bluuebunny/crossref_metadata_2025 huggingface dataset:
Consumption of a COX‐2 inhibitor stimulates muscle protein synthesis after resistance exercise in humans
Authors: Burd N A , Dickinson J M , LeMoine J K , Carroll C C , Haus J M , Hughes G , Sanders C , Trappe T A
Published: June 2021

We have previously shown that non‐specific blockade of the cyclooxygenase (COX) enzymes in human skeletal muscle eliminates the normal increase in muscle protein synthesis following resistance exercise. We tested the hypothesis that this COX‐mediated increase in postexercise muscle protein synthesis is specifically regulated by the COX‐2 isoform. Sixteen males (23 ± 1 y, 177 ± 2 cm, 81.5 ± 3.4 kg) were randomly assigned to one of two groups that received three doses of either a specific COX‐2 inhibitor (celecoxib; 200 mg per dose, 600 mg total) or a placebo during the 24 h following a single bout of resistance exercise. Skeletal muscle fractional synthesis rate (FSR) was measured at rest and 24 hours postexercise. Mixed muscle FSR was increased following exercise to a greater extent (206%, p<0.05) in the COX‐2 group (0.052 ± 0.014 %/h) as compared with the placebo group (0.017 ± 0.007 %/h). These results suggest that specific inhibition of the COX‐2 isoform in human skeletal muscle causes a compensatory response in muscle protein synthesis. These data also highlight the involvement of the COX pathways in the regulation of muscle protein synthesis following resistance exercise.

NIH R01 AG020532 (TT)
Inhibition of xanthine oxidase mediated reactive oxygen species during exercise attenuates mitochondrial biogenesis signalling in skeletal muscle
Authors: Wadley Glenn David , McConell Glenn
Published: June 2021

INTRODUCTION:

We tested the hypothesis that blocking the xanthine oxidase (XO)‐mediated increase in reactive oxygen species (ROS) with allopurinol during treadmill running in rats would inhibit phosphorylation of several key mitochondrial biogenesis signalling proteins.

METHODS:

Sprague Dawley rats rested (rest), exercised (Ex) or exercised following 30min pretreatment with 32 mg/kg of allopurinol by IP injection (Ex+allo). The exercise groups ran on a treadmill for 60 min (25m/min, 5% incline). Mitochondrial biogenesis signalling proteins were examined in the whole cell (phosphorylated p38 MAPK and AMPK) and nuclear fraction (phosphorylated CREB and ATF‐2) of the gastrocnemius via immunoblotting.

RESULTS:

Allopurinol treatment abolished the exercise‐induced phosphorylation of p38MAPK and ATF‐2 (P<0.05). There was a tendency (P=0.07) for exercise to increase phosphorylation of AMPK and allopurinol had no effect on this. CREB phosphorylation was not significantly different between groups.

CONCLUSION:

This data suggests that XO‐mediated increases in ROS during exercise are involved in some aspects of mitochondrial biogenesis signalling following exercise in skeletal muscle. Further studies are needed to examine if other aspects of the mitochondrial biogenesis pathway (eg PGC‐1 and NRF‐1) are also are also inhibited by allopurinol treatment during exercise.
Exposure to intermittent hypoxia leads to habituation of the arousal response in developing rodents
Authors: Darnall Robert A. , McWilliams Samual B. , Schneider Robert W.
Published: June 2021

Arousal from sleep is an important apnea recovery mechanism. Victims of the Sudden Infant Death Syndrome (SIDS) have evidence of chronic hypoxia and impaired arousal mechanisms. We hypothesized that repeated exposure to hypoxia would cause arousal habituation as evidenced by a lengthening of the time to arousal and an increase in arousal threshold. Four rat pups (P13‐P17) were exposed to 6 trials of 5% oxygen (3 min) alternating with room air (~6 min). Inspired oxygen concentration (IO2), oxyhemoglobin saturation (SAT), heart rate (HR), and respiratory rate were continuously recorded. Rat pups were studied at thermoneutrality on piezoelectric film to detect body movements and were continuously videotaped. Arousals were identified as a stereotypical sequence of forepaw extension, head lifting, and an increase in HR. During the 6 trials of hypoxia, the time to arousal from the onset of the decrease in IO2 increased from 31.8 ± 7.0 to 53.2 ± 3.1 sec (P=0.024) and the IO2 at which arousal occurred decreased from 12.9 ± 0.8 to 9.5 ± 0.6 % (P=0.003). Similarly, the time of arousal from the onset of the decrease in SAT increased from 18.6 ± 5.8 to 48.9 ± 3.7 sec (P=0.032). These results support the hypothesis that exposure to intermittent hypoxia results in habituation of the arousal response in developing rat pups. We speculate that hypoxia induced arousal habituation may be an important mechanism in SIDS. P01 HD36379.
The cysteine precursor L‐2‐oxothiazolidine‐4‐carboxylate does not delay diaphragm fatigue in vitro
Authors: Ferreira Leonardo F , Reid Michael B
Published: June 2021

Metabolism of L‐2‐oxothiazolidine‐4‐carboxylate (OTC; Procysteine®) by 5‐oxoprolinase promotes intracellular cysteine and glutathione synthesis. The latter are important antioxidants in muscles. Therefore, we hypothesized that OTC would inhibit fatigue of diaphragm in vitro. Diaphragm strips from adult male ICR mice (n = 14) were incubated for 1 hr at 37 ºC with buffer (control, C) or OTC (10 mM) in an organ chamber. One min after completing a standardized force‐frequency protocol, muscles were stimulated (50 Hz, 500 ms train duration) every 2 s for 10 min. Force was measured at the onset and end of the fatigue trial. A fatigue index was calculated as the ratio of final‐to‐initial force. Data are presented as mean ± sem. Force developed at the onset of the fatigue protocol (C = 12.3 ± 2.3 N/cm2, OTC = 11.3 ± 1.5 N/cm2; p = 0.30) and the fatigue index (C = 0.43 ± 0.03, OTC = 0.51 ± 0.06; p = 0.45) were not different between control and OTC‐treated muscles. These results show that 10 mM OTC does not diminish diaphragm fatigue in vitro, and suggest that OTC may not be an effective treatment for muscle fatigue in vivo.

