Clinical and metabolic effects associated with weight changes and obeticholic acid in non-alcoholic steatohepatitis.

BACKGROUND: In a 72-week, randomised controlled trial of obeticholic acid (OCA) in non-alcoholic steatohepatitis (NASH), OCA was superior to placebo in improving serum ALT levels and liver histology. OCA therapy also reduced weight. AIMS: Because weight loss by itself can improve histology, to perform a post hoc analysis of the effects of weight loss and OCA treatment in improving clinical and metabolic features of NASH. METHODS: The analysis was limited to the 200 patients with baseline and end-of-treatment liver biopsies. Weight loss was defined as a relative decline from baseline of 2% or more at treatment end. RESULTS: Weight loss occurred in 44% (45/102) of OCA and 32% (31/98) of placebo-treated patients (P = 0.08). The NAFLD Activity score (NAS) improved more in those with than without weight loss in both the OCA- (-2.4 vs -1.2, P<0.001) and placebo-treated patients (-1.2 vs -0.5, P = 0.03). ALT levels also improved in those with vs without weight loss in OCA- (-43 vs -34 U/L, P = 0.12) and placebo-treated patients (-29 vs -10 U/L, P = 0.02). However, among those who lost weight, OCA was associated with opposite effects from placebo on changes in alkaline phosphatase (+21 vs -12 U/L, P<0.001), total (+13 vs -14 mg/dL, P = 0.02) and LDL cholesterol (+18 vs -12 mg/dL, P = 0.01), and HbA1c (+0.1 vs -0.4%, P = 0.01). CONCLUSIONS: OCA leads to weight loss in up to 44% of patients with NASH, and OCA therapy and weight loss have additive benefits on serum aminotransferases and histology. However, favourable effects of weight loss on alkaline phosphatase, lipids and blood glucose seen in placebo-treated patients were absent or reversed on OCA treatment. These findings stress the importance of assessing concomitant metabolic effects of new therapies of NASH. Clinical trial number: NCT01265498.

Regulation of bone mineral density in the grey squirrel, Sciurus carolinensis: Bioavailability of calcium oxalate, and implications for bark stripping.

The damage caused when grey squirrels strip the outer bark off trees and ingest the underlying phloem can result in reduced timber quality or tree death. This is extremely costly to the UK forestry industry and can alter woodland composition, hampering conservation efforts. The calcium hypothesis proposes that grey squirrels ingest phloem to ameliorate a seasonal calcium deficiency. Calcium in the phloem predominantly takes the form of calcium oxalate (CaOx), however not all mammals can utilise CaOx as a source of calcium. Here, we present the results of a small-scale study to determine the extent to which grey squirrels can utilise CaOx. One of three custom-made diets containing calcium in varying forms and quantities (CaOx diet, Low-calcium carbonate (CaCO3 ) diet and Control diet) were fed to three treatment groups of six squirrels for 8 weeks. Bone densitometric properties were measured at the end of this time using peripheral quantitative computed tomography and micro-computed tomography. Pyridinoline-a serum marker of bone resorption-was measured regularly throughout the study. Bone mineral density and cortical mineralisation were lower in squirrels fed the CaOx diet compared to the Control group but similar to that of those on the Low-calcium diet, suggesting that calcium from calcium oxalate was not effectively utilised to maintain bone mineralisation. Whilst no differences were observed in serum pyridinoline levels between individuals on different diets, females had on average higher levels than males throughout the study. Future work should seek to determine if this apparent lack of ability to utilise CaOx is common to a large sample of grey squirrels and if so, whether it is consistent across all areas and seasons.

Perilymph pharmacokinetics of locally-applied gentamicin in the guinea pig.

