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Biomedical subjects

S Inouye

Publications and source records attributed to S Inouye.

At least 73 records · Page 4Linked to original sources

Identification of a substrate for Pkn2, a protein Ser/Thr kinase from Myxococcus xanthus by a novel method for substrate identification.

Eukaryotic cells contain a large number of protein Ser/ Thr kinases, which play important roles in signal transduction required for cell proliferation, differentiation, and stress response and adaptation. It is also known that some prokaryotes contain a family of protein Ser/Thr kinases. A major challenge in the characterization of these kinases is how to identify their specific substrates. Here we developed such a method using a protein Ser/Thr kinase, Pkn2 from Myxococcus xanthus, a Gram-negative soil bacterium. When Pkn2 is inducibly expressed in E. coli, cells are unable to form colonies on agar plates. This lethal effect of Pkn2 was eliminated in an inactive Pkn2 mutant in which the highly conserved Lys residue was changed to Asn, indicating that phosphorylation of a cellular protein(s) in E. coli resulted in growth arrest. Several clones from an E. coli genomic library were found to suppress the lethal effect when co-expressed with pkn2. Four out of seven multi-copy suppressors were identified to encode HU, (3 for HUalpha and 1 for HUB) a histone-like DNA binding protein. Purified HUalpha was found to be specifically phosphorylated by Pkn2 at Thr-59, and the phosphorylated HUalpha became unable to bind to DNA, suggesting that the phosphorylation of endogenous HU proteins by Pkn2 contributed at least in part to the lethal effect in E. coli. The present method termed the STEK method (Suppressors of Toxic Effects of Kinases) may be widely used for the substrate identification not only for prokaryotic protein Ser/Thr kinases but also for eukaryotic kinases.

Bacterial Proteins↗

Systemic allergic reactions to gelatin included in vaccines as a stabilizer.

Most of the children who showed systemic immediate-type reactions, including anaphylactic shock, to measles, mumps, rubella, and varicella vaccines had IgE antibodies to gelatin; thus we suspected that the allergic symptoms are caused by gelatin antigen, which is usually included in these live-virus vaccines as a stabilizer. We hypothesized that the anti-gelatin IgE is elicited by immunization with DTaP (diphtheria-tetanus-acellular pertussis) vaccines, which contained a small amount of gelatin as a spillover protein after purification of pertussis toxin. To test this hypothesis, we conducted a case-control study to determine whether children with anti-gelatin IgE had received gelatin-containing DTaP vaccines, and it was indeed found that all such children in the study had immunization histories that included the gelatin-containing DTaP vaccines. Based on these findings, the vaccine manufacturers had removed gelatin from all the DTaP and live-virus vaccines produced in Japan by 2000.

Chickenpox Vaccine↗

Highly specific recognition of primer RNA structures for 2'-OH priming reaction by bacterial reverse transcriptases.

A minor population of Escherichia coli contains retro-elements called retrons, which encode reverse transcriptases (RT) to synthesize peculiar satellite DNAs called multicopy single-stranded DNA (msDNA). These RTs recognize specific RNA structures in their individual primer-template RNAs to initiate cDNA synthesis from the 2'-OH group of a specific internal G residue (branching G residue). The resulting products (msDNA) consist of RNA and single-stranded DNA, sharing hardly any sequence homology. Here, we investigated how RT-Ec86 recognizes the specific RNA structure in its primer-template RNA. On the basis of structural comparison with HIV-1 RT, domain exchanges were carried out between two E. coli RTs, RT-Ec86 and RT-Ec73. RT-Ec86 (320 residues) and RT-Ec73 (316 residues) share only 71 identical residues (22%). From the analysis of 10 such constructs, the C-terminal 91-residue sequence of RT-Ec86 was found to be essential for the recognition of the unique stem-loop structure and the branching G residue in the primer-template RNA for retron-Ec86. Using the SELEX (systematic evolution of ligands by exponential enrichment) method with RT-Ec86 and primer RNAs containing random sequences, the identical stem-loop structure (including the 3-U loop) to that found in the retron-Ec86 primer-template RNA was enriched. In addition, the highly conserved 4-base sequence (UAGC), including the branching G residue, was also enriched. These results indicate that the highly diverse C-terminal region recognizes specific stem-loop structures and the branching G residue located upstream of the stem-loop structure. The present results with seemingly primitive RNA-dependent DNA polymerases provide insight into the mechanisms for specific protein RNA recognition.

