Influence of rotational speed on torque/force generation and shaping ability during root canal instrumentation of extracted teeth with continuous rotation and optimum torque reverse motion.

AIM: To evaluate how different rotational speeds affect the torque/force generation and shaping ability of rotary root canal instrumentation using JIZAI (MANI, Utsunomiya, Japan) nickel-titanium instruments in continuous rotation and optimum torque reverse (OTR) motion. METHODOLOGY: Mesial root canals of extracted mandibular molars were instrumented up to size 25, 0.04 taper using JIZAI instruments, and anatomically matched canals were selected based on geometric features of the canal [canal volume (mm3 ), surface area (mm2 ), length, 15°-20° curvature and radius of curvature (4-8 mm)] after micro-computed tomographic scanning. An automated root canal instrumentation and torque/force analysing device was programmed to permit a simulated pecking motion (2 s downward and 1 s upward at 50 mm min-1 ). The selected canals were prepared with size 25, 0.06 taper JIZAI instruments using continuous rotation or OTR motion and further subdivided according to the rotational speed (300 or 500 rpm, n = 10 each). Real-time clockwise/counterclockwise torque and downward/upward force were recorded using a custom-made torque/force analysing device. Then, the registered pre- and post-operative micro-computed tomographic datasets were examined to evaluate the canal volume changes and centring ratios at 1, 3, 5 and 7 mm from the apical foramen. Data were analysed using two-way analysis of variance or the Kruskal-Wallis test and Mann-Whitney U test with Bonferroni correction (α = 5%). RESULTS: Maximum upward force and clockwise torque were significantly smaller in 500 rpm groups than in 300 rpm groups (P < .05); however, no significant difference was found between continuous rotation and OTR motion (P > .05). OTR motion developed higher maximum counterclockwise torque than continuous rotation (P < .05). Maximum downward force, canal volume changes and centring ratios were not significantly different among all groups (P > .05). There was no file fracture in any of the groups. CONCLUSIONS: Under laboratory conditions using JIZAI instruments, a rotational speed of 500 rpm generated significantly lower maximum screw-in forces and torque values than rotational speed of 300 rpm. Continuous rotation and OTR motion performed similarly in shaping the canals.

Prevalence of aspirin resistance in patients with type 2 diabetes.

Aspirin resistance has been recognised to occur in patients with cardiovascular disease and is associated with poor clinical prognosis. The purpose of the present study was to evaluate the prevalence of aspirin resistance in 172 patients with diabetes mellitus type 2 (DM-2). Platelet function of 172 consecutive patients with type 2 diabetes on chronic aspirin therapy was evaluated. The effect of aspirin was assessed using the platelet function analyser (PFA-100) system, reporting platelet-dependent thrombus formation as the time required to close a small aperture in a biologically active membrane. Resistance to aspirin was defined as a normal collagen/epinephrine-induced closure time (82-165 s). Aspirin responders were defined when closure time was > or =300 s. Thirty-seven (21.5%) of the type 2 diabetic patients were found to be resistant to chronic aspirin therapy, 29 (16.9%) were semi-responders and 106 (61.6%) were responders. Univariate analysis revealed that aspirin non-responders were significantly younger (p<0.05) compared to aspirin responders. A significant number of type 2 diabetic patients are resistant to aspirin therapy. Aspirin resistance can be evaluated by point-of-care testing and should be recognised in diabetic patients that are treated for primary or secondary prevention.

Inhibition of the cardiac p38-MAPK pathway by SB203580 delays ischemic cell death.