Financial Support: AHA Postdoctoral Fellowship (0725334B) and NASA/NSBRI (MA00209)
Chemotherapeutic drug effects on spontaneous mobility and swimming endurance of mice
Authors: Pagala Murali K , Kharode Ishita , Dahan Nessy , Arama Leron
Published: June 2021

Muscle weakness and enhanced fatigue are common complaints of cancer patients undergoing chemotherapy. In order to evaluate the underlying mechanisms, the present study assessed the acute effects of the chemotherapeutic drug, doxorubicin, on spontaneous motor activity and swimming performance of mice. Before and after i.p. injection of doxorubicin at 25 mg/kg into each mouse, the number of spontaneous movements was monitored for 24 hr using Columbus Instruments Opto‐varimax‐mini activity meter, and the integrated swimming activity (V.sec) was recorded for 40 min using a digital swimming monitor (Pagala, 1999).

Doxorubicin, within the clinical dose range, significantly reduced the stereotypic, ambulatory, and thus the total number of spontaneous movements of the mice in a 24 hr period (Table 1). Doxorubicin also caused a significant reduction in the integrated swimming activity of the mice over a 40 min period (Table 1). These results indicate that chemotherapeutic agents may significantly decrease the motivation and endurance for physical activity.

Supported by a special grant from Maimonides Med. Ctr.
Chronic loss of KATP channel activity results in an increase Ca2+ sensitivity during tetanic contractions.
Authors: Gariépy Louise , Bercier Jean‐Philippe , Renaud Jean‐Marc
Published: June 2021

The ATP‐sensitive K+ channel (KATP channel) is essential in preventing large increases in unstimulated [Ca2+]i and unstimulated force during fatigue (Cifelli et al. 2007. J. Physiol. 582:843) because of an excess Ca2+ influx through L‐type Ca2+ channels. The objective of this study was to determine whether a difference in Ca2+ sensitivity could be observed in unfatigued KATP channel deficient muscle. FDB bundles from CD‐1 mice (wild type) and from null mice for the Kir6.2 gene (Kir6.2−/− mice), which encodes for the protein making the pore of the channel, were used in this study. Lowering the [Ca2+]e from 2.4 to 0.6 mM had no effect on the peak tetanic force‐stimulus strength relationship of wild type FDBs as expected from previous studies. However, the relationship was shifted toward higher voltages in Kir6.2−/− FDBs. Addition of verapamil, a L‐type Ca2+ channel blocker, significantly reduced peak tetanic force at 2 μM in wild type FDBs, while it did the same at 0.5 μM in Kir6.2−/−FDBs. Verapamil also caused greater fade in Kir6.2−/− than wild type FDBs. Acutely blocking KATP channel in wild type FDBs with 10 μM glibenclamide did not affect the verapamil sensitivity. The results suggest that a chronic loss of KATP channel activity results in an increased needs for Ca2+ influx to generate and maintain force during a tetanic contraction.
Distribution of KATP channel among the different fiber types in skeletal muscle.
Authors: Banas Krystyna , Renaud Jean‐Marc
Published: June 2021

The ATP‐sensitive K+ channel (KATP channel) is essential in preventing contractile dysfunctions during exercise and fatigue in skeletal muscle (Cifelli et al. 2007. J. Physiol. 582:843). The extent of the contractile dysfunctions varies in the order of FDB = EDL ≫ soleus. Our studies and others have shown that, in mouse, FDB is composed primarily of type IIA and IIX fibers, the EDL has primarily IIB and IIX while soleus is composed of I and IIA. The objective of this study was to test the hypothesis that the protein content for the KATP channel is in the order of I < IIA ≪ IIB = IIX. Fiber types were identified using specific antibodies for each of the four myosin isoforms while the KATP channel was quantify using an antibody against the Kir6.2 subunit, which forms the pore of the channel. The data showed large Kir6.2 protein content in type IIA fibers of FDB and soleus, while very low levels were found in these same fibers in EDL. Type IIB (EDL only) and type IIX fibers (EDL & FDB) contained large amount of Kir6.2, while type I (FDB and soleus) had the lowest content. It is concluded that the expression of the Kir6.2 subunit of the KATP channel not only vary among fiber types, but also for type IIA fibers among different muscles.

Research supported by an operating grant from the Natural Sciences and Engineering Council of Canada (NSERC).
Structural and mechanical roles of the intermediate filament syncoilin in skeletal muscle: implications for muscular dystrophy
Authors: Gokhin David Samuel , Bang Marie‐Louise , Zhang Jianlin , Bremner Shannon N. , Chen Ju , Lieber Richard L.
Published: June 2021

The muscle‐specific intermediate filament syncoilin has been implicated as a contributor to muscular dystrophy because it interacts with α‐dystrobrevin and desmin, both of which are components of the skeletal muscle lateral force transmission network. To probe the functional roles of syncoilin, a syncoilin‐knockout (SKO) mouse strain was developed, and the structural and mechanical properties of SKO muscle were compared to wild‐type (WT) muscle. Muscle fiber cross‐sectional area was identical in SKO and WT muscles, and SKO muscle showed no signs of pathological disruption. Functionally, maximal isometric stress was significantly reduced in SKO muscle (240±22 kPa) compared to WT muscle (304±13 kPa), indicating that syncoilin deficiency disrupts active force generation. However, both SKO and WT muscles exhibited identical vulnerability to eccentric contraction‐induced muscle injury. Passive mechanical testing of single muscle fibers revealed no significant reduction in Young's modulus in SKO fibers (68±6 kPa) relative to WT fibers (75±5 kPa). Therefore, syncoilin deficiency results in a mild phenotypic change in skeletal muscle, although the reduced maximal isometric stress observed in SKO muscle may predispose the muscle to a dystrophic condition. These data suggest a subtle role of syncoilin in muscle force transmission pathways. Supported by NIH and the Department of Veterans Affairs
Dietary Cholesterol and Skeletal Muscle Hypertrophy with Resistance Training: A Randomized Placebo‐Controlled Trial
Authors: Riechman Steven E , Lee Chang Woock , Gasier Heath G , Chikani Gentle
Published: June 2021

Background:

We recently reported a strong positive association of dietary cholesterol and skeletal muscle responses to resistance training (Riechman, 2007). To confirm these findings we conducted a randomized placebo controlled trial in which we supplemented the diet with whole egg or egg white (control).

