Linear electrical properties of the transverse tubules and surface membrane of skeletal muscle fibers

With the use of two intracellular microelectrodes and a circuit designed to compensate for the effects of stray capacitances around the electrodes, transfer impedance measurements were made at frequencies from 0.5 to 1000 c/s on frog sartorius muscle fibers bathed in 7.5 mM K Ringer solution. Complete AC cable analyses performed at 46, 100, 215, 464, and 1000 c/s showed that the fibers behaved as ideal one-dimensional cables having purely resistive internal impedances (R_i = 102 ± 11 Ω cm). Two circuits were considered for fiber inside-outside impedance, a four lumped parameter circuit and a parallel resistance and capacitance shunted by the input impedance of a lattice model for the T-system. Least squares fits to fiber input impedance phase angles were better with the latter circuit than with the former. With the use of the lattice model the specific capacitance of both the surface and transverse tubule membranes was found to be 1 µF/cm² and the internal resistivity of the tubules to be about 300 Ω cm.     

The Anisotropic Elastic Properties of the Sarcolemma of the Frog Semitendinosus Muscle Fiber

Tension and curvature of the sarcolemmal tube of the frog muscle fiber were measured at different extensions and were used to calculate the anisotropic elastic properties of the sarcolemma. A model was derived to obtain the four parameters of the elasticity matrix of the sarcolemma. Sarcolemmal thickness was taken as 0.1 μm. Over the range of reversible sarcolemmal tube extension, the longitudinal elastic modulus E_L = 6.3 × 10⁷ dyn/cm², the circumferential modulus E_c = 0.88 × 10⁷ dyn/cm², the longitudinal Poisson's ratio σ_L = 1.2, and the circumferential Poisson's ratio σ_c = 0.18. At tubular rest length E_L = 1.2 × 10⁷ dyn/cm². The sarcolemma is less extensible in the longitudinal direction along the fiber axis than in the circumferential direction. It can be extended reversibly to 48% of its rest length, equivalent to extending the intact fiber from a sarcomere length of 3 μm to about 4.5 μm. The sarcolemma does not contribute to intact fiber tension at fiber sarcomere lengths <3 μm, and between 3 and 4 μm its contribution is about 20%. It also exerts a pressure on the myoplasm, which can be calculated by means of the model. The longitudinal elastic modulus of the whole fiber is 1 × 10⁵ dyn/cm² at a sarcomere length of 2.33 μm.     

Electrotonic Interaction between Muscle Fibers in the Rabbit Ventricle

Transmembrane potentials were recorded simultaneously from pairs of ventricular fibers in an isolated, regularly beating preparation. A double-barrelled microelectrode was used to record the potentials from, and to polarize, one fiber. A single microelectrode was used to record from a distant fiber. The existence of two systems of fibers, termed P and V, was confirmed. Histological evidence for the existence of two types of fibers is also presented. Electrotonic current spread was observed within both systems, electrotonic interaction between the two systems was rare and always weak. In the case of those pairs of fibers showing electrotonic interaction, the distance for an e-fold decrease in magnitude of the electrotonic potentials was found to be from 300 to 600 µ in P fibers and from 100 to 300 µ in V fibers. However, no electrotonic interaction could be observed in the majority of V fiber pairs. Moreover, the magnitude of the electrotonic potential did not decay monotonically with distance in any one direction. It is concluded that the rabbit ventricle cannot be regarded as a single freely interconnected syncytium.     

Use of lipophilic cations to measure the membrane potential of oat leaf protoplasts

Uptake of the lipophilic cation triphenylmethylphosphonium into mesophyll protoplasts of oat (Avena sativa L. cv. “Garry”) approaches equilibrium at 3 to 4 hours. The resulting external and internal concentrations are then used with the Nernst equation to obtain a membrane potential of −62 millivolts, inside negative. Potentials calculated in this manner are depolarized by adding 2 mm sodium azide and 50 μm carbonyl cyanide m-chlorophenylhydrazone as well as by increasing the external proton and potassium concentrations. The depolarizations are qualitatively similar to those seen when oat mesoyphll cells are measured in situ with microelectrodes. It is concluded that due to the lack of turgor and fragility of protoplasts, estimations of their membrane potential may be made more reliably, under some conditions, with lipophilic cations than with microelectrodes.     

