Biomedical subjects
E Hashimoto
Publications and source records attributed to E Hashimoto.
Organization and nature of fortimicin A (astromicin) biosynthetic genes studied using a cosmid library of Micromonospora olivasterospora DNA.
The cloning of five DNA segments carrying at least seven genes (fms1, fms3, fms4, fms5, fms7, fms11, and fms12) that participate in fortimicin A (astromicin) biosynthesis was described previously. These DNA fragments were used to screen a cosmid library of genomic DNA in order to examine if these biosynthetic genes are clustered in Micromonospora olivasterospora. One cosmid clone (pGLM990) was obtained, which hybridized to all the probes. Complementation analysis, using mutants blocked at various steps and chimeric plasmids subcloned from pGLM990, showed that three additional genes (fms8, fms10, and fms13) are present in pGLM990. A gene conferring self-resistance to the antibiotic, which was independently cloned in Streptomyces lividans, using the plasmid vector pIJ702 was also found to be linked to the cluster of biosynthetic genes. Thus, at least ten biosynthetic genes and a self-defense gene are clustered in a chromosomal region of about 27 kb in M. olivasterospora. Interestingly, the fms8 gene which participates in the dehydroxylation step of fortimicin A biosynthesis was found to have homology with a neomycin resistance gene nmrA from the neomycin-producing Micromonospora sp. MK50. Studies using a cell-free extract of the fms8 mutant and its parent strain showed that the enzyme encoded by fms8 phosphorylates a biosynthetic precursor, fortimicin KK1, in the presence of ATP. Thus the dehydroxylation reaction is suggested to occur via the phosphorylation of the target hydroxyl group. DNA regions homologous to fms genes were found in Micromonospora sp. SF-2098 and Dactylosporangium matsuzakiense, both producers of fortimicin group antibiotics.
Self cloning in Micromonospora olivasterospora of fms genes for fortimicin A (astromicin) biosynthesis.
We have cloned the seven genes that are responsible for biosynthesis of the antibiotic fortimicin A (FTM A) using a recently developed self-cloning system that employes the plasmid vector pMO116 for Micromonospora olivasterospora. Five chimeric plasmids that restored FTM A production in M. olivasterospora mutants blocked at different biosynthetic steps were isolated by shotgun cloning. Secondary transformation using other non-producing mutants showed that two additional FTM A biosynthetic genes were included on these plasmids, and that at least four of the genes were clustered. Interestingly AN38-1, a non-producing mutant that had a defect in dehydroxylation of a precursor of FTM A, was complemented by the DNA fragment containing a neomycin resistance gene that had been cloned from a neomycin-producing strain (Micromonospora sp. FTM A non-producing strain) in the course of constructing the plasmid vector pMO116. These results clearly show that this novel gene cloning system in Micromonospora is of practical use.
Phosphorylation of histone H2A by protein kinase C and identification of the phosphorylation site.
In regenerating rat liver, nuclear protein histone H2A was shown to be phosphorylated on its amino-terminal serine residue [Sung et al. (1971) J. Biol. Chem. 246, 1358-1364], but the protein kinase which phosphorylates this residue has not been identified. To evaluate the possibility that protein kinase C can phosphorylate this residue, calf thymus histone H2A was 32P-labeled by incubation with [gamma-32P]ATP and highly purified protein kinase C from rat brain in the presence of calcium and phospholipid. About 1 mol of 32P was incorporated per mol of histone H2A and the Km and apparent Vmax of the reaction were calculated to be 2.1 microM and 0.35 mumol/min/mg, respectively. So histone H2A seemed to be a good substrate for protein kinase C. Further, the proteolytic phosphopeptides of 32P-labeled histone H2A were isolated by means of a series of column chromatographies and analyzed for their amino acid compositions. Comparison of the data with the known primary structure of histone H2A revealed their amino acid sequence as 1Ser-Gly-Arg. These data suggest that protein kinase C may be a candidate for the protein kinase which phosphorylates the amino-terminal serine residue of histone H2A during the regeneration of rat liver.
Inhibitory effect of mitoxantrone on activity of protein kinase C and growth of HL60 cells.
