Solution structure of hpTX2, a toxin from Heteropoda venatoria spider that blocks Kv4.2 potassium channel.

HpTX2 is a toxin from the venom of Heteropoda venatoria spider that has been demonstrated to bind on Kv4.2 potassium channel. We have determined the solution structure of recombinant HpTX2 by use of conventional two-dimensional NMR techniques followed by distance-geometry and molecular dynamics. The calculated structure belongs to the Inhibitory Cystin Knot structural family that consists in a compact disulfide-bonded core, from which four loops emerge. A poorly defined two-stranded antiparallel beta-sheet (residues 20-23 and 25-28) is detected. Analysis of the electrostatic charge anisotropy allows us to propose a functional map of HpTX2 different from the one described for kappa-conotoxin PVIIA, but strongly related to the one of charybdotoxin. The orientation of the dipole moment of HpTX2 emerges through K27 which could therefore be the critical lysine residue. Close to this lysine are a second basic residue, R23, an aromatic cluster (F7, W25, W30) and an hydrophobic side chain (L24). The high density in aromatic side chains of the putative functional surface as well as the lack of an asparagine is proposed to be the structural basis of the specificity of HpTX2 toward Kv4.2 channel.

Effects of fluoroquinolones on HERG currents.

We have investigated the effects of four fluoroquinolones on the human ether-à-go-go-related gene (HERG) mediated K(+) currents to evaluate their potential to induce QT-prolongation. HERG currents were measured from stably transfected Chinese hamster ovary (CHO) cells by means of the patch-clamp technique. Bath application of sparfloxacin, moxifloxacin and grepafloxacin produced an inhibition of HERG outward currents at -40 mV with EC(50) of 13.5+/-0.8, 41. 2+/-2.0 and 37.5+/-3.3 microg/ml, respectively. Current inhibitions were reversible after washout of the compounds. By contrast, ciprofloxacin at concentrations of up to 100 microg/ml did not effect HERG outward currents.

Wear induced by motion between bone and titanium or cobalt-chrome alloys.

We studied the wear generated by motion between polished and shot-blasted titanium-alloy (Ti-6Al-4V) or cobalt-chrome alloy (Co-Cr) surfaces and cortical bone in vitro. Semicircular sections of human proximal femoral cortex were reamed to fit metal cylinders of each alloy. The cylinders were then fitted in the bone, loaded and rotated in physiological saline. Ti-alloy resulted in more wear both of the bone and of the metal than did Co-Cr alloy. Metal wear was reduced and bone wear was increased by shot-blasting, a procedure which introduces surface residual stresses and roughens the metal surface. We conclude that when there is gross motion between a metal implant and bone, Ti-alloy is likely to generate more wear debris than Co-Cr alloy. The least wear both of bone and of metal was produced by polished Co-Cr.

[Bupivacaine 0.5% and 0.75% for peridural anesthesia in operations on the extremities]. / Bupivacain 0.5% und 0.75% zur Periduralanaesthesie bei Extremitätenoperationen.

The safety of bupivacaine in respect of its cardiotoxicity has been discussed recently. Especially the concentration of bupivacaine 0.75% has been heavily criticized, first of all in obstetrical anaesthesia. Therefore two groups of patients receiving bupivacaine 0.5% and bupivacaine 0.75% were compared according to complications and side effects related to the local anaesthetic. 749 patients undergoing varicose vein stripping were divided in two groups: 371 patients received bupivacaine 0.5% with adrenalin 1:200,000 and 378 patients bupivacaine 0.75% with POR-8 (0.1 i.U./ml) for epidural anaesthesia. The average age was 46 years (20-71 y.). The average height 168 cm (149-196 cm), and the average weight 72 kg (40-99 kg). There were 68.7% female and 31.3% male patients in these groups. The anaesthesia charts of these patients were evaluated retrospectively. The incidence of hypotension, breadycardia, tachycardia, extrasystoles, vomiting and muscle twirching were taken as side effects. The two groups were compared. The results showed in relation to age, risk-grouping and the dosage/kg no evidence of an increased cardiotoxicity of the higher concentration of bupivacaine. Thus bupivacaine 0.75% does not show any increase in complications or side effects as long as its application is done correctly.

[Can follow-up scintigraphy be a decision aid for therapy choice in Perthes disease?]. / Kann die Verlaufsszintigraphie eine Entscheidungshilfe für die einzuschlagende Therapie beim Morbus Perthes geben?

In the period from 1988 to 1982 91 patients with M. Legg-Calvé-Perthes received orthopaedic treatment. In 51 cases surgical treatment for femoral head containment by intertrochanteric derotation varus osteotomy was performed. 40 patients were treated conservatively. Most cases received a Thomas-splint to eliminate weightbearing on the affected hip, some used crutches. Out of this group we present 6 cases that showed during the time of their first examination the radiologic stages 1 and 2 according to Waldenström. At the time when the M. Perthes was diagnosed these patients received a radionuclide bone scan, which was repeated of the average 6 months later to aid in the choice of therapy. Two other cases are presented for comparison, who received the first bone scan in the Perthes stage 2 and 3 according to Waldenström (fragmentation stage) showing an activity defect and were then operated with a derotation varus osteotomy after the follow-up bone scan had not shown revascularisation. All patients that had a complete revascularisation in the follow-up bone scan during the fragmentation stage had a good final result after conservative treatment. For other cases, where the bone scan in the fragmentation stage shows a persisting lack of epiphysis perfusion, it is recommended to decide for surgical treatment with derotational varus osteotomy, since the development of "head-at-risk" signs is likely, which will not resolve after later surgery.

Screening lead compounds for QT interval prolongation.

