bluuebunny/crossref_metadata_2025 huggingface dataset:
Osmosensitive gene expression in C. elegans is regulated by conserved signaling mechanisms that control protein translation initiation
Authors: Lee Elaine , Strange Kevin
Published: June 2021

Osmotic homeostasis is fundamental for life. To cope with hypertonic stress (HS), C. elegans accumulates the organic osmolyte glycerol via increased expression of the glycerol synthesis enzyme GPDH‐1. We previously identified 122 rgpd (regulators of gpdh‐1) genes that when silenced constitutively activate gpdh‐1 expression and glycerol accumulation in the absence of HS. Fifty‐five rgpd genes play highly conserved roles in protein translation. Both HS and RNAi silencing of rgpd genes that encode aminoacyl‐tRNA synthetases and eukaryotic translation initiation factors (eIFs) dramatically reduce protein synthesis. Pharmacological inhibition of protein synthesis with cycloheximide or kasugamycin induces constitutive gpdh‐1 expression. eIF‐2α phosphorylation is a highly conserved signal for translation inhibition. Phospho‐eIF‐2α levels increase 2‐fold within 1 h of HS. Loss‐of‐function mutations in GCN kinases, which mediate eIF‐2α phosphorylation, inhibit HS induced gpdh‐1 expression. We suggest that components of the protein synthesis network function as part of an osmosensing and signaling circuit that detects cellular HS and activates downstream effector mechanisms including gpdh‐1 gene expression.
Structural and Functional Changes in the C‐terminal Regulatory Tail of the Na+/H+ Exchanger Mediate Phosphorylation Induced Regulation
Authors: Li Xiuju , Fliegel Larry
Published: June 2021

The Na+/H+ exchanger isoform 1 (NHE1) is a plasma membrane pH regulatory protein that removes one intracellular H+ in exchange for an extracellular Na+. NHE1 is inactive at alkaline intracellular pH but is active with decreasing pHi. The set point of the protein is altered by phosphorylation of the cytoplasmic tail. We examined the mechanism by which phosphorylation mediates NHE1 activation. In the full length protein, mutation of Ser770 and Ser771 to either Asp or Ala resulted in an inability to stimulate the protein by sustained intracellular acidosis. By using western blotting against the HA‐tagged full length NHE1 protein, we found that NHE1 proteins with the mutation S726/729D and S770/771A showed no difference in the digestion pattern or sensitivity to digestion compared with the wild type. However, the S770/771D mutant always appeared to be more sensitive to digestion with trypsin than controls. We examined the expressed C‐terminal regulatory region of NHE1 with and without phosphomimetic S770/771D mutations. These NHE1 proteins had varying sensitivity to trypsin digestion. This difference did not occur with phosphomimetic mutations at S703D, S726/729D. The results suggest that phosphorylation of NHE1 at Ser770 and Ser771 causes a significant changed in the conformation of the NHE1 regulatory tail that may affect NHE1 activity. Supported by Canadian Institutes of Health Research.
Normalization Of CFTR Brush Border Membrane Trafficking Defects In The Intestines Of Myosin 1a/6 Double Mutant Mice
Authors: Hoekstra Nadia Eritrea , Mark Mooseker , Ameen Nadia
Published: June 2021

Brush border myosins can move cargo, maintain membrane tension and regulate membrane trafficking. Insertion of CFTR channels into the intestinal brush border membrane (IBBM) is required for anion secretion. The mechanisms of CFTR delivery into the IBBM and its retrieval into trafficking compartments are poorly understood. Myosin 6 (Myo6), the only minus end motor, is the counterpart to the major plus end motor Myosin 1a (Myo1a) in the enterocyte BB. Absence of Myo6 leads to loss of intermicrovillar membrane (MVM) tension, increased surface CFTR and defective apical endocytosis (Traffic. 2007(8):998–1006; JBC, 285 (22), 2010), while absence of Myo1a leads to MVM defects due to lack of plus end tension. Preliminary studies indicate mistargeting of CFTR to the basolateral domain of villus enterocytes in Myo1aKO mice, that is associated with reduced anion transport. We hypothesized that the observed CFTR trafficking defects in Myo6 and Myo1a KO mice would be reversed in Myo1a/6 DKO mice. Preliminary studies reveal normalization of BB architecture in Myo1a/6 DKO mice. Immunolocalization studies of WT129 (WT) and Myo1a/6 DKO intestine reveal normalization of CFTR distribution in all segments. Immunoblots confirmed lack of alteration in CFTR expression between WT and Myo1a/6 DKO mice. Our data suggest that Myo6 works in tandem with Myo1a to regulate the trafficking of CFTR between subapical endosomes and the apical BBM.
Lithium and glycogen synthase kinase‐3 inhibition induces lysosomal degradation of aquaporin‐2
Authors: Edemir Bayram , Jardzejewski Marc , Schlatter Eberhard
Published: June 2021

Lithium (Li), used as treatment of bipolar disorders can lead to diabetes insipidus which shows decreased expression of aquaporin‐ 2 (AQP2) and polyuria. Li also reduces glycogen synthase kinase‐3 (GSK3) activity. GSK3beta deficient mice show decreased urinary concentration ability. The underlying cellular and molecular mechanisms are not fully understood. We used primary cultured inner medullary collecting duct (IMCD) cells, treated with Li or the GSK3 inhibitor SB216763. IMCD cells endogenously express AQP2 and other principal cell markers. AQP2 expression was verified by western blot analysis and realtime PCR. Treatment for 24h with Li or SB216763 reduced expression by 75%. Realtime PCR showed that Li treatment reduced AQP2 expression while SB216763 had no effect. The proteasom inhibitor MG132 or the lysosomal inhibitor bafilomycin (BF) reduced AQP2 mRNA but not protein expression. Li plus MG132 still reduced AQP2 expression. Li plus BF showed normal AQP2 expression. Furthermore in BF but not in MG132 treated cells AQP2 accumulated in vesicular structures. In conclusion we show that Li and GSK3 inhibition reduced AQP2 protein but only Li also mRNA expression. The accumulation of AQP2 after BF treatment indicates recycling of AQP2 in lysosoms. Li and GSK3 inhibition affect this lysosomal recycling thereby promoting lysosomal degradation of AQP2.
The multifaceted metabolism of reactive oxygen species (ROS) by mitochondria
Authors: Treberg Jason
Published: June 2021

