Stem cell factor has histamine releasing activity in rat connective tissue-type mast cells.

Stem cell factor (SCF) was documented to be involved in the growth of mast cells controlled by fibroblasts. We tested the effect of recombinant rat SCF on degranulation from rat peritoneal mast cells (connective tissue-type mast cells: CTMC). SCF induced histamine release (approximately 20% of total histamine content) in a dose-dependent fashion. The release response was relatively rapid and reached a maximum within 5 min. The release showed total dependence on the presence of extracellular phosphatidylserine (PTS). These results reveal that SCF has histamine releasing activity in CTMC.     

Unusual accumulation of copper related to induction of metallothionein in the liver of LEC rats.

Copper (Cu), iron (Fe), zinc (Zn) and manganese (Mn) levels in organs of LEC rats (Long-Evans rats with a cinnamon-like coat color), which develop spontaneous jaundice with hereditary hepatitis, were determined by instrumental neutron activation analysis method. Unusual accumulations of Cu in the liver of LEC rats were found, depending on the age of the animals, the metal concentration being more than approximately 20-40 times those of normal LEA rats (Long-Evans rats with an agouti coat color). Fe and Zn were also accumulated, in addition to Cu, significantly in the LEC rats. The unusual Cu accumulations in the liver of LEC rats were associated with the induction of metallothionein, estimated by radioimmunoassay method, in the liver of LEC rats, rather than that of superoxide dismutase, estimated by electron spin resonance -spin trapping method. These findings suggest that the unusual Cu accumulation in LEC rats is involved in the development of jaundice, hepatic injury and hepatocellular carcinoma.     

Estimation of structural changes in the cataractous rat lens using Raman spectroscopy.

For quantitative evaluation of cataract-related changes in lens proteins and lens water, the relative contents of water and SH residues and changes in the microenvironments of aromatic amino acid residues were quantitatively examined in cataract of the rat lens which had been induced by sodium selenite. Using Raman spectroscopy, results were compared with those of age-matched control lenses. The relative contents of water and SH residues decreased with increasing age in normal lenses from 3 to 8 weeks of age. In the cataractous lens, the relative water content increased constantly as compared with that of age-matched controls. The relative SH residue content continued to decline in the cataractous lenses of animals at every age. The microenvironments of tyrosine residues in cataractous lenses also changed progressively.     

Specific phosphorylation of the acidic central region of the N-myc protein by casein kinase II.

The central region of the N-myc protein has a characteristic amino acid sequence EDTLSDSDDEDD, which is very similar to those of particular domains of adenovirus E1A, human papilloma virus E7, Simian virus 40 large T, c-myc and L-myc proteins. Domains of these three viral oncoproteins have recently been shown to be specific binding sites for the tumor-suppressor gene retinoblastoma protein. We have noted that the sequence of serine followed by a cluster of acidic amino acids is exactly the same as that of a typical substrate of casein kinase II (CKII). Therefore, we investigated whether these nuclear oncoproteins are phosphorylated by CKII. For this purpose, we fused the beta-galactosidase and N-myc genes including this domain and expressed it in Escherichia coli cells. Several mutant N-myc genes, containing single amino acid substitutions in this domain, were also used to produce fused proteins. Strong phosphorylation by CKII was detected with the fused protein of wild-type N-myc. However, no phosphorylation of beta-galactosidase itself was observed and the phosphorylations of fused mutant proteins were low. Another fused N-myc protein containing most of the C-terminal region downstream of this acidic region was not phosphorylated by CKII. Analysis of phosphorylation sites in synthetic peptides of this acidic region identified the major sites phosphorylated by CKII as Ser261 and Ser263. On two-dimensional tryptic mapping of phosphorylated N-myc proteins, major spots of in vitro-labeled and in-vivo-labeled N-myc proteins were detected in the same positions. These results suggest that two serine residues of the acidic central region of the N-myc protein are phosphorylated by CKII in vivo as well as in vitro. The functional significance of this acidic domain is discussed.     

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.     

Eclosion hormone activity in haemolymph of eclosing silkmoth, Bombyx mori

The presence of eclosion hormone in the haemolymph of eclosing adults of the silkworm, Bombyx mori, was demonstrated to provide evidence that the hormone is involved in the induction of eclosion of this insect.The eclosion of the adult occurred within 55–75 min after light-on, when the pupae had been kept under conditions of 16 h light and 8 h dark during adult development. The hormone became detectable in the haemolymph 10–20 min after light-on, followed by eclosion 40 min later. The hormonal activity increased sharply to a maximal level (30 units/ml haemolymph) at 30 min prior to the eclosion and then fell rapidly: detectable eclosion hormone was present in the haemolymph for only 15–20 min. The hormone was partially purified from the haemolymph of eclosing animals and a 400-fold purification was achieved. The preparation was rather stable against heat treatment, and the molecular weight was estimated to be 5,000–10,000 by gel-filtration on a Sephadex G-50 (superfine). The origin of eclosion hormone in the haemolymph is discussed.     

Cerulenin resistance in a cerulenin-producing fungus. III. Studies on active-site peptides of fatty acid synthetase from Cephalosporium caerulens

Active-site peptides of acetyl transferase, condensing enzyme and acyl carrier protein in the neighborhood of the prosthetic group, 4'-phosphopantetheine, of Cephalosporium caerulens fatty acid synthetase were investigated. The enzyme was reacted with [14C]acetyl-CoA or [14C]iodoacetamide. 14C-Labeled enzyme was digested with pepsin, trypsin or both. 14C-Labeled peptides were isolated by several purification procedures. The amino acid sequence of the active site of condensing enzyme was determined to be Tyr-Gln-Val-Glu-Ser-Cys-Pro-Ile-Leu-Glu-Gly-Lys and that of acetyl transferase was Phe-Ser-Gly-Ala-Thr-Gly-His-Ser-Gln-Gly. The amino acid composition around the 4'-phosphopantetheine-carrying serine was determined to be Asx2, Thr, Ser, Glx3, Gly2, Ala, Ile, Leu3, and Lys. When these active-site peptides were compared with those of Saccharomyces cerevisiae synthetase, a high degree of homology was observed in the active-site peptides of the acetyl transferase and acyl carrier protein domains. However, that of the condensing enzyme domain gave lower homology. These findings may support the assumption that the low reactivity of cerulenin with C. caerulens synthetase is a consequence of the structure of the condensing enzyme domain.     

The mode of recognition of tumor antigens by noncytolytic-type antitumor T cells: Role of antigen-presenting cells and their surface class I and class II H-2 molecules

Summary The present study investigated the role of antigen-presenting cells (APC) in the activation of noncytolytic T cells against tumor antigens. The noncytolytic-type T cells exerted their antitumor effect by producing -interferon (IFN-) and by activating macrophages as the ultimate effectors. The production of IFN- by these noncytolytic T cells following the stimulation with tumor cells required the participation of Ia⁺ APC, since the depletion of APC from cultures of tumor-immunized spleen cells resulted in almost complete inhibition of the IFN- production. Both L3T4⁺ and Lyt-2⁺ subsets of T cells were capable of producing IFN-, and the requirement of APC for the production of IFN- was the case irrespective of whether noncytolytic T cells were of L3T4⁺ or Lyt-2⁺ phenotype. More importantly, it was demonstrated that the production of IFN- by L3T4⁺ and Lyt-2⁺ T cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to cultures. These results indicate that antitumor L3T4⁺ or Lyt-2⁺ noncytolytic T cells are activated for the IFN- production by recognizing tumor antigens in the context of self-class II or -class I H-2 molecules on APC.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan