bluuebunny/crossref_metadata_2025 huggingface dataset:
The Drosophila heart as a model for investigating human cardiomyopathies
Authors: Cammarato Anthony Ross , Ocorr Karen , Bodmer Rolf , Bernstein Sanford I.
Published: June 2021

Myosin is the molecular motor that drives muscle contraction. Alterations that affect the motor's kinetic and mechanical properties are associated with the pathogenesis of human cardiomyopathies. We investigated the influence of Drosophila melanogaster myosin mutations on the structure and function of fly hearts. Beating hearts expressing D45 (hypoactive) or Mhc5 (hyperactive) myosins were imaged using direct immersion DIC optics in conjunction with a high‐speed digital camera. Cardiac movements were monitored via edge tracings obtained from processed movies. Our novel image and computational analysis revealed that depressed motor function in D45 flies produced a dilatory cardiac response, similar to that found in vertebrates expressing myosin with specific dilated cardiomyopathy mutations. This suggests an apparently conserved pathological response to impaired motor function. Mhc5 hearts with enhanced myosin function showed phenotypes analogous to those seen in restrictive cardiomyopathy, suggesting the human disease could have similar origins. Our data suggest Drosophila is a valuable system for modeling and identifying mutations analogous to those associated with human cardiac disorders. We are currently exploiting the power of Drosophila to manipulate genetic interactions between sarcomeric components. Our goal is to suppress defects in hearts with distinct myofibrillar mutations.
Repetitive bouts of mechanical compression improve systemic markers of endothelial function in patients with coronary artery disease
Authors: Casey Darren Patrick , Conti C Richard , Nichols Wilmer W , Choi Calvin Y , Khuddus Matheen A , Braith Randy W
Published: June 2021

We hypothesized that repetitive mechanical compression (MC) of the lower limbs would lead to favorable changes in peripheral endothelial function in patients with coronary artery disease (CAD). To test this hypothesis, 35 1‐hour sessions of MC were administered to CAD patients (n=12). Each session consisted of sequential inflation and deflation of compressible cuffs wrapped around the calves and lower and upper thighs. Compressed air pressure (300mmHg) was applied in a sequence synchronized with the cardiac cycle. Eight (n=8) CAD patients served as sham controls (CON). Brachial (b) and femoral (f) artery flow‐mediated dilation (FMD) and plasma levels of nitric oxide metabolites (NOx) and asymmetric dimethylarginine (ADMA) were assessed pre and post intervention. The MC group demonstrated increases in b‐FMD (3.7 ± 0.2 vs. 5.7 ± 0.4 %, P < 0.05) and f‐FMD (2.7 ± 0.2 vs. 3.5 ± 0.3%, P < 0.05). Additionally, a 40% increase in NOx (22.8 ± 2.2 vs. 31.9 ± 4.5 [mu]mol/L, P < 0.05) and a 26% decrease in ADMA (0.62 ± 0.06 vs. 0.46 ± 0.03 [mu]mol/L, P < 0.05) were observed in the MC group. There were no significant changes in any outcome measures for the CON group. These results suggest that external mechanical compression of the lower limbs improves markers of peripheral endothelial function in CAD patients. This effect is likely attributed to increases in shear stress on the endothelium.

Supported by NIH R01 HL‐077571‐01
Regulation of skeletal muscle blood flow in the elderly: role of alpha‐1 adrenergic receptor sensitivity
Authors: Wray David Walter , Nishiyama Steven K. , Harris Ryan A. , Richardson Russell S.
Published: June 2021

In nine young (25 ± 1 yrs) and six older (72 ± 2 yrs) healthy volunteers, we evaluated changes in leg blood flow (ultrasound Doppler) during blood flow‐adjusted, intra‐arterial infusion of phenylephrine (PE, a selective alpha‐1 adrenergic agonist) at rest and during knee‐extensor exercise. Heart rate, arterial blood pressure, femoral artery diameter, and blood velocity were measured in all subjects at rest and exercise at 20, 40, and 60% of maximal work rate (WRmax). In a subset of subjects (n = 10), leg O2 consumption (VO2) and lactate efflux were also evaluated. At rest, PE reduced blood flow to a lesser degree in older (−37 ± 3%) compared to young (−54 ± 4%) subjects. During exercise, older subjects maintained a greater vasoconstrictor response to PE at 40% WRmax (−14 ± 3%, older; −7 ± 2%, young) and 60% WRmax (−11 ± 3%, older; −4 ± 3%, young), which may be attributed to lower absolute work rates in the older group. At a similar absolute work rate and leg VO2, vasoconstriction to PE was not different between groups (−14 ± 3%; older; −17 ± 5%, young). In both groups, blunting of PE‐mediated vasoconstriction was highly correlated with leg VO2 but not leg lactate efflux. Together, these data indicate that alpha‐1 adrenergic receptors become less sensitive during knee extensor exercise as a consequence of absolute work and the associated level of oxidative metabolism, with no effect of age per se.
Role of G‐protein coupled receptor kinase‐2 (GRK2) in the modulation of renal vascular response in SHR by PPAR
Authors: Yousefipour Zivar , Oyekan Adebayo , Newaz Mohammad
Published: June 2021