Mitochondrial reactive oxygen species (ROS) are frequently implicated in various disease states and environmental stressors. Mitochondrial ROS production is not a single process, but rather the net result of several processes including multiple sites of production and elimination. Moreover, the topology of ROS production by mitochondria differs by the site(s) involved which may also impact the relative importance of these products and as the antioxidant systems involved with their further metabolism. This research examines the importance of assay conditions when studying mitochondrial ROS production and consumption, and the interplay between these processes. Particular focus is paid to the influence of external factors such as temperature and pH on these processes. This research is supported by the Canada Research Chairs program and the Canada Fund for Innovation.
Protein profile in mitochondria of pigs selected for residual feed intake
Authors: Grubbs Judson Kyle , Fritchen Aaron , Harris Amanda , Huff-Lonergan Elisabeth , Gabler Nicholas K , Lonergan Steven M
Published: June 2021

The efficiency nutrients utilization depends partially on mitochondrial efficiency and function. Additionally, mitochondria are a prime source of reactive oxygen species. The purpose of this study was to compare mitochondria protein profile in pigs (n = 11 per line) divergently selected for feed efficiency (low or high residual feed intake, RFI). Mitochondria were freshly isolated from the longissimus dorsi muscle by differential centrifugation, leaving an enriched mitochondria pellet. The pellets were solubilized and 2D‐Differential In Gel Electrophoresis, pH 3–10 11cm strips in the first dimension and 12.5 % acrylamide gels in the second dimension, were performed. Proteins were identified by electrospray mass spectrometry (ESI/MS). ERO1α was found to be increased in high RFI pigs (P = 0.073). ERO1α impacts mitochondria permeability transition pore through production of reactive oxygen species. SLC41a2, a Mg transporter, was increased (P = 0.13), possibly impacting superoxide dismutase conversion of superoxide to hydrogen peroxide. Cytochrome P450 4A11, a potential source of oxidative stress, was found to be increased (P = 0.14) in high RFI pigs. These data tend to indicate less efficient, high RFI, pigs may be more prone to intracellular oxidative stress than low RFI pigs. This research was supported by USDA‐AFRI grant# 2010‐65206‐20670.
Involvement of orexin A and melanin concentrating hormone neurons of the lateral hypothalamic area in the food intake response to reduced glucose availability
Authors: Takamata Akira , Taguchi Sayumi , Ikeda Saori , Aida Eri , Morimoto Keiko
Published: June 2021

The purpose of the present study was to elucidate the involvement of melanin concentrating hormone (MCH) and orexin A neurons at the lateral hypothalamic area (LHA) in the regulation of food intake stimulated by reduced glucose availability. In the present study, we examined the food intake response and c‐Fos expression in the MCH and orexin A neurons after 2‐deoxy‐D‐glucose (2DG)‐induced glucoprivation or systemic insulin‐induced hypoglycemia. Nine‐week‐old male rats were injected with either 2‐deoxy‐D‐glucose (2DG; 400 mg/kg; i.v.) or insulin (5 units/kg; s.c.), and food and water intakes were measured. Two days after the behavioral test, we examined c‐Fos expression in the MCH and orexin A neurons in the LHA immunohistochemically after 2DG or insulin administration. Both MCH and orexin A neurons were located in the LHA, but these peptides were distinctly localized in the separate neurons. Both 2DG and insulin administrations increased food intake compared with saline administration, and induced c‐Fos expression in the orexin A neurons, but did not induce c‐Fos expression in the MCH neurons of the LHA. These data indicate that orexin A neurons play a role but MCH neurons are not involved in the food intake response to reduced glucose availability.
Bardet Biedl syndrome genes are required for autonomic control of the circulation
Authors: Morgan Donald A , Sheffield Val C , Rahmouni Kamal
Published: June 2021

Bardet Biedl syndrome (BBS) is a pleiotropic autosomal recessive disorder with several features including hypertension and cardiovascular disease. The 16 Bbs genes identified so far have been implicated in the regulation of several physiological functions. Here, we used the knock‐in Bbs1M390R mouse model that phenocopy human BBS to test the hypothesis that Bbs genes are important for autonomic function. Baroreflex sensitivity (BRS) was examined in conscious state using the responses of renal sympathetic activity, arterial pressure and heart rate (HR) to the sequential intravenous injection of sodium nitroprusside and phenylephrine (PE). Notably, the maximum gain of the baroreflex was reduced in the Bbs1M390R mice (0.8±0.5%) vs. controls (3.3±0.9%, P<0.01). Direct recording of baroreceptor activity from the aortic depressor nerve (ADN) during PE‐induced hypertension which provides another measure of BRS revealed attenuated ADN baroreflex response in Bbs1M390R mice (6±2%) vs. controls (12±3%, P<0.05). Finally, analysis of the central component of the baroreflex with electrical stimulation (40 Hz) of the ADN showed an attenuated HR reflex response in Bbs1M390R mice (−47±16 bpm) vs. controls (−88±15 bpm, P<0.03) indicating that the impairment in BRS in BBS mice is centrally‐mediated. Our data demonstrates that Bbs genes are critically involved in the autonomic regulation of the cardiovascular function.
PACAP causes long‐term increases in sympathetic nerve activity and is necessary for the sympathetic response to acute intermittent hypoxia
Authors: Farnham Melissa Mary-Jean , Pilowsky Paul M
Published: June 2021

Overall objective

To determine if the sustained elevation of splanchnic sympathetic nerve activity (sSNA), termed long‐term facilitation (LTF), following acute intermittent hypoxia (AIH) is due to intermittent, release of sympathoexcitatory peptides, such as pituitary adenylate cyclase activating polypeptide (PACAP).

