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10 kJ-Capacity Energy Storage Coil Made of MgB2 proposed in the Advanced Superconducting Power Conditioning System (ASPCS) was fabricated, and an electric current test was conducted with indirect liquid hydrogen cooling. This coil consists of three DP (double pancake) coils with an inner diameter of 400 mm and an outer diameter of 606 mm. The first DP was manufactured by the W&R (Wind and Reacted), and second and third DPs were manufactured by the R&W (React and Wind). A thermosiphon system is used for cooling the coils, in which the coolant is circulate without pump. For energization, several DC excitation and AC excitation tests were carried out with liquid hydrogen, and the rated current of 600 A was successfully achieved. The evaluation of both DC and AC losses were also performed, indicating needs to reduce the losses for stable operation under the indirect cooling conditions.

Chemotherapy is an adjuvant treatment for glioblastomas, however, chemotherapy remains palliative because of the development of multidrug resistance (MDR). Following prolonged chemotherapy, MDR protein 1 (MDR1) and CD133 increase in recurrent glioblastomas. CD133 positive (CD133+) glioma cancer stem-like cells (GCSCs) markedly promote drug resistance and exhibit increased DNA damage repair capability; thus they have a key role in determining tumor chemosensitivity. Although CD133, DNA-dependent protein kinase (DNA-PK), and MDR1 are elevated in CD133+ GCSCs, the relationship among these molecules has not been elucidated. In this study, MDR glioblastoma cell lines were created in response to prolonged doxorubicin chemotherapy. CD133, DNA-PK and MDR1 were markedly elevated in these cells. CD133 and DNA-PK may increase MDR1 via the phosphatidylinositol-3-kinase (PI3K)-Akt signal pathway. PI3K downstream targets Akt and nuclear factor (NF)-κB, which interacts with the MDR1 promoter, were also elevated in these cells. Downregulation of CD133 and DNA-PK by small interfering RNA, or inhibition of PI3K or Akt, decreased Akt, NF-κB and MDR1 expression. The results indicate that CD133 and DNA-PK regulate MDR1 through the PI3K- or Akt-NF-κB signal pathway. Consequently, a novel chemotherapeutic regimen targeting CD133 and DNA-PK in combination with traditional protocols may increase chemotherapeutic efficacy and improve prognosis for individuals who present with glioblastoma.

Study Design:A retrospective radiographic study with a minimum 2-year follow-up.Objective:To evaluate the relationships between the cervical articular facets’ morphology and the incidence of traumatic cervical spinal cord injury (CSCI) without major fracture or dislocation.Setting:Spinal Injuries Center, Japan.Methods:This study included 113 patients with traumatic CSCI without major fracture or dislocation. Eighty-four healthy volunteers without neurological deficits or cervical cord pathology on magnetic resonance imaging (MRI) were defined as control subjects. We used a plain sagittal radiograph to measure the facet sagittal angles (FSA) at four cervical segments in all the CSCI patients and controls. We defined the FSA as the angle between the inferior margin of the superior cervical spinal body and the inferior articular process of the superior vertebra.Results:Most frequent incidence of CSCI was seen at C3–4 segment (54%). With respect to CSCI at C3–4 segment, 55.7% of the subjects showed smallest FSA at C3–4 segment.Conclusion:Most of the traumatic CSCI at C3–4 segment showed raised cervical articular facets at C3–4 segment. On the basis of our results, we hypothesized that the raised cervical articular facets might have an important role in the etiology of traumatic CSCI. The cervical spinal cord at the C3–4 segment might receive the highest load during acute hyperextension of the cervical spine because of the C3–4 articular facets’ morphology.

Dehydroepiandrosterone (DHEA) is believed to have an anti-tumor effect, as well as anti-inflammatory, antioxidant, and anti-aging effects. To clarify the possible inhibitory action of DHEA on pituitary tumor cells, we tested the effects of DHEA, alone or in combination with the nuclear factor-κB (NF-κB) inhibitor parthenolide (PRT), on AtT20 corticotroph cell growth and function both in vitroand in vivo. We found that, in vitro, DHEA and PRT had potent inhibitory effects on pro-opiomelanocortin and NF-κB-dependent gene expression. They also suppressed the transcription activity of survivin, a representative anti-apoptotic factor, and induced apoptosis in this cell line. Furthermore, using BALB/C nude mice with xenografts of AtT20 cells in vivo, we found that the combined administration of DHEA and PRT significantly attenuated tumor growth and survivin expression. The treatment also decreased the elevated plasma corticosterone levels and ameliorated the malnutrition induced by tumor growth. Altogether, these results suggested that combined treatments of DHEA and PRT potently inhibit the growth and function of corticotroph tumor cells both in vitroand in vivo. This effect may, at least partly, be caused by the suppressive effects of these compounds, such as survivin and other inhibitor of apoptosis proteins, on NF-κB-mediated gene transcription.

