bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of the two‐pore domain potassium channel, TASK‐2, in rat pancreatic acinar cell zymogen granule membrane
Authors: Lotshaw David Paul , Grayburn W. Scott , Yasui Linda S. , Gasser Kenneth W.
Published: June 2021

Ion flux across pancreatic secretory vesicle membranes contributes to exocytotic secretion: mediating vesicular swelling involved in “kiss and run” exocytosis, promoting solubilization and release of vesicle content, and contributing to intravesicular pH changes. The present study implicates the pH sensitive TASK‐2 channel in one or more of these processes: TASK‐2 expression was documented and localized to zymogen granule membranes and secretion shown to be modulated by TASK‐2 selective drugs. TASK‐2 mRNA expression was demonstrated using SYBR Green real time PCR. Total RNA was isolated from purified acinar cells obtained by Percoll density gradient centrifugation of collagenase‐dispersed cells. Reverse transcription was performed on 1 μg of DNase‐treated total RNA using random decamer primers. TASK‐2‐specific forward and reverse primers amplified a single product from the cDNA based on dissociation curve analysis. Western blot analysis using an anti‐TASK‐2 antibody (Alomone) revealed two immunoreactive protein bands in a crude microsomal fraction migrating as a doublet at 54 and 55 kD (the predicted mass of rTASK‐2 is 58 kD); identical protein bands were obtained in Western blots of purified zymogen granule membranes. Confocal microscopy revealed that immunoreactivity was cytoplasmic and primarily localized to the apical pole. Finally, CCK‐induced amylase secretion from diced pancreatic tissue fragments was increased 100% by the TASK‐2 agonist, halothane, without affecting basal release and completely inhibited by the TASK‐2 inhibitor, clofilium, without affecting basal secretion.
Impaired prostaglandin E2 (PGE2) production in urothelial cells from an interstitial cystitis patient
Authors: Rastogi Prerna , Dorokhov Nikolay , Rickard Alice , McHowat Jane
Published: June 2021

SR22 cells are from the inflamed area of the bladder of a patient with interstitial cystitis (IC), a disease characterized by increased activated mast cells in the bladder wall and compromised urothelial function. Since the urinary content of tryptase is increased in IC, this study determined whether there was impairment of prostaglandin E2 (PGE2) release from SR22 cells in response to tryptase. Responses in SR22 cells were compared to responses observed in PD07i cells derived from a healthy bladder. Interestingly, although PAR‐2 density was significantly lower in SR22 cells, tryptase stimulation of either SR22 or PD07i resulted in a similar increase in phospholipase A2 activity and arachidonic acid release. However, SR22 cells did not show any increase in PGE2 release in response to tryptase, whereas there was a significant increase in PGE2 release from tryptase‐stimulated PD07i. The levels of COX‐1 and COX‐2 mRNA expression were equivalent, but mRNA expression of PGE synthase was considerably lower and of hydroxyprostaglandin dehydrogenase was considerably higher in SR22 compared to PD07i, suggesting that both decreased production and increased metabolism could responsible for the lack of PGE2 response. Since PGE2 is cytoprotective, these data may explain why IC is associated with impaired ability to resist cell damage and warrants further investigation.

Supported by DK66119 (JM)
Effect of thyrotropin‐releasing hormone on neuroventilation in bullfrogs
Authors: Richter M M , Brundage C M , Taylor B E
Published: June 2021

Thyrotropin‐releasing hormone (TRH), a peptide hormone that is found in all classes of vertebrates, is also synthesized in brainstem raphe neurons and may exert direct excitatory actions on ventilatory‐related neurons. The role of TRH as a neuroventilatory stimulant in amphibian species has received little attention. Neuroventilation and neuroventilatory responses of the in vitro en bloc bullfrog tadpole brainstem have been well characterized, and serve as an excellent model for assessing potential modulators of amphibian neuroventilation. TRH (0.1– 10μM) was bath applied to the in vitro en bloc brainstems of early‐stage tadpoles. Bath application of TRH increased the frequency of both lung and buccal bursts. These data indicate that TRH modulates activity in the amphibian ventilatory control network and may suggest functional homology of the amphibian and mammalian networks. Funded by NIH‐NINDS 2U54NS041069‐06A
Bursts of brown adipose tissue (BAT) metabolism underlie periodic fluctuations in body temperature (Tb) in rats: a newly discovered ultradian rhythm
Authors: Ootsuka Youichirou , Almioradian Abbas , Blessing William W.
Published: June 2021

Mammalian Tb exhibits ultradian rhythms. We have investigated whether BAT thermogenesis contributes to these rhythms in rats. Sprague Dawley rats (330–370 g) were instrumented under isoflurane anesthesia for continuous measurement of Tb (SubCue, in peritoneal cavity), interscapular BAT temperature (Data Sciences International, TA‐F40W/TP) and tail artery blood flow (Iowa Doppler probe around base of tail artery). A headpiece could be attached by flexible tubing to a swivel device. At least one week later conscious unrestrained rats were placed in a controlled temperature environment with reverse day‐night cycle lighting and ad libitum access to food and water. Ambient temperature was maintained constant for 24 hours, with different temperatures (18–30°C) on different days. Temperature of BAT increased by 0.9±0.2°C every 112±6 min (n=4 rats, 16 occasions), in both light and dark cycles, closely correlated with increases in Tb. Onset of iBAT activity was followed within a few minutes by a sudden increase in tail blood flow. Our results provide the first demonstration that sudden increases in heat production by BAT make a major contribution to ultradian rhythms in Tb. Supported by NHMRC.
Effects of Exercise Intensity and Duration on Arachidonic Acid Metabolism
Authors: Giordano Rose M , Newman John W , Ramos Marisa , Gale Barbara , Stebbins Charles L
Published: June 2021

