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C Kojima

Publications and source records attributed to C Kojima.

At least 19 recordsLinked to original sources
PubMed2006

Toxicity of a trivalent organic arsenic compound, dimethylarsinous glutathione in a rat liver cell line (TRL 1215).

BACKGROUND AND PURPOSE: Although inorganic arsenite (As(III)) is toxic in humans, it has recently emerged as an effective chemotherapeutic agent for acute promyelocytic leukemia (APL). In humans and most animals, As(III) is enzymatically methylated in the liver to weakly toxic dimethylarsinic acid (DMAs(V)) that is a major pentavalent methylarsenic metabolite. Recent reports have indicated that trivalent methylarsenicals are produced through methylation of As(III) and participate in arsenic poisoning. Trivalent methylarsenicals may be generated as arsenical-glutathione conjugates, such as dimethylarsinous glutathione (DMAs(III)G), during the methylation process. However, less information is available on the cytotoxicity of DMAs(III)G. EXPERIMENTAL APPROACH: We synthesized and purified DMAs(III)G using high performance TLC (HPTLC) methods and measured its cytotoxicity in rat liver cell line (TRL 1215 cells). KEY RESULTS: DMAs(III)G was highly cytotoxic in TRL 1215 cells with a LC(50) of 160 nM. We also found that DMAs(III)G molecule itself was not transported efficiently into the cells and was not cytotoxic; however it readily became strongly cytotoxic by dissociating into trivalent dimethylarsenicals and glutathione (GSH). The addition of GSH in micromolar physiological concentrations prevented the breakdown of DMAs(III)G, and the DMAs(III)G-induced cytotoxicity. Physiological concentrations of normal human serum (HS), human serum albumin (HSA), and human red blood cells (HRBC) also reduced both the cytotoxicity and cellular arsenic uptake induced by exposure to DMAs(III)G. CONCLUSIONS AND IMPLICATIONS: These findings suggest that the significant cytotoxicity induced by DMAs(III)G may not be seen in healthy humans, even if DMAs(III)G is formed in the body from As(III).

Animals↗
PubMed2001

Slow motion in the CAA*TTG sequence of a DNA decamer duplex studied by NMR.

In DNA duplexes, pyrimidine-purine steps are believed to be flexible or conformationally unstable. Indeed, several DNA crystal structures exhibit a multitude of conformations for CpA*TpG steps. The question arises of whether this structural flexibility is accompanied by dynamical flexibility, i.e., a question pertaining to the energy barrier between conformations. Except for TpA steps, slow motions on the microsecond-to-millisecond time scale have not been detected in duplexes until now. In the present study, such slow motion was investigated by 1H, 13C, and 15N NMR relaxation measurements on a DNA decamer d(CATTTGCATC)*d(GATGCAAATG). The DNA decamer was enriched with 15% 13C and 98% 15N isotopes for each adenosine and guanosine residue. Three lines of evidence support the notion of slow motion in the CAA*TTG moiety. Analysis of (15)N relaxation showed that the order parameter, S2, of guanosine imino NH groups was about 0.8, similar to that of CH groups for this oligomer. The strong temperature dependence of guanosine NH S2 in the CAA*TTG sequence indicated the presence of a large-amplitude motion. Signals of adenosine H8 protons in the CAA*TTG sequence were broadened in 2D 1H NOESY spectra, which also suggested the existence of slow motion. As well as being smaller than for other adenine residues, the 1H T2 values exhibited a magnetic field strength dependence for all adenosine H8 signals in the ATTTG*CAAAT region, suggesting slow motions more pronounced at the first adenosine in the CAA*TTG sequence but extending over the CAAAT*ATTTG region. This phenomenon was further examined by the pulse field strength dependence of the 1H, 13C, and 15N T1rho values. 1H and 13C T1rho values showed a pulse field strength dependence, but 15N T1rho did not. Assuming a two-site exchange process, an exchange time constant of 20-300 micros was estimated for the first adenosine in the CAA sequence. The exact nature of this motion remains unknown.

Base Pairing↗
PubMed2001

Solid-phase synthesis of selectively labeled DNA: applications for multidimensional nuclear magnetic resonance spectroscopy.

The solid-phase chemical synthesis method has a strong advantage over the enzymatic method for preparing selectively labeled DNA oligomers. Atom-specific and fully labeled 2'-deoxynucleosides are economically prepared with routinely available isotope precursors using this synthetic route. Special DNA oligomers prepared by advanced labeling techniques are needed for advanced NMR applications, and chemical synthesis is the method of choice to respond to such demands. As a summary of this chapter, two tables are given. Table I lists the labeled nucleosides reported to be available by chemical syntheses. Table II lists the NMR studies using labeled DNA oligomers that were prepared by chemical syntheses.

