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T Todo

Publications and source records attributed to T Todo.

At least 55 records · Page 3Linked to original sources
PubMed1998

A new Drosophila ultraviolet light-damaged DNA recognition endonuclease that selectively nicks a (6-4) photoproduct site.

We have previously described the purification of an ultraviolet light (UV) damage-specific DNA-binding protein from Drosophila melanogaster, designated D-DDB P1 [Nucleic Acids Res., 23 (1995) 2600-2607]. Here, we obtained highly purified D-DDB P1 from Drosophila Kc cells, and we found that D-DDB P1 is also a nuclease. D-DDB P1 can selectively bind to pyrimidine (6-4) pyrimidone photoproducts, and in the presence of Mg++, D-DDB P1 can catalyze an incision immediately on the 3' and 5' sides of the (6-4) photoproduct site.

Animals↗
PubMed1998

Neuronal localization of fibroblast growth factor-9 immunoreactivity in human and rat brain.

Fibroblast growth factor-9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting-type factor that exhibits a growth-stimulating effect on cultured glial cells. In order to elucidate the roles of FGF-9 in the central nervous system, we investigated in detail the distribution of FGF-9 proteins in the normal human and rat brains by immunohistochemistry using two different antibodies specific to FGF-9. In both human and rat, a strong expression of FGF-9 immunoreactivity was localized mainly in neurons throughout the normal brain. Immunoreactive glial cells were rarely encountered. In the human brain, strong and uniform immunoreactivity was observed in neurons of cerebral cortex, hippocampus, substantia nigra, motor nuclei of the brainstem, and Purkinje cell layer. A detailed mapping in the rat brain showed a distribution of FGF-9 immunoreactivity in a widespread population of neurons, though the intensity varied between different locations and even among the same nucleus. The most prominent expression in rat was observed in neurons of the mitral cell layer of the olfactory bulb, red nucleus, mesencephalic trigeminal nucleus, motor trigeminal nucleus, facial nucleus, reticular nucleus and Purkinje cell layer. These findings suggest that FGF-9 plays an important role in the central nervous system and may have a potential function closely connected to neurons in the normal brain.

Animals↗
PubMed1998

Cloning and characterization of a gene (UVR3) required for photorepair of 6-4 photoproducts in Arabidopsis thaliana.

UV radiation induces two major classes of pyrimidine dimers: the pyrimidine [6-4] pyrimidone photoproduct (6-4 product) and the cyclobutane pyrimidine dimer (CPD). Many organisms produce enzymes, termed photolyases, that specifically bind to these damage products and split them via a UV-A/blue light-dependent mechanism, thereby reversing the damage. These photolyases are specific for either CPDs or 6-4 products. A gene that expresses a protein with 6-4 photolyase activity in vitro was recently cloned from Drosophila melanogaster and Xenopus laevis. We report here the isolation of a homolog of this gene, cloned on the basis of sequence similarity, from the higher plant Arabidopsis thaliana. This cloned gene produces a protein with 6-4 photolyase activity when expressed in Escherichia coli. We also find that a previously described mutant of Arabidopsis (uvr3) that is defective in photoreactivation of 6-4 products carries a nonsense mutation in this 6-4 photolyase homolog. We have therefore termed this gene UVR3. Although homologs of this gene have previously been shown to produce a functional 6-4 photolyase when expressed in heterologous systems, this is the first demonstration of a requirement for this gene for photoreactivation of 6-4 products in vivo.

Amino Acid Sequence↗
PubMed1998

Expression and growth stimulatory effect of fibroblast growth factor 9 in human brain tumors.