Conformational Transitions of Subunit ? in ATP Synthase from Thermophilic Bacillus PS3

Subunit ɛ of bacterial and chloroplast F_OF₁-ATP synthase is responsible for inhibition of ATPase activity. In Bacillus PS3 enzyme, subunit ɛ can adopt two conformations. In the “extended”, inhibitory conformation, its two C-terminal α-helices are stretched along subunit γ. In the “contracted”, noninhibitory conformation, these helices form a hairpin. The transition of subunit ɛ from an extended to a contracted state was studied in ATP synthase incorporated in Bacillus PS3 membranes at 59°C. Fluorescence energy resonance transfer between fluorophores introduced in the C-terminus of subunit ɛ and in the N-terminus of subunit γ was used to follow the conformational transition in real time. It was found that ATP induced the conformational transition from the extended to the contracted state (half-maximum transition extent at 140 μM ATP). ADP could neither prevent nor reverse the ATP-induced conformational change, but it did slow it down. Acid residues in the DELSEED region of subunit β were found to stabilize the extended conformation of ɛ. Binding of ATP directly to ɛ was not essential for the ATP-induced conformational change. The ATP concentration necessary for the half-maximal transition (140 μM) suggests that subunit ɛ probably adopts the extended state and strongly inhibits ATP hydrolysis only when the intracellular ATP level drops significantly below the normal value.     

A simplified mass-transfer model for visual pigments in amphibian retinal-cone outer segments

When radiolabeled precursors and autoradiography are used to investigate turnover of protein components in photoreceptive cone outer segments (COSs), the labeled components—primarily visual pigment molecules (opsins)—are diffusely distributed along the COS. To further assess this COS labeling pattern, we derive a simplified mass-transfer model for quantifying the contributions of advective and diffusive mechanisms to the distribution of opsins within COSs of the frog retina. Two opsin-containing regions of the COS are evaluated: the core axial array of disks and the plasmalemma. Numerical solutions of the mass-transfer model indicate three distinct stages of system evolution. In the first stage, plasmalemma diffusion is dominant. In the second stage, the plasmalemma density reaches a metastable state and transfer between the plasmalemma and disk region occurs, which is followed by an increase in density that is qualitatively similar for both regions. The final stage consists of both regions slowly evolving to the steady-state solution. Our results indicate that autoradiographic and cognate approaches for tracking labeled opsins in the COS cannot be effective methodologies for assessing new disk formation at the base of the COS.Abbreviations used: A, area (μm²), COS, cone outer segment, D, mass diffusion coefficient (μm²/s), h_m, mass transfer coefficient (μm/s), L, cone outer segment length (μm), PDE, partial differential equation, r, radius (μm), t, time (s), T, plasmalemma thickness (μm), u, plasmalemma or disk region (axial) velocity (μm/s), V, volume (μm³), W, plasmalemma width (μm), x, axial direction, v, disk to plasmalemma velocity (μm/s), ρ₁, disk label density, ρ₂, plasmalemma label density, ϕ, nonvoid fraction     

Conduction of hyperpolarization along hamster feed arteries: augmentation by acetylcholine

The conduction of vasodilation along resistance vessels has been presumed to reflect the electrotonic spread of hyperpolarization from cell to cell along the vessel wall through gap junction channels. However, the vasomotor response to acetylcholine (ACh) encompasses greater distances than can be explained by passive decay. To investigate the underlying mechanism for this behavior, we tested the hypothesis that ACh augments the conduction of hyperpolarization. Feed arteries (n = 23; diameter, 58 +/- 4 microm; segment length, 2-8 mm) were isolated from the hamster retractor muscle, cannulated at each end, and pressurized to 75 mmHg (at 37 degrees C). Vessels were impaled with one or two dye-containing microelectrodes simultaneously (separation distance, 50 microm to 3.5 mm). Membrane potential (E(m)) (rest, approximately -30 mV) and electrical responses were similar between endothelium and smooth muscle, as predicted for robust myoendothelial coupling. Current injection (-0.8 nA, 1.5 s) evoked hyperpolarization (-10 +/- 1 mV; membrane time constant, 240 ms) that conducted along the vessel with a length constant (lambda) = 1.2 +/- 0.1 mm; spontaneous E(m) oscillations (approximately 1 Hz) decayed with lambda = 1.2 + 0.1 mm. In contrast, ACh microiontophoresis (500 nA, 500 ms, 1 microm tip) evoked hyperpolarization (-14 +/- 2 mV) that conducted with lambda = 1.9 +/- 0.1 mm, 60% further (P < 0.05) than responses evoked by purely electrical stimuli. These findings indicate that ACh augments the conduction of hyperpolarization along the vessel wall.     

