Search PubMed⌕ Search

Biomedical subjects

A Saito

Publications and source records attributed to A Saito.

At least 73 records · Page 4Linked to original sources

Streptomyces olivaceoviridis possesses a phosphotransferase system that mediates specific, phosphoenolpyruvate-dependent uptake of N-acetylglucosamine.

We recently described the ABC transporter Ngc (encoded by the ncgEFG operon) from Streptomyces olivaceoviridis, the first of its kind to be shown to transport N-acetylglucosamine and N,N'-diacetylchitobiose (chitobiose). A chromosomal mutant carrying a disruption of the ngcE gene, which encodes the sugar binding protein, was still able to transport N-acetylglucosamine. This phenotype can now be attributed to a functional phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS). Two adjacent homologous genes, ptsC1 and ptsC2, were identified, and deduced to encode proteins which are 56% identical and can be predicted to contain eight transmembrane regions. PtsC1 (432 amino acids) and PtsC2 (403 residues) each correspond to a single EIIC domain; such domains are otherwise known only in several bacterial multidomain permeases for glucose/mannose or N-acetylglucosamine. The C-terminal sequences of PtsC1 and PtsC2 correspond to the motifs LKTPGREP and LPTRGRES, respectively. The ptsB gene located upstream of ptsC1 is predicted to encode a homologue of the EIIB domains usually found in bacterial multidomain permeases. Physiological and biochemical analyses of ngcE mutants carrying disruptive insertions in ptsC1 or ptsC2 or both revealed that, when grown on N-acetylglucosamine, the membrane component PtsC2, unlike PtsC1, mediates PEP-dependent, specific (K(m)=5 micro M) transport of N-acetylglucosamine, but not of other hexoses. Cross-complementation of membrane and cytoplasmic fractions from the various mutants led to the conclusion that S. olivaceoviridis also expresses the functional soluble components HPr, EI and EIIA of the PTS system. During growth on xylose, uptake of this pentose occurred if ptsC1 or ptsC2 was intact, but not in a mutant containing disrupted forms of both genes.

Acetylglucosamine↗

Deterministic retrieval of surface waviness by means of topography with coherent X-rays.

A surface profile retrieval technique from multiple X-ray total reflection images taken at various distances with full coherent illumination is demonstrated. An experiment was performed using the 1 km-long BL29XU beamline at the SPring-8 facility, Japan. Obtained results are compared with results from the optical metrology technique (Fizeau's interferometer). Good agreement between X-ray and optical methods proves the validity of the current approach. Meanwhile, the sensitivity of the X-ray technique is several times higher than that of the standard one. This technique is well suited to the needs of characterizing grazing optics for new-generation X-ray sources.

Journal Article↗

The novel Streptomyces olivaceoviridis ABC transporter Ngc mediates uptake of N-acetylglucosamine and N,N'-diacetylchitobiose.

During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [(14)C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N, N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (K(D)=8.28 x 10(-9) M) and for chitobiose (K(D)=2.87 x 10(-8) M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (K(D)=4.5 x 10(-6) M), chitopentaose (K(D)=1.03 x 10(-6) M) and chitohexaose (K(D)=3.02 x 10(-6) M); the lowest value was measured for chitotriose (K(D)=19.4 x 10(-6) M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo.

ATP-Binding Cassette Transporters↗

Relationship between the stability of lysozymes mutated at the inside hydrophobic core and secretion in Saccharomyces cerevisiae.

The relationship between the stability of lysozymes mutated at the inside hydrophobic core and secretion was investigated to understand the optimal secretion of mutant lysozymes in the Saccharomyces cerevisiae. S91T mutant lysozyme increased in the methyl residue inside the core greatly increased the conformational stability. The secretion amount of S91T in S. cerevisiae increased greatly compared with wild-type lysozyme. On the other hand, I55V and T40S/I55V mutant lysozymes decreased in methyl residue inside the core brought about their unstable conformation. The secretion amounts of these unstable mutant lysozymes significantly decreased. In addition, the effect of glycosylation on the secretion of these mutants was investigated. The secretion amounts of glycosylated lysozyme S91T/G49N with stable hydrophobic core greatly increased compared with that of glycosylated lysozyme G49N, while those of mutant I55V/G49N and T40S/I55V/G49N with unstable hydrophobic core greatly decreased. These results indicate that the secretion amounts of mutant lysozymes increase in proportion to the hydrophobic core stabilities and that a similar good correlation was obtained with glycosylated lysozymes.