Methods:

3 groups of 50–69 year old generally healthy subjects (N=30) underwent 12 weeks of resistance exercise training (RET, 3x/week, 2–3 sets, 8–12 reps, 70% of max strength). Each group consumed 3.5 mg/kg lean/day (<200 mg/day, LC), 7.0 mg/kg lean/day (∼400 mg/day, MC), or 14.0 mg/kg lean/day (∼800 mg/day, HC) of cholesterol. Subjects were asked to complete 48 food records over the 12 wks to confirm dietary compliance. Body composition (DEXA) and maximal strength tests were conducted before and after the training.

Results:

Summary strength gain (chest press +leg press) was increased significantly (P<0.05) with increasing dietary cholesterol (HC=52±8%, MC=38±8%, LC=21±8%). The mean gain in lean mass was 1.7±1.0 kg (3.7%) where subjects also lost 0.5±1.7 kg of fat mass (1.6%). Changes in lean mass and fat mass between groups was not significantly different (P>0.05).

Conclusion:

Our results confirm our previous finding that high dietary cholesterol contributes to strength gain with resistance training; however, lean mass gains were inconsistent. Supported by US Poultry and Egg Association.
Estrogen Attenuation of Post‐Exercise Muscle Leukocyte Infiltration is not Receptor Mediated
Authors: Iqbal Sobia , Enns Deborah L. , Tiidus Peter M.
Published: June 2021

Previous work from our laboratory has demonstrated that estrogen will attenuate leukocyte infiltration into skeletal muscle following eccentric exercise. However the mechanisms by which estrogen exerts its effects are still uncertain. We investigated the role of estrogen receptors (ER) on muscle leukocyte infiltration following eccentric exercise through administration of the ER antagonist ICI 182,780 following downhill running to ovariectomized female rats with (E+) or without (E−) estrogen supplementation. At 24 and 48 h post‐exercise, soleus and white vastus muscles were removed and immunostained for HIS 48 (neutrophil) and ED1 (macrophage) positive cells. The increase in number of fibres positive for HIS 48 in soleus and positive for ED1 in the soleus and white vastus was significantly attenuated (p<0.05) in E+ relative to E− rats at 24 hours post‐exercise. E+ rats administered the ER agonist also had significantly (p<0.05) attenuated 24 h post‐exercise increases in HIS 48 and ED1 positive soleus and white vastus muscle fibres similar (p>0.05) to those without ER antagonist. This suggests that the ability of estrogen to attenuate post‐exercise leukocyte infiltration into skeletal muscle is not ER‐mediated.

Supported by NSERC Canada
Stretch‐like membrane permeabilization stimulates ERK2 phosphorylation dependent on calcium influx in C2C12 myotubes
Authors: Rahnert Jill Anne , Burkholder Thomas J
Published: June 2021

Mechanical stimulation of skeletal muscle is associated with hypertrophic signaling and increased membrane permeability. An increase in intracellular calcium may promote growth via ERK1/2 activation, and mechanical stretch may facilitate calcium influx either through stretch activated channels or by direct, physical damage of the membrane. These mechanisms differ in that, in addition to Ca2+ influx, membrane damage would allow efflux of cytoplasmic proteins, which might activate ERK1/2 by calcium‐independent pathways. We hypothesize that calcium influx is sufficient to cause stretch‐induced ERK phosphorylation. C2C12 cultures are treated with saponin to mimic membrane perforation or with A23187 to specifically facilitate calcium influx. In myotubes subjected to 15% mechanical stretch, ERK phosphorylation is increased to 161±45% (p=0.025) of control. Saponin treatment increases ERK phosphorylation to 489±290%, which is suppressed by 500uM EGTA. Similar to stretch, A23187 exposure increases ERK phosphorylation by 169±56% (p<0.001), but this is independent of EGTA. A23187 may also incorporate into intracellular membranes, and more complete calcium chelation with 5uM BAPTA‐AM completely blocked A23187‐induced ERK phosphorylation. We conclude that calcium entry is sufficient to stimulate activation of ERK implicating a role for calcium in initiating hypertrophic signaling.
Gender dymorphism in muscle leptin receptors
Authors: Guerra Borja , Fuentes Teresa , Delgado‐Guerra Safira , Guadalupe‐Grau Amelia , Olmedillas Hugo , Santana Alfredo , Dorado Cecilia , Calbet José A.L.
Published: June 2021

To determine if there is gender dymorphism in the expression of leptin receptors (OB‐R170, OB‐R128 and OB‐R98) and perilipin in skeletal muscle we obtained muscle biopsies from thirty‐six healthy men (age = 27.2 ± 1.1 yr, height = 176.5 ± 0.97 cm, body mass = 77.1 ± 1.9 kg, body fat = 18.1 ± 0.5%; mean ± s.e.m.) and thirty‐two females (age = 26.7 ± 1.2 yr, height = 165.3 ± 1.1 cm, body mass = 61.3 ± 1.5 kg, body fat = 28.1 ± 1.2%; mean ± s.e.m.). The expression of OB‐R protein, perilipin and tubulin were analysed by Western blot in muscle biopsies obtained from the m. vastus lateralis, and in subcutaneous adipose tissue of the thigh (n=10). While the OB‐R128 and OB‐R98 where found in the skeletal muscle and adipose tissue, the OB‐R170 isoform was only detected in skeletal muscle. Leptin receptor expression was 43% (OB‐R170, P<0.05), 153% (OB‐R128, P<0.05) and 28% (OB‐R98; P=0.07) higher in females than in males, as well as perilipin content (+99%, P<0.001) and was related to the plasma leptin concentration (r=0.57, P<0.001). Plasma leptin concentration was 85% higher in females than in the males (P<0.05). Perilipin content in the adipose tissue was similar in males (n=5) and females 5(n=5).