Altered functional differentiation of mesoangioblasts in a genetic myopathy

Mutations underlying genetic cardiomyopathies might affect differentiation commitment of resident progenitor cells. Cardiac mesoangioblasts (cMabs) are multipotent progenitor cells resident in the myocardium. A switch from cardiac to skeletal muscle differentiation has been recently described in cMabs from β-sarcoglycan-null mice (βSG^−/−), a murine model of genetic myopathy with early myocardial involvement. Although complementation with βSG gene was inconsequential, knock-in of miRNA669a (missing in βSG^−/− cMabs) partially rescued the mutation-induced molecular phenotype. Here, we undertook a detailed evaluation of functional differentiation of βSG^−/− cMabs and tested the effects of miRNA669a-induced rescue in vitro. To this end, cMabs were compared with neonatal cardiomyocytes (CMs) and skeletal muscle C2C12 cells, representative of cardiac and skeletal muscle respectively. Consistent with previous data on molecular patterns, electrophysiological and Ca²⁺-handling properties of βSG^−/− cMabs were closer to C2C12 cells than to CM ones. Nevertheless, subtler aspects, including action potential contour, Ca²⁺-spark properties and RyR isoform expression, distinguished βSG^−/− cMabs from C2C12 cells. Contrary to previous reports, wild-type cMabs failed to show functional differentiation towards either cell type. Knock-in of miRNA669a in βSG^−/− cMabs rescued the wild-type functional phenotype, i.e. it completely prevented development of skeletal muscle functional responses. We conclude that miRNA669a expression, ablated by βSG deletion, may prevent functional differentiation of cMabs towards the skeletal muscle phenotype.     

Improving consumption rate estimates by incorporating wild activity into a bioenergetics model

Consumption is the basis of metabolic and trophic ecology and is used to assess an animal''s trophic impact. The contribution of activity to an animal''s energy budget is an important parameter when estimating consumption, yet activity is usually measured in captive animals. Developments in telemetry have allowed the energetic costs of activity to be measured for wild animals; however, wild activity is seldom incorporated into estimates of consumption rates. We calculated the consumption rate of a free‐ranging marine predator (yellowtail kingfish, Seriola lalandi) by integrating the energetic cost of free‐ranging activity into a bioenergetics model. Accelerometry transmitters were used in conjunction with laboratory respirometry trials to estimate kingfish active metabolic rate in the wild. These field‐derived consumption rate estimates were compared with those estimated by two traditional bioenergetics methods. The first method derived routine swimming speed from fish morphology as an index of activity (a “morphometric” method), and the second considered activity as a fixed proportion of standard metabolic rate (a “physiological” method). The mean consumption rate for free‐ranging kingfish measured by accelerometry was 152 J·g⁻¹·day⁻¹, which lay between the estimates from the morphometric method (μ = 134 J·g⁻¹·day⁻¹) and the physiological method (μ = 181 J·g⁻¹·day⁻¹). Incorporating field‐derived activity values resulted in the smallest variance in log‐normally distributed consumption rates (σ = 0.31), compared with the morphometric (σ = 0.57) and physiological (σ = 0.78) methods. Incorporating field‐derived activity into bioenergetics models probably provided more realistic estimates of consumption rate compared with the traditional methods, which may further our understanding of trophic interactions that underpin ecosystem‐based fisheries management. The general methods used to estimate active metabolic rates of free‐ranging fish could be extended to examine ecological energetics and trophic interactions across aquatic and terrestrial ecosystems.     

Electromechanical coupling in tubular muscle fibers. II. Resistance and capacitance of one transverse tubule

In tubular muscle fibers of the yellow scorpion the transverse tubules are arranged in a radial symmetry. This particular morphology, enables one to derive values for electrical components of one transverse tubule (TT) by treating the TT as a core conductor rather than a complex network. The electrical properties of tubular muscle fibers were completely characterized and analyzed by measuring two independent functions of frequency, i.e., the characteristic impedance and the propagation function. The impedance of a single tubular muscle fiber was determined with microelectrodes over the frequency range 1 Hz to 1.5 kHz. The results were fitted to a possible equivalent circuit model which is based on morphological evidence. The average component values for this model are: Ri = 209 omega-cm, Rm, and RT = 980 omega-cm2 (referred to unit area of surface membrane), Cm and CT = 0.9 muF/cm2, and RL = 103 omega-cm. Relating the equivalent circuit to ultrastructure shows that the average component values are consistent with the hypothesis that the TT is open to the extracellular medium, the electrical capacity of surface and TT membranes is about 1 muF/cm2, and the spread of surface depolarization into the TT is attenuated by about 25%.     

