Chromosomal localization of three genes coamplified in the multidrug-resistant CHRC5 Chinese hamster ovary cell line

At least five gene classes are amplified in the multidrug-resistant CHO cell line CHRC5. Protein products have been identified for two classes; class 2 codes for the large membrane P-glycoprotein, whereas class 4 encodes the small cytoplasmic calcium-binding protein sorcin (V19). By DNA analysis we have shown previously that these five genes are linked in two groups: class 1 + 2 + 3; and class 4 + 5. By use of in situ hybridization with complementary DNAs derived from the resistant cell line we demonstrate here that genes from both linkage groups are amplified and situated together in each of two different chromosomal regions of the resistant Chinese hamster cell line. The positions of the amplicons correspond to cytogenetically identified homogeneously staining regions in an altered 7q+ chromosome and in a rearranged Z-7 [t(3;4)] chromosome. The native genes were mapped both in the CHRC5 line and in a normal diploid Chinese hamster cell strain, CHNF 86. We confirm the position of the class 2 gene on 1q26 and we show that class 4 and 5 genes are located in the same region of 1q. We conclude that the gene classes 2, 4, and 5 are closely juxtaposed in the normal Chinese hamster genome and comprise one amplicon in resistant cells. Our results are compatible with the hypothesis that multidrug resistance is due to overexpression of P-glycoprotein genes and that the other genes amplified in the CHRC5 line are coamplified because they happen to lie close to the P-glycoprotein genes.     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevis.

Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (approximately 62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals.     

Assessing the spatiotemporal evolution of neuronal activation with single-trial event-related potentials and functional MRI

The brain acts as an integrated information processing system, which methods in cognitive neuroscience have so far depicted in a fragmented fashion. Here, we propose a simple and robust way to integrate functional MRI (fMRI) with single trial event-related potentials (ERP) to provide a more complete spatiotemporal characterization of evoked responses in the human brain. The idea behind the approach is to find brain regions whose fMRI responses can be predicted by paradigm-induced amplitude modulations of simultaneously acquired single trial ERPs. The method was used to study a variant of a two-stimulus auditory target detection (odd-ball) paradigm that manipulated predictability through alternations of stimulus sequences with random or regular target-to-target intervals. In addition to electrophysiologic and hemodynamic evoked responses to auditory targets per se, single-trial modulations were expressed during the latencies of the P2 (170-ms), N2 (200-ms), and P3 (320-ms) components and predicted spatially separated fMRI activation patterns. These spatiotemporal matches, i.e., the prediction of hemodynamic activation by time-variant information from single trial ERPs, permit inferences about regional responses using fMRI with the temporal resolution provided by electrophysiology.     

Phase dependency of electrotonic spread of hyperpolarizing current pulses in the rabbit sinoatrial node

Electrotonic current spread in the SA node of the rabbit was measured by means of hyperpolarizing current pulses (1 to 10 microA, 60 ms), which were injected intracellularly through a K(+)-perfused suction electrode. The pulses were applied at the beginning, middle or end of the diastolic depolarization phase. The resulting membrane potential change of nodal fibers was measured with microelectrodes. Space constants were calculated by fitting single exponential curves to the data. The input resistance (Rin) of fibers at different sites in the SA node was measured by means of a double barrel microelectrode (current pulses 5.5 to 11 nA, 60 ms) to detect a change in the internal resistance during the diastolic depolarization phase. During diastole the average electrotonic potential increased by 30% (P less than 0.001), the increase of the space constant ranged from 9 to 183% (P less than 0.05). Rin however, did not change during diastole. It is concluded that the electrotonic spread increased phase dependently, due to an increase of membrane resistance; the internal resistance was not phase dependent.     