Methods

In urethane‐anaesthetized, artificially ventilated, male rats (n=18 Sprague–Dawley), we investigated the effect of 10, 45s, episodes of 10%O2–90%N2 every 5 min (AIH) on sSNA after intrathecal infusion of 10μl of vehicle, PACAP (300μM) or the PAC1/VPAC2 receptor antagonist, PACAP(6–38) (1mM). In a separate group of rats (n=6) without AIH, 10 doses of PACAP (10μL of 10μM) were given intrathecally every 5 min.

Results

Vehicle‐treated rats showed a 31.0±8.1% increase in sSNA 60min after AIH, which doubled to 67.7±14.1% in PACAP treated rats. Infusing the PACAP antagonist PACAP(6–38) prior to AIH abolished sympathetic LTF (0.1±2.7%). The responses were significantly different (P<0.0001; 2‐way ANOVA). Intermittent, sub‐threshold, infusion of PACAP (10×10μM) was as effective as AIH in causing LTF (sSNA increase of 38.±8.0%).

Conclusion

PACAP, acting via spinal PAC1/VPAC2 receptors, is sufficient to cause sympathetic LTF in the absence of AIH and necessary for the elaboration of sympathetic LTF following AIH. Support provided by Macquarie University, ARC and NHMRC.
Acid sensing ion channels (ASICs) in muscle skeletal afferents are heteromultimers with ASIC3 being a necessary component
Authors: Gautam Mamta , Benson Christopher J
Published: June 2021

ASICs are activated by extracellular acidosis, and in skeletal muscle afferents are thought to sense metabolic changes associated with ischemia and exercise. ASICs are formed as homo‐ or heterotrimers of several isoforms: ASIC1a, ‐1b, ‐2a, ‐2b, and ‐3, with each channel displaying distinct biophysical and pharmacological properties. To dissect the ASIC composition in muscle afferents, we studied the properties of pH‐evoked currents (amplitude, pH sensitivity, the kinetics of desensitization and recovery from desensitization, and pharmacological modulation) in isolated mouse skeletal muscle afferents that had been labeled using a retrograde tracer dye (DiI) injected into the gastrocnemius muscle from C57Bl6 (WT) and ASIC null mice using whole‐cell patch‐clamp. We found ASIC‐like currents in 60–70% of WT muscle afferents, and their fast kinetics of desensitization indicate that they are carried by heteromeric ASIC channels, with ASIC3 being a required component. Currents from ASIC1a −/− muscle sensory neurons were less pH sensitive and displayed faster recovery; those from ASIC2 −/− mice showed diminished potentiation by zinc; and currents ASIC3 −/− mice displayed markedly slower desensitization kinetics than those from WT mice. Finally, ASIC‐like currents were absent from triple null mice lacking ASIC1a, ‐2, and ‐3. We conclude ASICs in skeletal muscle afferents are heteromers of multiple subunits.
Evidence for chemosensitive fibers in the aortic depressor nerve in mice but not in rats
Authors: Stauss Harald Martin , Morgan Donald A , Rahmouni Kamal
Published: June 2021

A “J‐shaped” response of the aortic depressor nerve (ADN) to changes in aortic pressure has been described in rabbits. Specifically, at low pressure a paradoxical increase in ADN activity was noted during the diastolic phase of the cardiac cycle. We studied the physiological significance of this phenomenon. Lowering blood pressure (BP, Na‐nitroprusside) in anesthetized rats (n=4) abolished any ADN activity, suggesting that the “J‐shaped” response does not exist in rats. In anesthetized mice (spontaneously breathing 100% O2, n=11) total and systolic ADN activities mirrored BP changes elicited by Na‐nitroprusside and phenylephrine. In contrast, diastolic discharge activity showed an inverse relationship with BP, i.e., a decrease in BP caused an increase in diastolic ADN activity and vice versa. Electrical stimulation of the ADN (n=11 mice) during different phases of the cardiac cycle decreased BP and heart rate independently of the phase of the cardiac cycle, suggesting that central nervous system processing of ADN activity is independent of the cardiac cycle. Switching the inspiratory gas from 100% O2 to room air caused an immediate increase in diastolic ADN activity by 27.7±4.9% (n=4 mice) even before the onset of hypoxia‐induced hypotension. These results suggest that the ADN in mice (and possibly rabbits) but not in rats contains chemosensitive nerve fibers originating from glomus cells in the aortic bodies.
Imbalance of Angiotensin Receptor Expression and Function in the Spinal Cord: Potential Mechanism of Sympathetic Overactivity in CHF Rats
Authors: Chao Jie , Gao Guan , Zucker Irving H. , Gao Lie
Published: June 2021

In a recent study we showed that angiotensin II (AII) in the intermediolateral cell column (IML) of the spinal cord plays a critical role in the regulation of mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) in normal rats. In this study, the role of IML AII in sympatho‐excitation of chronic heart failure (CHF) rat induced by coronary‐ligation was studied. AT1R and AT2R expression in the IML was measured by Western blot and immunostaining. Compared with sham rats, CHF rats exhibited a higher AT1R and lower AT2R expression. Then effects of microinjecting AII, losartan (AT1R blocker), or PD123319 (AT2R blocker) into the IML were evaluated. AII evoked significantly stronger pressor and sympatho‐excitatory responses in CHF rats than that in sham rats (ΔMAP: 26 ± 1 vs 15 ± 2 mmHg, RSNA: 210 ± 9 vs 142 ± 6 % of baseline). Losartan significantly decreased basal RSNA and attenuated AII evoked responses in CHF rats, while PD123319 failed to affect MAP and RSNA. Finally, the effect of AII on electrophysiological properties of cultured IML neurons was assessed using whole‐cell patch clamp. AII significantly inhibited the K+ current (from 167 ± 9 to 39 ± 5 pA/pF at +80 mV) and elevated the resting membrane potential (from −55 ± 1 to −31 ± 1 mV). In conclusion, the change of AT1R and AT2R expression in the IML contributes to the sympatho‐excitation in CHF, possibly by elevating neuronal excitability via K+ current inhibition.
Differential regulation of sympathetic burst frequency and amplitude following acute hypoxia
Authors: Steinback Craig , Shoemaker J Kevin
Published: June 2021