G‐protein coupled receptor (GPCR) kinase‐2 (GRK‐2) has been impllicated in hypertension and GRK‐2 inhibition reduced blood pressure (BP). Peroxisome proliferators activated receptor γ (PPARγ), a transcription factor regulates GPCRs but the connection with GRK‐2 is not known. Here, we examined GRK‐2 role in PPARγ‐mediated improvement in SHR. SHR/WKY rats were treated with GW1929 (GW), a PPARγ ligand (0.5mg/kg; 2 month) and BP was monitored. Whole kidney perfusion was determined (laser dopler scanner) and renal vascular response was measured with or without heparin, a GRK‐2 inhibitor. In SHR, GW reduced BP (25±1%) and increased renal perfusion (59±3%). Phenylephrine (PE) vasoconstriction was greater in SHRs (15±1%) then WKY rats and GW abolished this. Heparin attenuated PE response in untreated but not GW‐treated SHR. GW enhanced Ach vasodilatation in SHR (41±1%) and this was improved by heparin (79±18%). GW enhanced SNP vasodilatation in SHR (35±6%) but remained unchanged with heparin. In SHR, GW enhanced PPARγ mRNA but reduced GRK‐2 mRNA. These results suggest that down‐regulation of PPARγ and up‐regulation of GRK‐2 may responsible for increased renal vascular reactivity of SHR and that PPARγ transcriptionally attenuated GRK‐2 function in hypertension.
Renal Endothelin Production is Blunted in the Dahl Salt Sensitive Rat
Authors: Speed Joshua S , Lamarca Babbette , Fournier Lillian M , Cockrell Kathy , Granger Joey P
Published: June 2021

Although it has been well established that the renal endothelin (ET‐1) system plays an important role in mediating salt excretion under high salt intake through activation of medullary ET‐B receptors, the role of this system in Dahl salt sensitive (DS) hypertension is unclear. The purpose of this study was to determine if the DS rat has abnormalities in the renal medullary endothelin system. The experiment consists of 4 groups, DS rats on low and high Na intakes, and Dahl salt resistant rats (DR) on low and high Na intakes. The results show a slight increase in urinary ET‐1 between the DS rats on low Na (13±2 pg/ml) versus high Na diet (29.8±5.5 pg/ml). Meanwhile, there was a 6‐fold increase in ET‐1 production in the DR rats (17.8±4 pg/ml on low Na diet versus 112±44pg/ml on high Na diet). Medullary ET‐1 production in DR rats on high Na diet was 70.9±5 pg/mg. Interestingly, DS rats produced 50% less medullary ET‐1 compared to DR on high Na (31 ± 2.8 pg/mg). Our results also indicate a downregulation of the ETB receptor in the DS rat (7.7±.8 pg/mg low Na, 2.4±.79 pg/mg high Na), while there is no significant change in arterial pressure after 7 days of a high sodium intake (DS low Na 131±2 mmHg, high Na 138±2 mmHg, DR low Na 135±3 mmHg, high Na 132±2 mmHg). These data indicate that a decrease in medullary ETB receptors and intrarenal production of ET‐1 in the DS rat could play an important role in the development of salt sensitive hypertension in the DS rat.
Upregulation of NHE3 in renal proximal tubule is associated with hGRK4 486V‐promoted salt sensitivity in transgenic mice
Authors: Wang Xiaoyan , Wang Zheng , Asico Laureano D , Escano Crisanto , Jose Pedro A
Published: June 2021

Salt‐sensitive hypertension in humans is associated with variants of GRK4. hGRK4γ 486V causes salt‐resistant mice (25% SJL/J, 75% CB57BL/6J) to become salt‐sensitive. In current studies, we measured blood pressure (BP), urinary sodium excretion (UNaV), and renal sodium transporters in the 486V transgenic mice (486V) and non‐transgenic littermates (NT) maintained on normal (0.9%, NS) or high (6%, HS) NaCl diet for 3 weeks. BPs were increased in 486V by HS but not affected in NT while UNaV was similar in both mouse strains on NS and HS, suggesting a blunted pressure‐natriuresis response. On NS, 486V mice had lower protein abundance of NHE3 (65±3, NT set as 100%) and αENaC (75±9) but greater abundance of NaPi2 (277±18), γENaC (164±34), and αNKA (196±28); No difference was found in NKCC2, NCC and βENaC. HS decreased NHE3 (47±6) and αENaC (67±8) only in NT, the expected normal response in salt‐resistant mice. HS did not decrease the other sodium transporters in either mouse strain. Because the activity of plasma membrane transporters is better reflected on the expression at the plasma membrane, we measured NHE3 in renal brush border membranes (BBM). On NS, BBM NHE3 was similar in 486V and NT mice (110±13 vs 100±27, respectively). On HS, it was dramatically increased in 486V mice (230±41) but not altered in NT mice (118±24). Our findings suggest that increased sodium transport in the proximal tubule may contribute to the blunted pressure‐natriuresis and salt‐sensitive hypertension in hGRK4γ 486V transgenic mice.
Continuous long‐term recording of renal (RSNA) and lumber (LSNA) sympathetic nerve activity and arterial pressure (AP) in conscious rats: Responses to chronic angiotensin II (AngII) administration
Authors: Yoshimoto Misa , Miki Kenju , Osborn John W
Published: June 2021

We measured long‐term RSNA, LSNA and AP responses to either AngII (150 ng/kg/min, s.c.) or vehicle (VEH) in rats consuming a high salt diet (2.0% NaCl). Rats were instrumented with an electrode on the renal or lumbar nerve and a telemetry transmitter for measurement of SNA and AP respectively. Beginning 7 days later, RSNA, LSNA and AP were measured continuously for 4 days before and 11 days during AngII or VEH. RSNA results: Control AP (mmHg) was similar in VEH (103 ± 2; N= 5) and AngII (105 ± 3; N=6) rats. On Day 1 of AngII, AP was increased (122 ± 6) whereas RSNA was decreased 26 % from control levels. AP and RSNA did not change in VEH rats on Day 1. On Day 10 of AngII, AP remained elevated (127 ± 9) and RSNA was reduced 30% from control. By day 10 of vehicle, AP was 103 ± 3 and RSNA was 121% of control levels. LSNA results: Baseline AP was similar in VEH (108 ± 3; N=3) and AngII (107 ± 4; N=4) rats. On Day 1 of AngII, AP increased (128 ± 6) and LSNA remained unchanged (94 ± 6% of control). By day 10 of AngII, AP remained elevated (151 ± 7) but LSNA was not different than control (91 ± 9 %). AP and LSNA did not change in VEH rats over the entire protocol. We conclude that AngII‐salt hypertension is associated with long‐term differential responses of renal and lumbar SNA. NIH Grant HL76312
Gene Regulation by Riboswitches
Authors: Breaker Ronald Ray
Published: June 2021

