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We present a digoxin-clarithromycin interaction in two patients in whom digoxin concentrations were unexpectedly increased. The ratio of renal digoxin clearance to creatinine clearance in one patient was lower during the concomitant administration of clarithromycin (0.64 and 0.73) than that after cessation of clarithromycin administration (1.30 +/- 0.20; mean +/- SD). Because P-glycoprotein could play an important role in the renal secretion of digoxin, we hypothesized that clarithromycin decreases renal digoxin excretion by inhibiting P-glycoprotein-mediated transport. Digoxin transport was evaluated with use of a kidney epithelial cell line, which expresses the human P-glycoprotein on the apical membrane by transfection with MDR1 complementary deoxyribonucleic acid. Clarithromycin inhibited the transcellular transport of digoxin from the basolateral to the apical side in a concentration-dependent manner and concomitantly increased the cellular accumulation of digoxin. These results suggest that clarithromycin may inhibit the P-glycoprotein-mediated tubular secretion of digoxin, and this interaction mechanism may contribute to an increase in the serum digoxin concentration.

1. Single axons of pontine nucleus neurons (PN axons) receiving cerebral input were stained intra-axonally with horseradish peroxidase (HRP) in the cerebellum of cats. The axonal trajectory of single PN axons was reconstructed from serial sections of the cerebellum and the brain stem. 2. Axons were penetrated in the white matter near the dentate nucleus, and, after electrophysiological identification, PN axons were injected iontophoretically with HRP. The identification criteria for the PN axons were 1) their direct responses to stimulation of the contralateral pontine nucleus (PN), 2) their synaptic activation from the contralateral cerebral cortex, and 3) the decrease in threshold for evoking direct spikes in stimulation of the PN by conditioning stimuli applied in the cerebral cortex. 3. Two hundred thirty-three axons were electrophysiologically identified as PN axons receiving the input from the cerebral cortex. Ninety-six of them were stained successfully with HRP, and reconstructions were made from 40 well-stained PN axons. All of them gave rise to mossy fibers and terminated in the granular layer of the cerebellar cortex as typical mossy fiber rosettes. Out of these, 22 gave axon collaterals to the dentate nucleus. Virtually all of the axon branches observed in the dentate nucleus were axon collaterals of mossy fibers from the PN to the cerebellar cortex. In 7 of these 22 PN axons, cell bodies were retrogradely labeled with HRP, and all of them were found in the contralateral PN. 4. The stained-stem axons arising from the PN ran medially in the pons, crossed the midline, and then ascended dorsocaudally in the branchium pontis. After passing in the white matter anterior to or lateral to the dentate nucleus, they entered into the cerebellar cortex. On their way, one to three axon collaterals were given off from parent axons to the dentate nucleus. The diameter of these collaterals was very thin (mean, 0.6 microns), compared with the large diameter of the parent axons (mean, 2.1 microns). 5. Some axon collaterals were very simple and had only one terminal branch with or without short branchlets, whereas others were more complex, and single axon collaterals ramified before forming a terminal arborization. Axon collaterals of single PN axons mainly spread mediolaterally or dorsoventrally in the frontal plane but had a very narrow rostrocaudal extension. 6. Terminal branches usually bore swellings en passant along their length and one terminal swelling at their end. The number of swellings per axon collateral ranged 23-180 (116 +/- 52, mean +/- SD).(ABSTRACT TRUNCATED AT 400 WORDS)

We describe here the construction of a library of small interfering RNA expression vectors targeted to a few hundred apoptosis-related genes and the application of this library to an investigation of thapsigargin (TG)-induced apoptosis. Thapsigargin triggers endoplasmic reticulum stress, with subsequent apoptosis, but the molecular mechanisms underlying this process are incompletely understood. Using our library, we identified three anti-apoptotic genes, namely, NOXA, E2F1, and MAPK1, in addition to already characterized genes in the apoptotic pathway. In contrast to proposals by others, our data revealed (i) that TG-induced apoptosis is associated with Apaf1 in a caspase-3-and caspase-9-independent manner; (ii) that the E2F1-PUMA pathway might be involved; and (iii) that the ERK pathway, via MAP3K8 (mitogen-activated protein kinase kinase 8), is required for the induction by TG of apoptosis. Our study demonstrates clearly that unexpected and novel genes can be identified effectively by our method, and it provides evidence for the efficacy and utility of the comprehensive analysis of signaling networks and pathways using a library of small interfering RNA expression vectors.Although the human genome has been sequenced, the functions of many genes remain unknown. Phenotypic analysis using gene silencing appears to be an effective strategy in efforts to understand gene functions, and exploitation of RNA interference (RNAi) 1 appears to be a novel and powerful approach to the silencing of specific genes (1). RNAi is an intrinsic and evolutionarily conserved phenomenon in plants and animals whereby double-stranded RNA induces the sequence-specific degradation of homologous RNA (2).Genome-wide RNAi screening in Caenorhabditis elegans and Drosophila cells, using libraries of double-stranded RNAs, has been demonstrated to be extremely useful in efforts to identify genes that regulate various processes and has enhanced our understanding of various biological processes (3-6). However, two major problems are associated with such screening in mammalian cells. First, double-stranded RNA induces an antiviral response that can result in cell death (7). However, Tuschl and co-workers (8) demonstrated that 21-and 22-nucleotide small interfering RNAs (siRNAs) can induce RNAi in the absence of an antiviral response in cultured mammalian cells. Subsequently, various groups, including our own, have developed systems for vector-mediated specific RNAi in mammalian cells (9 -16). The second problem is that the effectiveness of siRNA is strongly dependent on the target sites in target RNAs. To generate a high quality library, we developed an original algorithm that allows us to predict favorable target sites (17). Using this algorithm and our optimized siRNA expression system (17), we are now constructing an siRNA expression library that will encompass the complete array of transcripts from the human genome. In this study, for construction of our first siRNA library, we focused on an apoptotic pathway and generated a library targeted t...

