Chronic progressive cervical myelopathy with HTLV-I infection: Variant form of HAM/TSP?

OBJECTIVE: To investigate the role of human T-lymphotrophic virus type I (HTLV-I) infection in four patients who developed slowly progressive myelopathy with abnormal MRI lesions in the cervical cord levels. METHODS: Clinical and neuroradiologic examinations were performed, and the odds that an HTLV-I-infected individual of specified genotype, age, and provirus load had HTLV-I-associated myelopathy (HAM)/tropical spastic paraparesis (TSP) were calculated. RESULTS: Anti-HTLV-I antibodies were positive in both the serum and the CSF in all of the patients. Biopsied sample from spinal cord lesions showed inflammatory changes in Patient 1. Patient 2 had a demyelinating type of sensorimotor polyneuropathy. Two of the three patients examined showed high risk of developing HAM/TSP in virologic and immunologic aspects. CONCLUSION: These four cases may belong to a variant form of HAM/TSP, predominantly involving the cervical cord levels.

Identification of tyrosine residues involved in ligand recognition by the phosphatidylinositol 3-kinase Src homology 3 domain: circular dichroism and UV resonance Raman studies.

Src homology 3 (SH3) domains are small noncatalytic protein modules capable of mediating protein-protein interactions. We previously demonstrated that the association of a ligand peptide RLP1 (RKLPPRPSK) causes environmental and structural changes of Trp55 and some of seven Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain by circular dichroism (CD) and 235-nm excited UV resonance Raman (UVRR) spectroscopies [Okishio, N., et al. (2000) Biopolymers 57, 208-217]. In this work, the affected Tyr residues were identified as Tyr12, Tyr14, and Tyr73 by the CD analysis of a series of mutants, in which every single Tyr residue was replaced by a Phe residue. Among these three residues, Tyr14 was found to be a main contributor to the UVRR spectral change upon the RLP1 binding. Interestingly, CD and UVRR analyses revealed that RLP1 associates with the Y14F and Y14H mutants in different ways. These results suggest that Tyr14 plays a crucial role in the ligand recognition, and the amino acid substitution at Tyr14 affects the mode of PI3K SH3-ligand interaction. Our findings give an insight into how SH3 domains can produce diversity and specificity to transduce signaling within cells.

Formation, characterization, and reactivity of bis(mu-oxo)dinickel(III) complexes supported by a series of bis[2-(2-pyridyl)ethyl]amine ligands.

Bis(mu-oxo)dinickel(III) complexes supported by a series of bis[2-(2-pyridyl)ethyl]amine ligands have been successfully generated by treating the corresponding bis(mu-hydroxo)dinickel(II) complexes or bis(mu-methoxo)dinickel(II) complex with an equimolar amount of H(2)O(2) in acetone at low temperature. The bis(mu-oxo)dinickel(III) complexes exhibit a characteristic UV-vis absorption band at approximately 410 nm and a resonance Raman band at 600-610 cm(-1) that shifted to 570-580 cm(-1) upon (18)O-substitution. Kinetic studies and isotope labeling experiments using (18)O(2) imply the existence of intermediate(s) such as peroxo dinickel(II) in the course of formation of the bis(mu-oxo)dinickel(III) complex. The bis(mu-oxo)dinickel(III) complexes supported by the mononucleating ligands (L1(X) = para-substituted N,N-bis[2-(2-pyridyl)ethyl]-2-phenylethylamine; X = OMe, Me, H, Cl) gradually decompose, leading to benzylic hydroxylation of the ligand side arm (phenethyl group). The kinetics of the ligand hydroxylation process including kinetic deuterium isotope effects (KIE), p-substituent effects (Hammett plot), and activation parameters (Delta H(H)(*) and Delta S(H)(*)) indicate that the bis(muxo)dinickel(III) complex exhibits an ability of hydrogen atom abstraction from the substrate moiety as in the case of the bis(mu-oxo)dicopper(III) complex. Such a reactivity of bis(mu-oxo)dinickel(III) complexes has also been suggested by the observed reactivity toward external substrates such as phenol derivatives and 1,4-cyclohexadiene. The thermal stability of the bis(mu-oxo)dinickel(III) complex is significantly enhanced when the dinucleating ligand with a longer alkyl strap is adopted instead of the mononucleating ligand. In the m-xylyl ligand system, no aromatic ligand hydroxylation occurred, showing a sharp contrast with the reactivity of the (mu-eta(2):eta(2)-peroxo)dicopper(II) complex with the same ligand which induces aromatic ligand hydroxylation via an electrophilic aromatic substitution mechanism. Differences in the structure and reactivity of the active oxygen complexes between the nickel and the copper systems are discussed on the basis of the detailed comparison of these two systems with the same ligand.