Carbon Isotopes↗
PubMed2001

Sugar conformation of a stereospecific 2'-R or 2'-S deuterium-labeled DNA decamer studied with proton-proton J coupling constants.

The sugar conformation of a DNA decamer was studied with proton-proton 3J coupling constants. Two samples, one comprising stereospecifically labeled 2'-R-2H for all residues and the other 2'-S-2H, were prepared by the method of Kawashima et al. [J. Org. Chem. (1995) 60, 6980-6986; Nucleosides Nucleotides (1995) 14, 333-336], the deuterium labeling being highly stereospecific (> or = 99% for all 2''-2H, > or = 98% for 2'-2H of A, C, and T, and > or = 93% for 2'-2H of G). The 3J values of all H1'-H2' and H1'-H2'' pairs, and several H2'-H3' and H2''-H3' pairs were determined by line fitting of 1D spectra with 0.1-0.2 Hz precision. The observed J coupling constants were explained by the rigid sugar conformation model, and the sugar conformations were found to be between C3'-exo and C2'-endo with phi(m) values of 26 degrees to 44 degrees, except for the second and 3' terminal residues C2 and C10. For the C2 and C10 residues, the lower fraction of S-type conformation was estimated from JH1'H2' and JH1'H2'' values. For C10, the N-S two-site jump model or Gaussian distribution of the torsion angle model could explain the observed J values, and 68% S-type conformation or C1'-exo conformation with 27 degrees distribution was obtained, respectively. The differences between these two motional models are discussed based on a simple simulation of J-coupling constants.

Carbohydrate Conformation↗
PubMed2001

Synthesis of 5-substituted [N3-15N]-pyrimidine nucleosides: Developing model systems for NMR studies of substituent effects on the N-H...N hydrogen bond in duplex DNA.

The effects on various NMR parameters of substitutions, which may influence the hydrogen bond strengths of Watson-Crick-type base pairs, were investigated for DNA dodecamers containing 5-substituted-2'-deoxyuridine derivatives in oligomers, 5'-d(CGCGAATXCGCG)-3', where A and X were [ul-15N]-2'-deoxyadenosine and [3(-15)N]-2'-deoxyuridine derivatives. The substitution effects on the NMR parameters were linearly correlated with the pKa values of the 2'-deoxyuridine derivatives.

Base Sequence↗
PubMed2001

Effect of macrolides on the expression of HLA-DR and costimulatory molecules on antigen-presenting cells in nasal polyps.

To clarify the basis of macrolide therapy for improvement of chronic sinusitis, we investigated the effect of macrolides on the expression of HLA-DR and costimulatory molecules such as CD54 and CD80 on macrophages in nasal polyps. Nasal polyps taken from 54 patients who had or had not been treated with macrolides were immunohistochemically studied. The percentages of CD68-positive macrophages expressing HLA-DR or CD54 were not significantly different between patients treated with macrolides and those not treated with macrolides. However, among patients exhibiting no atopic predisposition, the number of CD80-positive macrophages was higher in patients treated with macrolides than in those not treated. In addition, the percentage of CD80-positive macrophages was negatively correlated with the percentage of infiltrating eosinophils in the polyps. These results demonstrate that macrophages act as antigen-presenting cells, expressing both major histocompatibility complex II and costimulators, and that the expression of CD80 may play a key role in the immune responses occurring in a nasal polyp. Macrolides may modulate the mucosal immune responses through CD80 expression.

Adolescent↗
PubMed2000

Solution structure of an RNA duplex including a C-U base pair.

The formation of the C-U base pair in a duplex was observed in solution by means of the temperature profile of (15)N chemical shifts, and the precise geometry of the C-U base pair was also determined by NOE-based structure calculation. From the solution structure of the RNA oligomer, r[CGACUCAGG].r[CCUGCGUCG], it was found that a single C-U mismatch preferred being stacked in the duplex rather than being flipped-out even in solution. Moreover, it adopts an irregular geometry, where the amino nitrogen (N4) of the cytidine and keto-oxygen (O4) of the uridine are within hydrogen-bonding distance, as seen in crystals. To further prove the presence of a hydrogen bond in the C-U pair, we employed a point-labeled cytidine at the exocyclic amino nitrogen of the cytidine in the C-U pair. The temperature profile of its (15)N chemical shift showed a sigmoidal transition curve, indicating the presence of a hydrogen bond in the C-U pair in the duplex.