Metabolites of the cytochrome P450 pathway may contribute to vasodilation of the vasculature of skeletal muscle during exercise. We determined effects of exercise intensity and duration on plasma concentrations of specific metabolites in the epoxyeicosatrienoic acid family. This allowed us to determine the threshold workload, optimal workload, and time period required to produce increases in plasma concentrations of these vasoactive substances. Fourteen healthy volunteers performed maximal exercise testing on a bicycle ergometer during visit 1. On separate days subjects performed exercise testing for 20 min at 30%, 60%, and 80% of their maximal workload. The last day consisted of 40min of exercise at 60% workload. Venous blood was obtained before, during and after exercise for analysis. Compared to baseline, significant increases were observed during the 80% workload at 20 min: 14,15 DHET (0.76±0.20 vs. 0.93±0.27 pmol/ml) and at 2 min post exercise: 11,12 DHET (0.64±0.22 vs. 0.71±0.24 pmol/ml). Mean 40 min values at 60% workload were: 14,15 DHET (0.79±0.22 vs. 0.91±0.31 pmol/ml)and at 2 min post: 14,15 EET (0.11± 0.05 vs 0.21 ±0.16 pmol/ml). Results suggest that these metabolites are released during short term high intensity or long term moderate intensity exercise.
In vivo mitochondrial function varies with muscle and training status in young adults
Authors: Larsen Ryan Godsk , Callahan Damien M. , Lanza Ian R. , Kent‐Braun Jane A.
Published: June 2021

Phosphorus magnetic resonance spectroscopy (MRS) was used to measure the effect of training status on skeletal muscle oxidative capacity (Vmax), an in vivo marker of mitochondrial function, in two morphologically‐distinct human muscles‐tibialis anterior (TA) and vastus lateralis (VL).

Methods: Healthy, untrained (7F, 7M, 26±1 yrs, mean±SE) and trained (7M, 1F, 28±1) adults performed a 16‐s and a 24‐s maximal voluntary isometric contraction of the TA and VL, respectively, while intracellular phosphocreatine (PCr) concentration was measured by MRS. The rate constant of post‐contraction PCr recovery (kPCr) was determined and used to calculate Vmax (kPCr · resting [PCr]).

Results: There were main effects of muscle and group on Vmax and kPCr such that oxidative capacity was higher in VL than TA (p≤0.046), and higher in trained than untrained (p<0.0001).

Conclusion: This study provides novel evidence of muscle‐specific differences in in vivo mitochondrial function that is independent of training status.
Contraction‐induced fatty acid uptake and oxidation are regulated in part via CaMKKβ‐dependent AMPK activation in perfused rat muscle
Authors: Abbott Marcia Jeanine , Turcotte Lorraine
Published: June 2021

Ca2+/calmodulin‐dependent protein kinase kinase (CaMKK) and AMP‐activated protein kinase (AMPK) have been individually implicated in the regulation of muscle substrate metabolism during exercise. The purpose of this study was to determine if CaMKKβ is involved in the regulation of FA metabolism during muscle contraction in rats. Rat hindlimbs were perfused at rest (n=16), with 3mM caffeine (n=15), with 2mM AICAR (n=16), or during moderate intensity muscle contraction (n=14), and with or without 5μM STO‐609, a CaMKKβ inhibitor. FA uptake and oxidation increased (P<0.05) by 83 and 68% with caffeine treatment, 54 and 94% with AICAR treatment and 78 and 126% with muscle contraction. STO‐609 abolished (P<0.05) caffeine‐ and contraction‐induced FA uptake and oxidation, but had no effect with AICAR treatment (P>0.05). CaMKKβ activity was increased (P<0.05) 102% by caffeine treatment and 136% by muscle contraction, but was not affected by AICAR treatment (P>0.05). STO‐609 prevented the caffeine‐ and contraction‐induced increase in CaMKKβ activity. Caffeine, AICAR and muscle contraction increased (P<0.05) AMPKα2 activity by 240%, 12‐fold and 496%, but did not affect (P>0.05) AMPKα1 activity. STO‐609 decreased (P<0.05) AMPKα2 activity by 54 and 52% during caffeine treatment and muscle contraction, but did not affect (P>0.05) AICAR‐induced activity. These results show the critical importance of Ca2+‐dependent signaling via CaMKKβ activation in the regulation of FA uptake and oxidation in contracting rat skeletal muscle.
Intermittent obstructive apnea in conscious rats
Authors: Schoorlemmer Guus H. M. , Rossi Marcio V. , Pedrino Gustavo R. , Cravo Sergio L. D.
Published: June 2021

We developed a new method to intermittently obstruct the airway in conscious rats to replicate the hypercapnea and respiratory effort characteristic of sleep apnea. A teflon tube (6 mm long, o.d. 2.5 mm, i.d. 2.1 mm) was inserted in the trachea of adult male rats. This tube contained the tip of an inflatable balloon catheter made from polyurethane tubing (10 cm long, o.d. 1.0 mm, i.d. 0.6 mm, Scientific Commodities, USA). The open end of the balloon catheter left the trachea through the incision, and was led subcutaneously to a metal connector that was externalized between the shoulder blades. Thus inflation of the balloon catheter closed the trachea without causing pain. Rats rapidly regained body weight after surgery and appeared healthy, although breathing occasionally was audible during the subsequent 1 – 2 months. Inflation of the balloon for 10 sec with 15 μL water caused forceful breathing movements. Heart rate became irregular and fell by up to 200 beats/min, and these effects were blocked by pretreatment with 3 mg atropine. Balloon inflation increased blood pressure by up to 40 mm Hg, and caused caused alertness, locomotion and rearing. These effects disappeared almost instantly after disinflation. Inflations repeated every 3 min gave reproducible cardiovascular changes. This method seems suitable for the analysis of the effects of chronic intermittent apnea. Financial support: FAPESP, CNPq.
Improvement of Ca2+ Transport and Muscle Relaxation in Skeletal Muscle From Sarcolipin Null Mice
Authors: Tupling A. Russell , Hussain Dawar , Trivieri Maria G. , Babu Gopal J , Backx Peter H. , Periasamy Muthu , MacLennan David H. , Gramolini Anthony O.
Published: June 2021