In summary, females express a greater amount of the long isoform of the leptin receptor in skeletal muscle. These results can not be explained by differences in fat tissue inside the skeletal muscle.
A theoretical model of the diffusion of glycolytic enzymes and other cytosolic proteins from rabbit skinned muscle fibers
Authors: Carlson Brian E. , Vigoreaux Jim O. , Maughan David W.
Published: June 2021

The rate of diffusion of proteins in the cytosol of muscle fibers is known to be considerably less than that of the same proteins in an aqueous solution. We measured the time course of diffusion of seven glycolytic and three other cytosolic proteins from skinned psoas muscle fibers under oil and transferred sequentially through drops of physiological relaxing solution. The rate cytosolic proteins diffused from the fibers into the stirred solution was significantly less than that in bulk water, especially at the onset. Collisions with the myofilament lattice hinder diffusion, and model fits to the data return an effective mesh size of ~12 nm, i.e., comparable to the distance between myofilaments. Binding to fixed structures and other proteins and the initially high concentration of cytosolic protein also reduce the rate of diffusion, but the initial lag in diffusion cannot be entirely accounted for by current models incorporating these features. A model fit to the diffusion time course of phosphoglucose isomerase (an exemplar protein) shows that, at the onset, steric hindrance and binding reduces the diffusion coefficient to 0.126 that in bulk water, 3.22e‐7 cm2/s, with a further reduction to 7.02e‐11 cm2/s due to protein crowding, which dissipates with time. We conclude that a complex mathematical description of the effect of protein crowding is required to represent the diffusion of proteins from skinned muscle fibers.
Amino acid sequence and phosphorylation status of myosin light chain MLC20 during vascular smooth muscle cell maturation
Authors: Graupner Gerhart , Zhang Kangling L , Injeti Elisha R , Pearce William J
Published: June 2021

Myosin light chain kinase (MLCK) is an important enzymatic regulator of vascular contractility that phosphorylates the regulatory protein, myosin light chain 20 (MLC20). We have recently demonstrated that the specific activity of MLCK is strikingly greater in fetal vs. adult sheep carotid arteries. Because cell‐specific splicing isoforms of myosin‐related genes have been identified in vascular smooth muscle, defining a late stage in the differentiation pathway from the synthetic to contractile phenotype, we tested the hypothesis that developmental alteration in MLCK activity may be at least partially dependent on developmental regulation of MLC20 isoforms. We also tested the hypothesis that developmental differences in baseline phosphorylation of MLC20 may influence MLCK activity. MLC20 was sequenced using a combination of biochemical purification and electron‐spray ionization‐mass spectrometry (ESI‐MS). Phosphorylation of MLC20 was quantified by urea gel electrophoresis and immunoblotting with antibodies against both total MLC and SER19‐phospho‐MLC. Our results demonstrate a high level of amino acid sequence conservation in both N‐terminal and C‐terminal regions of sheep MLC20 in comparison to other mammalian species and are consistent with the hypothesis that developmental shifts in baseline phosphorylation influence MLCK activity. Supported by NIH grants HD31226, HL54120, and HL64867
Cardiac expression of opioid genes in hibernation
Authors: Ackermann Laynez W , Denning Gerene M , Dickson Eric W
Published: June 2021

Hibernation is a state of markedly reduced body temperature and metabolism, and is characterized by profound resistance to hypothermia‐ and ischemia‐mediated injury. Hibernation is induced by pharmacologic delta opioids and blocked by delta opioid receptor antagonists. Despite evidence for a key role by opioids in hibernation, relatively little is known about the expression of opioid genes in hibernators. To address this question, we have partially sequenced woodchuck mRNAs for the opioid precursor proteins (preproenkephalin, pPENK; prodynorphin, pPD; propiomelanocortin, POMC) and the opioid receptors (delta, DOR; kappa, KOR; mu, MOR). In all cases, woodchuck mRNA sequences were highly homologous with human, rat, and mouse sequences, suggesting significant conservation of these proteins. Additionally, all six mRNAs were expressed in heart, and there appeared to be hibernation‐dependent changes in pPENK (4‐fold decrease) and DOR (2‐fold increase) mRNAs. Additional studies are underway to measure expression of these genes in tissues from summer‐active and hibernating animals. Understanding the molecular mechanisms by which hibernation protects the heart and other organs from ischemic injury may contribute to future therapeutic interventions that harness these remarkable protective effects. Funding for these studies was provided by the University of Iowa Department of Emergency Medicine.
Non‐uniform transmural remodeling in ovine chronic mitral regurgitation
Authors: Ennis Daniel B , Nguyen Tom C , Itoh Akinobu , Carlhäll Carl Johan , Oakes Robert A , Bothe Wolfgang , Liang David , Miller D Craig , Ingels Neil B
Published: June 2021

Objective:

Test the hypothesis that chronic “pure” mitral regurgitation (MR) induces non‐uniform transmural LV remodeling.

Methods:

Surgical creation of a ∼4.8mm hole in the posterior mitral leaflet created “pure” MR in 12 sheep (HOLE); 8 were sham operated (CNTL). Radiopaque transmural beads in the anterobasal wall were used to quantify epicardial (EPI) and endocardial (ENDO) remodeling strains in the circumferential (EC) and radial (ER) directions. Remodeling strains were calculated using end‐diastole (ED) 1 week (WK‐01) postoperatively as the reference and ED at 12 weeks (WK‐12) as the deformed configuration.

