bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of HepG2 and Huh7 cells using dimethyl sulfoxide
Authors: Mowbray Catherine Anne , Howard Alison , Morsman Janine , Hirst Barry H.
Published: June 2021

The secondary hepatocytes HepG2 and Huh7 are phenotypically poorly differentiated compared with hepatocytes in situ, limiting their use in drug development. This study assessed the efficacy of dimethyl sulfoxide (DMSO) to promote mature hepatocyte‐like differentiation. mRNA of two hepatocyte maturity markers (albumin (ALB) and transferrin (TF)), one of immaturity (α‐fetoprotein (AFP)), and key metabolic enzymes CYP3A4 and UGT1A1, was measured by qPCR. Responses to DMSO were normalised to confluent untreated cells (CUC). 1% DMSO increased ALB and TF mRNA in both cell lines with maximal expression at 15 to 20 days, followed by a return to control levels (eg. HepG2: ALB: confluent untreated cells (CUC): 1.0 ± 0.1; 15d: 6.6 ± 2.3, p<0.05. TF: CUC: 1.0 ± 0.2; 20d: 13.8 ± 0.6, p<0.05). DMSO did not reduce AFP mRNA in either cell line. CYP3A4 mRNA was low in both cell lines and not affected by DMSO. 2% DMSO increased UGT1A1 mRNA in both cell lines (HepG2: CUC: 1.0 ± 0.2; 5d: 2187.0 ± 153.0, p<0.05. Huh7: CUC: 1.0 ± 0.2; 28d: 4.0 ± 0.3, p<0.05). The data indicate these cell lines may effectively be differentiated to provide suitable models for early stage drug testing. Funded by BBSRC and Sanofi‐Aventis.
Effects of nitric oxide on HIF‐1α and FIH‐1 following Clostridium difficile toxin exposure
Authors: Lee Joshua Youngun , Hirota Simon , Traboulsi Danya , Beck Paul , MacDonald Justin
Published: June 2021

Clostridium difficile (C.diff) is a leading cause of infectious colitis in humans. C.diff toxins disrupt the intestinal epithelial barrier and induce the release of proinflammatory mediators. The hypoxia‐inducible factor‐1 (HIF‐1α) is a stress‐induced transcription factor that mediates mucosal and barrier protective genes. Nitric oxide (NO) and the factor‐inhibiting HIF‐1α (FIH‐1) modulate HIF‐1α expression and signaling, respectively. We hypothesize that NO produced during C.diff toxin‐induced intestinal injury is an important signal to activate HIF‐1α‐mediated protection against C.diff toxin‐induced damage.

The in vitro studies analyzed HIF‐1α and FIH‐1 levels during toxin exposure of Caco2 cells. Cells were challenged with both toxin and nitric oxide synthase (NOS) inhibitors to study NO effects on HIF‐1α/FIH‐1 expression. In vivo studies assessed ileal loop tissue from toxin‐treated wild‐type (WT) and inducible NOS knockout (iNOS KO) mice.

We have identified that HIF‐1α levels increase at least two‐fold in toxin‐treated cells. FIH‐1 levels also show a 35% decrease in response to toxin. This increase in HIF‐1α protein was suppressed with NOS inhibition. In vivo, serum NO levels increased with toxin exposure; a response which was abrogated in iNOS KO mice.

Further study is needed to demonstrate a direct effect of NO on HIF‐1α levels in response to C.diff toxin in vivo. Funding provided by CAG and CIHR.
Mitochondrial calcium flux modulates hypoxic vasoconstriction in sea lamprey dorsal aortas
Authors: Russell Michael
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) ensures optimal ventilation and perfusion matching in the normal lung, whereas global HPV due to altitude or disease leads to significant morbidity and mortality. Hypoxic vasoconstriction (HV) has been identified in aortas from every vertebrate class except Mammalia, suggesting a long history for HV outside respiratory tissues. We have studied the sea lamprey dorsal aorta (LDA) to identify the most basic signaling processes underlying HV. Previously we found that LDA efficiently cycled intracellular calcium (Ca2+i) during HV through an unknown mechanism. In the present study we hypothesized that mitochondria may be involved in Ca2+i flux during HV in isolated LDA. Using standard myography, we examined the effects of specific inhibitors of mitochondrial Ca2+i flux. HV was significantly reduced (77.4 ±7.3% of control, n=8) by the specific mitochondrial Na+/Ca2+ exchange inhibitor CGP37157 (2.5 μM) in calcium‐free (0 Ca2+o) buffer. Conversely, HV was augmented (127±11.9%, n=12) in the presence of the mitochondrial calcium uptake inhibitor RU360 (10 μM) in 2 mM Ca2+o. HV was significantly reduced (77.4 ±7.3%, n=8) in the presence of RU360 following a single hypoxic exposure in 0 Ca2+o. These results suggest that mitochondria regulate Ca2+i availability during hypoxia in isolated LDA. Supported by a grant from the MedCen Foundation of the Medical Center of Central Georgia.
Presence of mRNA for the vanilloid receptor in frog skeletal muscle
Authors: del Socorro Ortiz‐Mesina Monica , Huerta Miguel , Castro Elena , Montoya‐Perez Rocío , Saavedra‐Molina Alfredo , Sánchez‐Pastor Enrique , Urzúa Zorayda , Trujillo Xóchitl
Published: June 2021