Base Pairing↗
PubMed2000

Simple suppression of spurious peaks in TROSY experiments.

In (1)H-(15)N TROSY experiments of proteins and nucleic acids, where the second coherence transfer delay time tau' has been fixed as 5.6 ms, 1/(2(1)J(NH)), in order to achieve complete spin-state selection, spurious negative peaks are observed along the (15)N axes. These peaks are often annoyingly large, especially for nucleic acids. A simple product operator calculation, however, indicated that the shortening of the second delay time tau', which is next to the t1 period, would efficiently suppress these spurious peaks, without sacrificing the sensitivities of the TROSY peaks too much. We have shown for three systems, two 11- and 17-kDa proteins and one 8-kDa DNA duplex, that these spurious peaks can be effectively suppressed with delay times of 3.3 ms for the two proteins and 2.3 ms for the DNA. These delay times, optimized by trial and error, for the spurious peak suppression did not depend on the magnetic field strength and the temperature very much. Although the shortened tau' delay times attenuate the TROSY peak intensities by about 10 and 20% for the two proteins and the DNA, respectively, this simple modification will be useful for the quantitative uses of TROSY peaks and will result in cleaner spectra for various TROSY-based multiple resonance experiments.

Carrier Proteins↗
PubMed2000

Studies of physicochemical properties of N-H...N hydrogen bonds in DNA, using selective 15N-labeling and direct 15N 1D NMR.

15N-15N scalar coupling constants across base pair hydrogen bonds (2hJ(NN)) were studied using residue- and atom-specifically 15N labeled DNA oligomers. The N3 atom selectively 15N enriched 2'-deoxycytidine and thymidine, and the uniformly 15N enriched 2'-deoxyadenosine and 2'-deoxyguanosine, were chemically prepared and incorporated into two DNA oligomers, d(CGCGAATTCGCG)2 and d(CGCAAAAAGCG).d(CGCTTTTTGCG). This isotope labeling enabled us to determine the 2hJ(NN) value from the splitting of the 15N 1D spectrum. Additionally, it enabled the determination of 2hJ(NN) in D2O quite easily and highly quantitatively. The temperature and DNA sequence dependence were examined for these oligomers. The sequence dependence was not clear; however, a significant decrease of 2hJ(NN) was observed by elevating the temperature. This temperature dependence was not due to the hydrogen exchange, since the addition of 20 mM NH3 did not change the 2hJ(NN) values. The 2hJ(NN) values in D2O were somewhat smaller than those in H2O. As compared to our 15N 1D method, the quantitative HNN-COSY method gave systematically smaller 2hJ(NN) values in our system, due to the lower 15N fraction of our sample (79 and 88% for dA and the other nucleotides, respectively) and the insufficient power of the 15N RF pulse (B1 = 6.6 kHz). These systematic differences were recovered by theoretical correction of the 15N isotope fraction contribution, by using the composite 15N 180 degrees pulse in a quantitative HNN-COSY experiment.

Ammonia↗
PubMed1999

Protection of chickens against very virulent infectious bursal disease virus (IBDV) and Marek's disease virus (MDV) with a recombinant MDV expressing IBDV VP2.

To develop a herpes virus vaccine that can induce immunity for an extended period, a recombinant Marek's disease (MD) virus (MDV) CVI-988 strain expressing infectious bursal disease virus (IBDV) host-protective antigen VP2 at the US2 site (rMDV) was developed under the control of an SV40 early promoter. Chickens vaccinated with the rMDV showed no clinical signs and no mortality and 55% of the chickens were considered protected histopathologically after challenge with very virulent IBDV (vvIBDV), whereas all of the chickens vaccinated with the conventional IBDV vaccine showed no clinical signs and were protected. Chickens vaccinated with the CVI-988 or chickens in the challenge control showed severe clinical signs and high mortality (70-75%) and none of them were protected. Also, the rMDV conferred full protection to chickens against vvMDV just as the CVI-988 strain did, whereas 90% of the challenge control chickens died of MD. Antibody levels against IBDV and MDV following the vaccination increased continuously for at least 10 weeks. No histopathological lesions in the rMDV-vaccinated chickens and no contact transmission of the rMDV to their penmates were confirmed. These results demonstrate that an effective and safe recombinant herpesvirus-based IBD vaccine could be constructed by expressing the VP2 antigen at the US2 site of the CVI-988 vaccine strain.