Remodeling of gap junctions in mouse hearts hypertrophied by forced retinoic acid signaling

BACKGROUND: Beta-MHC-hRARalpha transgenic mice express a constitutively active (truncated) form of the human retinoic acid receptor which triggers development of dilated cardiomyopathy. In those hearts, we studied expression of gap junction proteins in relation to electrical impulse propagation. METHODS AND RESULTS: As compared to wildtype mice, hearts of 4-6 month old mice with 7-12 inserted hRARalpha copies are marked by an increased heart weight/body weight- and heart weight/tibia length ratio. 3-extremity lead ECGs revealed prolongation of the Q-j interval suggesting delayed ventricular activation. Mapping of electrical activity of epi- and endocardial left ventricular free wall revealed activation delay, increased heterogeneity in conduction and regional conduction block. Ventricular tachycardias did not occur spontaneously nor could be induced by ventricular pacing. Immunohistochemical analysis showed profound and heterogeneous redistribution and down-regulation of the gap junction protein connexin43 (Cx43) in the left ventricular free wall. Here, hRARalpha expression induced re-expression of the hypertrophic markers alpha-skeletal actin and beta-MHC, and in 3 out of 10 severely affected mice, re-expression of Cx40. Concomitant with changes in expression/distribution of Cx43, changes in expression and distribution of beta-catenin and N-cadherin (two other intercalated disk associated proteins) were observed. CONCLUSIONS: Beta-MHC-hRARalpha transgenic hearts show heterogeneous re-expression of (early) sarcomeric genes while expression of connexin43, N-cadherin and beta-catenin is down-regulated. We postulate that the resulting aberrant ventricular activation does not trigger development of lethal arrhythmias due to the small size of remaining healthy ventricular tissue where the transgene is not expressed.     

Regulation of myocardial connexins during hypertrophic remodelling.

Cardiac hypertrophic remodelling, initiated by signalling cascades in response to increased workload, injury or intrinsic disease, is initially adaptive. However, prolonged hypertrophy as a consequence of pathological stress leads to maladaptive changes that increase the risk for fatal ventricular arrhythmias. One of these changes is the remodelling of myocardial gap junctions, which provide for electrical coupling of adjacent cardiomyocytes. Myocardial gap junctions are composed of three connexin isotypes, connexin40 (Cx40), -43 (Cx43), and -45 (Cx45) and each display a characteristic developmental and regional expression pattern. Alterations in the distribution and expression of Cx43, the predominant isoform in the adult ventricles, has been the main focus of examination in humans, experimental animal models and cultured cardiomyocytes in response to hypertrophy. The molecular mechanisms and signalling pathways underlying these changes have been studied less thoroughly. In this review we summarize what is known about the remodelling of myocardial gap junctions during hypertrophy, the putative underlying mechanisms and functional consequences thereof.     

Effect of transmural vagal stimulation on electrotonic current spread in the rabbit sinoatrial node.

OBJECTIVE: The effect of vagal stimulation on the decay of electrotonic potential caused by intracellular current injection and on input resistance was measured in the sinoatrial node of isolated rabbit right atria. METHODS: Studies were performed on New Zealand White rabbits weighing approximately 2-3 kg. Vagal stimulation was achieved by transmural stimulation of intramural nerve fibres in the presence of propranolol. A K+ perfused suction electrode was used to inject hyperpolarising current pulses; input resistance was measured by means of a double barrel microelectrode. RESULTS: Vagal stimulation which caused a 14-20% increase of cycle length diminished electronic potential significantly by a decrease of membrane resistance. The input resistance of the sinoatrial node was not affected. Space constant values calculated by using either a one or a two dimensional model of electrotonic current spread were decreased on average by 13% and 14% respectively. CONCLUSIONS: The results from this study show that vagal stimulation which gave rise to a moderate negative chronotropic effect and marked changes in action potential configuration of nodal fibres affects the electrotonic interaction within the sinoatrial node. This may have consequences for the electrical activity and synchronisation of the sinoatrial nodal fibres.     

Rhythm perception: Speeding up or slowing down affects different subcomponents of the ERP P3 complex

The aim of this study was to investigate, by measuring the event related potential (ERP) P3 complex, whether the perception of small accelerations differs from that of small decelerations. Participants had to decide whether the last beat of a short sequence was presented 'too early' or 'too late'. Target beats were accelerated or decelerated with 0%, 2%, 5%, or 10%. Individuals differed in their capability to detect small tempo changes. We found that good responders were able to identify all tempo changes whereas poor responders were only able to identify large (10%) tempo changes. In addition, we found that tempo changes affected two subcomponents of the ERP P3 in good performers. Accelerations increased a late-P3 amplitude whereas decelerations increased an early-P3 amplitude. These results imply the principle possibility to measure differential P3 effects within one task. This is important for acquiring more refined knowledge concerning different subcomponents of the ERP P3 complex and the cognitive processes by which they are elicited.