Biomedical subjects

Y Sako

Publications and source records attributed to Y Sako.

At least 37 records · Page 2Linked to original sources

PubMed2000

Mitochondrial genetic code in cestodes.

The flatworm mitochondrial genetic code, which has been used for all species of the Platyhelminthes, is mainly characterized by AUA codon for isoleucine, AAA codon for asparagine and UAA codon for tyrosine. In eight species of cestodes (Echinococcus multilocularis, Echinococcus granlosus, Taenia solium Taenia saginata, Taenia hydatigena, Taenia crassiceps, Hymenolepis nama and Mesocestoides corti), the cytochrome c oxidase subunit I (COI) genes were partially sequenced to verify this genetic code. Comparison of the COI-encoding nucleotide sequences with those of human, sea urchin, fruit fly, nematode and yeast indicated that the assignments of AUA and AAA codons are adequate for cestodes. In addition, the nucleotide sequences of ATPase subunit 6 (ATP6) gene and its flanking region were compared to examine initiation and stop codons. In the related species of T. solium and T. saginata, the deduced amino acid sequences of ATP6 were homogeneous; however, the conversion of initiation codon AUG into GUG was observed in T. saginata. We also found the similar conversion in T. crassiceps. The C-terminal sequences of putative ATP6 proteins were highly conserved among the eight species and the stop codon UAG was altered to UAA in all Taenia species. The features of the gene-junctional region between NADH dehydrogenase subunit 4 (ND4) and glutamine tRNA (tRNAGln) genes also supported that UAA serves as a stop codon. Based on these results, we propose that the flatworm mitochondrial code should be modified for cestodes, particularly, in an initiating methionine codon (GUG) and a terminating codon (UAA).

Animals↗

PubMed2000

Single-molecule imaging of EGFR signalling on the surface of living cells.

The early events in signal transduction from the epidermal growth factor (EGF) receptor (EGFR) are dimerization and autophosphorylation of the receptor, induced by binding of EGF. Here we observe these events in living cells by visualizing single molecules of fluorescent-dye-labelled EGF in the plasma membrane of A431 carcinoma cells. Single-molecule tracking reveals that the predominant mechanism of dimerization involves the formation of a cell-surface complex of one EGF molecule and an EGFR dimer, followed by the direct arrest of a second EGF molecule, indicating that the EGFR dimers were probably preformed before the binding of the second EGF molecule. Single-molecule fluorescence-resonance energy transfer shows that EGF-EGFR complexes indeed form dimers at the molecular level. Use of a monoclonal antibody specific to the phosphorylated (activated) EGFR reveals that the EGFR becomes phosphorylated after dimerization.

Antibodies, Monoclonal↗

PubMed2000

Novel prenyltransferase gene encoding farnesylgeranyl diphosphate synthase from a hyperthermophilic archaeon, Aeropyrum pernix. Molecularevolution with alteration in product specificity.

Prenyltransferases catalyse sequential condensations of isopentenyl diphosphate with allylic diphosphates. Previously, we reported the presence of farnesylgeranyl diphosphate (FGPP) synthase activity synthesizing C25 isoprenyl diphosphate in Natronobacterium pharaonis which is a haloalkaliphilic archaeon having C20-C25 diether lipids in addition to C20-C20 diether lipids commonly occurring in archaea [Tachibana, A. (1994) FEBS Lett. 341, 291-294]. Recently, it was found that a newly isolated aerobic hyperthermophilic archaeon, Aeropyrum pernix, had only C25-C25 diether lipids, not the usual C20-containing lipids [Morii, H., Yagi, H., Akutsu, H., Nomura, N., Sako, Y. & Koga, Y. (1999) Biochim. Biophys. Acta 1436, 426-436]. In this report, we describe the isoloation from A. pernix of the novel prenyltransferase gene, fgs, encoding FGPP synthase. The protein encoded by fgs was expressed in Escherichia coli as a glutathione S-transferase fusion protein and produced FGPP as a final product. Phylogenetic analysis of fgs with other prenyltransferases revealed that the short-chain prenyltransferase family is divided into three subfamilies: bacterial subfamily I, eukaryotic subfamily II, and archaeal subfamily III. fgs is clearly contained within the archaeal geranylgeranyl diphosphate (GGPP) synthase group (subfamily III), suggesting that FGPP synthase evolved from an archaeal GGPP synthase with an alteration in product specificity.

Alkyl and Aryl Transferases↗

PubMed2000

Reconstitution of brefeldin A-induced golgi tubulation and fusion with the endoplasmic reticulum in semi-intact chinese hamster ovary cells.

