-Cys34). The similar spatial arrangements of the 'back side' of this inhibitor and the 'secondary contact region' of the other inhibitors with respect to the reactive site suggest an important common role of these regions in exhibiting inhibitory activity.     

Inhibitory effects of various synthetic substrates for aminopeptidases on phagocytosis of immune complexes by macrophages

The inhibitory effects of various 7-(aminoacyl)-4-methylcoumarinylamides (AA-MCA's), synthetic substrates for aminopeptidases, on phagocytosis of immune complexes by guinea pig peritoneal macrophages were investigated by measuring the intracellular uptake of sensitized 51Cr-sheep erythrocytes and 125I-alpha-amylase complexed with homologous IgG2 antibody. Among the AA-MCA's examined, MCA compounds of hydrophobic amino acids (Phe, Tyr, Leu, and Pro) were found to inhibit the intracellular uptake and digestion of immune complexes. Sucrose density gradient centrifugation of the homogenates of macrophages treated with the inhibitors for 1 h at 37 degrees C showed that they modulated the lysosomes, resulting in a decrease in buoyant density of the organella. These effects of the inhibitors on the buoyant density of the lysosomes as well as on the phagocytic activity of macrophages disappeared upon removal of the inhibitors from the cells by washing. Since none of 7-amino-4-methylcoumarin, L-phenylalanine, and bestatin methyl ester could significantly inhibit the phagocytosis of immune complexes by macrophages, the MCA compounds of hydrophobic amino acids appear to inhibit the phagocytosis as a consequence of their lysosomotropic nature.     

A unique trypsin-like protease associated with plasma membranes of rat liver

One way in which serum promotes survival of primary cultured hepatocytes is by inhibiting plasma membrane protease (Nakamura, T., Asami, O., Tanaka, K., and Ichihara, A. (1984) Exp. Cell Res. 155, 81-91). One of these proteases was solubilized from the plasma membranes of rat liver with 4% octyl glucoside and purified to a homogeneous state by affinity chromatography on bovine pancreatic trypsin inhibitor linked to Sepharose 4B. The protease had an apparent Mr = 120,000 by Sephacryl S-200 gel filtration and the Mr of its subunits was 30,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It appeared to be a glycoprotein. A high concentration of detergent was necessary to keep the protein soluble. The purified enzyme readily hydrolyzed synthetic tripeptide nitroanilides at sites adjacent to Arg or Lys residues, but did not degrade synthetic substrates of chymotrypsin, elastase, or aminopeptidase. It showed endopeptidase activity, hydrolyzing various proteins such as casein, hemoglobin, and denatured albumin. The enzyme was strongly inhibited by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bovine pancreatic trypsin inhibitor, leupeptin, antipain, and alpha 1-antitrypsin, but not by chymostatin, elastatinal, or inhibitors of carboxyl, thiol, or metallo proteases, suggesting that it is a seryl trypsin-like protease. This protease was found in plasma membranes of rat and mouse liver and in small amounts in those of kidney, but not in those of brain, red cells, Ehrlich ascites tumor, or two Morris hepatomas, suggesting that it may be involved in differentiated functions of normal hepatocytes.