Intratympanic gentamicin therapy is widely used clinically to suppress the vestibular symptoms of Meniere's disease. Dosing in humans was empirically established and we still know remarkably little about where gentamicin enters the inner ear, where it reaches in the inner ear and what time course it follows after local applications. In this study, gentamicin was applied to the round window niche as a 20 µL bolus of 40 mg/ml solution. Ten 2 µL samples of perilymph were collected sequentially from the lateral semi-circular canal (LSCC) at times from 1 to 4 h after application. Gentamicin concentration was typically highest in samples originating from the vestibule and was lower in samples originating from scala tympani. To interpret these results, perilymph elimination kinetics for gentamicin was quantified by loading the entire perilymph space by injection at the LSCC with a 500 µg/ml gentamicin solution followed by sequential perilymph sampling from the LSCC after different delay times. This allowed concentration decline in perilymph to be followed with time. Gentamicin was retained well in scala vestibuli and the vestibule but declined rapidly at the base of scala tympani, dominated by interactions of perilymph with CSF, as reported for other substances. Quantitative analysis, taking into account perilymph kinetics for gentamicin, showed that more gentamicin entered at the round window membrane (57%) than at the stapes (35%) but the lower concentrations found in scala tympani were due to greater losses there. The gentamicin levels found in perilymph of the vestibule, which are higher than would be expected from round window entry alone, undoubtedly contribute to the vestibulotoxic effects of the drug. Furthermore, calculations of gentamicin distribution following targeted applications to the RW or stapes are more consistent with cochleotoxicity depending on the gentamicin concentration in scala vestibuli rather than that in scala tympani.

Perilymph Kinetics of FITC-Dextran Reveals Homeostasis Dominated by the Cochlear Aqueduct and Cerebrospinal Fluid.

Understanding how drugs are distributed in perilymph following local applications is important as local drug therapies are increasingly used to treat disorders of the inner ear. The potential contribution of cerebrospinal fluid (CSF) entry to perilymph homeostasis has been controversial for over half a century, largely due to artifactual contamination of collected perilymph samples with CSF. Measures of perilymph flow and of drug distribution following round window niche applications have both suggested a slow, apically directed flow occurs along scala tympani (ST) in the normal, sealed cochlea. In the present study, we have used fluorescein isothiocyanate-dextran as a marker to study perilymph kinetics in guinea pigs. Dextran is lost from perilymph more slowly than other substances so far quantified. Dextran solutions were injected from pipettes sealed into the lateral semicircular canal (SCC), the cochlear apex, or the basal turn of ST. After varying delays, sequential perilymph samples were taken from the cochlear apex or lateral SCC, allowing dextran distribution along the perilymphatic spaces to be quantified. Variability was low and findings were consistent with the injection procedure driving volume flow towards the cochlear aqueduct, and with volume flow during perilymph sampling driven by CSF entry at the aqueduct. The decline of dextran with time in the period between injection and sampling was consistent with both a slow volume influx of CSF (~30 nL/min) entering the basal turn of ST at the cochlear aqueduct and a CSF-perilymph exchange driven by pressure-driven fluid oscillation across the cochlear aqueduct. Sample data also allowed contributions of other processes, such as communications with adjacent compartments, to be quantified. The study demonstrates that drug kinetics in the basal turn of ST is complex and is influenced by a considerable number of interacting processes.

The auditory nerve overlapped waveform (ANOW) originates in the cochlear apex.

Measurements of cochlear function with compound action potentials (CAPs), auditory brainstem responses, and otoacoustic emissions work well with high-frequency sounds but are problematic at low frequencies. We have recently shown that the auditory nerve overlapped waveform (ANOW) can objectively quantify low-frequency (<1 kHz) auditory sensitivity, as thresholds for ANOW at low frequencies and for CAP at high frequencies relate similarly to single auditory nerve fiber thresholds. This favorable relationship, however, does not necessarily mean that ANOW originates from auditory nerve fibers innervating low-frequency regions of the cochlear apex. In the present study, we recorded the cochlear response to tone bursts of low frequency (353, 500, and 707 Hz) and high frequency (2 to 16 kHz) during administration of tetrodotoxin (TTX) to block neural function. TTX was injected using a novel method of slow administration from a pipette sealed into the cochlear apex, allowing real-time measurements of systematic neural blocking from apex to base. The amplitude of phase-locked (ANOW) and onset (CAP) neural firing to moderate-level, low-frequency sounds were markedly suppressed before thresholds and responses to moderate-level, high-frequency sounds were affected. These results demonstrate that the ANOW originates from responses of auditory nerve fibers innervating cochlear apex, confirming that ANOW provides a valid physiological measure of low-frequency auditory nerve function.

Adding liquid feed to a total mixed ration reduces feed sorting behavior and improves productivity of lactating dairy cows.

