Biomedical subjects
Publications and source records attributed to N Misawa.
Alpha-hemolytic-like activity from Campylobacter jejuni was clearly apparent when the medium pH ranged from 6.0 to 6.5, but the hemolytic zones disappeared when the pH of the medium increased. Beta-hemolytic-like activity just beneath the bacterial growth appeared after prolonged incubation. The hemolytic activity was not influenced by the species of blood.
The Limulus amoebocyte lysate (LAL) test, a simple and rapid method, was applied for the evaluation of bacterial contamination of carcasses in a slaughterhouse. Twenty-five pork carcasses were examined for bacterial contamination, and we evaluated the correlation between the LAL test and the methods usually employed for detecting indicator organisms of total viable cells, coliforms or Staphylococcus aureus. The reciprocal of the highest dilution found to be positive was used as the LAL index. A high correlation was observed between the LAL index and the number of coliforms, but not between the index and the number of other organisms. The LAL test was accomplished within 2 hr after sampling, and thus may serve as a useful tool for the substitute indicator of coliforms among bacterial contamination in carcasses.
A DNA fragment which has been isolated previously from an Anabaena DNA expression library was subcloned. The corresponding protein was overexpressed in Escherichia coli. The recombinant enzyme was fully active in converting zeta-carotene into lycopene in vitro with neurosporene as an intermediate. A smaller fragment which still contained the active enzyme was sequenced. An open reading frame of 1497 bp was found coding for a protein consisting of 499 amino acids with the calculated molecular weight of 56,740. In a computer search of nucleotide sequences contained in the EMBL nucleotide sequence library, all the best-fitting comparisons were carotenoid desaturases. The highest similarity was found with the crtI phytoene desaturase genes of bacteria and the al-1 gene from Neurospora crassa. A much lower similarity was found with the pds genes coding for phytoene desaturase from cyanobacteria and higher plants. It is shown in protein similarity plots that the amino acid similarity of zeta-carotene desaturase to the latter is mainly limited to the N terminus of the polypeptides. In contrast, the protein similarity plots and a comparison of a conserved region clearly demonstrate that there is a strong relationship between zeta-carotene desaturase and the phytoene desaturases from various bacteria and fungi. Therefore we propose that the zeta-carotene desaturase gene is homologous to the crt I phytoene desaturase genes of bacteria and fungi.
Full length and truncated cDNA expression constructs of the phytoene synthase (psy) gene from tomato have been ligated into a pUC8 cloning vector. One of the truncated constructs was introduced into Escherichia coli carrying the Erwinia uredovora GGPP synthase gene. This transformant produced 15,15'-cis-phytoene, which was identified on the basis of its UV and IR spectral data, from geranylgeranyl diphosphate. The function of this gene product was further confirmed by in vitro assay using cell-free extract of E. coli harboring the construct. On transformation with the above constructs together with a plasmid containing the carotenoid gene cluster from E. uredovora devoid of the phytoene synthase (crtB) gene, yellow, carotenoid-containing, E. coli colonies were produced. The amounts of carotenoids synthesized by the transformed cells, related to the steady-state levels of psy mRNA, varied depending upon the psy constructs. The full-length psy clone produced 16-fold less carotenoids per unit amount of RNA than cells containing phytoene synthase without the first 114 N-terminal amino acids. Removal of further amino acids from the N-terminus caused a large decrease in carotenogenesis. A Western blot of ripe fruit stroma with a monoclonal antibody raised against phytoene synthase revealed a single protein band of apparent molecular mass 38 kDa. Based upon this immunological evidence, we conclude that the size of the transit peptide of phytoene synthase from ripe tomato fruit is approximately 9 kDa, corresponding to about 80 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)
A tissue culture assay system to detect Campylobacter jejuni cytotoxin was developed, and the effect of culture conditions on cytotoxin production was examined. Broth-culture filtrates from clinical isolates were used. The expression of cytotoxicity was dependent on the presence of serum in the culture medium. Although the titre of cytotoxin was low, the greatest cytotoxicity was revealed when newborn calf (NCS), adult bovine or goat serum was added to minimum essential medium. Moderate or weak cytotoxicity was observed when fetal calf (FCS), swine or human serum was added, and no cytotoxicity was seen with the addition of horse, rabbit, cat or chicken serum. Furthermore, the cytotoxic titre in serum-free culture (SFC) for Chinese hamster ovary (CHO) cells was higher than that with serum supplementation. CHO cells showed the highest sensitivity in FCS, NCS and SFC assay systems, whereas HeLa, Vero, HEp-2 and Intestine 407 cells were sensitive only in the NCS assay. Brucella broth was excellent for cytotoxin production, but iron supplementation had no effect. Cytotoxic expression in the three assay systems differed between stationary and stirred cultures. Our results on cytotoxin production by reference strains of C. jejuni did not agree with the original reports. These findings suggest that several different cytotoxins may be produced by C. jejuni, and that the assay system may be important for the expression of cytotoxic activity.