We explored the presence of the vanilloid receptor (TRPV1) in the frog skeletal muscle by identifying the mARN for TRPV1 by RT‐PCR using as positive control the rat muscle and exploring the action of capsaicin on caffeine evoked contractures. Tension recording experiments were carried out on bundles from the slow cruralis and the fast bundles from the extensor digitorum longus muscle. Tension evoked by caffeine (6 mM) in the absence or presence of capsaicin and capsazepine was compared. In the slow muscle fibers, capsaicin (5 and 10 μM) diminished the maximum tension until 91.24 ± 2.53, and 85.15 ± 1.2 % respecting to control. This effect was blocked after application of capsazepine (10 μM). In the fast muscle fibers, capsaicin (1 and 10 μM) diminished the maximum tension until 79.4 ± 1.84 and 71.37 ± 3.4 %, regarding to control. However, this effect was partially reverted in the presence of capsazepine (1 μM). mRNA for TRPV1 was detected in the frog slow cruralis muscle, but not for the fast EDL muscle. Capsaicin modulates tension in the slow skeletal muscle fibers possibly via TRPV1 receptor. Also, results in the fast skeletal muscle fibers suggest an independent effect of capsaicin on TRPV1 and possibly directly on some step of the E‐C coupling. Financed by CONACyT‐Mexico grant 83113 to MH. MOM, RMP, ZU received CONACyT fellowship.
Effect of Dietary Cholesterol on Blood Biomarkers during Resistance Training: Randomized Controlled Trial
Authors: Riechman Steven , Lee Chang‐Woock , Lee Teak , Chen Vincent , Bui Steve
Published: June 2021

To examine possible explanations for the observation that cholesterol is positively associated to skeletal muscle responses to resistance training, a randomized, double blind placebo controlled trial of dietary cholesterol administration during 12 weeks of resistance exercise training (RET, 3x/week, 2‐3 sets, 8–12 reps, 70% of max strength) in 50–65 year old generally healthy men and women was conducted. Fasting acute and chronic blood samples were collected to determine the association of the cholesterol administration and changes in blood biomarkers of select anabolic hormones, and inflammatory and metabolic markers that may help explain muscle responsiveness to cholesterol. Groups consumed either 3.5 mg/kg lean/day (<200 mg/day, LC, N=9), 7.0 mg/kg lean/day (~400 mg/day, MC, N=9), or 14.0 mg/kg lean/day (~800 mg/day, HC, N=9) of cholesterol via egg powder dissolved in a chocolate flavored food. Dietary cholesterol did not alter resting or mean testosterone (LC=5.5±0.9, MC=4.8±1.0, HC=2.9±1.1 ng/ml) but was positively associated to responsiveness to acute RET. Resting C reactive protein, insulin, cortisol, glucose, and c‐peptide were unaffected by RET or cholesterol. Though cholesterol is associated to muscle responsiveness RET, blood biomarkers do not provide sufficient clarification of the potential mechanism.
Elevated levels of phosphate enhance myosin driven actin filament velocity under acidic conditions
Authors: Stout Jordan Catherine , Guha Arunima , Harkey Jennifer , Debold Edward Patrick
Published: June 2021

Elevated levels of phosphate (Pi) and decreased pH (i.e. acidosis) are believed to contribute to muscular fatigue by directly inhibiting muscle's molecular motor, myosin. However, the mechanisms of inhibition are not fully understood at a molecular level. We determined the effects of elevated Pi (30mM) and low pH (6.5) at saturating ATP (2mM), separately and in combination, on myosin's ability to move actin in an in vitro motility assay (Vactin). Decreasing the pH without added Pi significantly (p<0.05) slowed Vactin (6.1 ± 0.2 at pH 7.4 vs. 1.6 ± 0.2 um/s at pH 6.5). The addition of Pi slightly increased Vactin at pH 7.4 (6.1 ± 0.2 at 0mM Pi vs. 6.8 ± 0.2 um/s at 30mM Pi), while at pH 6.5, Pi increased Vactin ~2‐fold (1.6 ± 0.2 at 0mM Pi vs. 3.3 ± 0.2 um/s at 30mM Pi). Repeating these experiments at sub‐saturating ATP showed that elevated Pi decreased Vactin below 1mM ATP at pH 7.4. However, at pH 6.5, elevated Pi continued to increase Vactin at all measured ATP concentrations (from 100uM to 2mM). These data indicate that the effects of Pi on myosin depend on both the pH and the ATP concentration. Since ATP affects the rigor lifetime and pH is believed to affect the ADP‐lifetime, these data imply that Pi may be able to rebind to myosin at more than one step in the cross‐bridge cycle. This work was funded by American Heart Association SDG grant (09SDG2260776) to Edward P. Debold
Increased intramuscular triglycerides are associated with increased AMPK alpha1 and cleaved SREBP1 in aged skeletal muscle
Authors: Morris Evan P , Rivas Donato A , Fielding Roger A
Published: June 2021

Increased intramuscular lipid content has previously been noted in lean and obese elderly persons and is thought to be associated with muscle attenuation, insulin resistance and decreased muscle quality. However, it is still unknown by what exact mechanisms these increases in intramuscular lipids occur and what effect mechanical overload has on lipid metabolic regulators in aged skeletal muscle. The purpose of this study was to determined the effect of 28 days of overload on the storage of intramuscular triglycerides (IMTG) and metabolic regulators (SREBP1, AMPK) of lipid synthesis in the plantaris of young (YNG; 6‐month old) and aged (OLD; 30‐month old) Fischer 344 x Brown Norway rats subjected to bilateral synergist ablation (SA) of two thirds of the gastrocnemius muscle or sham surgery (CON). There was a ~43% increase in IMTG in OLD CON (p<0.05 vs. YNG CON) and SA had no effect on IMTG levels in either group. There was a 41% increase in the expression of the active cleaved form of sterol‐regulatory binding protein 1 (SREBP1) in OLD (p<0.05 vs. YNG CON). SA had no effect on the expression of SREBP1 in OLD SA but was significantly increased in YNG SA (p<0.05 vs. YNG CON). There was no difference in the expression of the full form of SREBP1 between groups. There was a ~20% increase in the expression of the AMP‐activated protein kinase (AMPK) catalytic subunit α1 isoform in the OLD CON (p<0.05 vs. YNG CON). SA had no effect on the expression of AMPK α1 isoform in OLD SA but increased the expression in YNG SA ~20% (p<0.05 vs. YNG CON). There was no change in the expression of the AMPK α2 isoform between groups. These data suggest that the regulation of lipid metabolism is impaired in aging skeletal muscle and does not respond to mechanical overload via SA.
Molecular Identification and Quantitative Analysis of Voltage‐gated K+ Channels of Rat Hypothalamic Paraventricular Nucleus Neurons
Authors: LEE SEUL KI , LEE SORA , SHIN SEUNG YUB , RYU PAN DONG , LEE SO YEONG
Published: June 2021

