Biomedical subjects

O Nikaido

Publications and source records attributed to O Nikaido.

At least 37 records · Page 2Linked to original sources

PubMed1999

Conformational multiplicity of the antibody combining site of a monoclonal antibody specific for a (6-4) photoproduct.

The antigen binding site of monoclonal antibody 64M5, which possesses a high degree of affinity for DNA containing pyrimidine (6-4) pyrimidone photoproducts, were investigated by use of stable-isotope-assisted NMR spectroscopy. A variety of 64M5 Fab fragments specifically labeled with 13C and 15N at backbone amide groups were prepared. Extensive assignments of amide resonances originating from the variable region of 64M5 were made by using 2D-HN(CO) measurements along with recombination of the heavy and light chains of 64M5. On the basis of chemical shift changes of the amide resonances caused upon addition of d(T[6-4]T) and d(GTAT[6-4]TATG), the binding sites of 64M5 Fab for the (6-4) photodimer and for the oligodeoxynucleotides flanking it were identified. It was revealed that the L1 and L3 segments, which are responsible for the binding to (6-4) photodimer, exhibit conformational multiplicities in the absence of antigens, and take different conformations between the d(T[6-4]T) and d(GTAT[6-4]TATG)-bound forms. On the basis of spectral comparison with another Fab fragment with a similarity in the amino acid sequence of the VL domain of 64M5, we suggest that the conformational multiplicities observed in the present study is caused by a substitution of an amino acid residue at the position of a key residue in L3 canonical structure, which leads to a preferable effect on the antigen binding, and by a specific combination of L1 and L3 canonical structures.

Amino Acid Sequence↗

PubMed1999

Probing the interaction between a high-affinity single-chain Fv and a pyrimidine (6-4) pyrimidone photodimer by site-directed mutagenesis.

We have used site-directed mutagenesis to uncover the origin of the binding affinity differences exhibited by a series of monoclonal antibodies that recognize pyrimidine (6-4) pyrimidone photoproducts in the context of single- or double-stranded DNA. In this study, we have focused on two antibodies-64M3 and 64M5-that share the same binding specificity but differ in their affinities. We used single-chain Fv (scFv) derivatives for these studies since they can be easily expressed in Escherichia coli. To facilitate this, we also developed a simple, on-column refolding procedure for scFvs that is rapid and does not require high dilution. We took several precautions to ensure that the scFvs faithfully reflected the behavior of the parent monoclonal antibodies. Results obtained from chimeric scFvs constructed from 64M3 and 64M5 suggested that the higher affinity of the 64M5 antibody was mainly due to its VL region. Loop-grafting studies in which VH CDR loops of 64M3 were individually transplanted into 64M5 were consistent with this hypothesis. Since the VL sequences of 64M3 and 64M5 differ at only three positions (L30, L50, and L90), alanine-scanning mutagenesis was used to assess the importance of these three residues in DNA binding by 64M5. These studies highlighted the importance of all three VL CDR loops; furthermore, they suggested that photoproduct binding involved conformational changes within the VL region.

Alanine↗

PubMed1999

Development and characterization of a DNA solar dosimeter.

In this paper, we report the development and characterization of a solar ultraviolet (UV) dosimetry system that can be used as a film badge for radiation monitoring. DNA molecules are coated on a thin nylon membrane as a UV dosimeter. The membrane is sealed in a polyethylene filter envelope with silica gel to keep the humidity low. After exposure to UV or solar light, induced DNA damage is measured by an immunochemical reaction. The intensity of color developed during the immunological reaction can be correlated linearly with the irradiated UV dose delivered by an Oriel solar simulator within a limited dose range. We observe no effects of temperature on the level of damage induction. The membrane is proficient for measuring DNA damage for more than 21 days when stored at either 37 or 4 degrees C. The induced damage remains stable on the membrane for at least 22 days at both 37 and 4 degrees C. In addition to these indoor experiments, we report measurements of solar UV dose in outdoor experiments.