Effects of metal ions on physicochemical properties and redox reactivity of phenolates and phenoxyl radicals: mechanistic insight into hydrogen atom abstraction by phenoxyl radical-metal complexes.

Phenolate and phenoxyl radical complexes of a series of alkaline earth metal ions as well as monovalent cations such as Na+ and K+ have been prepared by using 2,4-di-tert-butyl-6-(1,4,7,10-tetraoxa-13-aza-cyclopentadec-13-ylmethyl)phenol (L1H) and 2,4-di-tert-butyl-6-(1,4,7,10,13-pentaoxa-16-aza-cyclooctadec-16-ylmethyl)phenol (L2H) to examine the effects of the cations on the structure, physicochemical properties and redox reactivity of the phenolate and phenoxyl radical complexes. Crystal structures of the Mg2+- and Ca2+-complexes of L1- as well as the Ca2+- and Sr2+-complexes of L2- were determined by X-ray crystallographic analysis, showing that the crown ether rings in the Ca2+-complexes are significantly distorted from planarity, whereas those in the Mg2+- and Sr2+-complexes are fairly flat. The spectral features (UV-vis) as well as the redox potentials of the phenolate complexes are also influenced by the metal ions, depending on the Lewis acidity of the metal ions. The phenoxyl radical complexes are successfully generated in situ by the oxidation of the phenolate complexes with (NH4)(2)[Ce4+(NO3)6] (CAN). They exhibited strong absorption bands around 400 nm together with a broad one around 600-900 nm, the latter of which is also affected by the metal ions. The phenoxyl radical-metal complexes are characterized by resonance Raman, ESI-MS, and ESR spectra, and the metal ion effects on those spectroscopic features are also discussed. Stability and reactivity of the phenoxyl radical-metal complexes are significantly different, depending on the type of metal ions. The disproportionation of the phenoxyl radicals is significantly retarded by the electronic repulsion between the metal cation and a generated organic cation (Ln+), leading to stabilization of the radicals. On the other hand, divalent cations decelerate the rate of hydrogen atom abstraction from 10-methyl-9,10-dihydroacridine (AcrH2) and its 9-substituted derivatives (AcrHR) by the phenoxyl radicals. On the basis of primary kinetic deuterium isotope effects and energetic consideration of the electron-transfer step from AcrH2 to the phenoxyl radical-metal complexes, we propose that the hydrogen atom abstraction by the phenoxyl radical-alkaline earth metal complexes proceeds via electron transfer followed by proton transfer.

Characterization of imidazolate-bridged dinuclear and mononuclear hydroperoxo complexes.