This study was designed to determine the effect of adding a molasses-based liquid feed (LF) supplement to a total mixed ration (TMR) on the feed sorting behavior and production of dairy cows. Twelve lactating Holstein cows (88.2±19.5 DIM) were exposed, in a crossover design with 21-d periods, to each of 2 treatment diets: 1) control TMR and 2) control TMR with 4.1% dietary dry matter LF added. Dry matter intake (DMI), sorting, and milk yield were recorded for the last 7 d of each treatment period. Milk samples were collected for composition analysis for the last 3 d of each treatment period; these data were used to calculate 4% fat-corrected milk and energy-corrected milk yield. Sorting was determined by subjecting fresh feed and orts samples to particle separation and expressing the actual intake of each particle fraction as a percentage of the predicted intake of that fraction. Addition of LF did not noticeably change the nutrient composition of the ration, with the exception of an expected increase in dietary sugar concentration (from 4.0 to 5.4%). Liquid feed supplementation affected the particle size distribution of the ration, resulting in a lesser amount of short and a greater amount of fine particles. Cows sorted against the longest ration particles on both treatment diets; the extent of this sorting was greater on the control diet (55.0 vs. 68.8%). Dry matter intake was 1.4 kg/d higher when cows were fed the LF diet as compared with the control diet, resulting in higher acid-detergent fiber, neutral-detergent fiber, and sugar intakes. As a result of the increased DMI, cows tended to produce 1.9 kg/d more milk and produced 3.1 and 3.2 kg/d more 4% fat-corrected milk and energy-corrected milk, respectively, on the LF diet. As a result, cows tended to produce more milk fat (0.13 kg/d) and produced more milk protein (0.09 kg/d) on the LF diet. No difference between treatments was observed in the efficiency of milk production. Overall, adding a molasses-based LF to TMR can be used to decrease feed sorting, enhance DMI, and improve milk yield.

Pro-neural miR-128 is a glioma tumor suppressor that targets mitogenic kinases.

MicroRNAs (miRNAs) carry out post-transcriptional control of a multitude of cellular processes. Aberrant expression of miRNA can lead to diseases, including cancer. Gliomas are aggressive brain tumors that are thought to arise from transformed glioma-initiating neural stem cells (giNSCs). With the use of giNSCs and human glioblastoma cells, we investigated the function of miRNAs in gliomas. We identified pro-neuronal miR-128 as a candidate glioma tumor suppressor miRNA. Decreased expression of miR-128 correlates with aggressive human glioma subtypes. With a combination of molecular, cellular and in vivo approaches, we characterize miR-128's tumor suppressive role. miR-128 represses giNSC growth by enhancing neuronal differentiation. miR-128 represses growth and mediates differentiation by targeting oncogenic receptor tyrosine kinases (RTKs) epithelial growth factor receptor and platelet-derived growth factor receptor-α. Using an autochthonous glioma mouse model, we demonstrated that miR-128 repressed gliomagenesis. We identified miR-128 as a glioma tumor suppressor that targets RTK signaling to repress giNSC self-renewal and enhance differentiation.

Relationship between diet and liver carcinomas in roe deer in Kielder Forest and Galloway Forest.

The winter diets of roe deer culled from Kielder Forest, in north-east England, where the incidence of liver carcinomas in roe deer is high, and Galloway Forest, in south-west Scotland, where the incidence of liver carcinomas is low, were compared by microhistological analysis of faeces. Both areas are planted with spruce forests but the diets of the deer from Kielder Forest were less varied and contained more spruce and heather than the diets of the deer from Galloway Forest.

Subcellular compartmentalization of E2F family members is required for maintenance of the postmitotic state in terminally differentiated muscle.

Maintenance of cells in a quiescent state after terminal differentiation occurs through a number of mechanisms that regulate the activity of the E2F family of transcription factors. We report here that changes in the subcellular compartmentalization of the E2F family proteins are required to prevent nuclei in terminally differentiated skeletal muscle from reentering S phase. In terminally differentiated L6 myotubes, E2F-1, E2F-3, and E2F-5 were primarily cytoplasmic, E2F-2 was nuclear, whereas E2F-4 became partitioned between both compartments. In these same cells, pRB family members, pRB, p107, and p130 were also nuclear. This compartmentalization of the E2F-1 and E2F-4 in differentiated muscle cells grown in vitro reflected their observed subcellular location in situ. We determined further that exogenous E2F-1 or E2F-4 expressed in myotubes at levels fourfold greater than endogenous proteins compartmentalized identically to their endogenous counterparts. Only when overexpressed at higher levels was inappropriate subcellular location for these proteins observed. At these levels, induction of the E2F-regulated genes, cyclins A and E, and suppression of factors associated with myogenesis, myogenin, and p21(Cip1) was observed. Only at these levels of E2F expression did nuclei in these terminally differentiated cells enter S phase. These data demonstrate that regulation of the subcellular compartmentalization of E2F-family members is required to maintain nuclei in a quiescent state in terminally differentiated cells.