Treatment of a bis(μ-diphenylsilyl) complex, {Cp*Ru(μ-η²-HSiPh2)}2(μ-H)(H) (2a, Cp* = η⁵-C5Me5), with trifluoroacetic acid in toluene resulted in introduction of a trifluoroacetoxy group on a bridging silicon atom with concomitant loss of one phenyl group as benzene, affording a mixed-bridging bis(μ-silylene) complex, {Cp*Ru(μ-H)}2{μ-SiPh(OCOCF3)}(μ-SiPh2) (3b), in moderate yield. Nucleophilic addition of the acetoxy group took place in the intermediate μ-silyl,μ-silylene complex (Cp*Ru)2(μ-η²-HSiPh2)(μ-SiPh2)(μ-CF3CO2)(μ-H)(H) (7), containing a bridging carboxylate group. The trifluoroacetoxy group of 3b underwent nucleophilic displacement at the bridging silicon atom, which afforded the mixed-bridging bis(μ-silylene) complexes {Cp*Ru(μ-H)}2(μ-SiPhX)(μ-SiPh2) (3c, X = OMe; 3d, X = OH) upon treatment with methanol and aqueous KOH, respectively. VT NMR studies of 3b, 3c, 3d, {Cp*Ru(μ-H)}2(μ-SiPhMe)(μ-SiMe2) (3e), and {Cp*Ru(μ-H)(μ-SiPhMe)}2 (3f-syn) revealed a linear correlation between the electronic nature of the substituents on the bridging silicon atom and the activation parameters of the hydride site-exchange process occurring in the bis(μ-silylene) complex.

Treatment of the triruthenium pentahydrido complex {Cp*Ru(μ-H)}3(μ3-H)2 (1; Cp* = η^5-C5Me5) with cycloalkene resulted in exclusive formation of the μ3-cycloalkyne complex {Cp*Ru(μ-H)}3(μ3-η²:η²(⊥)-CnH2n-4) (2a; n = 5, 2b; n = 6) as a result of vinylic C−H bond cleavage. VT NMR studies revealed fluxional behavior of the perpendicularly coordinated cycloalkyne ligand. Reaction of 2b with carbon monoxide afforded the 48-electron complex (Cp*Ru)3(μ-CO)(μ3-CO)(μ-H)(μ3-η²(∥)-C6H8) (3b) with a parallel-coordinated alkyne ligand.

A series of μ-silylene complexes was synthesized by way of sequential Si−H and Si−C bond scission of tertiary silanes on diruthenium centers generated from Cp*Ru(μ-H)4RuCp* (1; Cp* = η⁵-C5Me5). While both bis- and mono(μ-diphenylsilylene) complexes, {Cp*Ru(μ-SiPh2)(μ-H)}2 (3a) and {Cp*Ru(μ-H)}2(μ-SiPh2) (4a), were obtained in the case of Ph3SiH, the reactions of 1 with Ph2MeSiH and PhMe2SiH exclusively afforded bis(μ-silylene) complexes, {Cp*Ru(μ-SiPhMe)(μ-H)} (3b-syn/anti) and {Cp*Ru(μ-H)}2(μ-SiPhMe)(μ-SiMe2) (3c), respectively. The Si−C bond scission proceeded via prior π-coordination of the phenyl group of the intermediary formed silyl groups, which was supported by the fact that the reaction of 1 with (CH2&dbd;CH)RSiH2 (R = Ph, Me) afforded exclusively the mono(μ-silylene) complex {Cp*Ru(μ-H)}2(μ-SiEtR) (4b, R = Me; 4c, R = Ph) as a result of intramolecular coordination of the vinyl group. Reaction of a mixture of 3b-syn and 3b-anti with PMe3 afforded the bis(μ-silylene) complex {Cp*Ru(μ-SiPhMe)}2(PMe3)(H)2 (5), which adopted only the syn structure with respect to the two bridging silylene ligands. Isomerization of the anti to the syn form arose from rotation of the bridging silylene ligands, which was confirmed by the VT-NMR studies of 3b-syn and 3c. While reactions of 3 with H2 and CO afforded respectively the bis(μ-silyl) complexes {Cp*Ru(μ-η²-HSiR2)}2(μ-H)(H) (2) and bis(μ-silylene) complexes containing two terminal carbonyl groups {Cp*Ru(CO)(μ-SiR2)}2 (6), liberation of the bridging silylene ligand of 4 was observed during the reaction with H2 and CO, together with formation of the mono(μ-silylene) complex {Cp*Ru(CO)}(μ-CO)(μ-SiR¹R²) (8a, R¹ = R² = Ph; 8b, R¹ = Me, R² = Et; 8c, R¹ = Ph, R² = Et). X-ray diffraction studies were performed on 3c, 4a,b, 5, and 8a,b, and they clearly demonstrated the bridging silylene structures of these complexes.