The hypothalamic paraventricular nucleus (PVN), an important integrating site in regulating the neuroendocrine and autonomic nervous systems, is composed of heterogeneous neurons. These neurons are classified into two groups, type I and type II neurons, based on their electrophysiological properties. In the present study, we investigated the molecular identification of voltage‐gated K (Kv) channels. These are known to be associated with a delay in the onset of the first action potential, which is a distinctive characteristic of type I neurons.

In order to identify the mRNA expression profiles, the PVN neurons were divided into type I and type II neurons according to their electrophysiological properties. The recorded cells were harvested and single‐cell RT‐PCR analysis was performed. The single‐cell RT‐PCR analysis revealed that Kv1.2, Kv1.3, Kv1.4, Kv4.1, Kv4.2, and Kv4.3 were expressed in both type I and type II neurons. The relative quantitative analysis revealed that the expression density of Kv1.2, Kv4.2, and Kv4.3 were significantly higher in type I neurons than in type II neurons. These results suggest that several Kv channels are present in both type I and type II neurons and Kv1.2, Kv4.2 and Kv4.3 are the potential Kv channels responsible for determining the distinct electrophysiological properties. This work is supported by National Research Foundation of Korea (2009‐0067186)
Superoxide scavenging in the rostral ventrolateral medulla blunts the pressor response elicited by peripheral chemoreflex activation in conscious rats
Authors: Ribeiro Thaís Porto , do Socorro França‐Silva Maria , Porto‐Silva Clarice , Medeiros Isac Almeida , Braga Valdir A
Published: June 2021

Reactive oxygen species have been implicated in sleep obstructive apnea and hypertension. However, a link between oxidative stress and peripheral chemoreflex (PC) integration within the RVLM has never been investigated. Here, we tested the hypothesis that the pressor response induced by PC activation involves the angiotensin‐II/AT1R/superoxide pathway within the rostral ventrolateral medulla (RVLM). Seventeen male Wistar rats (260–300g) were implanted with bilateral guide‐canulae towards the RVLM four days prior the experiments. PC activation with potassium cyanide (80 μg/Kg, i.v.) produced a increase in blood pressure, which was attenuated 2 minutes after bilateral microinjection of Losartan (1 μM/50 nL), an AT1 receptor antagonist, in the RVLM (+54±4 vs +19±3 ΔmmHg P<0.05, n=6). Moreover, superoxide scavenging in the RVLM using a superoxide dismutase (SOD) mimetic, Tempol (50 nmol/50 nL), significantly blunted the pressor response to PC activation (+50±3 vs +18±3 ΔmmHg P<0.05, n=7), while saline did not (n=4). Taken together, these data suggest that the neurotransmission of the PC within the RVLM involves, at least in part, the activation of AT1 receptors and downstream superoxide formation.
Lesion of the organum vasculosum of the lamina terminalis (OVLT) does not impair the hypotension produced by chronic infusion of losartan in conscious rats
Authors: Vieira Alexandre Antonio , Nahey David , Collister John
Published: June 2021

Introduction

Several forebrain areas possess important mechanisms related to cardiovascular regulation including those mediating effects of angiotensin II (ANG II). We have previously demonstrated in conscious rats that lesions of the OVLT completely blocked the hypertension produced by chronic infusion of ANG II.

AIM

In the present study we tested the hypothesis that the OVLT is also necessary for the hypotension produced by chronic infusion of AT1 antagonist losartan.

Methods

Sham or OVLT Sprague‐Dawley lesioned rats (280–320 g, n=5/group) were instrumented with intravenous catheters and radiotelemetric blood pressure transducers. Rats were given 3 days of saline control infusion (7 mL/day) and were then infused with losartan (10 mg/kg/day) for 10 days, followed by 3 recovery days. Twenty‐four (24‐hour) average measurements of mean arterial pressure (MAP) and heart rate (HR) were made throughout the protocol. Daily water and food intake, as well as urinary output (UO) were measured.

Results

By day 8 of losartan infusion, MAP had decreased similarly (35 ± 3 mmHg) in sham rats and (39 ± 3 mmHg) in OVLT lesioned rats, without changes in HR. Moreover, OVLT lesions did not produce any changes in water and food intake and also in UO.

Conclusion and discussion

The integrity of the OVLT is not important for the hypotension produced by infusion of losartan in conscious rats. These results suggest that the OVLT might play an important role during elevation of plasma ANG II facilitating increases of blood pressure but is not involved with baseline effects of endogenous ANG II.

