Visualization of single molecules of RNA polymerase sliding along DNA.

Transcription requires that RNA polymerase binds to promoters buried in nonspecific sites on DNA. The search for promoters may be facilitated if the polymerase slides along the molecule of DNA. Single molecules of Escherichia coli RNA polymerase were visualized, and their movements on immobilized bacteriophage lambda and T7 DNAs were examined. Deviating from drifts by bulk flow, about 40 percent of the enzyme molecules moved along the extended DNA. The results provide direct evidence for sliding as a mechanism for relocation of the enzyme on DNA.

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.

Multiple conformational transitions of hen egg-white lysozyme in aqueous acetic acid solutions.

Circular dichroism measurements revealed that hen egg-white lysozyme underwent multiple conformational transitions upon the addition of acetic acid. The transitions were reversible as judged from complete recoveries of enzymatic activity, electrophoretic mobility in SDS-polyacrylamide gel, and of ellipticity. Two transitions, with the mid-concentrations of 26 and 38% (v/v), were observed with the CD spectra in the amide absorption region. The two transitions were essentially athermal in the temperature ranges, 0 to 25 degrees C for the former and -10 to 10 degrees C for the latter. The trough ellipticity for the product of the transition at the higher acetic acid concentration (DII form) very closely approached the value for the synthetic polypeptides in the beta-conformation as the temperature was lowered. Molecular weight measurements by sedimentation equilibrium indicated that the products were both monomeric. Measurements of CD spectra in the aromatic absorption region showed another transition, whose mid-concentration varied with temperature from 26% (v/v) (at about 25 degrees C) to 38% (v/v) (at -10 degrees C). A change in the hydrodynamic volume detectable by exclusion chromatography was associated with this transition only.

A branched pathway in the early stage of transcription by Escherichia coli RNA polymerase.

The time-course of synthesis of long and short (abortive) transcripts by Escherichia coli RNA polymerase was investigated under single round conditions in vitro. The synthesis of long RNA initiated at the lambda PR (with an altered leader sequence) or at the lacUV5 promoter was completed within five minutes, but abortive transcripts were continuously synthesized for at least 20 minutes. The results indicate the presence of transcription complexes that are capable only of abortive synthesis, and not of productive elongation. Consistently, only one in four of the RNA polymerase molecules that initially associated with the lambda PR promoter synthesized long RNA. The enzyme reisolated from productive complexes synthesized both long and abortive transcripts, behaving just like the original enzyme. This suggests that RNA polymerase is homogeneous with respect to its ability to synthesize the two types of product. Overall, the results indicate that many transcription complexes can be irreversibly trapped in vitro in an abortive cycle.

Morphological pathway of flagellar assembly in Salmonella typhimurium.

The process of flagellar assembly was investigated in Salmonella typhimurium. Seven types of flagellar precursors produced by various flagellar mutants were purified by CsCl density gradient protocol. They were characterized morphologically by electron microscopy, and biochemically by two-dimensional gel electrophoresis. The MS ring is formed in the absence of any other flagellar components, including the switch complex and the putative export apparatus. Four proteins previously identified as rod components, FlgB, FlgC, FlgF, FlgG, and another protein, FliE, assemble co-operatively into a stable structure. The hook is formed in two distinct steps; formation of its proximal part and elongation. Proximal part formation occurs, but elongation does not occur, in the absence of the LP ring. FlgD is necessary for hook formation, but not for LP-ring formation. A revised pathway of flagellar assembly is proposed based on these and other results.

Pathophysiological significance of coronary collaterals for preservation of the myocardium during coronary occlusion and reperfusion in anaesthetised dogs.

The significance of pre-existing coronary collaterals for preservation of the myocardium during coronary occlusion and reperfusion was studied in anaesthetised open-chest dogs. The extent of myocardial ischaemic damage was assessed by the accumulation of calcium in the central zone of the ischaemic area and the size of myocardial infarct by the p-nitroblue tetrazolium staining method. Myocardial creatine kinase release was considered as an index reflecting both the extent of ischaemic myocardial damage and the size of the infarct. Reperfusion with the arterial blood of a main branch of the left coronary artery following the 60 min occlusion aggravated the ischaemic myocardial damage, as evidenced by exaggerated cardiac irregularity, myocardial calcium accumulation, and creatine kinase release. The myocardial calcium content and the logarithm of the creatine kinase release during reperfusion were linearly related to the infarct size. These parameters of myocardial damage were inversely related to the retrograde blood flow through the peripheral end of the occluded artery or the ratio of the retrograde-to-orthograde flow, indicating that the pre-existing collaterals influence preservation of the ischaemic myocardium. Thus, it is suggested that the collateral flow available during an ischaemic episode determines the viability of the myocardial cells during ischaemia, and in turn during reperfusion.