Animals↗
PubMed1999

Structural effect of complete [Rp]-phosphorothioate and phosphorodithioate substitutions in the DNA strand of a model antisense inhibitor-target RNA complex.

Chemically modified DNA oligonucleotides have been crucial to the success of antisense therapeutics. Although such modifications are ubiquitous in the clinic, high-resolution structural studies of pharmaceutically relevant derivatives have been limited to only a few molecules. We have completed a high-resolution NMR structural study of three DNA.RNA hybrids with the sequence d(CCTATAATCC). r(GGAUUAUAGG). All hybrids contain an unmodified RNA strand, whereas the DNA strand of each hybrid contains one of three different sugar-phosphate backbone linkages at each nucleotide: (1) phosphate, (2) [Rp]-phosphorothioate, or (3) phosphorodithioate. The UV and NMR melting profiles revealed that the normal hybrid is more stable than the [Rp]-phosphorothioate, which in turn is more stable than the phosphorodithioate. Homonuclear two-dimensional nuclear Overhauser effect spectroscopy and double quantum-filtered correlation spectroscopy afforded nearly complete non-labile proton assignments. The three molecules show nearly equivalent chemical shifts, with the exception of H3' protons, which are shifted downfield in a manner that appears correlated with the degree of sulfur substitution at phosphate. All three hybrids exhibit unusually broad linewidths for deoxyribose protons H2' and H2".Distance restraints were calculated from NOE cross-peak intensities via a complete relaxation matrix approach using the program RANDMARDI. Detailed comparison of interproton distances from each hybrid indicates that the three molecules share a common structure, with neither strand in canonical A or B form. Correlation of R factors, calculated using the program CORMA with DNA H2'-base and H3'-base distances, revealed a relative increase in the population of B-type sugar conformations for deoxyriboses in the A+T-rich center of the hybrid sequence. It is widely known that the activity of enzymes which act upon DNA.RNA hybrid substrates (e.g. ribonuclease H) is impacted when the hybrids contain phosphorothioate or phosphorodithioate substitutions. The structural similarity of the three hybrids examined here suggests that factors other than global structure may mediate the activity of these enzymes.

Base Sequence↗
PubMed1999

Quantitative measurement of transverse and longitudinal cross-correlation between 13C-1H dipolar interaction and 13C chemical shift anisotropy: application to a 13C-labeled DNA duplex.

Measurement of both longitudinal and transverse relaxation interference (cross-correlation) between 13C chemical shift anisotropy and 13C-1H dipolar interactions is described. The ratio of the transverse to longitudinal cross-correlation rates readily yields the ratio of spectral densities J(0)/J(omegaC), independent of any structural attributes such as internuclear distance or chemical shift tensor. The spectral density at zero frequency J(0) is also independent of chemical exchange effects. With limited internal motions, the ratio also enables an accurate evaluation of the correlation time for overall molecular tumbling. Applicability of this approach to investigating dynamics has been demonstrated by measurements made at three temperatures using a DNA decamer duplex with purines randomly enriched to 15% in 13C.

Anisotropy↗
PubMed1999

Intermolecular contacts between the lambda-Cro repressor and the operator DNA characterized by nuclear magnetic resonance spectroscopy.

The specific interaction between lambda phage Cro repressor and the DNA fragment bearing the consensus sequence of operators has been studied using nuclear magnetic resonance (NMR). Using both 15N- and 13C/15N- labeled lambda-Cro in complex with unlabeled DNA, chemical shift assignments of the lambda-Cro-DNA complex were obtained using heteronuclear NMR experiments. Inter-molecular contacts between the protein and DNA were identified using heteronuclear filtered NOESY experiments. The inter-molecular contacts were supplemented with intra-protein and intra-DNA NOE constraints to dock lambda-Cro to the bent B-form DNA using restrained molecular dynamics. The structure of one of the subunits of lambda-Cro in the complex is essentially the same as that of the unbound form. In the complex, inter-molecular NOEs were observed between the "helix-turn-helix" region comprising the alpha2 and alpha3 helices of the lambda-Cro protein and the major groove of the DNA. The methyl group of Thr17 forms a hydrophobic contact with the methyl group of the thymine at base pair 1 in the DNA, and Val25 and Ala29 make hydrophobic contacts with the methyl group of the thymine at base pair 5. The presence and the absence of these contacts can explain the difference in the affinity of lambda-Cro to several variants of the operator sequence.

