Correction to: The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease.

In the original version of this article, the name of one of the authors is not correct. The correct name should be W. A. Linke, which is shown correctly in the authorgroup section above.

The Sydney Heart Bank: improving translational research while eliminating or reducing the use of animal models of human heart disease.

The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.

Antiferromagnetic Ordering of Magnetic Clusters Units in Nb(6)F(15).

We have studied the magnetic cluster compound Nb(6)F(15) which has an odd number of 15 valence electrons per (Nb(6)F(12))(3+) cluster core, as a function of temperature using nuclear magnetic resonance, magnetic susceptibility, electron magnetic resonance and neutron powder diffraction. Nuclear magnetic resonance of the (19)F nuclei shows two lines corresponding to the apical F(a-a) nucleus, and to the inner F(i) nuclei. The temperature dependence of the signal from the F(i) nuclei reveals an antiferromagnetic ordering at T < 5 K, with a hyperfine field of ~2 mT. Magnetic susceptibility exhibits a Curie-Weiss behavior with T(N) ~5 K, and µ(eff) ~1.57 µ(B) close to the expected theoretical value for one unpaired electron (1.73 µ(B)). Electron magnetic resonance linewidth shows a transition at 5 K. Upon cooling from 10 to 1.4 K, the neutron diffraction shows a decrease in the intensity of the low-angle diffuse scattering below Q ~0.27 Å(-1). This decrease is consistent with emergence of magnetic order of large magnetic objects (clusters). This study shows that Nb(6)F(15) is paramagnetic at RT and undergoes a transition to antiferromagnetic order at 5 K. This unique antiferromagnetic ordering results from the interaction between magnetic spins delocalized over each entire (Nb(6)F(12) (i))(3+) cluster core, rather than the common magnetic ordering.

Low-dose lidocaine suppresses experimentally induced hyperalgesia in humans.

BACKGROUND: The antinociceptive effects of systemically administered local anesthetics have been shown in various conditions, such as neuralgia, polyneuropathy, fibromyalgia, and postoperative pain. The objective of the study was to identify the peripheral mechanisms of action of low-dose local anesthetics in a model of experimental pain. METHODS: In a first experimental trial, participants (n=12) received lidocaine systemically (a bolus injection of 2 mg/kg in 10 min followed by an intravenous infusion of 2 mg x kg(-1) x h(-1) for another 50 min). In a second trial, modified intravenous regional anesthesia was administered to exclude possible central analgesic effects. In one arm, patients received an infusion of 40 ml lidocaine, 0.05%; in their other arm, 40 ml NaCl, 0.9%, served as a control. In both trials, calibrated tonic and phasic mechanical and chemical (histamine) stimuli were applied to determine differentially the impairment of tactile and nociceptive perception. RESULTS: Mechanical sensitivity to touch, phasic mechanical stimuli of noxious intensity, and heat pain thresholds remained unchanged after systemic and regional application of the anesthetic. In contrast, histamine-induced itch (intravenous regional anesthesia), axon reflex flare (systemic treatment), and development of acute mechanical hyperalgesia during tonic pressure (12 N; 2 min) of an interdigital web was significantly suppressed after both treatments. CONCLUSIONS: Increasing painfulness during sustained pinching has been attributed to excitation and simultaneous sensitization of particular Adelta- and C-nociceptors. This hyperalgesic mechanism seems to be particularly sensitive to low concentrations of lidocaine. These findings confirm clinical experience with lidocaine in pain states dominated by hyperalgesia.

[Predictability and precision of "target-controlled infusion" (TCI) of propofol with the "Disoprifusor TCI" system]. / Prädiktivität und Präzision einer "target-controlled infusion" (TCI) von Propofol mit dem System "Disoprifusor TCI".