The fungal metabolite brefeldin A (BFA) induces the disassembly of the Golgi complex in mammalian cells. The drug seems to accentuate tubule formation and causes the subsequent fusion with the endoplasmic reticulum (ER). To investigate the biochemical requirements and kinetics of BFA-induced Golgi disassembly, we have reconstituted the process of green fluorescent protein-tagged Golgi complex disassembly in streptolysin O-permeabilized semi-intact Chinese hamster ovary cells. For quantitative analysis of the morphological changes to the Golgi complex in semi-intact cells, we developed a novel morphometric analysis. Based on this analysis, we have dissected the BFA-induced Golgi disassembly process biochemically into two processes, Golgi tubule formation and fusion with the ER, and found that the formation is induced by only ATP and the residual factors in the cells and that the subsequent fusion is mediated in an N-ethylmaleimide-sensitive factor-dependent manner via Golgi tubules. Tubulation occurs by two pathways that depend on either microtubule integrity or exogenously added cytosol. In the presence of GTPgammaS, coat protein I inhibited the Golgi tubule fusion with the ER but showed no apparent effect on tubulation. Additionally, we analyzed the kinetics of tubulation and fusion independently in nocodazole-treated and -untreated semi-intact cells and found that tubulation is a rate-limiting step of the Golgi disassembly.

Animals↗

PubMed2000

Molecular characterization and diagnostic value of Taenia solium low-molecular-weight antigen genes.

Neurocysticercosis (NCC) caused by infection with the larvae of Taenia solium is an important cause of neurological disease worldwide. In order to establish an enzyme-linked immunosorbent assay (ELISA) for this infection using recombinant proteins, we carried out molecular cloning and identified four candidates as diagnostic antigens (designated Ag1, Ag1V1, Ag2, and Ag2V1). Except for Ag2V1, these clones could encode a 7-kDa polypeptide, and Ag2V1 could encode a 10-kDa polypeptide. All of the clones were very similar. Except for Ag2V1, recombinant proteins were successfully expressed using an Escherichia coli expression system. Immunoblot analysis of NCC patient sera detected recombinant proteins, but because reactivity to recombinant Ag1 was too weak, Ag1 was not suitable as an immunodiagnostic antigen. So, Ag1V1 and Ag2 were chosen as ELISA antigens, and the Ag1V1/Ag2 chimeric protein was expressed. Of 49 serum samples from NCC patients confirmed to be seropositive by immunoblot analysis, 44 (89.7%) were positive by ELISA. No assays of serum samples from patients with other parasitic infections recognized the Ag1V1/Ag2 chimeric protein. The Ag1V1/Ag2 chimeric protein obtained in this study had a high value for differential immunodiagnosis.

Amino Acid Sequence↗

PubMed2000

Chronic sulfonylurea treatment and hyperglycemia aggravate disproportionately elevated plasma proinsulin levels in patients with type 2 diabetes.

It is established that disproportionately elevated plasma proinsulin levels occur in patients with Type 2 diabetes. In the present study, multivariate analysis was performed to determine what factors contributed to the disproportionately elevated plasma proinsulin levels in Japanese patients with Type 2 diabetes (n=276). Results from univariate analysis showed that both fasting proinsulin/C-peptide ratio and proinsulin/IRI ratio were approximately 2-fold higher in patients with Type 2 diabetes than those in healthy nondiabetic subjects (n=45). In patients with Type 2 diabetes, both proinsulin/C-peptide ratio and proinsulin/IRI ratio were significantly positively correlated with fasting plasma glucose level (FPG) and HbA1c. Neither proinsulin/C-peptide ratio nor proinsulin/IRI ratio was significantly correlated with BMI. Sulfonylurea-treated subjects had a significant elevation in both proinsulin/C-peptide ratio and proinsulin/IRI ratio compared with diet-treated subjects, whereas nonsulfonylurea hypoglycemic agent-treated subjects did not. Multivariate analysis confirmed that sulfonylurea treatment and FPG were significant determinants of both fasting proinsulin/C-peptide ratio (P=0.006 and P=0.030, respectively) and proinsulin/IRI ratio (P=0.003 and P=0.016, respectively) in patients with Type 2 diabetes. These results imply that disproportionate hyperproinsulinemia may reflect an excessive overwork of beta cells under chronic sulfonylurea treatment as well as hyperglycemia.

Body Mass Index↗

PubMed2000

Neurocysticercosis and echinococcosis in Asia: recent advances in the establishment of highly reliable differential serodiagnosis for international collaboration.