Mitoxantrone, a new anthraquinone, showed inhibitory an effect on protein kinase C (PKC) activity. Its IC50 value was 4.4 micrograms/ml (8.5 microM), which is much lower than those of the well-known anthracyclines daunorubicin and doxorubicin, the IC50 values of which are more than 100 micrograms/ml (> 170 microM). Kinetic studies demonstrated that mitoxantrone inhibited PKC in a competitive manner with respect to histone H1, and its Ki value was 6.3 microM (Ki values of daunorubicin and doxorubicin were 0.89 and 0.15 mM, respectively), and in a non-competitive manner with respect to phosphatidylserine and ATP. Inhibition of phosphorylation by mitoxantrone was observed with various substrates including S6 peptide, myelin basic protein and its peptide substrate derived from the amino-terminal region. Their IC50 values were 0.49 microgram/ml (0.95 microM), 1.8 micrograms/ml (3.5 microM), and 0.82 microgram/ml (1.6 microM), respectively. Mitoxantrone did not markedly inhibit the activity of cyclic AMP-dependent protein kinase, casein kinase I or casein kinase II, at concentrations of less than 10 micrograms/ml. On the other hand, brief exposure (5 min) of HL60 cells to mitoxantrone caused the inhibition of cell growth with an IC50 value of 52 ng/ml (0.1 microM). In HL60 cells, most of the PKC activity (about 90%) was detected in the cytosolic fraction. When HL60 cells exposed to 10 micrograms/ml mitoxantrone for 5 min were observed with fluorescence microscopy, the fluorescence elicited from mitoxantrone was detected in the extranuclear area. These results indicated that mitoxantrone is a potent inhibitor of PKC, and this inhibition may be one of the mechanisms of antitumor activity of mitoxantrone.
Cloning and analysis of a gene (sms13) encoding sannamycin B-glycyltransferase from Streptomyces sannanensis and its distribution among actinomycetes.
A gene encoding sannamycin B-glycyltransferase (sms13) of Streptomyces sannanensis IFO 14239 was identified by cloning and complementation of S. sannanensis mutant SN13 which is blocked at the interconversion of sannamycins B and A. The cloned DNA fragment also permitted the conversion of fortimicin B to A both in S. sannanensis SN13 and Streptomyces lividans TK23. DNA sequences similar to sms13 were detected in all five producers of the fortimicin-group antibiotics, Micromonospora olivasterospora ATCC 21819 (fortimicin-producer), Micromonospora sp. strain SF-2098 ATCC 31580 (SF-2052), Dactylosporangium matsuzakiense ATCC 31570 (dactimicin), Streptomyces tenjimariensis ATCC 31603 (istamycin), and Saccharopolyspora hirsuta ATCC 20501 (sporaricin). This suggests that these genes of similar function from different genera were derived from a common ancestral gene.
Characterization of sannamycin A-nonproducing mutants of Streptomyces sannanensis.
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[Platelet adenylate cyclase activity in alcoholics].
Platelet adenylate cyclase (AC) activity in the long-term abstinent alcoholics, who maintained abstinence from alcohol for at least one year was examined. Guanine nucleotide [Gpp(NH)p] stimulated AC activity was not changed in platelet membrane from long-term abstinent alcoholics. However, the extent of AC activity induced by 250 mM ethanol in vitro, in the presence of 1 microM Gpp(NH)p, was significantly reduced in platelet membrane from long-term abstinent alcoholics. These results suggest that platelet AC activity may be a biological marker for alcoholics.
[Pattern analysis of protein electrophoresis data].
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[Clinical usefulness of measurement of creatine contents in human erythrocytes as an index of erythropoiesis].
The apparent half-life of erythrocytes in human blood have usually been measured using 51Cr or 32P-labeled erythrocytes. However, these methods are not suitable for routine methods for the determination of the half-life of erythrocytes because of requirement for both patient admission and complicated procedure. In 1967 Griffiths and Fitzpatrick suggested that creatine level in human packed erythrocytes reflected the mean-age of erythrocyte population. Fehr and Knob reported that erythrocyte creatine levels correlated closely with half-life of erythrocytes by means of 51Cr in severe and milder hemolytic anemias. In order to verify the clinical usefulness of measurement of creatine contents in human erythrocytes as an index of erythropoiesis, the number of reticulocytes counted by an automated reticulocyte counter (R-3000, Toa medical instruments) and creatine contents in erythrocytes measured by high performance liquid chromatography were compared. To evaluate the erythropoietic dynamics, we tried to determine the frequency distribution for individual erythrocyte cell age using erythrocyte creatine. The effect of erythrocyte life-span on hemoglobin A1c(HbA1c) was also appraised in normal blood sugar subjects. A significant increase (p less than 0.005) of erythrocyte creatine was found both in inactive autoimmune hemolytic anemia (AIHA) and in active AIHA. In severe AIHA, the frequency distribution of erythrocyte creatine shifted to the area of high level erythrocyte creatine, and showed a broad-pattern form corresponding to erythropoiesis. Negative correlation (r = -0.707, p less than 0.005) was revealed between erythrocyte creatine and HbA1c in normal blood sugar subjects.(ABSTRACT TRUNCATED AT 250 WORDS)
[A study on creatine uptake into human erythrocytes: relation to erythrocyte aging].
