Satoshi Mizuno

Irreversible inhibition of v-src tyrosine kinase activity by herbimycin A and its abrogation by sulfhydryl compounds.

Herbimycin A, an antibiotic which reverses Rous sarcoma virus transformation, inhibited irreversibly the auto- and trans-phosphorylation activities of p60v-src in in vitro immune complex kinase assays. The addition of a sulfhydryl compound such as dithiothreitol, 2-mercaptoethanol, glutathione (reduced form) or cysteine abolished the ability of herbimycin A to inactivate p60v-src kinase as well as the ability to reverse transformed cell morphology, whereas the addition of oxidized glutathione, cystine or methionine showed no effect. The sulfhydryl alkylating reagent N-ethylmaleimide also, although less effectively, inactivated p60v-src kinase activity in vitro. These results suggest the likelihood that sulfhydryl groups of p60v-src are involved in the inactivation of v-src tyrosine kinase activity by herbimycin A.

Induction of p27Kip1 degradation and anchorage independence by Ras through the MAP kinase signaling pathway

While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenic transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When rat normal fibroblasts were cultured in suspension without substrate attachment, the cell cycle arrested in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulated. Conditional expression of oncogenic Ras induced the G1-S transition of the cell cycle and significantly shortened the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevented the oncogenic Ras-induced degradation of p27Kip1. These results suggest that the loss of substrate attachment induces the cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. Furthermore, we have found that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2.

Inhibition of transforming activity of tyrosine kinase oncogenes by herbimycin A

We studied the effectiveness of herbimycin A, an inhibitor of the function of the temperature-sensitive src oncogene, to reverse the morphologies of chicken and mammalian cells transformed by various oncogenes. It was found that the antibiotic was effective against the cells transformed by tyrosine kinase oncogenes src, yes, fps, ros, abl, erbB, but did not reverse the transformed morphologies induced by oncogenes raf, ras, and myc. Moreover, decreases in phosphotyrosine content of the total cellular proteins and in 36K protein phosphorylation by herbimycin treatment supported the selective inhibition of the antibiotic on the transforming activity of tyrosine kinase oncogenes.

PURIFICATION AND PRIMARY AMINO ACID SEQUENCE OF A NOVEL NEUTROPHIL CHEMOTACTIC FACTOR LECT2

We purified a neutrophil chemotactic factor from a culture fluid of the PHA-activated human T-cell leukemia SKW-3 cells. The factor showed a 16-kDa basic protein by Tricin-SDS-polyacrylamide gel electorophoresis and analysis of amino acid composition. The primary amino acid sequence revealed that the chemotactic factor was significantly different from other known chemotactic factors, indicating a novel protein designated LECT2. The sequence revealed homology with the myb-induced myeloid protein-1 (Mim-1), which is expressed from gene in immature and normal granulocytes of chicken. Its biological function had not yet been identified. LECT2 and Mim-1 may be involved in the regulation of neutrophil functions in an as yet unidentified way.

Molecular cloning of human and bovine LECT2 having a neutrophil chemotactic activity and its specific expression in the liver.

We previously reported the purification and amino acid sequence of a novel neutrophil chemotactic protein termed LECT2 (leukocyte cell-derived chemotaxin 2). In this paper we report molecular cloning of human and bovine LECT2 cDNAs based on the amino acid sequence of the purified protein. The deduced amino acid sequence of human LECT2 (hLECT2) shows an 86% identity to bovine LECT2 (bLECT2). The deduced primary structures of LECT2 were highly homologous to the repeated units of Mim-1 protein (myb induced myeloid protein-1). The mim-1 gene is one of the known myb target genes and is specifically expressed in normal and transformed immature granulocytes in the chicken. Northern blot analysis of normal human tissues demonstrated that the hLECT2 gene is specifically expressed in the adult and fetal livers. In addition, several human hepatoma cell lines also expressed LECT2 mRNA, suggesting that hepatic cells in the liver produce LECT2 protein.

Macular Carotenoid Levels of Normal Subjects and Age-Related Maculopathy Patients in a Japanese Population

PURPOSE Macular carotenoid pigments composed of lutein and zeaxanthin are thought to affect the development of age-related maculopathy (ARM). Macular carotenoid levels were measured in normal Japanese subjects and Japanese patients with ARM. DESIGN Observational case-control series. PARTICIPANTS One hundred normal eyes of 100 normal subjects and 187 eyes of 97 patients with ARM; all were Japanese. The definitions of early ARM and late ARM (exudative age-related macular degeneration [AMD] and dry AMD) were used according to an accepted international classification system. METHODS Macular carotenoid levels were measured using resonance Raman spectroscopy. MAIN OUTCOME MEASURE Raman signal intensity generated from carbon-carbon double bond vibrations of lutein and zeaxanthin. RESULTS The mean (+/-standard deviation [SD]) macular carotenoid level in normal subjects was 1471+/-540 Raman counts. The macular carotenoid levels in normal subjects declined with age. The mean macular carotenoid level was 620+/-204 (+/-SD) in eyes with early ARM and 427+/-283 (+/-SD) in eyes with late ARM (equal to AMD). The macular carotenoid levels of early ARM and AMD were significantly lower than those in normal subjects older than 60 years (1100+/-340 [+/-SD]). No difference was revealed in carotenoid levels by the severity for ARM, type of AMD (exudative, atrophic, and disciform scar), or types of choroidal neovascularization (classic, minimally classic, occult, polypoidal choroidal vasculopathy), although small numbers in some groups weakened statistical power. Macular carotenoid levels were affected by the severity of macular disease in the opposing eye. The average for normal eyes where AMD was found in the opposite eye was significantly lower than that of normal eyes in the absence of AMD in the opposite eye (i.e., healthy volunteers older than 60 years). CONCLUSIONS Macular carotenoids decreased even in older healthy individuals. The ARM patients showed lower macular carotenoid levels than healthy people. Low macular carotenoid levels may be one of the risk factors of progression in ARM.