Sarcolipin (SLN) is an inhibitor of SERCA type pumps by direct binding. To demonstrate whether ablation of SLN in skeletal muscle might impair skeletal muscle function directly, we analyzed skeletal muscle contractility and Ca2+ uptake in SLN null mice (KO). SLN protein expression in wildtype mice (WT) was found to be highest in the quadriceps and gastrocnemius (G) followed by the diaphragm and soleus (Sol), whereas very little SLN expression was seen in the tibialis anterior or extensor digitorum longus (EDL). No SLN protein was detected in muscles from KO. Compared with WT, Ca2+ uptake was increased in the G and Sol of KO muscles, while no significant difference was observed in the EDL. Contractility measurements of WT and KO Sol and EDL muscles were performed. No differences were seen between WT and KO muscles in peak tetanic force; however, peak twitch force was lower in KO by ~28% but only in Sol. Maximum normalized (s‐1) rates of contraction (+dF/dt) and relaxation (−dF/dt) measured during a twitch contraction were significantly higher in Sol of KO compared with WT (+dF/dt, 50.2±5.9 in KO vs 30.9±1.0 in WT; −dF/dt, 7.2±0.5 in KO vs 5.2±0.5 in WT). There were no differences between KO and WT in +dF/dt or −dF/dt in EDL. These results show that SLN regulates muscle contractility in mouse slow twitch fibers by inhibiting SERCA function and reducing Ca2+ uptake.

Supported by HSFO (DHM, PHB, AOG), CIHR (DHM, PHB) and NSERC (ART).
Mitochondria's Role in Store‐Operated Calcium Entry
Authors: Barry Christina Jean , Cioffi Donna L. , Stevens Troy
Published: June 2021

Inflammatory agonists deplete calcium from the endoplasmic reticulum and activate calcium entry in a process referred to as store‐operated calcium (SOC). Calcium store depletion can be distinctly measured from calcium entry using a recalcification protocol, in which store depletion is activated in the presence of low extracellular calcium. Upon addition of high extracellular calcium, calcium enters through activated SOC channels. We loaded pulmonary artery endothelial cells with either a membrane‐localized calcium indicator (FFP‐18) or a cytosolic‐calcium indicator (Fura‐2). In the presence of 100 nM calcium, thapsigargin‐induced store depletion transiently increased both membrane and cytosolic calcium and returned to baseline. Upon addition of 5 mM extracellular calcium, SOC entry stably increased membrane calcium whereas cytosolic calcium transiently increased then slowly decreased. We sought to determine whether the decrease in cytosolic calcium was due to calcium uptake by mitochondria. Mitochondrial calcium uptake was inhibited using CCCP or antimycin and oligomycin. Inhibition of mitochondrial calcium uptake increased cytosolic calcium, but did not prevent the decline in cytosolic calcium observed following SOC entry. Thus, mitochondria sequester cytosolic calcium, but are not solely responsible for the decrease in cytosolic calcium measured during SOC entry. Supported by HL60024.
Phosphatidylinositol‐4, 5‐bisphosphate has an inhibitory action on native TRPC6 activity in arterial myocytes
Authors: Albert Anthony , Saleh Sohag , Large William
Published: June 2021

It is generally accepted that TRPC3/C6 and C7 cation channel proteins are activated by diacylglycerol (DAG) via a protein kinase C (PKC) independent pathway but there is no clear evidence on how this DAG‐ mediated process occurs. However a precursor of DAG, phosphatidylinositol‐4, 5‐bisphosphate (PIP2), may also have an important role in modulating the activity of different TRP channels (Voets & Nilius, 2007). We have shown in native rabbit mesenteric arteries that angiotensin II (Ang II) activates a channel, which TRPC6 proteins are likely to represent essential molecular components, through DAG acting via a PKC‐independent mechanism (Saleh et al, 2006) and the present work investigates the effect of PIP2 on this channel activity using whole‐cell and single channel recording.

Inclusion of PIP2 in the patch pipette solution and application of PIP2 to the cytosolic surface of inside‐out patches markedly reduced Ang II‐induced whole‐cell cation conductances and channel activity by over 80% at −50 mV. Moreover application of an anti‐PIP2 antibody increased TRPC6 channel activity induced by either Ang II or OAG in inside‐out patches. In cell‐attached patches bath application of the phosphatidylinositol kinase inhibitors wortmannin and LY294002 and the PIP2 scavenger poly‐lysine produced pronounced activation of TRPC6 channel activity.

These novel data indicate that PIP2, a substrate of DAG production, has a profound inhibitory action of TRPC6 channel activity in native arterial myocytes indicating that both hydrolysis of PIP2 and production of DAG may be important in activating channels from the TRPC3/C6/C7 subfamily of TRPC proteins.