In order to assess the relation between creatine uptake into human erythrocytes and erythrocyte aging, actual influx of creatine into erythrocytes and creatine contents in erythrocytes were measured by using method of high performance liquid chromatography and isotopic estimation of 14C-creatine concentration. Actual influx of creatine into erythrocytes was showed rapidly, in spite of constant contents of erythrocyte creatine under the condition to be incubated at 37 degrees C for approximate 4 hours in isotonic saline containing high concentration of creatine. Km values of the creatine uptake into young erythrocytes were evidently smaller than that into old erythrocytes. On the other hand, no significant difference of Vmax values was observed to be dependent on erythrocyte aging.
Identification of the phosphorylation sites of H2B histone by a catalytic fragment of p72syk from porcine spleen.
Phosphorylated sites of calf thymus H2B histone were investigated with a catalytic fragment of 72 kDa protein-tyrosine kinase (p72syk). Three of five tyrosine residues in H2B histone can be phosphorylated by this kinase. In this analysis, H2B histone was thoroughly phosphorylated in vitro with [gamma-32P]ATP and the kinase, and then digested with a lysylendopeptidase. The resulting radioactive phosphopeptides were separated by a reverse-phase column on high performance liquid chromatography. Subsequent sequential Edman degradation of the purified phosphopeptides revealed that 40Y, 83Y and 121Y were phosphorylated. 121Y is the major phosphorylated residue in H2B histone. No phosphorylation was detected in 37Y and 42Y. Although the consensus sequence was not defined from these analyses, our data suggest that higher-order structure(s) in addition to primary one may participate in recognition of H2B histone by this protein kinase.
Studies on protein kinase C tightly-bound to rat liver plasma membrane and its protease-activated form.
1. Rat liver plasma membrane contained two types of protein kinase C which could be extracted by Ca2(+)-chelator and detergent, respectively. The activities of these two enzymes were nearly equivalent. 2. The detergent-extracted protein kinase C, tightly-bound to membrane, was separated into two subtypes by hydroxyapatite column chromatography. Based on the elution profile and the Ca2+/phospholipid requirement, the major and the minor components were identified as type III and type II protein kinase C, respectively. 3. The detergent-extracted protein kinase C was converted to an active fragment with Mr 45,000 by limited proteolysis with trypsin. Incubation under physiological level of ionic strength increased the stability of this active enzyme and protected it from further inactivation by trypsin. 4. Phosphorylation of H1 histone by the protease-activated kinase was stimulated 1.5-2-fold by phosphatidylserine. However, this enzyme phosphorylated multiple proteins in rat liver subcellular fractions in Ca2(+)- and phospholipid-independent manner. 5. These results suggest that the protein kinase C (mainly type III enzyme) tightly-bound to rat liver plasma membrane may have important role through protein phosphorylation by the native or the protease-activated kinase.
A novel, highly efficient gene-cloning system for Micromonospora strains.
A highly efficient gene-cloning system for Micromonospora olivasterospora, a producer of the antibiotic fortimicin A (astromicin), suited to shotgun cloning has been developed. The system is supported by two new advancements accomplished in this study. One is the construction of novel plasmid vectors pMO116, pMO126, pMO133, pMO136, and pMO217, all consisting of replicons from newly found Micromonospora plasmids and selectable markers cloned from a neomycin-producing Micromonospora strain. The other advancement is the establishment of a new protocol for bacterial protoplasting in which some kinds of sugar alcohols are added in precultures. Such sugar alcohols were found to sensitize a wide taxonomical range of bacteria to lysozyme. The system is reproducible and reliable and has a high efficiency of more than 10(6) CFU/micrograms of DNA.
