bluuebunny/crossref_metadata_2025 huggingface dataset:
Stimulation of the renin angiotensin system (RAS) in an early stage of life causes sustained hypertension in rats
Authors: Heijnen Bart , van Essen Helma , Debets Jacques , Peutz‐Kootstra Carine , Lijnen Peter , Janssen Ben , Struijker‐Boudier Harry
Published: June 2021

Transient administration of inhibitors of the renin angiotensin system (RAS) during the prehypertensive period of spontaneously hypertensive rats leads to a long‐lasting lowering of blood pressure (BP) and a reduction of end organ damage. We tested if a transient stimulation of the RAS at young age would exert the opposite effect and permanently increases BP.

Studies were performed in Cyp1a1 Ren2 transgenic rats. In this model the production of mouse renin is enhanced when 0.3% Indole‐3 carbinol (I3C) is added to the diet. After withdrawal of I3C the production of mouse renin stops almost immediately. Cyp1a1 Ren2 rats received I3C‐treatment between 4 to 8 weeks of age (WOA) or between 30–34 WOA. Non‐treated rats served as control. BP follow‐up was performed via tailcuff and intra‐arterial BP was determined at 4, 8, 12 and 20 WOA or 34 and 38 WOA. Data are presented as mean ± SEM.

At 12 WOA, i.e. 4 weeks after I3C withdrawal, the I3C‐treated rats show a statistically significant increase in BP compared with control rats (156±18 versus 129±6 mmHg). This increase in BP is still present at 20 WOA, i.e. 12 weeks after I3C withdrawal (141±7 versus 125±6mmHg). In the adult rats (30–34 weeks), BP returned immediately to control values after stopping renin activation.

This study shows that, at least in rats, sustained hypertension can be induced by a transient stimulation of the RAS in an early stage of life.
Angiotensin II (AngII) diminishes the effect of SGK1 on WNK4‐mediated inhibition of ROMK1 channels
Authors: Yue Peng , Lin Daohong , Pan Chunyang , Sun Peng , Wang Wenhui
Published: June 2021

We used the perforated whole‐cell patch‐clamp to examine the effect of AngII on ROMK1 channels in HEK293T cells transfected with GFP‐ROMK1. Application of AngII inhibited ROMK1 channels in a dose‐dependent manner. The inhibitory effect of AngII on ROMK1 was abolished by losartan (AT1R antagonist), GF109203x (PKC inhibitor) or siRNA of c‐Src. The notion that c‐Src was involved in mediating the effect of AngII on ROMK1 channels was also suggested by the finding that AngII stimulated the tyrosine phosphorylation of c‐Src on tyrosine residue 416 and increased the tyrosine phosphorylation of ROMK1 channels. AngII failed to inhibit K channels in cells transfected with ROMK1 mutant (R1Y337A) in which the tyrosine phosphorylation site was mutated. In contrast, expression of WNK4 was still able to inhibit R1Y337A and the inhibitory effect of WNK4 was abolished by SGK1. However, although AngII had no effect on R1Y337A regardless of SGK1 presence, AngII restored the WNK4‐induced inhibition of R1Y337A in the presence of SGK1. We conclude that AngII inhibited ROMK1 channels by two mechanisms: stimulating the tyrosine phosphorylation of ROMK channels and enhancing the WNK‐induced inhibition of ROMK1. Since volume‐depletion increased aldosterone and AngII level, AngII could play an important role in suppressing K secretion while stimulating Na absorption in aldosterone‐sensitive nephron.
8‐CPT‐cAMP is a self‐inhibition reliever for ENaC expressed in Xenopus oocytes
Authors: JI HONG‐LONG , Molina Raul , Han Dong‐Yun , Zhao Run‐Zhen
Published: June 2021

The epithelial Na+ channels (ENaC) are up‐regulated by cAMP/PKA signal pathway to remove luminal fluid and salt in epithelial tissues. As a specific PKA activator, cell permeable 8‐CPT‐cAMP has been broadly applied to stimulate native ENaC activity in epithelial cells. Recently, it has been proposed that 8‐CPT‐cAMP may act as a ligand of ENaC with unknown mechanisms. To examine whether 8‐CPT‐cAMP stimulates ENaC by eliminating self‐inhibition, both wild type and mutated human αβγ ENaC were expressed in oocytes. Acutely perfusion but not cytosolic microinjection of 8‐CPT‐cAMP activated ENaC current in a dose‐dependent manner with an EC50 value of 49μM (n=5). 8‐CPT‐cAMP reversibly reduced external Na+ self‐inhibition (Ipeak/Isustained ratio, P<0.05). Using Zn2+ ions to modify external histidine residues abolished the eliminating effect of 8‐CPT‐cAMP on self‐inhibition. However, thiol modifying reagent did not exhibit any effect. Furthermore, we identified that γH233R was required for 8‐CPT‐cAMP induced stimulation of ENaC. In addition, 8‐CPT‐cAMP did not increase the channel activity associated with a high opening time mutant, βS520C. Our results suggest that in addition to serve as a key signal molecule of cGMP/PKA signaling pathway, 8‐CPT‐cAMP may be an ENaC opener by relieving self‐inhibition and increasing open probability.