Dinucleating ligands having two metal-binding sites bridged by an imidazolate moiety, Hbdpi, HMe(2)bdpi, and HMe(4)bdpi (Hbdpi = 4,5-bis(di(2-pyridylmethyl)aminomethyl)imidazole, HMe(2)bdpi = 4,5-bis((6-methyl-2-pyridylmethyl)(2-pyridylmethyl)aminomethyl)imidazole, HMe(4)bdpi = 4,5-bis(di(6-methyl-2-pyridylmethyl)aminomethyl)imidazole), have been designed and synthesized as model ligands for copper-zinc superoxide dismutase (Cu,Zn-SOD). The corresponding mononucleating ligands, MeIm(Py)(2), MeIm(Me)(1), and MeIm(Me)(2) (MeIm(Py)(2) = (1-methyl-4-imidazolylmethyl)bis(2-pyridylmethyl)amine, MeIm(Me)(1) = (1-methyl-4-imidazolylmethyl)(6-methyl-2-pyridylmethyl)(2-pyridylmethyl)amine, MeIm(Me)(2) = (1-methyl-4-imidazolyl-methyl)bis(6-methyl-2-pyridylmethyl)amine), have also been synthesized for comparison. The imidazolate-bridged Cu(II)-Cu(II) homodinuclear complexes represented as [Cu(2)(bdpi)(CH(3)CN)(2)](ClO(4))(3).CH(3)CN.3H(2)O (1), [Cu(2)(Me(2)bdpi)(CH(3)CN)(2)](ClO(4))(3) (2), [Cu(2)(Me(4)bdpi)(H(2)O)(2)](ClO(4))(3).4H(2)O (3), a Cu(II)-Zn(II) heterodinuclear complex of the type of [CuZn(bdpi)(CH(3)CN)(2)](ClO(4))(3).2CH(3)CN (4), Cu(II) mononuclear complexes of [Cu(MeIm(Py)(2))(CH(3)CN)](ClO(4))(2).CH(3)CN (5), [Cu(MeIm(Me)(1))(CH(3)CN)](ClO(4))(2)( )()(6), and [Cu(MeIm(Me)(2))(CH(3)CN)](ClO(4))(2)( )()(7) have been synthesized and the structures of complexes 5-7 determined by X-ray crystallography. The complexes 1-7 have a pentacoordinate structure at each metal ion with the imidazolate or 1-methylimidazole nitrogen, two pyridine nitrogens, the tertiary amine nitrogen, and a solvent (CH(3)CN or H(2)O) which can be readily replaced by a substrate. The reactions between complexes 1-7 and hydrogen peroxide (H(2)O(2)) in the presence of a base at -80 degrees C yield green solutions which exhibit intense bands at 360-380 nm, consistent with the generation of hydroperoxo Cu(II) species in all cases. The resonance Raman spectra of all hydroperoxo intermediates at -80 degrees C exhibit a strong resonance-enhanced Raman band at 834-851 cm(-1), which shifts to 788-803 cm(-1) (Deltanu = 46 cm(-1)) when (18)O-labeled H(2)O(2) was used, which are assigned to the O-O stretching frequency of a hydroperoxo ion. The resonance Raman spectra of hydroperoxo adducts of complexes 2 and 6 show two Raman bands at 848 (802) and 834 (788), 851 (805), and 835 (789) cm(-1) (in the case of H(2)(18)O(2), Deltanu = 46 cm(-1)), respectively. The ESR spectra of all hydroperoxo complexes are quite close to those of the parent Cu(II) complexes except 6. The spectrum of 6 exhibits a mixture signal of trigonal-bipyramid and square-pyramid which is consistent with the results of resonance Raman spectrum.

Heme structure of hemoglobin M Iwate [alpha 87(F8)His-->Tyr]: a UV and visible resonance Raman study.

Heme structures of a natural mutant hemoglobin (Hb), Hb M Iwate [alpha87(F8)His-->Tyr], and protonation of its F8-Tyr were examined with the 244-nm excited UV resonance Raman (UVRR) and the 406.7- and 441.6-nm excited visible resonance Raman (RR) spectroscopy. It was clarified from the UVRR bands at 1605 and 1166 cm(-)(1) characteristic of tyrosinate that the tyrosine (F8) of the abnormal subunit in Hb M Iwate adopts a deprotonated form. UV Raman bands of other Tyr residues indicated that the protein takes the T-quaternary structure even in the met form. Although both hemes of alpha and beta subunits in metHb A take a six-coordinate (6c) high-spin structure, the 406.7-nm excited RR spectrum of metHb M Iwate indicated that the abnormal alpha subunit adopts a 5c high-spin structure. The present results and our previous observation of the nu(Fe)(-)(O(tyrosine)) Raman band [Nagai et al. (1989) Biochemistry 28, 2418-2422] have proved that F8-tyrosinate is covalently bound to Fe(III) heme in the alpha subunit of Hb M Iwate. As a result, peripheral groups of porphyrin ring, especially the vinyl and the propionate side chains, were so strongly influenced that the RR spectrum in the low-frequency region excited at 406.7 nm is distinctly changed from the normal pattern. When Hb M Iwate was fully reduced, the characteristic UVRR bands of tyrosinate disappeared and the Raman bands of tyrosine at 1620 (Y8a), 1207 (Y7a), and 1177 cm(-)(1) (Y9a) increased in intensity. Coordination of distal His(E7) to the Fe(II) heme in the reduced alpha subunit of Hb M Iwate was proved by the observation of the nu(Fe)(-)(His) RR band in the 441.6-nm excited RR spectrum at the same frequency as that of its isolated alpha chain. The effects of the distal-His coordination on the heme appeared as a distortion of the peripheral groups of heme. A possible mechanism for the formation of a Fe(III)-tyrosinate bond in Hb M Iwate is discussed.

