[Ribosome recycling factor (RRF): a factor which disassemble the post-termination complex and reduces translational error].
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Amino Acid Sequence↗
Biomedical subjects
A Kaji
Publications and source records attributed to A Kaji.
Two point mutations in the U3 region of the long terminal repeat convert a subgroup A transformation-defective rous sarcoma virus to a cytopathic virus.
To elucidate the mechanism of cytopathicity of the transformation-defective avian retrovirus tdPH2010, we examined the function of two point mutations we had previously found in the long terminal repeat U3 region of this virus. Our previous studies showed that the U3 region was responsible for the cytopathic effects. These mutations were a G-to-T mutation at position -126 from the transcription start site and a G-to-A mutation at -23. Site-directed mutagenesis was performed on a noncytopathic, wild-type virus BSU to alter the nucleotides at these two positions, one at a time, to those of tdPH2010. Cell growth assay using the altered viruses revealed that host cell growth was retarded only when both of these mutations were present. The two additional mutations previously found in the direct repeat 1-polypurine tract (DR1-PPT) region of tdPH2010 were present also in the noncytopathic strain tdPH2013. Site-directed mutagenesis confirmed that these two mutations had indeed no role in the cytopathic effect of tdPH2010. None of these mutations influenced virus production from the infected cells. We conclude that the cytopathic effects by tdPH2010 are ascribed to the two point mutations in the U3 region.
Animals↗
Disassembly of the post-termination complex and reduction of translational error by ribosome recycling factor (RRF)-A possible new target for antibacterial agents.
In this paper, we briefly review RRF (ribosome recycling factor, previously called ribosome releasing factor) (for recent reviews covering historical background see (1, 2)).
Amino Acid Sequence↗
Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.
Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.
Alleles↗
Effects of high-lipase pancreatin on fecal fat, neutral sterol, bile acid, and short-chain fatty acid excretion in patients with pancreatic insufficiency resulting from chronic pancreatitis.
CONCLUSIONS: Steatorrhea was almost completely stopped and malabsorption of neutral sterols and short-chain fatty acids was reduced by treatment of high-lipase pancreatin in Japanese patients with pancreatic insufficiency whose dietary fat consumption is low. METHODS: Fifteen patients with chronic pancreatitis complicated by steatorrhea who consumed an average of 48 g of dietary fats a day were selected as subjects and given 3 g of high-lipase pancreatin (lipase, 379,800 USP U/g), at each meal (total daily dose is 9 g) for a mean duration of 28.5 d. Fecal output and fecal fat neutral sterol, bile acid, and short-chain fatty acid excretion were determined before and after the course of pancreatin therapy. RESULTS: Pancreatin administration resulted in significant reductions (P < 0.01) in fecal output (from 243.2 to 149.1 g), excretion of fecal fat, (from 12.3 to 3.9 g), animal sterols (from 816.3 to 604.6 mg), and short-chain fatty acids (from 52.6 to 18.5 mM). In contrast, no marked changes were recorded in fecal excretion of beta-sitosterol (a plant sterol), bile acids, or the hydroxy fatty acid fraction. Fecal fat and short-chain fatty-acid excretion showed strong correlations with fecal output.
Adult↗
Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex.
RF3 was initially characterized as a factor that stimulates translational termination in an in vitro assay. The factor has a GTP binding site and shows sequence similarity to elongation factors EF-Tu and EF-G. Paradoxically, addition of GTP abolishes RF3 stimulation in the classical termination assay, using stop triplets. We here show GTP hydrolysis, which is only dependent on the simultaneous presence of RF3 and ribosomes. Applying a new termination assay, which uses a minimessenger RNA instead of separate triplets, we show that GTP in the presence of RF3 stimulates termination at rate-limiting concentrations of RF1. We show that RF3 can substitute for EF-G in RRF-dependent ribosome recycling reactions in vitro. This activity is GTP-dependent. In addition, excess RF3 and RRF in the presence of GTP caused release of nonhydrolyzed fmet-tRNA. This supports previous genetic experiments, showing that RF3 might be involved in ribosomal drop off of peptidyl-tRNA. In contrast to GTP involvement of the above reactions, stimulation of termination with RF2 by RF3 was independent of the presence of GTP. This is consistent with previous studies, indicating that RF3 enhances the affinity of RF2 for the termination complex without GTP hydrolysis. Based on our results, we propose a model of how RF3 might function in translational termination and ribosome recycling.