DNA↗

PubMed1999

The relative expression of mutated XPB genes results in xeroderma pigmentosum/Cockayne's syndrome or trichothiodystrophy cellular phenotypes.

The human XPB DNA helicase is a subunit of the DNA repair/basal transcription factor TFIIH, involved in early steps of the nucleotide excision repair pathway. Two distinct clinical phenotypes, xeroderma pigmentosum associated with Cockayne's syndrome (XP/CS) and trichothiodystrophy (TTD), can be due to mutations in the XPB gene. In the present work, we studied cellular DNA repair properties of skin fibro-blasts from two patients mutated in the XPB gene: an XP/CS patient cell (XPCS2BA) with a T296C (F99S) transition and a TTD patient cell (TTD6VI) exhibiting an A355C (T119P) transversion. Both cells are clearly associated with different levels of alterations in their response to UV light. To establish the relationship between the relative expression level of these two alleles and DNA repair properties, we transfected SV40-transformed XPCS2BA (XPCS2BASV) cells with a plasmid (pTTD6VI) carrying the XPB-A355C cDNA and examined DNA repair properties after UV irradiation (cell survival, unscheduled DNA synthesis and kinetics of photoproduct removal) in stable transfectants. We isolated three clones, which express the XPB-A355C gene (Cl-5) or the XPB-T296C gene (Cl-14) or both genes (Cl-19). This con-stitutes a model system allowing us to correlate the relative expression levels of the XPB-A355C (TTD) and XPB-T296C (XP/CS) genes with various DNA repair properties. Overexpression of the XPB-A355C (TTD) gene in an XP/CS cell gives rise to a cellular phenotype of increased repair similar to that of TTD6VI cells, while equal expression of the two mutated genes leads to an intermediate cellular phenotype between XP/CS and TTD.

Cell Line, Transformed↗

PubMed1999

Crystal structures of the 64M-2 and 64M-3 antibody Fabs complexed with DNA (6-4) photoproducts.

Crystal structures of the 64M-2 antibody Fab fragment complexed with DNA photoproducts of dT(6-4)T and dTT(6-4)TT, and of the 64M-3 Fab fragment complexed with dT(6-4)T were determined. The 5'-thymine base of the bound dT(6-4)T ligand is in a half-chair conformation, and its base plane is nearly perpendicular to the planar 3'-pyrimidone base. The 64M-2 and 64M-3 Fabs have a common structure suitable for accommodating the dT(6-4)T ligand. In each of the antigen binding sites of the 64M-2 and 64M-3 Fabs, basic residues of His 35H and Arg 95H are located at the bottom of the binding pocket, and are hydrogen-bonded to the base moieties of dT(6-4)T. Two water molecules are involved in the interactions that intervene between the base moieties and the binding site. Aromatic residues of Trp 33H and Tyr 100iH form a side-wall of the pocket and are in van der Waals interactions with the base moieties. The Trp 33H side-chain is placed in parallel to the 3'-pyrimidone base, and the Tyr 100iH side-chain is nearly perpendicular to the 5'-thymine base. His 27dL, Tyr 32L, Leu 93L, and Ser 58H forming another side-wall are located in the vicinity of the sugar-phosphate backbone. In the 64M-2 Fab complex with dTT(6-4)TT, 5'- and 3'-side phosphate groups are also involved in interaction with Fab residues.

Crystallography, X-Ray↗

PubMed1999

High levels of 8-hydroxy-2'-deoxyguanosine appear in normal human epidermis after a single dose of ultraviolet radiation.