Denervation causes fiber atrophy and myosin heavy chain co-expression in senescent skeletal muscle

Although denervation has long been implicated in aging muscle, the degree to which it is causes the fiber atrophy seen in aging muscle is unknown. To address this question, we quantified motoneuron soma counts in the lumbar spinal cord using choline acetyl transferase immunhistochemistry and quantified the size of denervated versus innervated muscle fibers in the gastrocnemius muscle using the in situ expression of the denervation-specific sodium channel, Nav_1.5, in young adult (YA) and senescent (SEN) rats. To gain insights into the mechanisms driving myofiber atrophy, we also examined the myofiber expression of the two primary ubiquitin ligases necessary for muscle atrophy (MAFbx, MuRF1). MN soma number in lumbar spinal cord declined 27% between YA (638±34 MNs×mm⁻¹) and SEN (469±13 MNs×mm⁻¹). Nav_1.5 positive fibers (1548±70 μm²) were 35% smaller than Nav_1.5 negative fibers (2367±78 μm²; P<0.05) in SEN muscle, whereas Nav_1.5 negative fibers in SEN were only 7% smaller than fibers in YA (2553±33 μm²; P<0.05) where no Nav_1.5 labeling was seen, suggesting denervation is the primary cause of aging myofiber atrophy. Nav_1.5 positive fibers had higher levels of MAFbx and MuRF1 (P<0.05), consistent with involvement of the proteasome proteolytic pathway in the atrophy of denervated muscle fibers in aging muscle. In summary, our study provides the first quantitative assessment of the contribution of denervation to myofiber atrophy in aging muscle, suggesting it explains the majority of the atrophy we observed. This striking result suggests a renewed focus should be placed on denervation in seeking understanding of the causes of and treatments for aging muscle atrophy.     

PET/CT imaging of c-Myc transgenic mice identifies the genotoxic N-nitroso-diethylamine as carcinogen in a short-term cancer bioassay

Background

More than 100,000 chemicals are in use but have not been tested for their safety. To overcome limitations in the cancer bioassay several alternative testing strategies are explored. The inability to monitor non-invasively onset and progression of disease limits, however, the value of current testing strategies. Here, we report the application of in vivo imaging to a c-Myc transgenic mouse model of liver cancer for the development of a short-term cancer bioassay.

Methodology/Principal Findings

μCT and ¹⁸F-FDG μPET were used to detect and quantify tumor lesions after treatment with the genotoxic carcinogen NDEA, the tumor promoting agent BHT or the hepatotoxin paracetamol. Tumor growth was investigated between the ages of 4 to 8.5 months and contrast-enhanced μCT imaging detected liver lesions as well as metastatic spread with high sensitivity and accuracy as confirmed by histopathology. Significant differences in the onset of tumor growth, tumor load and glucose metabolism were observed when the NDEA treatment group was compared with any of the other treatment groups. NDEA treatment of c-Myc transgenic mice significantly accelerated tumor growth and caused metastatic spread of HCC in to lung but this treatment also induced primary lung cancer growth. In contrast, BHT and paracetamol did not promote hepatocarcinogenesis.

Conclusions/Significance

The present study evidences the accuracy of in vivo imaging in defining tumor growth, tumor load, lesion number and metastatic spread. Consequently, the application of in vivo imaging techniques to transgenic animal models may possibly enable short-term cancer bioassays to significantly improve hazard identification and follow-up examinations of different organs by non-invasive methods.     