UNLABELLED: In Germany a TCI-system for propofol (Disoprifusor-TCI) has been commercially available since spring 1997. We investigated the prediction error and precision of this TCI system as part of a multicentre study. Bias, precision, blood concentrations and dosage of propofol were compared with patients receiving propofol via a manually controlled infusion device. METHODS: After approval by the local Ethics Committee and written informed consent, 21 patients of ASA-classification I to III scheduled for major abdominal surgery received either a target controlled infusion (group T, Disoprifusor-TCI) or a manually controlled infusion (group M) of propofol. The propofol plasma concentrations were measured by HPLC. The prediction error for each measurement, the median prediction error (MDPE) or bias, the median absolute prediction error (MDAPE) or precision and the divergence (change of the prediction error over infusion time) were calculated for both groups. RESULTS: For all patients in group T (n = 12) the bias of the TCI system was 6.7% and the precision 27.5%. For 70% of all measured plasma concentrations the absolute prediction error was < or = 37%. The divergence was -5.4% per hour. For all patients in group M (n = 9) the bias was 44.2% and the precision 50%. The mean amount of propofol infused per kilogram body weight and hour was significant higher in T (9.0 +/- 1.2 mg/kg/h) than in M (6.6 +/- 1.2 mg/kg/h, p < 0.005). CONCLUSIONS: With a precision of 27.5% the investigated TCI system (Diprifusor-TCI) showed an acceptable inaccuracy, as for TCI-systems a median prediction error of +/- 30% has to be expected due to the inherent variability of pharmacokinetic parameters. Further studies will be necessary to find out whether the investigated TCI system for propofol may offer substantial advantages.

Complete identification of nonbridging phosphate oxygens involved in hammerhead cleavage.

Phosphorothioate interference analysis is suited for the rapid identification of structurally and functionally important phosphate groups. Previous interference studies, however, have been limited to the analysis of pro-Rp phosphate oxygens. We employed solid-phase oligonucleotide synthesis and modification interference analysis to investigate either of the nonbridging phosphate oxygens within the hammerhead ribozyme. Two novel sites of Sp phosphorothioate interference were identified at positions A6 and U16.1. The results from interference experiments were confirmed by single phosphorothioate substitutions at sites of interest. Metal rescue experiments revealed that direct metal ion coordination occurs in the functional ribozyme only at the site of cleavage and at the pro-Rp oxygen of position Ag. The new approach may be generally useful in rapidly evaluating the functional importance of phosphate groups in nucleic acids.

Comparison between three transmucosal routes of administration of midazolam in children.

Midazolam was applied transmucosally in 47 children randomly assigned to three different groups. Group N received 0.2 mg.kg-1 nasally, group R 0.5 mg.kg-1 rectally, and group S 0.2 mg.kg-1 sublingually. All groups were treated 60 min prior to a planned i.v. puncture with EMLA. Reliable and valid psychological parameters (such as emotional situation, shivering, awareness, respiratory rate and facial colour) were scored after premedication and before and after i.v. puncture, 20 min after premedication and until induction. A blood sample was drawn 10, 30 and 60 min after premedication and the levels of midazolam, alpha-hydroxy-midazolam, ACTH, glucose and cortisol were measured. In all three groups the plasma levels of midazolam 10 min after premedication were higher than 70 ng.ml-1 (accepted as a sedative level). 30 min after premedication the midazolam level in the sublingual group was statistically significantly higher than in the nasal group and the psychological parameters in all three groups were significantly changed (10 min after premedication). The psychological parameters were not significantly different between the three groups over the whole study. Sublingual premedication has some advantages (most readily accepted, highest plasma levels and lowest deviations) and could be the first choice in premedication of children. All three transmucosal applications are safe and well accepted, although nasal application was rejected by two of the children.

Role of adenosine in the hypoxic induction of vascular endothelial growth factor in porcine brain derived microvascular endothelial cells.

Hypoxia induced the mRNA expression of vascular endothelial growth factor (VEGF) in porcine brain derived microvascular endothelial cells (BMEC) in a time-dependent manner. Corresponding to the mRNA induction the protein level of VEGF was elevated during hypoxia. The adenosine A1 receptor antagonist 8-phenyltheophylline (8-PT) reduced the hypoxia-induced VEGF mRNA and protein expression significantly. The treatment of BMEC with cobalt chloride-known to activate an oxygen sensing mechanism similar to the one used by the erythropoietin gene-also induced the VEGF mRNA expression, but 8-PT did not reduce this VEGF induction. Although, earlier studies revealed that agents like phorbolester induced the VEGF mRNA expression, the specific inhibitor of the proteinkinase C (PKC) bisindolylmaleimide (BIM) did not reduce but enhanced the hypoxia-induced VEGF mRNA expression. These results indicate that the VEGF induction in BMEC can proceed through PKC-dependent and -independent pathways (like those acting via the putative oxygen sensor). Hypoxia in BMEC probably activates the PKC-dependent pathway mainly via adenosine which might be formed during hypoxia and thereby inhibits activation of PKC-independent, oxygen sensing, pathways. This suggestion was supported by the fact that hypoxia as well as adenosine increased the VEGF mRNA expression post-transcriptionally by enhancing the stability of the VEGF mRNA [corrected].