Current evidence suggests that the persistent sympathetic nerve activity (SNA), commonly observed following exposure to hypoxia (HX), is mediated by chemoreceptor sensitization and baroreflex resetting. Evidence in animals suggests that these reflexes may independently regulate the frequency (gating) and amplitude (neuronal recruitment) of SNA bursts. In humans (n=7) we examined the regulation of SNA following acute isocapnic HX (5 min; end‐tidal PO2 = 45 Torr) and euoxic hypercapnia (HC; 5 min; end‐tidal PCO2 = +10 from baseline). HX increased SNA burst frequency (21±7 to 28±8 Burst/min, P<0.05) and amplitude (99±10 to 125±19 au, P<0.05) as did HC (14±6 to 22±10 bursts/min, P<0.05 and 100±12 to 133±29 au, P<0.05 respectively). Burst frequency (26±7 bursts/min, P<0.05), but not amplitude (97±12 au), remained elevated 10 min post HX. Both frequency and amplitude decreased during recovery following HC. These data clearly indicate two separate mechanisms, related to HX, regulating the pattern and magnitude of sympathetic outflow in humans. This study was supported by NSERC.
Assessment of central chemosensitivity through simultaneous recording of single‐unit and whole‐nerve responses to CO2
Authors: Reed Mitchell , Iceman Kimberly , Harris Michael , Taylor Barbara
Published: June 2021

The isolated frog brainstem is an established preparation to quantify respiratory drive and the role of central CO2 sensitivity in control of breathing. Here, we establish a technique to quantify neuronal response to high CO2 (hypercapnia) and compare it to the concomitant response by the intact respiratory network. Recorded neurons were subsequently juxtacellularly labeled, facilitating identification of location and phenotype of respiratory CO2‐ sensitive neurons. Neurograms representing buccal and lung breathing were obtained with suction electrodes recording whole‐nerve activity in cranial nerves 5 and 7. Simultaneously, extracellular electrodes recorded single neurons in established CO2‐sensitive sites of the frog brainstem. Neural activity was recorded while brainstems were exposed to normocapnia and hypercapnia (1.5 and 5.0% CO2, balance O2). Neurons were then entrained to an injected current and juxtacellularly labeled with biotinamide. After electrophysiology experiments, brainstem slices were stained with streptavidin‐AlexaFluor and the recorded cells were imaged. Our results validate this technique, which is the first reported capacity to simultaneously assess CO2 sensitivity of the central network that controls breathing and a single neuron embedded in the network. We expect to use this technique to elucidate neural mechanisms responsible for respiratory drive. NSF IOS‐1022442
The neuroepithelial body microenvironment as vagal mechanotransducer in intrapulmonary airways
Authors: Lembrechts Robrecht , Brouns Inge , Schnorbusch Kathy , Pintelon Isabel , Timmermans Jean-Pierre , Adriaensen Dirk
Published: June 2021

Mechanical information collected by sensory nerve terminals in the airways is mainly conducted to the CNS by vagal myelinated nerve fibers. A majority of these terminals contacts densely innervated groups of neuroendocrine airway epithelial cells (neuroepithelial bodies; NEBs), prompting us to study the potential role of NEBs as key transducers of mechanical airway stimuli into activation of vagal sensory terminals. Mild hypo‐osmotic stimuli were used to study mechanosensitivity in a mouse lung slice model for live cell imaging (LCI) of NEBs, and evoked a selective Ca2+ rise in NEB cells. Immunofluorescence and LCI showed that TRPC5, a Ca2+ permeable hypo‐osmotically/mechanically gated TRP channel, was expressed on the apical membrane, and functionally involved in the osmomechanical activation and subsequent release of ATP by NEB cells. ATP may then activate ligand gated P2X2/3 ATP receptors expressed by the terminals of myelinated vagal afferents in the NEB microenvironment. These terminals additionally expressed the inhibitory mechanogated K2P channel TRAAK, likely serving a regulatory role in the indirect mechanical activation. The presented study characterized the NEB microenvironment as a functional unit for vagal airway mechanotransduction.

Support: IWT fellowship SB/81162 (RL); FWO grants G.0081.08 (DA) and G.0589.11N (DA, JPT); UA grants GOA BOF 2007 (DA) and KP BOF 2006 (IB)
Severe hypoxia in isolated adult turtle brainstems decreases respiratory motor burst clustering, frequency, and amplitude
Authors: Bartman Michelle Ellen , Johnson Stephen Mark
Published: June 2021

Responses of isolated amphibian and mammalian brainstems or slices to hypoxia are well characterized, but how isolated reptilian brainstems respond to severe hypoxia is unknown. Thus, we exposed isolated adult turtle brainstems to 4.0 h of hypoxia (bath PO2=36 ± 2 mmHg) while recording respiratory‐related motor bursts on hypoglossal nerves. In brainstems producing episodic discharge (n=6; baseline bursts/episode > 1.75), bursts/episode decreased from 2.3 ± 0.2 to 1.3 ± 0.2 (P<0.05) and burst frequency decreased by 32% (P<0.05) after 90 min of hypoxia. Burst amplitude steadily decreased and reached 64% of baseline (P<0.05) after 3.5 h of hypoxia. Burst shape did not appear to be altered. In brainstems producing singlet discharge (n=6; baseline bursts/episode < 1.25), burst amplitude decreased to 57% of baseline (P<0.05) after 4.0 h of hypoxia while other variables were unchanged. The decrease in bursts/episode observed in vitro was similar to the hypoxia‐induced decrease in breaths/episode observed in vivo, which required 5‐HT3 receptor activation (unpublished observations). Thus, we hypothesize that the hypoxia‐induced decrease in bursts/episode also requires 5‐HT3 receptor activation. Supported by T32 HL07654.
Oxygen sensing pathway: genetic adaptation at high‐altitude
Authors: Pasha MA Qadar , Mishra Aastha , Mohammad Ghulam
Published: June 2021

