Local dynamics coupled to hydration water determines DNA-sequence-dependent deformability.

Molecular dynamics (MD) simulations and quasielastic neutron scattering (QENS) experiments were conducted on two hydrated DNA dodecamers with distinct deformability: 5'CGCG[under AATT]̲CGCG3' and 5'CGCG[under TTAA]̲CGCG3'. The former is known to be rigid and the latter to be flexible. The mean-square displacements of DNA dodecamers exhibit so-called dynamical transition around 200-240 K for both sequences. To investigate the DNA-sequence-dependent dynamics, the dynamics of DNA and hydration water above the transition temperature were examined using both MD simulations and QENS experiments. The fluctuation amplitude of the AATT central tetramer is smaller, and its relaxation time is longer, than that observed in TTAA, suggesting that the AT step is kinetically more stable than TA. The sequence-dependent local base pair step dynamics correlates with the kinetics of breaking the hydrogen bond between DNA and hydration water. The sequence-dependent DNA base pair step fluctuations appear above the dynamical transition temperature. Together with these results, we conclude that DNA deformability is related to the local dynamics of the base pair steps, themselves coupled to hydration water in the minor groove.

The use of MRI in pre-operative evaluation of anterior talofibular ligament in chronic ankle instability.

OBJECTIVES: To evaluate the applicability of MRI for the quantitative assessment of anterior talofibular ligaments (ATFLs) in symptomatic chronic ankle instability (CAI). METHODS: Between 1997 and 2010, 39 patients with symptomatic CAI underwent surgical treatment (22 male, 17 female, mean age 25.4 years (15 to 40)). In all patients, the maximum diameters of the ATFLs were measured on pre-operative T2-weighted MR images in planes parallel to the path of the ATFL. They were classified into three groups based on a previously published method with modifications: 'normal', diameter = 1.0 - 3.2 mm; 'thickened', diameter > 3.2 mm; 'thin or absent', diameter < 1.0 mm. Stress radiography was performed with the maximum manual force in inversion under general anaesthesia immediately prior to surgery. In surgery, ATFLs were macroscopically divided into two categories: 'thickened', an obvious thickened ligament and 'thin or absent'. The imaging results were compared with the macroscopic results that are considered to be of a gold standard. RESULTS: Agreement was reached when comparison was made between groups, based on MRI and macroscopic findings. ATFLs were abnormal in all 39 cases and classified as ten 'thickened' and 29 'thin or absent'. As to talar tilt stress radiography, a clear cut-off angle, which would allow discrimination between 'thickened' and 'thin or absent' patients, was not identified. CONCLUSION: MRI is valuable as a pre-operative assessment tool that can provide the quantitative information of ATFLs in patients with CAI. Cite this article Bone Joint Res 2014;3:241-5.

Pharmacodynamic effects of chronic cigarette smoke exposure in spontaneously hypertensive rats.

In investigating the influence of chronic cigarette smoke exposure on hypertension, we compared the pharmacodynamic effects of enforced exposure to smoke on spontaneously hypertensive rats (SHR) with those on Wistar-Kyoto (WKY) rats. Chronic cigarette smoke exposure for 8 weeks decreased the elevated heart rate of mature male SHR to approximately the rate in WKY rats 24 h after smoke exposure. Both systolic and diastolic blood pressures also decreased slightly. However, WKY rats showed a marked rise in heart rate soon after exposure to cigarette smoke began, with no change in blood pressure, while the heart rate of SHR in the early stage remained similar to that of animals without exposure, although their blood pressure was clearly reduced. The body weight of both strains tended to decrease during smoke exposure, but the effect was more severe in SHR. Moreover, the effects of chronic smoke exposure were observed using retired, aged female SHR breeders. A decrease in body weight and heart rate, but not in blood pressure, was also recognized even in these mature animals. These effects gradually recovered after withdrawal from exposure. On the basis of these results, a profile of chronic cigarette smoke exposure under hypertension is discussed in this study.

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes.

Improved strains for the production of riboflavin (vitamin B2) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5'-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l(-1) for 72 h in a 5-l jar fermentor without any end product inhibition.

The effect of endothelin-1 on lipopolysaccharide-induced cyclooxygenase 2 expression in association with prostaglandin E(2).