Characterization of differentially expressed genes following brief cardiac ischemia.

For the study of ischemia-related adaptations we employed DDRT-PCR on mRNA obtained from heart regions undergoing brief (10') occlusions/reperfusions. We found and sequenced 52 differentially expressed clones that we further characterized using Northern analysis with RNA from a wide variety of tissues. We selected only clones (1) with a new sequence, (2) that showed a Northern signal, and (3) that exhibited organ-specific (heart, muscle) or (4) situation-specific (ischemia, non-specific stress) expression. We found two new heart-specific transcripts and three new stress-inducible genes. The transcripts with known sequences showed an expression pattern typical for repair processes after ischemia-reperfusion (ubiquitin, ATPases). About 20% of clones were truly differentially expressed.

[Cardiac gene expression after brief coronary occlusion]. / Kardiale Genexpression nach kurzen Koronarverschläussen.

In the pig short coronary occlusions induce molecular damage on the protein level in the myocardium, which elicit repair mechanisms by increased transcription and translation, including the activation of potential transcription factors (protooncogenes), genes involved in repair processes (heat shock genes) or calcium-binding genes. Additionally, some growth factors like insulin-like growth factor II show increased transcription in accordance with their function as trophic factors for reversibly injured myocardium. Changes in mRNA levels mostly are due to increased transcription rates and rarely due to prolonged half-life of the mRNA. However, at present our data do not allow us to conclude which genes are causative for myocardial stunning and/or ischemic preconditioning.

Nasal midazolam in children, plasma concentrations and the effect on respiration.

Twenty ASA 1 children, one to six years old, weighing 10-20 kg, scheduled for a combination of general and caudal anaesthesia received at random midazolam 0.2, 0.4, or 0.6 mg.kg-1 or NaCl 0.9% (control group) intranasally. Drug or NaCl 0.9% were administered in one nostril, after inhalation induction of anaesthesia, intubation without relaxant and caudal anaesthesia. Spontaneous respiration was via a circle system and fresh gas flow of 6 l.min-1 (N2O/O2 = 2:1), PEEP 5 cm H2O, endtidal halothane 0.4%. Immediately before and 2, 5, 8, 12, 16, 20, 30, 60 and 120 min after application of the drug 2.5 ml blood was sampled for plasma levels of midazolam. Endtidal CO2, respiratory rate, and oxygen saturation were recorded as long as the children were intubated. Endtidal CO2 and respiratory rate showed no statistical difference between the groups at any time, however, in the group receiving 0.6 mg.kg-1, endtidal CO2 increased significantly from 5.3 kPa (41 mm Hg) at the start to 5.9 kPa (45.5 mm Hg) after 30 min. Plasma levels of midazolam were detected 2 min after application in 10 of 15 patients. Median peak levels were found between 12 and 16 min. Medians of peak plasma levels showed no statistical difference between the three groups (0.2 mg.kg-1:111 ng.ml-1, 0.4 mg.kg-1:136 ng.ml-1, 0.6 mg.kg-1:277 ng.ml-1). After 30, 60 and 120 min medians of midazolam plasma concentration were significantly higher in the group 0.6 mg.kg-1.

Altered gene transcription following brief episodes of coronary occlusions.

This study was designed to elucidate whether previously observed enhanced mRNAs were due to accelerated transcription, enhanced mRNA stability or both mechanisms. We employed the nuclear run-on technique on myocardial nuclei and found the transcriptional induction of several genes, especially nuclear protooncogenes, Ca2+ regulating and heat shock protein genes.

Changes in gene expression following short coronary occlusions studied in porcine hearts with run-on assays.