Major photoproducts induced by carcinogenic ultraviolet (UV) radiation are the cylobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts (6-4 PPs). 8-Hydroxy-2 -deoxyguanosine (8-OHdG) is also a DNA base-modified product generated by reactive oxygen species in conditions of ultraviolet stress, Although UVB-induced CPDs and 6-4 PPs have been investigated in animal and human skin, little is known about the role of 8-OHdG in UVB-induced human skin damage or carcinogenesis. Normal human skin from three volunteers was exposed to UV radiation, and the time course of induction and removal of 8-OHdG was examined by immunohistochemical analysis with catalysed signal amplification on formalin-fixed paraffin sections. Formation of CPDs and 6-4 PPs was also examined by immunostaining on the same skin specimens. Control epidermis with no exposure to UV radiation showed little nuclear staining of 8-OHdG, but an increased level of 8-OHdG was clearly observed in epidermis biopsied after irradiation. Induced 8-OHdG can rapidly be removed from nucleus during the first 24-48 h, as the staining intensity diminished gradually, almost reaching the control level by 72-96 h after irradiation. Staining for CPDs or 6-4 PPs revealed induction of these photoproducts in human skin, although 6-4 PP-positive cells disappeared more rapidly than those that stained for CPDs or 8-OHdG. Together with protective effect of antioxidants, our results indicate that not only CPDs and 6-4 PPs but also 8-OHdG may play a significant part in UV carcinogenesis.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1999

Postnatal growth failure, short life span, and early onset of cellular senescence and subsequent immortalization in mice lacking the xeroderma pigmentosum group G gene.

The xeroderma pigmentosum group G (XP-G) gene (XPG) encodes a structure-specific DNA endonuclease that functions in nucleotide excision repair (NER). XP-G patients show various symptoms, ranging from mild cutaneous abnormalities to severe dermatological impairments. In some cases, patients exhibit growth failure and life-shortening and neurological dysfunctions, which are characteristics of Cockayne syndrome (CS). The known XPG protein function as the 3' nuclease in NER, however, cannot explain the development of CS in certain XP-G patients. To gain an insight into the functions of the XPG protein, we have generated and examined mice lacking xpg (the mouse counterpart of the human XPG gene) alleles. The xpg-deficient mice exhibited postnatal growth failure and underwent premature death. Since XPA-deficient mice, which are totally defective in NER, do not show such symptoms, our data indicate that XPG performs an additional function(s) besides its role in NER. Our in vitro studies showed that primary embryonic fibroblasts isolated from the xpg-deficient mice underwent premature senescence and exhibited the early onset of immortalization and accumulation of p53.

Animals↗

PubMed1999

Somatic cell mutation induced by sunlight in Drosophila.

There is ample epidemiological evidence showing that sunlight can cause skin cancer in the human. In experimental studies, simulated sunlight or UV lamps are used for demonstrating carcinogenesis and other biological effects. Little studies, however, have been performed using natural sunlight itself. In this work, we have examined the mutagenicity of natural sunlight in Drosophila. The Drosophila wing spot test is useful to detect somatic cell mutations. Third instar larvae in petri dishes were exposed to sunlight (ultraviolet region with < 290 nm wavelength cut off by a plastic cover) in the yard of Okayama University campus (north latitude: 34 degrees 39', east longitude: 133 degrees 55'). The sunlight was mutagenic in Drosophila larvae and produced pyrimidine dimers in their DNA. In the observed mutagenicity, there was dependence on the exposure period and UV fluence. During the two-year monitoring, the highest induction of mutant spot observed was 1.98 total spots/wing on June 25, 1998, and the lowest was 0.64 on December 29, 1998, while non-exposure spontaneous spots were 0.29 and 0.32 on these days, respectively. Thus, solar radiation was mutagenic both in summer and in winter.

Animals↗

PubMed1998

Mutational analysis of a function of xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair.

To analyze the function of the xeroderma pigmentosum group A (XPA) protein in strand-specific DNA repair, we examined repair of UV-induced cyclobutane pyrimidine dimer (CPD) in transcribed and non-transcribed strands of the dihydrofolate reductase gene of xeroderma pigmentosum group A (XP-A) cell line (XP12ROSV) which was transfected with various types of mutant XPA cDNA. The transfectant overexpressing mutant XPA with a defect in the interaction with either ERCC1, replication protein A (RPA), or general transcription factor TFIIH, showed more or less decreased repair of CPD in each strand in parallel, while in the transfectant overexpressing R207G (Arg207to Gly) mutant XPA derived from XP129, a UV-resistant XP12ROSV revertant, the rate of CPD repair was almost normal in each strand. We also examined the dose responses of the XPA protein on CPD repair in each strand by the modulation of the expression levels of wild-type or R207G mutant XPA using an inducible expression system, LacSwitchtrade mark promoter. There were good correlations between the rate of CPD repair in each strand and the amount of XPA protein produced in these Lac cells. Our results indicate that the XPA protein is equally important for the CPD repair in both transcribed and non-transcribed strands and that the R207G mutation found in XP129 may not be responsible for a selective defect in CPD repair in the non-transcribed strand in XP129.