Saleh SN et al (2006). J Physiol 577, 479–495. Voets T & Nilius B (2007). J Physiol 582, 939–944. Work supported by The Wellcome Trust.
Intravascular Pressure Activates Ryanodine Receptors and Elicits Calcium Waves in Cerebral Arterial Smooth Muscle Cells.
Authors: Brett Suzanne E , Bradley Karen N , Welsh Donald G
Published: June 2021

The study examined the effects of intravascular pressure on Ca2+ wave generation in cerebral arterial smooth muscle cells. Rat cerebral arteries stripped of endothelium and loaded with fluo‐4 were mounted in a pressure myograph. Ca2+ waves were monitored with a spinning disk confocal microscope coupled to an ICCD that captured images at a rate of 10 frames per second. Elevating intravascular pressure (15 to 80 mmHg) increased Ca2+ wave frequency (0.56±0.3 to 8.47±0.5 events min−1) and the proportion of firing cells (6.98±2.9 to 66.43±8.7%). The generation and maintenance of these asynchronous events was largely voltage‐independent since Diltiazem (30 μM; L‐type Ca2+ channel blocker) application and manipulations of resting membrane potential had only a limited effect on pressure‐induced Ca2+ waves. Ryanodine (50 μM) application blocked Ca2+ wave generation in cerebral arterial smooth muscle cells and attenuated pressure‐induced vasoconstriction. In closing, mechanical stimuli are capable of activating ryanodine receptors and augmenting the generation of Ca2+ waves in a manner that is independent of resting membrane potential. While Ca2+ waves are asynchronous among neighboring smooth muscle cells, their generation contributes to the maintenance of myogenic tone in the cerebral circulation. Supported by the Natural Science and Engineering Research Council of Canada.
Kidney‐associated brown adipose tissue. Potential relationship to etiology of genetic hypertension in the SHR (spontaneously hypertensive rat)
Authors: Thomas Joanna , Sweeney Derina , Liu Jia , Breen Laura , Printz Morton P
Published: June 2021

Brown adipose tissue (BAT) is found proximal to many neonate and adult mammalian organ systems. Initial studies of SHR and normotensive BNLx strains found differential expression of uncoupling protein 1 (UCP1) mRNA in the medullary‐pelvic area of the kidney and an association with level of blood pressure. AIMS: The study aims were to identify the location of kidney‐associated BAT, analyze differences in BAT morphology and gene expression and test association with hypertension. METHODS: Immunofluorescent microscopy (IFM) and H&E staining were used to examine cell morphology, perilipin and UCP1 protein staining in 4 and 12 week animals. RT‐PCR quantified expression of genes important to BAT and lipid metabolism and test if different between SHR and BNLx. Cell functional differences were probed by cold exposure experiments. RESULTS: BAT was localized to the kidney ureter‐pelvic junction in proximity to the ureter adventitia, renal nerves and blood vessels. Striking differences were found in BAT cell structure and size between the strains. BAT of both strains responded differentially to cold exposure (morphology and UCP1 expression) but other studies found defective SHR response to ephedrine. CONCLUSIONS: We conclude BAT is located immediately proximal to the major vascular and nerve conduits to the kidney which may enable BAT to affect renal functions. Finding distinct cellular and functional differences between pre‐hypertensive 4 wk old genetically hypertensive and normotensive strains implicate BAT as potentially related to hypertension development. Future studies are warranted into the role of kidney‐associated BAT with regards to the pathology of hypertension and metabolic syndrome. Supported by NIH HL‐35018.
Different effect of acute heart failure on stepwise frequency bands of QRS complex in electrocardiogram
Authors: Matsushita Shonosuke , Tokunaga Chiho , Enomoto Yoshiharu , Kanemoto Shinya , Watanabe Yutaka , Hiramatsu Yuji
Published: June 2021

We found that high frequency component of QRS complex in EKG decrease in acute heart failure after open‐heart surgery. It became one of the indexes to recognize degree of heart failure electrically. However, which frequency bands of QRS complex are most sensitive to heart failure has not been identified. To clarify this, relationship between voltage of stepwise frequency bands (1–20Hz, 20–40Hz, 40–80Hz, >80Hz) of QRS complex and left ventricular pressure (LVP) was examined in 4‐minopyridine (4‐AP) induced rat heart failure model. LVP was decreased by use of 4‐AP (from 82+/−23 to 37+/−20 mmHg: p<0.005). Although lower frequency bands (1–20Hz, 20–40Hz) were unchanged, higher frequency bands (40–80Hz, >80Hz) were decreased from 4.1+/−2.0 to 1.6+/−1.4 (mV)^2 (p<0.01), from 3.0+/−1.2 to 0.3+/−0.4 (p<0.0001) respectively when LVP decreased. After recovery of LVP, voltage of high frequency both bands returned to pre‐treatment level. Most sensitive frequency bands to heart failure was bands of >80Hz. Since Ca2+ overload to cardiomyocytes decreased high frequency component of QRS complex from our previous study, elevation of intracellular Ca2+ concentration during heart failure may pivotal role in this phenomenon.
Bioenergetics of adaptation to the environmental hypoxia of high altitude at rest and exercise
Authors: Reynafarje Baltazar Davila
Published: June 2021

The effect of hypoxia on the mechanism of energy transduction has been studied by determining the rates of O2 uptake and energy generation in both humans and guinea‐pig mitochondria under resting and active metabolic conditions. The following findings were made. 1) The O2 level required for half maximal rates of ATP syntesis (Km for O2)is higher than 0.03 mM or 20 torr. 2)The so‐called lactate paradox of high altitude adaptation is no more than an improved efficiency in the mitochondrial process of energy transduction. 3)The amount of ATP formed or energy generated per atom of O2 consumed by guinea‐pig mitochondria or humans during strenuous physical exercise is significantly higher in natives to high altitude than in those from sea level. 4) The mitochondrial rates of ATP synthesis are orders of magnitude higher than under state‐3 metabolic conditions provided the membrane is fully reduced, a conditions that is optimally attained during short periods of maximal hypoxia. It is concluded that athletes improve the reduced state of their muscles by enhancing the hypoxia during the warm up period, and that the most important factor in determining the extent and rates of energy transduction an ATP syntheis is not the proton gradient or the level of ADP but the level of O2 and the conformational state of the mitochondria
Regulation of Renal Angiotensin Converting Enzyme 2 (ACE2) Activity by Angiotensin II (AngII)
Authors: Liu Jun , Ji Hong , Kraus Carl , Zheng Wei , Wu Xie , Sandberg Kathryn
Published: June 2021