Support: Capes Foundation/Brazil, process number: BEX 1819/09‐7
Effect of simulated microgravity on sympathetic outflow to the mesentery during an orthostatic challenge
Authors: Stecyk Jonathan A. W. , Kaufman Susan
Published: June 2021

Female astronauts suffer from orthostatic hypotesion upon return to a normal gravitational field. Recent work from our laboratory has shown that female rats exposed to 2 weeks of simulated microgravity (hindlimb unloading; HU) also experience orthostatic hypotension during an active orthostatic challenge (head‐up tilt; HUT; i.e., volunatary mounting of a 45° slope). The simulated microgravity abolishes a reflex mesenteric vasoconstriction that occurs in response to HUT, in part due to a decreased sensitivity of mesenteric arteries to adrenergic stimulation. We examined if HU also attenuates sympathetic outflow to the mesentery induced by HUT. Virgin female rats were chronically instrumented with recording electrodes (Telemetry Research) on a nerve fiber branching from the coelic ganglion and innervating the mesentery, as well as a blood pressure transducer (DSI). Nerve activity, mean arterial pressure (MAP) and heart rate (fH) responses to HUT were measured prior to, immediately following and 3 h post‐HU. Compared to pre‐HU, HUT immediately following HU induced increased sympathetic outflow to the mesentery in both amplitude and frequency, whereas MAP and fH responses were similar (immediate and sustained increases). Thus, increased nerve activity compensated for deficient mesenteric vascular constriction. However, by 3 h post‐HU, the increased nerve activity was attenuated and the tachycardia less. We propose that an inability to maintain a compensatory increase in sympathetic outflow to the mesentery in face of an impaired end organ response to adrenergic stimulation contributes to orthostatic hypotesion in females following simulated microgravity. AHFMR
Application of Principles and Metrics of Operations Management to Water Processing in the Kidney
Authors: Kishore Bellamkonda K. , Schmidt Glen
Published: June 2021

We applied the principles and metrics of Operations Management (OM), a discipline of business schools, to water processing by the nephron. The unique architecture and arrangement of nephrons and blood vessels in the kidney are comparable to the design of a recycling plant that processes raw materials into products using several hundreds of thousands of identical processing units. The units (nephrons), their stations (nephron segments) and machines (channels and transporters), supply (blood), and delivery (urine) lines and reclaiming mechanisms (reabsorption) are very efficiently arranged and coordinated:

#
Station
Machine(s)
Function
Max. Capacity (% of Total)
Control

1
Glomerulus
GBM
Filtration
180L/d(100%)
Yes

2
Proximal Tubule (PT)
AQP1
Reclamation
~ 67%
No

3
Descending Thin Limb (DTL)
AQP1
Reclamation
~ 15
No

4
Collecting Duct (CD)
AQP2, AQP3. and AQP4
Reclamation
~ 15–17%
Yes

5
Medullary Thick Ascending Limb (mTAL)
NKCC2 (BSC1)
Driving Shaft
N/A
Yes

The key metrics are: Process Capacity – sum of the capacities of all nephrons; Capacity Utilization ‐ ~ 0.3 under normal conditions; Flow Rate – 125 ml/min at glomerulus, decreases to ~ 1 ml/min to the end; Flow Time – variable; Cycle Time – for whole body blood ~ 5 min; Process Time – 0.8 min/L of blood; Feedback Control – through macula densa. Using published data from knockouts of specific AQPs, we also determined changes in the above metrics. Thus, application of principles and metrics of OM will provide insights into renal function and modeling in health and disease from a different perspective, and may open a new discipline in renal physiology.
Bradykinin B2 receptor activates extracellular signal regulated kinase in renal cells from a mouse model of polycystic kidney disease
Authors: Bunni Marlene A , Morinelli Thomas A , Bell Phillip D , Raymond John R , Garnovskaya Maria N
Published: June 2021

The vasoactive peptide bradykinin (BK) is a mitogen for normal renal cells. We explored the effects of BK in orpk cilia (+) and orpk cilia (−) collecting duct cell lines, which were derived from a mouse model of polycystic kidney disease (PKD). mRNAs for BK B1 and BK B2 receptors were demonstrated by RT‐PCR in both cell lines. To study BK signaling pathways, we employed fluorescent measurements of intracellular Ca2+, a Cytosensor microphysiometer to measure changes in extracellular pH as a reflection of activation of the Na+/H+ exchanger (NHE), and Western blotting to assess extracellular signal regulated kinase (ERK) activation.

Exposure to 100 nM BK resulted in rapid elevation of intracellular Ca2+, caused a ≥ 20% increase in NHE activity, and a ≥ 200% increase in ERK phosphorylation. While BK‐induced Ca2+ signals and NHE activation were similar in orpk cilia (+) and orpk cilia (−) cell lines, BK‐induced ERK phosphorylation was less pronounced in orpk cilia (−) cells. The BK B2 receptor antagonist and an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor blocked BK‐induced ERK phosphorylation suggesting a role for the B2 receptor‐induced EGFR transactivation in the mitogenic ERK pathway. We speculate that cilium is required for effective transactivation of EGFR and ERK activation by BK B2 receptors in PKD cells. These studies are important because of proposed role of ERK pathway in pathophysiology of PKD.
Differential regulation of kidney RAS genes by high sodium intake in lean and obese Zucker rats
Authors: Samuel Preethi , Ali Quaisar , Sabuhi Rifat , Hussain Tahir
Published: June 2021

Renal tissue Renin Angiotensin System (RAS) plays an important role in many diseases including hypertension. We evaluated transcriptional changes between beneficial components AT2, ACE2, and Mas R of RAS alongside deleterious components AT1A/B, ACE1 and renin in kidney cortex (KC) following 2‐wks of 8% high sodium diet (HSD) in lean and obese Zucker rats (LZR and OZR). We hypothesize that RAS contributes to the pathogenesis of hypertension via a differential regulation of beneficial vs deleterious components of renal RAS in response to HSD. The HSD treatment increased the MAP in both the LZR (16 mmHg) and OZR (21 mmHg) the increase being greater in OZR. The expression data as shown in table below revealed that increased relative expression pattern of renin, ACE1, and AT1B could be contributing towards elevated MAP in HSD fed animals. Further, higher change in MAP of HSD fed OZR seem to be resulting from increased expression of the deleterious component and concomitant decrease in the expression of beneficial components. The study suggests that decrease in expression of beneficial components may be responsible for the un‐opposed actions of the deleterious components of RAS contributing to the greater blood pressure (BP) in obesity, highlighting the importance of careful evaluation of the role of RAS in BP regulation.