OBJECTIVE: Fibroblast growth factor 9 (FGF-9) is a relatively new member of the FGF family isolated from the conditioned medium of a human glioblastoma cell line as a secreting type factor that exhibits a growth-stimulating effect on primary glial cells. To elucidate the roles of FGF-9 in human brain tumors, the expression and biological activities of FGF-9 were studied using culture cells and surgically obtained tumor specimens. METHODS: Measurement of FGF-9 and basic FGF in conditioned media of cell cultures was performed by using a sandwich enzyme immunoassay. The mitogenic effect of FGF-9 was evaluated by cell growth studies. FGF-9 expression in vivo was demonstrated by immunohistochemistry. RESULTS: One of 4 glioma cell lines and 4 of 16 human meningiomas examined actually secreted detectable amounts of FGF-9 proteins. In comparison, basic FGF production was detected from 3 of 4 glioma cell lines and 11 of 16 human meningiomas. Similarly to basic FGF, recombinant human FGF-9 significantly stimulated the in vitro cell proliferation in three of four glioma cell lines investigated in a dose-dependent manner. A time course growth study using U87 MG cells revealed an accelerated growth stimulation by FGF-9 after Day 4. The growth stimulatory activity was also shown in three of four human meningiomas studied. Moderate to strong immunoreactivity for FGF-9 was observed in 40 (82%) of 49 human brain tumors examined irrespective of origin, tumor type, grade of malignancy, or whether initial or recurrent. In contrast, strong immunostaining was localized in neurons in the normal human cerebral cortex. CONCLUSION: The present findings suggest that FGF-9 may be involved in the biology of human brain tumors with a possible importance in tumor cell growth. Whether the growth factor is more generally involved in oncogenesis of human tumors awaits further investigation.

Brain↗
PubMed1997

Binding and catalytic properties of Xenopus (6-4) photolyase.

Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6-4]T in double-stranded DNA with high affinity (KD = 10(-9)) and to T[6-4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (KD = approximately 2 x 10(-8)). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (koff = 2.9 x 10(-5) s-1). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (phi) of repair is approximately 0.11. The fully reduced form (E-FADH-) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis, syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.

Animals↗
PubMed1997

Identification of the midbrain locomotor nuclei and their descending pathways in the teleost carp, Cyprinus carpio.

In order to identify the mesencephalic spinal pathways for initiation of swimming in the carp, we employed electrical and chemical microstimulation of the mesencephalic tegmentum. Electrical stimulation of the midbrain in decerebrate carp produced bilateral or unilateral rhythmic movements of the tail. Bilateral alternating movements were induced by stimulation with the lowest threshold currents to the brain region just beneath the third ventricle at the level of the mid mesencephalon. The region included the nucleus of medial longitudinal fasciculus (Nflm), the medial longitudinal fasciculus (flm), the red nucleus (Nrb). To specify the nuclei of the origin of the descending pathway, we microinjected 0.1 M L-glutamic acid to the region. Both bilateral and unilateral tail movements were induced, the majority being the latter. The unilateral movements were accompanied with tail flips toward the ipsilateral side of stimulation sites. The smallest injection volume required for initiation of the movement was recorded at the Nflm. Bilateral tail movements were produced only by injections into the medial region between the nucleus of the both sides. The present results imply a crucial role of Nflm neurons in the initiation of swimming Nflm neurons on one side project through flm to the ipsilateral spinal cord along its entire length and regulate activities of the individual central pattern generators.

Animals↗
PubMed1997

Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase.

Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development. Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts [(6-4)photoproducts] are the two major photoproducts produced in DNA by UV irradiation. Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts [(6-4)photolyase]. (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism. The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family. Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity. Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD. This is the first indication of FAD as the chromophore of (6-4)photolyase.

Animals↗
PubMed1997

Motor neurons in human and rat spinal cord synthesize fibroblast growth factor-9.

Fibroblast growth factor (FGF)-9, initially referred to as a glia-activating factor, is a recently identified member of the FGF family. In the present study we demonstrated that spinal cord motor neurons and dorsal root ganglion neurons were strongly immunostained with specific antibodies to FGF-9 in human and rat tissues. By in situ hybridization using digoxigenin-labeled antisense probe to FGF-9 mRNA, we found specific signals in these neurons in rat. By immunoblotting analysis, we detected a 30/29 kDa doublet band in human spinal cord proteins, which corresponded to the doublet band of originally isolated FGF-9 from culture media. Our results indicate that these neurons synthesize FGF-9.

