Cell-to-cell distances between tumor-infiltrating inflammatory cells have the potential to distinguish functionally active from suppressed inflammatory cells.

Beyond their mere presence, the distribution pattern of inflammatory cells is of special interest. Our hypothesis was that random distribution may be a clear indicator of being non-functional as a consequence of lack of interaction. Here, we have assessed the implication of cell-to-cell distances among inflammatory cells in anal squamous cell carcinoma and a possible association with survival data. Thirty-eight patients suffering from anal carcinoma were studied using tissue microarrays, double staining immunohistochemistry, whole slide scanning and image analysis software. Therapy consisted of concurrent radiochemotherapy. Numbers of stromal and intraepithelial tumor-infiltrating inflammatory cells (TIC) and the distances between cells were quantified. Double-staining of FoxP3(+) cells with either CD8(+), CD1a(+) or CD20(+) cells was performed. Measured cell-to-cell distances were compared to computer simulated cell-to-cell distances leading to the assumption of non-randomly distributed and therefore functional immune cells. Intraepithelial CD1a(+) and CD20(+) cells were randomly distributed and therefore regarded as non-functional. In contrary, stromal CD20(+) cells had a non-random distribution pattern. A non-random distance between CD20(+) and FoxP3(+) cells was associated with a clearly unfavorable outcome. Measured distances between FoxP3(+) cells were distinctly shorter than expected and indicate a functional active state of the regulatory T cells (Treg). Analysis of cell-to-cell distances between TIC has the potential to distinguish between suppressed non-functional and functionally active inflammatory cells. We conclude that in this tumor model most of the CD1a(+) cells are non-functional as are the intraepithelial CD20(+) cells, while stromal CD20(+) cells and FoxP3(+) cells are functional cells.

Three-dimensional analysis of tumour vascular corrosion casts using stereoimaging and micro-computed tomography.

OBJECTIVE: In order to perform effective translational research for cancer therapy, we need to employ pre-clinical models which reflect the clinical situation. The purpose of this study was to quantitatively compare the vascular architecture of human colorectal cancer and experimental tumour models to determine the suitability of animal models for vascular studies and antivascular therapy. METHODS: In this study we investigated the three-dimensional properties of colonic tumour vasculature in both human clinical tissues (normal mucosa control [n=20], carcinoma [n=20] and adenoma [n=6]) and murine colorectal xenografts (LS147T [n=6] and SW1222 [n=6]). Scanning Electron Microscope Stereoimaging (SEM) and X-ray Micro-Computed Tomography (Micro-CT) methods were employed for 3D analyses of the vascular corrosion casts from these tissues. RESULTS: Morphological measurements showed that there were significant differences in the underlying morphology in the different tissues. Of the studied xenografts, LS147T is more consistently similar to the vascular architecture of the human carcinoma than SW1222. The only reversal of this is for the inter-vessel distance. CONCLUSION: While SEM stereoimaging provided better surface detailed resolution of the corrosion casts, it was complimented by the fully 3D micro-CT method. Comparison made between the xenografts and clinical tumours showed that the LS147T xenografts shared many similarities with the clinical tumour vasculature. This study provides insight into how to select the most suitable pre-clinical models for translational studies of clinical cancer therapy.

Data standards for minimum information collection for antibody therapy experiments.

Research groups developing antibody therapies generate diverse data sets; the value of these sets would be compounded when shared or amalgamated. A complete amalgamation of diverse data sets requires data standards for information collection during experiments. We propose to define elements of the data standards in the form of common data elements (CDEs) in order to clarify each experiment's targets and data values. We have created a set of core information elements which we suggest should be collected from antibody therapy experiments. We propose these as a basis for community consultation with a view to defining a set of data standards which can be developed under the auspices of the Antibody Society.

Computational function assignment for potential drug targets: from single genes to cellular systems.

Biomedical science is currently undergoing an epoch-marking transition from its classical phase to the post-genome era. The outstanding success of world-wide genome sequencing efforts, evidenced by the recent publication of the draft of the human genome, together with the completion of several genomes of eukaryotic model organisms and the availability of microbial genome sequences, is opening up data sources of unprecedented scale for drug discovery. Furthermore, the elucidation of genome expression states through transcriptomic and proteomic techniques is playing a crucial role in the characterisation of disease at the molecular level. At the same time, our still very limited knowledge of the biological functions of genes and proteins at different levels of cellular organisation is preventing full exploitation of the available data. This review will discuss current computational techniques for function prediction based on the sequence-structure-function paradigm. Newly emerging approaches aimed at gaining an expanded understanding of function through integration of data from various sources and modelling of complex cellular systems will also be highlighted.