Gene
LZR C‐LZR HSD
OZR C‐OZR C
OZR C‐OZR HSD

AGT
Nc
*up
*dn

AT2
Nc
**up
**dn

ACE2
Nc
*dn
**dn

Mas R
Nc
*up
**dn

AT1A
Nc
Nc
Nc

AT1B
*up
Nc
Nc

ACE1
Nc
*up
**up

Renin
*up
*up
Nc

The data analyzed by one‐way ANOVA and Student t test. n= 6 to 7 in each group, p value of <0.05, <0.005 is indicated by * & ** respectively. No change (Nc), downregulation (dn) and upregulation (up).
Higher renal AT1 receptor affinity exaggerates Ang II induced Na/K‐ATPase stimulation in spontaneously hypertensive rats.
Authors: Javkhedkar Apurva Arvind , Lokhandwala Mustafa F , Banday Anees Ahmad
Published: June 2021

Angiotensin II (Ang II) via renal Ang II type 1 receptors (AT1 R) regulates proximal tubular Na reabsorption by acting on Na/K‐ATPase (NKA) and thus plays an important role in regulation of blood pressure (BP). Recent evidences suggest that oxidative stress modulates the function of renal AT1 R during hypertension. The aim of the present study is to investigate if oxidative stress modulates renal AT1 R signaling in spontaneously hypertensive rats (SHR). We performed experiments in 12 weeks old SHR and Wistar Kyoto (WKY) rats served as control. Our results show that in addition to increased BP there is oxidative stress in proximal tubules, as evidenced by increase in protein carbonylation, in SHR compared to WKY rats. Western blot analysis showed equal AT1 R protein expression in SHR and WKY rats. 125I‐sar‐ANG II binding showed an increase in affinity of AT1 R in SHR, but no change in receptor numbers. In SHR there was enhanced AT1R‐ G protein‐coupling and stimulation of protein kinase C activity. In WKY rats, incubation of proximal tubules with Ang II caused stimulation of NKA at low (10−10M) concentration but not at high (10−6M) concentration. However in SHR we observed stimulation of NKA at both concentrations of Ang II.

These data suggest that in SHR oxidative stress is associated with increased renal AT1 R affinity which leads to enhanced Ang II signaling resulting in greater NKA stimulation which contributes to hypertension.
Variation of urinary sulfate in relation to renal arylsulfatase A and B in rat models of hypertension
Authors: Kotlo Kumar U , Bhattacharyya Sumit , Danziger Robert S , Tobacman Joanne K
Published: June 2021

The enzyme arylsulfatase B (ARSB; N‐acetylgalactosamine‐4‐sulfate) removes the sulfate group from the non‐reducing end of the sulfated glycosaminoglycans chondroitin‐4‐sulfate and dermatan sulfate. Recent work in renal epithelial cells in cell culture has demonstrated that ASB activity regulates cellular chondroitin‐4‐sulfate (C4S) and kininogen content and bradykinin release in the spent media. In experiments in salt‐sensitive (SS), salt‐resistant (SR), and spontaneously hypertensive (SHR) rats, measurements of urinary sulfate and bradykinin and cellular arylsulfatase A (ARSA) and arylsulfatase B (ARSB) activity and chondroitin‐4‐sulfate content demonstrate significant relationships. Significant differences in the ARSA and ARSB activity (in nmol/mg protein/hr) were demonstrated between the SS (151 ± 8; 106 ± 10), SR (180 ± 9; 132 ± 10) and SHR (227 ± 10; 161 ± 10) rats, in contrast to no differences in the steroid sulfatase, galactose‐6‐sulfatase, or iduronate‐2‐sulfatase activity. (Iduronate‐2‐sulfatase activity was absent.) Urinary sulfate was highly correlated with the ARSB (r=0.96) and the ARSA (r=0.96) activity. Urinary bradykinin was inversely associated with the C4S (r= −0.88). Since ARSB activity is affected by chloride and phosphate, as well as other anions, and ARSA can remove 3‐sulfate groups from cerebroside sulfate, as well as from sulfated tyrosine residues, the study findings suggest new approaches to mechanisms of blood pressure regulation. VA Merit Review funding
Multimodal learning styles are preferred by students of dental physiology
Authors: SOTO JUAN MANUEL SOLIS , NAJERA ROSA ISELA SANCHEZ , MEESTER IRENE
Published: June 2021

There are several ways of analyze the ways of learning, one of the most accepted is the VARK questionnaire. In this work we investigated the preferred way of learning of students of the course of physiology of the faculty of odontology of the UANL of Monterrey, Mexico. The VARK questionnaire was apply to students of the course of physiology (n= 323). The importance of differences in gender (63% female), age (71% 16–19 years old, 29% 20<23 years old) and standard of living (low, medium, high) with respect to learning style preference was also investigated. The preferred learning styles were multimodal (MM: 39.8%), kinesthetic (K: 28.2%), visual (V: 22%), aural (A: 8.6%), and read/write (RW: 1.4%). Each group (gender, age, standard of living) displayed similar learning preferences as the above reported, with the mayor differences observed in the less preferred learning styles. Males preferred RW above A (14.18 and 9.66%, respectively), while the opposite was observed in female (9.75% and 14.7, respectively). Students of odontology, in the course of physiology of the north of Mexico have multimodal preference of learning followed by the kinesthetic style. Courses should be prepared according these preferences to improve student performance.
Transient kinetic analysis of the elongation mode of Dengue Virus RNA polymerase domain
Authors: Jin Zhinan , Deval Jerome , Johnson Kenneth A. , Swinney David C.
Published: June 2021