Neurocysticercosis (NCC) and echinococcosis, caused by the larval stage of taeniid cestodes, are recognized as major parasitic zoonoses threatening human life worldwide. Cystic echinococcosis (CE), caused by Echinococcus granulosus, has well been known to be more widely distributing in Europe and Asia (Eurasia) than alveolar echinococcosis (AE) caused by E. multilocularis. However, it has recently been found that AE is more widely distributing or spreading in Eurasia. Furthermore, NCC caused by Taenia solium is also spreading in Eurasia. Due to the lack of reliable methodology for diagnosing these zoonotic cestodiases worldwide, prevalence rates of these diseases are extremely underestimated. Our group has been working for the establishment of differential serodiagnosis and molecular diagnosis of AE. CE and NCC as international collaboration projects sponsored by the Ministry of Education, Japan from 1994 until 2000 at least. In this paper, we introduce (1) the most recent original work on the establishment of differential serodiagnoses of NCC, AE and CE, (2) international collaboration work on epidemiology of these diseases in several countries, and discuss (3) what we can and should do for the control of such global parasitic diseases. It is stressed that international collaboration or cooperation work on the control of parasitic diseases is only successful based on the original scientific contribution of high standard.

Animals↗

PubMed1999

A novel phosphoglycolipid archaetidyl(glucosyl)inositol with two sesterterpanyl chains from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1.

The structures of two novel polar lipids (AGI and AI) of an aerobic hyperthermophilic archaeon, Aeropyrum pernix, were elucidated. AGI and AI were the only two major lipids and accounted for 91 mol% and 9 mol%, respectively, of total polar lipids of this organism. The core lipid of A. pernix total lipids consisted solely of 2,3-di-O-sesterterpanyl-sn-glycerol (C25,25-archaeol). The molecular weights of the free acid forms of AGI and AI were shown by FAB-mass spectrometry to be 1196 and 1034, respectively. AI was completely hydrolyzed by phosphatidylinositol-specific phospholipase C, while AGI was not hydrolyzed at all under the same condition for the hydrolysis of AI. The molar ratio of phosphate, myo-inositol, and glucose in AGI was 1.0:0.97:0.95. The positions of linkages between myo-inositol and glucose, and between myo-inositol and phosphate in AGI were determined by NMR analyses of intact AGI and glucosylinositol prepared from AGI. Finally, it was concluded that the structures of AGI and AI were 2,3-di-O-sesterterpanyl-sn-glycerol-1-phospho-1'-(2'-O-alpha-D-glu cosyl)- myo-inositol (C25,25-archaetidyl(glucosyl)inositol) and 2,3-di-O-sesterterpanyl-sn-glycerol-1-phospho-myo-inositol (C25,25-archaetidylinositol), respectively. This is the first example that a core lipid of whole polar lipids is composed of only one species C25,25-archaeol in one organism and that glucosylinositol is found in a polar lipid as a polar head group.

Crenarchaeota↗

PubMed1999

Purification and characterization of an intracellular heat-stable proteinase (pernilase) from the marine hyperthermophilic archaeon Aeropyrum pernix K1.

A novel intracellular serine proteinase from the marine aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) that we designated pernilase was purified by ammonium sulfate precipitation, anionic-exchange chromatography, affinity chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 50 kDa as determined by SDS-PAGE. The proteinase had a broad pH profile (pH 5-10) with an optimum pH of 9.0 for peptide hydrolysis. The optimum temperature for enzyme activity was 90 degrees C. The enzyme was strongly inhibited by diisopropyl fluorophosphate (DFP) and phenylmethyl sulfonylfluoride (PMSF), suggesting that it corresponds to a serine proteinase. The enzyme was highly resistant to the reducing agents dithiothreitol and 2-mercaptoethanol but sensitive to the denaturing reagents guanidine-HCl and urea and also to the detergent sodium dodecyl sulfate (SDS). Pernilase showed high substrate specificity for Boc-Leu-Gly-Arg-MCA peptide. Thermostability of this enzyme showed half-lives of 85min at 100 degrees C and 12 min at 110 degrees C.

Amino Acid Sequence↗

PubMed1999

Molecular cloning and characterisation of 23-kDa piroplasm surface proteins of Theileria sergenti and Theileria buffeli.

The cDNA encoding a 23-kDa piroplasm membrane protein (p23) of Theileria sergenti Chitose (C)-type was isolated and its nucleotide sequence was determined. The gene encodes a polypeptide of 223 aa with a 28 residue N-terminal signal sequence and a hydrophobic, valine-rich, C-terminal transmembrane domain, as deduced from its nucleotide sequence. Southern blot hybridisation analysis proved that p23 gene was a single copy gene and had allelic forms of the gene in the parasite population. By PCR, the open reading frames of T. sergenti Ikeda (I)-type and Theileria buffeli (B)-type p23 were amplified from genomic DNA and their nucleotide sequences were also determined. Comparison of C-type sequence with that of I-type and B-type revealed 90.5% and 93.5% sequence similarity, respectively, at the aa level. These results suggest that a conserved molecule in these benign Theileria spp. could be a candidate antigen for the development of an anti-piroplasm vaccine.