GTP Phosphohydrolase-Linked Elongation Factors↗
A single amino-acid substitution in gag p19 protein (MA) of Rous sarcoma virus suppresses virus production from infected cells.
Gag p19 protein (MA) of the transformation-defective Rous sarcoma virus mutant, tdPH2010, has a point mutation at nucleotide 376 (G to A) that results in an amino-acid change at residue 126 of p19 (Glu to Lys). This single amino-acid change is the cause of the aberrantly fast migration of this protein on SDS-polyacrylamide gels. To study the biological significance of the mutation, we introduced this mutation into a transformation-defective derivative of the molecularly cloned Rous sarcoma virus, SRA2, and examined its effect on virus replication. The virus possessing the mutation in its gag p19 gene had 50% slower replication as measured by the amount of reverse transcriptase as well as gag p27 protein (CA) in the culture media. Glu at position 126 appears to be important for efficient production of Rous sarcoma virus in vitro.
Amino Acid Substitution↗
Endovascular treatment of intractable oronasal bleeding associated with severe craniofacial injury.
BACKGROUND: Severe craniofacial injury may cause intractable oronasal bleeding, which is refractory to conventional treatments. This study will evaluate the efficacy of endovascular treatment for such oronasal bleeding. METHODS: Nine males between the ages of 19 and 62 years who had intractable oronasal bleeding resulting from severe craniofacial injuries received treatments of transarterial embolization using Gelfoam pledgets, polyvinyl alcohol particles, or platinum coils. We then reviewed their clinical and neuroradiologic characteristics retrospectively. RESULTS: In all but one case, angiography demonstrated bleeding points as extravasation. These bleeding points were multiple in seven cases. Except for bleeding from ethmoidal arteries, selective embolization was successful. In all cases, intractable oronasal bleeding was controlled. Patient survival was not directly related to oronasal bleeding, but rather was strongly correlated with associated brain injuries. CONCLUSION: Endovascular treatment is an acceptable treatment for intractable oronasal bleeding associated with severe craniofacial injuries when conventional treatments have failed.
Adult↗
Relationship between the structure and cytotoxic activity of asterriquinone, an antitumor metabolite of Aspergillus terreus, and its alkyl ether derivatives.
Asterriquinone (ARQ) is an antitumor metabolite of Aspergillus terreus IFO 6123. In this study we synthesized several ARQ alkyl ethers and studied their cytotoxic activity against mouse leukemia P388 cells. The dissociation constant was also similar among the ARQ monoalkyl ethers. There was a good correlation between the hydrophobicity of ARQ monoalkyl ethers estimated by high performance liquid chromatography and the intracellular content accumulated for 60 min. ARQ monoalkyl ethers were cytotoxic, but ARQ dimethyl ether was not, as previously reported. The cytotoxicity of the ARQ monoalkyl ether derivatives was increased with extension of the alkyl chain length. The cytotoxicity was closely correlated with the intracellular content. The strongest ARQ derivative, ARQ monohexyl ether (ARQHex), formed more DNA-interstrand cross-links in the cells than ARQ. After treatment of P388 cells with ARQ and ARQHex for 6 h, a nucleosomal ladder pattern, as well as the appearance of degraded DNA, observed by flow cytometry, indicated that these compounds caused cellular apoptosis. Moreover, ARQ and ARQHex accumulated in the cells at the G1 phase of the cell cycle. These results indicated that ARQ monoalkyl ethers increased cytotoxicity, according to their membrane permeability based on the hydrophobicity, and they caused apoptotic cell death, as did ARQ.