ACE2 is a monocarboxypeptidase that counteracts the vasoconstrictor effects of AngII by converting AngII to Ang[1‐7] in kidney and other tissues. We studied renal ACE2 in AngII‐induced hypertension in male (M) and female (F) mice after developing a sensitive assay for ACE2 activity in the mouse kidney. While no sex differences were found in ACE2 Km, under control (Ctr) conditions, ACE2 Vmax (maximum enzyme velocity) was 1.9‐fold greater in M vs F [relative fluorescence units (RFU): M‐Ctr, 1550±16 vs F‐Ctr, 823±120, p<0.0001]. After AngII infusion (800 ng/min/kg x 7d), ACE2 Vmax was increased in both sexes though the magnitude of this increase was greater in F [RFU: M‐AngII, 1890±87 vs F‐AngII, 1200±58, n=5; p<0.0001]; AngII increased renal ACE2 activity by 22% in M vs 45% in F. Mean arterial pressure (MAP) was measured by radiotelemetry. After AngII infusion, peak MAP was higher in M vs F mice [MAP (mm Hg): M‐AngII, 145±5.5 vs F‐AngII, 132±3.8]. This study demonstrates that renal ACE2 activity is up‐regulated by infusing the AngII substrate in both sexes. Up‐regulation of renal ACE2 may be a compensatory feedback mechanism for counteracting the increase in plasma AngII and MAP induced by AngII infusion. The finding that the magnitude of this effect is greater in F vs M raises the possibility that these differences in renal ACE2 feedback contribute to why M mice have higher MAP than F after AngII infusion (R01 AG19291).
Hydraulic conductivity and solute permeability of an in vitro blood‐brain barrier (BBB) model
Authors: Fu Bingmei , Li Guanglei , Simon Melissa , Shi Zhongdong , Cancel Limary , Morrison Barclay , Tarbell John
Published: June 2021

The BBB is a dynamic interface between the circulation system and the central nervous system (CNS). It serves as a barrier to potentially harmful molecules, and a facilitator for transport of nutrients to ensure an optimal microenvironment in CNS. However, it also provides a major obstacle for drug delivery to the brain for the treatment of CNS disorders. It is thus very important to investigate the transport properties of the BBB. In our study, an in vitro BBB model was developed by culturing mouse brain microvessel endothelial cells (bENd3, ATCC, VA) into monolayer on a Transwell system (0.4 μm pore size, 4x106 pores/cm2, Corning, NY). Using a bubble tracker system and an automated fluorometer system, we quantified the hydraulic conductivity (Lp) and solute permeability (P) of this in vitro BBB model, respectively. The measured Lp is 1.5 (±0.34, n=2) x10−7cm/s/cmH2O. The measured P to TMR‐Dextran 70K and P to TMR‐Dextran 10K are 0.57 (±0.13, n=8) and 1.6 (±0.34, n=6) ×10−6 cm/s, respectively. While P values of this in vitro model for both solutes are only several times larger than the in vivo data (Yuan et al., 2008), Lp is two orders of magnitude larger. Our results suggest that the basement membrane and astrocyte processes, which surround the endothelium, contribute significantly to the BBB permeability. A co‐culture system with these components needs to be developed. Supported by the Andrew Grove Foundation.
Calcium‐dependent and ‐independent mechanisms in the response to oxygen of the chicken ductus arteriosus
Authors: van Cleef Anne NH , Zoer Bea , Cogolludo Angel L , Perez‐Vizcaino Francisco , Villamor Eduardo
Published: June 2021

We aimed to investigate the relative contribution of voltage‐ and store‐operated Ca2+ channels (VOCC and SOCC respectively), intracellular Ca2+ stores and Ca2+ sensitization in O2‐induced contraction of chicken ductus arteriosus (DA). Isolated DA rings from 15 and 19‐d chicken fetuses (total incubation 21‐d) were mounted in a myograph and exposed to normoxia. In the 19‐d DA, O2‐induced contraction was reduced by the absence of extracellular Ca2+ (28.7% of control contraction) or by the presence of the L‐type VOCC antagonist nifedipine (10 μM; 45.0%); the sarcoplasmic reticulum Ca2+ ATPase inhibitors thapsigargin (0.1 μM; 80.7%) and cyclopiazonic acid (0.1 μM; 22.2%); the SOCC inhibitor SKF 96365 (10 μM; 41.7%); the blocker of Ca2+ release from IP3‐sensitive stores 2‐APB (0.5 μM; 44.0%); and the Rho‐kinase inhibitor hydroxyfasudil (1 μM; 62.3%). The combination of nifedipine, thapsigargin, SKF 96365 and hydroxyfasudil abolished O2‐induced contraction. The L‐type VOCC agonist BAY K8644 contracted the 19‐d DA in a concentration‐dependent manner. This contraction was not affected by O2 tension. The 15‐d DA showed a very weak O2‐induced contraction and did not respond to BAY K8644. In conclusion, our results indicate that Ca2+ entry through VOCC and SOCC, Ca2+ release from intracellular stores and Rho‐kinase‐mediated Ca2+ sensitization contribute to O2‐induced contraction of the chicken DA. Functional immaturity of VOCC appears to play a role in the lack of O2 responsiveness in the immature chicken DA.

