bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute hypotension/hypertension in vitro causes temporary post‐receptor up/down regulation of subsequent arterial contractions
Authors: Bednarek Melissa Lynn , Miner Amy S , Alvarez Silvina M , Speich John E , Ratz Paul H
Published: June 2021

Recent studies suggest that smooth muscle length (L)‐tension (T) curves are dynamic. Using in vitro rabbit femoral artery rings we determined whether vascular smooth muscle (VSM) displays L‐history adaptation. The 2nd of 2 sequential isometric contractions induced after release from the optimum L for muscle contraction (Lo) to 0.8‐fold Lo was 1.1‐fold that of the 1st. If the muscle was stretched briefly from 0.8‐ to 1.2‐ and 1.4‐fold Lo between the 1st and 2nd contractions at 0.8‐fold Lo, the 2nd contraction was 0.9‐fold and 0.4‐fold, respectively, of the 1st. This down‐regulation was not due to tissue damage. The generation of two full L‐T curves, one produced by incrementally increasing muscle L (0.4 ‐ 1.2‐fold Lo UP) , the other by incrementally decreasing muscle L (1.2 ‐ 0.4‐fold Lo DN), revealed that active T and passive T were stronger when starting from a shorter muscle L. The maximum difference between active T UP and active T DN was at 0.6‐fold Lo and between passive T UP and passive T DN was at 1.2‐fold Lo. Preliminary data suggest that this L‐history adaptation was not caused by alterations in cell calcium nor myosin light chain phosphorylation. Together, these data support the hypothesis that numbers of crossbridges rather than crossbridge (thick filament) regulation may play the predominant role in VSM L‐history adaptation. Support: R01HL061320
Serotonin depletion near end‐stage disease does not impair ventilation in a rat model of amyotrophic lateral sclerosis (ALS)
Authors: Nichols Nicole L. , Johnson Rebecca A. , Nashold Lisa J. , Mitchell Gordon S.
Published: June 2021

In rodent models of ALS (SOD1G93A rat), ventilation is maintained until late in disease progression, despite a paucity of surviving respiratory motor neurons. Thus, surviving motor neurons compensate for motor neuron loss and preserve ventilation. We tested the hypothesis that 5‐HT is necessary for such compensatory plasticity by measuring ventilatory capacity following 5‐HT depletion near disease end stage in SOD1G93A rats. When SOD1G93A rats decreased body mass 15%, mutants (and wild type litter mates) were given para‐chlorophenylalanine (PCPA; 300 mg/kg IP) or vehicle injections (0.9% NaCl, 7.5 ml/kg IP) for 2 days. Ventilatory measurements were made during baseline, hypoxic (10.5% O2), hypercapnic (7% CO2) and hypoxic/hypercapnic (10.5% O2/7% CO2) conditions via whole‐body plethysmography (n=3‐5 per group). PCPA had no effect (or actually increased) minute ventilation and tidal volume in all conditions studied in both mutant and wild type rats. PCPA decreased breathing frequency (f) in wild type rats; although f was lower in untreated mutant rats (vs wild type), PCPA had no additional effect. Our data provide no evidence that 5‐HT depletion impairs the capacity to increase ventilation in end‐stage SOD1G93A rats. However, we cannot rule out a role for 5‐HT in the induction (vs maintenance) of compensatory plasticity in SOD1G93A rats.

[Supported by NIH NSO57778.]
Abnormal Neurovascular Control During Exercise After Myocardial Infarction
Authors: Rondon Maria Urbana P. B. , Martinez Daniel G. , Nicolau Jose C , Lage Rony L , Laterza Mateus C. , Trombetta Ivani C. , Matos Luciana D.N.J. , Negrao Carlos E.
Published: June 2021

Objective

We hypothesized that muscle sympathetic nerve activity (MSNA) responses during exercise would be exacerbated whereas forearm blood flow responses would be reduced in patients post myocardial infarction (MI).

Methods

We studied 23 patients (52±2 yr; ejection fraction= 56±1%), 30‐days after MI and 8 age‐matched healthy controls (C, 46±1 yr), during handgrip exercise. MSNA was measured by microneurography, forearm blood flow by venous occlusion plethysmography, and blood pressure by oscillometric method.

Results

Baseline MSNA was higher (57±4 vs. 35±2 bursts/100HB, P=.006), and forearm vascular conductance (FVC) was lower (1.62±0.1 vs. 2.97±0.4 units, P<.002) in MI patients when compared with C. No significant difference in mean blood pressure between groups was found (90±5 vs. 86±2 mmHg, P=.61). MSNA bursts/100HB similarly increased during peak exercise in both groups (P<.001). However, the MSNA levels were significantly higher in MI patients than in C (66±4 vs. 47±3 bursts/100HB, P=.01). FVC significantly increased during exercise in C (2.60±0.3 vs. 4.13±0.6 units, P<.001), but not in MI patients in whom FVC was unchanged (1.48±0.1 vs. 1.53±0.2 units, P=.99).

