Involvement of brain oxidation in the cognitive impairment in a triple transgenic mouse model of Alzheimer's disease: noninvasive measurement of the brain redox state by magnetic resonance imaging.

Oxidative stress is considered to be related to the onset and/or progression of Alzheimer's disease (AD), but there is insufficient evidence of its role(s). In this study, we evaluated the relationships between the brain redox state and cognitive function using a triple transgenic mouse model of AD (3 × Tg-AD mouse). One group of 3 × Tg-AD mice started to receive an α-tocopherol-supplemented diet at 2 months of age and another group of 3 × Tg-AD mice was fed a normal diet. The levels of α-tocopherol, reduced glutathione, oxidized glutathione, and lipid peroxidation were decreased in the cerebral cortex and hippocampus at 4 months of age in the 3 × Tg-AD mice fed a normal diet. These reductions were abrogated by the supplementation of α-tocopherol in the diet. During Morris water maze testing, the 3 × Tg-AD mice did not exhibit cognitive impairment at 4 months of age, but started to show cognitive dysfunction at 6 months of age, and α-tocopherol supplementation suppressed this dysfunction. Magnetic resonance imaging (MRI) using 3-hydroxymethyl-proxyl as a probe showed decreases in the signal intensity in the brains of 3 × Tg-AD mice at 4 months of age, and this reduction was clearly attenuated by α-tocopherol supplementation. Taken together, these findings suggest that oxidative stress can be associated with the cognitive impairment in 3 × Tg-AD mice. Furthermore, MRI might be a powerful tool to noninvasively evaluate the increases in reactive radicals, especially those occurring during the early stages of AD.

Performance enhancement of semiconductor devices by control of discrete dopant distribution.

As semiconductor devices are scaled down to the nanometre level, random dopant fluctuation in the conducting channel caused by the small number of dopant atoms will significantly affect device performance. We fabricated semiconductor devices with random discrete dopant distribution in the drain side and then evaluated how well we could control the drain current of the devices. The results showed that the drain current in devices with the dopant distribution in the drain side was several per cent higher than that in devices with the dopant distribution in the source side. We believe that this increase in current is caused by the suppression of injection velocity degradation in the source side. The capability to control the location of individual dopant atoms enhances drain current and, therefore, the performance of nanodevices. Accurately controlling both the amount and the positioning of dopant atoms is critical for the advancement of true nanoelectronics.

Biomechanical evaluation of anterior spinal instrumentation systems for scoliosis: in vitro fatigue simulation.

STUDY DESIGN: A biomechanical study was designed to assess the bone-screw interface fixation strength among five anterior spinal instrumentation systems for scoliosis before and after a fatigue simulation. OBJECTIVES: The objectives of the current study were twofold: 1) evaluate the static (initial) strength at the bone-screw interface and 2) evaluate dynamic (post fatigue) strength of the bone-screw interface after a fatigue simulation to investigate a possible mechanism for postoperative loss of correction. SUMMARY OF BACKGROUND DATA: Although the recent advancement of anterior instrumentation for scoliosis has permitted shorter fusion segments and improved surgical correction, the loss of correction over the instrumented segments still has been reported in one-rod systems. Little is known about the mechanism for loss of correction. METHODS: Twenty-five fresh-frozen calf spines (T6-L6) were used. A total of five instrumentation systems included the following: Anterior ISOLA (ISOLA), Bad Wildungen Metz (BWM), Texas Scottish Rite Hospital system (TSRH), Cotrel-Dubousset Hoph (CDH), and Kaneda Anterior Scoliosis System (KASS). Screw pullout and rotational tests in the sagittal plane using a single vertebra were performed to investigate bone-screw interface fixation strength before and after a fatigue simulation. To simulate cyclic loading that the spine could undergo in vivo, a fatigue simulation using compressive-flexion loading up to 24,000 cycles was carried out. RESULTS: Mean maximum tensile pullout force decreased in the following order: KASS > CDH > BWM > TSRH > ISOLA (F = 29.91, P < 0.0001). KASS blunt tip screw was 26% stronger in pullout force than KASS sharp tip screw (P < 0.05). The one-rod system demonstrated a positive correlation between pullout force and both bone mineral density and screw insertional torque. For fatigue analysis the rotational strength at the most cephalad and caudal segments significantly decreased after a fatigue simulation in the one-rod system (P < 0.05). The two-rod system showed no significant decrease after a fatigue simulation. CONCLUSIONS: Simulating the cyclic loading to the construct, screw loosening at the bone-screw interface was produced in the one-rod system. This screw loosening may elucidate one mechanism for loss of correction in the one-rod system. The two-rod system may have the potential to minimize the risk of loss of correction.