We demonstrated previously that endothelin-1 (10(-14) to 10(-8) M) promotes lipopolysaccharide-induced cyclooxygenase 2 expression and prostaglandin E(2) production through endothelin ET(B) receptors effects which are up-regulated by lipopolysaccharide. In the present study, we confirmed these findings and showed that prostaglandin E(2) (10(-6) to 10(-5) M) inhibited the lipopolysaccharide plus endothelin-1-induced cyclooxygenase 2 expression more profoundly as compared to its inhibition of the lipopolysaccharide-induced cyclooxygenase 2 expression. The endothelin ET(B) receptor selective antagonist, N-cis-2, 6-dimethylpiperidino-carbonyl-L-gamma-methyl-leucyl-D-L-methoxy carbon yl-tryptophanyl-D-norleucine (BQ788), partly inhibited this suppression. Interestingly, the expression of endothelin ET(B) receptors in macrophages was increased by lipopolysaccharide plus prostaglandin E(2) (10(-8) to 10(-5) M) about 1.6-fold compared with that evoked by lipopolysaccharide stimulation alone. We also showed that treatment with endothelin-1 at 10(-14) M (15 min) elevated an intracellular cyclic AMP concentration in macrophages stimulated by lipopolysaccharide or lipopolysaccharide plus prostaglandin E(2) (10(-6) M) for 6 h, and the elevation in the latter cells was more pronounced. These results suggested that endothelin-1 shows an opposite modulation of lipopolysaccharide-induced cyclooxygenase 2 expression in macrophages through endothelin ET(B) receptors, depending on the level of extracellular prostaglandin E(2), and the changes of intracellular cyclic AMP by endothelin-1 may be involved in this mechanism.

Modulation by endothelin-1 of lipopolysaccharide-induced cyclooxygenase 2 expression in mouse peritoneal macrophages.

We investigated the modulation by endothelin-1 of lipopolysaccharide-induced cyclooxygenase 2 expression and prostaglandin E2 production by mouse peritoneal macrophages. Our previous report showed that endothelin-1 at concentrations above 10(-11) M induced cyclooxygenase 2 expression through mainly endothelin ET(B) receptors and that an endothelin ET(B) receptor-mediated process was not involved in cyclooxygenase 2 activation in macrophages stimulated by lipopolysaccharide for 4 h. In the present study, when macrophages were stimulated by lipopolysaccharide for 12 h in the presence of endothelin-1 (10(-15) to 10(-8) M), cyclooxygenase 2 expression and prostaglandin E2 production were enhanced by 1.2- to 1.6-fold. The endothelin ET(B) receptor selective antagonist, BQ788 (N-cis-2,6-dimethylpiperidino-carbonyl-L-gamma-methyl-leucyl-D-L-m ethoxycarbonyl-tryptophanyl-norleucine), significantly inhibited this synergistic effect of endothelin-1. In addition, the cyclooxygenase 2-selective inhibitor, NS398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide), also suppressed this effect. Western blot analysis showed that the endothelin ET(B) receptor was up-regulated by lipopolysaccharide in a time- and concentration-dependent manner, and that this up-regulation was inhibited by NS398. From these results, we conclude that endothelin-1 promotes lipopolysaccharide-induced cyclooxygenase 2 activation in the delayed phase through endothelin ET(B) receptors up-regulated by lipopolysaccharide.

Analysis of circadian blood pressure rhythm and target-organ damage in stroke-prone spontaneously hypertensive rats.

OBJECTIVE: We compared diurnal patterns of blood pressure in Wistar-Kyoto (WKY) rats, spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SHRSP), and analyzed the relationship between the change in diurnal patterns of blood pressure and target-organ damage in SHRSP. MATERIALS AND METHODS: Blood pressure, heart rate and motor activity in the three groups of rats were continuously monitored by radiotelemetry, from 1100 h on the first measuring day to 1300 h on the third measuring day. The left ventricular weight and the ratio of beta-myosin heavy chain to alpha-myosin heavy chain in the cardiac left ventricle, morphological changes in the glomerular basement membrane in the kidney, 24 h urinary protein excretion and brain weights were also measured in 10-, 12- or 17-week-old SHRSP. RESULTS: The SHR circadian blood pressure rhythm exhibited a pattern which peaked during the rats' active (light-off or dark) phase, but the peak time was a little closer to the resting (light-on) phase compared with that for WKY rats. Although the circadian blood pressure rhythm for 10-week-old SHRSP was similar to that observed for SHR, the patterns in 12- and 17-week-old SHRSP were shifted further towards the resting phase. Heart and left ventricular weight increased with the progression of hypertension. The ratio of beta- to alpha-myosin heavy chain in the left ventricle was higher in 12- and 17-week-old SHRSP than in 10-week-old SHRSP. Brain weight was increased significantly in 17-week-old SHRSP compared with 10- and 12-week-old SHRSP. Increased urinary protein excretion and morphological changes in the glomerular basement membrane in the kidney were observed in 12- and 17-week-old SHRSP. CONCLUSIONS: These data suggest that SHRSP have an abnormal circadian blood pressure rhythm associated with hypertensive target-organ damage. This rat strain may therefore be a useful model in which to investigate the mechanisms responsible for the alteration in the circadian blood pressure rhythm, and to analyze the relationship between the abnormal circadian rhythm and target-organ damage.