Dengue viral RNA polymerase replicates its positive single‐stranded RNA genome in two phases: de novo initiation and elongation. The slow and inefficient initiation during replication masks the elongation mode. The aim of this work was to develop methodology to characterize the mechanism of elongation. Formation of the elongation complex required preincubation of the enzyme with a double stranded RNA (primer/template) for 1 hour at 37 °C in the presence of magnesium. The elongation complex incorporated a correct nucleotide at a rate of ~20 s−1. The fidelity of the polymerase, ~ 2 x 10−5, was determined by measuring the specificity of incorporation of correct and incorrect nucleotides. The reaction pathway comprised of enzyme and RNA binding followed by nucleotide binding and incorporation was studied by transient kinetic methods. The microscopic rate constants for each step of the pathway were obtained by global data fitting and computer simulation.
Novel adaptations of the pinniped (Mammalia:Carnivora) jaw anatomy to aquatic prey‐capture
Authors: Jones Katrina Elizabeth , Ruff Chris , Goswami Anjali
Published: June 2021

Biomechanical models of jaw anatomy in terrestrial mammals often focus on mastication as the driving force for adaptation. As marine mammals, pinnipeds (e.g.seals) use their jaws primarily for prey‐capture; but not for mastication. Here we reveal the functional adaptations of jaw anatomy to novel marine feeding behavior; filter feeding, suction feeding and “grip and tear” feeding.

We measured 100 mandibles, representing 25 of 34 extant species, using a digitizer to capture co‐ordinate data for 15 homologous landmarks. Relevant linear measurements were extracted from the landmark data. We then used X‐ray data to reconstruct cortical thickness to calculate section modulus (strength estimate) for four species (n=25). We compared the specialist feeders for each measure, based on expected mechanical scaling.

Filter feeders displayed a very long toothrow, which increases sieve area, and a highly elliptical mandibular symphysis to prevent wishboning during the expulsion of water. The suction feeding jaw was shorter with a weaker mandible, lower bite force and diminutive canines, due to the reduced requirement for biting. Grip and tear feeders were adapted for a large gape (longer jaw), strong biting, and a posteriorly directed muscle vector for capturing large prey. This study elucidates novel adaptation of mammal jaw anatomy to the biomechanical challenges of prey‐capture in the marine realm.
Lectin‐dependent quality control of the Na,K‐ATPase maturation in the ER
Authors: Vagin Olga , Tokhtaeva Elmira , Munson Keith , Sachs George
Published: June 2021

To evaluate the role of N‐glycan‐lectin interactions in maturation of the Na,K‐ATPase β2 subunit, the effects of removing all 8 or selected N‐glycans on trafficking of the β2 and its binding to calnexin were studied in MDCK cells. N‐glycan‐dependent binding of the β2 to calnexin persists for several hours after completing translation, suggesting involvement of the ER glucosyltransferase UGGT1 that is known to induce additional rounds of calnexin‐assisted folding of misfolded glycoproteins by re‐glucosylating N‐glycans next to local folding defects exposing hydrophobic amino acids. The β2 is retained in the ER when calnexin binding is prevented by castanospermine. Homology modeling of the β2 using the crystal structure of the α1‐β1 Na,K‐ATPase shows the presence of a relatively hydrophobic cluster proximal to N‐glycans 2 and 7. Combined, but not separate, removal of N‐glycosylation sites 2 and 7 dramatically impairs calnexin binding and prevents the export of the β2 from the ER. Similar effects are observed with hydrophilic substitution of two hydrophobic amino acids in the cluster, suggesting that exposure of these residues in incompletely folded conformers results in UGGT1‐catalyzed re‐glucosylation of N‐glycans 7 and 2, allowing post‐translational calnexin‐assisted re‐folding, which, in turn, is required for maturation of the Na,K‐ATPase β2 subunit.

Supported by NIH grants DK077149 and DK058333.
Purification and Characterization of Hyalin by Chromatography
Authors: Stancu Keith M. , Carroll Edward J.
Published: June 2021

Hyalin is a large glycoprotein found in the extracellular hyaline layer sea urchin embryos. During gastrulation it mediates organization of the archenteron required to form the digestive system. Hyalin consists of a family of glycoproteins with 11.6, 9.6, 8.8, and 6.4S values, with an 11.6S species as a major component. The research goal is to determine the amino acid sequence and fully characterize the 11.6S species with respect to carbohydrate to protein linkages and biologically active structural motifs. Hyalin was collected by calcium chloride precipitation of a cell surface extract of embryos. Sephacryl S‐500 HR resin was used in a 100mL column (1.5 X 53.5 cm) to further isolate the 11.6S species. Several standards were run to allow for the determination of the molecular weight (mw) by plotting a standard curve of mw versus elution volume/void volume using thyroglobulin, apoferritin, β‐amylase, alcohol dehydrogenase, and bovine serum albumin. The molecular weight of the 11.6S species was determined to be 1,300,000. Native gel electrophoresis results showed the 11.6S protein contains two isomers which differ by charge. Initial anion exchange experiments using DEAE sepharose resin allowed for the separation of these two isomers. The two different components eluted at 0.6M and 1.1M NaCl concentrations with good resolution. These methods of purification will provide the material for further structural analysis.
Crystal structure of Helicobacter pylori MinE, a cell division topological specificity factor
Authors: Youn Hyung‐Seop , Lee Jung‐Gyu , An Jun Yop , Eom Soo Hyun
Published: June 2021