We report that SB203580 (SB), a specific inhibitor of p38-MAPK, protects pig myocardium against ischemic injury in an in vivo model. SB was applied by local infusion into the subsequently ischemic myocardium for 60 min before a 60-min period of coronary occlusion followed by 60-min reperfusion (index ischemia). Infarct size was reduced from a control value of 69.3 +/- 2.7% to 36.8 +/- 3.7%. When SB was infused systemically for 10 min before index ischemia, infarct size was reduced to 36.1 +/- 5.6%. We measured the content of phosphorylated p38-MAPK after systemic infusion of SB and Krebs-Henseleit buffer (KHB; negative control) and during the subsequent ischemic period using an antibody that reacts specifically with dual-phosphorylated p38-MAPK (Thr180/ Tyr182). Ischemia with and without SB significantly increased phospho-p38-MAPK, with a maximum reached at 20 min but was less at 30 and 45 min under the influence of the inhibitor. The systemic infusion of SB for 10 min before index ischemia did not significantly change the p38-MAPK activities (compared with vehicle, studied by in-gel phosphorylation) < or =20 min of ischemia, but activities were reduced at 30 and 45 min. Measurements of p38-MAPK activities in situations in which SB was present during in-gel phosphorylation showed significant inhibition of p38-MAPK activities. The systemic infusion of SB significantly inhibited the ischemia-induced phosphorylation of nuclear activating transcription factor 2 (ATF-2). Using a specific ATF-2 antibody, we did not observe significant changes in ATF-2 abundance when nuclear fractions from untreated, KHB-, and SB-treated tissues were compared. We investigated also the effect of local and systemic infusion of SB on the cardioprotection induced by ischemic preconditioning (IP). The infusions (local or systemic) of SB before and during the IP protocol did not influence the infarct size reduction mediated by IP. The observed protection of the myocardium against ischemic damage by SB points to the negative role of the p38-MAPK pathway during ischemia.

Okadaic acid and anisomycin are protective and stimulate the SAPK/JNK pathway.

We report that okadaic acid (OA), a known inhibitor of Ser/Thr phosphatases, protects pig myocardium against ischemic injury in an in vivo model and stimulates the activities of stress-activated protein kinases/c-Jun N-terminal kinases (SAPKs/JNKs). When OA was directly infused into the subsequently ischemic myocardium for 60 min before a 60-min period of coronary occlusion followed by reperfusion, infarct size was reduced from a control value of 83.4 +/- 2.8% of the risk region to 40.7 +/- 9.1%. When OA was infused for 10 min before a 5-min occlusion and during 45 min thereafter, infarct size was reduced to 26.5%. In a separate set of similar experiments, we pretreated pig hearts in vivo with the protein-synthesis inhibitor and known activator of SAPK/JNK, anisomycin (AN), and found that this compound also significantly reduced infarct size from 83.4 +/- 2.8.1% to 48.1 +/- 5.1%. For in vitro assays, OA (600 nM), AN (500 microM), or solvent (KHB) were locally infused into the left ventricular myocardium, and biopsies from in situ beating hearts were obtained after 10, 30, and 60 min of infusion. The activities of Ser/Thr phosphatases (PPases), especially PP-2A, were significantly decreased after OA infusion. OA infusion increased the activity (in-gel phosphorylation of N-terminal c-Jun1-135) of both 46- and 55-kDa SAPK/JNKs (twofold to threefold, 30 and 60 min of infusion), and this increase correlated well with the observed decrease of PPase activities. Western blot analysis with a phosphospecific SAPK/JNK (Thr 183/Tyr 185) antibody showed an increased content of the phosphorylated forms after OA treatment. We observed significant stimulation of SAPK/JNK activity also after AN treatment (threefold to fourfold, after 30 min of infusion). In contrast to the SAPK/JNKs, the infusion of both OA and AN did not significantly change the activities and phosphorylation of extracellular signal-related kinases (ERKs) and p38-MAPK. The findings that the protective effect of both OA and AN correlates with increased activity of SAPK/JNKs suggest the involvement of these enzymes in the mechanism of cardioprotection.

Binding of the stress protein alpha B-crystallin to cardiac myofibrils correlates with the degree of myocardial damage during ischemia/reperfusion in vivo.