Anti-Infective Agents↗

Transplantation for accidental acute high-dose total body neutron- and gamma-radiation exposure.

Accidental exposure to acute high-dose total body neutron radiation is rare. We report a 35-year-old man exposed to a total body dose of 5.4 Gy neutron- and 8.5-13 Gy gamma-radiation in a radiation criticality accident. He received a blood stem cell transplant from his HLA-identical sister. There was bone marrow recovery with complete donor chimerism. Random chromatid breaks were observed in donor cells suggesting a bystander effect of neutron exposure. The subject died 82 days after the accident (75 days post transplant) from multi-organ failure.

Adult↗

Gelatin-specific cellular immune responses persist for more than 3 years after priming with gelatin containing DTaP vaccine.

BACKGROUND: Gelatin-specific cell-mediated immunity develops in subjects inoculated with gelatin containing DTaP vaccine. However, it is not yet known whether such established sensitization to gelatin disappears or persists with time. OBJECTIVE: The aim of this study was to follow the patients with gelatin sensitization elicited by DTaP vaccination for their lymphocyte responsiveness and IgE, IgG antibody specific to gelatin over several years and to compare the activities with those at the time of enrollment into the study. METHODS: We studied 28 subjects who developed positive lymphocyte proliferation test (LPT) after receiving gelatin containing DTaP vaccine and eight subjects who had a negative LPT after inoculation of non-gelatin DTaP. Determination of IgE, IgG antibodies and specific lymphoproliferative response directed against gelatin were performed at enrollment and on follow up. RESULTS: None of the subjects had antibody to gelatin at enrollment and none developed gelatin IgE or IgG during follow-up. There was no significant difference in the SIs of the subjects receiving gelatin DTaP (P = 0.150, 95% CI, -0.198-0.032), whereas lymphocyte activity to gelatin increased between enrollment and follow-up in the subjects with non-gelatin DTaP (P = 0.011, 95% CI, 0.063-0.338). CONCLUSION: Gelatin-specific lymphocyte activity persists at comparable levels for more than 3 years in subjects who acquire a positive LPT response to gelatin after receiving primary DTaP vaccine containing gelatin. Furthermore, five out of eight subjects initially with negative LPT to gelatin have been shown to acquire specific LPT with time.

Diphtheria-Tetanus-acellular Pertussis Vaccines↗

Detection of genes expressed in primary colon cancers by in situ hybridisation: overexpression of RACK 1.

AIMS: The isolation of various genes that are expressed in a region specific manner is considered useful for research in molecular pathology. In situ hybridisation (ISH) was used in a screening procedure to isolate these genes efficiently, using colon cancer as a model. METHODS: Suppression subtractive hybridisation (SSH) between colon cancer tissue samples and corresponding non-cancerous tissues was performed. Genes showing high expression in the cancers were selected using macro-DNA array analysis. As a final screening procedure, conventional ISH was performed to isolate genes expressed specifically in colon cancers. RESULTS: Sixty nine clones were selected by SSH and macro-DNA array analyses. These clones were then analysed by ISH to examine their expression patterns. ISH screening revealed that all the clones screened showed more intense signals in colon cancers than in non-cancerous tissues. Among them, RACK 1, which is a protein kinase C receptor and a homologue of the G protein beta subunit, was expressed intensely in colon cancer cells. RACK 1 expression was evaluated in multiple samples by ISH, and the results confirmed that RACK 1 was universally overexpressed in cells of all 11 colon cancers examined. CONCLUSIONS: Many genes, including RACK 1, expressed in colon cancer cells can be isolated efficiently by this method, and their precise expression pattern can be evaluated. These results indicate that ISH is an excellent technique for systemic screening of genes expressed in a region specific manner.