In gram negative bacteria, proper placement of the FtsZ ring, mediated by nucleoid occlusion and the activities of the dynamic oscillating Min proteins MinC, MinD and MinE, is required for correct positioning of the cell division septum. MinE is a topological specificity factor that counters the activity of MinCD division inhibitor at the mid‐cell division site. Its structure consists of an anti‐MinCD domain and a topology specificity domain (TSD). Previous NMR analysis of truncated Escherichia coli MinE showed that the TSD domain contains a long α‐helix and two antiparallel β‐strands, which mediate formation of a homodimeric α/β structure. Here we present the crystal structure of full‐length Helicobacter pylori MinE and redefine its TSD based on that structure. The N‐terminal region of the TSD (residues 19–26), previously defined as part of the anti‐MinCD domain, forms a β‐strand (βA) and participates in TSD folding. In addition, H. pylori MinE forms a dimer through the interaction of anti‐parallel βA‐strands. Moreover, we observed serial dimer‐dimer interactions within the crystal packing, resulting in the formation of a filamentous structure. We therefore redefine the functional domain of MinE and propose that a multimeric filamentous structure is formed through anti‐parallel β‐strand interactions. This work was supported by the “Systems biology infrastructure establishment grant” provided by GIST.
Individual growth and career planning for graduate students and postdoctoral fellows (10.1)
Authors: Clifford Philip , Fuhrmann Cynthia
Published: June 2021

Do you want to find a rewarding and satisfying career? Of course! How do you find the right path, especially when there are so many career directions doctoral level scientists can follow? For instance, there are more than 50 potential job titles for biomedical PhD scientists listed at myIDP.sciencecareers.org. Attend this thought‐provoking and interactive workshop taught by myIDP coauthors to learn about a logical, step‐by‐step process for selecting the best career trajectory for you. You will also be provided with helpful tools for exploring your career options and setting goals to move you to the next stage in your career.
Stimulating attitudes of inquiry (100.1)
Authors: White Harold
Published: June 2021

Much of contemporary science education focuses on what we know, but neglects the evidence supporting the conclusions that fill textbooks. Students thus often learn information as abstractions disconnected from their experience. They rarely encounter the observations, clever experiments, and controversies that generate new ideas and refute old. Problem‐based learning (PBL) is an approach to education that encourages students to identify what they don’t know, ask questions, and actively pursue knowledge based on their curiosity and through collaboration with other students. Rather than attempt to cover massive amounts of information, PBL seeks to foster life‐long habits of learning through in‐depth exploration and fundamental understanding of carefully selected problems that reveal basic concepts and interdisciplinary connections. Problems based on the primary literature with emphasis on classic articles expose students not only to the logic of research but introduce them to the history of science, the people who made important discoveries, and the culture science.
Chimeric apolipoproteins as novel plasma cholesterol lowering agents (1002.2)
Authors: Ibe Nnejiuwa , Lek Mark , Beck Wendy , Weers Paul , Narayanaswami Vasanthany
Published: June 2021

Apolipoprotein E3 (apoE3, 299 residues, 34 kDa) and apolipoprotein A‐I (apoA‐I, 243 residues, 28 kDa) play significant roles in regulating plasma cholesterol levels, by virtue of their abilities to facilitate cholesterol uptake by binding the low density lipoprotein receptor (LDLr) and promoting cholesterol efflux from macrophages, respectively. We aim to generate a chimeric apolipoprotein by combining the LDLr binding region in the N‐terminal domain (NT) of apoE3 (residues 1‐191) with the cholesterol efflux‐promoting segment in the C‐terminal domain (CT) of apoA‐I (residues 181‐243). Overlap extension PCR was employed to fuse the nucleotide sequence encoding apoE3 NT with apoA‐I CT, followed by ligation into a pET20b(+) vector. The DNA sequence of the apoE3‐NT/apoA‐I‐CT chimera was verified. Overexpression of the chimera in E. coli yielded ~40 mg/L of purified protein. SDS‐PAGE and Western blot analyses confirmed the presence of a protein band of ~31 kDa, and MALDI‐TOF mass spectrometry revealed a mass of 30,977 Da (expected mass: 30,975 Da). Preliminary circular dichroism data of the chimera revealed α‐helical character, similar to apoE3 and apoA‐I. Further biophysical, spectroscopic and, lipid‐, LDLr‐binding and cholesterol efflux functional assays will be performed. The chimera has the potential to be used as a powerful cholesterol‐lowering agent to protect against cardiovascular disease.

Grant Funding Source: Supported by the NIH #GM105561, #GM089564, and MARC grant T34GM008074‐22
Fluorinated aromatic amino acids distinguish side chain cation‐π interactions from membrane insertion (1002.5)
Authors: Roberts Mary , He Tao , Gao Jianmin
Published: June 2021

Peripheral membrane proteins use a variety of motifs to bind transiently to membranes. For aromatic amino acids that are critical for membrane binding, two types of interaction can be envisioned: side chain insertion into the hydrocarbon milieu or formation of an amino acid π ‐ choline cation complex with phosphatidylcholine (PC). Distinguishing between these two can be difficult. However, site specific incorporation of fluorinated tyrosine or phenylalanine should differentiate these two binding modes. A fluorinated aromatic ring is more hydrophobic, favoring insertion, but the electrostatics of the ring have been changed such that the center is electropositive, destabilizing cation‐π complexes. A variant of S. aureus phosphatidylinositol‐specific phospholipase C that has an enhanced affinity for PC (N254Y/H258Y) was used to test this. If H258Y, a key residue in a helix that approaches the membrane surface, is replaced by either pentafluoro‐Phe or difluoro‐Tyr, binding affinity for PC‐rich membranes (measured by FCS) is lost. In contrast, when Phe249, a residue in a loop thought to insert into membranes, is replaced by pentafluoro‐Phe, the binding affinity is increased. Mutated Tyr that do not participate in membrane binding show no change in membrane binding affinities of the protein. This strategy offers the ability to test the role of Tyr and Phe in any peripheral membrane protein that can be expressed in E. coli.