Stress proteins are assumed to protect cells against various kinds of stresses including ischemia. In this study, we focused on the behaviour of the most abundant myocardial stress protein, alpha B-crystallin, during ischemia and reperfusion of the pig heart in vivo, alpha B-crystallin constitutes 1-2% of the soluble protein pool and underwent, during severe but reversibly damaging ischemia (25 min), complete translocation to the Z-line area of myofibrils. Irreversibly damaging ischemia (60 min) was accompanied by extreme stretching of the majority of myofibrils, and by concomitant extension of alpha B-crystallin localization from the Z-line area to I-bands. This I-band shift correlated with displacement of the T12 epitope of titin from the vicinity of Z-lines into I-bands, indicating that the primary binding sites for alpha B-crystallin might also be located in juxtaposition to Z-lines and move into the I-bands during extreme sarcomeric stretching. During reperfusion after 25 min of ischemia, alpha B-crystallin disappeared rapidly from myofibrils: whereas reperfusion after irreversibly damaging ischemia (60 min) resulted in dissociation of alpha B-crystallin only from those myofibrils and myocardiocytes that were still able to contract, and alpha B-crystallin remained bound to the overstretched, damaged myofibrils no longer capable of contraction. The time course of translocation of alpha B-crystallin to myofibrils during ischemia correlated with phosphorylation of approximately 20% of the entire alpha B-crystallin pool. However, disappearance of alpha B-crystallin from myofibrils during reperfusion was not accompanied by dephosphorylation, indicating that phosphorylation alone does not explain myofibrillar binding of alpha B-crystallin. Ischemia-induced myofibrillar targeting of alpha B-crystallin probably requires additional structural and posttranslational modifications of myofibrillar components in juxtaposition to I-bands.

Ischemia-induced phosphorylation and translocation of stress protein alpha B-crystallin to Z lines of myocardium.

It is becoming clear that stress proteins play a role in various aspects of postischemic myocardial recovery and that the cytoskeleton of cardiac myocytes is an important determinant for cellular survival during ischemia and energy depletion. In the present study, we addressed the question of whether the cytoskeleton-binding stress protein alpha B-crystallin may be involved in early cellular responses of rat and porcine myocardium to ischemia. Immunostaining and subcellular fractionation revealed a rapid ischemia-induced redistribution of alpha B-crystallin from a cytosolic pool to intercalated disks and Z lines of the myofibrils. This striking translocation of alpha B-crystallin from the cytosol to sites of the myofibrillar system that are known to be sensitive to ischemia-reperfusion injury was accompanied by a rapid shift of a fraction of alpha B-crystallin to a more acidic isoelectric point. This shift is caused by alpha B-crystallin phosphorylation, as identified by its augmentation in the presence of phosphatase inhibitors (vanadate, fluoride) and comigration of the acidic alpha B-crystallin form with the phosphorylated B1 form of lenticular alpha B-crystallin. In view of the chaperone-like function of alpha B-crystallin in conjunction with its high level of constitutive expression in the myocardium (1-2% of soluble protein content), we consider alpha B-crystallin an excellent candidate to play a role in early aspects of the protection of the myocardial contractile apparatus against ischemia-reperfusion injury.

Intramyocardial infusion of FGF-1 mimics ischemic preconditioning in pig myocardium.