OBJECTIVE: Several reports have described antipituitary antibodies by immunofluorescent or immunoblotting methods in patients with lymphocytic hypophysitis. However, with the exception of the pituitary hormones, individual antigens specific for the pituitary gland have not been studied. To understand the pathogenesis of lymphocytic hypophysitis and to diagnose this disease efficiently, we studied the presence of autoantibodies against three pituitary-specific proteins, GH and two novel pituitary-specific proteins, namely, pituitary gland specific factor 1a (PGSF1a) and PGSF2. DESIGN: Seventeen patients with lymphocytic hypophysitis, all of whom had pituitary enlargement (5 with lymphocytic adenohypophysitis and 12 with lymphocytic infundibuloneurohypophysitis, including 3 of the latter group proven by biopsy), and 14 patients with hypopituitarism without pituitary enlargement (10 with isolated ACTH deficiency and 4 with idiopathic TSH deficiency) were studied, and compared with 11 patients with non-functioning pituitary macroadenoma, 31 patients with other autoimmune diseases, and 36 healthy controls. METHODS: The presence of each antibody was studied by radioligand assay using recombinant human (35)S-labeled protein. RESULTS: Three (18%) patients with lymphocytic hypophysitis having pituitary enlargement, five (36%) patients with hypopituitarism without pituitary enlargement and three (9.7%) patients with other autoimmune diseases were positive for one or more of the antibodies studied. CONCLUSIONS: Anti-human GH, anti-PGSF1a, and anti-PGSF2 antibodies were detected in patients with lymphocytic hypophysitis and other hypopituitarism, but were not detected in patients with non-functioning pituitary macroadenoma. Detection of these antibodies may be useful for the diagnosis of lymphocytic hypophysitis.

Reaction of the triruthenium pentahydride complex {Cp*Ru(μ-H)}3(μ3-H)2 (1; Cp* = η⁵-C5Me5) with acetylene was investigated, in which the μ3-ethylidene−μ3-η²(∥)-ethyne complex {Cp*Ru(μ-H)}3(μ-CMeH){μ3-η²(∥)-CH&dbd;CH} (2) was produced via formation of the intermediary bis(μ-vinyl) complex {Cp*Ru(μ-H)}3(μ-η¹:η²-CH&dbd;CH2)2 (3). An α-C−H bond of the μ-ethylidene ligand of 2 underwent oxidative addition upon thermolysis to yield the μ3-ethylidyne−μ3-η²(∥)-ethyne complex (Cp*Ru)3(μ-H)2(μ3-CCH3){μ3-η²(∥)-CH&dbd;CH} (5), which showed fluxional behavior of both the μ3-ethyne and the hydride ligands. Further C−H bond cleavage of the μ3-ethylidyne ligand, affording the μ-vinylidene−μ3-η²(∥)-ethyne complex (Cp*Ru)3(μ-H)(μ3-C&dbd;CH2){μ3-η²(∥)-CH&dbd;CH} (6), was also investigated. Treatment of 5 with an excess amount of acetylene resulted in C−C bond formation at the Ru3 core, and the μ3-ethylidyne−μ3-η³-diruthenaallyl complex (Cp*Ru)3{μ3-η¹:η³:η¹-C(H)C(H)CCH3}(μ3-CCH3)(μ-H) (7) was obtained. The molecular structures of 5 and 7 were determined by single-crystal X-ray diffraction studies.

The diruthenium complex {Cp*Ru(μ-H)}2{μ-η²:η²-HSiMe2(CH&dbd;CH2)} (5a; Cp* = η⁵-C5Me5) containing a μ-vinylsilane ligand was prepared by the reaction of Cp*Ru(μ-H)4RuCp* (1) with dimethylvinylsilane. The μ-η²:η²-coordination mode of dimethylvinylsilane was confirmed by means of ¹H, ¹³C, and ²⁹Si NMR and IR spectroscopy. The Si−C(sp²) bond of 5a was readily cleaved upon thermolysis with liberating hydrogen and resulted in formation of the μ-silylene, μ-ethylidyne complex (Cp*Ru)2(μ-SiMe2)(μ-CCH3)(μ-H) (8) via the μ-silyl, μ-vinyl complex (Cp*Ru)2(μ-η²-HSiMe2)(μ-η²-CH&dbd;CH2) (9a) as an intermediate. The reaction of 1 with trimethylvinylsilane rendered cleavage of both Si−C(sp²) and Si−C(sp³) bonds, which resulted in formation of 8. Treatment of 8 with carbon monoxide led to formation of the μ-ethylidene complex by migration of the hydride to the bridging carbon; thus {Cp*Ru(CO)}2(μ-CHMe)(μ-SiMe2) (10) was obtained. The reaction of 8 with ethylene afforded the μ-vinyl, μ-silylene complex (Cp*Ru)2(μ-η²-CH&dbd;CH2)(μ-SiMe2)(μ-H)(CH2&dbd;CH2) (11). The reaction of 8 with molecular hydrogen resulted in a re-formation of 1 with liberating dimethylethylsilane, which showed formation of the Si−C bond on the dinuclear system. The molecular structures of 5a, 8, and 10 were determined by single-crystal X-ray diffraction studies.

A method for the easy isolation and direct sequencing of N-terminally blocked peptide in proteins refractory to N-terminal sequencing was developed. It is based essentially on tandem enzymatic treatments of the protein with trypsin and carboxypeptidase B, and selective isolation of the N^α-blocked peptide using ion-exchange chromatography. The chromatographic step was optimized for picomole amounts of sample and very short elution times by placing a thin layer of the resin over the membrane of an ultrafiltration tube. The isolated fraction can be analyzed directly using MALDI or ESI mass spectrometry. The method was applied to several recombinant and natural N-terminal acetylated proteins. A critical discussion on the intrinsic limitations of the method is also given.