Conclusion

Sympathetic outflow is augmented and FVC is blunted during exercise in patients with MI. These findings highlight the abnormal neurovascular control during physiological maneuvers in patients with recent MI.
Mild skin surface cooling does not increase arterial stiffness in humans
Authors: Hess Kari L. , Wilson Thad E. , Gao Zhaohui , Sauder Charity L. , Ray Chester A. , Monahan Kevin D.
Published: June 2021

To test the hypothesis that mild skin surface cooling acutely increases central arterial stiffness and alters timing and amplitude of central wave reflections we measured aortic pulse wave velocity (PWV), central augmentation index, and transit time of the reflected wave before and during 20 min of cooling (water perfused suit) in 12 adults (25 ?1 years old, mean ?SE). Cooling decreased skin temperature (Δ‐3.6 ±0.4° C; P<0.05) without effecting sublingual temperature. Arterial blood pressure increased during cooling (Δ10±1 and Δ8±1 mmHg for systolic and diastolic blood pressure, respectively; P<0.05). Arterial stiffness, measured as aortic PWV, was unchanged (6.1±0.2 vs. 6.6±0.3 m/s for baseline and cooling, respectively; P>0.40) by cooling. In contrast timing and amplitude of central wave reflections were modified by cooling as assessed by measures of augmentation index (6±2% vs. 14±4%; P<0.05) and transit time of the reflected wave (169±7 vs. 152±5 ms; P<0.05). None of these measures changed during a separate control trial. Collectively, these data indicate that mild skin surface cooling does not acutely increase central arterial stiffness (aortic PWV) although it does modify the timing and amplitude of central wave reflections. These latter responses likely result from cold induced splanchnic, renal, and cutaneous vasoconstriction.

Support: NIH AG24420, HL77670, M01 RR10732
Glucose modulation of postganglionic sympathetic neurons innervating blood vessels
Authors: Damon Deborah H.
Published: June 2021

The present study tested the hypothesis that elevated glucose modulates postganglionic sympathetic neurons innervating blood vessels by studying effects of hyperglycemia on norepinephrine (NE) synthesis (tyrosine hydroxylase (TH)), NE release, and NE uptake (NE transporter (NET)). Effects were studied in vivo in rats made hyperglycemic (313+ 18.3 mg/dl) with STZ (50 mg/kg). In this model, elevated glucose increased TH (western analysis) in postganglionic sympathetic neurons innervating tail arteries (n = 5). Elevated glucose also increased basal NE release (tritiated‐NE released/tritiated‐NE taken up) from isolated rat tail arteries. NE released in 25 mM glucose (4.0+1.02%) was greater than that released in 5 mM glucose (1.78+0.15%; n = 4; p < 0.05). Hyperglycemia also modulated cultured postganglionic sympathetic neurons. NET (western analysis) in neurons grown in 25 mM glucose was markedly increased compared to that in neurons grown in 5 mM glucose (n = 3). These data clearly indicate that elevated glucose affects the function of postganglionic sympathetic neurons innervating blood vessels. The in vitro data suggest that glucose directly affects postganglionic sympathetic neurons, suggesting that sympathetic ganglia are more than passive relays. These findings may have important implications for sympathetic control of cardiovascular function under hyperglycemic conditions. HL076774, NS043316
Evidence of an essential role of NHERF1 and NHEF2 in G‐protein coupled receptor regulation of CFTR‐mediated intestinal anion secretion in vivo
Authors: Singh Anurag Kumar , Riederer Brigitte , de Jonge Hugo R. , Donowitz Mark , Weinman Edward J. , Kocher Olivier , Hogema Boris M. , Seidler Ursula
Published: June 2021

Heterologous expression studies have demonstrated that the PDZ‐proteins NHERF1 and NHERF2 modulate CFTR interaction with other proteins. To study their biological roles in vivo in an epithelium that highly expresses CFTR as well as NHERF1 and 2, we investigated the effect of NHERF1 and 2 ablation on CFTR‐dependent duodenal HCO3− secretion. Proximal duodenum of anesthetized mice was perfused in situ, and HCO3− secretion was determined by back‐titration. Co‐localization of CFTR, β2‐adrenergic receptor and LPA2 receptors, as well as PDZ proteins in the luminal membrane of crypt duodenocytes were established by Western analysis of isolated brush border membranes, immunohistochemistry and laser micro‐dissection followed by q‐PCR (quantitative‐PCR). NHERF1 ablation strongly reduced basal as well as forskolin (FSK)‐stimulated HCO3− secretory rates, and completely prevented β2‐adrenergic stimulation. NHERF2 deletion significantly augmented FSK‐stimulated HCO3− secretion, and completely prevented the inhibitory effect of LPA. We conclude that NHERF1 and 2 differentially modulate basal and agonist‐mediated duodenal HCO3− secretion in vivo in a CFTR‐dependent fashion. NHERF1 is an obligatory linker for β2‐adrenergic stimulation of CFTR, and strongly augments cAMP‐mediated stimulation. NHERF2 confers inhibitory signals, i.e. as a coupling factor between inhibitory LPA receptor signalling and CFTR.
Electrophysiological changes in Slc26a3 (down‐regulated in adenoma, Dra) knockout intestine are associated with epithelial cell alkalinity
Authors: Clarke Lane L , Horak R Zachary , Simpson Janet E , Walker Nancy M
Published: June 2021

Dra is the major apical membrane anion exchanger in the small and large intestine. Studies of recombinant DRA have proposed an electrogenic 2Cl−:1HCO3− stoichiometry (J. Gen. Physiol. 127: 511, 2006). However, in Dra knockout mice (DraKO) loss of Cl− absorption is associated with only small increases in transepithelial short‐circuit current (Isc). To investigate the electrophysiological changes resulting from loss of Dra, microelectrode analysis, intracellular pH (pHi) microfluorimetry and Ussing chamber studies were performed on DraKO and wild‐type (WT) intestine. Microelectrode studies revealed that the apical membrane potential (Va) of cecal epithelium was paradoxically greater in the DraKO and Va depolarization induced by extracellular Cl− (Cl−o) removal was not different than WT. Studies of cystic fibrosis mouse intestine indicated that Va depolarization during Cl−o removal was CFTR dependent and eliminated by high [K+]. Microfluorimetry studies revealed alkaline pHi in DraKO small and large intestinal epithelia. Ussing chamber experiments of DraKO small intestine found that increased Isc was abolished by epithelial acidification. We conclude that intestinal Dra demonstrates an electroneutral stoichiometry (e.g., 1Cl−:1HCO3−) and that increased Isc in DraKO intestine is associated with epithelial alkalinity that may activate basolateral membrane K+ channels. Support: NIDDK and CFF.
Structural Studies of Human Plasma Membrane Anion Exchanger, AE1, Expressed in Saccharomyces cerevisiae
Authors: Bonar Pamela Therese , Casey Joseph R.
Published: June 2021