Supported by HL087017 and HL095435.
Biophysical features of a new cloned delta ENaC subunit
Authors: Han Dongyun , Nie Hongguang , Zhao Runzhen , Su Xuefeng , Ji Honglong
Published: June 2021

The epithelial sodium channels (ENaC) is the critical rate‐limiting determinant in maintaining airway and lung lining fluid. δ ENaC (δ1) has been detected in human lung epithelial cells in addition to α, β, and γ subunits. Here we cloned a splicing variance of δ1 counterpart, termed as δ2 ENaC, in human bronchial epithelial cell line 16HBE140. Compared with δ1 subunit, δ2 ENaC possesses 66 extra amino acid residues in its cytosolic amino terminal tail. To characterize the biophysical and pharmacological features of the δ2 ENaC, we comparably examined the whole‐cell and single channel properties associated with δ1βγ and δ2βγ hENaC expressed in Xenopus oocytes. The relative cation selectivity ratio of Na+/Li+/K+ (1/0.69/0.09) for δ2βγ ENaC was similar with that of δ1βγ ENaC (1/0.64/0.07). And there was no significant difference in external Na+ affinity between δ1βγ and δ2βγ hENaC. The apparent dissociation constant for amiloride (Kiamil), EC50 value of capsazepine, proton gating kinetics for δ2βγ ENaC, however, significantly differed from those of δ1βγ ENaC. Single‐channel patch clamp results revealed that unitary conductance, single channel activity (NPo), and gating kinetics varied between δ1βγ and δ2βγ ENaC. Our data demonstrate that heterologously expressed δ1 and δ2 ENaC channels are segregated by their biophysical and pharmacological properties, at least partially accounting for the diverse native epithelial Na+ channels in lung epithelial cells. Supported by HL087017 and HL095435.
Activation of peripheral mu‐opioid receptor attenuates the augmented exercise pressor reflex seen in rats with chronic femoral artery occlusion
Authors: Tsuchimochi Hirotsugu , McCord Jennifer L , Kaufman Marc P
Published: June 2021

The sensory arm of the exercise pressor reflex arc is comprised of group III and IV afferents. Recently, opioid receptors have been shown to be expressed on them. Although the stimulation of opioid receptors in the central nervous system has been shown to attenuate the exercise pressor reflex, the effect of stimulation of peripheral opioid receptors on the reflex is unknown. We therefore tested the hypothesis that activation of peripheral μ‐opioid receptors attenuates the reflex pressor response to static contraction in decerebrated rats. The pressor responses to static contraction were compared before and after injection of the μ‐opioid receptor agonist DAMGO into the abdominal aorta of rats in which one femoral artery was occluded 72 hours previously (n=8) and in other rats whose femoral arteries were freely perfused (n=8). The pressor response to contraction in rats whose femoral artery was ligated was attenuated by DAMGO (1 μg) from Δ 37 ± 4 mmHg to Δ 20 ± 4 mmHg (P<0.001). This inhibitory effect was antagonized by pretreatment with 100 μg of naloxone (Δ 29 ± 5 mmHg before and Δ29 ± 5 mmHg after, P = 0.9). In contrast, DAMGO had no effect on the pressor response to contraction in rats whose femoral arteries were freely perfused (Δ19 ± 4 mmHg before and Δ 21 ± 3 mmHg after, P = 0.9). We conclude that stimulation of peripheral μ‐opioid receptors attenuates the exercise pressor reflex in rats with “chronic” femoral arterial ligation.
Caloric Restriction Attenuates Many Age‐Related Changes in Skeletal Muscle Mitochondrial Physiology
Authors: Lanza Ian R , Li Eileena , Jakaitis Daniel , Ali Bushra , Nair K. Sreekumaran
Published: June 2021

Mitochondrial function declines with age but increases with caloric restriction (CR). Mitochondria and muscle fibers were prepared from young (8–11mo) and old (22–24mo) AL‐fed and old CR mice. State 3 respiration was lower in OAL vs. YAL, but preserved in OCR (table). Normalizing to protein content eliminated the age‐effect in AL mice but amplified the effect of CR, indicating that CR enhances the intrinsic mito function. ROS production in isolated fibers increased with age in AL but not CR mice (table). Coupling was reduced in OAL but not OCR. These results indicate that mito capacity declines with age in situ and in vitro, apparently driven largely by decreased mitochondrial protein content. CR increases mito capacity and efficiency independently of mito protein content.