In maize (Zea mays L.) two leaf-specific ferredoxin (Fd) isoproteins, Fd I and Fd II, are distributed differentially in mesophyll and bundle-sheath cells. A novel cDNA encoding the precursor of Fd II (pFD2) was isolated by heterologous hybridization using a cDNA for Fd I (pFD1) as a probe. The assignment of the cDNAs to the Fds was verified by capillary liquid-chromatography/electrospray ionization-mass spectrometry. RNA-blot analysis demonstrated that transcripts for Fd I and Fd II accumulated specifically in mesophyll and bundle-sheath cells, respectively. The mature regions of pFD1 and pFD2 were expressed in Escherichia coli as functional Fds. Fd I and Fd II had similar redox potentials of -423 and -406 mV, respectively, but the Km value of Fd-NADP+ reductase for Fd II was about 3-fold larger than that for Fd I. Asparagine at position 65 of Fd II is a unique residue compared with Fd I and other Fds from various plants, which have aspartic acid or glutamic acid at the corresponding position as an electrostatic interaction site with Fd-NADP+ reductase. Substitution of asparagine-65 with aspartic acid increased the affinity of Fd II with Fd-NADP+ reductase to a level comparable to that of Fd I. These structural and functional differences of Fd I and Fd II may be related to their cell-specific expression in the leaves of a C4 plant.

We recently showed that a nuclear location signal (NLS)‐containing karyophile forms a stable complex with cytoplasmic components for nuclear pore‐targeting The complex, termed nuclear pore‐targeting complex (PTAC), contained two essential proteins of 54 and 90 kDa, respectively, as estimated by electrophoresis. In this study, we found that the 54 kDa component of PTAC is the mouse homologue of Xenopus importin (m‐importin). Cytoplasmic injection of the antibodies raised against recombinant m‐importin showed an inhibitory effect on nuclear import of a karyophile in living mammalian cells. A portion of cytoplasmically injected antibodies migrated rapidly into the nucleus, indicating dynamic movement of this protein across the nuclear envelope. Moreover, the injected antibodies co‐precipitated the karyophile, in an NLS‐dependent manner, with endogenous m‐importin in the cytoplasm. These results provide in vivo evidence that m‐importin is involved in nuclear protein import through association with a NLS in the cytoplasm before nuclear pore binding.

A heterotrimeric G‐protein in vertebrate photoreceptor cells is called transducin (T alpha beta gamma), whose gamma‐subunit is a mixture of two components, T gamma‐1 and T gamma‐2. T gamma‐2 is S‐farnesylated and partly carboxyl methylated at the C‐terminal cysteine residue, whereas T gamma‐1 lacks the modified cysteine residue. To elucidate the physiological significance of the double modifications in T gamma, we established a simple chromatographic procedure to isolate T gamma‐1, methylated T gamma‐2 and non‐methylated T gamma‐2 on a reversed phase column. Taking advantage of the high and reproducible yield of T gamma from the column, we analyzed the composition of T gamma subspecies in the T alpha‐T beta gamma complex which did not bind with transducin‐depleted rod outer segment membranes containing metarhodopsin II. The binding of T alpha‐T beta gamma with the membranes was shown to require the S‐farnesylated cysteine residue of T gamma, whose methylation further enhanced the binding. This synergistic effect was not evident when T alpha was either absent or converted to the GTP‐bound form which is known to dissociate from T beta gamma. Thus we concluded that a formation of the ternary complex, T alpha‐T beta gamma‐metarhodopsin II, is enhanced by the farnesylation and methylation of T gamma. This suggests that the double modifications provide most efficient signal transduction in photoreceptor cells.

Allophycocyanin (APC) α- and β-subunits were isolated from Anabaena cylindricaand their amino acid sequences were determined. The α- and β-subunits consisted of 160 and 161 amino acid residues, respectively, and lacked tryptophan. The β-subunit contained an unknown derivative of aspartic acid. The sequences were compared with those of other allophycocyanins.

In an abnormal fibrinogen with impaired fibrin monomer polymerization designed as fibrinogen Osaka II, we have identified substitution of Arg by Cys at position 275 of the gamma chain. This Cys is linked to a free cysteine molecule by a disulfide link as evidenced by fast atom bombardment mass spectrometry. This finding was supported by identification of a single cysteine released from isolated abnormal fragment D1 upon reduction. This unique cystine structure at the mutation site has not been reported heretofore in any abnormal protein including fibrinogen. The substitution may well perturb the structure required for fibrin monomer polymerization, specifically that assigned to the carboxyl-terminal D domain of fibrinogen. Indeed, isolated fragment D1 with the Cys substitution failed to inhibit thrombin-mediated clotting of normal fibrinogen and normal fibrin monomer polymerization, while normal fragment D1 inhibited them markedly. Our data seem to provide supporting evidence that the putative polymerization site(s) assigned to the D domain of fibrinogen may be structure-dependent, including the carboxyl-terminal segment of the gamma chain as well as a contiguous region that contains the gamma 275 residue.