Riboswitches are structured RNAs usually located in the untranslated regions (UTRs) of bacterial mRNAs that bind metabolites and control gene expression. We have recently examined a series of riboswitches that carry newfound aptamer structures and that control gene expression using previously uncharacterized mechanisms. For example, several riboswitch classes have been discovered that sense the coenzyme S‐adenosylmethionine (SAM) and discriminate against the demethylated analog S‐adenosylhomocysteine (SAH). However, we have discovered a widespread riboswitch class that preferentially recognizes SAH versus SAM, and controls genes required to recycle SAH. We also have identified a large and well‐conserved RNA element that functions as a riboswitch for molybdenum coenzyme (Moco). These findings indicate that numerous metabolite‐sensing riboswitches likely remain undiscovered. Furthermore, we have determined that some mRNAs in eukaryotes use riboswitches to control alternative splicing. The diversity of riboswitch aptamer types and the diversity of mechanisms used by these molecular switches to control gene expression demonstrate the enormous structural and functional potential of RNA that is harnessed by modern organisms.
A novel pharmacologic alternative to ganglionic blockade: cardiovascular responses to systemic terbutaline
Authors: Peterson Elisha , Eisenach John H , Wilkins Brad W , Nicholson Wayne T , Johnson Christopher P , Joyner Michael J , Hesse Christiane
Published: June 2021

The ganglionic blocker trimethaphan (TMP) is no longer produced; alternatives are needed to inhibit baroreflex control of the circulation during systemic hemodynamic studies. We tested the hypothesis that combination anti‐cholinergic glycopyrrolate (Gly) and α2 receptor agonist dexmedetomidine (Dex) would inhibit baroreflex buffering in response to vasodilation to a similar degree as ganglionic blockade with TMP. In 2 separate trials, 5 males received an I.V. bolus and infusion of Gly‐Dex, and 11 males received TMP infusion. To offset the fall in blood pressure with TMP, phenylephrine (PE) was used to raise pressure to pre‐TMP baseline. Terbutaline was then infused for 15 minutes at 2 doses (33 and 67 ng/kg/min). Heart rate (HR), blood pressure (BP, oscillometric cuff for Gly‐Dex, brachial arterial line for TMP+PE), and cardiac output (CO, acetylene breathing) were recorded. Resting HR was higher with Gly‐Dex (mean ± SEM: 93±7mmHg) than TMP+PE (74±3mmHg, p<0.001) but resting BP, CO, stroke volume (SV), and total peripheral resistance (TPR) were similar between Gly‐Dex and TMP+PE. The cardiovascular responses to terbutaline were similar between Gly‐Dex and TMP+PE, except for the increase in CO which was significantly greater in the TMP + PE group. We conclude that Gly‐Dex is a viable pharmacologic alternative to TMP in physiologic measures of BP and TPR during vasodilator infusions.
Mast Cell Stabilization added to Beta‐Adrenergic Receptor Blockade has beneficial effects on Left Ventricular Function, Remodeling and Isolated Cardiomyocyte function in Canine Mitral Regurgitation
Authors: Pat Betty M , Killingsworth Cheryl , Denney Thomas , Powell Pam , Tillson Michael , Dillon A. Ray , Dell'Italia Louis J
Published: June 2021

Left ventricular (LV) dilatation in volume overload of isolated mitral regurgitation (MR) is marked by increased adrenergic drive and loss of extracellular matrix (ECM). Because mast cells are increased throughout the course of MR and may promote ECM degradation, we hypothesized that β1‐receptor blockade (β1‐RB) + mast cell stabilization (MCS) attenuates LV remodeling after 4 months of canine MR. β1‐RB +/− MCS did not attenuate the 30% reduction in ECM induced by MR. Three‐dimensional (3‐D) magnetic resonance imaging showed a 75% increase in LV end‐diastolic volume and a 45% increase in LV 3‐D radius/wall thickness (R/T) in MR +/− β1‐RB vs. normals. MCS did not attenuate LV dilatation but improved LV end‐systolic (ES) volume and 3‐D LVES R/T, resulting in improved LV fractional shortening. β1‐RB + MCS prevented cardiomyocyte elongation caused by MR + β1‐RB alone and normalized Ca++ transients, which were decreased in MR alone. In addition, isoproterenol induced Ca++ transients were improved with MCS. LV peak + dP/dtmax was significantly decreased in MR + β1‐RB and normalized by MCS. β1‐ RB + MCS reversed cardiomyocyte elongation and improved LV chamber function compared to β1‐RB alone without improving ECM loss, suggesting that MCS has a direct effect on cardiomyocyte remodeling and function in isolated MR. Supported by NIH RO1 HL54816 and SCCOR P50HL077100.
Muscarinic potassium channels play a significant role in the negative chronotropic response with or without background sympathetic tone
Authors: Mizuno Masaki , Kamiya Atsunori , Kawada Toru , Sugimachi Masaru
Published: June 2021

