Application of laser tweezers to studies of the fences and tethers of the membrane skeleton that regulate the movements of plasma membrane proteins.
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Animals↗
Biomedical subjects
Y Sako
Publications and source records attributed to Y Sako.
Acquired thermotolerance and temperature-induced protein accumulation in the extremely thermophilic bacterium Rhodothermus obamensis.
Temperature-induced changes in thermotolerance and protein composition were examined in heat-shocked cells and high-temperature-grown cells of the extremely thermophilic bacterium Rhodothermus obamensis. The survival at temperatures superoptimal for growth (90 and 95 degrees C) was enhanced in both heat-shocked cells and high-temperature-grown cells relative to that of cells grown at optimal temperatures. In a comparison of protein composition using two-dimensional gel electrophoresis, putative heat shock proteins (HSPs) and high-temperature growth-specific proteins (HGPs) were detected. N-terminal amino acid sequence analysis revealed that the putative HSPs were quite similar to the ATP-binding subunits of ABC transporters and the HGPs were proteins corresponding to domains II and III of elongation factor Tu. These results suggested that this extreme thermophile has developed temperature-induced responses that include increased survival under hyperthermal conditions, changes in protein composition, and also the production of novel HSPs.
Amino Acid Sequence↗
Molecular characterization and postsplicing fate of three introns within the single rRNA operon of the hyperthermophilic archaeon Aeropyrum pernix K1.
The single rRNA operon (arnS-arnL) of the hyperthermophilic archaeon Aeropyrum pernix K1 was sequenced. The DNA sequence data and detailed RNA analyses disclosed an unusual feature: the presence of three introns at hitherto undescribed insertion positions within the rRNA genes. The 699-nucleotide (nt) intron Ialpha was located at position 908 (Escherichia coli numbering [H. F. Noller, Annu. Rev. Biochem. 53:119-162, 1984]) of the 16S rRNA, while the 202-nt intron Ibeta and 575-nt intron Igamma were located at positions 1085 and 1927 (E. coli numbering), respectively, of the 23S rRNA. They were located within highly conserved sites which have been implicated as crucial for rRNA function in E. coli. All three introns were remarkably AT rich (41.5 to 43.1 mol% G+C) compared with the mature rRNAs (67.7 and 69.2 mol% G+C for 16S and 23S rRNAs, respectively). No obvious primary sequence similarities were detected among them. After splicing from rRNA transcripts in vivo, a large quantity of intronic RNAs were stably retained in the linear monomeric form, whereas a trace of topoisomeric RNA molecules also appeared, as characterized by their behavior in two-dimensional gel electrophoresis. Secondary structural models of the Ialpha-, Ibeta-, and Igamma-containing rRNA precursors agree with the bulge-helix-bulge motif. Two of the introns, Ialpha and Igamma, contained open reading frames whose protein translation exhibited no overall similarity with proteins reported so far. However, both share a LAGLI-DADG motif characteristic of homing endonucleases.
Amino Acid Sequence↗
Cloning, sequence analysis, and expression in Escherichia coli of a gene coding for an enzyme from Bacillus circulans K-1 that degrades guar gum.
A 2,048-bp nucleotide sequence containing a gene coding for an enzyme that degraded guar gum from Bacillus circulans K-1 was identified by polymerase chain reaction walking. This G-gene consisted of 1,551 nucleotides coding for a protein with Mr 55,242. The enzyme was overexpressed in Escherichia coli JM109 cells by the cloning the G-gene downstream of the lac Z promoter of pUC19. The molecular mass of recombinant G-enzyme estimated by SDS-PAGE was 62 KDa, close to that from strain K-1. Analysis of the recombinant enzyme showed GalNAc, Xyl, GlcNAc, Man, Glc, and Gal to account for 1.7%, 14.4%, 6.1%, 3.2%, 54.2%, and 10.4%, respectively, of the total monosaccharides. Polyacrylamide gel electrophoresis of this enzyme with staining gave a red band. The results suggested that the sugars accounted for the differences in the molecular masses. The recombinant enzyme had two kinds of N-terminal sequences, Thr-Met-Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile and Ile-Thr-Pro-Ser-Phe-Ala-Ser-Gly-Phe-Tyr-Val-Ile-Gly-Thr. Comparison of these sequences with the deduced N-terminal sequence coded for the G-gene showed that the amino acid, first Met, of the lac Z gene or the next residues Thr-Met in the recombinant enzyme were absent in the native enzyme. Methionines near and at the N-terminus of the mature protein probably were digested by methionine aminopeptidases of E. coli after translation. The properties of recombinant G-enzyme were similar to those of the enzyme from K-1 cells.