Relation of atrial refractoriness to upper and lower limits of vulnerability for atrial fibrillation/flutter following implantable ventricular defibrillator shocks.

BACKGROUND: Implantable ventricular cardioverter defibrillator (ICD) shocks can cause atrial fibrillation/flutter (AF). This study investigated the pathogenesis of AF after ICD shocks in a canine model. METHODS AND RESULTS: The study was conducted in 8 dogs. In 5 dogs (group 1), truncated exponential (8 ms, 78% tilt) monophasic and biphasic shocks were delivered through a bipolar epicardial (patch) or endocardial lead. After the last S1 of atrial pacing at a cycle length of 350 ms, shocks of 0.1 to 7.6 A (0.005 to 27.7 J) were delivered, timed to the atrial effective refractory period (AERP). Ventricular defibrillation thresholds were also determined. In 3 dogs (group 2), the effect of the open versus closed chest technique on AF induction was tested in the endocardial biphasic shock configuration. AF was induced in all 8 dogs and in all waveforms and configurations. Mean AF duration was 11.5+/-6 s, with a mean ventricular rate of 184+/-37 bpm. Ventricular shocks could induce AF only if they were timed between an AERP of -60 to 40 ms, -40 to 60 ms, -40 to 60 ms, and -20 to 60 ms in the epicardial monophasic, epicardial biphasic, endocardial monophasic, and endocardial biphasic configurations, respectively. The mean+/-SD of the upper limit of vulnerability (ULV) for AF induction (in J) was 5. 2+/-0.6, 3.5+/-0.4, 5.2+/-1.2, and 2.5+/-0.1 for the epicardial monophasic, epicardial biphasic, endocardial monophasic, and endocardial biphasic configurations, respectively (P<0.05). The lower limit of vulnerability (LLV) was 0.8+/-0.1, 0.8+/-0.1, 0.9+/-0, and 0.6+/-0 for the epicardial monophasic, epicardial biphasic, endocardial monophasic, and endocardial biphasic configurations, respectively (P=NS). The ventricular defibrillation threshold (in J) for all wave forms and configurations was higher than the ULV (P<0. 05). CONCLUSIONS: (1) An atrial LLV and ULV exist for ventricular ICD shock-induced AF; (2) the shock-induced AF is related to both shock intensity and its timing to AERP; and (3) avoiding this atrial window of vulnerability may minimize the risk of post-ICD shock AF.

Regulation of expression and activity of distinct pRB, E2F, D-type cyclin, and CKI family members during terminal differentiation of P19 cells.

The cell cycle regulatory proteins, which include cyclin-dependent kinases (cdks), cdk inhibitors (CKIs), cyclins, and the pRB, and E2F families of proteins, constitute a network of interacting factors which govern exit from or passage through the mammalian cell cycle. While the proteins within these families have similar structural characteristics, each family member exhibits distinct expression patterns during embryogenesis and distinct biological activities. In order to begin to understand the tissue-specific roles of these interacting factors, we determined the expression pattern and activity of the pRB, E2F, cyclin, cdk, and CKI families of cell cycle regulatory proteins during retinoic acid-induced (neuronal pathway) and DMSO-induced (cardiac muscle pathway) differentiation of the pluripotent murine embryonal carcinoma cell line, P19. We demonstrate here that P19 terminal differentiation causes lineage-specific changes in the expression and activity of distinct members of the E2F, pRB, cyclin, and CKI families. Furthermore, dynamic changes in the activities of these cell cycle regulatory proteins occur through several overlapping mechanisms, culminating in repression of DNA-binding activity by all of the E2F family members as cells terminally differentiate.

Defibrillation efficacy using high-frequency switching to proportion current among simultaneous shock pathways.