Amino Acid Sequence↗

PubMed1999

A cambialistic SOD in a strictly aerobic hyperthermophilic archaeon, Aeropyrum pernix.

The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperthermophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6 kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-reconstituted SOD was observed at 105 degrees C even after 5 h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400 mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe.

Aerobiosis↗

PubMed1999

Molecular characterization of a Haemaphysalis longicornis tick salivary gland-associated 29-kilodalton protein and its effect as a vaccine against tick infestation in rabbits.

The use of tick vaccines in mammalian hosts has been shown to be the most promising alternative tick control method to current use of acaricides, which suffers from a number of limitations. However, the success of this method is dependent on the identification, cloning, and in vitro expression of tick molecules involved in the mediation of key physiological roles with respect to the biological success of a tick as a vector and pest. We have sequenced and characterized a Haemaphysalis longicornis tick salivary gland-associated cDNA coding for a 29-kDa extracellular matrix-like protein. This protein is expressed in both unfed and fed immature and mature H. longicornis ticks. The predicted amino acid sequence of p29 shows high homology to sequences of some known extracellular matrix like-proteins with the structural conservation similar to all known collagen proteins. Immunization with the recombinant p29 conferred a significant protective immunity in rabbits, resulting in reduced engorgement weight for adult ticks and up to 40 and 56% mortality in larvae and nymphs that fed on the immunized rabbits. We speculate that this protein is associated with formation of tick cement, a chemical compound that enables the tick to remain attached to the host, and suggest a role for p29 as a candidate tick vaccine molecule for the control of ticks. We have discussed our findings with respect to the search of tick molecules for vaccine candidates.

Amino Acid Sequence↗

PubMed1999

Two family B DNA polymerases from Aeropyrum pernix, an aerobic hyperthermophilic crenarchaeote.

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'-->5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80 degrees C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95 degrees C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.

Amino Acid Sequence↗

PubMed1999

Cloning of a cysteine proteinase gene of Theileria sergenti.

A cDNA encoding cysteine proteinase of Theileria sergenti was isolated from a piroplasm cDNA library and its nucleotide sequence was determined. The gene encodes a polypeptide of 402 amino acids with predicted molecular mass of 46.4 kDa. Analysis of the predicted amino acid sequence revealed a number of features common to known cysteine proteinases. Southern blot analysis showed that the cysteine proteinase gene was likely to be a single copy per genome.

Amino Acid Sequence↗

PubMed1999

Expression of a major piroplasm surface protein of Theileria sergenti in sporozoite stage.

A 32 kilodalton major piroplasm surface protein (MPSP) is expressed abundantly on the surface of intraerythrocytic piroplasms of Theileria sergenti and is considered to be a candidate antigen for vaccine development against piroplasmosis. In this study, transcripts of MPSP gene were detected in an expression cDNA library prepared from T. sergenti-infected tick salivary glands. Expression of MPSP in the sporozoite stage was also confirmed by immunoblot analysis. Its expression at the sporozoite and intraerythrocytic stages gives scope for possible induction of protective immunity being targeted at both stages by immunization with recombinant MPSP.

Animals↗

PubMed1998

Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton.

Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

Anion Exchange Protein 1, Erythrocyte↗

PubMed1998

Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.

The translational movement of E-cadherin, a calcium-dependent cell-cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and alpha-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm phi colloidal gold or 210-nm phi latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell-cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 micron by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 x 10(-10) and 2.1 x 10(-10) cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 micron2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 x 10(-10) cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/micron, respectively, indicating a difference in the skeletal structures that produce these two effects.

Animals↗

PubMed1998

Structure of the erythrocyte membrane skeleton as observed by atomic force microscopy.

The structure of the membrane skeleton on the cytoplasmic surface of the erythrocyte plasma membrane was observed in dried human erythrocyte ghosts by atomic force microscopy (AFM), taking advantage of its high sensitivity to small height variations in surfaces. The majority of the membrane skeleton can be imaged, even on the extracellular surface of the membrane. Various fixation and drying methods were examined for preparation of ghost membrane samples for AFM observation, and it was found that freeze-drying (freezing by rapid immersion in a cryogen) of unfixed specimens was a fast and simple way to obtain consistently good results for observation without removing the membrane or extending the membrane skeleton. Observation of the membrane skeleton at the external surface of the cell was possible mainly because the bilayer portion of the membrane sank into the cell during the drying process. The average mesh size of the spectrin network observed at the extracellular and cytoplasmic surfaces of the plasma membrane was 4800 and 3000 nm2, respectively, which indicates that spectrin forms a three-dimensionally folded meshwork, and that 80% of spectrin can be observed at the extracellular surface of the plasma membrane.

Cell Fractionation↗