Grant Funding Source: NIH GM60418
Binding of microfibril‐associated glycoprotein 2 to extracellular microfibrils is promoted by proprotein convertase cleavage (1005.3)
Authors: Connett Breanna , Perez Edgar , Donovan Lauren , Hogrebe Gregory , Lai Khang , Withers Gordon , Miyamoto Alison
Published: June 2021

Microfibrils are extracellular matrix fibers whose major components are the fibrillin proteins (Fbn‐1, ‐2, ‐3), which are important for tissue mechanical stability and elasticity. MAGP2 is a secreted protein found on specific microfibrils. Following MAGP2 secretion, PC cleavage occurs at the RLRR consensus site near the MAGP2 carboxyl‐terminus (CT). Since PC cleavage of Fbn‐1 is required for microfibril incorporation, we investigated the role PC cleavage plays in the localization of MAGP2 on microfibrils using immunocytochemistry. Transient expression of wild‐type MAGP2 in mouse ovarian epithelial cells that produce abundant Fbn‐2 microfibrils resulted in weak, but detectable extracellular MAGP2 fibers. Abundant MAGP2 fibers were generated when only the CT half of MAGP2 was expressed, confirming that the matrix‐binding domain in the CT half of MAGP2 is responsible for fiber localization. Mutation of the CT half of MAGP2 to prevent PC cleavage showed a small, but significant, decrease in extracellular MAGP2 fibers, indicating that PC cleavage promotes MAGP2‐microfibril binding. Preliminary data suggest that MAGP2 may bind preferentially to Fbn‐2 fibers, and that soluble MAGP2 can bind to microfibrils. Understanding the normal maturation and function of MAGP2 may be relevant to both angiogenesis and ovarian carcinoma progression, since previous studies implicate MAGP2 in both processes.
Mannose‐based inhibitors of Escherichia coli adhesion: design and applications (1007.2)
Authors: Bouckaert Julie
Published: June 2021

Structure‐thermodynamic relationships in protein‐carbohydrate interactions provide important decision makers in the design and maturation of bacterial ant‐adhesives. Many interactions in our innate defense depend on the specific recognition, of a high enough density of a glycan sequence, to evoke immune reaction. Bacteria hijack glycan receptors that are reminiscent of a physiological state of a mamalian cell that indicates a suitable habitat for the completion of their pathogenic cycle. The interruption of this process is envisioned using anti‐adhesives that selectively block bacterial adhesion but do not interfere with the immune system.

The understanding of the biophysical basis of host‐pathogen interactions to empower intervention can be studied using purified adhesins and synthetic multivalent inhibitors. We measured affinities and stoichiometries using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). The geometry of a multivalent complex was determined using small angle X‐ray scattering (SAXS) and was found to agree with the stoichiometry and molecular size derived from ITC and DLS data respectively. Stunningly, a correlation could be found between the dispersion part of interaction energies (quantum chemical calculations), ITC‐derived entropies and crystal structure electron densities of inhibitors bound to FimH.

Grant Funding Source: Supported by ANR‐12‐BSV5‐0016‐01
Characterization of an O‐GlcNAc‐binding protein (1007.3)
Authors: Groves Jennifer , Zachara Natasha
Published: June 2021

O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc) is a dynamic post‐translational modification of nuclear, cytoplasmic, and mitochondrial proteins. While O‐GlcNAc has been demonstrated to modulate numerous cellular processes, the molecular mechanisms by which O‐GlcNAc mediates protein function are not well defined. One mechanism by which O‐GlcNAc may regulate protein function is by promoting protein‐protein interactions. In this study we have investigated whether the intracellular hyaluronan‐binding protein p32 binds proteins in an O‐GlcNAc‐dependent manner. Using ELISA and dot blotting techniques we have demonstrated that p32 binds GlcNAc‐conjugates preferentially to other sugar‐conjugates in vitro. Suggesting that p32 interacts with O‐GlcNAc in vivo, p32 enriches O‐GlcNAc‐modified proteins similar to the O‐GlcNAc‐specific antibody RL2. Finally, our data demonstrate that overexpression of p32 fused to the biotin ligase BirA results in a biotinylation pattern that can be modulated by manipulating O‐GlcNAc levels. Preliminary studies suggest that one candidate protein bound by p32 in an O‐GlcNAc‐dependent manner is the O‐GlcNAc transferase (OGT), the enzyme responsible for the addition of O‐GlcNAc. Taken together, these data suggest that p32 may contain an O‐GlcNAc‐binding motif that dictates its ability to interact with a subset of intracellular proteins such as OGT.

Grant Funding Source: Supported by the National Heart, Lung, and Blood Institute (P01HL107153)
Deciphering the dynamic regulation of phosphoinositide 3‐kinases downstream of G‐protein coupled receptors and receptor tyrosine kinases (1008.1)
Authors: Burke John , Vadas Oscar , Perisic Olga , Williams Roger L
Published: June 2021

>Class I phosphoinositide 3 kinases (PI3K) are activated downstream of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs), mediating a number of physiological, and pathophysiological processes including inflammation, and tumour growth. We have determined the molecular basis for the activation of PI3Ks downstream of GPCRs and RTKs by applying a synthesis of hydrogen deuterium exchange mass spectrometry (HDX‐MS), X‐ray crystallography and biochemical/cellular assays. HDX‐MS provides information on the conformation and solvent accessibility of protein amide hydrogens, and has allowed us to interrogate the structural dynamics of these protein complexes on a membrane surface.

The class I PI3Ks are composed of four different catalytic isoforms (p110α, p110β, p110δ, and p110γ), and only the p110β and p110γ isoforms can be activated directly by Gβγ subunits derived from GPCRs. However, this interaction only occurs on a membrane surface. By biochemically reconstituting PI3K with Gβγ on a membrane surface and carrying out HDX‐MS experiments we were able to determine the molecular interface of both p110β and p110γ with Gβγ subunits. Intriguingly we find that membrane binding causes conformational changes at the Gβγ interface, revealing a key dynamic role for membrane binding in priming activation downstream of GPCRs. Mutations that disrupted the Gβγ interface prevented oncogenic transformation, revealing a key role of GPCR signalling in mediating oncogenic transformation through PI3Ks.