Blotting, Northern↗

Glyco-western blotting: biotinylated dermatan sulfate as a probe for the detection of dermatan sulfate binding proteins using western blotting.

Dermatan sulfate was biotinylated through the free amino residue of the core protein. The lysates of mouse and human lung culture cells were electrophoresed and blotted to a nitrocellulose membrane. The membrane was blocked successively with bovine serum albumin, avidin and biotin, and then treated with biotinylated dermatan sulfate followed by visualization using alkaline phosphatase conjugated avidin and its substrates. More than 30 bands were observed on the membrane when 1 microgram/ml of biotinylated dermatan sulfate was used. The binding was prevented by an excess of dermatan sulfate but not chondroitin sulfate A or heparan sulfate. Some of the bands resisted washing with high salt concentration buffer. 60 kDa heat shock protein was found to be a dermatan sulfate binding protein upon two dimensional electrophoresis.

Animals↗

Monocyte chemoattractant protein-1-dependent increase of V alpha 14 NKT cells in lungs and their roles in Th1 response and host defense in cryptococcal infection.

To elucidate the role of NKT cells in the host defense to cryptococcal infection, we examined the proportion of these cells, identified by the expression of CD3 and NK1.1, in lungs after intratracheal infection with Cryptococcus neoformans. This population increased on day 3 after infection, reached a peak level on days 6-7, and decreased thereafter. In Valpha14 NKT cell-deficient mice, such increase was significantly attenuated. The proportion of Valpha14 NKT cells, detected by binding to alpha-galactosylceramide-loaded CD1d tetramer, and the expression of Valpha14 mRNA increased after infection with a similar kinetics. The delayed-type hypersensitivity response and differentiation of the fungus-specific Th1 cells was reduced in Valpha14 NKT cell-deficient mice, compared with control mice. Additionally, elimination of this fungal pathogen from lungs was significantly delayed in Valpha14 NKT cell-deficient mice. Production of monocyte chemoattractant protein (MCP)-1 in lungs, detected at both mRNA and protein levels, increased on day 1, reached a peak level on day 3, and decreased thereafter, which preceded the increase in NKT cells. Finally, the increase of total and Valpha14(+) subset of NKT cells after infection was significantly reduced in MCP-1-deficient mice. Our results demonstrated that NKT cells, especially Valpha14(+) subset, accumulated in a MCP-1-dependent manner in the lungs after infection with C. neoformans and played an important role in the development of Th1 response and host resistance to this fungal pathogen.

Animals↗

Trail antigen in Eimeria stiedai sporozoites associated with a thrombospondin-related motif and the entry of cultured cells.

In order to examine the antigenic similarity and specificity of the trail antigen of Eimeria stiedai and Etp 100, a microneme protein of Eimeria tenella, monoclonal antibodies to the trail antigen of E. stiedai sporozoites were selected by an indirect immunofluorescent antibody method. The monoclonal antibody of one clone, 3D10, reacted with the anterior portion of non-fixed sporozoites. By immunoblotting, the monoclonal antibody was found to react with a 100 kDa antigen of E. stiedai sporozoites, and a 117 kDa antigen of E. tenella sporozoites and merozoites. It was also found to react with a recombinant protein with thrombospondin-/properdin-like motifs homologous to E. tenella microneme protein Etp 100. The monoclonal antibody significantly inhibited the penetration of E. stiedai sporozoites into cultured rabbit hepatobiliary epithelial cells. These results suggest that E. stiedai sporozoites have a trail antigen, located in the anterior region on the outer surface of the sporozoites, which has an epitope with thrombospondin-/properdin-like motifs similar to E. tenella microneme protein Etp 100. This protein may play an important functional role in the process of penetration of host cells.

Amino Acid Sequence↗

A strong association between HLA-DR9 and gelatin allergy in the Japanese population.

The frequency of HLA class I and II phenotypes was determined among 23 patients with positive gelatin IgE, eight of whom developed anaphylaxis, 18 patients who did not have gelatin IgE but who experienced non-immediate reactions after exposure to gelatin. HLA-DR9, which is unique to Orientals, was present in 56.5% of the gelatin IgE positive patients, as compared to control population frequency of 24% (P < 0.002). In the non-immediate reaction group, who did not generate IgE, phenotype distribution resembled controls. HLA-DR9 positive individuals have a relative risk of 4.1 for developing gelatin allergy with positive IgE.