[Systematization of clinical laboratory--some problems caused from specimen transport through laboratory reports].
Through error and trials we have designed an ideal system for the clinical laboratory. Truly, it has been a very difficult task requiring our long-term experience working in the clinical laboratory. For example, if we purchase new examination equipment without any consideration or if we decide what type of equipment to introduce according to the common advice of the purchase committee of the hospital or the medical school, then we cannot design an ideal system of laboratory examinations and are forced to invest a large sum of money in vain. Moreover, the use of innumerable examination containers or test tubes which are disposable, can become a financial burden to the hospital. We have been trying to design a system of laboratory automation for more than ten years and have been successful in designing not only a specimen transport system using conveyer-belts but also various kinds of examination robotic systems. This report describes our own examples of designing a system of laboratory automation.
[A follow-up study of pupillary dynamics in patients with ocular hypertension and primary open angle glaucoma].
The authors previously reported light reflex abnormalities and adrenergic supersensitivity to topical epinephrine (DPE; dipivalyl epinephrine) of the pupils in patients with ocular hypertension (OH) and those with primary open angle glaucoma (POAG). In this investigation, we attempted to reconfirm pupillary light reflex abnormalities we reported previously, and to investigate the relationship between the normotensive effect of 0.1% DPE and the pupillary light reflex abnormalities in OHs and POAGs. A total of 11 OHs and 11 POAGs under good oculotensive control with neither mydriatics nor miotics were examined. They were measured by an open-loop photically stimulated infrared videopupilogram, and were neurologically diagnosed by comparing the simulated patterns of the light reflex made by topical autonomic agents. When we considered the progress of the stage of POAG, both OHs and POAGs showed satisfactory reproducibility of pupillary light reflex abnormalities. Other cases in which light reflexes altered, showed worsening of the visual field deficit, which was prominent when OHs developed into POAG. OHs with significant reduction of intraocular pressure (IOP) after topical administration of 0.1% DPE showed various kinds of abnormal pupillary light reflexes. On the contrary, cases with little reduction of IOP after 0.1% DPE instillation showed normal pupillary light reflexes in OHs and afferent pupillary defect on POAGs.
Protease-activated protein kinase C in rat liver.
In regenerating rat liver, an elevated protein kinase activity was detected which phosphorylated ribosomal protein S6 and histones. The properties of this enzyme were closely similar with those of protease-activated protein kinase C with Mr 45,000. During the study of the mechanism of proteolytic activation, type III protein kinase C (encoding alpha-sequence) was shown to be subjected to limited proteolysis by trypsin-like protease and converted to protein kinase M in ionic strength- and pH-dependent manner. This reaction was stimulated in the presence of Ca2+ and phospholipid under slightly higher ionic strength condition than physiological level (greater than 140 mM NaCl) and alkaline pH (7.5-8.0). These results suggest that activation of Na+/H+ exchanger in plasma membrane may trigger this type of proteolytic activation of protein kinase C. In addition to protein kinase M, another type of protease-activated kinase with Mr 80,000 was detected when limited proteolysis of protein kinase C was performed on inactive form of this enzyme (in the absence of either Ca2+ or phospholipid or both activators) under lower ionic strength condition. The molecular mass of this active enzyme was slightly smaller (approximately 200) than that of native protein kinase C. However, it is not clear at this time whether this small fragment was released from amino-terminal or carboxy-terminal domain to make protein kinase C partially active in the absence of Ca2+ and phospholipid. Although it has been proposed that proteolytic degradation of protein kinase C is involved in down regulation of this enzyme, the physiological significance of these two types of protease-activated forms of protein kinases in liver has remained obscure.
Comparison of substrate recognition by protein kinase C (type III) between rat liver cytosolic and particulate fractions.
1. Phosphorylation of rat liver endogenous substrates by protein kinase C (type III) was compared between cytosolic and particulate (mitochondria, microsomes and plasma membrane) fractions. 2. The rate and the maximum level of protein phosphorylation were several-fold higher in particulate fractions than in cytosolic fraction. 3. Protein phosphorylation in cytosolic fraction was dependent on both Ca2+ and phospholipid, but only Ca2+ was necessary in phosphorylation of particulate fractions. 4. These results suggest that protein kinase C (type III) has much more target proteins in particulate fractions rather than in cytosolic fraction and Ca2+ was important regulator in particulate protein phosphorylation.