Although a larger decrease in the steady‐state heart rate (HR) in response to more potent tonic vagal stimulation (VS) is mediated by the muscarinic potassium (KACh) channels, the effect of background sympathetic tone on the contribution of the KACh channels to the negative chronotropic response remains to be elucidated. In anesthetized rabbits with sinoaortic denervation and vagotomy, we examined the HR response to stepwise vagal stimulation (VS) (5, 10, 15 and 20 Hz). A selective KACh channel blocker tertiapin attenuated the HR decrease to less than half of that observed under control condition. A concomitant tonic cardiac sympathetic stimulation (5 Hz) significantly augmented the HR reduction both in control (91±16 to 130±27 bpm, @20Hz VS, mean±SD, P<0.01) and tertiapin (30±12 to 67±13 bpm, P<0.01) condition. The contribution of KACh channels, assessed by the attenuation of the HR reduction, was 60±24 and 63±17 bpm in the absence and the presence of background sympathetic tone, respectively. In conclusion, muscarinic potassium channels play a significant role in the negative chronotropic response with or without background sympathetic tone.
Beta‐2 adrenergic receptor haplotype and echocardiographic indices in heart failure patients
Authors: Eisenach John H , Snyder EM , Hulsebus ML , Turner ST , Johnson BD
Published: June 2021

We recently reported altered cardiovascular structure and function in heart failure (HF) patients based on the Arg16/Gly and Gln27/Glu beta‐2 adrenergic receptor (ADRB2) polymorphisms. The purpose of this study was to extend the echocardiographic analysis based on haplotype combination of ADRB2 at nucleotides −654, 46 79, 252, 491, 523, in a larger database of HF patients (n=126, age 56±1yr, LV ejection fraction, LVEF 29±1%, BMI 28±0.4kg/m2, mean±SEM) and healthy controls (n=100, age 50±2yr, LVEF 63±0.7%, BMI 25±0.3kg/m2). The most common haplotypes in both HF and control were 2, 8, and 12 (2=AACGCC, 8=GGCACA, and 12=GGGGCC, for nucleotides −654, 46, 79, 252, 491, and 523). Cardiac function did not differ according to haplotype pairs in the control subjects. In HF, the widest discrepancies were between haplotype pair 2/2 (which includes the Arg16+Gln27 homozygotes) and haplotype pair 12/12 (which includes the Gly16+Glu27 homozygotes), respectively: 2/2 displayed lower cardiac index (2.3±0.1 v 2.7±0.1 l/min/m2), greater left atrial diameter (53±2 v 47±2 mm), shorter LV decel time (185±11 v 224±14 msec), and greater right ventricular pressure (44±2 v 33±2 mmHg) p<0.05 for all). These data suggest that in HF, haplotype variation of the ADRB2 is associated with differences in cardiac function, and SNP positions 16 and 27 remain major loci of interest.

Support: K23RR‐17520, HL‐71478.
Epidermal Growth Factor Receptor activates ERK but not JNK through a Ras independent pathway during cardiomyocyte stretch
Authors: Duquesnes Nicolas , Morel Eric , Lezoualc’h Frank , Crozatier Bertrand
Published: June 2021

We evaluated the Mitogen Activated Protein kinases (MAPK) pathways activated during stretch, a major determinant of ventricular hypertrophy, in neonatal rat ventricular cardiac myocytes in culture. The activations of Ras, ERK and JNK peaked 15 min after the initiation of stretch, suggesting a cause‐effect relationship. A dominant negative form of Ras did not inhibit stretch‐induced ERK and JNK activations showing that Ras was not involved in MAPK stimulation. Among the pathways that activate MAPK, tyrosine kinases were shown to play a major role. Genistein, a tyrosine kinase inhibitor, significantly decreased both ERK and JNK activations during stretch but did not inhibit Ras. The epidermal growth factor receptor (EGFR) is one of the tyrosine kinase receptors. We examined its role using a specific blocker, AG1478. It decreased both basal ERK phosphorylation through the Ras‐Raf1‐MEK cascade and stretch‐induced ERK activation independently of this cascade. In contrast with its effects on ERK, AG1478 did not significantly modify JNK phosphorylation both in basal conditions and during stretch. In addition, our data show that the PI3 kinase‐Akt /PKB pathway is downstream of the EGFR, participating in ERK but not JNK activation. We conclude that the EGFR acts on ERK phosphorylation through 2 different pathways: in basal conditions through the Ras pathway and during stretch through the PI3kinase pathway.
Effects of Bedrest, Aging and Exercise Training on the Dynamic Starling Mechanism
Authors: Shibata Shigeki , Hastings Jeff , Bhella Paul , Pacini Eric , Krainski Felix , Palmer Dean , VanGundy Tiffany , Creson Daniel , Fu Qi , Zhang Rong , Levine Benjamin D.
Published: June 2021

We assessed how physical activity and/or aging modify the dynamic Starling mechanism. Spectral transfer function gain between beat‐to‐beat changes in left ventricular end‐diastolic pressure (LVEDP) and stroke volume (SV) was used as an index of the dynamic Starling mechanism. Beat‐to‐beat pulmonary artery diastolic pressure (Swan‐Ganz catheter) was used as an index of beat‐to‐beat LVEDP. Beat‐to‐beat SV was calculated from finger arterial pressure with the Modelflow (Portapres). 1) A cross‐section of sedentary individuals 20–80 yrs old showed a linear relationship between the dynamic Starling mechanism index and age (Index=−0.022*Age+1.98, R=0.61 P<0.001). 2) Dynamic Starling mechanism indices in two elderly subjects (60 and 75 yrs old), who had been training (>150min/wk) for more than 25 yrs were 1.19 and 1.32. These were substantially higher than those predicted from their age. 3) A total of 14 subjects underwent 5‐wk head down bedrest; 11 with and 3 without exercise countermeasure (Ex and non‐Ex). Both the Ex and the non‐Ex showed a significant decrease of the dynamic Starling mechanism after bedrest while the magnitude of the decrease was lower in the Ex (−33%) than in the non‐Ex (−64%). These findings suggest unique parallels between bedrest deconditioning and aging in cardiovascular function; exercise training may be an effective countermeasure against both bedrest and aging. Supported by NSBRI and NIH
Misconceptions about Edward Jenner and the Eradication of Smallpox
Authors: Schneider Suzanne
Published: June 2021

In the 18th century smallpox was a scourge that infected 60% and killed 10% of England's population. On May 14, 1796 a young English doctor persuaded a local farmer to allow his 8 yr‐old son, James Phipps, to be infected, first with cowpox, then with smallpox. The boy did develop cowpox but was resistant to smallpox. The boy was the first to be “vaccinated” until 1979, when the World Health Organization declared smallpox eradicated. There are several misconceptions about this story.