YAL
OAL
OCR

Fibers (pmol/s/m g tiss)
CI
54.0±4.1
38.9±1.6*
50.8±3.0†

CII
44.4±3.4
36.9±1.4*
45.0±2.3†

CI+II
63.4±5.5
51.8±2.5*
67.0±3.4†

Mito (pmol/s/m g tiss)
CI
38.9±1.8
21.5±2.6*
30.7±2.0†

CII
30.6±0.9
19.6±1.2*
24.7±1.4†

CI+II
59.0±1.6
34.0±1.9*
46.7±2.7†

Mito (pmol/s/m g prot)
CI
8350±635
7071±1082
10422±863§

CII
6536±414
6309±506
8408±676§

CI+II
12601±762
11063±1156
15949±1381§

H2O2 (pM/s/mg tiss)
CI
10.0±1.8
30.8±3.7*
15.0±2.0†

CI+II
141.4±20.9
187.2±53.8
101.1±13.6†

P:O
State IV to III
2.94±0.03
2.71±0.02*
2.92±0.05†

Means±SEM.

P<0.05 OAL vs YAL,

P<0.05 OCR vs OAL,

P<0.05 OCR vs. OAL & YAL.
Effects of β‐hydroxy‐β‐methylbutyrate (HMB) on Muscle IGF‐I and MGF mRNA Expression in Aged Female Rats during 10‐Week Resistance Training
Authors: Park Young‐Min , Lee Sang‐Rok , Wilson Jacob M. , Henning Paul C. , Ugrinowitsch Carlos , Zourdos Michael C. , Arjmandi Bahram H. , Rathmacher John A. , Kim Jeong‐Su
Published: June 2021

HMB intake during resistance training (RT) has been shown to improve muscle mass and function. Our purpose was to determine whether HMB intake would enhance RT‐induced improvements in muscular function and mitogenic responses in aged animals. Sixteen, 19‐month old, Sprague‐Dawley female rats were randomly divided into 3 groups: control (CON), HMB (0.46 g/kg/d) and non‐HMB. After CON was sacrificed as a baseline, the HMB and non‐HMB groups underwent intense ladder climbing exercise training every third day for 10 wks with weights attached to the tail. Maximum strength was assessed pre‐, mid‐ and post‐RT by measuring the maximum weight (g) the animal could climb to the top of ladder with one time. RT‐PCR was used to analyze IGF‐I and MGF mRNA expression in gastrocnemius and soleus muscles. There was a main time effect (+238%; pre 263±16 vs. post 890±28 g) and group × time effect (HMB +265% vs. non‐HMB +214%) for maximum strength (p<0.05). MGF mRNA expression increased in both HMB (+32%) and non‐HMB (+40%) after 10‐wk RT as reflected by the main group effect (p<0.05) with no between group differences. Although the main group effect for IGF‐I only approached significance (p=0.1), post hoc analysis revealed significant increases only in the HMB group (+33%, p<0.05). Our findings suggest that HMB intake during RT induces greater strength gains in aged individuals maybe by increasing muscle mitogenic responses (IGF‐I mRNA).
A session of strength exercise induced cardiac hypertrophy by AT1 receptor‐AKT‐mTOR signaling pathway
Authors: Soares Melo Stéphano Freitas , Amadeu Marco Aurélio , Fernandes Tiago , Carmo Everton Crivoi , Magalhães Flávio Castro , Mendes Barreti Diego Lopes , Reno Socci Ursula Paula , Alves Cleber Rener , Roque Redondo Fernanda Roberta , Brum Patrícia Chakur , Oliveira Edilamar Menezes
Published: June 2021