Interactions of phosphatidylinositol 3-kinase Src homology 3 domain with its ligand peptide studied by absorption, circular dichroism, and UV resonance raman spectroscopies.

Absorption, circular dichroism (CD), and UV resonance Raman (UVRR) spectroscopies were applied to selectively examine the environmental and structural changes of Trp and Tyr residues in the phosphatidylinositol 3-kinase (PI3K) SH3 domain induced by ligand association. Comparison of the spectra of PI3K SH3 in the presence or absence of its ligand peptide RLP1 (RKLPPRPSK) indicated that RLP1 binding changed the environment of Trp55 of the SH3 to be more hydrophilic and its H bonding weaker and that of Tyr residues to be more hydrophobic. The D21N mutant (Asp21 --> Asn) of the SH3 yielded a UV CD distinct from that of the wild type, and its spectral changes induced by RLP1 binding were smaller and different from those of the wild type in absorption, CD, and UVRR spectra, suggesting that the mutation of conserved Asp21 affected the conformation of the ligand binding cleft and thus might lead to the decrease in the ligand affinity. These data provide direct evidence for the occurrence of environmental and structural changes of PI3K SH3 by the association of a ligand and the D21N mutation.

A novel diiron complex as a functional model for hemerythrin.

Diiron(II) complexes with a novel dinucleating polypyridine ligand, N,N,N',N'-tetrakis(6-pivalamido-2-pyridylmethyl)-1,3-diaminopropan-2-ol (HTPPDO), were synthesized as functional models of hemerythrin. Structural characterization of the complexes, [Fe2II(Htppdo)(PhCOO)](ClO4)3 (1), [Fe2II(Htppdo)((p-Cl)PhCOO)](ClO4)3 (2), [Fe2II(Htppdo)((p-Cl)PhCOO)](BF4)3 (2') and [Fe2II(tppdo)((p-Cl)PhCOO)](ClO4)2 (3), were accomplished by electronic absorption, and IR spectroscopic, electrochemical, and X-ray diffraction methods. The crystal structures of 1 and 2' revealed that the two iron atoms are asymmetrically coordinated with HTPPDO and bridging benzoate. One of the iron centers (Fe(1)) has a seven-coordinate capped octahedral geometry comprised of an N3O4 donor set which includes the propanol oxygen of HTPPDO. The other iron center (Fe(2)) forms an octahedron with an N3O3 donor set and one vacant site. The two iron atoms are bridged by benzoate (1) or p-chlorobenzoate (2). On the other hand, both Fe atoms of complex 3 are both symmetrically coordinated with N3O4 donors and two bridging ligands; benzoate and the propanolate of TPPDO. Reactions of these complexes with dioxygen were followed by electronic absorption, resonance Raman and ESR spectroscopies. Reversible dioxygen-binding was demonstrated by observation of an intense LMCT band for O2(2-) to Fe(III) at 610 (1) and 606 nm (2) upon exposure of dioxygen to acetone solutions of 1 and 2 prepared under an anaerobic conditions at -50 degrees C. The resonance Raman spectra of the dioxygen adduct of 1 exhibited two peaks assignable to the nu(O-O) stretching mode at 873 and 887 cm(-1), which shifted to 825 and 839 cm(-1) upon binding of (18)O2. ESR spectra of all dioxygen adducts were silent. These findings suggest that dioxygen coordinates to the diiron atoms as a peroxo anion in a mu-1,2 mode. Complex 3 exhibited irreversible dioxygen binding. These results indicate that the reversible binding of dioxygen is governed by the hydrophobicity of the dioxygen-binding environment rather than the iron redox potentials.