Amino Acid Sequence↗
Identification of B cell epitopes of a 30 kDa Babesia equi merozoite surface protein.
A 30 kDa immunodominant surface antigen (p30) of Babesia equi has been used as a diagnostic antigen. The B cell epitopes on this molecule recognized by horse sera and monoclonal antibody (MAb) against p30, 36/133.97, were determined. A synthetic peptide of p30 with amino acid sequence of 123FYQEVLFKGFEAV135 exhibited strong positive reaction with the infected horse sera. In contrast, MAb 36/133.97 recognized different region of p30, as peptide synthesized with amino acid sequence of 27ASGAVVDFQLESI39 reacted strongly. In competitive inhibition ELISA, the binding of MAb 36/133.97 to recombinant p30 was inhibited by horse antibodies, although they did not recognize same or an overlapping epitope. The data on B cell epitopes in this study may be important in improving serodiagnostic methods of B. equi infection.
Amino Acid Sequence↗
Differentiation and quantification of Theileria sergenti piroplasm types using type-specific monoclonal antibodies.
Antigenic properties of two representative allelic products of the major piroplasm surface protein (MPSP) of Theileria sergenti were studied. Sera from cattle infected with either of Ikeda and Chitose types of the parasite reacted strongly with homologous but weakly with heterologous recombinant antigens in immunoblotting. Monoclonal antibodies (MoAbs) produced against the both allelic products of MPSP parasites reacted only to the immunizing antigen. These results suggested that crossreactivity between two allelic products is very low inspite of relatively high homology in their amino acid sequences. Double staining of parasitized erythrocyte smear using type-specific MoAbs by an indirect immunofluorescent assay revealed that the set of MoAbs was useful for quantitative and differential detection of each type of parasite in mixed population.
Animals↗
[Slowly progressive IDDM with rheumatoid arthritis and Hashimoto disease in high elderly].
We report a 79-year-old woman case of slowly progressive IDDM (SPIDDM) with rheumatoid arthritis (RA) and Hashimoto disease. High titer of anti-glutamic acid decarboxylase antibody (GAD) with a value of 16,400 U/ml (normal value: less than 5 U/ml) and deteriorated secretion of insulin, and clinical course led to the diagnosis of SPIDDM. Both anti-islet cell and anti-insulin antibodies were negative. One year prior to the diagnosis, at 78 years of age, she was newly diagnosed with NIDDM and had been medicated with sulfonylurea and voglibose, resulting her glucose levels well-controlled. Four months before admission, a gradual increase of plasma glucose was noticed, while oral hypoglycemic agents were fully administrated. On admission, her glycemic control was revealed as follows; a fasting blood glucose level of 458 mg/dl and an HbA1 C level of 14.3%. Urinary CPR was 22.5 micrograms day. Her insulin secretion was proved not to be induced with intravenous glucagon injection. Hyperinsulinemic euglycemic glucose clamp test showed the normal glucose uptake ratio; 9.5 mg/kg/min. Moderate doses of subcutaneous insulin (20 units daily) were effective on her diabetes control. She was newly diagnosed with Hashimoto disease that required thyroid hormone replacement 50 micrograms per day after having developed NIDDM. High titer of anti-thyroglobulin antibody (46.9 U/ml) and anti-thyroid peroxidase antibody (81.5 U/ml) were observed. The patient had been medicated for RA with anti-inflammatory drugs since her early seventieth. Rheumatoid factor was elevated to 127.7 IU/L and, anti-nuclear antibody (x 80) and anti-DNA antibody (x 80) were present. It may be of interest that a specific phenotype of HLA; A24 (9) and DR9 recognized to be susceptible to IDDM was detected in the high-elderly onset SPIDDM. Taken together HLA typing with her history of both RA and Hashimoto disease, our case may provide the information to the mechanism of pathogenesis of SPIDDM. Furthermore, to out knowledge, this is the first case of SPIDDM in the aged; 75-year-old or more.
Aged↗
[Multi-photon excitation fluorescence microscopy and its application to the studies of intracellular signal transduction].