Contribution of endothelin-1 to disruption of blood-brain barrier permeability in dogs.

We examined the effect of intracisternal application of endothelin-1 (ET-1) on the permeability of fluorescein into the cerebrospinal fluid (CSF) in beagle dogs in order to evaluate its role in disruption of blood-brain barrier (BBB) permeability seen in the subarachnoid hemorrhage animal model. Intracisternal application of their autologous blood for producing a canine two-hemorrhage model revealed an enhanced fluorescein permeability into the CSF together with the development of cerebral vasospasm. A single dose of ET-1 (40 pmol/animal) significantly increased penetration of fluorescein compared with that in normal dogs. Although its magnitude was much less than that in the two-hemorrhage model after the first administration of ET-1, the second challenge of the same dose of ET-1 with a 48-h interval produced marked disruption of BBB permeability similar to those in the animal model. Moreover, the ET-1-induced enhancement of fluorescein permeability into the CSF was completely prevented by intracisternal pretreatment with an endothelin ET(A)-receptor selective antagonist, S-0139 (0.03 mg/kg), as were the ET-1-induced cerebral vasoconstriction and behavioral changes as previously reported. Thus, we conclude that ET-1 acting on the adventitial site of brain in dogs contributes to the disruption of BBB permeability via endothelin ET(A)-receptor mediation.

Cyclooxygenase 2 expression by endothelin-1-stimulated mouse resident peritoneal macrophages in vitro.

Macrophages have been shown to produce endothelin and to play a role in the pathogenesis of neural damage after cerebral ischemia or vasospasm after subarachnoid hemorrhage. Cyclooxygenase 2 is induced during inflammation following brain insult and participates in inflammation-mediated neurotoxicity. However, it has not yet been established how endothelin-1 acts on cyclooxygenase 2 expression in macrophages. In the present study, we examined the effects of endothelin-1 on cyclooxygenase 2 expression and prostaglandin E2 production, and the effects of endothelin ET(A) and ET(B) receptor antagonists. Stimulation by endothelin-1 ranging from 10(-11) to 10(-9) M time and dose dependently increased the production of prostaglandin E2 and the expression of cyclooxygenase 2 protein without changing that of cyclooxygenase 1 protein, an effect which was inhibited by dexamethasone, nonsteroidal anti-inflammatory drugs and the selective endothelin ET(B) receptor antagonist, BQ788 (N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-L-me thoxycarbonyl-tryptophanyl-D-norleucine). The selective endothelin ET(A) receptor antagonist, BQ123 [cyclo (D-Trp-D-Asp-Pro-D-Val-Leu)] also inhibited these reactions, but its potency was less than that of the selective endothelin ET(B) receptor antagonist. Endothelin ET(A) and ET(B) receptor antagonists had no effects on cyclooxygenase 2 protein expression and prostaglandin E2 production in lipopolysaccharide-stimulated macrophages. We conclude that endothelin-1 increases cyclooxygenase 2 protein expression and prostaglandin E2 production via mainly endothelin ET(B) receptors and partly endothelin ET(A) receptors in macrophages; however, lipopolysaccharide increases both cyclooxygenase 2 protein expression and prostaglandin E2 production in pacrophages without involving endothelin ET(A) or ET(B) receptor-mediated processes.

Profiles of an intravenously available endothelin A-receptor antagonist, S-0139, for preventing cerebral vasospasm in a canine two-hemorrhage model.

We examined the prophylactic effect of a novel nonpeptide endothelin (ET) A-receptor selective antagonist, S-0139, using a canine two-hemorrhage model and an ET-1-induced cerebral vasospasm model. The agent markedly prevented cerebral vasospasm in the canine two-hemorrhage model when given intracisternally or intravenously by continuous daily dosing. An efficacious intravenous method was to apply a relatively high initial dose followed by daily sustaining administration at a much lower dose, which alone would have been ineffective. The need for sustaining dosing may imply daily successive attacks of ETs in the cerebral vessel compartment after the introduction of autologous blood into the subarachnoid space. A small amount of S-0139 was detected from the cerebrospinal fluid (CSF) with an apparent lag time after its disappearance from the plasma following intravenous dosing of 0.83 mg/kg/min for 12 min, however, cerebral vasoconstriction induced by ET-1 dosing from the adventitial side was clearly inhibited during such a lag period. Moreover, its movement into the CSF was greatly enhanced after the application of autologous blood to the animals. From these results, we conclude that ET-1 play a major role in producing delayed cerebral vasospasm in this canine two-hemorrhage model, and S-0139 effectively antagonizes the action of ET-1 even by intravenous treatment because it moves easily into the cerebral vessel compartment from plasma.