We have engineered a conventional yeast, Saccharomyces cerevisiae, to confer a novel biosynthetic pathway for the production of beta-carotene and lycopene by introducing the bacterial carotenoid biosynthesis genes, which are individually surrounded by the promoters and terminators derived from S. cerevisiae. beta-Carotene and lycopene accumulated in the cells of this yeast, which was considered to be a result of the carbon flow for the ergosterol biosynthetic pathway being partially directed to the pathway for the carotenoid production.
Carotenoids with cyclic end groups are essential components of the photosynthetic membrane in all known oxygenic photosynthetic organisms. These yellow pigments serve the vital role of protecting against potentially lethal photo-oxidative damage. Many of the enzymes and genes of the carotenoid biosynthetic pathway in cyanobacteria, algae and plants remain to be isolated or identified. We have cloned a cyanobacterial gene encoding lycopene cyclase, an enzyme that converts the acyclic carotenoid lycopene to the bicyclic molecule beta-carotene. The gene was identified through the use of an experimental herbicide, 2-(4-methylphenoxy)triethylamine hydrochloride (MPTA), that prevents the cyclization of lycopene in plants and cyanobacteria. Chemically-induced mutants of the cyanobacterium Synechococcus sp. PCC7942 were selected for resistance to MPTA, and a mutation responsible for this resistance was mapped to a genomic DNA region of 200 bp by genetic complementation of the resistance in wild-type cells. A 1.5 kb genomic DNA fragment containing this MPTA-resistance mutation was expressed in a lycopene-accumulating strain of Escherichia coli. The conversion of lycopene to beta-carotene in these cells demonstrated that this fragment encodes the enzyme lycopene cyclase. The results indicate that a single gene product, designated lcy, catalyzes both of the cyclization reactions that are required to produce beta-carotene from lycopene, and prove that this enzyme is a target site of the herbicide MPTA. The cloned cyanobacterial lcy gene hybridized well with genomic DNA from eukaryotic algae, thus it will enable the identification and cloning of homologous genes for lycopene cyclase in algae and plants.
Geranylgeranyl pyrophosphate (GGPP) synthase from Erwinia uredovora was overexpressed in Escherichia coli and purified to homogeneity from solubilized inclusion bodies. In this protein the first 13 N-terminal amino acids were replaced by 16 other amino acids resulting from the cloning vector pUC18. Nevertheless, the enzyme showed activity after purification which could be stimulated sixfold by appropriate activation conditions. The homogeneous enzyme was used to study substrate and product specificity as well as to determine Km values for isopentenyl pyrophosphate, dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), and farnesyl pyrophosphate (FPP). Reaction rates and Km values indicate that FPP and GPP are the genuine allylic substrates for GGPP synthase, but not DMAPP. Independent of the allylic substrate employed, GGPP was the only reaction product of the enzymatic reaction.
In this work the Neurospora crassa al-3 gene function was determined. Geranylgeranyl pyrophosphate (GGPP) synthase activity was measured in al-2 FGSC 313 and al-3 RP100 FGSC 2082 mutant strains by in vitro synthesis methods. This experiment showed that al-3 RP100 mutant expresses a reduced GGPP synthase activity. The mutated al-3 gene was cloned and sequenced; a single missense mutation was found changing serine into asparagine. Genetic complementation was performed by Escherichia coli transformation, with clusters of crt genes from Erwinia uredovora. Carotenoid accumulation was observed in E. coli transformants when the N. crassa al-3 gene substitutes the GGPP synthase gene (crtE) in the carotenogenic crt cluster. Cell-free studies with E. coli transformants gave direct evidence of the function of the al-3 protein as GGPP synthase and indicated that a short-chain prenylpyrophosphate, such as dimethylallyl pyrophosphate, is the genuine substrate.
Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides. This enzyme shows diversity concerning its function and amino acid homology among various organisms. The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter. This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants. The production and processing of the corresponding protein could be demonstrated by Western blotting. Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids. A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of beta-carotene. In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon.