Cell Line↗

PubMed1998

31P NMR study of the interactions between oligodeoxynucleotides containing (6-4) photoproduct and Fab fragments of monoclonal antibodies specific for (6-4) photoproduct.

A 31P nuclear magnetic resonance (NMR) study of the interactions between oligonucleotides containing the (6-4) photoproduct and the Fab fragments of monoclonal antibodies (64M3 and 64M5) recognizing the (6-4) photoproduct is reported. The 31P chemical shift data indicate that backbone conformation of (64) adduct is affected by the presence of flanking oligodeoxynucleotides, and (6-4) adducts with different backbone conformations are accommodated in the antigen binding sites of these antibodies. It was also revealed that epitopes for these antibodies consist of not only the (6-4) adduct but the flanking di- or tri-deoxynucleotides on both the 5' and 3' sides as well.

Antibodies, Monoclonal↗

PubMed1998

Antibodies specific for (6-4) DNA photoproducts: cloning, antibody modeling and construction of a single-chain Fv derivative.

We have investigated a series of four monoclonal antibodies that specifically recognize pyrimidine (6-4) pyrimidone photoproducts. One of these antibodies (64M4), bound all four possible pyrimidine-pyrimidone photoadducts with equal affinities whereas the others (64M2, 64M3 and 64M5) were selective for TC and TT sequences. In addition, 64M5 had the highest binding affinity for photodamaged DNA of the four [T. Mori et al., Photochem. Photobiol. 54 (1991) 225-232]. To help understand the differences between these antibodies, we have cloned and sequenced the variable region genes from all four. Comparing these sequences revealed that all four were highly similar to one another, although there were some differences in potential antigen-contact regions. To assess the influences of these sequence differences at the structural level, computer models were constructed for all four antibodies. Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. A single-chain Fv derivative of 64M5 was therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested. This derivative could be isolated from Escherichia coli using two chromatographic steps and possessed the same binding specificity as the parent monoclonal antibody.

Amino Acid Sequence↗

PubMed1998

ATM mutations in patients with ataxia telangiectasia screened by a hierarchical strategy.

ATM has been identified as a gene that is responsible for ataxia telangiectasia (AT), a pleiotropic disorder of autosomal recessive inheritance. While many mutations of this gene in AT patients of various ethnicities have been reported, data on Japanese patients are scarce. In this report, we present the results of a thorough survey of ATM mutations in 14 unrelated AT patients, with an emphasis on Japanese subjects. We used a hierarchical strategy in which we extensively analyzed the entire coding region of the cDNA. In the first stage, point mutations were sought by PCR-SSCP in short patches. In the second and third stages, the products of medium- and long-patch PCR, each covering the entire region, were examined by agarose gel electrophoresis to search for length changes. We found a total of 15 mutations (including 12 new) and 4 polymorphisms. Abnormal splicing of ATM was frequent among Japanese, and no hotspot was obvious, suggesting no strong founder effects in this ethnic group. Eleven patients carried either one homozygous or two compound heterozygous mutations, one patient carried only one detectable heterozygous mutation, and no mutation was found in two patients. Overall, mutations were found in at least 75% of the different ATM alleles examined. Possible reasons for the inability to detect mutations in some patients are discussed.

Ataxia Telangiectasia↗

PubMed1998

Enhanced human tumor cell transplantability in a new congenic immunodeficient mouse; KSN-BNX.