Can correlated mutations in protein domain families be used for protein design?

Evidence from diverse studies, such as protein design experiments and analysis of the emergence of drug resistance in human immunodeficiency virus 1 (HIV-1), indicates that protein function can be diminished or altered by mutations at positions distant from the classic 'functional' site. Furthermore, results from correlation analysis of the ligand-binding domain of nuclear receptors suggest that mutation events at positions distributed throughout a protein domain may be involved in functional diversification during the evolution of homologous domain families. This review explores potential applications for a protein design procedure based on correlated substitutions.

Classification and phylogenetic relationships of African tilapiine fishes inferred from mitochondrial DNA sequences.

African cichlid fishes are composed of two major lineages, the haplochromines and the tilapiines. Whereas the phylogenetic relationships of the haplochromines have been studied extensively, primarily because of their spectacular adaptive radiations in the Great Lakes of East Africa, little is known about the relationships among the tilapiine species, despite the fact that they have become an important component of African, indeed world, aquaculture. To remedy this situation, molecular phylogenetic analysis of tilapiine fishes was undertaken. A segment of mitochondrial DNA encompassing the terminal part of the tRNA(Pro) gene and the most variable part of the control region was amplified by the polymerase chain reaction with DNA samples isolated from 42 tilapiine species, and the amplification products were subjected to heteroduplex analysis and sequencing. Phylogenetic trees based on 68 sequences revealed the existence of 11 sequence groups and 11 single-sequence branches. The groups, designated Ti1 through Ti11, were distinguished by specific combinations of diagnostic substitutions, formation of monophyletic clusters, and separation by genetic distances in excess of 0.04. Although the relationships among the groups could not be resolved, the sequences separated Oreochromis and Sarotherodon from Tilapia, as defined by Trewavas. The Oreochromis sequences clustered with the Sarotherodon sequences and thus supported the hypothesis that the mouthbrooding behavior of the tilapiine fishes evolved only once from the substrate-spawning behavior. Since on phylogenetic trees the O. alcalicus (sub)species were always separated from O. amphimelas by other Oreochromis species, it was concluded that the adaptation to life in water with a high salt concentration and high pH values evolved independently at least twice in the tilapiine fishes. The tilapiines diverged from the haplochromines more than 8 million years ago; most of the intragroup divergences among the tilapiines took place an estimated 1.1 to 6 million years ago.

Macrophage migration inhibitory factor of the parasitic nematode Trichinella spiralis.

cDNAs were obtained for macrophage migration-inhibitory factor (MIF)/L-dopachrome methyl ester tautomerase homologues from the parasitic nematodes Trichinella spiralis (TsMIF) and Trichuris trichiura (TtMIF). The translated sequences, which were partly confirmed by sequencing of proteolytic fragments, show 42 and 44% identity respectively with human or mouse MIF, and are shorter by one C-terminal residue. Unlike vertebrate MIF and MIF homologues of filarial nematodes, neither TsMIF nor TtMIF contain cysteine residues. Soluble recombinant TsMIF, expressed in Escherichia coli showed secondary structure (by CD spectroscopy) and quaternary structure (by light-scattering and gel filtration) similar to that of the trimeric mammalian MIFs and D-dopachrome tautomerase. The catalytic specificity of recombinant TsMIF in the ketonization of phenylpyruvate (1.4x10(6) M(-1) x s(-1)) was comparable with that of human MIF, while that of p-hydroxyphenylpyruvate (9.1x10(4) M(-1) x s(-1)) was 71-fold lower. TsMIF showed high specificity in tautomerization of the methyl ester of L-dopachrome compared with non-esterified L-dopachrome (>87000-fold) and a high kcat (approximately 4x10(4) s(-1). The crystal structure, determined to 1.65 A (1 A=0.1 nm), was generally similar to that of human MIF, but differed in the boundaries of the putative active-site pocket, which can explain the low activity towards p-hydroxyphenylpyruvate. The central pore was blocked, but was continuous, with the three putative tautomerase sites. Recombinant TsMIF (5 ng/ml-5 pg/ml) inhibited migration of human peripheral-blood mononuclear cells in a manner similar to that shown by human MIF, but had no effect from 5 to 500 ng/ml on anti-CD3-stimulated murine T-cell proliferation. TsMIF was detected in supernatants of T. spiralis larvae cultured in vitro at 6 ng/ml (55 ng/mg total secreted protein). In conclusion TsMIF has structural, catalytic and cell-migration-inhibitory properties which indicate that it is partially orthologous to mammalian MIF.