Biomechanical evaluation of stand-alone interbody fusion cages in the cervical spine.

STUDY DESIGN: An in vitro biomechanical investigation of the immediate stability in cervical reconstruction. OBJECTIVES: The purpose of this study was to compare the segmental stability afforded by the interbody fusion cage, the anterior locking plate, and the "gold standard" autograft. SUMMARY OF BACKGROUND DATA: Recently, interbody fusion cage devices have been developed and used for cervical reconstruction, but to the authors' knowledge no studies have investigated the biomechanical properties of the stand-alone interbody cage device in the cervical spine. METHODS: Using six human cervical specimens, nondestructive biomechanical testing were performed, including axial rotation (+/-1.5 Nm, 50 N preload), flexion/extension (+/-1.5 Nm) and lateral bending (+/-1.5 Nm) loading modes. After C4-C5 discectomy, each specimen was reconstructed in the following order: RABEA cage (cage), tricortical bone graft (autograft), cervical spine locking plate system (plate). Unconstrained three-dimensional segmental range of motion at C4-C5 and above and below were evaluated. RESULTS: In flexion/extension, the plate demonstrated significantly lower range of motion than did the cage and the autograft (P < 0.005), and the cage showed a significantly higher range of motion than did the intact spine (P < 0.05). Under axial rotation, the plate indicated a significantly lower range of motion than did all other groups (P < 0.05). No significant differences were indicated in lateral bending. Adjacent to C4-C5, an increased range of motion was observed. CONCLUSIONS: The increased motion adjacent to C4-C5 may provide an argument for acceleration of disc degeneration. From the biomechanical point of view, this study suggests that the cervical interbody fusion cage should be supplemented with additional external or internal supports to prevent excessive motion in flexion-extension.

Biomechanical and morphologic evaluation of a three-dimensional fabric sheep artificial intervertebral disc: in vitro and in vivo analysis.

STUDY DESIGN: We have developed a new artificial intervertebral disc consisting of triaxial three-dimensional fabric for the sheep lumbar spine. To clarify the characteristics of the new implant, a series of biomechanical tests and morphologic evaluations were conducted. OBJECTIVES: To investigate the static, viscoelastic, and fatigue properties of the three-dimensional fabric disc in comparison with natural sheep disc and to evaluate their biomechanical and morphologic alteration in vivo. SUMMARY OF BACKGROUND DATA: In its human dimensions the three-dimensional fabric disc revealed mechanical properties similar to a natural human disc. METHODS: The disc-body units from sheep spine and the sheep three-dimensional fabric discs underwent tensile-compressive (200 N), torsional (5 Nm), and creep-recovery tests (30 minutes-30 minutes, 200 N). After fatigue loading (2 million, compressive 200 N) the biomechanical changes and the debris were investigated. For in vivo evaluation after placing in the sheep psoas muscles for 6 months, the surface of the three-dimensional fabric disc was evaluated using macroscopy and scanning electron microscopy, followed by previous biomechanical tests. RESULTS: The behavior of the sheep three-dimensional fabric disc was similar to that of natural sheep disc in tensile-compressive and creep-recovery tests. In torsional testing the behavior of natural sheep disc was more rigid than that of the sheep three-dimensional fabric disc. After fatigue loading there was no biomechanical change and no debris detected. Six months after surgery no morphologic deterioration was observed nor were there changes in biomechanical parameters. CONCLUSIONS: The sheep three-dimensional fabric disc exhibited biomechanical and morphologic biostability, appropriate viscoelasticity, and excellent fatigue properties. The three-dimensional fabric disc has a potential for clinical application of human intervertebral disc replacement.