We introduced two mutant genes (beige; bg that induces the deficiency of natural killer (NK) activity and xid that decreases the production of immunoglobulin) into KSN nude mice with high reproductive performances. We produced KSN bg/bg(nu/nu) (KSN-bg), KSN-xid/xid(nu/nn) (KSN-xid), KSN xid/xid,bg/bg(nu/nu) (KSN-BNX) and KSN-nu/+ (KS) mice by back-cross (cross-intercross method). All strains showed as high a reproductivity rate as the parental KSN mice. KSN-xid and KSN-BNX mice had a reduced percentage of B220 positive cells in the spleens compared to KSN and KSN-bg mice, but they showed increased percentages of Thy-1 and asialo GM1 positive cells. The serum immunoglobulin concentrations of KSN-BNX were as low as KSN-xid. Both KSN-bg and KSN-BNX mice showed deficient NK activity in spleens, whereas KSN-xid mice showed an elevated NK activity. Compared to nude mice, the growth of both human tumor cell TCO-1 and BxPc-3 transplanted subcutaneously was enhanced in KSN-BNX mice. However Panc-1 cells that was rejected in nude mice was not accepted in KSN-BNX mice. Liver metastasis of human pancreatic tumor cells; Capan-1, BxPc-3 and MIAPaCa-2 were studied. No significant difference was observed in the percentage of metastasis formed mice between nude and KSN-BNX mice.

Animals↗

PubMed1998

Human melanocytes and keratinocytes exposed to UVB or UVA in vivo show comparable levels of thymine dimers.

Epidemiology shows a relationship between solar exposure and all types of skin cancer. Understanding the mechanisms of skin cancer requires knowledge of the photomolecular events that occur within the relevant epidermal cell types in vivo. Studies to date have focused on UVR-induced DNA lesions in keratinocytes, the majority epidermal cell population which gives rise to most skin cancers. Malignant melanoma, arising from melanocytes (5%-10% of epidermal cells), accounts for most skin cancer deaths. We report on new techniques to detect DNA photolesions in human epidermal melanocytes in situ. Previously nonexposed buttock skin of volunteers of skin types I/II was exposed to clinically relevant doses of narrow bandwidth UVB (300 nm) and UVA (320 nm, 340 nm, 360 nm) radiation. Biopsies were taken immediately afterwards and processed for routine histology. Microscope sections were prepared and double-stained with fluorescent-tagged monoclonal antibodies for thymine dimers and melanocytes. UVR dose-response curves for dimer levels within melanocyte nuclei were determined by image analysis and compared with dimer levels in adjacent basal cell keratinocytes. Our data show that UVB and UVA readily induce thymine dimers in melanocytes at levels that are comparable with those found in adjacent keratinocytes. This new technique will enable melanocyte specific studies, such as DNA repair kinetics, to be done in vivo.

Adult↗

PubMed1998

The similarity of action spectra for thymine dimers in human epidermis and erythema suggests that DNA is the chromophore for erythema.

The location of DNA photodamage within the epidermis is crucial as basal layer cells are the most likely to have carcinogenic potential. We have determined the action spectra for DNA photodamage in different human epidermal layers in situ. Previously unexposed buttock skin was irradiated with 0.5, 1, 2, and 3 minimal erythema doses of monochromatic UVR at 280, 290, 300, 310, 320, 340, and 360 nm. Punch biopsies were taken immediately after exposure and paraffin sections were prepared for immunoperoxidase staining with a monoclonal antibody against thymine dimers that were quantitated by image analysis. Dimers were measured at two basal layer regions, the mid and the upper living epidermis. The slopes of dose-response curves were used to generate four action spectra, all of which had maxima at 300 nm. Dimer action spectra between 300 and 360 nm were independent of epidermal layer, indicating comparable epidermal transmission at these wavelengths. Furthermore, we observed 300 nm-induced dimers in dermal nuclei; however, there was a marked effect of epidermal layer between 280 and 300 nm, showing relatively poor transmission of 280 and 290 nm to the basal layer. These data indicate that solar UVB (approximately 295-320 nm) is more damaging to basal cells than predicted from transmission data obtained from human epidermis ex vivo. The epidermal dimer action spectra were compared with erythema action spectra determined from the same volunteers and ultraviolet radiation sources. Overall, these spectral comparisons suggest that DNA is a major chromophore for erythema in the 280-340 nm region.