The angiotensin II type I (AT1) receptor has an important participation in the development of cardiac hypertrophy (CH). Previously, we showed that AT1 receptor participates in the cardiac hypertrophy induced by resistance training in rats (Barauna, et. al., 2008). We studied AT1 receptor signaling pathways related to the CH in rats submitted to a session of resistance exercise (SRE). Male Wistar rats were divided into six groups: control (CO); exercised and killed 5 minutes after exercise (Ex 5); exercised and killed 30 minutes after exercise (Ex 30); control treated with Losartan (CO Los); treated with Losartan, exercised and killed 5 minutes after the exercise (Ex Los 5); treated with Losartan, exercised and killed 30 minutes after training (Ex Los 30). The exercise consisted of 4 sets of 12 repetitions with 80% overload of work. The results show that phosphorylation activity of AKT in group Ex 5 and Ex 30 increased 63% and 62%, respectively, compared to CO, whereas the phosphorylation of mTOR and ERK 1/2 was increased 65% and 59%, respectively, compared to CO, only in the group Ex 30. Furthermore, these effects were blocked by losartan treatment in groups Ex Los 5 and Ex Los 30. However, to JNK1/2, p38 and p70S6K proteins it were not observe any difference between the groups. These results, together with ours previous data shows that the AT1 receptor has an role in the activation of AKT pathway/mTOR and ERK 1/2 after a SRE.
Cardiac enlargement during pregnancy may involve new myocyte formation
Authors: Foster Sara , Miller Christopher , Bilko Andrey , Injaian Allison , Bailey Beth A
Published: June 2021

Cardiac enlargement that develops during pregnancy is an important yet under‐studied adaptation to physiologic stress. In light of recent evidence of resident stem cell pools in the heart, we tested the hypothesis that pregnancy‐induced cardiac enlargement may involve both myocyte hypertrophy and new myocyte formation. 6 nonpregenant age matched controls (C) and 6 mice in late pregnancy (LP, 17–19 days) were studied. Hearts were formalin fixed and 5μ sections were examined. Laminin staining was used to identify cell perimeter and determine myocyte cross‐sectional area (MCSA). Wet weights of LP hearts were 23% greater than C (240±9 vs 195±13mg). Increases in MCSA were not proportional to increases in heart mass. MCSA of LP (262±3μ2, n=225) was 11% greater than of C (236± 3μ2, n=225). Analysis of sections from different regions of the heart showed different degrees of MCSA hypertrophy. Apex, mid and basal sections were analyzed from all hearts. No difference in MCSA among sections was found in C. MCSA of all sections were greater in LP than C, but MCSA of the mid‐heart in LP (275 ±4μ2, n=75) were greater than MCSA of apex (252± 5μ2, n=75) and base (259 ±4μ2, n=75). Myocyte hypertrophy during pregnancy is not sufficient to account for the whole‐heart enlargement suggesting that new myocytes are generated during pregnancy. Hypertrophy is not evenly distributed during pregnancy suggesting an unequal distribution of wall stress.
Interaction of GABA and glutamate inputs in the dorsal motor nucleus of the vagus
Authors: Babic Tanja , Browning Kirsteen N , Travagli R Alberto
Published: June 2021

The dorsal motor nucleus of the vagus (DMV) is pivotal in the regulation of gastric motility. Microinjection of bicuculline (BIC) into the DMV increases gastric motility whereas kynurenic acid (KYN) has no effect unless preceded by microinjection of BIC. The aims of this study were to determine the effects of BIC and KYN on DMV neurons in a slice preparation.

Using whole cell patch clamp recordings in 67 DMV neurons, we investigated the effects of BIC and KYN, or their combination, on the firing rate of DMV neurons.

Perfusion with BIC increased the firing frequency in 51% of neurons and decreased it in 30%, leaving the remaining neurons unaffected. Following application of BIC, KYN either decreased (60%) or increased (40%) their firing frequency. When KYN was applied first, the firing rate was decreased in 57% and increased 17% neurons. Further perfusion with BIC had no additional effect in most neurons that responded to KYN.

These data confirm in vivo studies demonstrating that DMV neurons are under tonic GABAergic control. In contrast to these in vivo studies, though, DMV neurons also responded to KYN, suggesting that they receive tonic glutamatergic input. These data suggest that those DMV neurons regulating gastric motility may be the ones affected by a tonic GABAergic input, whereas the circuits that receive a glutamatergic input may be the ones involved in functions other that gastric motility.

Supported by DK 55530
Activity of Neurons in the Rostral Ventrolateral Medulla (RVLM) of Conscious Cats
Authors: Barman Susan M , Cotter Lucy A , DeStefino Vincent J , Reighard Derek A , Yates Bill J
Published: June 2021