Conformational switching of Escherichia coli RNA polymerase-promoter binary complex is facilitated by elongation factor GreA and GreB.

BACKGROUND: The initiation arrest at a modified lambdaPR promoter is caused by irreversible divergence of the reaction pathway into productive and arrested branches. Escherichia coli GreA and GreB induce cleavage of the nascent transcript and relieve arrest in elongation. They also reduce abortive synthesis at several promoters and relieve initiation arrest. Their mechanism of action during initiation, and its relationship to the branched initiation pathway are unknown. RESULTS: The Gre factors mitigated initiation arrest only when they were added to the binary complex of the holoenzyme bound to the lambdaPR promoter, prior to RNA synthesis. They exerted little effect when they were added to ternary initiation complexes. They accelerated the exchange of the binary complex with its free components by 6-9-fold. When they are present, a high concentration of the initiating nucleotide increased yield of the full-length transcript, whereas a low concentration did not. CONCLUSIONS: All the results presented above can be explained by a model where the productive and arrested pathways diverge at the binary complex stage. The Gre factors relieve the initiation arrest by introducing reversibility between subspecies of the binary complex that are precursors of the two pathways. RNA cleavage is unlikely to cause relief of initiation arrest.

Direct observation of DNA rotation during transcription by Escherichia coli RNA polymerase.

Helical filaments driven by linear molecular motors are anticipated to rotate around their axis, but rotation consistent with the helical pitch has not been observed. 14S dynein and non-claret disjunctional protein (ncd) rotated a microtubule more efficiently than expected for its helical pitch, and myosin rotated an actin filament only poorly. For DNA-based motors such as RNA polymerase, transcription-induced supercoiling of DNA supports the general picture of tracking along the DNA helix. Here we report direct and real-time optical microscopy measurements of rotation rate that are consistent with high-fidelity tracking. Single RNA polymerase molecules attached to a glass surface rotated DNA for >100 revolutions around the right-handed screw axis of the double helix with a rotary torque of >5 pN nm. This real-time observation of rotation opens the possibility of resolving individual transcription steps.

Biomechanical comparison of five different atlantoaxial posterior fixation techniques.

STUDY DESIGN: Five different reconstructions of the atlantoaxial complex were biomechanically compared in vitro in a nondestructive test. OBJECTIVES: To determine whether non-bone graft-dependent one-point fixation affords stability levels equivalent to three-point reconstructions. SUMMARY OF BACKGROUND DATA: Previous investigations have demonstrated that three-point fixation, using bilateral transarticular screws in combination with posterior wiring, provide the most effective resistance to minimize motion around C1-C2. However, placement of transarticular screws is technically demanding. Posterior wiring techniques affording one-point fixation have failure rates of approximately 15%, with failure considered to be secondary to structural bone graft failures. One-point, non-bone graft-dependent fixations have not been tested. METHODS: Eight human cervical specimens, C0-C3 were loaded nondestructively. Unconstrained three-dimensional segmental motion was measured. The reconstructions tested were two one-point fixations, one two-point fixation, and two three-point fixations. RESULTS: Under axial rotation two and three-point reconstructions provided better stiffness than the one-point reconstructions (P < 0.05). During flexion-extension, higher stiffness levels were observed in one- and three-point fixations when compared with the intact spine (P < 0.05). In lateral bending no significant differences were observed among the six groups, although the trend was that reconstructions including transarticular screws provided greater stability than one-point fixations. CONCLUSION: The current findings substantiate the use of three-point fixation as the treatment of choice for C1-C2 instability. [l: atlantoaxial fixation, biomechanics, cervical spine, instability, spinal instrumentation, transarticular screws]

Roles of functional loops and the C-terminal segment of a single-stranded DNA binding protein elucidated by X-Ray structure analysis.