INTRODUCTION: Multiple-pathway electrode configurations generally allow improved current distribution over the heart and lower defibrillation thresholds than single-pathway systems. However, current distributions using multiple pathways are largely determined by electrode type and location. We hypothesized that switching the current among multiple pathways at high frequency (HF) could allow the switching duty cycle to control the proportion of time-averaged current flowing in each pathway, thus permitting altered (possibly improved) defibrillation efficacy using the same electrodes and shock waveform. METHODS AND RESULTS: In dogs, we measured the current (I50) for 50% defibrillation success using catheter electrodes in the right ventricular apex (cathode) and superior vena cava (A-pathway anode) and a subcutaneous patch on the left chest wall (B-pathway anode). In group 1 (N = 7), we measured I50s for shocks that used HF to proportion 10% to 90% of the current to the A-pathway. Shocks with 10% to 30% of the current in the A-pathway had significantly lowr I50s than nonproportioned shocks using all three electrodes. However, the resistance differed among single and simultaneous pathways so energy did not necessarily parallel these changes. In group 2 (N = 6), we measured I50s for shocks to the B-pathway alone, for nonproportioned shocks to A and B, and for shocks that proportioned 80% of the current to the B-pathway using either HF, sequential, or amplitude proportioning methods. All proportioning methods had similar I50s that were significantly lower than the I50 for nonproportioned shocks to A and B and that were comparable to shocks to the B-pathway alone. CONCLUSIONS: Shocks with most current proportioned to the B-pathway had lower defibrillation currents than nonproportioned shocks using both pathways. Thus, defibrillation efficacy was changed by HF proportioning without changing the electrodes or shock waveform. These findings suggest that HF proportioning may be a method to improve defibrillation.

Refractory period extension during ventricular pacing at fibrillatory pacing rates.

Refractory period extension (RPE) has been proposed as a basic mechanism for defibrillation but it remains unclear if RPE exists at the fast rates associated with ventricular fibrillation. In 7 pentobarbital anesthetized dogs, we measured refractory periods with and without 8 ms rectangular transcardiac shocks at left ventricular pacing rates of 200-600 beats/min. To achieve these high rates, an incremental rate pacing method was used to produce pacing train timing sequences requiring 4.5-27 seconds. A variably timed premature stimulus followed the last stimulus in each pacing train. To determine refractoriness, a 128 electrode array (4 x 4 cm) was used to detect the presence, or absence of an activation sequence sweeping away from the pacing site. At each rate, a control refractory period (RPc) was measured and refractory periods were also measured for 8 and 12 V/cm shocks with coupling intervals of 60% to 90% of RPc. RPc decreased as the rate increased with a minimum RPc of 94 ms at a rate of 600 beats/min (100 ms cycle length). RPE/RPc versus shock coupling interval was similar at all pacing rates. RPE/RPc increased with increased coupling interval or higher shock intensity. We conclude that during ventricular pacing at fibrillatory rates tissue is nearly always in a refractory state; that RPE exists at fibrillatory activation rates; and that RPE/RPc versus shock coupling interval does not vary strongly with pacing rate. These findings support the hypothesis that RPE contributes to defibrillation.

Refractory interval after transcardiac shocks during ventricular fibrillation.

BACKGROUND: Measurements of refractory period extension by shocks during ventricular pacing at fast rates predict that all tissue should be refractory for a brief interval after shocks during fibrillation. This study experimentally determined whether a refractory interval was present just after a shock during fibrillation. METHODS AND RESULTS: In pentobarbital-anesthetized dogs, rectangular monophasic (4-ms) or biphasic (2.5/1.5-ms) shocks were followed with a 2-ms postshock stimulus (PSS) delivered to the defibrillation electrodes. We measured the effect of PSS on the shock current (I50) required for 50% defibrillation success. In group 1 (n = 6), a 1.0-A PSS had no effect on I50 when delivered up to 35 ms after monophasic shocks but greatly increased I50 when delivered at 50 to 90 ms. A 0.5-A PSS had no effect at any timing. In group 2 (n = 6), we compared 1.0-A PSSs after monophasic and biphasic shocks. The effect of PSS after monophasic shocks was similar to group 1. After biphasic shocks, PSS at the same timings had similar effects but caused even greater increases in I50. CONCLUSIONS: We conclude that after both monophasic and biphasic shocks during fibrillation, there is a postshock interval during which the heart is refractory to the refibrillating effect of PSS. The interval is shorter for biphasic than for monophasic shocks with the same duration and defibrillation efficacy. These findings support the refractory period extension hypothesis for defibrillation and suggest that propagating depolarization activity is absent immediately after defibrillation shocks but that it develops again at the end of the refractory interval or later.