Carbon Isotopes↗
PubMed1998

Risk factors for recurrent and residual cholesteatoma in children determined by second stage operation.

OBJECTIVE: To clarify the risk factors for the development of recurrent and residual cholesteatoma in children. METHODS: We studied 84 ears of 83 children aged 10 years or younger who underwent a second stage operation 1 year after primary surgery with a canal wall reconstruction procedure, and analyzed the clinical risk factors for recurrent and residual cholesteatoma. RESULTS: Recurrent cholesteatoma was detected in 21 ears (25%) and residual cholesteatoma was noted in 35 (42%) of 84 ears. With respect to recurrent cholesteatoma, significant risk factors were determined to be male gender, pars flaccida type of cholesteatoma and the association of otitis media with effusion either in the side affected by cholesteatoma or on the opposite side. On the other hand, congenital type of cholesteatoma was a significant negative risk factor. With respect to residual cholesteatoma, the only risk factor was a posterosuperior type of cholesteatoma. Residual cholesteatoma was sometimes found even when the surgeon had declared complete removal of the cholesteatoma matrix at the time of primary surgery. CONCLUSIONS: High incidence of recurrent and residual cholesteatoma was noted at the second stage operation. Occurrence of recurrent cholesteatoma is closely related to eustachian tube dysfunction. Thin and highly proliferative cholesteatoma matrix in children may be responsible for high occurrence of residual cholesteatoma. Therefore, planned staged surgery is preferable to single stage surgery for the treatment of pediatric cholesteatoma.

Child↗
PubMed1998

DNA duplex dynamics: NMR relaxation studies of a decamer with uniformly 13C-labeled purine nucleotides.

Dynamics in a DNA decamer duplex, d(CATTTGCATC). d(GATGCAAATG), were investigated via a detailed 13C NMR relaxation study. Every 2'-deoxyadenosine and 2'-deoxyguanidine was chemically enriched with 15% 13C and 98% 15N isotopes. Six nuclear relaxation parameters [R(13Cz), R(1Hz), R(2(1)Hz13Cz), R(13Cx), R(2(1)Hz13Cx) and steady-state 13C¿1H¿ NOE] were measured at 600 MHz and three were measured at 500 MHz (1H frequency) for the CH spin systems of sugar 1', 3', and 4' as well as base 8 and 2 positions. A dependence of relaxation parameter values on chemical position was clearly observed; however, no sequence-specific variation was readily evident within our experimental error of approximately 5-10%, except for 3' and 5' termini. It was demonstrated that the random 15% 13C enrichment effectively suppressed both scalar and dipolar contributions of the neighboring carbons and protons on the relaxation parameters. To analyze dynamics via all observed relaxation parameters, full spectral density mapping (1992, J. W. Peng and G. Wagner, J. Magn. Reson. 98, 308) and the "model-free" approach (1982, Lipari and Szabo, J. Am. Chem. Soc. 104, 4546) were applied complementarily. A linear correlation between three spectral density values, J(omegaC), J(omegaH - omegaC), and J(omegaH + omegaC) was observed in plots containing all measured values, but not for the other spectral density terms including J(0). These linear correlations reflect the effect of overall motion and similar internal motions for each CH vector in the decamer. The correlations yielded two correlation times, 3-4 ns and 10-200 ps. One value, 3-4 ns, corresponds to the value of 3.3 ns obtained for the overall isotropic tumbling correlation time determined from analysis of 13C T1/T2 ratios. The possibility of overall anisotropic tumbling was examined, but statistical analysis showed no advantage over the assumption of simple isotropic tumbling. Lack of correlations entailing J(0) implies that a relatively slow chemical exchange contributes to yielding of effective Jeff(0) values. Based on spectral density mapping and the T1/T2 ratio analysis, three basic assumptions were initially employed (and subsequently justified) for the model-free calculation: isotropic overall tumbling, one internal motion, and the presence of chemical exchange terms. Except for terminal residues, the order parameter S2 and the corresponding fast internal motion correlation time were determined to be about 0.8 +/- 0.1 and 20 +/- 20 ps, respectively, for the various CH vectors. Only a few differences were observed between or within sugars and bases. The internal motion is very fast (ps-ns time scale) and its amplitude restricted; e.g., assuming a simple wobble-in-a-cone model, the internal motion is restricted to an angular amplitude of +/-22. 5 degrees for each of the 1', 3', 4', 2, and 8 positions in the purine nucleotides in the entire duplex.

Adenosine↗