Anion exchanger 1 (AE1), which is expressed in the renal and erythrocyte membranes, functions to exchange chloride for bicarbonate across the plasma membrane. While there is a high‐resolution structure of the N‐terminal cytoplasmic domain of AE1, there is no high‐resolution structure of the 55 kDa AE1 membrane domain. We have developed an expression and purification system for AE1, which takes advantage of S. cerevisiae as a large‐scale expression system of properly folded homogeneous AE1. The expression construct (AE1‐Rho) consists of the AE1 membrane domain, amino acids 388 to 911 of human AE1, followed by the 9 C‐terminal amino acids of rhodopsin. Milligram quantities of AE1‐Rho have been purified in the presence of the detergent fos‐choline, by a 1D4 antibody affinity column, which recognizes the 9 C‐terminal amino acids of rhodopsin tagged to the AE1 membrane domain. AE1‐Rho is non‐glycosylated and prodominantly dimeric, as determined by respectively endoglycosidase F treatment and size exclusion chromatography. AE1‐Rho secondary structure was assessed by circular dichroism. The functional activity of AE1‐Rho was measured by radioactive sulfate efflux assays in reconstituted proteoliposomes. This research is supported by the Canadian Institutes of Health Research.
Topology of the C‐terminal Transmembrane Region of NBCe1‐A Differs from AE1
Authors: Zhu Quansheng , Kao Liyo , Liu Weixin , Newman Debra , Orozco Nicholas , Azimov Rustam , Pushkin Alexander , Abuladze Natalia , Kurtz Ira
Published: June 2021

Sodium‐dependent bicarbonate cotransporter 1 (NBCe1) and sodium‐independent chloride/bicarbonate exchanger 1 (AE1) belong to the SLC4 transport family of membrane proteins that mediate bicarbonate transport. The extensively analyzed topology of AE1 highlights the structural and functional importance of the C‐terminal transmembrane region, which is considered to form the anion transport site. Residues in the C‐terminal transmembrane region of NBCe1‐A and AE1 are 40% homologous, however, no information is available on the topology of this region in NBCe1‐A. In this study, we performed introduced cysteine scanning mutagenesis coupled with sulfhydryl specific chemistry in the C‐terminal transmembrane region of NBCe1‐A. One hundred sixty cysteine mutations were individually introduced between Ala800 and Asp960 on a NBCe1‐A construct in which five cytoplasmic endogenous cysteines were changed to serine. These mutations completely covered the C‐terminal transmembrane region of NBCe1‐A, which includes the two proposed re‐entrant loops in AE1. The location of introduced cysteine residues were determined by whole cell labeling with two cysteine specific reagents: membrane permeant biotin maleimide (BM) and membrane impermeant MTS‐TAMRA. Of 160 introduced cysteine mutants, 31 were labeled with BM and 10 were labeled with MTS‐TAMRA. Our data demonstrate that topology of the C‐terminal transmembrane region of NBCe1‐A differs from AE1 in the following aspects: 1) NBCe1‐A lacks the two AE1 re‐entrant loops; 2) NBCe1‐A has 5 rather than 4 transmembrane helices (TM) in this region; 3) the average size of NBCe1‐A C‐terminal TMs is greater than AE1. On the basis of results, we propose a new topology model for the C‐terminal transmembrane region of NBCe1‐A.

Supported by NIH
NADPH oxidase signaling regulates Epithelial Sodium Channel (ENaC) activity
Authors: Park Michael S , Takemura Yoshizumi , Self Julie L , Helms My N
Published: June 2021

In general, over‐production of superoxide (O2−) in the kidneys leads to serious renal disorders. However, when released in low quantities in a controlled manner, O2− may serve as an important signaling molecule in epithelial cells. Presently, we examine whether sodium transporting epithelial cells can control release of O2− , through regulated NADPH oxidase activity, in order to regulate ENaC in renal epithelia. First, we showed that A6 distal nephron cells express each of the critical NADPH oxidase (NOX) components that catalyze the production of O2− (membrane bound gp91phox and p22phox; cytosolic p67 phox and p47 phox subunits; and small G protein Rac1) using western blot. Since complete activation of NOX is believed to occur following Rac1activation, we then tested whether inhibition or activation of Rac1 influenced ENaC activity. We report that, indeed, 1μM NSC23766 inhibition of Rac1 significantly decreases single channel measurements of ENaC activity from 1.16+.27 to .50+.11 (mean NPo values+SE, n=6, p<0.05). Conversely, 10ng/mL EGF activation of Rac1 significantly increased ENaC NPo values, on average, from .93+.25 to 1.30+.31 in 6 independent observations (p<0.05). HPLC measurements verify that Rac1 inhibition decreases O2− release. Very briefly, we conclude that NADPH‐oxidase induced O2− signaling is a new pathway for ENaC regulation.
Roles of Insulin Receptor Substrates (IRS) in renal function and renal hemodynamics.
Authors: Hashimoto Seiji , Maoka Tomochika , Kawata Tetuya , Mochizuki Toshio , Koike Takao
Published: June 2021