Anaphylaxis↗

Specific IgA antibody response to coproantigens of Cryptosporidium parvum in serum and saliva of calves after experimental infection.

The antibody response to coproantigens of Cryptosporidium parvum was examined in saliva and sera of calves experimentally infected with C. parvum. Coproantigens of C. parvum with approximate molecular masses of 17, 15 and less than 14kDa were found in the feces of infected calves on day 3 or later, and 60 and 23kDa coproantigens observed between days 4 and 9 post-infection, respectively. The antibody reactivity to the coproantigens was mainly attributable to IgA class antibodies in saliva and was detectable during the convalescent phase of infection. A 15kDa protein isolated from the feces of infected calves by immunoaffinity adsorption using a monoclonal anti C. parvum antibody was recognized by IgA antibodies present in the saliva during the convalescent phase of infection. These results suggest that this coproantigen may be released from C. parvum sporozoites and may induce IgA antibody production in the mucosal immune system of infected calves.

Animals↗

A 14-3-3 protein homologue is expressed in feline enteroepithelial-stages of Toxoplasma gondii.

Fourteen cDNA clones encoding epitopes of proteins of Toxoplasma gondii feline enteroepithelial-stages parasites were isolated and expressed in Escherichia coli in an effort to determine the antigenecity of the parasites. Sequence analysis showed that four of the cDNA clones had a 930-bp open-reading frame encoding a product showing similarity to the 14-3-3 protein mRNA sequence.(1) Southern hybridization of DIG-labeled positive clone with T. gondii genomic DNA cleaved with EcoRI, BamHI and HindIII resulted in one or two bands in each case. In an immunofluorescence assay, polyclonal and monoclonal antibodies raised against the expressed protein showed strong reactivity with feline enteroepithelial-stages parasites and sporozoites. In a complementation assay in which a plasmid carrying the protein-coding region of the isolated cDNA was introduced into a Saccharomyces cerevisiae mutant, strain DS9-22, the expressed protein showed complementation of the function of the 14-3-3 protein in yeast transformants. These findings suggest that T. gondii parasites produce a protein showing partial homology with members of the 14-3-3 protein family and this protein is expressed in feline enteroepithelial-stages parasites.

14-3-3 Proteins↗

Semantic cueing effects on word retrieval in aphasic patients with lexical retrieval deficit.

We examined lexical access in word production, focusing on lexical retrieval deficit in aphasic patients. These patients were asked to name one picture at a time and we investigated how semantic cueing affected their ability to retrieve the word. When the subjects experienced a retrieval failure, they were presented with one type of cue, and then they tried to name the target picture again. Four types of cues were used: phonological, semantic related, semantic category member, and baseline cue. Both phonological and semantically related cues improved performance. In contrast, category member cues had no effects on releasing the retrieval block. These results can be explained by an interactive activation model in word production (Dell & O'Seaghdha, 1991).

Adult↗

Anaplastic meningioma with papillary, rhabdoid, and epithelial features: a case report.

A 74-year-old man manifested disturbed consciousness and right hemiparesis. Computed tomography revealed a left frontal parasagittal meningeal tumor with extensive peritumoral brain edema and skull invasion. Subtotal removal was performed. Five years later, he underwent two more operations of massive recurrences. Pathological studies revealed anaplastic meningioma with two different histological areas. One was an epithelial and meningothelial area, and the other was a papillary and rhabdoid area. In the papillary and rhabdoid area, small tumor cells with a high nucleus/cytoplasm ratio proliferated densely around the dilated central capillaries with a pseudopapillary pattern. Many rhabdoid cells (vimentin ++, cytokeratin AE1/AE3 +, epithelial membrane antigen [EMA] + +) tended to be distributed far from the central capillaries. There were many mitotic figures near the central vessels. Dense MIB1-positive nuclei were also observed near the central vessels. The trabecular pattern of the tumor cells in the epithelial area was quite different from the histological features of chordoid meningioma.

Aged↗