Edward Jenner was not the first to use cowpox to prevent smallpox. Other doctors in England were doing the same, but Jenner brought attention to this procedure. He self‐published his results after his original paper was rejected.

His experiments seem unethical. Actually, his procedures were not unusual for his times. Even today his work would pose an interesting risk/benefit dilemma. Jenner remained friends with James, who was a pall bearer at his funeral.

Jenner theorized the virus started with an infected horse hoof disease. Later it was discovered that the virus passes from rodent to cat to cow to milkmaid.

Jenner was a small country doctor, astute but with little education. Jenner, from a prestigious family, apprenticed with a local doctor at the age of 13 for seven years. He then trained in medicine at St. George's Hospital in London. After 2 years he returned home to became a gentleman doctor and well‐known naturalist. Lack of medical diploma was not unusual, with only two official medical schools in England.

Smallpox is eradicated. The virus does exist today, in frozen vials stored in Atlanta and Moscow. The virus is preserved to develop vaccines in case the virus reappears or is unleased by terrorists.
The activation of immunoreceptor NKG2D in MPTP‐induced animal models stimulates the pathogenesis of Parkinson's disease
Authors: Tay Samuel SW , Chao Yinxia , He Beiping
Published: June 2021

Neuroinflammation in Parkinson's disease (PD) involves the activation of microglia, complement receptor system and possibly systemic natural killer (NK) cells, suggesting that innate immunity has an important role in the pathogenesis of this disease. We examined NK cell activity and the expression of its regulatory molecules in the substantia nigra of MPTP induced PD models and compared the results with those of healthy controls. Expression of the immunoreceptor NKG2D was significantly increased in the PD models, thereby inducing dopaminergic neurons in the substantia nigra compacta (SNc) to become susceptible to NK cells after the latter bind to receptors on their target cells. It is hypothesized that the activation by NK cells via their target iimunoreceptor NKG2D stimulates neuroinflammation leading to PD in MPTP‐induced animal models.

Supported by an Academic Research Grant R181‐000‐096‐112 from the National University of Singapore.
A preliminary examination of astrocyte morphology across multiple human forebrain nuclei
Authors: Krilowicz Beverly L. , Nienhuis Robert , Thannickal Thomas , Siegel Jerome
Published: June 2021

Multiple physiological roles for astrocytes have emerged over the last two decades, though little attention has focused on astrocyte morphology. In this study we address this omission and supply data that describe differences in astrocyte populations across 14 human forebrain nuclei, including sites in the thalamus, hypothalamus and basal ganglia. 40 um coronal sections from the human forebrain (n=4 brains) were stained with antibody to GFAP and sections from 2 of the brains were also counterstained with H+E. Stereoinvestigator software was used to randomly sample sites within each nucleus using accepted stereological techniques. Individual brain and forebrain nuclei had significant effects on both mean astrocyte size and density (P<0.001), as well as the distributions of astrocyte size and density (P<0.001). Our results suggest that human astrocyte morphology differs significantly across the 14 forebrain nuclei examined in this study. These results suggest that astrocytes exhibit regional anatomical differences that may correlate with differences in their physiological roles associated with established differences in neuronal physiology in these nuclei. (CSULA Sabbatical Leave Award to BLK.)
Nok‐mediated RPE integrity is essential for retinal epithelial polarity and cytoarchitecture
Authors: Wei Xiangyun
Published: June 2021

In vertebrates, the retinal pigment epithelium (RPE) and the retina are derived from different parts of the optical vesicle. Previous studies suggest that the RPE plays a role in the development of retina by unknown mechanisms. The recent findings of mutations of epithelial polarity genes such as nok that cause defects in both the RPE and the retina further energizes the research on whether and how the RPE participates in retinal development. Here, we generated a transgenic fish line (pt106) in which Nok function is restored in the RPE but not in the neural retina. The characterization of pt106 revealed that the RPE plays an essential role in maintaining proper polarity of the retinal neuroepithelium. This polarity maintenance is largely dependent on Nok‐mediated RPE integrity but not on retinal expression of Nok. In addition, we found that the restoration of RPE integrity has a dramatic positive effect on retinal cellular pattern formation during neurogenesis. Our results suggest a pathway that governs the cellular pattern formation of the neural retina: Nok‐mediated RPE integrity ensures the retinal neuroepithelium to polarize correctly; the correct retinal epithelial polarity then underlies proper migration and spatial arrangements of retinal cells during neurogenesis. Our study is the first to show the essentiality of RPE integrity for retinal neuroepithelial polarity and cellular pattern formation.
Immortalized keratinocytes that overexpress H‐ras produce an invasive, randomized epithelium in organotypic culture
Authors: Vaughan Melville B. , Andrews Capri M. , Wright Woodring E. , Shay Jerry W.
Published: June 2021

Ras proteins affect proliferation and expression of collagen‐degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen‐rich basement membrane. Therefore we asked whether overexpression of H‐ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent. Previously described immortalized foreskin keratinocytes were engineered to overexpress the H‐ras protein. Skin equivalents, composed of fibroblast‐contracted collagen gels overlaid with keratinocytes, were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to proliferation (Ki67), differentiation (involucrin, keratin‐17, p63) and basement‐membrane formation (collagen IV, laminin‐5) were studied. Results showed that H‐ras keratinocytes produced an invasive, randomized epithelium most apparent in the lower strata. The superficial strata retained somewhat normal characteristics, however. Studies such as these may be helpful to understand cancer progression so that treatment or even prevention can be developed.
ECCENTRIC FORCES DURING RESISTANCE EXERCISE IN A REHABILITATION SETTING: ARE THEY SUPRA‐MAXIMAL?
Authors: Foreman K. Bo , Dibble Lee E. , Marcus Robin L. , Droge John , Ward Andrew , LaStayo Paul C.
Published: June 2021