Activity of the retinoblastoma family proteins, pRB, p107, and p130, during cellular proliferation and differentiation.

Genetic evidence from retinoblastoma patients and experiments describing the mechanism of cellular transformation by the DNA tumor viruses have defined a central role for the retinoblastoma protein (pRB) family of tumor suppressors in the normal regulation of the eukaryotic cell cycle. These proteins, pRB, p107, and p130, act in a cell cycle-dependent manner to regulate the activity of a number of important cellular transcription factors, such as the E2F-family, which in turn regulate expression of genes whose products are important for cell cycle progression. In addition, inhibition of E2F activity by the pRB family proteins is required for cell cycle exit after terminal differentiation or nutrient depletion. The loss of functional pRB, due to mutation of both RB1 alleles, results in deregulated E2F activity and a predisposition to specific malignancies. Similarly, inactivation of the pRB family by the transforming proteins of the DNA tumor viruses overcomes cellular quiescence and prevents terminal differentiation by blocking the interaction of pRB, p107, and p130 with the E2F proteins, leading to cell cycle progression and, ultimately, cellular transformation. Together these two lines of evidence implicate the pRB family of negative cell cycle regulators and the E2F family of transcription factors as central components in the cell cycle machinery.

Defibrillation using a high-frequency series of monophasic rectangular pulses: observations and model predictions.

INTRODUCTION: Capacitor-discharge type waveforms are practical for defibrillation devices but may not be optimum. Discharging a capacitor as a series of high-frequency (HF) pulses may allow effective waveform shaping by modulating the pulses. This approach could lead to improved defibrillation by allowing waveforms that would otherwise be unachievable with a capacitor-discharge approach. However, little is known about defibrillation with HF. METHODS AND RESULTS: In open chest pentobarbital anesthetized dogs, we measured defibrillation thresholds for continuous rectangular waveforms with 5-, 10-, and 20-msec durations and for 10- and 20-msec long series of HF rectangular pulses. HF series had a 50% "on-time" duty cycle at 100 Hz to 20 kHz. At 1 kHz and above, defibrillation with HF required the same time-averaged current but approximately twice the peak current and energy as defibrillation with continuous waveforms having the same envelope duration. At lower frequencies, defibrillation peak current and energy approached values required for the continuous waveforms. While waveforms were not actually filtered, the heart responded as though the HF series were low-pass filtered. A filtered effective waveform model with a 3.7-msec time constant predicts these HF data and makes reasonable predictions for various continuous waveform shapes. CONCLUSION: Defibrillation is possible using HF pulses up to 20 kHz and has a frequency response similar to a low-pass filter. A filtered effective waveform model predicts these HF results and may help explain how waveforms influence defibrillation efficacy. While the unmodulated HF pulsing used in this study increased defibrillation requirements, these findings support the concept that HF pulse modulation can be used to change the effective shape of a waveform, which could permit more efficacious waveform shapes and a net reduction of thresholds.

Effects of flecainide, encainide, and clofilium on ventricular refractory period extension by transcardiac shocks.

OBJECTIVE: The mechanisms by which pharmacological agents alter electrical defibrillation are not fully understood. It has been proposed that, in addition to directly stimulating tissue, defibrillation may involve refractory period extension (RPE) produced by the shock. Accordingly, pharmacological agents might modulate defibrillation by altering RPE. This study examined the effect of Class I and Class III antiarrhythmic agents on RPE by transcardiac shocks. METHODS: In four groups of pentobarbital anesthetized dogs, RPE was measured during rapid ventricular pacing before and after administration of either the Class I agents flecainide (n = 7) or encainide (n = 7), the Class III agent clofilium (n = 7), or vehicle (n = 5). Measurements included QRS duration during sinus rhythm and a conduction time, QTC interval and refractory period, and RPE for 4- to 10-V/cm shocks delivered 20-80 ms before the end of the tissue absolute refractory period. For the 6-V/cm shocks, the interval after the shock during which tissue remained refractory (RIAS) was also computed. RESULTS: Drugs affected QRS duration, conduction time, QTC, and refractory period ( without shocks) in accordance with their anticipated Class I and Class III actions. Without drugs, significant RPE was observed in all animals for all shocks delivered 40 ms or less before the end of the refractory period. Clofilium, encainide, and flecainide had a tendency to increase RPE but only clofilium produced a significant increase. For 6-V/cm shocks with different timings, the minimum RIAS was found to be approximately 43 ms, and occurred for shocks given 20-30 ms before the end of the refractory period. CONCLUSIONS: At drug dosages that produced moderate Class III ( approximately equal to 15%) or strong Class I (approximately equal to 35%) effects, only the Class III agent significantly increased RPE and RIAS. Thus, in addition to altering tissue excitability, the effect of antiarrhythmic agents to increase RPE and the minimum RIAS may help explain their influence on defibrillation threshold.