We have reported previously that renal hemodynamic abnormalities exist in the prediabetic stage of type II diabetic rats. At this stage these rats have insulin resistance. It is well known that insulin resistance is frequently associated with renal abnormalities, but the mechanism underlying this association has remained speculative. Although insulin is known to modify renal hemodynamics, little is known about the roles of insulin receptor substrates (IRS1, IRS2) in the renal actions of insulin. To address this issue, we investigated in wild type (WT), IRS1‐deficient (IRS1‐/‐), and IRS2‐deficient (IRS2‐/‐) mice. IRS2‐/‐ mice had elevated blood pressure and glucose level as expected. 24‐h urine collections revealed that creatinine clearance did not significantly differ between these groups. Fractional excretion of sodium was significantly lower in IRS1‐/‐ and IRS2‐/‐ compared to WT mice. Albuminuria was found in IRS1‐/‐ and IRS2‐/‐ groups. We assessed autoregulatory responses of total renal blood flow (RBF), and of superficial (SBF) and deep renal cortical blood flow (DBF) to stepwise reductions of renal perfusion pressure (RPP) induced by a manual clamp on the abdominal aorta. RBF, SBF and DBF fell significantly more in IRS1‐/‐ and IRS2‐/‐ than in WT mice. These results indicate that IRS plays a major role in the stimulation of renal functions and renal hemodynamics in type II diabetes.
iTRAQ Proteomics Approaches Reveal Novel Mitochondrial Targets of Estrogen Deficiency in Aged Female Rat Heart
Authors: Korzick Donna Hope , Lancaster Timothy S , Jefferson Sarah J , Tomicek Nanette J
Published: June 2021

Background

The incidence of MI rises sharply at the time of menopause, implicating loss of estrogen (E2) in age‐related increases in ischemic injury.

Purpose

To identify new mitochondrial protein targets for the treatment of acute MI in aged women using a state‐of‐the‐art proteomics approach. We hypothesized that E2 deficiency exacerbates age‐dependent disruptions in mitochondrial proteins in female rat heart.

Methods

Targeted proteomic analyses were conducted in mitochondria isolated from left ventricles of adult (6 mo) and aged (24 mo) F344 ovary‐intact or ovariectomized (OVX) rats using the newly released isobaric tags for relative and absolute quantification (iTRAQ) 8plex labeling and mass spectrometry (n=4‐5/group).

Results

Initial multi‐dimensional protein identification technology (MudPIT) analyses revealed n=638 distinct mitochondrial proteins. With iTRAQ, significant group differences (false discovery rate < 5%) were observed in proteins related to cellular metabolism, ion transport, oxidative stress and cell death regulation. Effects of age‐associated E2 deficiency were primarily limited to proteins which regulate oxidative stress, and were not observed in adult OVX.

Conclusions

Proteomic analysis through iTRAQ is effective in identifying new candidate proteins for further in‐depth study of age‐ and E2‐dependent alterations in mitochondrial cardioprotective signaling.
Regulation of ciliary beat frequency by sex hormones in guinea pig Fallopian tube: interactions between estrogen and progesterone
Authors: Nakahari Takashi , Nishimura Ayumi
Published: June 2021

Fallopian tube CBF was measured using a microscope connecting a high‐speed camera (500 Hz). β‐Estradiol‐benzoate (βE2‐benz) and medroxy‐progesterone (m‐Prog) were administered by subcutaneous injection for 1.5 days. βE2‐benz changed CBF in a dose dependent manner; a low dose (2 mg/day) increased CBF (14.5 Hz), whereas a high dose (20 mg/day) decreased it (10.83 Hz). An inhibitor of βE2 (2 mg/day, ICI‐182,780) maintained CBF at 12 Hz, and the further addition of βE2‐benz did not change CBF. m‐Prog (4 mg/day) decreased CBF (12.8 Hz), and an inhibitor of progesterone (50mg/day, mifepristone) maintained CBF at 15 Hz, and the further addition of m‐Prog did not change CBF. The addition of both inhibitors increased CBF to 17.4 Hz, and then, the addition of m‐Prog, instead of mifepristone, decreased it to 11 Hz, however, the further addition of βE2‐benz, instead of ICI 182,780, increased to 17.5 Hz. Thus, βE2‐benz inhibited the m‐Prog action. Moreover, m‐Prog shifted the CBF dose response curve of βE2‐benz to the right, suggesting m‐Prog decreased the CBF sensitivity to βE2‐benz. In conclusion, Fallopian tube CBF was regulated by interactions between estrogen and progesterone. The experiments were approved by the Animal Research Committee of our College.
Investigating the Role of Proteases in Aggressiveness of Inflammatory Breast Cancer
Authors: Victor Bernadette , Sloane Bonnie
Published: June 2021

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer with a three‐year survival rate of 42% versus 85% for other breast cancers. IBC is an aggressive, metastatic and highly angiogenic form of locally advanced breast cancer that invades into lymphatics where it forms large cellular aggregates. Here we performed live‐cell imaging using a SUM149 model of human inflammatory breast cancer. We hypothesize that the invasion of IBC is dependent on proteolytic activity at the tumor cell surface and that proteases contribute to the formation of tumor cell aggregates within the lymphatics. We assessed proteolytic activity using our functional assay for proteolysis. Quantification on a per cell basis throughout the volume of IBC structures was performed. Briefly, cells were grown in 3D reconstituted basement membrane (rBM) overlay cultures in which the lower layer of rBM is mixed with DQ‐collagen IV. Fluorescent cleavage products arising from the degradation of the collagen IV substrate were imaged on a Leica TCS SP5 laser scanning confocal microscope. Fluorescence intensities from 3D reconstructions of optical sections were quantified using Metamorph and Volocity software. Incubating the 3D rBM overlay cultures of SUM149 with inhibitors that target different classes of protease indicated that matrix metalloproteases and perhaps the cysteine protease cathepsin B play roles in the invasiveness of IBC.
Autoantibody against AT1 receptor from preeclamptic patients induces rats' endothelial injury
Authors: Yang Lihong , Zhang Shuli , Zheng Ronghua , Yan Li , Zhao Rongrui , Liu Huirong
Published: June 2021