OBJECTIVE: Brief coronary occlusions cause upregulation of expression in a wide variety of genes. These changes in tissue mRNA concentration could have been produced by transcriptional or post-transcriptional events. The aim of this study was to discriminate between increased transcription and changes in mRNA stability using run-on assays with isolated myocyte nuclei. METHODS: Myocyte nuclei isolated from ischaemic/reperfused and normal myocardium were incubated with labelled ribonucleotides. The radioactive RNA was then hybridised with specific cDNA probes and slot blots were autoradiographed. RESULTS: There was increased transcriptional activity for the proto-oncogenes c-myc, c-jun, jun-B, and jun-D. There were marked increases in transcriptional activity for sarcoplasmic Ca(2+)-ATPase, calmodulin, phospholamban, and calsequestrin. Strong transcriptional activity was found for the ubiquitin and heat shock protein (hsp27, hsp70) genes, and for PAI-1 and GAPDH. The transcription for the beta myosin heavy chain gene was not altered. CONCLUSIONS: Changes in the tissue concentration of mRNA species following brief coronary occlusion and reperfusion are most often the result of altered transcriptional activity. Increased c-fos mRNA concentrations observed in earlier studies cannot be explained by transcriptional activity of myocytes during reperfusion. Calmodulin is strongly transcribed but tissue concentration stays constant. The overall pattern of gene expression is indicative of damage at the molecular level, and calcium binding proteins (among perhaps many others) are in need of repair.

Enhanced gene expression of calcium regulatory proteins in stunned porcine myocardium.

OBJECTIVE: Increasing evidence points to a molecular disturbance of Ca2+ homeostasis in stunned myocardium. The aim of this study was therefore to investigate the expression of mRNAs for Ca2+ binding proteins related to the sarcoplasmic reticulum in a porcine model of myocardial stunning. METHODS: In 22 anaesthetised pigs, stunning was achieved by one or two cycles of 10 min left anterior descending coronary artery occlusion and reperfusion. Hearts were excised at various timepoints of the protocol. Total RNA was extracted from stunned (experimental) as well as normally perfused (control) myocardium. RESULTS: Northern blot analysis using radioactive cDNA probes revealed that the Ca(2+)-ATPase mRNA levels increased 1.6-fold compared to the control value at 90 min of the second reperfusion. The steady state level of phospholamban mRNA rose 2.5-fold at 180 min of reperfusion. A 2.3-fold increase in calsequestrin mRNAs was observed after 90 min of the second reperfusion. The calmodulin and alpha, beta myosin heavy chain mRNA levels were unchanged. A glyceraldehyde-3-phosphate dehydrogenase cDNA probe served as a reference system. Nuclear run-on assays showed increased transcription for Ca(2+)-ATPase and calsequestrin at 90 min of reperfusion, supporting the view that increased mRNA levels seen with northern hybridisation were due to increased transcription of the respective gene. CONCLUSIONS: The results suggest specific repair mechanisms of stunned myocardium and point to the involvement of calcium regulatory proteins related to the sarcoplasmic reticulum in the pathogenesis of myocardial stunning.

Expression of heat shock proteins in the normal and stunned porcine myocardium.

OBJECTIVE: The aim was to examine the expression of ubiquitin (Ub), 27 kDa heat shock protein (hsp27), and hsp60 mRNA in normal and briefly ischaemic and reperfused porcine myocardium: METHODS: The left anterior descending coronary artery was occluded for two periods of 10 min separated by 30 min of reperfusion. After the second occlusion the myocardium was reperfused up to 210 min. Tissue from ischaemic, ischaemic-reperfused, and non-ischaemic regions of the heart was analysed by northern and slot blot hybridisation and nuclear run-on transcription assays employing radiolabelled cDNA probes for Ub, hsp27, and hsp60, as well as by western blot using monoclonal antibodies recognising Ub protein conjugates and antiserum recognising hsp27. RESULTS: Systolic wall thickening was significantly decreased at 30 min reperfusion after both occlusions and remained depressed at longer periods of reperfusion. Using northern blot hybridizations, several mRNAs encoding Ub, 0.9 kb mRNA encoding hsp27, and 2.2 kb mRNA encoding hsp60 were detected in sham operated, non-ischaemic, and ischaemic myocardial tissues. Densitometric analysis of northern and slot blot hybridisation signals showed significant increase of basal tissue levels of Ub mRNA in stunned regions only during the 30 min of the second reperfusion period. Increased levels of hsp27 mRNA in stunned tissue were already noted at the first ischaemic period and were sustained compared to control during the subsequent periods of reperfusion. Changes in hsp60 mRNA tissue levels were not observed during ischaemia and subsequent reperfusions. Transcription of the Ub and hsp27 genes was increased during 30 and 120 min of the second reperfusion period. The transient enhancement of tissue levels of Ub mRNA was associated with temporary formation of new Ub-protein conjugates. However, the increased synthesis of mRNA encoding hsp27 was not followed by changes of hsp27 protein content in myocardial tissue. CONCLUSIONS: The findings support the hypothesis that molecular damage occurs in stunned myocardium; however, the target molecules remain to be recognised.