Coronary circulatory failure and thromboxane A2 release during coronary occlusion and reperfusion in anaesthetised dogs.

Attempts were made to demonstrate release of vasoactive substances from the heart during coronary occlusion (for 60 min) and reperfusion (for 60 min), and to clarify the pathophysiological significance of them. Vasoactive substances were detected by superfusion of rabbit aortic and dog coronary arterial strips with great coronary venous blood. Plasma thromboxane (TX) B2 was radioimmunologically assayed. Gradually developing, sustained contraction of both vascular strips was noted during coronary occlusion and reperfusion, while a transient contraction in rabbit aortic and relaxation in dog coronary arterial strips were seen immediately after reperfusion. The TXB2 released into the great coronary venous blood significantly increased during occlusion and reperfusion. Indomethacin treatment of the dog abolished the sustained contraction of both vascular strips and TXB2 release. The transient contraction of rabbit aorta after reperfusion was inhibited by phenoxybenzamine. Reactive hyperaemia following a 60 min occlusion was significantly depressed, as compared with that following 30 s to 30 min occlusion, and the depression was alleviated by indomethacin and imidazole. These results suggest that catecholamine(s) and TXA2 are released during coronary occlusion and reperfusion, and that the latter might be responsible for the coronary circulatory failure during reperfusion of irreversibly damaged myocardium.

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.

Studies on scavengers of active oxygen species. 1. Synthesis and biological activity of 2-O-alkylascorbic acids.

A novel series of 2-O-alkylascorbic acids (5a-u) was synthesized, and their scavenging activities against active oxygen species as well as their suppressive effects on the arrhythmias in rat heart ischemia-reperfusion models were evaluated. Some 2-O-alkylascorbic acids (5e-1) exhibited potent inhibiting activities against lipid peroxidation in rat brain homogenates and in alleviating effects in the ischemia-reperfusion models. Studies on the structure-activity relationship demonstrated that a free 3-enolic hydroxyl group and the longer alkyl chains substituted on the 2-hydroxyl group of ascorbic acid were beneficial for the biological and pharmacological activities. 2-O-Octadecylascorbic acid (5k, CV-3611), one of the most potent and promising compounds, markedly inhibited lipid peroxidation (IC50 = 4.3 X 10(-6) M) and alleviated myocardial lesions induced by ischemia-reperfusion at an oral dose of 1 mg/kg in rats.

Endothelin-1 analogues substituted at both position 18 and 19: highly potent endothelin antagonists with no selectivity for either receptor subtype ETA or ETB.

Novel endothelin-1 (ET-1) analogues which are highly potent endothelin antagonists at both receptor subtype ETA and ETB are reported. The replacement of Asp18 with the Thr18 and of Ile19 with a hydrophobic amino acid whose side-chain branches on the gamma-carbon such as Leu, cyclohexylalanine, and gamma-methylleucine (gamma-MeLeu) resulted in loss of or significantly decreased the biological activity of ET-1, while high affinity for the ETA (IC50 = 0.42-0.70 nM) and ETB (IC50 = 0.17-0.43 nM) receptor was retained. These compounds were shown to have high antagonist activities in ET-1-induced vasoconstriction of porcine coronary artery (pA2 7.4-7.7) and in Sarafotoxin S6c-induced vasoconstriction of rabbit pulmonary artery ([Thr18, gamma-MeLeu19]ET-1: pA2 8.4). Among these compounds, [Thr18, gamma-MeLeu19]ET-1 has the desirable characteristic of possessing no agonist activity at either receptor subtype.

alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetate. Large Hammett's rho constant and participation of histidine-acylated intermediate.