Pre‐eclampsia occurs in about 3‐5% of pregnancies causing maternal, neonatal morbidity and mortality. The autoimmune antibody against the second extracellular loop of angiotensin II receptor type 1(AT1‐AA) might involve vascular injury in preeclamptic patients. No evidence shows the relationship between AT1‐AA and blood pressure. Current study demonstrated AT1‐AA is high frequency, high titer. Our further studies clarified that AT1‐AA induced endothelial dysfunction in vitro. Present study aims to investigate whether AT1‐AA could induce endothelial injury in vivo. First, anti‐AT1R autoantibody negative female rats (180‐200g) were immunized with peptides 165‐191 (IHRNVFFIINTNITVCAFHYESQNSTL). AT1‐AA rose and peaked at week 8 (OD, 2.81±0.07 vs 0.43±0.05, P<0.01). ET‐1 activity was performed by EIA Kit. A significant increase occurred at week 3, peaked at week 9 (OD492, 0.38±0.03 vs 0.01±0.01;P<0.01). Abdominal aortas were isolated from immunized rats. ICAM‐1 fluorescence levels is 5.9616 ±0.7194 vs 1.8213±0.5014, P<0.01. Results suggest that overexpression of AT1‐AA is a risk factor in pregnant women and may play a causative role in the development of preeclamptic patients. AT1‐AA may induce endothelial injury in vivo and contribute to vascular dysfunction. It suggested protecting endothelial may provide a new tool for the treatment of preeclampsia involving the production of autoantibody.
Diets enriched in stearic acid do not depress cardiac function
Authors: Thomas Candice , Smart Eric J
Published: June 2021

Diabetes is associated with an increased risk of congestive heart failure and heart disease, however, the effects of specific fatty acids on the development of these diseases are unknown. To determine the effects of dietary fats on cardiac function in the context of diabetes, db/db mice (a model of type 2 diabetes) were fed diets enriched in palmitic, linoleic, or stearic acid (40% kcal) for 8 weeks. Left ventricular cardiac output was measured with a Millar pressure/volume catheterization system. All of the mice gained the same amount of weight, however, mice fed the stearic acid diet had significantly lower blood glucose levels and consumed less food. Mice fed diets enriched in palmitic or linoleic acid had decreased cardiac function with a heart rate of 298‐376 beats/min, cardiac output of 6568‐6759 μl/min, stroke work of 1215‐1319 mmHg*μl, dP/dt max of 4961 to 6147 mmHg/sec and dP/dt min of ‐3990 to ‐5886 mmHg/sec. In contrast, mice fed a diet enriched in stearic acid had a heart rate of 503 bpm, cardiac output of 9936 µl/minute, stroke work of 1518 mmHg*μl, dP/dt max of 7353 mmHg/sec and dP/dt min of ‐6725 mmHg/sec. The data demonstrate that palmitic and linoleic acid enriched diets negatively impacts heart function in diabetics whereas stearic acid did not. These data suggest that the type of fatty acids consumed by diabetics can differentially impact cardiac function.
Non‐lumenal lymphatic/blood endothelial cell connections exist at the capillary level in unstimulated adult rat mesenteric microvascular networks
Authors: Robichaux Jennifer L. , Tanno Eleanor , Murfee Walter Lee
Published: June 2021

Recent observations from our laboratory suggest that lymphatic and blood endothelial cells physically interact at the capillary level in adult microvascular networks. The objective of this study was to 1) examine the endothelial cell structure and phenotypes at lymphatic/blood vessel connections and 2) to determine whether physical connections provide sites of fluid conductance. Mesenteric tissues were harvested from adult male Wistar rats (350 25 g) and labeled for PECAM and LYVE‐1, Prox‐1, or Podoplanin. Direct lymphatic/blood endothelial cell connections were supported by continuous PECAM junctional labeling in < 1 µm optical sections using confocal microscopy. While PECAM labeling identified both lymphatic and blood capillaries at locations of direct lymphatic/blood endothelial cell connections, LYVE‐1, Prox1, and Podoplanin expression did not extend along the blood capillary segments. PECAM positive lymphatic sprouts, structurally similar to blood capillary sprouts, also lacked observable lymphatic marker labeling. At 21/21 sites of connections, BSI‐lectin labeling via intra‐mesenteric artery injection did not identify the lymphatic vessel. These observations suggest that non‐lumenal lymphatic/blood endothelial cell connections exist in unstimulated adult microvascular networks and highlight the potential for lymphatic/blood endothelial cell plasticity.
Temporally controlled overexpression of cardiac‐specific PI3Kα‐ a novel transgenic mouse model
Authors: Tseng Yi‐Tang , Yano Naohiro , Dahdah John , Cao Tram , Tseng Andy , Zhao Ting C , Padbury James F
Published: June 2021

Cardiac PI3K signaling plays important roles in cardiac growth and is highly regulated during development with highest levels found during the fetal‐neonatal transition period and lowest in the adult. Prolonged constitutive activation of PI3Kα in the heart results in hypertrophy. To examine the impact of temporal overexpression of PI3Kα in the heart, we have engineered a tetracycline transactivator (tet‐off) controlled inducible transgenic mouse model for cardiac‐specific overexpression of PI3Kα. Cardiac PI3K activity and Akt phosphorylation were significantly increased in adult mice after transgene induction following removal of doxycycline for 2 weeks. Heart weight to body weight ratio was not changed and there were no signs of cardiomyopathy. Overexpression of PI3Kα resulted in increased left ventricular (LV) developed pressure, LV dP/dt max and LV dP/dt min, but not heart rate, as assessed in Langendorff hearts. Mice overexpressing PI3Kα also had increases in the levels of Ca2+‐regulating proteins, including the L‐type Ca2+ channels, ryanodine receptors, and SERCA2a. Thus, temporally controlled overexpression of cardiac PI3Kα does not induce hypertrophy or cardiomyopathy but results in increased contractility, probably via increased expression of multiple Ca2+‐regulating proteins. These distinct phenotypes suggest a fundamental difference between transgenic mice with temporal or prolonged activation of cardiac PI3Kα. (NIH 1 P20 RR018728)
Extracellular β‐NAD (Beta‐nicotinamide adenine dinucleotide) Participates in the Regulation of Endothelial Cell (EC) Permeability
Authors: Umapathy Nagavedi S. , Verin Alexander D.
Published: June 2021