Glutathione peroxidase (GPx) was inactivated by S-nitroso-N-acetyl-D, L-penicillamine (SNAP), a nitric oxide donor (Asahi, M., Fujii, J., Suzuki, K., Seo, H. G., Kuzuya, T., Hori, M., Tada, M., Fujii, S., and Taniguchi, N. (1995) J. Biol. Chem. 270, 21035-21039). The structural basis of the inactivation was studied. We also show that 3-morpholinosydnonimine N-ethylcarbamide, a peroxynitrite precursor, as well as synthetic peroxynitrite also inactivated bovine GPx. The degree of incorporation of a sulfhydryl reagent, n-octyldithionitrobenzoic acid, into GPx decreased after pretreatment with SNAP as evidenced by mass spectrometry. To identify the modification site of this enzyme by SNAP, both SNAP-pretreated and untreated GPxs were reacted with n-octyldithionitrobenzoic acid and digested with lysylendopeptidase, and the resulting peptides were subjected to mass spectrometry. This technique identified a bridge between two peptides, one of which contains Sec45 at the catalytic center and Cys74, and the other contains Cys91. Although there are two possible combinations, selenocysteine 45 (Sec45) and Cys91 or Cys74 and Cys91, the tertiary structure of GPx indicates that a cross-link between Sec45 and Cys91 is more feasible. This is consistent with the experimental evidence that SNAP specifically inactivates GPx, in which Sec45 forms the catalytic center. Thus, we conclude that SNAP mainly oxidized Sec45 to form a selenenyl sulfide (Se-S) with a free thiol, leading to the inactivation of the enzyme. These data suggest that nitric oxide and its derivatives directly inactivate GPx in a specific manner via the production of a selenenyl sulfide, resulting in an increase in intracellular peroxides that are responsible for cellular damage.

A mass spectrometric method was applied to protein S, a development-specific protein of Myxococcus xanthus, in order to verify the amino acid sequence deduced from the nucleotide sequence of its gene. On examining proteolytic digests of the protein by fast atom bombardment mass spectrometry without separation of individual peptides, signals corresponding to individual peptides were observed in the mass spectra. The mass values of the observed signals were correlated to the theoretical mass values calculated from the predicted amino acid sequence, thereby identifying the peptides and proving the accuracy of the sequence. The previous finding that protein S is the product of the second gene of the two tandemly repeated genes on the M. xanthus chromosome ( Inouye , S., Franceschini , T., and Inouye , M. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6829-6833) is now unambiguously confirmed.

We summarize data from some of our recent studies on in vitroand in vivomodulation of interleukin-1 (IL-1) receptors in the mouse brain-endocrine-immune axis by stress and infection. Ether-laparotomy stress in mice resulted in a selective increase in pituitary IL-I receptors and a significant decrease in pituitary receptors for corticotropin-releasing factor (CRF), a major regulator of the endocrine response to stress. Intraperitoneal injection of rat/human CRF mimicked the effects of stress and resulted in a dramatic increase in [125I]IL-1α binding in the pituitary; [125I]IL-1α binding in the hippocampus, spleen, and testis was unaffected by stress or CRF treatment. Glucocorticoid treatment with dexamethasone alone did not alter [125I]IL-1α, binding but significantly inhibited CRF-induced upregulation of IL-1 receptors in the pituitary. The intracellular mechanism(s) involved in stress and CRF-induced upregulation of IL-1 receptors in the pituitary gland were examined by evaluating the effects of treatment of AtT-20 mouse pituitary corticotroph cells with a variety of neuroendocrine mediators of stress. CRF, forskolin, and isoproterenol (β2adrenergic receptor agonist) produced dose-dependent increases in cAMP production and [125I]IL-1α binding. In contrast, somatostatin and dexamethasone significantly inhibited CRF-stimulated increase of cAMP production and [125I]IL-1α binding, suggesting a primary role for cAMP in the regulation of pituitary IL-1 receptors. Next, we investigated the modulation of LL-1 β levels and IL-1 receptors following infection of mice with the endotoxin, lipopolysaccharide (LPS). Acute administration of low doses of endotoxin (30 μg LPS/mouse) dramatically increased IL-1 β levels and reciprocally decreased [125I]IL-1α binding in peripheral tissues (pituitary, testis, liver, and spleen) but not in brain (hippocampus). This effect appeared to be dose related since higher doses of endotoxin (300 μg LPS/mouse) significantly decreased [125I]IL-1α binding in both peripheral tissues and brain. Endotoxin induced modulation of the IL-1 system was also dependent on the treatment regimen since two low-dose LPS injections (at 0 and 12 h) increased IL-1 β concentrations and decreased [125I]IL-1α binding in both central and peripheral tissues. These data provide further support for a role for IL-1 in coordinating brain-endocrine-immunoresponses to stress and infection.