The importance of somatic mutations in the V(lambda) gene 2a2 in human monoclonal anti-DNA antibodies.

2a2 is the most commonly rearranged gene in the human V(lambda )locus. It has been postulated that certain immunoglobulin genes (including 2a2) are rearranged preferentially because their germline sequences encode structures capable of binding to a range of antigens. Somatic mutation could then increase the specificity and affinity of binding to a particular antigen. We studied the properties of five IgG molecules in which the same heavy chain was paired with different light chains derived from 2a2. The pattern of somatic mutations in 2a2 was shown to be crucial in conferring the ability to bind DNA, but two different patterns of mutation each conferred this ability.Computer-generated models of the three-dimensional structures of these antibodies illustrate the ability of 2a2 to form a DNA binding site in different ways. Somatic mutations at the periphery of the DNA binding site were particularly important. In two different light chains, mutations to arginine at different sites in the complementarity determining regions (CDRs) enhanced binding to DNA. In a third light chain, however, mutation to arginine at a different site blocked binding to DNA.

Prediction of interaction partners for orphan nuclear receptors by prior-based protein sequence profiles.

We present a prior-based profile method for the prediction of protein-protein interaction partners that is here applied to the nuclear receptor superfamily. In this method, the diagnostic features are locally encoded in the physicochemical properties of residues in the interaction surface that are conserved in all proteins belonging to the defining set. The procedure models the positional variation based on that observed in the defining set and a prior-based substitution matrix derived from over 20,000 highly conserved positions in a set of 147 functional protein families. The method clusters sets of nuclear receptors known to interact with retinoid X receptor or corepressor proteins with predictive sets of receptors in C. elegans and higher metazoans. The method effectively reduces the search space of all possible interactions and yields experimentally testable predictions. Applications of this novel approach extend to interaction prediction problems in general, particularly to those that are not amenable to analysis by the rigid-body approximation.

The origin and age of haplochromine fishes in Lake Victoria, east Africa.

According to a widely held view, the more than 300 species of haplochromine cichlid fishes in Lake Victoria (LV), East Africa, originated from a single founder species in less than 12,000 years. This view, however, does not follow from the published geological and molecular evidence. The former does indeed suggest that the LV basin dried out less than 15,000 years ago, but it does not provide any information about the species that re-colonized the new lake or that remained in the rivers draining the area. The molecular evidence is inconclusive with respect to the origin of the LV haplochromines because cichlids from critical regions around LV were not adequately sampled; and as far as the age of the LV haplochromines is concerned, it in fact led to an estimate of 250,000-750,000 years old. In the present study, mitochondrial DNA (control region) variation was determined by heteroduplex and sequencing analyses of more than 670 specimens collected at widely distributed East African riverine and lacustrine localities. The analyses revealed the existence of seven haplogroups (I-VII) distinguishable by characteristic substitutions. All endemic LV samples tested fell into one of these haplogroups (V) which, however, was also found to be present at various other localities, both riverine and lacustrine, outside LV. Within this haplogroup, four subgroups (VA through VD) could be distinguished, two of which (VB and VC) were represented in LV and at other localities. The great majority of the LV haplochromine species could be classified as belonging to the VC subgroup, which was found only in LV and in the rivers draining into it. Hence, while the endemic haplochromine species of LV could not have originated from a single founding population, the lake does harbour a large species flock which probably arose in situ.

The PWWP domain: a potential protein-protein interaction domain in nuclear proteins influencing differentiation?

Upon characterization of WHSC1, a gene mapping to the Wolf-Hirschhorn syndrome critical region and at its C-terminus similar to the Drosophila ASH1/trithorax group proteins, we identified a novel protein domain designated PWWP domain. To gain insight into its structure, evolutionary conservation and its potential functional role, we performed database searches to identify other PWWP domain-containing proteins. We retrieved 39 proteins, and a multiple alignment shows that the domain spans some 70 amino acids. It is present in proteins of nuclear origin and plays a role in cell growth and differentiation. Due to its position, the composition of amino acids close to the PWWP motif and the pattern of other domains present, we hypothesize that the domain is involved in protein-protein interactions.