The late detection of cardiotoxic side effects, such as QT prolongation, induced by compounds of pharmacological interest can dramatically impede drug discovery and development projects, and consequently increase their cost. The launch of new drugs with undetected cardiotoxic side effects could have hazardous consequences and could trigger lethal cardiac dysrhythmias in patients. It is desirable, therefore, to test for the potential cardiotoxic side effects of compounds at an early stage of drug development. Electrophysiological test systems and cellular-based fluorometric high-throughput assays are now available for cloned human cardiac ion channels. These test systems are important tools in the preclinical safety evaluation of drugs and newly developed compounds.

Single voltage-gated K+ channels and their functions in small dorsal root ganglion neurones of rat.

1. Single voltage-activated K+ channels were investigated by means of the patch-clamp technique in small dorsal root ganglion (DRG) neurones in 150 microns thin slices of new-born rat DRG. It was found that K+ conductance in small DRG neurones is formed by one type of fast inactivating A-channel and four types of delayed rectifier K+ channels, which could be separated on the basis of their single-channel conductance, kinetics and sensitivity to external tetraethylammonium (TEA). 2. Potassium A-channels were observed at relatively moderate density. They were weakly sensitive to TEA and activated between -70 and +20 mV. The conductance of A-channels was about 40 pS for inward currents in symmetrical high-K+ solutions with external 5 mM TEA added to suppress other types of K+ channels. The time constant of channel inactivation (tau in) was 18.8 ms at -70 mV and 6 ms at potentials positive to -20 mV. 3. A fast delayed rectifier (DRF) channel with a conductance of 55 pS in symmetrical high-K+ solutions was the most frequent type of K+ channel. The channel activated in a broad potential range between -50 and +60 mV and demonstrated a fast deactivation within 1-3 ms after potential return to -80 mV in high-Ko+ solution. The tau in value was 90-150 ms at positive membrane potentials. The single-channel current amplitudes were blocked to 55% by 1 mM TEA. 4. Three further types of delayed rectifier K+ channels were called DR1-, DR2- and DR3- channels. Their single-channel conductances for inward currents in symmetrical high-K+ solutions were distributed between 30 and 44 pS. The channels activated in almost the same voltage range between -60 and -10 mV. Deactivation of the channels at -80 mV lasted tens of milliseconds. The channels were separated on the basis of their sensitivities to TEA. DR1-channel currents were reduced to 50% in the presence of 1 mM TEA, DR2-channel currents were reduced to about 50% by 5 mM TEA, whereas the amplitudes of currents through DR3-channels were almost unaffected by 5 mM TEA. 5. Addition of external 1 and 5 mM TEA to whole cells under current-clamp condition depolarized the cell membrane, lowered the threshold for action potential firing, prolonged action potential duration and reduced the amplitude of after-hyperpolarization. 6. It is concluded that potassium A-, DRF-, DR1-, DR2- and DR3-channels play multiple roles in the excitability of DRG neurones. Possible influences of these channels on the shape of the action potential, its firing threshold and the resting membrane potential of small DRG neurones are discussed.

Na+-activated K+ channels in small dorsal root ganglion neurones of rat.

1. Whole-cell Na+-activated K+ (KNa) channel currents and single KNa channels were studied with the patch-clamp method in small (20-25 micrometer) dorsal root ganglion (DRG) neurones in slices of rat dorsal root ganglia. 2. The whole-cell KNa channel current was identified as an additional K+-selective leakage current which appeared after cell perfusion with internal solutions containing different [Na+]. The concentration for half-maximal activation of KNa channel current was 39 mM and the Hill coefficient was 3.5. At [Na+]i above 12 mM, KNa channel current dominated the unspecific leakage current. The ratio of maximum KNa channel current to unspecific leakage current was 45. 3. KNa channel current was not activated by internal Li+. It was suppressed by external 20 mM Cs+ but not by 10 mM tetraethylammonium. 4. Single KNa channels with a conductance of 142 pS in 155 mM external K+ (K+o)-85 mM internal K+ (K+i) solutions were observed at a high density of about 2 channels micrometer-2. 5. In two-electrode experiments, a direct correlation was seen between development of whole- cell KNa channel current and activation of single KNa channels during perfusion of the neurone with Na+-containing internal solution. 6. Under current-clamp conditions, KNa channels did not contribute to the action potential. However, internal perfusion of the neurone with Na+ shifted the resting potential towards the equilibrium potential for K+ (EK). Varying external [K+] indicated that in neurones perfused with Na+-containing internal solution the resting potential followed the EK values predicted by the Nernst equation over a broader voltage range than in neurones perfused with Na+-free solution. 7. It is concluded that the function of KNa channels has no links to firing behaviour but that the channels could be involved in setting or stabilizing the resting potential in small DRG neurones.

Early detection and differentiation of periprosthetic fluid accumulation after vascular reconstructive surgery.

Ultrasonographic duplex scanning is used widely to screen the abdominal arterial system and to detect lesions in the extracranial cerebral arteries. Based on clinical studies of 11,712 vascular reconstructions, this report describes the early recognition of fluid accumulation around vascular prostheses. The clinical examination may suggest the typical complications that arise after vascular surgery such as hematoma, pseudoaneurysm, lymphocele, abscess, or perigraft cyst. Duplex scanning, combined with puncture and aspiration, proved to be of great benefit in differentiating the specific type and extent of the complication. Special attention is paid to the so-called perigraft reaction, thought to be a result of an aseptic biological incompatibility to synthetic vascular grafts. It is emphasized that ultrasonographic routine follow-up after vascular surgery is essential for early diagnosis, especially since each complication suspected requires specific therapeutic treatment to prevent exacerbation because of inadequate treatment.