A detailed examination of the mechanism of the hydrolysis of phenyl acetates by alpha-chymotrypsin [EC 3.4.21.1] was carried out. The effective deacylation rate constants of some phenyl acetates obtained by titration of the acetyl-enzyme decreased at low substrate concentrations and showed anomalous pH dependences and solvent isotope effects. The transient kinetics of deacylation of the acetyl-enzyme were biphasic. A spectrum and a breakdown rate similar to those of acetylimidazole were observed when the acetyl-enzyme was denaturated with sodium dodecyl sulfate. These results indicate the participation of histidine-acylated enzyme, which woud account for the anomalous phenomena previously found in this system, including a large value of Hammett's rho. The relation between the substrate activation and the two intermediates is discussed.

A method for analyzing enzyme kinetics with substrate activation and inhibition and its application to the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

A general kinetic method was developed to analyze enzyme-catalyzed systems complicated by the presence of activation or inhibition by substrate. The method was applied to the alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis of p-chlorophenyl and p-methoxyphenyl acetates. Deacylation rate constants which were not complicated by substrate activation were obtained. The analysis shows that the abnormal substituent dependence of kcat in the steady state hydrolysis is due not to substrate activation but to inappropriateness of the two-step mechanism or the existence of more than one acetyl-enzyme intermediate.

Substituent effects on substrate activation and Michaelis-Menten Kinetic parameters in the alpha-chymotrypsin-catalyzed hydrolysis of phenyl acetates.

The effects of substituents on the steady state and pre-steady state kinetics in alpha-chymotrypsin [EC 3.4.21.1]-catalyzed hydrolysis were studied using substituted phenyl acetates. In the steady state hydrolysis, substrate activation, which had been observed and studied previously for p-nitrophenyl acetate, was also observed for p-bromo, p-chloro-, and m-methylphenyl acetates. Little activation was observed for p-acetyl-, m-nitro-, p-methyl-, and p-methoxyphenyl acetates. Addition of p-dichlorobenzene increased kcat for all substrates examined and greatly diminished the substrate activation for the activatable substrate(s) to activator binding site(s). The value of kcat decreased in accordance with increase of the sigma-value of substituents. On the other hand, kcat/Km (app) showed an opposite sigma- dependence, as was previously observed. In pre-steady state measurements, little burst was observed for more electron-donating substituents than m-nitro. The sigma dependence of kcat is apparently not consistent with the prediction derived from that of kcat/Km (app) on the basis of the usual two-step mechanism with a common acetyl-enzyme intermediate.

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.

Catch-relaxing peptide isolated from Mytilus pedal ganglia.

A peptide that relaxes catch tension of the anterior byssus retractor muscle of Mytilus edulis was purified from pedal ganglion extracts of the mussel. Its primary structure was determined to be H-Ala-Met-Pro-Met-Leu-Arg-Leu-NH2. This peptide was found to have not only catch-relaxing action on the byssus retractor muscle but also modulatory actions on contractions in various molluscan muscles.

Cardiovascular effects of endothelin in dogs: positive inotropic action in vivo.

Endothelin, administered i.v. to anesthetized dogs, dose dependently increased the cardiac output, left ventricular systolic pressure (LVSP), and maximum upstroke velocity (max dp/dt) of the LVSP for about 10 min without changing the heart rate. Thereafter the cardiac output decreased to below the control level but max dp/dt decreased to the control level. The arterial pressure and total peripheral resistance showed an initial, transient decrease followed by a sustained increase. These results suggest that endothelin has positive inotropic and long-lasting vasoconstrictive effects preceded by transient vasodilatation in vivo.

Ontogeny of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding sites in the rat brain.

Endogenous levels of pituitary adenylate cyclase-activating polypeptide (PACAP) and its binding site densities were measured in eight brain regions in rats of different ages (2-240 days) by sandwich-enzyme immunoassay and autoradiography. PACAP levels were quite low at day 2, peaked in 30-60 days, and then remained constant in most regions. Such ontogenetic changes are similar to those of vasoactive intestinal polypeptide (VIP) and classical neurotransmitters. PACAP-binding sites were already dense at day 2 and varied only slightly up to day 240. These results suggest that PACAP may have modulatory effects on brain development.

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.

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.