Animals↗
Studies on the cytotoxicity of asterriquinone derivatives.
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Animals↗
[A case of Salmonella subdural empyema developed in chronic subdural hematoma].
A case of Salmonella subdural empyema developed in chronic subdural hematoma (Infected Subdural Hematoma; ISH) was reported. A 64-year-old man had been in a nearby hospital due to myelodysplastic syndrome with cerebral infarction for two months. His condition there had been almost uneventful. But spike fever occurred and the patient became drowsy two days before his transfer to our medical center. His consciousness level deteriorated progressively and CT scan showed a right chronic subdural hematoma. He had had no history of head trauma in the previous two months. On admission to our center, his consciousness level was semicoma with anisocoria. An emergency operation was performed via a single burr hole initially. From the burr hole, old bloody fluid accompanied by yellowish pus was obtained. Thus so-called ISH was diagnosed and the craniotomy was carried out. Gram stain of the specimen revealed gram negative rods. Although an epileptic state developed after the operation, it was controlled by barbiturate coma therapy for 3 days, followed by phenytoin administration. Fever subsided gradually with antibiotics sensitive to the bacteria and his anisocoria disappeared on the 4th postoperative day. In this case, Salmonella enteritidis was detected from bacterial culture both of the specimen and of the arterial blood. Salmonella enteritidis might have been implanted on the capsule of the chronic subdural hematoma by bacteremia derived from immunological dysfunction due to myelodysplastic syndrome. In conclusion, the possibility of ISH should be considered in chronic subdural hematoma patients with immunological dysfunction.
Chronic Disease↗
Inhibition of HIV type 1 RNA dimerization by antisense DNA corresponding to the 17-nucleotide sequence downstream from the splice donor site of HIV type 1 RNA.
HIV-1 RNA dimerization involves at least two key regions, one located upstream from the splice donor (SD) site, and the other located downstream from the SD site. To determine the precise location and the mechanism of action of the downstream region, we constructed a model system using a synthetic HIV-1 RNA fragment (HXB2, 455-1146), which dimerized at relatively low salt concentrations (100 mM KCl, 1 mM MgCl2). We tested in this system antisense DNAs that are complementary to both the upstream and downstream regions of HIV-1 RNA for their possible inhibitory effects on dimerization. Antisense DNAs complementary to nucleotides 773-789 located downstream from the SD site effectively inhibited dimerization of HIV-1 RNA. These inhibitory antisense DNAs hybridized with the dimer form of HIV-1 RNA, and dissociated the dimer into monomers. However, antisense DNAs complementary to the region upstream from the SD site did not hybridize with the dimer, although they inhibited RNA dimerization and also dissociated the preformed dimer.
Base Sequence↗
Tetrahydronaphthalene lignan compounds as potent anti-HIV type 1 agents.
Anti-HIV-1 activity of tetrahydronaphthalene (THN) derivatives of lignan compounds was studied. THN derivatives prevented cell death caused by HIV-1 infection in MT-4 cells. They also inhibited giant cell formation by HIV-1 in Sup-T1 cells, and p24 production in HIV-1-infected H9 cells. The 50% effective concentration (ED50) of the most active compound, 1737 [5,6,7-trimethoxy-4-(3,4,5-trimethoxyphenyl)-1,3,3a,4,9,9a-hexahydron aphtho[2,3-c]thiophene], for inhibition of the cytopathic effects of HIV-1 infection ranged from 0.15 to 0.8 microM. The 50% cytotoxic concentration (CC50) of compound 1737 measured by the viability of MT-4 cells was 58 microM, indicating a selective index (CC50/ED50) of 70-400. Substitution of the phenyl ring with other structures markedly decreased cytotoxicity but did not affect the antiviral activity of the compounds. This resulted in compounds with a high selective index. One such compound was 1738 [7-methoxy5,6-methylenedioxy-4-(4-benzyloxy-3-methoxyphenyl)1,3,3a ,4,9,9a-hexahydronaphtho[2,3-c]thiophene], with a selective index higher than 770. The time-of-addition experiment indicated that these compounds acted at or near the reverse transcription step of the HIV-1 life cycle. THN derivatives inhibited HIV-1 reverse transcriptase (RT) in vitro at a concentration of 1 microM. Resistant viruses selected in the presence of THN derivatives showed some degree of cross-resistance to other nonnucleoside RT inhibitors, but not to the nucleoside RT inhibitor, AZT. THN derivatives failed to inhibit replication of pyridinone- and nevirapine-resistant HIV strains. However, compound 1737 inhibited replication of a TIBO-resistant strain more effectively than the wild-type HIV-1. Consistent with this result, compound 1737 also inhibited TIBO-resistant RT more effectively than the wild-type RT in vitro. These results suggested that THN derivatives interact with RT in a manner similar to but slightly different from that of other nonnucleoside HIV-1 RT inhibitors.