Oxidation of Benzyl Alcohol with Cu(II) and Zn(II) Complexes of the Phenoxyl Radical as a Model of the Reaction of Galactose Oxidase.

Profound insights into the catalytic mechanism of galactose oxidase (GO) are offered by new models of the active form of the metalloenzyme. The important role of the Cu(II) center in the oxidation of benzyl alcohol to benzaldehyde by the Cu(II)-phenoxyl radical complex of ligand 1 has been revealed by comparison with the reactivity of the corresponding Zn(II)-phenoxyl radical complex; py=2-pyridyl.

UV resonance Raman studies of alpha-nitrosyl hemoglobin derivatives: relation between the alpha 1-beta 2 subunit interface interactions and the Fe-histidine bonding of alpha heme.

Human alpha-nitrosyl beta-deoxy hemoglobin A, alpha(NO)beta(deoxy), is considered to have a T (tense) structure with the low O(2) affinity extreme and the Fe-histidine (His87) (Fe-His) bond of alpha heme cleaved. The Fe-His bonding of alpha heme and the intersubunit interactions at the alpha 1-beta 2 contact of alpha(NO)-Hbs have been examined under various conditions with EPR and UV resonance Raman (UVRR) spectra excited at 235 nm, respectively. NOHb at pH 6.7 gave the UVRR spectrum of the R structure, but in the presence of inositol-hexakis-phosphate (IHP) for which the Fe-His bond of the alpha heme is broken, UVRR bands of Trp residues behaved half-T-like while Tyr bands remained R-like. The half-ligated nitrosylHb, alpha(NO)beta(deoxy), in the presence of IHP at pH 5.6, gave T-like UVRR spectra for both Tyr and Trp, but binding of CO to its beta heme (alpha(NO)beta(CO)) changed the UVRR spectrum to half-T-like. Binding of NO to its beta heme (NOHb) changed the UVRR spectrum to 70% T-type for Trp but almost R-type for Tyr. When the pH was raised to 8.2 in the presence of IHP, the UVRR spectrum of NOHb was the same as that of COHb. EPR spectra of these Hbs indicated that the Fe-His bond of alpha(NO) heme is partially cleaved. On the other hand, the UVRR spectra of alpha(NO)beta(deoxy) in the absence of IHP at pH 8.8 showed the T-like UVRR spectrum, but the EPR spectrum indicated that 40-50% of the Fe-His bond of alpha hemes was intact. Therefore, it became evident that there is a qualitative correlation between the cleavage of the Fe-His bond of alpha heme and T-like contact of Trp-beta 37. We note that the behaviors of Tyr and Trp residues at the alpha 1-beta 2 interface are not synchronous. It is likely that the behaviors of Tyr residues are controlled by the ligation of beta heme through His-beta 92(F8)-->Val-beta 98(FG5)-->Asp-beta 99(G1 )-->Tyr-alpha 42(C7) or Tyr-beta 145(HC2).

The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma. The protonation of an active site histidine is hindered by pi-pi interactions.

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 A resolution) and reduced form (at 1.8 A resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His87 (equivalent to His90 in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals pi-pi stacking between Phe12 and His90, suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His90 (the "adjacent" site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.

Manganese intoxication during total parenteral nutrition: report of two cases and review of the literature.

We report two cases of manganese (Mn) intoxication during total parenteral nutrition including manganese (Mn). Both patients showed parkinsonism with psychiatric symptoms and elevated serum Mn levels. T1-weighted magnetic resonance images (MRI) revealed symmetrical high intensity lesions in the globus pallidus. Discontinuation of Mn supplementation and levodopa treatment improved the symptoms and MRI abnormalities in the both patients. Thus, careful attention should be paid to the long-term intravenous administration of Mn.

Quaternary structure sensitive tyrosine residues in human hemoglobin: UV resonance raman studies of mutants at alpha140, beta35, and beta145 tyrosine.

Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA [Nagai, M., et al. (1996) J. Mol. Struct. 379, 65-75; Huang, S., et al. (1997) Biochemistry 36, 6197-6206], but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone. To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here. The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions. The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond. The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible. In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift. In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected. This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes. It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145). These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments.