Results:

MR grade was greater in HOLE (∼3) than CNTL (<1) (P<0.001) at WK‐01 and WK‐12. LVED volume index increased in HOLE from WK‐01 to WK‐12 (P<0.05) and LV mass index was greater in HOLE at WK‐12 (P<0.01). HOLE EC was greater in ENDO than EPI (0.09±0.12 vs. 0.02±0.07, P<0.05) and HOLE EC in ENDO was greater than CNTL EC in ENDO (0.09±0.12 vs. −0.01±0.06, P<0.05). ER was less in EPI than ENDO for HOLE (−0.23±0.13 vs. −0.04±0.23, P<0.05) and CNTL (−0.15±0.07 vs. 0.15±0.44, P<0.05).

Conclusion:

EC and ER remodeling strains demonstrated significant transmural non‐uniformity. This non‐uniformity may arise from transmural differences in tissue stress. Such a transmural imbalance may underlie the decrease in LV torsion associated with chronic MR.

Support:

NIH HL29589/HL67025 (DCM) and HL087614 (DBE).
Myofibrillogenesis in the skeletal muscle cells of the zebrafish
Authors: Sanger Joseph W. , Wang Jushuo , Holloway Beth , Sanger Jean M.
Published: June 2021

We have proposed a three step model of myofibrillogenesis based on fixed and living cultured avian muscle cells. We have used Fluorescence Recovery After Photobleaching (FRAP) to study the dynamics of Z‐band proteins in these cells. To determine if similar processes exist inside living animals we have examined embryonic zebrafish where there is a temporal progression of myofibril formation that can be followed as myotomes increase in length from the youngest to the oldest myotomes. The earliest zebrafish myotomes exhibited small periodic z‐bodies of alpha‐actinin that were arrayed along overlapping actin fibers in patterns identical to premyofibrils in avian muscles. In young myotomes, myosin II was present in overlapping bundles that resembled myosin patterns in avian nascent myofibrils. A‐bands and Z‐bands were present in mature myofibrils in the elongated cells in older myotomes. However, at the ends of these cells the patterns of alpha‐actinin and myosin II resembled the arrangement seen in younger myotomes, suggesting that as the cells elongated, premyofibrils and nascent myofibrils were involved in the extension of myofibrils. FRAP experiments in zebrafish muscle showed recovery profiles of the proteins that were similar to those found for avian Z‐bands. Our experiments indicate that a stepwise model of myofibrillogenesis and the dynamics of Z‐band proteins occur in zebrafish. Grant support by MDA and NIH.
Ultrastructural features of a human placenta with acute chorioamnionitis
Authors: Meek Bill Dean , Hyde Scott , Collins Madison , McClain Oza
Published: June 2021

Portions of several human placentas were processed for light and transmission electron microscopy at the OSU‐CHS electron microscopy lab. Such placentas were characterized as 120g with acute placental abruption, acute chorioamnionitis, increased fetal nucleated red blood cells, villous edema and focal acute intravillous hemorrhages. In our study of the placenta, we were able to define the various parts of the placenta: decidual basal plate, chorionic plate, and chorion frondosum. The chorionic plate is the extra‐embryonic mesoderm of the chorion, bordering the amnionic cavity and lined by the amnion. The chorionic plate represents the embryonic contribution to the placenta. The chorionic plate is the site of histological abnormalities or pathology in these cases. This area was infiltrated with structures having a dark center and a clear surrounding area as revealed by light microscopy. These structures varied in size from 2–4 μm. Macrophages and neutrophils contained several dense spherical structures ranging in size from 0.2–0.5 μm. Several aggregates of these dense spherical bodies were observed. Some larger granular vesicles measured about l μm in size. Literature studies indicate a strong correlation with stages of the parachlamidiae organism. Further ultrastructural and histochemical analysis of these placental structures is currently in progress. OSU‐CHS Intramural Funding
Mechanism of DNA translocation in a replicative hexameric helicase
Authors: Joshua‐Tor Leemor , Enemark Eric J.
Published: June 2021

The E1 protein of papillomavirus is a replicative hexameric ring helicase belonging to the AAA+ family. Based on structures of the DNA‐binding domain of E1 complexes with DNA, we suggested a mechanism for DNA strand separation. However, the mechanism that couples the ATP cycle to DNA translocation has been unclear. I will present the crystal structure of the E1 hexamer with single‐stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA‐binding hairpins of each subunit form a spiral staircase that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP‐, ADP‐, and apo‐ configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP, and apo states while the associated DNA‐binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single‐stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.
The codes of “non‐coding” RNA: Translating the epigenome of acquired tolerance for recognition of functional self
Authors: Wissler Josef H.
Published: June 2021

AIM: Tolerance is a process of tissue indifference or non‐reactivity towards matter normally exciting adverse or immune responses. It concerns all living world in recognition of functional self and discrimination from damaged / junk self and nonself matter with many basic and clinical problems alike. As evident from paradigmatic LeDouarin phenomena and peptidome complexity, intrinsic epigenetic [non‐Mendelian] variation mechanisms are still not understood. Hence, functions of endogenous [oligo‐]RNA / RNP were investigated on tolerance of neovascular patterns [Wissler et al., Protides Biol. Fluids 34: 517–536, 1986; Materialwiss. Werkstofftech. 32: 984–1008, 2001; Ann. N. Y. Acad. Sci. 961: 292–297, 2002; 1022: 163–184, 2004; Mol. Biol. Cell Suppl. 18: 124, 2007; FASEB J. 21: 519.5, 2007]. RESULTS: Bioactive edited, modified, redox‐ and metalloregulated small hairpin shRNA [<200 bases] were found and sequenced. By metal ion‐structured 5’CUG3’‐hairpin loops, they may address homologous domains shared in proteofactors related to [epi]genetic information indexing of the epigenome, growth and metabolic syndrome, termed K/R3H [K/RxxxH], i.e. ‐t/s/xK/R/q/n/hxxxH/y/n/q/e/d/r/kx7–9h/xx7–9h/xx5–20K/R/q/n/e/h‐ with accessory basic [R/K]n, R/K‐zipper, SR/K/RS and/or HxxxH/y/n/q segments and relationship to other canonical domains [HxxxH, ATCUN, CxxxC, fingers, L‐zipper, Rossmann‐fold]. CONCLUSIONS: Such RNA are suitable as bioaptamers for fine tuning tolerance. They are earliest outputs in transcription [~98%], translation [~2%] and in estimated recognition repertoires, superior in diversity / specificity [>~1017] to immune proteins [~1013]. For tolerance, all attributes make them efficient and competent to recognize, address and integrate information flow on all possible molecular shapes back to genomic mechanisms.
Breathing: much more than just the intake of fresh air
Authors: Morris John Frederick
Published: June 2021