Supported by "Fundación de Investigación Médica Mutua Madrileña".
Noninvasive imaging of hemorrhage‐induced global ischemia with a transgenic mouse expressing luciferase coupled to hypoxia‐inducible factor (HIF1α)
Authors: Bowman Phillip , Bynum James , Kiang Juliann G. , Baer David , Johnson Joanna , Dubick Michael
Published: June 2021

Hemorrhagic shock leads to global ischemia, but available blood is distributed unevenly to the body's organs and it is generally accepted that blood is shunted to those organs that maintain critical functions. The organs that become most ischemic and their role in hemorrhagic shock, however, have not been determined. We used the recently described, genetically engineered mouse FVB.129S6‐Gt(ROSA)26Sortm1(HIF1α/luc) Kael/J* (Jackson Laboratories) which has a luciferase gene fused to the region of HIF1α that binds to von Hippel‐Lindau protein in an oxygen‐dependent manner generating a reporter that can be used to monitor oxygen availability in intact tissues. Thus more light would be emitted from ischemic tissue. To determine if this mouse could be used to identify organs affected by hemorrhagic shock, 40 per cent of the calculated blood volume was removed via the submaxillary vein, and the mice were injected with luciferin, anesthetized with isoflurane, and imaged in the Xenogen IVIS 100 Imaging system as a function of time after hemorrhage. The ventral surface of these mice exhibited increased light emitted from the region of the intestine that was most prominent at 3‐6 h after hemorrhage but still evident at 72 h. This technique should allow more detailed studies of those tissues most affected by hemorrhagic shock.

*Safran, M.,et. al., Proc Natl Acad Sci U S A 2006;103:105‐110.
Change in vitamin D status is associated with improved physical performance over one year: the LIFE‐P study
Authors: Houston Denise K , Tooze Janet , Neiberg Rebecca , Hausman Dorothy , Johnson Mary Ann , Nicklas Barbara , Miller Mike , Marsh Anthony , Bartali Benedetta , Newman Anne , Blair Steven , Kritchevsky Stephen B
Published: June 2021

Vitamin D insufficiency is common among older adults and may play a role in musculoskeletal function; however, few studies have examined the longitudinal association between vitamin D and physical function. Thus, we examined the association between vitamin D status and change in vitamin D status and physical performance in the Lifestyle Interventions and Independence for Elders Pilot (LIFE‐P) Study. Plasma 25(OH)D and physical performance using the short physical performance battery (SPPB) score were assessed at baseline, 6 and 12 months. Mixed models were used to examine the associations between 25(OH)D status (<50 nmol/L vs. ≥50 nmol/L) and SPPB in models adjusted for demographics, intervention group, season, physical activity and BMI (n=368). The mean age of the participants was 76.7 yrs; 68% were women, 76% were white, and 50% were vitamin D sufficient (≥50 nmol/L) at baseline. Vitamin D sufficiency was associated with higher SPPB scores at baseline (Mean difference (SE): 0.35 (0.16), p=0.03). Going from vitamin D insufficient to sufficient was associated with improved SPPB scores at follow‐up (0.57 (0.22), p=0.01). In conclusion, sufficient vitamin D status and change from insufficient to sufficient vitamin D status were associated with better physical performance. Supported by the NIA (U01 AG22376); the WFU OAIC (P30 AG021332); K01 AG030506; and the Sticht Foundation.
Caveolin‐1 scaffolding domain peptides inhibits endothelial cell proliferation
Authors: Fang Kai , Terracciano Justin , Kevil Christopher G
Published: June 2021

Angiogenesis is the process of new blood vessel sprouts from existing capillary networks with endothelial cell proliferation serving an essential role in this process. Caveolin‐1 protein is a plasma membrane protein governing caveolae formation that interacts with several signaling receptors and when over‐expressed can inhibit endothelial cell proliferation. However, the molecular mechanism of caveolin‐1 inhibition of endothelial proliferation is not known. Here we demonstrate that the Cav‐1 scaffolding domain (AP‐Cav amino acids 82‐101) significantly inhibits VEGF stimulation of Erk 1/2 phosphorylation and BrdU incorporation of endothelial cells compared to control AP peptide. Our studies demonstrate that the caveolin‐1 scaffolding domain peptide functionally inhibits endothelial cell proliferation which may be useful in designing novel types of angiogenesis inhibitors.
The relationship between variable SHH signaling and the severity of structural defects in the face and brain
Authors: Young Nathan M. , Hu Diane , Chong H. Jonathan , Hallgrímsson Benedikt , Liu Wei , Marcucio Ralph
Published: June 2021

Embryonic shape plays an important role in the etiology of craniofacial birth defects, yet how morphological variation is generated early in development and contributes to disease incidence is poorly understood. Here we propose a model in which there is a predictable relationship between variation in Shh signaling, the underlying cellular and molecular processes that control growth, embryonic craniofacial shape, and the severity of facial defects. To test these predictions, we induced variation in Shh signaling in chick embryos using a neutralizing antibody (5E1), imaged embryos with µCT, and calculated the relationship of craniofacial shape to dosage. Our preliminary results demonstrate a continuous range of phenotypes, with a fully penetrant phenotype at the highest dose of cells and progressively more variable and less severe phenotypes at lower doses. These results confirm that the Shh pathway acts in a predictable manner on the incidence and severity of craniofacial dysmorphology, particularly in the brain and face. Ongoing analyses are focused on linking these results explicitly to variation in gene expression and cell proliferation. Ultimately, understanding how genetic and developmental mechanisms interact to generate phenotypic variation, from healthy to diseased, will enable both the prediction of facial malformations in utero and corrective genetic therapies in human embryos.
Stem Cell‐based Therapies for Treating Myocardial Infarction
Authors: Lyons Gary Edward
Published: June 2021

Cell therapy is a potential new means of treating patients with myocardial infarction (MI) and chronic heart disease. Published stem cell research suggests that cell therapy can enhance cardiac repair and improve cardiac function. A variety of cell‐based therapies have been tested to treat rodent models of MI. Cells from bone marrow, adipose tissue, and embryonic stem cells and their derivatives have been reported. All have shown some improvement in cardiac function as shown by echocardiographic measurements. In addition to forming cardiac cell types such as myocytes, endothelial cells and vascular smooth muscle, stem cells also have paracrine effects that reduce post‐MI remodeling. Recent reports have shown that the heart has its own population of stem cells. Isolation and expansion of these cells would permit heart regeneration without the risk of immune rejection. Challenges that remain are finding the optimal donor cells, determining the mechanism by which engrafted cells survive, differentiate and improve cardiac function, and optimizing cell delivery methods.
Incorporation into the pre‐replication complex activates the Mcm2‐7 replicative DNA helicase for phosphorylation by the S‐phase kinase, Cdc7‐Dbf4
Authors: Bell Stephen P , Francis Laura I , Randell John C. W. , Takara Thomas J
Published: June 2021