Mutation induction and RBE of low energy neutrons in V79 cells.

We have examined the neutron energy dependency of cell killing and mutation induction at the hprt locus in Chinese hamster V79 cells. Monoenergetic neutrons at 0.32, 0.57, and 1.2 MeV were generated at the Hiroshima University Radiobiological Research Accelerator (HIRRAC) Facility, and were used to irradiate cells. The variation in RBE with neutron energy for the end points of cell survival and hprt mutation induction was observed. When compared to 137Cs gamma-rays, all neutron energies were more effective at both cell killing and induction of mutation. Over the range of the neutron energies examined, we found that cytotoxicity increased as the energy decreased from 1.2 to 0.32 MeV. In comparison to gamma-rays, RBEs for cell lethality at 10% survival were 5.7, 6.7, and 7.6 for 1.2, 0.57, and 0.32 MeV, respectively. Mutation induction, on the other hand, was highest at 0.57 MeV with a gradual decrease at 1.2 and 0.32 MeV. RBEs for mutation induction were 9.7, 19.4, and 13.9 for 1.2, 0.57, and 0.32 MeV neutrons. We isolated independent V79 cell mutants at the hprt locus from untreated and neutron-exposed cells and determined the genetic changes underlying the mutation by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. Preliminary results are suggestive of a specific relationship between deletion pattern and neutron energy.

An east Asian perspective of mind-body.

This paper addresses a need to re-examine the mind-body dualism established since Descartes. Descartes' dualism has been regarded by modern philosophers as an extremely insufficient solution to the problem of mind and body, from which is derived a long opposition in modern epistomology between idealism and empiricism. This dualism, bifurcating the region of spirit and matter, and the dichotomous models of thinking based on this dualism, have long dominated the world of modern philosophy and science. The paper examines states of conscious experience from an East Asian perspective allowing analysis on achieved supernormal consciousness rather than a focus on "normal" or "subnormal." The nature of the "transformation" of human consciousness will be studied both philosophically, as a transformation from "provisional" dualism to non-dualism, and neurophysiologically. The theoretical structure of the transformation will, in part, be examined through the model provided by a Japanese medieval Zen master, Takuan Sôhô. Then, to verify Takuan's theoretical explanation, toposcopic analysis of electroencephalographs will be presented of the performance of individuals practicing the martial arts technique of tôate.

The DNA sequence of gamma-glutamyltranspeptidase gene of Bacillus subtilis (natto) plasmid pUH1.

The gamma-glutamyltranspeptidase (gamma-GTP) gene of Bacillus subtilis (natto) plasmid designated pUH1, which is responsible for polyglutamate production, has been cloned and the nucleotide sequence determined. The sequence contains a single open-reading frame stretching for 1260 bp with a relative molecular mass of 49,356. Putative -35 and -10 sequences, TTCAAA and TATTAT, were observed as the consensus sequence for the promoter recognized by the sigma 43 RNA polymerase of B. subtilis, and the ribosome binding site, the sequence of which was AACGAG, was complementary to the binding sequence of B. subtilis 16S rRNA except for one base. The amino acid sequence of the gene with the segment of putative protein C403 of staphylococcal plasmid pE194 indicates homology, whereas that with Escherichia coli and mammalian gamma-GTPs does not show any similarity at all.

Molecular structure of the replication origin of a Bacillus subtilis (natto) plasmid, pUH1.

The structure of a 2.0-kb BstEII DNA sequence necessary and sufficient for the replication of a 5.7-kb Natto plasmid, pUH1, which is responsible for gamma-polyglutamate production by Bacillus subtilis (natto), has been characterized by using a trimethoprim resistance gene derived from B. subtilis chromosomal DNA as a selective marker. The 2.0-kb DNA sequence contains an open reading frame, rep, stretching for 999 bp; a promoter region for rep expression; and a possible replication origin for the plasmid upstream of the promotor. The predicted Rep protein has highly homologous amino acid sequences with rep14 of pFTB14 in B. amyloliquefaciens, RepB of pUB110, and protein A, which is necessary for pC194 replication in staphylococci throughout the protein molecule, but is not homologous with RepC of staphylococcal plasmid pT181.