Teaching lower respiratory system functional anatomy to medical students involves the same principles as for any other part or system. I highlight four: understanding rather than learning; use of modern imaging; focus on details relevant to common clinical conditions; active learning via problem solving. Anatomy can have so much detail that, if one starts with structure, students easily get lost. Rather, I start with function and then give only what structure is necessary for understanding the functions and their mechanisms (e.g. transport of gas to/from alveoli; exchange of gas in alveoli; moistening/warming incoming air; removal of contaminants to clean inspired air or dislodge material; maintaining the structure of the alveolar membrane). Teaching of structure must anticipate how students will see anatomy in clinical practice and link gross anatomy with the ever more sophisticated imaging available. Many common clinical conditions affect the lower respiratory tract itself ‐ asthma, bronchitis, lung cancer; others such as pneumothorax point up the need to link in the chest wall and pleural cavity. Oxford takes a 2‐stage approach: a basic 1st year course to inform learning in physiology, and a brief, clinically focused 3rd year course just before clinical training.
Roles of c‐Src and PKC in production of persistent calcium sparklet activity
Authors: Gulia Jyoti , Gui Peichun , Chao Jun‐Tzu , Navedo Manuel F , Santana Luis F , Davis Michael J
Published: June 2021

The L‐type calcium channel (Cav1.2) is regulated by multiple kinases, including PKA and c‐Src, which phosphorylate the Cav1.2 C‐terminus at residues S1901 and Y2122 (Cav1.2c), respectively, to enhance Ca2+ entry. PKC also enhances Cav1.2 current under some conditions and promotes persistent Cav1.2 Ca2+ sparklet activity (quantal Ca2+ entry events); however, the PKC phosphorylation site is unclear. We addressed two questions using TIRF microscopy to measure Ca2+ sparklets in patch clamped HEK cells expressing Cav1.2c. 1) Does PKC produce persistent calcium sparklet activity through phosphorylation of S1901? 2) Is c‐Src involved in the production of persistent sparklet activity?

Persistent sparklets (indicating a prolonged channel opening state) were defined as nPs > 0.2, where n= # of quantal levels and Ps= probability that a given sparklet site was active. Our results suggest that both PKC and c‐Src promote persistent calcium sparklet activity, possibly by phosphorylating residue Y2122 rather than S1901 on Cav1.2c.
Angiotensin (1‐7) downregulation of AQP1 in rat proximal cell is mediated by ACE and ACE2 balance
Authors: Palomino Zaira , Casarini Dulce Elena , Jung Flavia F
Published: June 2021

Aquaporin 1 (AQP1) is the major constitutively expressed water channel in the renal proximal tubule and thin descending limb where nearly 80% of water is reabsorbed. However, the factors regulating its expression are still elusive, we have shown that Angiotensin (1‐7) [Ang (1‐7)] downregulates AQP‐1 expression in immortalized rat proximal tubule cells (IRPTC). In this study, we investigated Bradykinin (Bk), Prostaglandin E2 (PGE2), Angiotensin Converting Enzyme I (ACE) and Angiotensin Converting Enzyme 2 (ACE2) as possible mediators of Ang (1‐7) effect on AQP1. After the time points stimulation release of Bk and PGE2 induced by stimulation with Ang‐(1‐7) was measure by HPLC and EIA respectivelly. There was a significant elevation of Bk (1.53 vs 0.43 nM/mg) and PGE2 (169.1 vs. control 23.3 pg/mL) release. Enzymatic activity showed that Ang (1‐7) inhibited the enzymatic activity of ACE ( control:0.46 ± 0.02 vs 5min: 0.04 ± 0.001 nM/min/mg) , and stimulated ACE2 activity (control: 180 ± 33.88 vs. 120 min: 1041.9 ± 77.2 nM/min/mg). This study demonstrates that the direct effect of Ang‐(1‐7) is mediated by release of BK and PGE2, as well as, inhibition of ACE and stimulation of ACE2.
Modeling approaches to the estimate of plasma variables from exhaled volatile organic compounds
Authors: Oliver Stacy R , Newcomb Robert L , Blake Donald R , Ngo Jerry , Lee Jane S , Meinardi Simone , Rosa Jaime S , Galassetti Pietro R
Published: June 2021

Exhaled volatile organic compounds (VOC) are potential biomarkers of biological processes, such as glucose (GL), insulin (INS), and lipid (FFA) metabolism. The accuracy of VOC‐derived estimates of plasma variables, though, depends on analytical methods and mathematical modeling. Here we report evolving modeling approaches in consecutive studies with simultaneous collection of plasma and exhaled gases at multiple time points, during 2‐4 h fluctuations of plasma GL, INS and FFA.

a) Plasma GL estimate from multi‐linear regression (MLR) of exhaled ethanol + acetone, after GL ingestion; avg R of measured vs estimated GL = .70

b) Plasma GL estimate from MLR of ethanol, acetone, methyl nitrate + 3 aromatic gases after i.v. GL bolus (scaling back models from 6 to 4 gases, see figure); avg R = .91

c) Plasma GL, INS, FFA estimates from MLR of 4‐gas clusters during continuous i.v. GL infusion; avg Rs = .86 (GL), .92 (INS), .89 (FFA)

d) Plasma INS estimate from MLR of a 5‐gas cluster + time, using residual values for gases (instead of breath‐room air differences used in a, b, c); avg R = .91 (same regression equation for all subjects, unlike a, b, c, where individual equations were used).