Although numerous studies in decerebrate or anesthetized animals of several species have considered the activity of neurons in the RVLM, to date no neurophysiological studies of this region have been conducted in conscious animals. The goal of the present study was to determine the firing patterns of RVLM neurons in conscious cats, and to compare the neuronal activity in this preparation to that previously documented in anesthetized animals. Recovery surgery was performed to implant a recording chamber on the caudal aspect of the skull, as well as a telemetric blood pressure recording device (Data Sciences Intl.) and probes to measure blood flow from the carotid artery (Transonic Systems). Blood pressure and blood flow measurements were used to establish whether units displayed cardiac‐related activity. Few (<25%) RVLM neurons exhibited robust cardiac‐related firing, but the mean coherence value relating unit activity to the arterial pulse (0.45±0.07) was higher than in anesthetized animals. Moreover, the mean spontaneous firing rate of RVLM neurons with cardiac‐related activity was over 6 times higher in awake cats than in anesthetized animals. Many RVLM neurons that lacked cardiac‐related firing displayed multifaceted patterns of rhythmic activity. These data suggest that the activity of RVLM neurons is highly complex in awake felines.
Morphological restoration of pituitary thyrotrope population by thymulin gene therapy in nude mice
Authors: Martines Eliana Vanesa , Reggiani Paula , Goya Rodolfo , Cónsole Gloria
Published: June 2021

Thymulin secretion is strongly influenced by the neuroendocrine system. Thymulin also possesses hypophysiotropic activity which suggests that this metallopeptide may play an important role in thymic‐pituitary axis, particularly during early life. The aim of the present study was to evaluate the histomorphological changes of neonatal thymulin gene therapy on the thyrotrope population of adult nude mice. We used an adenoviral vector expressing a synthetic gene for thymulin termed RAd‐FTS. On postnatal day 1, each experimental heterozygous (nu/+) and homozygous (nu/nu) pup of both sexes received a single bilateral i.m. injection of RAd‐FTS or RAd‐GFP, a control vector. On postnatal days 51–52, the mice were sacrificed, and their pituitaries were immediately dissected, fixed and immunostained. Morphometry was performed by means of an image‐analysis system. The calculated parameters were: volume density (VD), cell density (CD), and cell size (μm2). Serum thymulin levels were measured by a bioassay. It was observed that neonatal thymulin gene therapy in the athymic mice restored their serum thymulin levels. The histometrical analysis revealed that the treatment prevented the reduction in thyrotrope cell, volume density, and cell size in athymic mice. Our data suggests that thymulin gene therapy may be an effective strategy to approach deficits associated with endocrine thymus dysfunction. Research support provided by Universidad Adventista del Plata.
Release of short answer questions prior to an exam has a minimal impact on student performance
Authors: Selsby Joshua T
Published: June 2021

The purpose of this investigation was to determine the extent to which prior knowledge of short answer (SA) questions impacts exam performance in an introductory undergraduate physiology class. This class is taught each semester and covers 12 systems in a 16 week semester. Exams consist of multiple choice (MC) and SA sections. My hypothesis was that SA performance would be enhanced by prior knowledge of SA questions. To test this hypothesis, topics relevant to SA questions were sent to students 24–72 hours prior to exams. Over the course of semester one, this was done for the final two exams (of four). In the subsequent semester it was done for the first two exams, but not the remainder. Supplying students with exam material either slightly increased or did not increase SA scores. Interestingly, MC scores were consistently improved to a greater extent than SA scores. As a result, the overall scores of students receiving information regarding exam material were consistently and significantly improved by approximately 8% when compared to students that did not. The summation of these findings suggests that providing students with exam material prior to exams provides students with a minor advantage. It may also have the added benefit of reducing exam anxiety and improving instructor evaluations while maintaining exam rigor.
Teaching Inquiry Based Physiology in High Schools
Authors: Anjur Sowmya S
Published: June 2021

Students participated in inquiry‐based physiology labs to better understand the structure‐function relationship of major body systems. Students measured their respiration, blood pressure and heart rate while at rest (sitting), standing, lying down, and after 5 and 20 minutes of cardiovascular exercise. Students performed t‐tests on their data, which showed significant correlation during exercise. In a separate experiment, students tested their patellar, plantar, ankle‐jerk and pupillary reflexes under similar conditions, and used inquiry to decide which impulses originated from the central or peripheral nervous system, and to understand and correlate the relationship of these results with the previous experiment. Such modified experiments followed by inquiry based discussions gave students a better insight into understanding the relationship between anatomy and physiology of the organ systems of the body, and their significance to real‐world applications than traditional lab exercises alone.
Exercise mitigates the stunting effect of cold temperature on limb elongation in mice by increasing solute delivery to the growth plate
Authors: Serrat Maria A. , Williams Rebecca M. , Farnum Cornelia E.
Published: June 2021