Hemin control of globin synthesis: Action of an inhibitor formed in the absence of hemin on the reticulocyte cell-free system and its reversal by a ribosomal factor

Abstract The inhibitor of globin synthesis formed by incubation of the ribosome-free supernatant fraction of rabbit reticulocytes may be partially purified by precipitation at pH 5. It is inactivated by incubation with trypsin and is therefore presumed to be a protein. Incubation of a cell-free incorporating system with inhibitor results in disaggregation of the polyribosomes, indicating a block at the site of initiation. There is a lag phase of about 2 min before any effect on the polyribosome profile becomes evident. This suggests either that this time is required for interaction between the inhibitor and a component of the hemoglobin-synthesizing system or that the pool of a component past the block must be depleted before the inhibition becomes evident. The inhibitor blocks the synthesis of the α- and β-chains of hemoglobin equally. It also inhibits the formation of non-hemoglobin proteins, although there is a 10-min delay before inhibition of synthesis of these proteins becomes evident. A factor isolated by KCl extraction of reticulocyte ribosomes and eluted with 0.15 M KCl from a DEAE-cellulose column can overcome the action of the inhibitor. This fraction is required for protein synthesis by a reconstituted system containing the KCl-extracted reticulocyte ribosomes.

Hemin control of globin synthesis: Effect of a translational repressor on Met-tRNAf binding to the small ribosomal subunit and its relation to the activity and availability of an initiation factor

Abstract The block in peptide chain initiation brought about by hemin deficiency of the rabbit reticulocyte lysate system is accompanied by a marked decrease in Met-tRNA f bound to the small ribosomal subunit. Under conditions of hemin supplementation, a similar reduction of Met-tRNA f binding is caused by the addition of the translational repressor which is formed by incubation of the ribosome-free supernatant fraction in the absence of hemin. Both types of inhibition can be reversed by an initiation factor obtained from reticulocyte ribosomes. The activity of a suboptimal concentration of initiation factor in promoting Met-tRNA f binding is incompletely inhibited by a large dose of repressor. In a partial system, where translation is blocked by cycloheximide, the repressor has no effect on Met-tRNA f binding promoted by added initiation factor. Cycloheximide increases the Met-tRNA f bound under conditions of hemin supplementation and also permits its slow accumulation under conditions of hemin deficiency. This is attributed to its action in blocking both translation and the resulting turnover of Met-tRNA f bound to the small ribosomal subunit. No change in the distribution of initiation factor between different ribosome particles was found under inhibited conditions. The results indicate that the hemin-controlled repressor has no direct effect on either the activity or the availability of the initiation factor.

Inhibition of DNA-dependent RNA polymerase reaction of Escherichia coll by an antimicrobial antibiotic, streptovaricin

Abstract 1. 1. The effect of an antimicrobial antibiotic, streptovaricin, was studied on the DNA-dependent RNA polymerase (EC 2.7.7.6) reaction of Escherichia coli. 2. 2. Streptovaricin inhibited the RNA polymerase reaction of E. coli; 2 μg/ml of streptovaricin showed 60 % inhibition of the reaction. However, when streptovaricin was added to the reaction mixture after a 2-min incubation, little inhibition was demonstrated. 3. 3. The inhibition by streptovaricin pertained to the type of non-competitive nature as to streptovaricin and DNA primer. The similar relation was obtained between streptovaricin and the substrate, CTP. 4. 4. No binding of streptovaricin to DNA was demonstrated by any assay methods employed. 5. 5. The effect of streptovaricin was studied on DNA-dependent RNA-synthetic reaction by RNA polymerase which was extracted from Ehrlich ascites carcinoma cells in a soluble state. Streptovaricin showed no inhibition of the reaction.

Inhibition of an initiation codon function by hemin deficiency and the hemin-controlled translational repressor in the reticulocyte cell-free system.

The trinucleotide codon, AUG, promotes the combination of reticulocyte 40S and 60S native ribosomal subunits as well as recombination of those derived by dissociation factor. This interaction is inhibited by hemin deficiency or the hemin-controlled repressor and results in the loss of methionine from ribosomal bound met-tRNAf. The locus of inhibition among the partial reactions of peptide chain initiation is between met-tRNAf and initiation codon binding to the 40S ribosomal subunit and peptide bond formation.