Extracellular β‐NAD is known to increase intracellular calcium concentrations [Ca2+]I, inositol 1,4,5‐trisphate (IP3) and cAMP in different cell types and by different mechanisms. Since β‐NAD is released extracellularly in EC, we hypothesized that it could participate in the regulation of EC permeability. Our data demonstrate that β‐NAD increases the transmonolayer electrical resistance (TER) of human pulmonary artery EC (HPAEC) monolayers in a concentration‐dependant manner indicating barrier enhancement. Importantly, simultaneous addition of β‐NAD and the potent edemagenic agent, thrombin, significantly attenuated thrombin‐induced EC permeability representing the barrier‐protective effect of β‐NAD. Immunofluorescence studies reveal more pronounced staining of cell‐cell junctional protein VE‐Cadherin at the cellular periphery signifying increased tightness of cell‐cell contacts after β‐NAD stimulation. Recent reports suggest that extracellular β‐NAD is an agonist for P2Y1 and P2Y11 purinoceptors and our data demonstrate their moderate (P2Y1) and high expression (P2Y11) in HPAEC. Interestingly, specific siRNA studies indicate the participation of both of these purinoceptors in β‐NAD‐induced TER increase. In addition, β‐NAD restored microtubule disruption induced by Nocodazole as evidenced by ECIS (Electrical cell‐substrate impedance sensing) and Immunofluorescence.
Porous PEGDA hydrogels promote hMSC survival and eventual differentiatability
Authors: Gemeinhart Richard A , Keskar Vandana
Published: June 2021

Superporous poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds have the potential to overcome the problems of difficulty seeding cells into the scaffold. The interconnected pore network of the superporous hydrogels (SPH) not only allows cells to attach to the interior porous structure but also facilitates nutrient exchange within cells in the construct. We have shown that cells hMSC can rapidly and uniformly loaded in to the scaffold, can survive for up to a month, and can mineralize the tissue (Tissue Engineering, in press). Very interestingly, hMSCS, but not HUVECs, adhere within the porous network in an RGD‐peptide independent manner. In addition to this ability, hMSCs retain ability to differentiate within the porous network to cell types that appear as chondrocyte, osteoblast, and adipocyte upon stimulation with appropriate medium. MSCs treated with a specific inducing media or untreated controls do not exhibit markers for the cell types other than the appropriate type of the inducing medium, or no cell markers for untreated control cells.
Variations in hepatic arterial supply
Authors: Canby Craig A. , Nelson Daniel W.
Published: June 2021

The arterial supply to the liver is highly variable with variations occurring slightly less than 50% of the time. Variations occur, for example, when hepatic arteries are replaced or accessory arteries are present. Clinically, it is important to appreciate and recognize these hepatic arterial variations during radiologic and surgical procedures in the upper abdomen to affect positive patient outcomes. Consequently, the purpose of our study was to examine the frequency of variations in the hepatic arterial supply. Our study examined 84 cadavers for the presence of replaced and accessory arteries and the variant origin of the common hepatic artery (CHA). The most frequent pattern was type 2 (16.3%) which is characterized by the left gastric artery issuing an accessory vessel (n=13) or replaced left hepatic artery. A type III pattern [superior mesenteric artery (SMA) issuing a replaced right hepatic (n=13) or accessory artery] occurred 14% of the time. Infrequently, a replaced right hepatic artery originated from the gastroduodenal (GD) artery (3.5%) or celiac trunk (2.3%). In one instance, an accessory artery sprung from the GD. Lastly, the CHA was observed issuing from the aorta (2.3%) and SMA (1.2%). In summary, our study observed 35 instances of hepatic arterial variations (40.7%). The frequent occurrence of these variations underscores the importance of properly planning clinical procedures in the upper abdomen.
Tracking the Anatomical Contents of the Quadrilateral Space: Clinical Implications
Authors: Frumberg David Benjamin , Beckman Michael Joseph , Marquez Samuel
Published: June 2021

The quadrilateral space (QS), demarcated by osseo‐muscular borders, is the gateway for the axillary nerve (AN) and posterior circumflex humeral artery (PCHA) between anterior and posterior scapular regions. QS Syndrome is the condition whereby hypertrophy of associated musculature causes AN entrapment and compression of PCHA, potentially resulting in deltoid weakness, tenderness over the shoulder region, and pain radiating to the arm. This anatomical study assessed, identified, and categorized AN and PCHA transmission through the QS using a mixed‐sex sample size of 43 cadavers to determine the strength of association between AN and PCHA. Results showed AN present in all 86 QSs, while 89.5% contained PCHA. Variable arterial patterns observed were PCHA passing inferior to the teres major insertion on the humerus, originating from a common stem with the profunda brachii artery (9.3%) or from its own branch off the brachial artery (1.1%). One cadaver unilaterally possessed a PCHA in the QS and a branch from the profunda brachii artery. All unilateral arterial deviations occurred on the right side while two cadavers possessed bilateral deviations with PCHA arising from the profunda brachii artery. A full understanding of arterial variation in the QS region may help clinical characterization of QS Syndrome, and may obviate the need for arteriography to minimize postoperative bleeding and yield successful rehabilitation outcomes.