Ambient temperature and physical activity modulate bone elongation in mammals, but mechanisms underlying this plasticity are not understood. Longitudinal bone growth occurs in cartilaginous plates, which receive nutritional support via transport of solutes from the vasculature. We subjected weanling mice to temperature and wheel activity to test the hypothesis that activity and high temperature promote bone lengthening by increasing solute delivery to growth plates. We show that exercise mitigates the stunting effect of cold temperature on limb elongation after only 11 days of wheel running. Runners all had significantly lengthened limbs regardless of temperature, while non‐runners had shorter limbs that correlated with housing temperature. Tail length was impacted only by temperature, indicating the exercise effect was localized to limb bones and was not a systemic reaction. In vivo multiphoton imaging of systemically‐introduced tracers revealed enhanced solute delivery to tibial growth plates of wheel‐running mice. The temperature effect was minimal, suggesting that solute transport predominates the exercise growth response. These results provide evidence for the role of solute transport in exercised‐augmented limb elongation and are important for understanding limb length variation among mammals in different environments. Supported by NIH‐RO1AR052003‐04 and AAA Postdoctoral Fellowship.
OA/Gpnmb Mutation Impairs Osteoblast and Osteoclast Differentiation and Function
Authors: Belcher Joyce Yvonne , Rico Mario , Ablemagid Samir , Monroy Alexandra , Popoff Steven , Safadi Fayez
Published: June 2021

Osteoactivin/Gpnmb (OA/Gpnmb) is a glycoprotein identified in bone, and later found to be strongly localized to bone‐forming osteoblasts. There exists in mice a naturally occurring mutation for OA/Gpnmb, which is attributable to premature stop codon resulting in a truncated 150 amino acid OA/Gpnmb protein. Micro‐CT analysis of aged mice show decreased bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness (Tb. Th.) in OA/Gpnmb mutant mice when compared to wild‐type, implying that OA/Gpnmb mutant mice are more osteoporotic. Immunoflourescent analysis has shown OA/Gpnmb protein in mutant osteoblasts (OB) is retained in the peri‐nuclear/cis‐Golgi area. Electron microscopy analysis of osteoblasts showed no morphological differences in mutant osteoblasts versus control, suggesting OA/Gpnmb retention is not cytotoxic. qPCR mRNA and western blot analyses showed increased RANKL expression in OB from OA/Gpnmb mutant. Co‐culture of bone marrow cells showed marked increase in osteoclast (OC) numbers and size in mutant cultures when compared to WT. Treatment of WT and Mutant osteoblasts with recombinant OA/Gpnmb protein resulted in enhanced osteoclast number and size in vitro in WT and but not in mutant OB, suggesting an inherent defect to in mutant compared to WT OC. Collectively, these data suggest OA/Gpnmb acts as a regulator of osteoblast‐osteoclast differentiation and function.

Grant Funding Source: NIAMS
Regenerative medicine based on muscle stem cells
Authors: Huard Johnny , Mankin Henry J.
Published: June 2021

Members of the Stem Cell Research Center (SCRC) have isolated various populations of myogenic cells from the postnatal skeletal muscle of normal mice by means of the cells' adhesion characteristics, proliferation behavior, and myogenic and stem cell marker expression profiles. Most of these cell populations have displayed characteristics similar to those of skeletal muscle satellite cells; however, we also have identified a unique population of muscle‐derived stem cells (MDSCs). The MDSCs exhibit long‐term proliferation and high self‐renewal abilities, increased resistance to stress, and multipotency with the ability to differentiate, in vitro and in vivo, into a variety of tissue types including: muscular (skeletal and cardiac), neural, endothelial, osteogenic, and chondrogenic lineages. In contrast to other myogenic cell types, MDSCs are very efficient at engrafting and regenerating a variety of musculoskeletal tissues due to their increased ability to survive post‐implantation because of their high expression of anti‐oxidants. MDSCs activate host cells and are influenced by environmental cues released within injured tissues, which have been shown to impact the MDSCs regenerative capacity in various tissues. Some of the results regarding the crosstalk between the donor cells and host cells/tissues will be presented. Potential strategies are being explored to increase angiogenesis and prevent scar tissue formation within injured tissues as a means to further improve the regenerative potential of these cells. Finally recent results will be presented that suggest that the blood vessel walls harbor several cell types, including myo‐endothelial cells and pericytes that are likely the place of origin of the murine MDSCs discussed above. These human blood vessels derived cells are being tested for their regenerative potential in various tissues. The results outlined above open new avenues by which researchers could use muscle stem cell–based gene therapy and tissue engineering to improve tissue regeneration.
DNA‐binding properties and telomere function of hnRNP A3
Authors: Huang Pei‐Rong , Hong Shu‐Chen , Chen Chi‐Yuan , Wang Tzu‐Chien
Published: June 2021