Isolation and sequencing of cDNA clones coding for the catalytic unit of glucose-6-phosphatase from two haplochromine cichlid fishes.

Complementary DNA clones coding for the catalytic unit of the enzyme glucose-6-phosphatase (G6Pase) were obtained from Haplochromis nubilus and Haplochromis xenognathus, two cichlid fish species from Lake Victoria. The translated sequence of these two cDNAs identifies a polypeptide consisting of 352 amino acid residues and showing a 54.4% similarity to the human form of G6Pase. The amino acid sequences of the two fish species are identical. The comparison of the fish amino acid sequence with the corresponding sequences of rat, mouse, and human G6Pase revealed that the amino acid residues, which are involved in G6Pase catalysis in humans, are also conserved in fish G6Pase. Northern blot analysis showed that G6Pase is expressed at the same level in 6- and 10-day-old fish. A three base pair insertion/deletion polymorphism was found in the 3'-untranslated region of the fish G6Pase gene. The polymorphism will be a useful marker in a phylogenetic study of Lake Victoria cichlids.

Evolutionary constraint networks in ligand-binding domains: an information-theoretic approach.

Ligand-binding sites in homologous protein domains can diverge greatly during evolution. This poses a particularly interesting problem in those cases where the ligand-binding site is situated in, or close to, the domain core, or where ligand-docking induces dramatic conformational changes. These features are present in many receptors and enzymes; the hormone-binding domain of the nuclear receptors for steroids and retinoids, for example, exhibits both characteristics. It is therefore of great interest to determine how binding sites for diverse ligands evolve in core regions of structurally dynamic domains. Are evolutionary changes locally restricted to the ligand-binding site, or are they distributed throughout the domain? We describe here an information-theoretic approach for the study of covariation between ligand-contacting residues and compensatory mutations that preserve the structural integrity and the conformational dynamics of ligand-binding domains. We apply this method to the analysis of the nuclear receptor ligand-binding domain and show that the ligand-contacting residues in the hormone-binding pocket are evolutionarily linked to an extensive network of covarying positions.

Persistence of neutral polymorphisms in Lake Victoria cichlid fish.

Phylogenetic trees for groups of closely related species often have different topologies, depending on the genes used. One explanation for the discordant topologies is the persistence of polymorphisms through the speciation phase, followed by differential fixation of alleles in the resulting species. The existence of transspecies polymorphisms has been documented for alleles maintained by balancing selection but not for neutral alleles. In the present study, transspecific persistence of neutral polymorphisms was tested in the endemic haplochromine species flock of Lake Victoria cichlid fish. Putative noncoding region polymorphisms were identified at four randomly selected nuclear loci and tested on a collection of 12 Lake Victoria species and their putative riverine ancestors. At all loci, the same polymorphism was found to be present in nearly all the tested species, both lacustrine and riverine. Different polymorphisms at these loci were found in cichlids of other East African lakes (Malawi and Tanganyika). The Lake Victoria polymorphisms must have therefore arisen after the flocks now inhabiting the three great lakes diverged from one another, but before the riverine ancestors of the Lake Victoria flock colonized the Lake. Calculations based on the mtDNA clock suggest that the polymorphisms have persisted for about 1.4 million years. To maintain neutral polymorphisms for such a long time, the population size must have remained large throughout the entire period.

v-erbA oncogene initiates ultrastructural changes characteristic of early and intermediate events of meiotic maturation in Xenopus oocytes.

The growth-promoting properties of the retroviral v-erbA oncogene, a highly mutated version of the chicken thyroid hormone receptor (TR) alpha, have so far exclusively been linked to dominant repression of the antimitogenic roles of TR and retinoic acid receptors. Here we show that when expressed in Xenopus oocytes v-ErbA induced ultrastructural changes characteristic of early and intermediate events of meiotic maturation by activating gene transcription. v-ErbA-induced maturation events occurred without activation of the cAMP/maturation-promoting factor signal pathway and were arrested prior to meiotic spindle formation. The effects of v-ErbA were not mimicked by a dominant negative in vitro-generated mutant of human TR, suggesting that v-ErbA can contribute to cell cycle reentry by interference with regulatory pathways distinct from those involving TR. Interestingly, a portion of v-ErbA expressed in oocytes was present at the cytoplasmic fibrils of the nuclear pore complexes, suggesting that in addition to its intranuclear function v-ErbA may modulate nucleocytoplasmic transport.