Anti-HIV Agents↗
Human lymphotoxin mutein lacks hypotensive activity but has higher in vivo antitumor activity than lymphotoxin or tumor necrosis factor.
A serious drawback of tumor necrosis factor alpha (TNF) as a clinical antitumor agent is that it also has hypotensive activity. To overcome this problem, derivatives of its sister cytokine lymphotoxin (TNF-beta or LT) were prepared. One of them, mutein 2 (Mut2) has a deletion of amino acids 1-7 but contains substituted amino acids, Met-Phe-Pro at positions 8-10 of the mature human LT. This mutein has no hypotensive activity at the maximum dose (10 mg/kg) tested on rats. In contrast, a much lower dose (1 mg/kg) of TNF and LT caused a significant blood pressure drop. In vivo studies revealed that Mut2 was more effective than TNF or LT against MethA (a mouse tumor line) as judged by the therapeutic ratio [calculated as LD50 (dose that kills 50% of the animals)/ED50 (dose that reduces the tumor size by 50%)]. With five other different mouse tumors and two different human tumors, Mut2 was also effective and the effectiveness was comparable or superior to that of TNF or LT. These results suggest the possibility that this derivative may be usable as a clinical antitumor agent without the serious side effects associated with TNF.
Animals↗
Can pancreatic steatorrhea be diagnosed without chemical analysis?
CONCLUSION: Visual observation of feces, considering fecal output, is considered to be an excellent method of detection of steatorrhea when judged by experienced doctors. METHODS: Feces from 192 patients with untreated chronic pancreatitis or under pancreatic enzyme therapy were investigated. Feces were collected for three consecutive days and homogenized with water. Fecal samples were freeze-dried and analyzed for fatty acids by gas chromatography (GLC). The quantity of fat was calculated from the amount of fatty acid to obtain daily fecal fat excretion. Comparison of GLC method with van de Kamer method gave a significant (p < 0.01) positive correlation with correlation coefficient of 0.916 (n = 38). Steatorrhea was defined as fecal fat excretion exceeding 5 g/d. Mild steatorrhea was defined as 5-10 g/d, and severe steatorrhea as more than 10 g/d. RESULTS: Three visual identification items were used to consider fecal output exceeding 200 g/d: fecal fat concentration exceeding 4%, appearance, and odor. The results were compared with the results from GLC method. Detection of steatorrhea by means of visual properties was the most accurate, and correlation coefficient was 0.843 (p < 0.01) by Spearman's rank correlation test. This detection method was also significantly effective for differentiation of normal stool from mild and severe steatorrhea. The sensitivity and specificity were 89.3 and 91.1%, respectively, indicating a favorable result.
Celiac Disease↗
The U3 region of the long terminal repeat of a subgroup A transformation-defective rous sarcoma virus (tdPH2010) converts a noncytopathic virus to a cytopathic virus.