MLR models of 4‐6 residual gas values and time can therefore accurately estimate blood variables related to glucose metabolism, providing the developmental basis of a non‐invasive methodology for diabetes screening, diagnosing and monitoring.
Nitrosative stress induces inhibition of protein kinase C‐mediated vascular contractile response in mouse aorta
Authors: Choi Hyehun , Tostes Rita C.A. , Webb R. Clinton
Published: June 2021

S‐Nitrosylation is a ubiquitous protein modification in redox‐based signaling. This modification forms S‐nitrosothiol (SNO) from nitric oxide (NO) on cysteine residues. Dysregulation of (S)NO signaling, nitrosative stress, leads to impairment of cellular function. Protein kinase C (PKC) is an important signaling protein that plays a role in the regulation of vascular function. We hypothesized that S‐nitrosylation of PKC in vascular smooth muscle would inhibit its contractile activity. Aortic rings from C57BL6 mice were treated with denitrosylation inhibitors, auranofin or 1‐chloro‐2,4‐dinitrobenzene (DNCB) for 1 hour and propylamine propylamine NONOate (PANOate) as a NO donor for 20 minutes. Contractile responses of aorta to phorbol‐12,13‐dibutyrate (PDBu), a PKC activator, were attenuated by auranofin, DNCB and PANOate (vehicle Emax = 390 ± 19.6 %, auranofin Emax = 136 ± 10.7 %, PANOate Emax = 119 ± 6.4 %, auranofin and PANOate Emax = 6 ± 2.1 %, DNCB and PANOate Emax = 16 ± 7.1 %, *p<0.001 vs. vehicle). S‐Nitrosylation of total protein and PKCα was increased by auranofin and PANOate as compared to control protein. These data suggest that PKC is inactivated by S‐nitrosylation and this modification inhibits contractile responses to PDBu. Since S‐nitrosylated PKC could not phosphorylate the target signaling proteins related to contraction, signal transduction was impaired.
Role of viscosity and fermentability of dietary fibers on satiety‐related hormones in rats
Authors: Schroeder Natalia , Gallaher Daniel D , Marquart Len
Published: June 2021

The objective of this study was to determine the effect of two dietary fiber characteristics ‐small intestinal contents viscosity and large intestinal fermentability ‐on satiety‐related hormones in rats. Diets contained fiber sources that were either not viscous, somewhat viscous, or highly viscous, and either highly fermentable or non‐fermentable, in a 2 X 3 factorial design. The dietary fiber content was 8% (w/w). Blood was drawn in the fasting state after 35 days. At 42 days, rats were food‐deprived for 15 hours then fed 4 g of their respective diets. Two h later, plasma and small intestinal contents were collected. In the fed state, rats fed non‐fermentable fibers had significantly greater plasma GLP‐1 concentration than fermentable fibers. In the fasted state, among non‐fermentable fibers, viscosity had no effect on GLP‐1 concentration. However, among fermentable fibers, greater viscosity reduced GLP‐1 concentration. In the fed state, rats fed fermentable fibers had a significantly greater plasma ghrelin concentration than non‐fermentable fibers. In the fasted state, ghrelin concentration was influenced by a complex interaction between viscosity and fermentability. Plasma leptin was unaffected by either viscosity or fermentability. In conclusion, fiber fermentability alters short‐acting satiety‐related hormones such that non‐fermentable fibers may increase satiety. (Support by GTC Nutrition)

Grant Funding Source

Industry
Spinal nitric oxide synthase activity is necessary for phrenic long‐term facilitation following intermittent hypoxia
Authors: MacFarlane Peter M , Mitchell Gordon S
Published: June 2021

Acute intermittent hypoxia (AIH) elicits a serotonin‐dependent form of respiratory plasticity known as phrenic long‐term facilitation (pLTF). Although reactive oxygen species (ROS) from spinal NADPH oxidase activity are necessary for AIH‐induced pLTF, little is known about the role of reactive nitrogen species (RNS). Since RNS, and specifically nitric oxide (NO), are necessary for other forms of neuroplasticity, including ventilatory LTF in unanesthetized mice (Kline et al., 2002), we hypothesized that spinal nitric oxide synthase (NOS) activity and NO are required for pLTF. To test this hypothesis, the NOS inhibitor, L‐NAME, was injected intrathecally over the cervical spinal cord (C4) prior to AIH (3, 5‐min episodes, 10% O2) in anesthetized, paralyzed and ventilated rats. In control, vehicle treated rats (aCSF, 12µl volume), AIH elicited a progressive increase in integrated phrenic nerve burst amplitude (ie. pLTF) lasting 60 min post‐AIH. However, pLTF was attenuated in rats pre‐treated with L‐NAME (10mM; 12µl volume; p<0.05), suggesting spinal NOS activity/NO is necessary for AIH‐induced pLTF. The specific role of NO is not yet known.