Residents of Himalayas have evidence of selection of genetic variants and the genes of oxygen sensing pathway have emerged favorite candidates. Candidate gene and genome‐wide association studies (GWAS) have substantiated the involvement of endothelial PAS domain‐containing protein 1 (EPAS1) and HIF‐1 prolyl hydroxylase 2 (EGLN1) in adaptation to and disorders of hypobaric hypoxia. The case‐control design enumerated the selection of EGLN1 T allele of rs480902 and C allele of rs479200 in HA population (P=4.01E‐07). Conversely, the variants were overrepresented in HAPE (P<0.05). Other two candidates of this pathway namely endothelial nitric oxide synthase (NOS3) and endothelin‐1 (ET‐1) also associated. The alleles 298Glu of 298Glu/Asp and 4b of 4b/4a polymorphisms of NOS3, G of 2288G/T polymorphism and longer‐repeats of (CT)n–(CA)n repeat of ET‐1 were overrepresented in natives (P≤0.05); whereas, alleles 298Asp, 4a, T, and shorter‐repeats of respective gene polymorphisms were associated with HAPE (P≤0.05). Plasma NO, ET‐1 levels differentially associated with the variants. These studies clearly demonstrate the evolutionary selection of genetic variants in Tibetans that helped them achieve physiological profile similar to normoxic condition. This work was funded by Council of Scientific and Industrial Research, India.
Ion channel regulation by the Lkb1‐AMPK signalling pathway: the key to carotid body activation by hypoxia and metabolic homeostasis at the whole body level
Authors: Evans A. Mark , Hardie D. Grahame , Peers Chris , Kumar Prem , Wyatt Christopher N.
Published: June 2021

Our recent investigations provide further support for the proposal that, consequent to inhibition of mitochondrial oxidative phosphorylation, activation of AMP‐activated protein kinase (AMPK) mediates carotid body excitation by hypoxia. Consistent with the effects of hypoxia, intracellular dialysis from a patch pipette of an active (thiophosphorylated) recombinant AMPK heterotrimer (α2β2γ1) or application of the AMPK activators AICAR and A769662: (1) Inhibited BKCa currents and TASK K+ currents in rat carotid body type I cells; (2) Inhibited whole‐cell currents carried by KCa1.1 and TASK3, but not TASK1 channels expressed in HEK293 cells; (3) Triggered carotid body activation. Furthermore, preliminary data suggest that either conditional knockout of Lkb1 in type I cells or global knockout of the catalytic α1 and α2 subunit of AMPK, respectively, markedly attenuated the ventilatory response of mice to hypoxia. Accumulating evidence therefore suggests that the Lkb1‐AMPK signalling pathway is necessary for hypoxia‐response coupling by the carotid body, and serves to regulate oxygen and therefore energy supply at the whole body level.
Ascorbic acid attenuates sympathetic activation and endothelial dysfunction induced by short‐term intermittent hypoxia in humans
Authors: Leuenberger Urs A. , Linton-Frazier Latoya , Spilk Samson , Hogeman Cynthia
Published: June 2021

In healthy humans, short‐term intermittent hypoxia (IH) raises sympathetic nerve activity mirroring the sympathetic activation seen in patients with obstructive sleep apnea. To explore the role of oxidative stress, we measured blood pressure (BP), sympathetic activity (microneurography) and flow‐mediated brachial artery dilation before and following IH (30 episodes, O2 saturation nadir ~82%) with and without the antioxidant ascorbic acid (50 mg/kg iv) in healthy subjects (age 26±1 yrs; n=13). During saline infusion, IH produced a sustained increase in sympathetic activity (pre vs. post 20.0±1.8 vs. 25.2±1.8 bursts/min; P<0.05) and a small rise of mean BP (pre vs. post 86±2 vs. 89±3 mmHg; P<0.05). In contrast, on a separate day during infusion of ascorbic acid, IH did not raise sympathetic activity (pre vs. post 20.2±2.1 vs. 20.5±2.6 bursts/min; P=NS) or BP (pre vs. post 88±2 vs. 89±2 mmHg; P=NS). Moreover, whereas during saline infusion IH reduced flow‐mediated dilation (pre vs. post 6.8±1.3 vs. 3.2±1.6%; P<0.05), with ascorbic acid this effect was not seen (pre vs. post 6.0±1.2 vs. 6.1±1.4%; P=NS, n=8). These data suggest indirectly that oxidative stress may play a role in the sympathetic activation and vascular dysfunction induced by IH.

Supported by P01 HL077670 and UL1 RR033184.
Na/Ca exchanger‐1 mediates hypertrophy of cultured neonatal mouse cardiomyocytes in chronic intermittent hypoxia
Authors: Chen Ling , Wu Guizhen , Zhang Jin
Published: June 2021

Cardiac‐specific Na/Ca exchanger‐1 (NCX1) knockout (KO) mice is resistant to chronic intermittent hypoxia (CIH)‐induced cardiac hypertrophy in vivo (Chen et al. J Appl Physiol 2010; 109:1675). We studied the effect of CIH on NCX protein expression and cell size in primary cultured neonatal cardiomyocytes from either wild type (WT) or KO mice. CIH was produced by introducing mixed gases containing 0% or 21% O2 at a periodicity of 20 and 20 min, which achieved a nadir pO2 10 and peak 120 mmHg at the bottom of culture media. Daily exposure for CIH included 12h CIH, followed by 12h 21% O2. Control cells were cultured only in 21% O2. At the end of 5‐day exposure, WT with CIH had significantly greater NCX protein expression (~60%) and cell cross sectional area (1158 ± 181 vs 856 ± 98 μm2, P < 0.05) compared to WT control. Cell area in KO with CIH was 917 ± 138 μm2, which is significantly smaller than in WT with CIH (P< 0.05), but not significantly different from either WT or KO controls. NCX protein expression was not significantly different between KO groups, which are ~25% of that in WT control.