The purpose of this study was to compare eccentrically induced force production during a rehabilitative eccentric resistance exercise to forces produced during a maximal isometric force exercise. Twenty‐three (13 female, 10 male) unimpaired, health professions graduate students participated in a single force‐testing session lasting 30 min. The testing session included lower extremity (LE) extension exercises on a recumbent ergometer (Eccentron, Denver, CO). The first two LE exercises consisted of maximal isometric efforts and the second two were characterized as “hard” eccentric efforts at 18 rpm. Forces at the footplate and knee joint angles were recorded. Results indicated that the average force during a “hard” eccentric effort (male: 444.11N ± 83.84, female: 257.92N ± 38.21) were 54% greater (p<0.001) than the force induced with a maximal isometric effort (male: 288.17 ± 52.71, female: 167.27N ± 29.46) regardless of sex or joint angle. Furthermore, the eccentric force (male: 315.16N ± 116.03, female: 194.71N ± 59.31) produced at the same knee joint angle as the maximum isometric force (male: 288.17N ± 52.71, female: 167.27N ± 29.46) were 13% greater (p<0.04). In conclusion, forces induced in the lower extremity during a “hard” eccentric resistance exercise effort, are 13‐54% greater than maximal isometric forces.
Neurovascular Anatomy of the Embryonic Quail Hindlimb
Authors: Bentley Matthew T , Poole Thomas J
Published: June 2021

Blood vessel and nerve development in the vertebrate embryo possess certain similarities in pattern and molecular guidance cues. In order to study the specific influence of shared guidance molecules on nervous and vascular development, an understanding of normal neurovascular anatomy must be in place. The avian embryo is an excellent system for the study of neurovascular development and has traditionally focused on the chick and the quail forelimb. We have studied the pattern of nervous and vascular development in the quail hindlimb using immunohistochemistry and fluorescently labeled intravital injection combined with confocal and epifluorescent microscopy. The developmental patterns of major nerves and blood vessels of embryonic hindlimbs between the stages E2.75 and E6.5 are described. A high degree of spatial congruency was found between major nerves and blood vessels of the quail hindlimb. The spatial pattern of nervous development in the quail hindlimb was essentially identical to that of the chick, but was found to proceed at a slightly more rapid pace. This study provides the anatomical basis necessary for future studies examining the role of Sema3A on the congruency of nerves and blood vessels entering the embryonic hindlimb bud.
Quality control of mRNA export: An evolutionarily conserved zinc finger protein mediates preferential export of properly processed mRNA to the cytoplasm
Authors: Corbett Anita H , Kelly Seth M , Fasken Milo B , Leung Sara W , Stewart Murray
Published: June 2021

For gene expression to occur, properly processed mRNA transcripts must be exported from the nucleus to the cytoplasm for translation of the information they encode. While many RNA binding proteins interact transiently with transcripts to mediate processing steps, others remain associated and regulate subsequent steps in post‐transcriptional gene expression. Studies in our laboratory provide insight into how one mRNA binding protein couples mRNA processing and export. The essential yeast Nab2 protein is an evolutionarily conserved zinc finger protein required both for proper polyadenylation and mRNA export. Our recent work reveals that Nab2 binds specifically to polyadenosine RNA through a novel zinc finger‐dependent mechanism. The conserved N‐terminal domain of Nab2, which assumes a PWI‐fold found in other mRNA binding proteins, then mediates interactions with Mlp1 located at the inner nuclear basket of the nuclear pore. Our recent resolution of the structure of the Nab2 N‐terminal domain allowed us to design and exploit Nab2 mutants to demonstrate that interaction between Nab2 and Mlp1 is important for proper mRNA export. Supported by a grant from the NIH.
Cwc24p, a novel S. cerevisiae RING‐finger protein, affects U3 snoRNA splicing and rRNA processing
Authors: Goldfeder Mauricio Barbugiani , Oliveira Carla Columbano
Published: June 2021

Ribosome biogenesis involve the extensive processing of a precursor rRNA 35S by approximately 150 trans‐acting factors, giving rise to the mature rRNAs. Among these factors are snoRNPs (small nucleolar ribonucleoproteins), that are formed by a guide snoRNA (that base‐pairs with target sites within the pre‐rRNA) and evolutionarily conserved proteins. In this work we present the functional characterization of Cwc24p, a novel S. cerevisiae RING‐finger that interacts with Nop17p (a box C/D snoRNP assembly factor) and with Cef1p (a splicing factor, component of the NTC complex). Consistent with the interaction data, Cwc24p localizes to the cell nucleus. Using a conditional strain (GAL1::CWC24) we observed a small defect in rRNA processing upon depletion of Cwc24p. In order to investigate the involvement of Cwc24p in splicing, a splicing‐specific microarray analysis was conducted and revealed that depletion of Cwc24p leads to the accumulation of both U3 pre‐snoRNAs. U3 snoRNA, which is transcribed from two intron‐containing genes in yeast, is involved in the early cleavages of 35S pre‐rRNA, and the defective splicing of pre‐U3 detected in cells depleted of Cwc24p may be responsible for the accumulation of the 35S precursor rRNA. These results led us to the conclusion that Cwc24p is involved in pre‐U3 snoRNA splicing, indirectly affecting pre‐rRNA processing.