The single-stranded DNA (ssDNA) binding protein from Escherichia coli (EcoSSB) plays a central role in DNA replication, recombination and repair. The tertiary structure of EcoSSB was determined at 2.2 A resolution. This is rather higher resolution than previously reported. Crystals were grown from the homogeneous intact protein but the EcoSSB tetramer in the crystals contains truncated subunits lacking a part of the C-terminal. The structure determined includes biologically important flexible loops and C-terminal regions, and revealed the existence of concavities. These concavities include the residues important for ssDNA binding. An ssDNA can be fitted on the concavities and further stabilized through interactions with the loops forming flexible lids. It seems likely to play a central role in the binding of ssDNA.

Polymerase arrest at the lambdaP(R) promoter during transcription initiation.

During transcription initiation by Escherichia coli RNA polymerase, a fraction of the homogeneous enzyme population has been kinetically shown to form two types of nonproductive complexes at some promoters: moribund complexes, which produce only abortive transcripts, and fully inactive ternary complexes (Kubori, T., and Shimamoto, N. (1996) J. Mol. Biol. 256, 449-457). Here we report biochemical isolation of the complexes arrested at the lambdaP(R) promoter and an analysis of their structure by DNA and protein footprintings. We found that the isolated promoter-arrested complexes retain a stoichiometric amount of sigma(70) subunit. Exonuclease III footprints of the arrested complexes are backtracked compared with that of the binary complex, and KMnO(4) footprinting reveals a decrease in the melting of DNA in the promoter region. Protein footprints of the retained sigma(70) have shown a more exposed conformation in region 3, compared with binary complexes. This feature is similar to that of the complexes arrested in inactive state during transcription elongation, indicating the existence of a common inactivating mechanism during transcription initiation and elongation. The possible involvement of the promoter arrest in transcriptional regulation is discussed.

Attenuation of endogenous oxidative stress-induced cell death by cytochrome P450 inhibitors in primary cultures of rat hepatocytes.

This study reports an examination of the effects of endogenous oxidative stress on primary cultures of rat hepatocytes. To produce endogenous oxidative stress, 3-amino-1,2,4-triazole (ATZ) and mercaptosuccinic acid (MS), which are known to inhibit catalase and glutathione peroxidase activities, respectively, were used. When ATZ or MS was used alone, the extent of cell injuries was negligible, but a combination of the two agents resulted in cell death as assessed by trypan blue exclusion after 24 h of incubation. Cell death was accompanied by an approximately 5.8-fold the increase in the levels of thiobarbituric acid reactive substances, and showed chromatin condensation and DNA fragmentation. These deleterious effects were time dependent in that no significant change was detected up to 6 h. Treatment with SKF or 1-aminobenzotriazole, which are inhibitors of cytochrome P450, greatly attenuated this cell death as well as prevented chromatin condensation and DNA fragmentation. N(G)-monomethyl-L-arginine at 1 mM had no inhibitory effects on these changes. These findings suggest that endogenous oxidative stress under these conditions induced cell death that resembles apoptosis and that endogenous oxidative stress was directly related to the cytochrome P450 enzyme system in this system.

GroEL is involved in activation of Escherichia coli RNA polymerase devoid of the omega subunit in vivo.