An abrupt conductor motion is one of the dominant origins of a premature quench in a high-performance, high-current-density, magnet. A model based on microscopic slip phenomena is proposed to estimate the disturbance energy due to conductor motion from the mechanical properties of the conductors and the winding configurations, and the stability of the conductor against the disturbance. Stability of a rectangular-cross-section conductor is better than that of a round-cross-section conductor. To improve the stability by reducing the disturbance energy, the aspect ratio (for the rectangular conductor) and the diameter (for the round conductor) must be increased.<>

A potent anticoagulant, anti-lipopolysaccharide (LPS) factor, found in limulus hemocytes inhibits the LPS-mediated activation of limulus coagulation cascade and shows an antibacterial action against R-types of Gram-negative bacteria (Morita, T., Ohtsubo, S., Nakamura, T., Tanaka, S., Iwanaga, S., Ohashi, K., and Niwa, M. (1985) J. Biochem. (Tokyo) 97, 1611-1620). The complete amino acid sequence of this substance was determined by sequencing the peptides obtained by selective proteolytic cleavage. The NH2-terminal end of anti-LPS factor was pyroglutamic acid. Anti-LPS factor had two variant residues at position 36 and the COOH-terminal end, respectively. The following sequence was assigned to anti-LPS factor, and it was also confirmed by fast atom bombardment mass spectrometry. less than EGGIWTQLALALVKNLATLWQSGDFQFLGHE (formula; see text) Limulus anti-LPS factor consisted of a single chain of 102 residues with 2 half-cystines in disulfide linkage. Its NH2-terminal region up to 20 residues was highly hydrophobic, and positively charged residues were clustered mainly within the disulfide loop. By searching the homologous sequence in known protein sequences with that of anti-LPS factor, we found a structural homology between anti-LPS factor and alpha-lactalbumin/lysozyme family.

A biosurfactant termed arthrofactin produced by Arthrobacter species strain MIS38 was purified and chemically characterized as 3-hydroxydecanoyl-D-leucyl-D-asparagyl-D-threonyl-D- leucyl-D-leucyl-D-seryl-L-leucyl-D-seryl-L-isoleucyl-L-isoleucyl-L-as paragyl lactone. Surface activity of arthrofactin was examined, with surfactin as a control. Critical micelle concentration values of arthrofactin and surfactin were around 1.0 x 10(-5) M and 7.0 x 10(-5) M at 25 degrees C, respectively. Arthrofactin was found to be five to seven times more effective than surfactin. The minimum surface tension value of arthrofactin was 24 mN/m at a concentration higher than the critical micelle concentration. According to the oil displacement assay, arthrofactin was a better oil remover than synthetic surfactants, such as Triton X-100 and sodium dodecyl sulfate. Arthrofactin is one of the most effective lipopeptide biosurfactants.

Proteolytic digests of interleukin-2 from a human leukemic T-cell line produced by Escherichia coli carrying a recombinant DNA were analyzed by fast atom bombardment mass spectrometry. The mass values of intense signals observed in the mass spectrum were consistent with peptides predicted from the nucleotide sequence of cDNA for human interleukin-2, an indication that the protein with the predicted amino acid sequence was produced by E. coli. BrCN and proteolytic digests of interleukin-2 obtained from cultured cells were also examined by fast atom bombardment mass spectrometry. The observed mass values were identical with those from interleukin-2 from E. coli except for that of the NH2-terminal sequence, in which the Thr residue at position 3 was bound to a sugar moiety. The mass spectra of the digests of the two interleukin-2 preparations and synthetic peptides with sequences from 117 to 128 and 121 to 128 predicted from the nucleotide sequence of cDNA for a human interleukin-2 indicated that Cys residues at positions 58 and 105 are linked by a disulfide bond and that the Cys residue at position 125 is free.

The amino acid sequence of the ubiquinone binding protein (QP-C) in the cytochrome bc1 region of the mitochondrial electron transfer chain was determined by analysis of peptides obtained by cyanogen bromide cleavage and staphylococcal protease digestion of succinylated derivatives. It was found to consist of 110 amino acid residues and its amino terminus to be blocked by an acetyl group, as determined by mass spectrometry of the amino-terminal peptide and a comparison with peptides chemically synthesized on high-performance liquid chromatography. The molecular weight of this ubiquinone binding protein including the acetyl group was calculated to be 13,389. The predicted secondary structure of QP-C has alpha-helical content of about 50% and QP-C was classified as an “all-alpha” or “alpha + beta” protein. This is the first report describing the amino acid sequence of the ubiquinone binding protein. A comparison of this sequence with that of the 14-kDa subunit of the yeast ubiquinol-cytochrome c reductase complex from the nucleotide sequence showed these two sequences to be quite similar.

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).

The gamma-subunits of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) are isoprenylated and alpha-carboxyl methylated at their COOH-terminal cysteine residues. These modifications are necessary for membrane attachment of the beta gamma complex, but a requirement of an additional factor has been proposed for the stable binding. We explored a possible contribution of the blocked amino terminus of beta-subunits of bovine photoreceptor G-protein, transducin (T alpha/T beta gamma = Gt alpha/beta 1 gamma 1), and of three beta gamma complexes (beta 1 gamma 2, beta 1 gamma 3, and beta 1 gamma 7) purified from bovine brains. Structural analyses revealed that every beta 1-subunit has an N-acetylated serine, which is unlikely to contribute to the membrane association. Since neither protease nor heat treatment of photoreceptor membranes affected the membrane binding of T beta gamma, it seems unlikely that rhodopsin (or other membrane proteins) serves as an anchor protein for accepting T beta gamma. In fact, T beta gamma bound to phospholipid large unilamellar vesicles (LUVs), of which the polar head groups strongly influenced the binding: T beta gamma alone showed 2-fold higher binding for negatively charged phosphatidylserine-LUVs than for neutral phosphatidylcholine (PC)-LUVs, while the affinity of T alpha/T beta gamma complex for the phosphatidylserine-LUVs was lower than that for the PC-LUVs. These results indicate that 1) an ionic interaction between T beta gamma and membrane surface plays an important role in the stable membrane association, and 2) the domain(s) of T beta gamma responsible for the association would be different between trimeric and dissociated states. We also found that synthetic peptides corresponding to the COOH-terminal region of T gamma inhibited T alpha-T beta gamma interaction only when the peptides were isoprenylated. This suggests that the isoprenyl moiety is located at the contact site between the subunits, not at the membrane-binding domain, when T beta gamma is complexed with T alpha.