Adult↗

PubMed1998

Specificities and rates of binding of anti-(6-4) photoproduct antibody fragments to synthetic thymine photoproducts.

Pyrimidine (6-4) pyrimidone photoproducts are some of the major DNA photolesions induced by ultraviolet (UV) light. A monoclonal antibody (64M5) specific to a (6-4) photoproduct has been established and the corresponding single-chain antibody (64M5scFv) has been prepared. In this study, we characterized the ligand selectivities of 64M5 and 64M5scFv using synthetic octadeoxynucleotides containing either a central cis-syn cyclobutane thymine dimer (T[c,s]T), the (6-4) photoproduct of TpT (T[6-4]T), or its Dewar isomer (T[Dewar]T) by means of enzyme-linked immunosorbent assays (ELISA). Both 64M5 and 64M5scFv recognized T[6-4]T, but not the other photoproducts. We synthesized several biotinylated oligonucleotides of different lengths containing (T[6-4]T) to analyze the effects of the antigen size on the binding rates of an antigen binding fragment (64M5Fab) and 64M5scFv by means of surface plasmon resonance. The association rate constants for oligonucleotides of different sizes containing T[6-4]T as to 64M5Fab were found to be almost the same (1.9-5.6 x 10(5) M(-1) x s(-1)), while the dissociation rate constant for the largest oligonucleotide (d8mer, 8.0 x 10(-5) s(-1)) was significantly smaller than that for the d2mer (4.2 x 10(-2) s(-1)). These results indicate that 64M5Fab recognized the d2mer as the epitope and that the binding affinity for T[6-4]T depended on the flanking oligonucleotides. The dissociation rate constants for 64M5scFv as to the antigen analogs were almost the same as those for the various T[6-4]T-oligonucleotides as to 64M5Fab, suggesting that the conformations of these antibody binding regions are pretty similar to each other.

Antibody Specificity↗

PubMed1998

The role of surface lysines in pyrimidine (6-4) pyrimidone photoproduct binding by a high-affinity antibody.

The roles of four VH surface lysine side-chains in antibody recognition of DNA containing a central pyrimidine (6-4) pyrimidone photoproduct was investigated. Three of the four lysines (at positions H62, H64 and H66) are strongly conserved; however, the presence of lysine at position H68 was highly unusual. Each lysine was replaced individually by alanine; in addition, a quadruple mutant was also created. Studies of the binding kinetics revealed that these lysine residues mainly influenced the association phase. Our results suggest that these side chains help to guide the DNA polyanion to the antigen binding pocket by electrostatic effects.

Antibody Affinity↗

PubMed1998

A comparison of UVB-carcinogenesis between nude mice and nude beige mice.

To gain an insight into the relationship between UVB-carcinogenesis and natural killer activity, we examined ultraviolet light-induced carcinogenesis in mice with high natural killer, activity (KSN) and mice with natural killer deficiency (KSN-bg). We exposed mice six times a week to three levels of daily ultraviolet B (UVB) doses; 320, 160 and 0 J/m2/day. During the latency period of skin tumor development in KSN mice, we detected no suppression of the natural killer activity at both 320 and 160 J/m2/day. Even at 1340 J/m2/day, we could not detect any significant suppression of NK activity in KSN mice. When we irradiated spleen cells in vitro, we observed NK activity suppression. Next, we compared the carcinogenic effects of UVB-irradiation on KSN and KSN-bg mice. At 320 J/m2/day, we detected no significant differences between them. In contrast, at 160 J/m2/day, KSN-bg mice showed a significantly higher rate of skin tumor induction than KSN mice (p < 0.05). Most UVB-induced tumors were squamous cell carcinoma, the rest were spindle cell carcinoma, papilloma and mixed type. Our results suggest that NK activity plays a protective role against UVB-carcinogenesis from low daily-doses of UVB-irradiation.

Animals↗