Animals↗
PubMed1997

Molecular evolution of the photolyase-blue-light photoreceptor family.

The photolyase-blue-light photoreceptor family is composed of cyclobutane pyrimidine dimer (CPD) photolyases, (6-4) photolyases, and blue-light photoreceptors. CPD photolyase and (6-4) photolyase are involved in photoreactivation for CPD and (6-4) photoproducts, respectively. CPD photolyase is classified into two subclasses, class I and II, based on amino acid sequence similarity. Blue-light photoreceptors are essential light detectors for the early development of plants. The amino acid sequence of the receptor is similar to those of the photolyases, although the receptor does not show the activity of photoreactivation. To investigate the functional divergence of the family, the amino acid sequences of the proteins were aligned. The alignment suggested that the recognition mechanisms of the cofactors and the substrate of class I CPD photolyases (class I photolyases) are different from those of class II CPD photolyases (class II photolyases). We reconstructed the phylogenetic trees based on the alignment by the NJ method and the ML method. The phylogenetic analysis suggested that the ancestral gene of the family had encoded CPD photolyase and that the gene duplication of the ancestral proteins had occurred at least eight times before the divergence between eubacteria and eukaryotes.

Amino Acid Sequence↗
PubMed1997

Characterization of a human homolog of (6-4) photolyase.

(6-4)Photolyase catalyzes light-dependent repair of UV-induced pyrimidine (6-4) pyrimidone photoproducts. A human cDNA clone which has high sequence homology to the (6-4)photolyase gene (H64PRH gene) was identified. In this paper we also isolated a genomic clone corresponding to the H64PRH cDNA and mapped it to chromosome 12q24.1 by fluorescence in situ hybridization (FISH). Northern-blot analysis revealed transcription of this gene in all human tissues examined. The H64PRH protein was overproduced in E. coli, partially purified and characterized. Like (6-4)photolyase, the enzyme contains two chromophores, one of which is FAD. However, the enzyme does not show any detectable photolyase activity.

Chromosome Mapping↗
PubMed1997

Non-mutagenic repair of (6-4)photoproducts by (6-4)photolyase purified from Drosophila melanogaster.

The (6-4)photoproduct DNA photolyase ((6-4)photolyase) repairs UV-induced pyrimidine (6-4) pyrimidone photoproduct ((6-4)photoproduct, pyr[6,4]pyr) in a light dependent manner. Drosophila (6-4)photolyase was purified to near homogeneity from Drosophila embryonic cells and is shown to be a 62 kDa protein as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified (6-4)photolyase repairs (6-4)photoproducts induced at 5'-CC-3' site (C[6,4]C) as well as T[6,4]T and T[6,4]C. Photoreactivation of (6-4)photoproduct constructed in M13 phage eliminates the replication block and abolishes induced mutagenesis in E. coli cells, suggesting that the (6-4)photolyase repairs the photoproduct to the unmodified form.

Animals↗
PubMed1997

Photoreactivating enzyme for (6-4) photoproducts in cultured goldfish cells.

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6-4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6-4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6-4) photoproducts and its dissociation from (6-4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of protein(s) to (6-4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA photoreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6-4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6-4) photolyase in cultured goldfish cells as in Drosophila, Xenopus and Crotalus.

Animals↗
PubMed1996

Putative human blue-light photoreceptors hCRY1 and hCRY2 are flavoproteins.

Recently, a human cDNA clone with high sequence homology to the photolyase/blue-light photoreceptor family was identified. The putative protein encoded by this gene exhibited a strikingly high (48% identity) degree of homology to the Drosophila melanogaster (6-4) photolyase [Todo et al. (1996) Science 272, 109-112]. We have now identified a second human gene whose amino acid sequence displays 73% identity to the first one and have named the two genes CRY1 and CRY2, respectively. The corresponding proteins hCRY1 and hCRY2 were purified and characterized as maltose-binding fusion proteins. Similar to other members of the photolyase/blue-light photoreceptor family, both proteins were found to contain FAD and a pterin cofactor. Like the plant blue-light photoreceptors, both hCRY1 and hCRY2 lacked photolyase activity on the cyclobutane pyrimidine dimer and the (6-4) photoproduct. We conclude that these newly discovered members of the photolyase/photoreceptor family are not photolyases and instead may function as blue-light photoreceptors in humans.