In maize, a small multigene family encodes the cytosolic isoforms of glutamine synthetase (GS), and five cDNAs, designated pGS1a, pGS1b, pGS1c, pGS1d, and pGS1e, have been cloned (Sakakibara, H., Kawabata, S., Takahashi, H., Hase, T., and Sugiyama, T. (1992) Plant Cell Physiol. 33, 49-58; Li, M., Villemur, R., Hussey, P. J., Silflow, C. D., Gantt, J. S., and Snustad, D. P. (1993) Plant Mol. Biol. 23, 401-407). This report describes the identification and enzymatic characterization of the cytosolic isoforms of GS in maize roots, namely GS1 and GSr. The purified isoforms, as well as recombinant enzymes that had been overexpressed in Escherichia coli, were analyzed by capillary liquid chromatography/electrospray ionization-mass spectrometry, and GS1 and GSr were identified as the products of the GS1a/GS1b and GS1c/GS1d genes, respectively. Upon the addition of ammonia to the culture medium, significant amounts of GSr accumulated and a preferential increase in GS synthetase activity, as compared to GS transferase activity, was found in the root extract. Assays with the purified recombinant enzymes confirmed that the specific biosynthetic and synthetase activities of GSr were 1.6-fold higher than those of GS1. Marked differences in stability were also found between the two isoforms: GSr was more sensitive to heat than GS1 and octameric aggregates of the subunits of GSr were easily dissociated to monomers than those of GS1 at low concentrations of Mn2+ and Mg2+ ions. These characteristics of the ammonia-induced isoform of GS seem to be physiologically important for the primary assimilation of external ammonia by roots.

We have recently described a unique trisaccharide linked to a serine residue in the first epidermal growth factor-like domains of bovine blood coagulation factors VII (Ser-52) and IX (Ser-53) (Hase, S., Kawabata, S., Nishimura, H., Takeya, H., Sueyoshi, T., Miyata, T., Iwanaga, S., Takao, T., Shimonishi, Y., and Ikenaka, T. (1988) J. Biochem. (Tokyo) 104, 867-868). The sugar chain identified in these clotting factors consists of 1 mol of hexose (glucose (Glc] and 2 mol of pentose (xylose (Xyl]. We report here that human factors VII and IX and protein Z and bovine protein Z also contain such carbohydrate moieties linked to a serine residue at the same position found in bovine factors VII and IX. A glycopeptide derived from each of these proteins was subjected to amino acid sequence and component sugar analyses and fast atom bombardment mass spectrometric analysis. The results indicate that the glycopeptide derived from human factor IX contains 1 mol each of Glc and Xyl. The reducing end of this disaccharide was identified as Glc by analyzing the disaccharide generated by hydrazinolysis. In contrast, human factor VII and protein Z yielded two different glycopeptides which contained Glc and Xyl at molar ratios of 1:1 and 1:2, respectively, suggesting microheterogeneity of these O-linked sugar chains. Bovine protein Z glycopeptide contained 1 mol of Glc and 2 mol of Xyl. These sugar compositions were confirmed by analyses of the intact proteins. In relation to the trisaccharide sugar chain previously discovered in bovine factors VII and IX, these findings indicate the existence of a Xyl2-Glc-Ser and a Xyl-Glc-Ser structure in the first epidermal growth factor-like domains of human factors VII and IX and protein Z in addition to that of bovine protein Z. Whether these carbohydrate moieties contribute to the biological activities of these proteins is unknown.

Recoverin, a new member of the EF-hand superfamily, plays a critical role in the light/dark adaptation of retinal rods by regulating rhodopsin phosphorylation in a Ca(2+)-dependent manner. Recoverin is composed of four isoforms, each of which is modified at its N terminus by myristate (C14:0) or its structurally related fatty acid (C12:0, C14:2, or C14:1). Although the N-fatty acylation is implicated in protein-membrane and protein-protein interactions, the functional difference among the recoverin isoforms and the significance of the heterogeneous acylation have not been defined. Here we separated the heterogeneous recoverin into three fractions, C14:0-recoverin, C14:1-recoverin, and a mixture of C14:2- and C12:0- (C14:2/C12:0-) recoverin to evaluate the individual properties. Recoverin in every fraction bound Ca2+ as assessed by fluorescence spectroscopy and inhibited the light-dependent rhodopsin phosphorylation in the same range of free Ca2+ concentration (0.3-0.8 microM). However, the magnitude of the inhibition at higher Ca2+ concentration was different among the isoforms and ranked in the same order of the hydrophobicity of the N-fatty acyl groups: C14:0 > C14:1 > C14:2/C12:0. These results indicate that the diverged hydrophobicity of the recoverin N terminus plays an important role in the interaction with the membranes and/or its target protein but not with Ca2+.