Conclusion

NCX1 mediates cell hypertrophy in cultured neonatal cardiomyocytes following CIH exposure.
The role of Hsp90‐R2TP complex in snoRNP assembly and ribosomal RNA processing
Authors: Houry Walid A
Published: June 2021

Box C/D snoRNPs, which typically consist of a snoRNA and four protein components (Nop1, Nop56, Nop58 and Snu13), play an essential role in the modification and processing of pre‐rRNA. Earlier, we had demonstrated that the Hsp90‐R2TP (Rvb1‐Rvb2‐Tah1‐Pih1) complex is involved in box C/D snoRNP biogenesis and assembly and, consequently, influences pre‐rRNA processing. Rvb1 and Rvb2 are AAA+ helicases, while Tah1 and Pih1 are Hsp90 cofactors/interactors. We had shown that the stability of the R2TP complex is modulated by Hsp90. More recently, we found, unexpectedly, that the activity of the R2TP complex is controlled by the dynamic subcellular redistribution of its protein components in response to growth conditions and nutrient limitation. In vigorously growing cells, the R2TP complex localizes in the nucleoplasm and interacts tightly with box C/D snoRNPs through Pih1, whereas, this interaction is significantly reduced in poorly growing cells. The R2TP complex is found to affect snoRNP levels by stabilizing Nop58. Our data suggest the presence of an unconventional communication pathway in which the Hsp90 chaperone stabilizes and promotes the assembly of the R2TP complex which in turn stabilizes and promotes the assembly of the box C/D snoRNP complexes. Our progress in this regard will be described in addition to our ongoing efforts aimed at the structural characterization of the R2TP complex.
Dynamics of the SRP cycle: From scanning to targeting
Authors: Wintermeyer Wolfgang , Holtkamp Wolf , Lee Sejeong , Senyushkina Tamara , Bornemann Thomas , Rodnina Marina V.
Published: June 2021

Inner‐membrane proteins in Escherichia coli are synthesized on ribosomes that are bound to the translocation pore (translocon) and enter the membrane co‐translationally. Ribosomes are targeted to the translocon by the signal recognition particle (SRP) and the SRP receptor, FtsY. Targeting is initiated when SRP is recruited to ribosomes that expose a signal‐anchor sequence (SAS). Here we report the kinetics of the interaction of SRP with ribosomes in different functional states and with FtsY, as followed by stopped‐flow monitoring FRET between fluorophores in SRP and the ribosome or FtsY. Rapid SRP binding to the ribosome is followed by two rearrangements the forward rate constants of which are independent of the functional state of the ribosomes. Conversely, ribosome‐SRP complex dissociation is very slow with ribosomes exposing an SAS, whereas the complex with non‐translating ribosomes dissociates rapidly. SAS‐induced stabilization of SRP on the ribosome is correlated with an acceleration of receptor binding. Thus, SRP can rapidly scan all ribosomes until it encounters a ribosome exposing an SAS, where SRP binding is strongly stabilized and membrane targeting is promoted by accelerating targeting complex formation with FtsY and, presumably, the translocon. The formation of the targeting complex is correlated with the lengthening of the nascent peptide within the exit tunnel of the ribosome.
Mitogen activated protein kinases mediate endothelin regulation of tyrosine hydroxylase in the olfactory bulb
Authors: Giannoni Florencia , Nabhen Sabrina Laura , Guil Maria Julia , Hope Sandra Ingrid , Bianciotti Liliana Graciela , Vatta Marcelo Sergio
Published: June 2021

Endothelins (ETs) function as neuromodulators in different brain areas including the olfactory bulbs (OB) which are connected with regions that regulate the cardiovascular activity and mood. We previously reported that ETs increase tyrosine hydroxylase (TH) activity through several pathways like PKA, PKC, and CaMKII. TH activity modifications are due to an augment in protein total levels, its phosphorylation forms at Ser 19, 31 and 40 as well as the level of TH mRNA. It is also known that ERK1/2 are late effect proteins which are known to phosphorylate TH and modulate its activity. In the present study we aimed to determine if ERK1/2 mediated ETs regulation of TH expression in the OB. ET‐1 and ET‐3 increased ERK1/2 expression by 39% and 42%, respectively, but no changes were observed in phosphorylated ERK1/2. In the presence of PD98059 (ERK1/2 inhibitor) the increase of TH mRNA induced by ETs was abolished. In addition, the effect of ETs in total protein levels was partially blocked by ERK inhibitor presence. However, the level of TH phosphorylation at Ser 40, were totally reduced. These findings indicate that ETs modulated TH mRNA and protein levels through ERK1/2. Since Ser 40 phosphorylation has the major impact on TH activation, we suggest that ERK1/2 might be increasing TH activity by this mechanism. (ANPCyT, CONICET, UBA)
Altered muscular activation patterns and force feedback after spinal cord injury
Authors: Niazi Irrum Fawad , Nichols T. Richard , Howland Dena. R
Published: June 2021

One of the major clinical problems associated with spinal cord injury (SCI) is the lack of animal's ability to maintain weight support during standing or walking. We hypothesize that following SCI there is reorganization of the postural limb reflexes driven by stretch and load receptors in addition to loss of excitatory inputs from supraspinal structures to spinal interneurons that could result in altered intermuscular interactions. In control decerebrate animals there is a proximal to distal gradient in the strength of inhibitory force feedback among the limb muscles. We propose that pathways mediating this inhibition may remain inhibitory after spinal hemisection but their strengths may change to a distal to proximal gradient in the animal resulting in a loss of weight support. We also propose that ventral horn pathways are important for regulating the distribution of intermuscular force feedback. To test our hypothesis we are studying two types of SCI, namely dorsal and lateral hemisections, at both acute and chronic time points post‐injury. We measured intermuscular force feedback using combinations of stretches applied to pairs of muscles. Our most recent evidence supports our hypothesis and shows that the intermuscular force feedback is reversed after lateral but not dorsal hemisections.