Effects of an endothelin ET(A)-receptor antagonist, S-0139, on cerebral vasospasm and behavioral changes in dogs intracisternally administered endothelin-1.

The effects of an endothelin ET(A)-receptor selective antagonist, S-0139, were examined using dogs given endothelin-1 (ET-1) into the subarachnoid space. ET-1 at 40 pmol apparently constricted the basilar artery in anesthetized dogs and caused various grades of ataxia, facial clonus, nystagmus and other features in conscious dogs, partially mimicking those which have been reported for conscious rats. S-0139 could completely inhibit both the vasoconstriction and behavioral changes. It could also alleviate the behavioral changes caused by ET-1 in conscious dogs when given after the severe ataxia. We concluded that ET-1 in the subarachnoid space produces behavioral changes via endothelin ET(A)-receptor mediation similar to its cerebral vasoconstricting action, at least, in dogs.

Dual effects of lisinopril on puromycin aminonucleoside nephrosis in unilaterally nephrectomized rats.

The therapeutic effects of angiotensin converting enzyme inhibitor, lisinopril, on puromycin aminonucleoside (PAN)-induced nephrosis were investigated using unilaterally nephrectomized rats. Lisinopril showed potent dual effects on PAN nephrosis. Lisinopril treatment (50 mg/l in drinking water) from day 5 or day 9 reduced urinary protein excretion and suppressed the development of glomerular sclerosis at 8 weeks after PAN injection (150 mg/kg, i.p.), indicating a therapeutic effect on the nephrosis. Recovery of decreased anionic charge sites on the glomerular basement membrane was involved, at least in part, in the therapeutic action of lisinopril against proteinuria. On the other hand, oliguria and progressive azotemia derived from continuous deterioration of the renal function was induced if the treatment of lisinopril was started on the same day as PAN injection. The renal dysfunction induced by simultaneous administration of lisinopril with PAN could be abolished by combination dosing with sarcosine, an angiotensin II (AII)-receptor agonist. These results indicate that lisinopril treatment attenuates proteinuria by ameliorating the anionic charge barrier on the glomerular basement membrane and that it also protects against the development of chronic renal disease with segmental glomerular sclerosis, although AII depletion during the acute nephrotic stage exacerbates the renal damage in PAN nephrosis of unilaterally nephrectomized rats.

Pharmacological studies on a new antihypertensive agent, S-2150, a benzothiazepine derivative: 2. Hypotensive effects in normotensive and hypertensive rats.

S-2150 is a new 1,5-benzothiazepine derivative possessing both calcium channel-blocking and alpha 1-adrenoceptor-blocking effects. In isolated rat thoracic aorta precontracted with KCl (18 mM), the 50% inhibitory concentration (IC50) value was 190 nM for S-2150, which was similar to that of diltiazem. In aorta precontracted with phenylephrine (0.3 microM), IC50 values of S-2150 and diltiazem were 29 nM and > 10 microM, respectively. The relative contribution of calcium channel-blocking and alpha 1-adrenoceptor-blocking activities to hypotension was determined by using anesthetized rats before and after masking of the alpha 1-receptors with prazosin. The hypotensive effect of S-2150 [0.3 and 1 mg/kg intravenously (i.v.)] was attenuated by 40% after prazosin treatment, whereas that of diltiazem was not. In conscious spontaneously hypertensive rats (SHRs), renal hypertensive rats, and normotensive rats, S-2150 [10, 30, and 60 mg/kg orally (p.o.)] caused dose-dependent hypotensive effects. The effect of S-2150 was 4-7 times more potent than that of diltiazem. There were no changes in the hypotensive effects with consecutive administration of S-2150 during 6-8 weeks in SHRs and stroke-prone SHRs (SHRSPs). In SHRSPs, S-2150 reduced the mortality by stroke and small arterial hyperplasia in abdominal organs and also ameliorated renal excretory function. These results suggest that S-2150 may be a useful antihypertensive agent possessing both calcium-antagonistic and alpha 1-adrenoceptor-blocking effects.