Grant Funding Source

DO NOT HAVE FUNDING
Tracking Nasal Diversity in Multi‐Regional Populations: Insights into the Adaptive Significance of the Upper Respiratory System
Authors: Qureshi Naema , Gannon Patrick J , Obourn Chelsea , Márquez Samuel
Published: June 2021

The nasal complex (nose and paranasal sinuses) contributes a critical role in respiration by treatment of inspiratory air at the gates of the upper respiratory tract before the proper exchange of gases can take place in the alveolar region. Key components of this complex, the maxillary sinuses (MS), are implicated in a role for air conditioning of inspired air (Gannon et al, 1997). Differences in skeletal and soft tissue anatomy of the nasal region have been shown to be distinct but none have factored in a morphometric approach. This study included four human population groups from Africa, Asia, Europe and North America. We measured internal and external nasal parameters (ENP) including piriform dimensions, MS and endocranial volumes. Results showed: 1) piriform aperture area (PAA) variable did not have the power to discern populations; 2) MS volumes determined significant differences between groups living in cold and hot climatic conditions (p < 0.05) but did not strongly discern populations; 3) external linear nasal measures displayed a strong parametric value for discriminating between groups; and 4) integration of MS volume and PAA with ENP was the strongest variable suite to distinguish population groups. Our results suggest that the variables we employed may be an integrated functional suite of characters that reflects adaptations of the nasal complex during migrations across a wide range of climatic conditions.

Grant Funding Source

NA
The interscale skin of the intermandibular‐gular regions of Alligator mississippiensis serves as a distribution system for the conditioning secretion of the gular gland
Authors: Hopkins Brooke Andriane , Homberger Dominique G.
Published: June 2021

The skin of alligators is covered by plate‐like, non‐imbricate and hard‐cornified scales that are separated by a network of soft‐cornified interscale skin. The width of the interscale skin is correlated with the local expansibility of the skin. In most of the intermandibular‐gular region, the skin needs to stretch significantly when large prey items are swallowed to allow for the expansion of the pharynx. The interscale skin is widest along the mandibular rami where it forms longitudinal folds and channels that radiate rostrally from the paired gular gland orifices. The surface of these interscale channels is studded with small scale bumps. These wide channels are interconnected by a network of smaller, stretchable channels, whose surface structures are regionally distinct. The structured surface of the channels may enhance capillary action of the oily gular gland secretion as it is massaged through the channels by movements of the intermandibular‐gular skin during deglutition. This configuration of the intermandibular‐gular skin suggests that the channels serve to distribute the oily secretion of the gular gland to condition the stretchable interscale skin so as to keep it pliable and elastic. In contrast, the unyielding interscale skin in the area that subtends the hyoid and larynx is very narrow and has a smooth surface structure.
Nuclear pore complex structure and assembly involve the ER membrane protein Rtn1
Authors: Lazarus Michelle Denise , Dawson Renee , Wente Susan
Published: June 2021

Nucleocytoplasmic transport occurs exclusively through nuclear pore complexes (NPCs) which have been implicated in proper embryonic development. Although structural studies have resolved the complex NPC architecture, the mechanism that directs pore membrane fusion and NPC biogenesis remains unclear. Reticulons (RTNs) and Yop1/DP1 are conserved membrane protein families required to form and maintain the tubular endoplasmic reticulum (ER). Remarkably, whereas NPC membrane proteins such as Pom34 and Pom152 are not required for viability, an rtn1δ yop1δ pom34δ pom152δ quadruple null (δ) mutant is lethal. This suggests that Rtn1 and/or Yop1 play a role in NPC structure, assembly and/or maintenance. This lethality is rescued by expression of wild type RTN1, as well as by the more nuclear localized point mutant rtn1‐K48I. The exogenous expression of RTN1 and rtn1‐K48I also rescues a lethal pom34δ nup59δ double mutant. Nup59 is a structural NPC protein linked to NPC assembly. These results indicate that Rtn1 has at least a partial redundancy in function with NPC structural and transmembrane proteins. In addition, this suggests a function for the reticulons at the NE that is independent of RTNs role at the tubular ER. We hypothesize that RTNs and/or Yop1/DP1 stabilize the membrane curvature of the nuclear pore during NPC assembly.

Grant Funding Source

IRACDA and NIH
SLC/CCR7 enhances the maturation of semi‐mature BMDCs by the pNF‐¿B p65 pathway
Authors: Liang Chun‐min , Zhou Shuang , Wu Wen‐qing , Zhong Cui‐ping
Published: June 2021

The chemokine receptor CCR7 is highly expressed in dendritic cells (DCs), T cells, and other immune effector cells. The high‐affinity ligand that can bind to CCR7 is the secondary lymphoid tissue chemokine (SLC). The SLC/CCR7 axis plays an important role in the immune system by inducing the chemotaxis and migration of immune effector cells. In this study, we found that SLC also directly enhances the maturation of semi‐mature bone‐marrow‐derived dendritic cells (BMDCs), with increased expression of MHC II and CD86 (B7‐2) and enhanced antigen‐presenting cell (APC) function. pNF‐Bp65 was involved in this mechanism. The results indicated that there are cross‐talk signals underlying the SLC/CCR7 axis between the chemotaxis and maturation of BMDCs. Interestingly, we also found that while Ca2+ mobilization and subsequent migration of semi‐mature BMDCs could be totally inhibited by PTX, PTX only partially inhibited the maturation of semi‐mature BMDCs. The findings further suggested that the SLC/CCR7 axis uses different signaling modules to regulate its multiple functions in BMDCs.