Human telomeric DNA consists of hundreds to thousands of tandem repeat hexanucleotides (TTAGGG) and is bound by telomere‐associated proteins to form specialized structure that maintains the integrity of chromosome. These telomere‐associated proteins serve as structural and/or regulatory components for telomere functions. Dysfunctional telomeres are known to trigger growth arrest, senescence, and apoptosis. Previously, we have identified hnRNP A3, a poorly understood member of the hnRNPs A/B, as a novel telomere‐associated protein that binds specifically to single‐stranded telomeric DNA. In this study, the DNA‐binding properties of hnRNP A3 in vitro as well as its putative role of telomere maintenance in vivo were investigated. Here, we showed that the minimal binding sequence (MBS) for hnRNP A3 is 5′‐TAGGGTTAGGG‐3′, and the position of MBS in the single‐strand DNA had little or no effect on the binding by hnRNP A3. The consensus binding sequence in the 11‐mer MBS was characterized as 5′‐[T/C]AG[G/T]NN[T/C]AG[G/T]N‐3′. Furthermore, confocal microscopy and chromatin‐immunoprecipitation (ChIP) studies revealed that hnRNP A3 are associated with telomere in vivo. Knocking‐ down the expression of hnRNP A3 had no effect on telomere length maintenance and did not affect cell proliferation capacity. However, overexpression of hnRNPA3 resulted in the production of steady‐state short telomeres in several cell lines. These findings indicate that hnRNP A3 can participate in telomere function and acts as a negative regulator of telomere length maintenance. This work was supported by research grant CMRPD 170541.
Multiphoton Imaging – A Powerful Tool For Tissue‐State Diagnosis In Regenerative Medicine
Authors: Schenke‐Layland Katja
Published: June 2021

Interactions of cells with the ECM are the basis of many fundamental biological processes including the formation and organization of tissue structures and the remodeling and turnover of the preexisting tissue matrix due to disease or aging processes. A detailed analysis of the ECM is of great value in gaining structural and diagnostic information, particularly with regard to efforts in the field of regenerative medicine. Multiphoton imaging has become a powerful method for the artifact‐free evaluation of ECM structures with submicron resolution and without the need for invasive tissue processing. In our studies, we employed this imaging modality to determine the structural features of ECM in native and in vitro‐engineered tissues and organs, as well as to monitor ECM formation in stem cell cultures and bio‐inspired tissue‐equivalents. We were further able to quantify ECM damage and show that it translates into in vivo failure. Although the ability of the minimal‐invasive measurement of fluorescence and second harmonic generation intensities based on natural intrinsic fluorophores by optical sectioning has advanced our understanding of ECM structures in their native microenvironment and their role in tissue function and homeostasis, the development of miniaturized multiphoton micro‐endoscopes, which can be applied to internal organs, will further help the field of regenerative medicine.
Responses of mice lacking α1AMPK to acute and chronic thermogenic challenges
Authors: Bauwens Jake Daniel , Lindholm Christopher R. , Ertel Rebecca L. , Mulligan Jacob D. , Schmuck Eric G. , Saupe Kurt W.
Published: June 2021

While activity of α1 isoform of AMP‐activated protein kinase (AMPK) is higher in brown adipose tissue (BAT) than any tissue yet reported, its role in BAT is unknown. Our goal was to elucidate AMPK's in vivo role in BAT, specifically in acute non‐shivering thermogenesis (NST) and chronic (14 days at 4° C) cold‐induced hyperphagia and UCP1 upregulation. C57Bl/6 wild type (WT) and α1AMPK knockout (KO) mice were studied (6–8/group). During acute NST (IP injection of the β3‐adrenergic agonist CL 316,243) core temperature increased similarly (≈0.8°C) in WT and KO mice. Thus α1AMPK is not obligatory for acute NST. In a separate study, chronic cold‐induced hyperphagia was nearly identical in WT and KO mice (≈ 2.5 fold increase in food intake). Chronic cold caused less upregulation of UCP1 in KO (1.9‐fold) than in WT (4‐fold) mice, but did not cause a lower body temperature in KO mice. Therefore α1AMPK is not obligatory for hyperphagia or other adaptations that maintain body temperature near normal during chronic cold exposure. There was little if any compensatory upregulation of α2AMPK in α1AMPK KO mice. Overall our data indicate that high levels of α1AMPK activity in BAT do not play a key role in major aspects of thermoregulation, and instead suggest a currently undefined role for AMPK in BAT physiology.
How insulin binds to the insulin receptor: a synthetic chemical biology approach
Authors: Xu Bin , Hua Qing‐xin , Huang Kun , Chu Ying‐Chi , Hu Shi‐Quan , Whittaker Jonathan , Katsoyannis Panayotis G. , Weiss Michael A.
Published: June 2021