This work is supported by NIH grant HD32571, 1 I01 RX000243‐01A1, State of Florida BSCIRTF
Intramyocardial injection of heart tissue‐derived extracellular matrix improves cardiac function in a rat myocardial infarction model
Authors: Dai Wangde , Gerczuk Paul , Zhang Yuanyuan , Kay Gregory L. , Jyrala Aarne J. , Kloner Robert A.
Published: June 2021

Tissue‐derived decellularized extracellular matrix (ECM) particles were made from hearts of Fischer rats. One week after left coronary ligation in Fischer rats, saline or ECM particle suspension (75 ìl) was directly injected into the myocardial infarcted area. At 6 weeks after injection, left ventriculography demonstrated that LV ejection fraction was significantly greater in the ECM‐treated group (56.7±1.4%, n=19) than in the saline‐treated group (52.4±1.5%, n=17; p=0.043). Echocardiography showed that fractional shortening was significantly increased in the ECM‐treated group (26.2±2.2%) compared to the saline group (17.8±1.5%; p=0.0034). ECM implantation significantly increased the transmural thickness of the infarcted LV wall (0.602±0.029 mm) compared to the saline group (0.484±0.03 mm, p=0.0084). Infarct expansion index was significantly lower in the ECM‐treated group (1.053±0.051) compared to the saline group (1.382±0.096, p=0.0058). No injected ECM were identified within the infarcted area in H&E, and picrosirius red staining, suggesting that native tissue regenerated into the implanted ECM. There was a trend for smaller post‐mortem LV volume (349±12 ìl) in the ECM‐treated group compared with 371±11 ìl in the saline group. Implantation of heart tissue‐derived decellularized ECM thickens the LV infarcted wall and improves LV function after myocardial infarction in rats.
Extensive In vivo Metabolite‐Protein Interactions Revealed by Large‐Scale Systematic Analyses
Authors: Snyder Michael , Li Xiyan , Gianoulis Tara , Yip Kevin , Gerstein Mark
Published: June 2021

Extensive effort has been made to systematically characterize protein‐protein and DNA‐protein interactions. Natural small compounds comprise the majority of the cellular molecules and bind proteins as substrates, products, cofactors and ligands. However, a large scale investigation of in vivo protein‐small metabolite interactions has not been performed. We have developed a mass spectrometry assay for the large scale identification of in vivo protein‐hydrophobic small metabolite interactions in yeast and used it to analyze compounds that bind ergosterol biosynthetic proteins and protein kinases. 17–20% of these proteins bind small metabolites; a few interactions are known, but the vast majority are novel. Importantly, many key regulatory proteins such as protein kinases bind small metabolites. Ergosterol was found to bind many proteins and may function as a general regulator. It is required for the full activity of Ypk1, a homolog of mammalian AKT/SGK1 kinase family. Overall, our study defines potential key regulatory steps in lipid biosynthetic pathways and suggests small metabolites may play a hitherto underappreciated role as general regulators of protein activity and function.
Mechanisms of metabolic diversification in plants
Authors: Osbourn Anne Elisabeth
Published: June 2021

Groups of non‐homologous functionally‐related genes are commonplace in bacteria, where they are mostly organised as operons. Gene clusters are rare in eukaryotes and usually consist of paralogs that have evolved through gene duplication. However clusters of functionally‐related but non‐homologous genes have been discovered in eukaryotic microbes and animals, and are now emerging as a new theme in plant biology. So far nine such clusters have been found in plants, all encoding genes for the synthesis of secondary metabolites associated with biotic/abiotic stress tolerance. These secondary metabolic gene clusters are amongst the most diverse and rapidly evolving features of plant genomes and provide tantalizing links with adaptive genome plasticity in microbes and animals. The fact that such clusters are often restricted to narrow taxonomic bands marks them as among the most diverse and rapidly evolving features of plant genomes, as such providing excellent read‐outs for investigating genome plasticity and mechanisms of adaptive evolution. The study of these clusters will yield new insights into the rules and mechanisms governing the formation, function, maintenance and dissipation of operon‐like metabolic gene clusters in plants, and into plant genome dynamics and the factors that shape genome architecture.
Mobilization of very small embryonic‐like stem cells by G‐CSF in MACO mice
Authors: Zheng Min , Wang Jian
Published: June 2021

Objective

To investigate the effect of G‐CSF on neural restoration and on mobilization of very small embryonic‐like stem cells(VSEL‐SC) in bone marrow of focal cerebral ischemia mouse.

Methods

MCAO model was produced by filament occlusion method. 50¦Ìg/(kg.d) G‐CSF were given by subcutaneous injection for 5 days. Neurological scale was evaluated. The number of VSEL‐SC (CXCR4+Lin‐CD45‐) in peripheral blood was checked by fluorescence‐activated cell sorting analysis (FACS). Level of stromal derived factor‐1(SDF‐1) in plasma and infarct cerebral tissue were checked by ELISA. Expression of SDF‐1 in ischemia area was checked by immunohistochemical staining.

Results

G‐CSF treatment significantly decreased the neurological scale of MCAO model mice compared with controls. G‐CSF could mobilize VSEL‐SC in the bone marrow of focal cerebral ischemia mice into peripheral blood. The concentration of SDF‐1 in plasma and in cerebral tissue significantly increased in G‐CSF treated group. Expression of SDF‐1 in infarct region significantly increased in G‐CSF treated group.