Grant Funding Source

National Science Foundation of China No.30500280
Sepsis induces pathological changes in the spinal cord
Authors: Davies D. Ceri , Bradley Eleanor M. , Szewczy Kinga A. , Cope Celia , Osabutey Casmiel K. , Moss Raymond
Published: June 2021

Critical illness polyneuropathy is a poorly understood complication of sepsis. To investigate the effect of sepsis on the spinal cord, caecal ligation and puncture was used to induce sepsis in rats. Approximately 22 h later, the rats were given a lethal dose of anaesthetic, perfused through the heart with heparinised saline, then 2.0 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2‐ 7.4), at a pressure of 110 mm Hg. The lumbar spinal cord was removed and processed into Spurr's resin. Axial semithin and ultrathin sections of the ventral lumbar spinal cord were then cut and examined by light and transmission electron microscopy. Sepsis resulted in oedema surrounding microvessels and the central canal and a loss of glycoproteinaceous material from the lumen of the central canal. Central canal ependymal cells demonstrated nuclear condensation, cytoplasmic dilution, swelling of intracellular organelles, loss of microvilli and cilia. Morphometric analysis revealed that in septic rats, the mean cross‐sectional area of the lumen of the central canal was approximately 2.8 x larger (P < 0.05), ependymal cell cross‐sectional area was doubled (P < 0.001) and ependymal cell number increased by about a third (p < 0.01) compared to sham‐operated controls. These changes in the spinal cord are likely to have a profound effect on peripheral nerve function and may underlie critical illness polyneuropathy.
Human ELG1 regulates the level of monoubiquitinated PCNA through interactions with PCNA, USP1, and polymerase η
Authors: Myung Kyungjae , Banerjee Soma , Yang Kailin , Sikdar Nilabja , Lee Kyoo‐young , Cohn Martin , Wincovitch Stephen , Pak Evgenia , Nakanishi Koji , Jasin Maria , Dutra Amalia , D'Andrea Alan
Published: June 2021

The level of monoubiquitinated PCNA in chromatin is closely linked with DNA damage bypass during DNA replication. However, it remains unclear how the level of monoubiquitinated PCNA in chromatin is regulated. Here, we demonstrate that human ELG1 (hELG1) regulates the level of monoubiquitinated PCNA in chromatin, through its interactions with PCNA, the tanslesion synthesis DNA polymerase η, and the deubiquitinating enzyme, USP1. The level of hELG1 is induced during recovery from DNA damage, resulting in the assembly of distinct hELG1 foci at stalled DNA replication forks. Furthermore, hELG1 foci start to appear where foci of DNA polymerase η exist. Targeted gene knockdown of hELG1 by shRNAs enhanced homologous recombination and resulted in genomic instabilities and hypersensitivity to DNA damaging agents. Taken together, hELG1 suppresses genomic instability by reducing monoubiquitinated PCNA from chromatin after DNA damage bypass through its interaction with and stabilization of USP1.
Oxidative stress induces degradation of mitochondrial DNA
Authors: Shokolenko Inna N , Venediktova Natalia , Bochkareva Alexandra , Wilson Glenn L. , Alexeyev Mikhail F
Published: June 2021

Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nuclear DNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that single strand breaks and abasic sites prevail over mutagenic base lesions in ROS‐damaged mtDNA. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugarphosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions.
Enzymatic mechanism of human apurinic/apyrimidinic endonuclease against a THF AP site model substrate
Authors: Mundle Sophia T , Delaney James C , Essigman John M , Strauss Phyllis R
Published: June 2021

The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo) is a major factor in the maintenance of the integrity of the human genome. There are estimates that this enzyme is responsible for eliminating as many as 105 potentially mutagenic and genotoxic lesions from the genome of each cell every day. Furthermore, inhibition of AP endonuclease may be effective in decreasing the dose requirements of chemotherapeutics used in the treatment of cancer as well as other diseases. Therefore, it is essential to accurately and directly characterize the enzymatic mechanism of AP endo. Here we describe specifically designed double stranded DNA oligomers containing tetrahydrofuran (THF) with a 5' phosphorothioate linkage as the abasic site substrate. Using H218O during the cleavage reaction and leveraging the stereochemical preferences of AP endo and T4 DNA ligase for phosphorothioate substrates, we show that AP endo acts by a one‐step associative phosphoryl transfer mechanism on a THF‐containing substrate. Supported by National Institutes of Health Grant CA 72702 (to P.R.S.), and NIH Grant CA80024 and NIEHS Grant P30 ES002109 (to J.M.E.)
Functional Diversification and Evolution of Animal Zinc‐Dependent Alcohol Dehydrogenases
Authors: Julián‐Sánchez Adriana , Magis‐Weinberg Lucía , Riveros‐Rosas Héctor
Published: June 2021

Zinc‐dependent alcohol dehydrogenases (ADHs) comprise one of the best studied group of enzymes. Vertebrates possess eight distinct classes (I‐VIII) of ADHs, based upon sequence homology, catalytic activity and gene expression features. However, because many independent duplications occurred along the evolution of vertebrates, the identification of orthologous groups of proteins is not convincingly resolved. Since the number of ADH sequences in animals has been duplicated after the last analysis of these enzymes, a new phylogenetic analysis was performed.

Results provide a comprehensive scenario, where is clear that ancestral class III ADH give rise in first place to the class I ADH, which is present in all classes of vertebrates. Later, class II and VII emerged by duplication of class III ADH before the amphibian‐amniota split, but interestingly, class VII ADH was lost in mammals. Finally, classes IV to VI have a more recent origin, because the duplication that gave rise to these three classes occurred just after the appearance of mammals. Class VIII ADH is restricted only to amphibians.

The complex ADHs enzymogenesis in vertebrates is a consequence of the diverse physiological roles developed by these enzymes, as responsible to synthesize or catabolize several endogenous metabolites that regulate growth, metabolism, differentiation and neuroendocrine functions. Supported by DGAPA‐UNAM grant IN208308