Highly purified Escherichia coli RNA polymerase contains a small subunit termed omega that has a molecular mass of 10 105 Da and is comprised of 91 amino acids. E. coli strains lacking omega (omega-less) are viable, but exhibit a slow-growth phenotype. Renaturation of RNA polymerase isolated from an omega-less mutant, in the presence of omega, resulted in maximum recovery of activity. The omega-less RNA polymerase from omega-less strains recruits the chaperonin, GroEL (unlike the wild-type enzyme), suggesting a structural deformity of the mutant enzyme. The GroEL-containing core RNA polymerase interacts efficiently with sigma70 to generate the fully functional holoenzyme. However, when GroEL was removed, the enzyme was irreversibly nonfunctional and was unable to bind to sigma70. The damaged enzyme regained activity after going through a cycle of denaturation and reconstitution in the presence of omega or GroEL. GroES was found to have an inhibitory effect on the core-sigma70 association unlike the omega subunit. The omega subunit may therefore be needed for stabilization of the structure of RNA polymerase.

Bile acids influence hepatic chemiluminescence in normal and oxidative-stressed rats.

The aim of the present study was to clarify whether bile acids influence chemiluminescence (CL) in the liver in vivo. Hepatic CL was determined on the surface of the liver of anaesthetized rats by using a photon counter. In normal rats, hepatic CL was significantly decreased 30 min after enteral administration of chenodeoxycholic acid (CDCA) or deoxycholic acid (DCA), but returned to its initial level 3 h later, after part of the CDCA administered was metabolized. Ursodeoxycholic acid (UDCA) and cholic acid had no effect on CL. In contrast, hepatic CL was markedly increased 30 min after CDCA or DCA administration in rats given either buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, or diethyldithiocarbamate (DDC), an inhibitor of both superoxide dismutase and glutathione peroxidase. Chenodeoxycholic acid further increased the CL of BSO- or DDC-treated rats during inhalation of oxygen via a tracheal cannula. Coadministration of UDCA eliminated the effects of CDCA on the hepatic CL of normal and BSO- or DDC-treated rats with or without oxygen inhalation. We conclude that cytotoxic bile acids, such as CDCA, increase CL in the antioxidants-depleted or oxidative-stressed liver in vivo, but that UDCA prevents CDCA from developing CL.

Reduction in abortive transcription from the lambdaPR promoter by mutations in region 3 of the sigma70 subunit of Escherichia coli RNA polymerase.

Transcription initiation by Escherichia coli RNA polymerase at most promoters is associated with a reiterative synthesis and release of short abortive RNA products. We have investigated the mechanism of abortive RNA synthesis by using holoenzymes containing mutant sigma70 subunits with changes in region 3 (S506F and P504L), which reduce the ratio of abortive to full-length products. Binary complexes formed by these mutant enzymes at a modified lambdaPR promoter contained a smaller fraction of open complexes than for normal polymerase, suggesting an involvement of region 3 in melting duplex DNA or in maintenance of the open complex. The half-lives of the majority of binary complexes formed by the mutant enzymes were less than 1 min, in contrast to 30 min for the wild-type complexes. The time courses of transcription and pulse-labeling assays showed that moribund complexes, which generate only abortive products (Kubori, T., and Shimamoto, N. (1996) J. Mol. Biol. 256, 449-457), were formed by the mutant enzymes. However, they accumulated to a lesser extent than for the wild-type enzyme, due both to faster dissociation and conversion into inactive complexes. This is the main cause of the low degree of abortive transcription displayed by the mutant enzymes on this promoter.

Physical interference between escherichia coli RNA polymerase molecules transcribing in tandem enhances abortive synthesis and misincorporation.

Transcription initiation is accompanied with iterative synthesis and release of short transcripts. The molar ratio of enzyme to template was found to be critical for the amounts and distribution of the abortive products synthesized by Escherichia coli RNA polymerase from several promoters. At a high ratio abortive synthesis of 4-8 nt were enhanced at thelambda P R promoter. Removing excess RNA polymerase just before initiation, achieved by washing immobilized transcription complexes, prevented this enhancement. At this high ratio synthesis of an unexpected 6 nt transcript was enhanced when the enzyme stalled at position +32, but not when it stalled at position +73. This transcript had misincorporations at its fifth and sixth positions, probably due to slippage. Hydroxyl radical footprinting of the complex stalled at +32 in the presence of excess enzyme showed that more than one molecule of RNA polymerase was tandemly bound to the same DNA. These results suggest that: (i) when RNA polymerase molecules are tandemly transcribing the same DNA, transient collisions enhance abortive synthesis by the trailing molecule; (ii) when the leading polymerase stalled in the initially transcribed region blocks progression of the trailing polymerase, the latter can commit misincorporations, probably due to slippage synthesis.