The SM 22 promoter directs tissue-specific expression in arterial but not in venous or visceral smooth muscle cells in transgenic mice.

The transcriptional signals underlying smooth muscle differentiation are currently unknown. We report here the complete sequence and characterization of the single mouse gene for the smooth muscle-specific protein SM 22 and the transcriptional activity of its promoter in cultured smooth muscle cells in vitro and in transgenic mice. In the transgenic animals, promoter constructs ranging in length from 445 to 2126 bp directed reporter expression initially in the heart and the somites of embryos and subsequently in the arteries of the vascular system, but in none of the visceral muscles, nor in the veins. Expression in the heart was spatially restricted to the presumptive right ventricle and outflow tract and disappeared in the adult. Likewise, expression in the somites was only transitory and was not observed after about 14.5 days post coitum in the embryo. In the adult mouse, SM 22 promoter activity persisted in the smooth muscle cells of the arteries and was still notably absent from other smooth muscles, despite the ubiquitous presence of the endogenous SM 22 protein. These findings on the transcriptional activity of a smooth muscle promoter in vivo reveal the existence of different differentiation programmes for smooth muscle cells in the veins and the arteries and raise the expectation of a further subdivision of programmes among the visceral muscles.

Constitutive transactivation by the thyroid hormone receptor and a novel pattern of activity of its oncogenic homolog v-ErbA in Xenopus oocytes.

In Xenopus oocytes, the rat thyroid hormone receptor alpha (rTR alpha), but not its oncogenic homolog v-ErbA, constitutively activated thyroid hormone (T3)-responsive reporter genes at four positive thyroid hormone-responsive elements (TREs). At a subset of the positive TREs tested, the addition of T3 resulted in a further enhancement of reporter gene activation. In contrast, both rTR alpha and v-ErbA functioned as constitutive activators when bound to the clone 122 TREs, which are induced by unliganded TR in mammalian cells. Therefore, the responses of the ligand-independent activation domains of TR and v-ErbA to cell-specific and TRE-mediated induction are not equivalent. Coexpression of the human retinoid X receptor alpha (hRXR alpha) enhanced both ligand-dependent and ligand-independent activation functions of rTR alpha and human TR beta (hTR beta) at a palindromic TRE (TREp). An endogenous TR activity mediated T3 induction of TREp, while being repressed by an in vitro-generated dominant negative mutant of TR. T3-mediated gene activation, by exogenous or endogenous TR, was repressed by v-ErbA at three positive TREs, but not at the TRE from the third intron of the rat GH gene (rGH3 TRE). Interestingly, preinjection of nuclear protein extract from anterior pituitary cells converted v-ErbA into a constitutive activator at rGH3 TRE. The pituitary-specific factor Pit-1/GHF-1 or hRXR alpha did not induce activation by v-ErbA at rGH3 TRE, suggesting that the dominant negative phenotype of v-ErbA can be abolished by direct or indirect interactions with other nuclear factors.

Stereoselective electrophysiological effects of propafenone in Langendorff perfused guinea pig hearts.