The intraspinal morphology of single lateral vestibulospinal tract (LVST) axons was investigated with the method of intra-axonal staining with horseradish peroxidase (HRP) and three-dimensional reconstruction of the axonal trajectory. Axons penetrated in the ventral funiculus at C5-C8 were identified as LVST axons by their monosynaptic responses to stimulation of the ipsilateral vestibular nerve and by their direct responses to stimulation of the ipsilateral Deiters' nucleus and LVST. Reconstructions were made from 34 well-stained LVST axons. Of these, 23 terminated in the brachial segments (C5-Th1) and the other 11 projected below Th2. These axons were traced over distances of 2.9-16.3 mm rostrocaudally. Within these lengths, one to seven axon collaterals (mean +/- S.D., 3.2 +/- 2.0, N = 19) were given off at right angles from the stem axons of LVST axons terminating in the brachial segments. The mean diameters of stem axons and primary collaterals were 4.5 microns and 1.6 micron, respectively. In the gray matter, collaterals ramified successively, pursued a delta-like path, and terminated mainly in laminae VII and VIII or lamina IX. The rostrocaudal extension of a single collateral was very restricted (mean +/- S.D., 760 +/- 220 microns, N = 16), in contrast to the extensive dorsoventral and mediolateral extent of the terminal arborization. There were usually gaps between adjacent collateral arborizations from the same stem axons, since the intercollateral distances ranged from 400 to 4,300 microns (mean = 1,490 microns). LVST axons terminating in brachial segments were divided into two groups--a medial group and a lateral group--on the basis of their projection sites in the transverse plane of the gray matter. The axons of the medial type had their main projection to laminae VII and VIII of Rexed, while those of the lateral type terminated in lamina IX. The terminal arborizations of the medial type LVST axons were mainly distributed over lamina VIII, where synaptic boutons appeared to make contact with proximal dendrites or somata of medium-sized and large neurons in the ventromedial nucleus and also in the medial portion of lamina VII adjacent to the central canal and dorsal to lamina VIII. Five out of 15 medial type axons had a bilateral projection. One or two collaterals of each of these axons crossed the midline through the anterior commissure and terminated in lamina VII or VIII. It was concluded that the contralateral projection was sparse.(ABSTRACT TRUNCATED AT 400 WORDS)

This study provides a useful design concept for the development and introduction of patient-specific navigational templates for placing PSs.

To reveal patterns of input from the six semicircular canals to motoneurons of various neck muscles and their relationship to the mechanical actions of individual neck muscles, patterns of input to neck motoneurons of the longissimus and the semispinalis muscle groups were investigated in the upper cervical spinal cord of anesthetized cats. Intracellular potentials were recorded from motoneurons of the longissimus muscle group (obliquus capitis superior muscle, OCS; splenius muscle, SPL; longissimus muscle, LONG) and the semispinalis muscle group (biventer cervicis muscle, BIV; complexus muscle, COMP), and effects of separate electrical stimulation of the six ampullary nerves on them were analyzed in each preparation. Neck motoneurons usually received convergent inputs from all of the six ampullary nerves, and motoneurons that supplied a particular muscle had a homogeneous pattern of input from the six ampullary nerves. Two different patterns of input were identified for motoneurons of these two muscle groups; one pattern for motoneurons of the longissimus muscle group and the other pattern for motoneurons of the semispinalis muscle group. Motoneurons of the OCS, the SPL, and the LONG muscles received excitation from the three contralateral ampullary nerves and inhibition from the three ipsilateral ampullary nerves. BIV and COMP motoneurons received excitation from the bilateral anterior canal nerves (ACNs) and the contralateral canal nerve (LCN) and inhibition from the bilateral posterior canal nerves (PCNs) and the ipsilateral LCN. Latencies of postsynaptic potentials (PSPs) evoked by stimulation of each of the six ampullary nerves indicated that the earliest component of excitatory PSPs (EPSPs) and inhibitory PSPs (IPSPs) was disynaptic in these motoneurons. However, trisynaptic IPSPs were evoked by stimulation of the contralateral PCN in a considerable number of BIV and COMP motoneurons. In OCS, SPL, and LONG motoneurons, all of the excitation from the contralateral and all of the inhibition from the ipsilateral ampullary nerves were mediated through the ipsilateral medial longitudinal fascicle (MLF). In BIV and COMP motoneurons, disynaptic excitation from the contralateral ACN and LCN and disynaptic inhibition from the ipsilateral LCN and bilateral PCNs were mediated through the ipsilateral MLF, whereas disynaptic excitation from the ipsilateral ACN was mediated through the ipsilateral lateral vestibulospinal tract. The patterns of semicircular canal input to neck motoneurons of these two muscle groups are related closely to the mechanical actions of the individual neck muscles and the optimal stimulus to the semicircular canals such that the connections will tend to stabilize head positions in response to head perturbations.