Functional domains of Escherichia coli single-stranded DNA binding protein as assessed by analyses of the deletion mutants.

A series of C- and N-terminal deletion mutants of Escherichia coli single-stranded DNA binding protein (SSB) was constructed, purified, and characterized in terms of ability to self-multimerize and to bind to DNA. High-performance gel filtration chromatography revealed that the amino acids 89-105 play a key role in the maintenance of homotetramer for native SSB of 177 amino acids. Interestingly, all of the N-terminal deletion mutants studied here were eluted as octamers, indicating that the N-terminal 11 residues are involved in the prevention of the formation of octamers. The binding of SSB and its deletion mutant proteins to single-stranded d(T)n was examined by gel mobility shift assay and circular dichroism spectroscopy. C-terminal deletion mutant proteins, SSB1-135 and SSB1-115, maintained high affinity and may be wrapped by single-stranded DNA (ssDNA) in the same way as in the case of native SSB. In contrast, deletion of the C-terminal region (residues 89-115) or N-terminal region (residues 1-11) caused a dramatic decrease in the binding affinity. Furthermore, two different stoichiometries of SSB in the complexes with d(T)64, but not with d(T)32, were observed for native SSB, SSB1-135, SSB1-115, and SSB37-177, suggesting that the (SSB)65 and (SSB)35 binding modes, as previously demonstrated [Lohman, T. M., & Overman, L. B. (1985) J. Biol. Chem. 260, 3594-3603; Bujalowski, W., & Lohman, T. M. (1986) Biochemistry 25, 7799-7802], occurred at lower and higher SSB concentrations, respectively. A functional map for SSB molecule was presented and discussed.

Regions of the Escherichia coli primary sigma factor sigma70 that are involved in interaction with RNA polymerase core enzyme.

BACKGROUND: The sigma factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation. Most sigma factors share several regions with significant homology in their amino acid sequences (regions 1-4). Some primary sigma factors carry a large nonconserved segment between regions 1 and 2. The binding of an sigma factor to the core enzyme alters the structure and properties of the sigma factor, but little is known about the binding mechanism and subsequent reactions. In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli sigma70 by binding to core enzyme and promoter DNA. RESULTS: A segment between regions 1.1 and 1.2, and that in region 3.2, were preferentially cleaved by hydroxyl radicals. Upon binding to the core enzyme, segments in regions 1.1, 2, 3 and 4 were substantially protected, while cleavage at a small segment in region 4.2 was weakly enhanced. In a binary complex of holoenzyme and promoter DNA, additional segments in regions 2.4 and 4.2 were protected, while the protection at region 1.1 disappeared. The nonconserved acidic region of sigma70 in the holoenzyme became hypersensitive upon binding to DNA. CONCLUSIONS: These results suggest that not only the conserved region 2, but also regions 1.1, 3 and 4 of the sigma factor are involved in binding to the core enzyme. The nonconserved acidic region is likely to be more exposed by further binding of sigma factor to promoter DNA.

Structure of a replication-terminator protein complexed with DNA.

The crystal structure of the Escherichia coli replication-terminator protein (Tus) bound to terminus-site (Ter) DNA has been determined at 2.7 A resolution. The Tus protein folds into a previously undescribed architecture divided into two domains by a central basic cleft. This cleft accommodates locally deformed B-form Ter DNA and makes extensive contacts with the major groove, mainly through two interdomain beta-strands. The unusual structural features of this complex may explain how the replication fork is halted in only one direction.