Conclusion

The beneficial effect of G‐CSF may be related to the mobilization of VSEL‐SC and the promotion of SDF‐1 expression in the infarct tissue of MACO mice.

This work was supported by National Scientific Foundation Committee of China ( 31100985)
Topical Treatment with the Opioid Antagonist Naltrexone Facilitates Closure of Full‐Thickness Wounds in Diabetic Rats by Accelerating Neovascularization
Authors: Zagon Ian S , Immonen Jessica A , McLaughlin Patricia J
Published: June 2021

Patients with type 1 diabetes (T1D) are at risk for complications from aberrant wound healing that can lead to debilitating ulcers and amputation. The pathophysiology of diabetic wound repair is not understood. Delays in healing of cutaneous full‐thickness wounds in T1D rats can be reversed by blockade of endogenous opioids and their receptors. We hypothesized that blockade of opioid‐receptor pathways by topical application of naltrexone (NTX) accelerates angiogenesis in wounds of T1D rats. Six‐weeks after induction of hyperglycemia, rats received 5 mm diameter full thickness wounds. Topical NTX (or sterile water controls) was administered 3x/day, and tissues evaluated with markers for angiogenesis. On day 3 of treatment, the number of blood vessels positive for FGF and VEGF were 3‐fold greater in T1D rats exposed to NTX compared to T1D controls. On days 5 and 8, there was a 2.7‐fold increase in VEGF stained vessels in NTX‐treated T1D wounds in contrast to T1D controls, and wounds treated with NTX in T1D rats had comparable numbers of blood vessels to those of non‐diabetic animals. Development of smooth muscle tissue in vasculature (measured by α‐SMA) was increased over 2‐fold on days 8 and 10 in wound tissue of NTX‐treated T1D rats relative to T1D controls. These results suggest that there is an exaggerated role of endogenous opioid pathways in the pathogenesis of T1D wound repair, and treatment with an opioid antagonist can restore wound closure by accelerating angiogenesis in T1D rats. Supported by American Diabetes Association grant 7‐09‐BS‐128.
Characterization of hepatocyte‐specific SRSF1 (SF2/ASF) knockout mice
Authors: Langiewicz Magda , Sen Supriya , Webster Nicholas J
Published: June 2021

Alternative splicing is widely believed to have a major impact on almost all biological processes since it increases proteome complexity and thereby controls protein function. SRSF1 (SF2/ASF) is a member of the SR protein family, which are essential for constitutive splicing and fulfilling regulatory functions in alternative splicing. Previously, we have shown that SRSF1 binds to the alternatively spliced exon 11 of the insulin receptor (INSR) and promotes its incorporation in hepatoma cells. To demonstrate the physiological importance of alternative splicing to the regulation of metabolism, we created a hepatocyte‐specific knockout of SRSF1 (SRSF1LKO) by crossing SRSF1 flox/flox mice with albumin‐cre mice, which specifically express the cre recombinase in adult hepatocytes. Mice are born in the expected mendelian ratio and do not show any growth retardation unlike the hepatocytes specific SRSF3 knockout that we have previously characterized. We will present data showing efficient tissue specific deletion of the SRSF1 allele, the reduction in SRSF1 expression, and alterations in INSR splicing. We will also present data on the metabolic and physiological parameters and liver morphology between SFSR1LKO and littermate control mice, as well as alterations in global gene expression and splicing by exon array analysis. SFSR1LKO is an important model to study alternative splicing in vivo.

Supported by a Merit Review Award from the Department of Veterans Affairs.
Age‐dependent regulation of non‐coding RNAs in male germ cell development
Authors: Luk Alfred CS , Ng Shuk Han , Chan Wai-Yee , Lee Tin-Lap
Published: June 2021

Parental aging is a global problem that poses higher risk of genetics disorders for the offspring. While maternal aging has been exclusively studied, the effects of paternal aging remains largely elusive. Despite advances in functional genomics, majority of observations were taken from traditional genetics studies. Here we would like to examine the effect of paternal aging in male reproduction through studying the development of male germ cells. Rather than turning to known gene regulation, we identified non‐coding RNAs (ncRNAs) that demonstrate age‐dependent developmental regulation in male germ cell development by applying integrative analysis of our previous established GermSAGE and TileMapper resources. The findings strongly suggested the pivot role of ncRNAs in male germ cell development, and are expected to substantially change the concepts of ncRNA function in developmental, reproductive and stem cell biology.
Role of actin polymerization during early s‐looping of the vertebrate embryonic heart
Authors: Chu-LaGraff Quynh , Benn Kevan , LaGraff J. Luc , Ramasubramanian Ashok
Published: June 2021

During vertebrate development, the embryonic heart undergoes sequential morphogenesis to transition from c‐looping to s‐looping morphology. C‐looping begins at stage 10 (36 hrs.) and finishes by stage 12 (48 hrs.) when the heart tube gets its characteristic c‐shaped bend. Early s‐looping begins shortly thereafter and finishes by stage 16 (56 hrs.), when the primitive atrium moves superior to the primitive ventricle. Previous studies indicated that actin polymerization is essential for cell‐shape changes generating the characteristic bend of c‐looping. Here we extend the study to investigate the role of actin polymerization and orientation in driving the next phase of heart morphogenesis, the early s‐looping stage. Whole 48‐hr chicken embryos are isolated and cultured in varying concentrations of Cytochalasin D, an actin polymerization inhibitor, until the end of early s‐looping. Treated cultures are fixed, stained with Phalloidin‐Alexa 488 and wheat germ agglutininâ€” Alexa 594, and imaged using confocal microscopy. We will present results from these inhibitor studies detailing the three‐dimensional intracellular arrangement of actin and how this promote changes in overall heart morphology during early s‐looping. Research supported by NIH‐AREA 1F32HL079764.