The present study was focused on the stereoselective electrophysiological effects of (R)- and (S)-propafenone.HCl evaluated in isolated Langendorff perfused guinea pig hearts. Conduction intervals were measured using an ECG-recording method of high resolution. Refractory periods of the different parts of the myocardium were determined by stimulation with premature stimuli, as well as by stimulation with increasing pacing rate (rate-dependent/refractory periods). Drug concentrations of 0.1, 1 and 3 microM were tested. Both compounds induced a dose-dependent increase in AV-nodal, His-bundle, and intraventricular conduction time which reached significance (p less than 0.01) following 3 microM of either compound. Sinus rate was also dose-dependently and significantly reduced. (R)- and (S)-propafenone.HCl induced a marked prolongation of the rate-dependent refractory period of sino-atrial (by 140 +/- 22%, p less than 0.01 and by 141 +/- 14%, p less than 0.01, respectively) and AV-nodal (by 34 +/- 22%, p less than 0.01 and by 42 +/- 15%, p less than 0.01, respectively) conduction and of the atrial (by 182 +/- 21%, p less than 0.01 and by 195 +/- 15%, p less than 0.01, respectively) and ventricular (by 93 +/- 16%, p less than 0.01 and by 88 +/- 16%, p less than 0.01, respectively) myocardium. The effective refractory periods evaluated by stimulation with premature stimuli were also significantly prolonged under the influence of (R)- and (S)-propafenone.HCl, except the ventricular myocardial refractoriness by (R)-propafenone.HCl (increase to 114 +/- 23%, n.s.). Both compounds showed a strong rate-dependence of their effects and, thus, the refractory periods evaluated by stimulation with increasing pacing rate were significantly more prolonged than the refractory periods evaluated by stimulation with premature stimuli. The main difference between the effects of (R)- and (S)-propafenone.HCl on the cardiac electrical activity is the lack of effect of (R)-propafenone.HCl on the ventricular myocardial refractoriness evaluated by stimulation with premature stimuli.

Acute effects of the ACE inhibitor lisinopril on cardiac electrophysiological parameters of isolated guinea pig hearts.

Angiotensin-converting enzyme (ACE) inhibitors are of benefit in life-threatening ventricular arrhythmias in patients with congestive heart failure and ventricular tachycardia caused by the onset of myocardial ischemia as well as by reperfusion of an ischemic area. The aim of the present study was to investigate whether direct electrophysiological effects are responsible for these observations. Therefore, we investigated the electrophysiological effects of lisinopril on the whole cardiac conduction and pacemaker system in isolated guinea pig hearts perfused by the method of Langendorff at concentrations of 0.01, 0.1, 1, and 10 microM. Lisinopril did not affect heart rate, atrioventricular, His bundle, or intraventricular conduction at any of the concentrations tested. Likewise the frequency-dependent QT duration was unchanged. At a concentration of 10 microM, lisinopril prolonged effective refractory period evaluated by premature beats (46 +/- 15%, n = 8, p less than 0.01) as well as the rate-dependent effective refractory period (31 +/- 12, n = 8, p less than 0.01) of the atrioventricular conduction. These effects can be explained by lisinopril's action as a minor calcium antagonist at a toxic concentration of 10 microM. The present results show that electrophysiological parameters are not substantially altered by lisinopril. Therefore, several other mechanisms such as the unloading of the left ventricle and/or the suppression of noradrenalin release and the electrolyte (potassium and magnesium) repletion and/or regression of left ventricular hypertrophy as long-term effects may play a major role in the antiarrhythmic efficacy of ACE inhibitors.

Comparison of acute electrophysiological effects of amiodarone and its metabolite desethylamiodarone in Langendorff perfused guinea pig hearts.

During long-term treatment with amiodarone, slowing of conduction through the atrioventricular node, a prolongation of the QT-interval, and a prolongation of the atrial and ventricular myocardial refractoriness always developed. During short-term treatment, these effects were not found, except for depression of the AV-nodal conduction. This led to the suggestion that the electrophysiological effects of amiodarone during long-term treatment might be partly the result of the accumulation of its metabolite desethylamiodarone. Therefore, we examined the electrophysiological effects of amiodarone and desethylamiodarone on conduction and refractoriness in isolated spontaneously beating guinea pig hearts perfused by the method of Langendorff. Within 1 h of perfusion, desethylamiodarone caused a more pronounced prolongation of the AV-nodal, His-bundle, and intraventricular conduction intervals than did amiodarone. Desethylamiodarone, but not amiodarone led to a prolongation of the QT-interval. The refractoriness of sinoatrial-, AV-nodal conduction, and of the atrial myocardium were significantly more prolonged by amiodarone than by desethylamiodarone. Both compounds showed a comparable strong rate-dependent effect on AV-nodal refractoriness. The ventricular refractoriness was similarily prolonged by either compound. These results show that for the class-III effects (i.e., prolongation of repolarization period) observed under chronic treatment of amiodarone the metabolite desethylamiodarone may be responsible. Desethylamiodarone also exerts more pronounced effects on the fast-channel-dependent parts of the conduction system than does amiodarone, a fact indicated by a higher prolongation of His-bundle and intraventricular conduction.