Previous studies on the mRNA and protein level suggested a cardioprotective role of FGF-1. These presumed actions of FGF-1 and FGF-2, as well as the underlying mechanisms, were investigated in this study. Human recombinant FGF-1 (0.5 microgram/ml, 20 microliters/min) and FGF-2 (2 micrograms/ml) were applied by means of direct intramyocardial infusion (IM) for 60 min prior to a 60 min LAD-occlusion and 120 min reperfusion. Myocardial infarction compared to the region at risk was significantly decreased by FGF-1 and FGF-2 treatment (FGF-1: 51.8 +/- 7.7%, respectively. FGF-2: 57.3 +/- 6.5% v control 83.4 +/- 2.8%, P < 0.05). The increase in survival time was about 33 min, and equalled that of ischemic preconditioning. This effect was caused by the mitogenic part of the molecule, since infusion of a truncated version of FGF-1 (0.5-1 microgram/ml), lacking mitogenicity but maintaining hemodynamic activity, did not induce cardioprotection (78.3 +/- 0.73% v control 83.4 +/- 2.8%). Suramin (0.5 microgram/ml) prevented the observed cardioprotection (77.0 +/- 1.2% v control 83.4 +/- 2.8%) proving that the cardioprotective effect is receptor-mediated. Genistein (0.5 microgram/ml), an inhibitor of tyrosine kinases, abolished the cardioprotection as well (77.2 +/- 2.4% v control: 83.4 +/- 2.8%). Immunohistochemical staining revealed an uptake and translocation of exogenous FGF-1 to a (peri-)nuclear localization in myocytes and into non-myocytes for FGF-2. We conclude that both FGF-1 and FGF-2 are cardioprotective (FGF-1 being more active on a molar basis), and mimic ischemic preconditioning. Their actions are receptor-mediated and receptor activation is involved. Uptake and transport to a (peri-)nuclear localization, seems to be a pathway of minor relevance, since it could not be blocked by tyrosine kinase receptor inhibition. Tyrosine kinase-coupled receptor occupation in general is not protective as demonstrated by the lack of effect with VEGF-infusion.

Angiogenesis but not collateral growth is associated with ischemia after femoral artery occlusion.

It remains unclear whether capillary sprouting (angiogenesis) and in situ growth of muscular collateral arteries share the same or different molecular mechanisms. To study the role of ischemia in these two forms of vascular proliferation, we measured tissue flows and maximum collateral conductances in hindlimbs of 22 rabbits previously subjected to either acute, 7-day, 21-day, or no femoral artery occlusion. After 1 wk of femoral artery occlusion, corkscrew collaterals were observed radiographically in the thigh. These collaterals showed histochemical evidence for active proliferation of endothelial and smooth muscle cells. Maximum collateral conductance increased sixfold in the 1st wk. Perfusion deficits, however, were only observed in the distal adductor muscles (region of collateral reentry). In the lower leg, which suffered from a profound perfusion deficit, conductance increased in the absence of any visible collateral arteries but with evidence for capillary proliferation. This study therefore demonstrates that upon femoral artery occlusion angiogenesis occurs in regions of profound ischemia, whereas no direct correlation can be drawn between ischemia and collateral artery development.

Insulin-like growth factor-II delays myocardial infarction in experimental coronary artery occlusion.

OBJECTIVE: We have previously shown that short pulses of myocardial ischemia cause increased mRNA expression of the insulin-like growth factor II (IGF-II) gene. The expression of IGF-II precedes the expression of its binding protein 5 (IGFBP-5). The cardioprotective actions of the IGF-II peptide and of its binding protein 5 as well as the underlying mechanisms were investigated in this study. METHODS AND RESULTS: Human recombinant IGF-II (0.25 microgram/ml) was applied by means of direct intramyocardial infusion (IM) for 60 min prior to a 60 min LAD occlusion and 120 min reperfusion. Myocardial infarction, compared to the region at risk, was significantly decreased by IGF-II treatment, whereas infusion of Krebs-Henseleit buffer did not show any protective effect (IGF-II, 78.75 +/- 1.51%; control, 100%; P < 0.005). A comparable degree of cardioprotection was observed after infusion of an equipotent concentration of recombinant human insulin (0.02 IU/ml; 88.25 +/- 1.45%; P < 0.05). Lavendustin A (100 microM), an inhibitor of protein tyrosine kinases, prevented the observed cardioprotection. The protective effect of IGF-II was lost when IGFBP-5 was simultaneously infused. CONCLUSION: IGF-II, a peptide that binds to the insulin receptor and whose mRNA is rapidly transcribed by cardiac myocytes following ischemic stress, is cardioprotective and mimics ischemic preconditioning. Its observed actions are probably based on its metabolic effects and are mediated by the insulin or the IGF-I receptor. IGFBP-5, whose expression follows IGF-II's expression with a short delay, inhibits the cardioprotection afforded by IGF-II and may thus account for the limited temporal duration of ischemic preconditioning.