A cationic peptide, designated tachyplesin, was isolated from acid extracts of horseshoe crab (Tachypleus tridentatus) hemocyte debris. It consists of 17 residues and the structure determined by Edman degradation is: (formula; see text) The carboxyl-terminal end of this peptide was identified as arginine alpha-amide, and the whole sequence including the alpha-amide was also confirmed by fast atom bombardment mass spectrometry, indicating a mass value of 2263. Tachyplesin inhibits growth of both Gram-negative and -positive bacteria at low concentrations and formed a complex with bacterial lipopolysaccharide. Tachyplesin seems likely to act as antimicrobial peptide for self-defense in the horseshoe crab against invading microorganisms.

Proteolytic digests of biologically active fractions of recombinant human leukocyte interferon A expressed in large quantities in Escherichia coli were analyzed by fast atom bombardment mass spectrometry and high-performance liquid chromatography. The values observed in the mass spectra of digests of the major fraction of recombinant human leukocyte interferon A with trypsin and Staphylococcus aureus protease V8 accounted for 93% of the amino acid sequences of human leukocyte interferon A predicted from the nucleotide sequence of the gene encoding the protein, indicating that the major fraction of recombinant human leukocyte interferon A was expressed with the same amino acid sequence as that translated from the nucleotide sequence of the gene encoding the protein. Mass spectrometry of proteolytic digests of two minor fractions of recombinant human leukocyte interferon A and mass and amino acid analyses of their high-performance liquid chromatography fractions showed that the amino group of the N-terminal amino acid residue of interferon was in part acetylated, and the Cys-1 and Cys-98 residues were oxidized to cysteic acid or linked to glutathione. These findings suggest that amino acid residues in recombinant proteins prepared in large quantities in E. coli are modified post-translationally.

The complete amino acid sequence of a low molecular weight peptide from the hemolymph of the housefly Musca domestica L., which had been determined to competitively inhibit phenol oxidase (PO; monophenol, dihydroxy-phenylalanine:oxygen oxidoreductase; EC 1.14.18.1) in the nM range, was unambiguously established by employing both automatic Edman degradation and mass spectrometry. The physiologically active peptide, which was designated phenol oxidase inhibitor (POI), has an observed molecular weight of 4213.1 +/- 0.2 by electrospray ionization mass spectrometry. The relatively short and structurally dense peptide contained 38 amino acid residues rich in cysteine and lysine. Comparison of the observed and calculated molecular mass indicates that apparently all six cysteine residues form disulfide bridges. Interestingly, sequence analyses of both the intact and protease-digested S-pyridylethylated POI showed that one of the two tyrosine residues (Tyr-32) is hydroxylated to a 3,4-dihydroxyphenylalanine (dopa) residue. This agreed with the increase of 16 mass units observed in mass spectrometric measurements. This was further verified by submission of free L-dopa to the sequencer, which gave a retention time consistent with the atypical peak observed at the Edman cycle of the peptide containing dopa. This study demonstrates the existence of a biologically active, dopa-containing peptide among the insects. Since the POI activity was most prominent in aged pupae, especially pharate adults, the POI may play an important role in smoothing the way of adult emergence through hindering excessive melanization, as well as hardening, of cuticular proteins under the epicuticle.

Treatment of the triruthenium pentahydrido complex {Cp*Ru(μ-H)}3(μ3-H)2 (1; Cp* = η⁵-C5Me5) with substituted alkynes results in the exclusive formation of the μ3-alkyne complex {Cp*Ru(μ-H)}3(μ3-η²:η²(⊥)-RCCR‘) (2a, R = Ph, R‘ = H; 2b, R = C(CH3)3, R‘ = H; 2c, R = Ph, R‘ = CH3), in which the alkyne adopts a perpendicular coordination mode relative to one of the Ru−Ru edges, as revealed by an X-ray structure determination. The acetylenic carbon located inside the trinuclear core is shown to appear at particularly high magnetic field in the ¹³C NMR spectra.

Thermolysis of a triruthenium complex containing a nido-ruthenacyclopentadiene ligand {Cp*Ru(μ-H)}3(CH&dbd;CMe-CR&dbd;CH) (3a, R = H; 3b, R = Me, Cp* = η⁵-C5Me5) affords an open-form Ru3 cluster (Cp*Ru)2{Cp*Ru(CH&dbd;CMe-CR&dbd;CH)}(μ-H) (4a, R = H; 4b, R = Me) as a result of partial Ru−Ru bond cleavage. Re-formation of an Ru3 triangle accompanied with C−C bond cleavage of the C4 fragment was performed by prolonged heating of 4 to afford a μ3-methylidyne-μ3-diruthenaallyl complex (Cp*Ru)3(μ-H)(μ3-CH)(μ3-CRCMeCH) (5a, R = H; 5b, R = Me).

Oxidation of the triruthenium cluster 2 bearing a face-bridging allyl fragment results in the formation of a μ3-C3 ring on the Ru3 plane as a consequence of bimetallic reductive C−C coupling. Both structural and spectral data of 5 indicate carbenic character of the carbon atoms within the newly formed face-bridging C3 ring.