Circadian rhythm of plasma uric acid and handling stress-induced hyperuricemia in conscious cebus monkeys.

An apparent circadian rhythm of plasma uric acid and the effect of handling stress on plasma uric acid level in conscious cebus monkeys were demonstrated. The lowest level of plasma uric acid in the circadian rhythm occurred early in the morning and the highest, before bedtime at night. With experimental handling stress, the plasma uric acid level rose to much more than the maximum level of the circadian rhythm. Stress-induced hyperuricemia could be inhibited without an increase of urinary uric acid excretion by the minor tranquilizer diazepam at doses of more than 1 mg/kg, p.o. On the other hand, benzbromarone at 20 mg/kg, p.o. significantly inhibited the hyperuricemia with a hyperuricosuric effect, while probenecid at 50 mg/kg, p.o. had no effect on either the increased plasma uric acid or urinary uric acid excretion. Accordingly, it is concluded that the plasma uric acid level in conscious cebus monkeys easily fluctuates with experimental conditions and that the animals can be utilized to evaluate the hypouricemic and hyperuricosuric property of benzbromarone-like agents.

Hyperuricemia induced by the uricosuric drug probenecid in rats.

Stimulation of uric acid production by the well-known uricosuric drug probenecid was studied using potassium oxonate-treated rats and eviscerated rats subjected to functional hepatectomy. In oxonate-treated rats, probenecid was hyperuricosuric, increasing the glomerular-filtered amounts of uric acid and causing marked hyperuricemia. This could be completely blocked by combination dosing with allopurinol, an inhibitor of xanthine oxidase. In eviscerated rats subjected to functional hepatectomy, probenecid also increased plasma uric acid and urinary uric acid excretion, but when given together with allopurinol, the increase of plasma uric acid was abolished with a remarkable increase of plasma hypoxanthine and xanthine. When probenecid was given by combination dosing with propranolol, a beta adrenoceptor antagonist, the hyperuricemia was also completely blocked. Thus, probenecid is concluded to stimulate uric acid production, probably via some interaction with endogenous catecholamine, resulting in hyperuricemia in rats, although it is a practical hypouricemic drug in humans.

Stop-flow studies on tubular transport of uric acid in rats.

A stop-flow technique using pyrazinoic acid(PZO)-treated and -untreated rats was devised to evaluate drug effects on bi-directional transport of uric acid in the tubules. Constant venous infusion of test drugs to PZO-untreated rats was used to estimate their inhibitory effects on urate secretion, while their inhibitory effects on urate reabsorption was studied by intravenous administration as a bolus to PZO-treated rats. Probenecid, tienilic acid and R-(+)-enantiomer of S-8666, which is the uricosuric component of a new uricosuric diuretic, decreased the (Tua/Pua)/(Tin/Pin) value in the distal and proximal tubules by inhibiting urate secretion in PZO-untreated rats. On the other hand, all of these drugs increased the (Tua/Pua)/(Tin/Pin) value in the tubules in PZO-treated rats, which suggested that they also inhibited the reabsorptive flux of urate. This stop-flow technique in rat kidneys showed the possibilities of bi-directional inhibition by these drugs of urate transport in the tubules.

Effect of 711389-S, a new antiarrhythmic agent, on myocardial energy metabolism in guinea-pigs and rats.

The effect of 711389-S, a new antiarrhythmic agent, on myocardial energy metabolism was investigated using anaesthetized guinea-pigs and rats. 711389-S elevated the adenylate energy charge and phosphorylation potential in normal guinea-pig myocardium. Large doses also increased the myocardial lactate content with ECG abnormalities. The close relationship between rate-pressure product and the myocardial energy state under 711389-S treatments showed the suppression of energy consumption due to a decrease of work output. In guinea-pigs with arrhythmic myocardia induced by intravenous infusion of ouabain, 711389-S prevented the loss of high-energy phosphate compounds and the acceleration of anaerobic glycolysis concomitant with the effective antiarrhythmic property. In ischaemic myocardium produced by ligation of the coronary artery in rats, 711389-S suppressed the decreases of creatine phosphate, NAD+ and adenylate energy charge. Moreover, this agent effectively blocked the incidence of ventricular arrhythmias at an early stage following the ligation. In all of these actions, 711389-S was more effective than disopyramide, which is in the same class of antiarrhythmics. 711389-S was concluded to be a favourable antiarrhythmic agent offering beneficial action against arrhythmic and ischaemic metabolic changes in the myocardium.