Intramyocardial infusion of tool drugs for the study of molecular mechanisms in ischemic preconditioning.

Many of the new tool drugs useful for the study of molecular mechanisms of ischemic preconditioning (IP) are very valuable in in vitro systems but produce undesired side-effects after systemic injection in intact animals that limit their applicability. Our aim was to develop an experimental in vivo model that allows the use of said drugs in sufficiently high local concentrations, but avoiding at the same time the systemic side-effects. Several techniques were combined to study regional damage or protection as a result of local drug infusion such as nuclear staining, NADH fluorescence, fluorescent microspheres and tetrazolium salts. In open-chest pigs, the intramyocardial infusion (20 microliters/min) of the adenosine A1-receptor agonist N6-cyclohexyladenosine (0.3 mmol) for 10 min prior to a 60-min LAD-occlusion and 120-min reperfusion mimicked IP by exerting a local protection (n = 9, p < 0.001). Krebs-Henseleit buffer (negative control) was without protective effect. IP's cardioprotection was locally prevented by the intramyocardial application of the adenosine A1-receptor antagonist cyclopentyltheophylline (1 mmol, infused during IP; n = 6, p < 0.001) but not by KHB. The protein kinase C (PKC)-inhibitors staurosporine (100 nmol, n = 6) or bisindolylmaleimide (BIS, 25 mumol, n = 9) did not prevent IP locally. The PKC activator phorbol myristate acetate (PMA, 1 mumol, n = 6) was ineffective in preventing ischemic injury and increased the amount of necrosis in IP, whereas BIS exerted a local myocardial protection (n = 9, p < 0.001). In conclusion, the new model of intramyocardial infusion appears to be useful for the investigation of IP's signal transduction. Our data support the role of the adenosine A1-receptor in IP, but suggest that inhibition instead of activation of PKC may protect ischemic myocardium from infarction.

Salvage of jeopardized myocardium by ischemic preconditioning: is the quest over?

Helmholtz is quoted to have said that if he'd had any influence in creation he would have returned the human eye to its maker for revisions. The same could be said of the heart with its only very rudimentary ability to defend itself against ischemia. Ischemia was obviously not a problem during evolution: Early man did not live much longer than prime time for reproduction and no selection bias existed to prevent vascular diseases, an affliction of later life. In spite of this natural disadvantage of aged males the number of existing although not very efficient defense mechanisms is surprisingly large. It is the general belief that the knowledge of these mechanisms may lead to the development of new therapies that hopefully improve the imperfect product of natural selection.

Chemotherapy for patients with adenocarcinoma of the pancreas.

Adenocarcinomas of the pancreas are diagnosed at an advanced, non-curable stage in most patients. In addition to local relapse or progression, distant metastases determine the poor prognosis, resulting in a median survival of less than 6 months in most studies of patients with locally advanced or metastatic pancreatic cancer. None of the cytotoxic drugs available show impressive activity in treatment of this disease. Therefore, chemotherapy is not recommended in pancreatic cancer. In selected cases, 5-fluorouracil-based therapy - with or without simultaneous radiation - may result in tumor responses. With a once-a-week outpatient protocol of folinic acid and 5-fluorouracil, we observed a progression-free period from 15 to 42 weeks in nine of 19 patients treated without any serious adverse effects, confirming reports of the weak, but well-tolerated activity of this combination. To alter the prognosis for patients with advanced adenocarcinomas of the pancreas, new drugs with more activity have to be developed and tested in well-designed trials.

[The role of adenosine in "ischemic preconditioning"]. / Die Rolle von Adenosin bei "ischemic preconditioning".

Repeated short coronary occlusions trigger probably more than one pathway leading to increased tolerance toward ischemia. Adenosine plays probably a very important but not an exclusive role. It may act via opening of A1-receptor-operated K(+)-channels that lead to hyperpolarization of the cell, and it has antiadrenergic actions. A proposed action of adenosine via protein kinase C is not confirmed. Trophic factors are expressed during short periods of ischemia and contribute to survival, probably via inhibition of apoptosis.