The essential S‐phase kinase, Cdc7‐Dbf4, acts at eukaryotic origins of replication to trigger a cascade of protein associations that activate the Mcm2‐7 replicative helicase. Also known as Dbf4‐dependent kinase (DDK), this kinase is limiting in cells and acts at individual origins of replication at the time of initiation. Here we address the mechanisms that control the specificity of DDK action. We show that DDK preferentially targets a conformationally distinct sub‐population of Mcm2‐7 complexes that are tightly linked to the origin DNA during pre‐replicative complex (pre‐RC) formation. This targeting requires DDK to tightly associate with Mcm2‐7 complexes in a Dbf4‐dependent manner. Importantly, we find that DDK association with and phosphorylation of origin‐linked Mcm2‐7 complexes require prior phosphorylation of the pre‐RC. Our findings provide insights into the mechanisms that ensure that DDK action is spatially and temporally restricted to the subset of origin‐bound Mcm2‐7 complexes that will drive replication fork movement during S phase and suggest new mechanisms that could regulate origin activity.

This research was supported by funding from the National Institute of General Medical Sciences, the Howard Hughes Medical Institute, and the Leukemia and Lymphoma Society.
Innovative method to collect and analyze dietary intake data using still photographs
Authors: Humphries Kathleen , Pepper Alison C. , Seekins Tom W.
Published: June 2021

Visual imaging technologies open doors to new ways of collecting and analyzing dietary intake information. Currently digital images are used entrepreneurially, and to a minor degree in research. However, validity of the nutrient analyses is lacking and standardized training for photo interpreters needed. We show evaluation data from our RD training program on the dietary intake method, the photo taking and analysis protocols, and validity data from a recent study. Thirty‐two RD raters interpreted and analyzed a reliability series of 30 test meals. Their analyses at the food group level were compared to known meal contents. RD ratings were not clinically different for meal content or quantities for any food group. Mean differences between measured food amounts and RD reported amounts were: grains, .18 serving; vegetables, .64 serving; fruit, .01 serving; dairy, .10 serving; protein, .14 serving; discretionary calories, .11 serving.
Anti‐obesity effects of amino acid in high‐fat diet induced obese mice
Authors: Kobayashi Hisamine , Hirabayashi Yuri , Murakami Hitoshi , Ueda Takeo
Published: June 2021

Anti‐obesity effects of dietary supplemented amino acid have been reported. Dietary supplementation of glutamine, alanine, or leucine decreases high‐fat diet induced obesity in C57BL/6J mice. It was reported that histidine supplementation suppressed food intake and fat accumulation in Wistar rats. And it was shown that dietary arginine supplementation reduced fat mass in Zucker diabetic fatty rats. However there is no information about the anti‐obesity effects of other amino acids than above mentioned. Therefore, we conducted this series of experiments to compare the anti‐obesity effects of 20 kinds of proteinogenic amino acids. Male C57BL/6J mice were fed a high‐fat diet supplemented with either one of 20 proteinogenic amino acids (1‐6 %) for 4 to 8 weeks. Body weight, food intake, epididymal fat weight, and plasma triglyceride concentration were measured. Results showed that supplementation with any one of cystine (3%), histidine (5%), leucine (3%), lysine HCl (3%), threonine (3%), and tryptophan (3%) suppressed body weight gain and visceral fat deposition. Reduction of food intake was observed in cystine and histidine group but not in leucine, lysine, threonine, and tryptophan group. Plasma triglyceride concentration reduced only in lysine group. In conclusion, dietary supplementation of any one of leucine, lysine, threonine, and tryptophan prevents high‐fat diet induced obesity without reduction in food intake, which suggests increased energy expenditure. Among these amino acids, lysine is the most potent amino acid to reduce adiposity, since it decreases diet induced obesity as well as hyperlipidemia.
Oxidative Stress is a Mediator of Endoplasmic Reticulum Stress in Liver Cells
Authors: Gentile Christopher L , Flory Kale , Wei Yuren , Wang Dong , Pagliassotti Michael J
Published: June 2021

Lipids, particularly saturated lipids, induce endoplasmic reticulum (ER) stress in liver cells, although the mechanisms by which they do so are unclear. In the present study, we examined whether administration of the antioxidants taurine or alpha‐tocopherol could reduce lipid‐induced ER stress in liver cells. Primary rat hepatocytes and H4IIE liver cells (n=3 for all conditions) were incubated in a control medium containing 8mM of glucose or the same medium supplemented with albumin‐bound, long‐chain saturated (palmitate) or unsaturated (oleate) fatty acids (250 and 500 uM; 6 or 16 hrs) with and without taurine (0.25‐1.00%) or alpha‐tocopherol (VitE; 200 uM). Oleate did not induce ER stress in either primary hepatocytes or H4IIE cells regardless of dose or incubation time. Conversely, palmitate increased multiple markers of ER stress in both cell types. Taurine reduced palmitate‐induced ER stress in a dose‐dependent manner, reaching a ~50% reduction at the highest (1%) dose. VitE reduced palmitate‐induced ER stress by approximately 50%. These data suggest that lipid‐induced ER stress in liver cells is mediated, in part, by oxidative stress.
Nanomechanics of intrinsically disordered proteins in signaling pathways
Authors: Wang Kuan , Forbes Jeffrey G , Wittebort Richard J , Yadavalli Vamsi K , Tsai Wanxia L
Published: June 2021