A rectifying electrotonic synapse in the central nervous system of a vertebrate

The adductor muscles of the pectoral fins of the hatchetfish Gasteropelecus are innervated by bilateral pools of about 40 motoneurons which lie primarily in the first spinal segment. A pair of giant fibers on each side of the medulla send processes ventroposteriorly to the motoneuron pools. Electrophysiological evidence indicates that giant fibers are presynaptic to ipsilateral motoneurons, but not to contralateral ones. Transmission across the giant fiber, motoneuron synapse is electrically mediated as is indicated by direct measurement of electrotonic spread in either direction across the synapse, and by the extremely short latency of the giant fiber postsynaptic potentials (PSP's) in the motoneuron. The coupling resistance across the synapse was calculated from measurements of input and transfer resistance. The coupling resistance rectifies in such a way as to facilitate spread of depolarization from giant fiber to motoneuron, and to oppose transmission in the opposite direction. As a consequence of rectification, the giant fiber PSP in a motoneuron is augmented by hyperpolarization of the motoneuron. The coupling resistance calculated on the basis of this effect is in good agreement with calculations from input and transfer resistance data. Rectification at the electrotonic synapses may permit the motoneurons to act in small swimming movements as well as to fire synchronously in an extremely fast escape reflex mediated by Mauthner and giant fibers.     

Mechanisms of Transition from Normal to Reentrant Electrical Activity in a Model of Rabbit Atrial Tissue: Interaction of Tissue Heterogeneity and Anisotropy

Experimental evidence suggests that regional differences in action potential (AP) morphology can provide a substrate for initiation and maintenance of reentrant arrhythmias in the right atrium (RA), but the relationships between the complex electrophysiological and anatomical organization of the RA and the genesis of reentry are unclear. In this study, a biophysically detailed three-dimensional computer model of the right atrial tissue was constructed to study the role of tissue heterogeneity and anisotropy in arrhythmogenesis. The model of Lindblad et al. for a rabbit atrial cell was modified to incorporate experimental data on regional differences in several ionic currents (primarily, I_Na, I_CaL, I_K1, I_to, and I_sus) between the crista terminalis and pectinate muscle cells. The modified model was validated by its ability to reproduce the AP properties measured experimentally. The anatomical model of the rabbit RA (including tissue geometry and fiber orientation) was based on a recent histological reconstruction. Simulations with the resultant electrophysiologically and anatomically detailed three-dimensional model show that complex organization of the RA tissue causes breakdown of regular AP conduction patterns at high pacing rates (>11.75 Hz): as the AP in the crista terminalis cells is longer, and electrotonic coupling transverse to fibers of the crista terminalis is weak, high-frequency pacing at the border between the crista terminalis and pectinate muscles results in a unidirectional conduction block toward the crista terminalis and generation of reentry. Contributions of the tissue heterogeneity and anisotropy to reentry initiation mechanisms are quantified by measuring action potential duration (APD) gradients at the border between the crista terminalis and pectinate muscles: the APD gradients are high in areas where both heterogeneity and anisotropy are high, such that intrinsic APD differences are not diminished by electrotonic interactions. Thus, our detailed computer model reconstructs complex electrical activity in the RA, and provides new insights into the mechanisms of transition from focal atrial tachycardia into reentry.     

Exclusion of Protein from High Polymer Media: I. Derivation of Probability Distribution for the Number of Fiber Centers Within Any Sphere of Radius r

Ogston's (1958) fiber model based on Poisson's distribution function gives the average number of fibers making contact and no contact inside a sphere of radius r. The probability of penetration of spherical particles within a fibrous network was derived from the moment generating function [Formula: see text] A is the number of particles that intrude into a sphere of radious r. α(μ) is the probability that a particle, whose center is μ units away from the origin, intrudes into a sphere of radius r. A has a Poisson distribution with a mean value E(A) = 4πνα(μ)μ² dμ. The theoretical derivation of the distribution function of A gives Ogston's fiber model.     

Ion Selectivity in the KcsA Potassium Channel from the Perspective of the Ion Binding Site

To understand the thermodynamic exclusion of Na⁺ relative to K⁺ from the S₂ site of the selectivity filter, the distribution P_X(ɛ) (X = K⁺ or Na⁺) of the binding energy (ɛ) of the ion with the channel is analyzed using the potential distribution theorem. By expressing the excess chemical potential of the ion as a sum of mean-field 〈ɛ〉 and fluctuation μ^ex_flux,X contributions, we find that selectivity arises from a higher value of μflux,Na+ex relative to μflux,K+ex. To understand the role of site-site interactions on μ^ex_flux,X, we decompose P_X(ɛ) into n-dependent distributions, where n is the number of ion-coordinating ligands within a distance λ from the ion. For λ comparable to typical ion-oxygen bond distances, investigations building on this multistate model reveal an inverse correlation between favorable ion-site and site-site interactions: the ion-coordination states that most influence the thermodynamics of the ion are also those for which the binding site is energetically less strained and vice versa. This correlation motivates understanding entropic effects in ion binding to the site and leads to the finding that μ^ex_flux,X is directly proportional to the average site-site interaction energy, a quantity that is sensitive to the chemical type of the ligand coordinating the ion. Increasing the coordination number around Na⁺ only partially accounts for the observed magnitude of selectivity; acknowledging the chemical type of the ion-coordinating ligand is essential.     