Amino Acid Sequence↗

Identification and characterization of five cspA homologous genes from Myxococcus xanthus.

Escherichia coli contains a large CspA family consisting of nine homologues, in which four are cold-shock inducible and one is stationary-phase inducible. Here, we demonstrate that Myxococcus xanthus possesses at least five CspA homologues, CspA to CspE. Hydrophobic residues forming a hydrophobic core, and aromatic residues, which are included in functional motifs RNP-1 and RNP-2 involved in binding to RNA and ssDNA, are well conserved. These facts suggest that M. xanthus CspA homologues have a similar structure and function as E. coli CspA. However, in contrast to the E. coli CspA family, the expression of M. xanthus csp genes as judged by primer extension analysis is not significantly regulated by temperature changes, except for cspB of which expression was reduced to less than 10% upon heat shock at 42 degrees C. Such constitutive expression of the csp genes may be important for M. xanthus, a soil-dwelling bacterium, to survive under conditions of exposure to various environmental changes in nature.

Amino Acid Sequence↗

Superradiant rayleigh scattering from a bose-einstein condensate

Rayleigh scattering off a Bose-Einstein condensate was studied. Exposing an elongated condensate to a single off-resonant laser beam resulted in the observation of highly directional scattering of light and atoms. This collective light scattering is caused by the coherent center-of-mass motion of the atoms in the condensate. A directional beam of recoiling atoms was built up by matter wave amplification.

Journal Article↗

Specific IgG to gelatin in children with systemic immediate- and nonimmediate-type reactions to measles, mumps and rubella vaccines.

We examined anti-gelatin IgG in sera of children who suffered from systemic adverse reactions upon immunization with gelatin-containing live virus vaccines. In the group of 30 children who had immediate-type reactions and anti-gelatin IgE, 30 (100%) had anti-gelatin IgG and 29 (96%) had anti-gelatin IgG4. In another group of 75 children who had nonimmediate-type reactions and no anti-gelatin IgE, 22 (29%) had anti-gelatin IgG and six (8%) had IgG4. The IgG positivity well correlated with the lymphocyte proliferation assay positivity. In contrast, as a negative control, all 24 children who had no allergic reaction to live virus vaccines had no anti-gelatin IgG and IgG4. The results suggest that immune-response to gelatin may play a role in the pathogenesis of systemic nonimmediate-type reactions to the live virus vaccines.

Animals↗

Distribution and developmental changes of adenylate kinase isozymes in the rat brain: localization of adenylate kinase 1 in the olfactory bulb.

Changes in the levels of three adenylate kinase isozymes (AK1, AK2, and AK3) in the rat brain during development were investigated by immunoblot analysis. The levels of AK1 and AK3 of the whole brain increased after birth, while AK2 was detected only in the early embryonic period. In the adult rat brain, high levels of AK1 were present in the olfactory bulb. Immunohistochemical analysis showed that AK1 was found predominantly in the olfactory nerve layer and the glomerular layer. In the olfactory bulb, AK1 gene expression was enhanced in the early postnatal days, whereas it remained low in the cerebellum during the first 10 postnatal days. These results suggest that the AK isozymes are involved in neuronal maturation and regeneration. The understanding of the physiological actions of adenosine and ATP as neurotransmitters/neuromodulaters in the central nervous system has improved. ATP and adenosine receptors have been found to be widely distributed over the whole brain, although the intra- and extracellular metabolism of these compounds has not been well elucidated.

Adenylate Kinase↗

Parasite infection and Japanese cedar pollinosis in monkeys.

To evaluate the relationship between helminthic parasite infection and sensitization to Japanese cedar (Cryptomeria japonica; CJ) pollen allergens in Japanese monkeys (Macaca fuscata), we examined the parasite infection, presence of anti-pollen allergen IgE and development of pollinosis. Serum samples and fecal specimens were taken from 169 monkeys in five troops, and the presence of IgE antibody for CJ pollen allergen and of helminthic parasite eggs in their feces was examined. Of the 169 monkeys, helminthic parasite eggs from 138 monkeys were detected. The frequency of anti-CJ IgE (19%) and the level of total IgE did not differ significantly between the monkeys with and without parasite eggs. We examined the presence of anti-CJ IgE and pollinosis symptoms in 31 monkeys of a troop; six of the monkeys demonstrated anti-CJ IgE and pollinosis symptoms. Five of these six monkeys had parasite eggs. We found that the monkeys that demonstrated anti-CJ IgE and pollinosis symptoms had helminthic parasite infections. These data suggest that helminthic parasite infection does not reduce the development of clinical signs of CJ pollinosis in Japanese monkeys.