Supported by FAPESP and CAPES
Interactions of C‐ and N‐domains of mammalian mitochondrial translational initiation factor 3 with mammalian mitochondrial 28S subunits and their role in initiation complex formation
Authors: Haque Md. Emdadul , Spremulli Linda L
Published: June 2021

Bacterial initiation factor 3 (IF3) is organized into two domains (N‐domain and C‐domains) separated by a linker region. The C‐domain harbors the basic functions of IF3 with the N‐domain enhancing the binding of this factor to the small subunit. Mitochondrial IF3 (IF3mt) is also composed of N‐ and C‐domains. However, the N‐domain is preceded by an extension and the C‐domain is followed by an extension. To investigate the roles of these extended domains in IF3mt, constructs of the N‐ and C‐domains of IF3mt with and without the connecting linker were prepared and their activities tested. The Kd's for the binding of full length IF3mt and its C‐domain with and without the linker to mitochondrial 28S subunits are 30, 60 and 67 nM, respectively indicating that much of the ribosome binding interactions are mediated by the C‐domain. The N‐domain binds to 28S subunits with a 10‐fold lower affinity. Removal of the N‐domain drastically reduces the activity of IF3mt in the dissociation of mitochondrial 55S ribosomes although the C‐domain itself retains some activity. This residual activity depends significantly on the linker region. The N‐domain alone has no effect on the dissociation of ribosomes. The C‐domain with the linker has very similar activity as full length IF3mt in initiation complex formation. Surprisingly, the N‐domain with the linker significantly inhibits initiation complex formation. (Supported by NIH Grant GM32734)
StIP‐1/Elp2 regulation of DeltaF508‐CFTR vesicular exocytosis and of HSP70 expression
Authors: Suaud Laurence , Rubenstein Ronald C
Published: June 2021

Sodium 4‐Phenylbutyrate (4PBA) improves the intracellular trafficking of deltaF508‐CFTR in Cystic Fibrosis (CF) epithelial cells. In IB3‐1 CF bronchiolar epithelial cells, 1 mM 4PBA transiently increases the expression of StIP‐1, a STAT‐3 interacting protein. StIP‐1 is identical to Elongator protein 2 (Elp2); Elongator is a protein complex that facilitates mRNA transcription by RNA polymerase II. In immunofluorescence experiments, overexpression of StIP‐1 in IB3‐1 cells increased deltaF508‐CFTR trafficking to the plasma membrane even in the absence of 4PBA treatment. In contrast, overexpression of a mutant of StIP‐1 blocked the 4PBA‐stimulated improvement of deltaF508‐CFTR trafficking. StIP‐1 was observed to colocalize with a 58k golgi protein (Golgi marker) as well as with Snap25/23 (vesicle trafficking marker). These data suggest that StIP‐1 may regulate deltaF508‐CFTR trafficking along the exocytic pathway. As Elongator also directly regulated Hsp70 expression in yeast (Hanq et al., 2007), we tested whether StIP‐1/Elp2 regulates Hsp70 expression, which may regulate deltaF508 trafficking. In T84 cells transfected with siRNA targeting StIP‐1/Elp2, StIP‐1/Elp2 protein expression was reduced by about 50% and Hsp70 expression was reduced by 80% compared with control cells. These data suggest that StIP‐1/Elp2 may also influence deltaF508‐CFTR trafficking by regulating HSP 70 expression.
Integrin‐dependent and ‐independent potentiation of L‐type Calcium Current (Cav1.2) by cell stretch
Authors: Gui Peichun , Zamponi Gerald W , Davis George E , Davis Michael J
Published: June 2021

Recent studies in our lab using both native and heterologously expressed calcium channels (Cav1.2) indicate that L‐type calcium current is potentiated by activation of a5b1 integrin and requires PKA‐ and Src‐ phosphorylation of Cav1.2 C‐terminal residues. To test whether Cav1.2 channels are mechanosensitive and the possible role of integrins in this process, patch clamp methods were used to investigate the properties of Cav1.2 expressed in HEK 293 cells during intracellular application of positive pressure or more physiological, longitudinal two‐pipette stretch of a flexible membrane onto which cells were plated. The membrane was coated with either fibronectin or poly‐lysine to assess integrin‐dependent or ‐independent responses, respectively. In cells expressing the smooth muscle calcium channel isoform (Cav1.2b) alone or in combination with b2a/a2d‐1 accessory subunits, positive pipette pressures of 10~30 mmHg resulted in 20~45% increases in peak inward current. Similar results were found for the neuronal L‐type channel isoform Cav1.2c. Longitudinal stretch between 110 to 130% of resting cell length induced 25 to 60% graded increases in Cav1.2c current. The magnitude of the increase was significantly greater on fibronectin‐coated substrate than on poly‐lysine coated substrate. Preliminary findings showed that native L‐type calcium currents from VSMC exhibit similar mechanical sensitivity. Our results suggest that the Cav1.2 channel subunit shows a limited amount of intrinsic mechanosensitivity and that enhanced channel activity in response to longitudinal stretch may involve the integrin‐extracellular matrix axis.
Psalmotoxin‐1 docking to human Acid Sensing Ion Channel 1
Authors: Qadri Yawar J , Berdiev Bakhrom K , Song Yuhua , Lippton Howard L , Fuller Catherine M , Benos Dale J
Published: June 2021

Psalmotoxin‐1 (PcTx‐1) is a peptide toxin found in the venom of the Trinidad Chevron tarantula. It potently and specifically inhibits Acid Sensing Ion Channel 1 (ASIC‐1) containing channels. The activation of ASIC‐1 channels may lead to the sensation of pain and neuronal death during ischemia. This toxin may thus be a novel therapeutic reagent for the treatment of stroke or pain states. Using computational methods, we describe a docking site of PcTX‐1 with ASIC‐1 where the toxin interacts with the channel primarily through electrostatic interactions. Further analysis of the energetics of binding recapitulates single‐channel bilayer data which shows synthetic PcTX‐1 interacts with hASIC‐1 to stabilize a long‐closed state of the channel. Examining the binding as a function of pH suggests that the toxin‐channel interaction decreases as pH is lowered, an observation noted in prior radioligand binding studies. With the elucidation of the toxin/channel interface, this work will lead to the design of small molecule inhibitors that can mimic the effects of this peptide. This work is supported by NIH Grant DK37206 and the AMA foundation.
Increased nuclease activity in response to high NaCl
Authors: Dmitrieva Natalia I. , Burg Maurice B.
Published: June 2021