Effects of exercise on myocardial adenylate cyclase and Gi alpha expression in senescence.

The present study investigated the influence of physical training on alterations leading to reduced adenosine 3',5'-cyclic monophosphate (cAMP) formation in aged myocardium. Senescent (28-mo-old) rats underwent a moderate treadmill exercise program and were compared with sedentary controls. In myocardial membranes from aged rats, isoprenaline-stimulated adenylate cyclase activity (AC) was reduced compared with that in young animals and was accompanied by an increase of pertussis toxin substrates (17.5%) and an increased amount of immunodetectable inhibitory guanine nucleotide-binding proteins (Gi alpha; 72%). Physical training reduced the amount of Gi alpha proteins (30-35%) in young and old animals and enhanced only isoprenaline-stimulated AC in the aged rats, and basal and 5'-guanylylimidodiphosphate [Gpp(NH)p]- as well as isoprenaline-stimulated AC in young rats. Physical training or aging had no effect on the number of beta-adrenoceptors or m-cholinoceptors or on forskolin-stimulated AC in either group. The amount of immunodetectable stimulatory guanine nucleotide-binding proteins (Gs alpha) using an antiserum raised against the COOH-terminal peptide of Gs alpha (RMHLRQYELL) was unchanged in either condition. It is concluded that enhanced Gi alpha expression might be one mechanism leading to depressed cAMP formation in aged myocardium. Depressed AC and increased Gi alpha can be partially reversed by physical training, especially in young myocardium. Gi alpha might serve as a regulator of cardiac AC in a variety of physiological and pathophysiological situations in the absence of beta-adrenoceptor changes.

Laboratory evaluation of scilliroside used as a rodenticide against the lesser bandicoot rat, Bandicota bengalensis.

The toxicity and efficacy of the acute rodenticide scilliroside was evaluated in the laboratory against the lesser bandicoot rat, Bandicota bengalensis. The acute oral LD50 and LD95 doses for males were 0 . 8 mg/kg and 2 . 5 mg/kg respectively, and for females were 0 . 5 mg/kg and 1 . 6 mg/kg, respectively. When caged bandicoots were given a choice between plain and poison baits, the optimum concentration of scilliroside was found to be 0 . 05%. Symptoms of poisoning appear from 22 to 34 min after feeding starts and the latency pattern indicated an abrupt ceasing to feed at these points. Death occurred from 2 h to as long as 6 days after poisoning, following prolonged convulsive seizures. There appears to be aversion to scilliroside at all concentrations in food baits. Maximum mortality attained on free-choice feeding on scilliroside was 90%. Despite these disadvantages, the material may have merit as an alternative rodenticide to zinc phosphide where acute toxicants are to be used.

The susceptibility of Bandicota bengalensis from Rangoon, Burma to several anticoagulant rodenticides.

The baseline susceptibility of the lesser bandicoot rat, Bandicota bengalensis, from Rangoon, Burma, to five anticoagulant rodenticides was established with no-choice feeding in the laboratory. The susceptibility of lesser bandicoots to the several poisons (brodifacoum, difenacoum, diphacinone, coumatetralyl, and warfarin) was such that they were offered at a 0.001% concentration. B. bengalensis was most susceptible to brodifacoum, and in descending order, difenacoum, coumatetralyl, diphacinone and warfarin. In comparison with Rattus norvegicus on warfarin at 0.005%, B. bengalensis proved more susceptible. Feeding tests at 0.005% concentration indicated that a 1-day feeding on brodifacoum and difenacoum would result in complete mortality, whereas coumatetralyl and warfarin would require 4 days feeding to a 100% kill. Brodifacoum and difenacoum are recommended at 0.002-0.005% bait concentrations and coumatetralyl at 0.005--0.01% concentrations for the control of B. bengalensis in the field in Rangoon. The use of any anticoagulant material in rat control should be alternated with acute toxicants to retard the possible development of anticoagulant resistance.