Allergens↗

The in situ regeneration and extraction of recombinant aequorin from Escherichia coli cells and the purification of extracted aequorin.

Recombinant apoaequorin expressed in the periplasmic space of Escherichia coli cells was regenerated into aequorin and extracted from the cells, simultaneously, using a buffer that contained coelenterazine. Due to the mild extraction conditions, the impurities in the extract were minimal. Thus, the purification of extracted aequorin could be accomplished in only two steps, anion-exchange chromatography and hydrophobic interaction chromatography, simply by adsorption and elution in both steps. The purified recombinant aequorin was pure, based on various data, including HPLC analysis and light-emitting activity. The yield of purified aequorin was 25-35 mg from 600 ml of culture, which was over 75% of the total amount of apoaequorin expressed in E. coli cells.

Aequorin↗

IgE reactivity to alpha1 and alpha2 chains of bovine type 1 collagen in children with bovine gelatin allergy.

BACKGROUND: Anaphylactic reactions to measles, mumps, and rubella vaccines, including gelatin as a stabilizer, have been reported. It had been found that most of these reactions to live vaccines are caused by the bovine gelatin included in these vaccines. Gelatin mainly includes denatured type I collagen, which consists of alpha1 and alpha2 chains. OBJECTIVE: The current study was designed to investigate the IgE reactivity to alpha1 and alpha2 chains of bovine type I collagen in gelatin-sensitive children. METHODS: Serum samples were taken from 10 children who had anaphylaxis to the vaccines and high levels of specific IgE to bovine gelatin. Bovine type I collagen was isolated from bovine skin and then separated to alpha1 and alpha2 chains by column chromatography. IgE reactivity to denatured type I collagen and its alpha1 and alpha2 chains was analyzed by immunoblotting, ELISA, and histamine release from the mast cells passive sensitized with IgE antibodies in pooled serum of the children. RESULTS: All children had specific IgE to bovine type I collagen. Furthermore, IgE antibodies in their sera reacted with the alpha;2 chain but not with the alpha1 chain. Similarly, the mast cells sensitized with pooled sera in the children showed alpha2 chain-specific histamine release but not alpha1 chain-specific histamine release. CONCLUSION: In gelatin allergy denatured bovine type I collagen is a major allergen and IgE-binding sites exist in the alpha2 chain of type I collagen.

Anaphylaxis↗

Molecular and serological characterization of adenovirus genome type 7h isolated in Japan.

In 1996, three adenovirus type 7 (Ad7) strains were isolated from children with fever and upper respiratory diseases in Japan. Restriction endonucleases (REs) analysis and PCR amplification of the E3 7.7 kDa ORF revealed that these strains were genotype Ad7h and closely related to an Argentine Ad7h strain, which has been reported to be highly virulent and so far predominant only in South America. These strains showed weak cross-neutralizing activity and specific haemagglutination-inhibition activity to Ad3 antiserum. The present findings suggest that Ad7h in South America has spread to other parts of the world. Since the seroprevalence to Ad7 in the current Japanese population is very low due to the absence of Ad7 circulation in Japan for decades, Ad7 outbreak as a typical case of re-emerging infectious diseases is a cause for serious concern.

Adenovirus Infections, Human↗

Electronic network for monitoring travellers' diarrhoea and detection of an outbreak caused by Salmonella enteritidis among overseas travellers.

The Traveller's Diarrhoea Network, by which the Infectious Disease Surveillance Center is electronically connected with two major airport quarantine stations and three infectious disease hospitals, was launched in February 1988 in Japan. The data on travellers' diarrhoea detected is reported weekly by e-mail. Two clusters of infection among travellers returning from Italy were reported by two airport quarantine stations at the end of September 1998. A total of 12 salmonella isolates from 2 clusters were examined. All were identified as Salmonella enteritidis, phage type 4 and showed identical banding patterns on pulsed-field gel electrophoresis. A case-control study showed that the scrambled eggs served at the hotel restaurant in Rome were the likely source of this outbreak. This outbreak could not have been detected promptly and investigated easily without the e-mail network. International exchange of data on travellers' diarrhoea is important for preventing and controlling food-borne illnesses infected abroad.