High NaCl increases DNA breaks. The increase of breaks persists as long as NaCl remains elevated, but rapidly decreases when NaCl is lowered. The mechanism is uncertain. Nucleases are enzymes that break DNA during normal DNA processing (such as replication and transcription), during DNA repair and during apoptosis. In the present studies we tested for activation of nucleases in response to high NaCl. Activities of nucleases in extracts from HeLa cells exposed to 300 and 400 mosmol/kg (NaCl increased) were estimated by single cell electrophoresis (Comet assay). In this assay, broken DNA from cells that are embedded in agarose migrates under the action of an electrophoretic field, forming the "tail" of a "comet". The amount of DNA that migrates out of a nucleus and the distance that it migrates are measures of DNA fragmentation. Cells grown at 300 mosmol/kg were embedded in agarose and permeabilized, then incubated with protein extracts from cells remaining at 300 mosmol/kg or exposed to 400 mosmol/kg for 2 hours. Extracts from cells exposed to 400 mosmol/kg caused a 70% increase in Comet tail moment and doubling of the total Area of the Comet image, indicating increased DNA fragmentation. We propose that activation of nucleases may contribute to the high NaCl‐induced increase of DNA breaks. Supported by the Intramural Program of NHLBI.
Reversal Reaction in Leprosy Is Associated with Increased CXCL10 Production
Authors: Scollard David M. , Fowlkes Natalie , Martinez Alejandra , Chaduvula Mehervani , Nath Indira , Stryjewska Barbara , Williams Diana
Published: June 2021

Leprosy usually follows an indolent course, but is complicated in many patients by a systemic inflammatory syndrome termed 'Reversal Reaction' (RR) that appears to be a spontaneous enhancement of cellular immunity. RR is a major cause of neuritis and other morbidity in leprosy, but the mechanisms underlying this clinical syndrome are unknown, and no laboratory tests are available to diagnose or monitor it. Real‐time quantitative PCR revealed elevated expression of CXCL10 mRNA in skin biopsies during RR compared to pre‐RR biopsies in 7/7 patients. Immunohistochemical staining of skin biopsies did not identify an increase in the number of leukocytes strongly positive for CXCL10 or its receptor, CXCR3, but suggested an overall increase in staining for CXCL10. Measurements of serum levels of CXCL10 by ELISA in sequential, monthly specimens from 20 borderline (BL and BT) patients revealed significantly elevated levels of this chemokine associated with RR (p = 0.03). Together, these findings suggest that increased CXCL10 is a characteristic of cutaneous and systemic manifestations of RR. These findings may offer new possibilities for laboratory support in the diagnosis and monitoring of RR, and suggest that studies of the regulation of CXCL10 may provide further insight into the mechanisms responsible for this complication of leprosy.
A genome‐wide linkage analysis approach to find disease‐causing gene of Inherited Intracranial Aneurysm
Authors: Park Sung‐Hoon , Hong Seong‐Chul
Published: June 2021

Intracranial aneurysms (IAs) is characterized by abnormal bulging of one of arteries in the brain. The disease is believed to be caused by many environmental and genetic factors. In an attempt to elucidate the genetic factor contributing the etiology of IAs, a genome‐wide linkage analysis was conducted to find the locus for the disease‐causing gene of IAs. By careful investigation of the inheritance pattern of 600 IA patients at either Chonbuk National University Hospital (Chonju, South Korea) or Hana Hospital (Ilsan, South Korea), we recruited 5 families inheriting IAs dominantly. Entire genomes of the families were genotyped at 10 cM interval using ABI Linkage Mapping Set. A parametric method for linkage analyses was employed for each markerand calculated two‐point using LINKAGE package (v5.1). A lod score maximum of Zmax = 3.034 at θ= 0.00 was obtained with marker D8S549 located on chromosome 8p22. And a maximum multipoint LOD score was 3.067 on the 8p22 locus. Our results clearly imply that IAs in Korea have a genetic locus in 8p22.
Evaluation of vascular endothelial staining by various antibodies in human and xenograft tumors using three different methods of fixation
Authors: Breckenkamp Ann E , Mintze Karen , Barnes Versie , Vance Tasha , Pierson Nicholas , Pangallo Jenna , Sandusky George
Published: June 2021

Abstract

Light microscopy was used to compare the staining of various antibodies against the vascular endothelial markers CD31, CD34, CD105, EphB2, and EphB4 in human and xenograft tissues that were fixed in three different fixatives: 4% paraformaldehyde (PF), 10% Neutral Buffered Formalin (NBF), or Zinc‐Tris (ZT). Since the ability of an antibody to bind to its target molecule depends on the fixation method, it would be useful to identify antibodies that stain tumor vasculature regardless of which fixative was used to preserve the tissue. Each antibody evaluated was optimized in ZT fixed tissues, and the protocol was used to stain all samples, except for two antibodies that did not stain ZT fixed tissues. Bethyl Laboratories Rabbit Anti‐Human CD31 (#IHC‐00055) and Cell Signaling Mouse Anti‐Human CD34 (#3569) gave excellent staining in human tissues prepared in 4% PF, 10% NBF, and ZT, in contrast to the other antibodies evaluated. The Bethyl antibody also gave moderate staining in HCT116 xenografts prepared in all three fixatives, making it the only antibody evaluated that was useful for staining vasculature in human breast, colon, and lung tissues and xenografts for all three fixatives.