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Animals↗
An extremely heat-stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1.
An extracellular metalloproteinase, which we had designated aeropyrolysin, from the aerobic marine hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820), was purified by ammonium sulfate precipitation, anionic exchange chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 52 kDa as determined by SDS-PAGE. The proteinase had a broad pH optimum (pH 5-9) with a maximal activity at pH 6-8 for azocasein hydrolysis. The optimum temperature for enzyme activity was 100 degrees C in the absence of 1 mM CaCl2 and 110 degrees C in the presence of 1 mM CaCl2. The enzyme activity was completely inhibited by EDTA and EGTA, indicating that it was a metalloproteinase. The enzyme was highly resistant to the denaturing reagents urea, guanidine-HCl, dithiothreitol, 2-mercaptoethanol and SDS. The enzyme also showed a high activity with the metalloproteinase specific substrate MOCAc-Pro-Leu-Gly-Leu-A2pr(Dnp)-Ala-Arg-NH2. The enzyme was extremely thermostable showing half-lives of 2.5 h at 120 degrees C and 1.2 h at 125 degrees C in the presence of 1 mM CaCl2. These results indicate that this enzyme is one of the most thermostable extracellular proteinases reported to date.
Caseins↗
Control of Theileria sergenti infection by vaccination.
Bovine piroplasmosis caused by Theileria sergenti is a major cause of economic loss in grazing cattle in Japan. Infected calves show chronic anaemia with intraerythrocytic piroplasms and occasionally die in severe cases. We found that parasite stocks and isolates consist of genetically and antigenically mixed populations. To differentiate parasite populations bearing 3 allelic forms of p32/34, an immunodominant piroplasm surface protein, 3 sets of oligonucleotide primers were designed to amplify either of 3 alleles by polymerase chain reaction (PCR). By using this allele-specific PCR, we found that the majority of T. sergenti-infected calves in Japan harbored mixed parasite populations bearing C and I type parasites. To control Theileria infection, we produced 2 vaccine candidates: recombinant baculovirus p32 and synthetic peptide containing Lys-Glu-Lys (KEK) motif. Immunization with either recombinant p32 or synthetic peptide containing KEK sequences with Freund's complete adjuvant resulted in low parasitemia and reduced the clinical symptoms compared to control calves. Interestingly, the parasite with the p32 allelic form corresponding to the one used as the immunogen was suppressed.
Amino Acid Sequence↗
Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1.
Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6-17, which enhances the contrast by a factor of 6-21: (2) a lower rate of photobleaching by a factor of 2-4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations. probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.6-phi pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-phi pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.
Animals↗
Extremely thermostable phosphoenolpyruvate carboxylase from an extreme thermophile, Rhodothermus obamensis.
Phosphoenolpyruvate carboxylase (PEPC) was purified from an extremely thermophilic bacterium, Rhodothermus obamensis, growing optimally at 80 degrees C, which had recently been isolated from a shallow marine hydrothermal vent in Japan. The native enzyme was a homotetramer of 400 kDa in molecular mass, as estimated by gel filtration chromatography, and the subunit exhibited an apparent molecular mass of 100 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum temperature for enzyme activity was 75 degrees C. The enzyme exhibited an absolute requirement for divalent cations and a pH optimum of 8.0. The enzyme was extremely thermostable and there was no loss of enzyme activity on incubation for 2 h at 85 degrees C. The enzyme exhibited a positive allosteric property with acetyl-CoA and fructose 1,6-bisphosphate, and a negative one with L-aspartate and L-malate. These effectors affected not only the thermophilicity but also the thermostability of the enzyme, and the substrate, co-factors, and salts increased the thermostability as well. The extrinsic thermostabilization might be a possible mechanism for adaptation of the enzyme to high temperature.
Acetyl Coenzyme A↗
Purification, properties, and N-terminal amino acid sequences of guar gum-degrading enzyme from Bacillus circulans K-1.