Adult↗

IgE reactivity and cross-reactivity of Japanese monkeys (Macaca fuscata) to Japanese cedar (Cryptomeria japonica) and cypress (Chamaecyparis obtusa) pollen allergens.

BACKGROUND: The natural occurrence of Japanese cedar (Cryptomeria japonica, CJ) pollinosis has been reported in Japanese monkeys (Macaca fuscata). However, the reactivity to Japanese cypress (Chamaecyparis obtusa, CO) pollen allergens in these monkeys has not yet been reported. OBJECTIVES: The present study was designed to investigate the reactivity to CO pollen allergens in monkeys sensitized to CJ pollen allergens. METHODS: Serum samples from 40 monkeys naturally sensitized to CJ pollen allergens were collected from four troops. We measured the specific IgE to CO pollen allergens and examined the reactivity to the allergens by intradermal test. Cross-reactivity between CJ and CO pollen allergens was examined by ELISA inhibition method. Furthermore, we examined the sensitivity to the allergens by histamine release assay from leucocytes. RESULTS: All 40 monkeys had specific IgE to crude and purified major allergens (Cha o 1) of CO pollen. The monkeys showed a positive reaction to CO pollen allergens in the intradermal test. Allergenic cross-reactivity between Cha o 1 and Cry j 1 (a major allergen in CJ pollen) was also observed. Specific histamine release to both the major allergens was noted in two monkeys with CJ pollinosis. CONCLUSION: Japanese monkeys sensitized to Japanese cedar pollen allergens also demonstrate reactivity to Japanese cypress pollen allergens.

Allergens↗

Sensitization to grass, ragweed, mugwort pollen allergens in Japanese monkeys (Macaca fuscata): preliminary report.

BACKGROUND: The natural occurrence of Japanese cedar (CJ, Cryptomeria japonica) pollinosis has been reported in Japanese monkeys (Macaca fuscata). Furthermore, most of these monkeys with CJ pollinosis have immunoglobulin (Ig) E sensitization to Japanese cypress (Chamaecyparis obtusa) pollen. However, specific IgE to other pollens has not yet been reported. OBJECTIVES: The present study was designed to investigate IgE sensitization of Japanese monkeys to grass, ragweed, and mugwort pollen. METHODS: Serum samples from 47 monkeys as a general population in one troop were collected at random. We measured specific IgE to grass, ragweed and mugwort pollen. Next, 10 monkeys with CJ pollinosis from the same troop were also examined for their IgE sensitization to grass, ragweed, and mugwort pollen. RESULTS: Of 47 monkeys, 13 (28%) had specific IgE to CJ pollen, 15 (32%) to grass pollen, five (11%) to ragweed pollen, and three (6%) to mugwort pollen. Furthermore, CJ pollinosis monkeys seemed to be sensitized to these pollen allergens with higher frequency; of 10 monkeys, 10 (100%) had specific IgE to CJ pollen, six (60%) to grass pollen, four (40%) to ragweed pollen, and two (20%) to mugwort pollen. CONCLUSION: Japanese monkeys had specific IgE to grass, ragweed, and mugwort pollen in addition to CJ pollen.

Allergens↗

Reactivity of the immunoglobulin E in bovine gelatin-sensitive children to gelatins from various animals.

It has been reported that most children who showed anaphylaxis to measles, mumps and rubella vaccines containing bovine gelatin as a stabilizer have anti-bovine gelatin IgE. The present study was designed to investigate the reactivity of IgE in bovine gelatin-sensitive children to gelatins from various animals, and the antigenic cross-reactivity between the gelatins. Serum samples taken from 10 children who showed anaphylaxis to vaccines containing bovine gelatin were used in this study. The level of anti-bovine gelatin IgE in these serum samples ranged from 11.0 to 251 Ua/ml. The IgE in most of the children reacted to kangaroo and mouse gelatins, to which they had had little or no exposure as a food or a vaccine stabilizer. The IgE binding to kangaroo and mouse gelatins was completely inhibited by bovine gelatin, whereas reciprocal inhibition was not complete, indicating that antigenic cross-reactivity is present between the mammalian gelatins. Only one child had strong IgE reactivity to fish gelatins, and this reactivity was not inhibited by bovine gelatin, indicating that no antigenic cross-reactivity exists between bovine and fish gelatins. Most of the children who displayed sensitivity to bovine gelatin showed IgE reactivity to other mammalian gelatins. This reactivity may be due primarily to the antigenic cross-reactivity between mammalian gelatins.

Anaphylaxis↗