Amino Acid Sequence↗
PubMed1996

Purification and characterization of Drosophila melanogaster photolyase.

Animal-type photolyases have very limited sequence homology to microbial-type photolyases. We wanted to find out whether the two types of enzymes have different or similar biochemical and photochemical properties. In particular, the chromophore/cofactor composition of animal photolyases is of special interest since the presence and nature of a second chromophore in these enzymes are not known in contrast to the microbial photolyases which contain FAD cofactor, and folate or deazaflavin as second chromophores. We overproduced the Drosophila melanogaster photolyase in Escherichia coli using the cloned gene. The enzyme contains FAD and folate and thus belongs in the folate class of enzymes but with an action spectrum peak at 420 nm.

Absorption↗
PubMed1996

Molecular cloning and characterization of Japanese eel estrogen receptor cDNA.

A cDNA encoding the Japanese eel, Anguilla japonica, estrogen receptor (ER) was isolated and sequenced. This cDNA contains a complete open reading frame encoding 573 amino acid residues, and the molecular weight of this protein is calculated to be 63417. The amino acid sequence of the eel ER shows high homology of the DNA binding (80%) and ligand binding (55%) domains with those of other species. However, the other domains show greatly reduced homology (10-20%). When the cDNA was ligated to the pSVL vector and transfected into COS7 cells, a protein was produced that had high affinity for estradiol-17 beta (E2) and specifically bound estrogens. Northern analysis showed that three ER mRNAs with lengths of 5.6, 3.8 and 1.2 kb were expressed in eel liver. Their expression was E2 inducible, with the 5.6 kb mRNA being strongly dependent on E2 stimulation.

Amino Acid Sequence↗
PubMed1996

Similarity among the Drosophila (6-4)photolyase, a human photolyase homolog, and the DNA photolyase-blue-light photoreceptor family.

Ultraviolet light (UV)-induced DNA damage can be repaired by DNA photolyase in a light-dependent manner. Two types of photolyase are known, one specific for cyclobutane pyrimidine dimers (CPD photolyase) and another specific for pyrimidine (6-4) pyrimidone photoproducts[(6-4)photolyase]. In contrast to the CPD photolyase, which has been detected in a wide variety of organisms, the (6-4)photolyase has been found only in Drosophila melanogaster. In the present study a gene encoding the Drosophila(6-4)photolyase ws cloned, and the deduced amino acid sequence of the product was found to be similar to the CPD photolyase and to the blue-light photoreceptor of plants. A homolog of the Drosophila (6-4)photolyase gene was also cloned from human cells.

Amino Acid Sequence↗
PubMed1996

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) inhibits the binding activity of T4 endonuclease V to UV-damaged DNA.

3-Amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) is a mutagen/carcinogen derived from cooked foods which enhances the induction of mutations and chromosome aberrations by UV without microsomal activation. These co-mutagenic effects are considered to arise from inhibition of DNA excision repair at the incision step. However, the inhibition mechanism has not been clarified. In this study we show, using agarose gel electrophoresis, that Trp-P-1 inhibits incision by T4 endonuclease V, which cleaves DNA at the site of cyclobutane dimers. Trp-P-1 also inhibits the binding of this enzyme to UV-damaged DNA in a gel shift assay. In addition, the results of DNA unwinding assay with topoisomerase I suggest that Trp-P-1 intercalates into DNA molecules. The known intercalators ethidium bromide and acriflavine demonstrate similar effects in these experiments. However, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which showed no co-mutagenic effects in our previous study, does not demonstrate such effects. These results suggest that Trp-P-1 changes DNA conformation by intercalation, causing inhibition of binding of repair enzymes to UV-damaged DNA, and this in turn leads to inhibition of DNA excision repair and to co-mutagenic effects.

Carbolines↗