Expression and activity of the retinoblastoma protein (pRB)-family proteins, p107 and p130, during L6 myoblast differentiation.

The activity of the E2 F-family of transcription factors is tightly linked to control of the cell cycle. p107 and p130, two closely related members of the retinoblastoma protein-family of negative cell cycle regulators, modulate the activity of the E2f-family proteins by direct interaction with these factors. To understand the role of p107 and p130 in progression through or exit from the cell cycle, we have characterized the expression, phosphorylation state, cyclin-binding, and E2f-binding activity of p107 and p130 during terminal differentiation of rat myoblast cells into immature skeletal muscle (myotubes). In exponentially growing L6 myoblasts, p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 is phosphorylated in a cell cycle-dependent manner, and E2f-site binding complexes containing p107 can be observed throughout the cell cycle. During differentiation of L6 cells, p107 levels are reduced, while p130 protein levels are increased 8-fold. Despite both p107 and p130 becoming hypophosphorylated during myogenesis, the E2F-site DNA-binding complexes containing p107 observed in exponentially growing myoblasts are quantitatively replaced in myotubes with complexes containing only p130. In myotubes, p107 is not associated with E2f-family proteins that are capable of binding DNA. The failure to observe p107-containing complexes in myotubes appears to be due to the differentiation-specific induction of both p130 and cyclin D3, p107 is found in complexes with cyclin D3 in myotubes, and the addition of exogenous cyclin D3 or p130 to lysates from undifferentiated L6 cells was able to disrupt p107-containing E2F-site binding complexes. In myotubes, p130 also forms complexes with cyclin D3 as well as cyclin E, cdk2, and cdk4. We are able to copurify cyclin D3 with cyclin E from myotubes, indicating the presence of a macromolecular complex containing both cyclin E and cyclin D3 simultaneously bound to p130. Thus, in myoblasts, p107 is normally involved in regulation of E2f-family proteins during cell cycle progression, while p130 is a differentiation-specific regulator of E2f activity. Our results also provide evidence that the apparent positive regulator of cell cycle progression, cyclin D3, has a function in terminally differentiated muscle cells.

Characteristics of multiple-shock defibrillation.

INTRODUCTION: A new method for defibrillation allows two shocks to be combined to defibrillate with reduced current by adjusting their separation according to the cycle length of the fibrillation event. We investigated various aspects of this new method to better understand its characteristics and applicability to defibrillation. METHODS AND RESULTS: In 48 pentobarbital-anesthetized dogs, we measured the current for 50% defibrillation success using the new method with sequences of rectangular shocks. Group 1 studied the role of shock total duration and found that two-shock sequences followed a strength-duration curve similar to, but below, that for single shocks. Group 2 studied the role of amplitude and duration balance between shocks and found that two-shock sequences with equal shocks performed best. Group 3 studied whether the new method could be used with either biphasic waveforms or sequential shock pathways. Current reduction for the combined methods equaled the product of current reduction by each method, demonstrating that these methods can be effectively combined. Group 4 extended the method to include three-shock and four-shock sequences and found that a fourth shock did not further improve defibrillation. The optimum three-shock sequence required 33% lower current (P < 0.002) and 34% greater energy (P < 0.095 = NS) than a single shock. CONCLUSIONS: The new method allows defibrillation to be distributed over several fibrillatory cycles and has an improved strength-duration relationship. Two- or three-shock sequences using equal shocks permit a substantial reduction of defibrillation current that can be combined with the reduction for biphasic and sequential methods. Thus, the method may have application in low-current defibrillation devices.