Supported by NIH HL‐80209 and the Francis Family Foundation (PMM)
Arterial pulse modulated activity of pontine respiratory‐modulated neurons depends on vagal inputs in decerebrate cats
Authors: Dick Thomas E , Shannon Roger , Lindsey Bruce G , Nuding Sarah C , Segers Lauren S , Baekey David M , Morris Kendall F
Published: June 2021

The expression of respiratory‐modulated activity in the dorsolateral pons depends on vagal inputs (Dick TE et al. J Physiol 586: 4265‐4282, 2008). This activity is thought to reflect the relative strength of modulated to non‐modulated inputs, which is estimated byη2 statistic so increased η2 values reflect greater balance of respiratory‐modulated input probably due to the absence of pre‐synaptic gating and/or changes in recurrent inhibition. We hypothesized that increased respiratory‐modulation would affect expression of arterial pulse modulated activity. Ensemble recordings of neuronal activity were obtained before and after vagotomy in decerebrate, paralyzed and ventilated cats. We measured the δ2 statistic, which is based on the η2 but assesses the strength of the arterial‐pulse modulation. Expression of arterial‐pulse modulated activity was meager (28% of the neurons & δ2=0.11±0.11) and decreased with vagotomy (19% & δ2=0.09±0.07). Specifically, neurons that became highly respiratory modulated after vagotomy (η2 > 0.5, I‐Aug neurons) did not express arterial‐pulse modulation though they had weak arterial pulse modulation prior to vagotomy. Other neurons became pulse modulated after vagotomy even when their respiratory modulation increased. In summary, cardiorespiratory coupling is evident in the dl pons and is vagal dependent. NIH grants NS‐19814, HL‐49813, HL‐63042 and NS46062
Central vs. peripheral sites of action of antitussive agents in anesthetized guinea pigs
Authors: Chou Yang‐Ling , Canning Brendan J
Published: June 2021

Dextromethorphan (DEX), SKF10047, codeine, DAMGO, mexiletine and baclofen have all been reported to prevent cough, in part, through direct peripheral effects on airway sensory nerves. We evaluated the ability of these drugs to modify coughing evoked by citric acid (CA) applied topically to the tracheal mucosa of anesthetized GPs. Drugs were applied topically to the tracheal mucusa (10‐100 μM) or were microinjected (0.1‐500nmol) bilaterally into nTS. CA (0.001‐2M) applied topically in ascending doses and in 100 μL aliquots evoked 13±2 coughs cumulatively (n=36). Mexiletine applied topically to the tracheal mucosa nearly abolished CA evoked coughing (3±1 coughs; n=6). None of the other drugs had any effect on cough when applied topically to the tracheal mucosa (n≤4). By contrast, all 6 drugs studied dose dependently inhibited (90‐100%) cough when microinjected bilaterally into the nTS sites of cough receptor termination (n=3‐6). Microinjection of the drugs into adjacent nTS locations was without effect on cough (n≤3). The data indicate that baclofen, DEX, SKF10047, DAMGO and codeine prevent cough receptor dependent cough by actions in the CNS and not in the periphery.
Effects of Hypoxia on Fos Expression in RVLM‐Projecting Neurons in the Nucleus of the Solitary Tract (NTS)
Authors: King Luise T , Heesch Cheryl M , Kline David D , Hasser Eileen M
Published: June 2021

Peripheral chemosensory afferents responding to arterial oxygen levels terminate in the NTS, where afferent input is integrated, modified, and then sent to rostral ventrolateral medulla (RLVM) neurons. The cellular phenotype in the presumptive pathway remains largely unexplored. We hypothesized that hypoxia increases NTS neuronal activity primarily in RVLM‐projecting NTS cells. Rats (n=6) were microinjected with a fluorescent retrograde tracer into the RVLM, allowed to recover, and then exposed to normoxia (N) or hypoxia (H, 10% O2) for 3hr. Immunohistochemistry was performed using markers for neuronal activation (Fos) and catecholaminergic cells (tyrosine hydroxylase, TH). NTS neurons that were positive for Fos, TH, or tracer were counted unilaterally in 5 sections ±360 µm from calamus scriptorius. Hypoxia increased the number of Fos immunoreactive (IR) cells per section (H: 43±7 vs N: 5±1; p<0.05). In hypoxic rats 10±5% of Fos‐IR cells projected to the RVLM. In addition 12±1% of NTS cells expressing Fos were TH‐IR yet none of these projected to RVLM. Of RVLM‐projecting NTS cells, the percent that was Fos‐IR increased after hypoxia (H: 14±5 vs. N: 0±0%; p<0.05). A greater portion of TH‐IR cells also expressed Fos in hypoxic rats (H:23±1 vs N: 2±2%; p<0.05). Thus, hypoxia appears to increase neuronal activation in separate populations of catecholaminergic and RVLM projecting cells in the NTS. HL55306, HL085108
Ovarian failure contributes to the onset of the metabolic syndrome
Authors: Romero‐Aleshire Melissa Jill , Diamond‐Stanic Maggie Keck , Hoyer Patricia B , Brooks Heddwen L
Published: June 2021

Factors comprising the metabolic syndrome occur with increased incidence in post‐menopausal women. To investigate the effects of ovarian failure (OF) on the metabolic syndrome female B6C3F1 mice were fed a high‐fat diet (HF) and treated with VCD (which causes OF by selectively destroying ovarian pre‐antral follicles). After 12 weeks, HF/VCD mice gained more weight than cycling HF mice (HF/VCD 46.8±1 vs HF 41.3±1g, P<0.05), had an impaired glucose tolerance (AUC HF/VCD 17378±1140 vs HF 13455±643 mg/dl*min, P<0.05), had significantly higher insulin levels (HF/VCD 10.1±1 vs HF 5.4±1 ng/ml, P<0.05) and were more insulin resistant than HF cycling mice (HOMA/IR HF/VCD 113 ±20 vs HF 56 ±17 mg/dl* μU/ml, P<0.05). Both HF groups were hypercholesterolemic (HF 255±6, HF/VCD 258±10, vs Con 148±4 mg/dl). In addition, even when on a normal diet, ovarian failure alone induced an impaired glucose tolerance which was reversed with estrogen replacement (AUC VCD 141444±937 vs cycling 11160±1101 mg/dl*min, P<0.05). The VCD model of menopause provides a physiologically relevant means of studying the effects of ovarian failure on the progression of the metabolic syndrome.