A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus. This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli. The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization. Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis. The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E. coli cell suspension. On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined. An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps. Upon removal of urea, desaturase activity was restored. The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. FAD was involved in desaturation, whereas NAD and NADP were inhibitory. This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state.
The first committed step in the biosynthetic pathway of carotenoids in plants and algae is the conversion of geranylgeranyl pyrophosphate (GGPP) to prephytoene pyrophosphate (PPPP), which is converted to phytoene. We have cloned the gene pys that encodes the enzyme phytoene synthase in the cyanobacterium Synechococcus PCC7942. The co-expression of pys in cells of Escherichia coli together with the gene crtE from Erwinia uredovora, which encodes geranylgeranyl pyrophosphate synthase, resulted in accumulation of phytoene. This result indicates that phytoene synthase is a single polypeptide enzyme that catalyzes the 2-step reaction from GGPP to phytoene. The deduced amino acid sequence of pys is highly conserved with that of pTOM5, a tomato cDNA that is differentially expressed during fruit ripening. These findings suggest that pTOM5 encodes phytoene synthase in tomato.
The role of carotenoid genes crtB and crtE has been functionally assigned. These genes were cloned from Erwinia into Escherichia coli or Agrobacterium tumefaciens. Their functions were elucidated by assaying early isoprenoid enzymes involved in phytoene formation. In vitro reactions from extracts of E. coli carrying the crtE gene or a complete carotenogenic gene cluster in which crtB was deleted showed an elevated conversion of farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP). These results strongly indicate that the crtE gene encodes GGPP synthase. Introduction of the crtB gene into A. tumefaciens led to the conversion of GGPP into phytoene. This activity was absent in similar transformants with the crtE gene. Thus, the crtB gene probably encodes phytoene synthase, which was further supported by demonstration that phytoene accumulated in E. coli harboring both the crtB and crtE genes.
The Erwinia uredovora crtB, crtE, crtI, and crtY genes required for beta-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized. The transconjugants of Z. mobilis and A. tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 micrograms of beta-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.
The most important function of carotenoid pigments, especially beta-carotene in higher plants, is to protect organisms against photooxidative damage (G. Britton, in T. W. Goodwin, ed., Plant Pigments--1988, 1988; N. I. Krinsky, in O. Isler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). beta-Carotene also functions as a precursor of vitamin A in mammals (G. A. J. Pitt, in I. Osler, H. Gutmann, and U. Solms, ed., Carotenoids--1971, 1971). The enzymes and genes which mediate the biosynthesis of cyclic carotenoids such as beta-carotene are virtually unknown. We have elucidated for the first time the pathway for biosynthesis of these carotenoids at the level of enzyme-catalyzed reactions, using bacterial carotenoid biosynthesis genes. These genes were cloned from a phytopathogenic bacterium, Erwinia uredovora 20D3 (ATCC 19321), in Escherichia coli and located on a 6,918-bp fragment whose nucleotide sequence was determined. Six open reading frames were found and designated the crtE, crtX, crtY, crtI, crtB, and crtZ genes in reference to the carotenoid biosynthesis genes of a photosynthetic bacterium, Rhodobacter capsulatus; only crtZ had the opposite orientation from the others. The carotenoid biosynthetic pathway in Erwinia uredovora was clarified by analyzing carotenoids accumulated in E. coli transformants in which some of these six genes were expressed, as follows: geranylgeranyl PPiCrtB----prephytoene PPiCrtE----phytoeneCrtI---- lycopeneCrtY----beta-caroteneCrtZ----zeaxanthinCrtX--- -zeaxanthin-beta- diglucoside. The carotenoids in this pathway appear to be close to those in higher plants rather than to those in bacteria. Also significant is that only one gene product (CrtI) for the conversion of phytoene to lycopene is required, a conversion in which four sequential desaturations should occur via the intermediates phytofluene, zeta-carotene, and neurosporene.
We have successfully produced transgenic mice that carry the ret oncogene driven by a mouse mammary tumor virus promoter/enhancer. Mammary and salivary gland adenocarcinomas were developed in a stochastic fashion in these mice. Moreover, premalignant tumors with hyperplastic and dysplastic lesions of Harderian glands and male reproductive tracts frequently occurred at young ages. High expression of the transgene was closely associated with the development of these tumors although the levels of the transgene expression were variable among individuals. In addition, large amounts of phosphotyrosine-containing proteins were detected in cell lysates from mammary and salivary adenocarcinomas by immunoblotting with the anti-phosphotyrosine antibody.
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