Key players in the contractile machineries of muscle and nonmuscle cells possess intrinsically disordered domains that perform essential force and signal transduction functions. These disordered domains exist as ensembles of rapidly equilibrating conformations that manifest as unique elasticity when subjected to external force that in turn regulates protein interactions. We are performing systematic structural, nanomechanical and protein interaction analysis of intrinsically disordered domains in the force generating and bearing molecules with NMR, CD, atomic force microscopy of single molecules and the identification of force‐relevant structural transitions by Steered Molecular Dynamics simulations. For the giant elastic protein titin, the dynamic breaking and rejoining of salt bridges are major force generating events that modulate PEVK elasticity and binding to SH3 domain. For the giant ruler protein nebulin, mechanical unfolding of a helices and re‐orientation of salt bridges accompanied its stretch to full length (1µ) that compresses and stiffens actin filaments. For the motor protein myosin, the unfolding of the disordered coiled coil of S2 appears to be an integral part of the active force generating events. We propose that the shuffling of long‐range, non‐stereospecific salt bridges provide a facile mechanism to tether and untether the flexible chains of intrinsically disordered proteins, which in turn affect elasticity as well as control the accessibility of protein‐protein interaction to these "nanogel" in signaling pathways.
Imaging Real‐Time Gene Expression in Living Cells
Authors: Singer Robert H. , Shav‐Tal Yaron , Darzacq Xavier , de Turris Valeria , Gruenwald David , Chao Jeffrey , Lionnet Timothee , Shenoy Shailesh
Published: June 2021

We developed a system using fluorescent proteins to analyze gene expression in real‐time, and to follow individual mRNPs. An array of genes coding for a functional mRNA that contains 24 repeats of the MS2 coat protein binding motif combined with the MS2 coat protein fused to GFP or YFP allowed us to analyze the kinetics of transcription in real time and to detect single molecules of RNA in live cells. In these studies we used photo‐bleaching of GFP‐labeled mRNAs and of a YFP‐pol II fusion protein and photoactivation of paGFP labeled mRNA. Analysis of the complex process of transcription using fluorescent polymerase as well as fluorescent MS2 proteins provided an opportunity to model the kinetic steps of RNA synthesis. These results yield rate constants for each of the steps of promoter assembly, initiation and elongation. They demonstrate that transcription is inefficient and that polymerases can elongate faster than thought, but can pause stochastically. RNAs can then be followed as single molecules in the nucleus and cytoplasm. The analysis demonstrated that subsequent RNA movements were not directed, but governed by rules of simple diffusion. The kinetics of their transport through nuclear pores yielded a biexponential kinetics. Once in the cytoplasm, mRNAs diffuse but also can be directed to their destinations by virtue of a zipcode motif in the RNA. A zipcode binding protein (ZBP1) is essential for this localization and inhibition of translation. β‐actin can thusly be synthesized at sites of active F‐actin polymerization in migrating fibroblasts or growing neurons. Supported by NIH‐EB, GM.
Structure, reactivity and function of bacterial nitric oxide synthases
Authors: Crane Brian R , Sudhamsu Jawahar , Patel Bhumit , Davydov Roman , Hoffman Brian M
Published: June 2021

Nitric oxide synthases (NOSs) from higher organisms are heme‐based mono‐oxygenases that catalyze the conversion of L‐arginine (L‐arg) to nitric oxide (NO), a potent signaling molecule and cytotoxic agent. Bacterial homologs of mammalian NOSs (mNOSs) have recently been characterized in a subset of mostly gram‐positive bacteria. Crystallographic and spectroscopic analyses of bacterial NOSs bound to substrates, intermediates and inhibitors provide insights into structural and chemical properties important for this enzyme family to produce NO. In particular, NOSs isolated from thermophilic bacteria have slower reaction profiles than their mesophilic counterparts. This property has proved useful for the characterization of oxy‐heme species. The biological roles of bacterial NOS appear to differ from those of mNOS, but overall, they are not well understood. Nevertheless, studies of bacterial NOSs show great promise for unraveling new functions for NO in prokaryotes.
Dynamics of PIP2 regulation and metabolism seen with PIP2‐sensitive ion channels and optical probes.
Authors: Hille Bertil , Falkenburger Björn H. , Jensen Jill Bodily , Suh Byung Chang
Published: June 2021

KCNQ2/3 K+ channels require PIP2 to function. Activation of muscarinic M1 receptors (M1R) turns these channels off by Gq, PLC activation, and PIP2 depletion (Suh & Hille, 2002). To monitor steps of this signaling cascade and plasma membrane PIP2 dynamics, we use FRET pairs, translocation of probes, and the channel current in intact living tsA cells. M1Rs acting through PLC deplete PIP2, make DAG, and shut channels in ~10 s. Following FRET pairs in the receptor signaling pathway shows that receptor activation takes <0.1 s, receptor‐G‐protein interactions ~0.3 s, G protein activation ~2 s, and PLC is activated shortly after. Thus most of the delay in signaling to channels is the time it takes PLC to deplete PIP2 pools. Recovery of PIP2 after M1R agonist takes 100‐200 s. This recovery is wortmannin sensitive so PI(4)P needs to be regenerated as well as PIP2. Recovery time shortens with overexpression of PI 4‐kinase but not PIP 5‐kinase. PIP2 can also be depleted in a few seconds by activating a PIP2 5‐phosphatase with voltage steps (VSP) or with chemical dimerization, generating PI(4)P rather than IP3/DAG. Recovery from the perturbation by VSP takes only 15 s and becomes even faster if PIP 5‐kinase is overexpressed. It is not wortmannin sensitive. Thus the PIP 5‐kinase step in the synthesis of PIP2 is normally significantly faster than the PI 4‐kinase step. Supported by NIH grants RO1 NS08174, RO1 GM83913, T32 GM07108, and the HFSP.