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂⁻ consumption rates decreased from 0.68 and 0.64 μmol cm⁻² h⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm⁻² h⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

Ligands of Thermophilic ABC Transporters Encoded in a Newly Sequenced Genomic Region of Thermotoga maritima MSB8 Screened by Differential Scanning Fluorimetry

The chromosome of Thermotoga maritima strain MSB8 was found to have an 8,870-bp region that is not present in its published sequence. The isolate that was sequenced by The Institute for Genomic Research (TIGR) in 1999 is apparently a laboratory variant of the isolate deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM 3109) in 1986. This newly sequenced region from the DSMZ culture was located between TM1848 (cbp, cellobiose phosphorylase) and TM1847 (the 3′ end of a truncated ROK regulator). The new region contained seven genes: a beta glucosidase gene (bglA), three trehalose ABC transporter genes (treEFG), three xylose ABC transporter genes (xylE2F2K2), and the 5′ end of a gene encoding the ROK regulator TM1847. We present a new differential scanning fluorimetry method using a low pH that was necessary to screen potential ligands of these exceptionally thermostable periplasmic substrate-binding proteins. This method showed that trehalose, sucrose, and glucose stabilized TreE, and their binding was confirmed by measuring changes in intrinsic fluorescence upon ligand binding. Binding constants of 0.024 μM, 0.300 μM, and 56.78 μM at 60°C, respectively, were measured. XylE2 ligands were similarly determined and xylose, glucose, and fucose bound with K_d (dissociation constant) values of 0.042 μM, 0.059 μM, and 1.436 μM, respectively. Since there is no discernible phenotypic difference between the TIGR isolate and the DSMZ isolate despite the variance in their genomes, we propose that they be called genomovars: T. maritima MSB8 genomovar TIGR and T. maritima MSB8 genomovar DSM 3109, respectively.     

Reovirus Induction of and Sensitivity to Beta Interferon in Cardiac Myocyte Cultures Correlate with Induction of Myocarditis and Are Determined by Viral Core Proteins

Reovirus-induced acute myocarditis in mice serves as a model to investigate non-immune-mediated mechanisms of viral myocarditis. We have used primary cardiac myocyte cultures infected with a large panel of myocarditic and nonmyocarditic reassortant reoviruses to identify determinants of viral myocarditic potential. Here, we report that while both myocarditic and nonmyocarditic reoviruses kill cardiac myocytes, viral myocarditic potential correlates with viral spread through cardiac myocyte cultures and with cumulative cell death. To address the role of secreted interferon (IFN), we added anti-IFN-α/β antibody to infected cardiac myocyte cultures. Antibody benefited nonmyocarditic more than myocarditic virus spread (P < 0.001), and this benefit was associated with the reovirus M1 and L2 genes. There was no benefit for a differentiated skeletal muscle cell line culture (C2C12 cells), suggesting cell type specificity. IFN-β induction in reovirus-infected cardiac myocyte cultures correlated with viral myocarditic potential (P = 0.006) and was associated with the reovirus M1, S2, and L2 genes. Sensitivity to the antiviral effects of IFN-α/β added to cardiac myocyte cultures also correlated with viral myocarditic potential (P = 0.004) and was associated with the same reovirus genes. Several reoviruses induced IFN-β levels discordant with their myocarditic phenotypes, and for those tested, sensitivity to IFN-α/β compensated for the anomalous induction levels. Thus, the combination of induction of and sensitivity to IFN-α/β is a determinant of reovirus myocarditic potential. Finally, a nonmyocarditic reovirus induced cardiac lesions in mice depleted of IFN-α/β, demonstrating that IFN-α/β is a determinant of reovirus-induced myocarditis. This provides the first identification of reovirus genes associated with IFN induction and sensitivity and provides the first evidence that IFN-β can be a determinant of viral myocarditis and reovirus disease.