The molecular basis of the cytopathic effects (CPE) of the transformation-defective avian retrovirus mutant, tdPH2010, was studied. tdPH2010 is a subgroup A virus isolated from the Schmidt-Ruppin (NY) subgroup A strain (SRA(NY)). Subgroup A avian retroviruses are generally considered noncytopathic. Integrated tdPH2010 was molecularly cloned from infected quail cells. A noncytopathic, transformation-defective control strain, BSU, was created by deleting the src gene from the molecularly cloned wild type SRA(NY) virus. Chimeras between tdPH2010 and BSU were constructed and viruses were recovered from transfected chick embryo fibroblasts. Growth curves of cells infected with chimeric viruses indicated that the long terminal repeat (LTR) of tdPH2010 converts BSU to a cytopathic virus. Nucleotide sequencing revealed two point mutations unique to tdPH2010 in the U3 region of LTR at positions -126 and -23 from the transcription start site. Both mutations were located inside or near the promoter/enhancer elements of U3. The mutation at -126 (G to T) converted one of the very well-conserved pentanucleotide repeat (PRE) motifs from GGTGG to GGTGT. The other at -23 (G to A) is located next to the TATA box. The G at this position is conserved in all other known avian retrovirus promoters.
Animals↗
Lysophosphatidylcholine stimulates leukocyte rolling and adherence in rat mesenteric microvasculature.
Synthetic exogenous lysophosphatidylcholine (LPC) was used to induce leukocyte-endothelial cell interaction, utilizing intravital microscopy in the rat mesenteric microvasculature. Superfusion of the rat mesentery with 10 microM LPC significantly and time dependently increased leukocyte rolling and adherence compared with control rats. Moreover, LPC superfusion did not alter systemic blood pressure or mesenteric venular shear rate. Administration of either an anti-P-selectin monoclonal antibody (PB1.3 at 1 mg/kg i.v.) or a nitric oxide (NO) donor (CAS-1609, 10 microM superfusion) markedly attenuated LPC-induced leukocyte rolling and adherence (P < 0.01 from LPC alone). Immunohistochemical localization of P-selectin and intercellular adhesion molecule-1 (ICAM-1) expression on mesenteric venules was significantly increased after exposure to LPC compared with mesenteries superfused with phosphatidylcholine (P < 0.001). Flow cytometric analysis indicated that 10 microM LPC significantly (P < 0.05) increased P-selectin fluorescence on rat platelets. Furthermore, direct measurement of NO release from rat aortic vascular endothelium was significantly (P < 0.05) inhibited by 10 microM LPC. Thus LPC induces in vivo leukocyte rolling and adherence in the mesenteric rat microvasculature by increasing the surface expression of P-selectin and ICAM-1. Because inhibition of endothelial NO release promotes P-selectin expression, LPC-induced rolling and adherence may be mediated via reduced NO release.
Animals↗
Blood purification for crush syndrome.
At least 372 people developed crush syndrome after they were injured by the Great Hanshin-Awaji Earthquake. Of these, 23 were transferred to Osaka City General Hospital from the disaster area. The serum creatinine kinase (CK) of each of the 23 patients exceeded 10,000 IU/L. Sixteen of these patients were treated with various methods of blood purification including hemodialysis (HD), plasma exchange (PE), and continuous hemodiafiltration (CHDF). The effectiveness on each method of blood purification was evaluated in this study based on the clearance of myoglobin and the length of time until recovery from acute renal failure (ARF). None of the patients died, and none suffered from ARF longer than 2 months. The length of time required for blood purification was significantly correlated with the serum CK and myoglobin levels on admission. The serum myoglobin levels decreased linearly regardless of the method of blood purification used. Our findings showed that the severity of ARF that occurred in association with crush injury was proportional to the amount of crushed muscle and that once ARF had developed, the clearance of myoglobin was not affected by any of the blood purification methods tested including HD, PE, and CHDF. Therefore, the method of blood purification employed for crush syndrome should be selected for its effectiveness in treating ARF, rather than the elimination of myoglobin.
Acute Kidney Injury↗