A guar gum-degrading enzyme of the newly isolated Bacillus circulans K-1 was purified to an electrophoretically homogeneous state. The molecular weight of the purified enzyme was 62,000 by SDS-PAGE. The purified enzyme was separated into a least six isozymes by isoelectric focusing and the pI of these isozymes were 5.4, 5.5, 5.6, 5.8, 6.0, and 6.2, respectively. The N-terminal amino acid sequences of the typical three of these proteins were all the same, Ala-Ser-Gly-Phe-Tyr-Val-Ser-Gly-Thr-Lys-Leu-Asp-Ala-Thr-Gly-Gln-Pro-Phe- Val- Met-Arg. The enzyme was most active at pH 6.9 and at 64 degrees C. The enzyme was activated slightly by Al3+ and inhibited strongly by Sn2+ and Zn2+, N-bromosuccinimide, 2-mercaptoethanol, and ethylenediamine-tetraacetic acid.
Amino Acid Sequence↗
[Two-photon excitation fluorescence microscopy].
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Microscopy, Fluorescence↗
Vaccine development against Theileria parasite.
Bovine piroplasmosis caused by Theileria sergenti is a major cause of economical loss in grazing cattle in Japan. We found that parasite stocks and isolates consist of genetically and antigenically mixed population. To differentiate parasite populations bearing 2 allelic forms of p32, an immunodominant piroplasm surface protein, 2 sets of oligonucleotide primers were designed to amplify either of the 2 alleles by polymerase chain reaction (PCR). By using this allele-specific PCR, we found that the majority of T. sergenti-infected calves in Japan harbored mixed parasite populations with C and I type parasites. Amino acid sequence of p32 contains Lys-Glu-Lys (KEK) motif which is one of tripeptide necessary for malaria parasite to invade erythrocytes. We produced 2 vaccine candidates, recombinant baculovirus p32 and synthetic peptide containing KEK motif. Immunization of either recombinant p32 or synthetic peptide containing a KEK sequence with adjuvant resulted in low parasitemia and reduced the clinical symptoms compared to control calves. Interestingly, parasites with a p32 allelic form corresponding to one used as the immunogen were suppressed. Therefore, a cocktail vaccine containing KEK peptides derived from C and I type parasites is desired for control Theileria parasite infection in Japan.
Amino Acid Sequence↗
Purification and characterization of phosphoenolpyruvate carboxylase from the hyperthermophilic archaeon Methanothermus sociabilis.
Phosphoenolpyruvate carboxylase (PEPC) was purified for the first time from hyperthermophilic archaeon Methanothermus sociabilis, growing autotrophically with an optimum at 88 degrees C. The optimum temperature for enzyme activity was similar to that for growth and was 85 degrees C. The native enzyme was a homotetramer of 240 kDa molecular mass and the subunit displayed an apparent molecular mass of 60 kDa. The archaeal PEPC was insensitive to various metabolites which are known as allosteric effectors for most bacterial and eucaryal counterparts. The enzyme showed extreme thermostability such that there remained 80% of the enzyme activity after incubation for 2 h at 80 degrees C. These results implied that archaeal PEPC was significantly different from bacterial and eucaryal entities.
Archaea↗
Cell surface organization by the membrane skeleton.
Single-particle tracking and laser tweezers have facilitated the observation of the mechanics of molecular interactions in the plasma membrane of living cells at the level of single (or a few) molecules at nanometer/piconewton precision. These techniques have recently revealed that the membrane skeleton provides both confining and binding effects on the movement of membrane proteins, and that it can play a pivotal role in the molecular organization of the plasma membrane, especially in the formation of special membrane domains.
Biomechanical Phenomena↗
Aeropyrum pernix gen. nov., sp. nov., a novel aerobic hyperthermophilic archaeon growing at temperatures up to 100 degrees C.
A novel aerobic hyperthermophilic archaeon was isolated from a coastal solfataric vent at Kodakara-Jima Island, Japan. The new isolate, strain K1, is the first strictly aerobic organism growing at temperatures up to 100 degrees C. It grows optimally at 90 to 95 degrees C, pH 7.0, and a salinity of 3.5%. The cells are spherical shaped and 0.8 to 1.2 microns in diameter. Various proteinaceous complex compounds served as substrates during aerobic growth. Thiosulfate stimulates growth without producing H2S. The core lipids consist solely of C25-isopranyl archaeol (glycerol diether). The G + C content of the genomic DNA is 67 mol%. Phylogenetic analysis based on 16S rRNA sequence indicates that strain K1 is a new member of Crenarchaeota. On the basis of our results, the name Aeropyrum pernix gen. nov., sp. nov. is proposed (type strain: K1; JCM 9820).
Archaea↗