How insulin binds to the insulin receptor has long been the subject of speculation, and the active conformation of insulin and its mode of receptor binding remain elusive. Here, functional surfaces of insulin are probed by photo‐scanning mutagenesis of an extensive set of azido‐derivatives constructed by chemical synthesis. We demonstrate by residue‐specific photo cross‐linking that diabetes‐associated mutations occur at receptor‐binding sites. Receptor contacts are circumferential, suggesting that insulin is encaged within its receptor. Detailed mapping of photo‐products to the hormone‐binding domains of the insulin receptor demonstrates that the L1 domain and carboxy‐terminal insert domain are the major contact sites in the receptor. Our photo‐scanning approach not only reveals that consecutive residues in insulin contact widely separated sequences in the receptor, but also that the B‐chain beta‐strand acts as an arm to insert between receptor domains. Detachment of such a receptor‐binding arm consequently enables engagement of surfaces otherwise hidden in the free hormone. Photo‐scanning studies also identify a non‐classical receptor‐binding surface in insulin and so demarcate a novel interface. To our knowledge, this is the first comprehensive photo‐scanning study on a protein. (Supported by NIH grants DK56673 to PGK and DK40949 to MAW)
Apm‐1 interferes with transcriptional factor MIZ‐1 to repress the p21CIP1 gene
Authors: Lee Kyung‐Mi , Choi Won‐Il , Yoon Jae‐Hyeon , Hur Man‐Wook
Published: June 2021

The BTB/POZ family proteins have been implicated in many biological processes, including tumorigenesis, DNA damage responses, cell cycle progression and development. We collected the amino acid sequence information of all 183 human BTB‐POZ domain proteins. Among those proteins, MIZ‐1 regulates expression of several genes, and which can be repressed by c‐Myc. Previous studies identified that MIZ‐1 is associated with regulation of p21 and p15INK4B and binds to the two proximal regulatory elements of p21 promoter. We found that MIZ‐1 regulates transcription of not only p21 but also the other genes of p53 pathway, and induced biological functions of p21.

To investigate biological functions of particular POZ domain‐POZ domain interaction involving MIZ‐1, we choose Apm‐1, a potentially important protein interaction partner of MIZ‐1. Apm‐1 is a ubiquitously expressed BTB/POZ‐domain class transcription factor and represses transcription of p21 and ARF, which requires p53. Our co‐immunoprecipitation and GST‐fusion protein pull‐down assay showed that MIZ‐1 interacts with Apm‐1 via their POZ domains directly. We also found that Apm‐1 and MIZ‐1 coexist in the proximal p21 promoter by ChIP. Also reporter assay, quantitative PCR analysis, MTT and FACS analysis suggested that Apm‐1 interferes with transcription activator and cellular regulatory functions of MIZ‐1.
Purification of cortactin by anion exchange chromatography
Authors: Kruchten Anne E , Arns Katherine
Published: June 2021

Cortactin is a cytoskeletal actin‐binding protein that is upregulated in several types of metastatic cancers. Standard purification of cortactin from E. coli involves expression of the protein as a His‐tagged protein followed by affinity purification with nickel resin. This purification is sometimes followed by anion exchange chromatography, which appears to result in a cortactin protein with enhanced activity in actin polymerization assays compared to cortactin that is only affinity purified. To determine how further purification by anion exchange chromatography alters the activity of the protein, we analyzed KCl elution fractions of cortactin on native polyacrylamide gels and found each elution fraction contained a different band pattern. These differences suggest that purification by anion exchange separates proteins of unique structures due to folding or modification of the protein. Future work includes biochemical analysis of the structure and modifications of cortactin in the elution fractions. This work was supported by an M.J. Murdock Charitable Trust Faculty Start Up Grant to A.E. Kruchten and a Linfield College Faculty Student Collaborative Research Award to A.E. Kruchten and K. Arns.
Investigation of the Mechanism of Hydrogen Sulfide Activation of Protein Kinase C
Authors: Buck Lauren , Stahelin Robert V
Published: June 2021

Hydrogen Sulfide (H2S) has been shown to be an endogenously produced second messenger, affecting a variety of physiological and cell signaling processes. It is present at micromolar levels within the body and is produced via two key enzymes in the cysteine biosynthesis pathway: cystathione‐β synthase, and cystathione‐γ lyase. Despite its prior reputation as a noxious gas, H2S has been shown to have significant involvement in protection of cardiac tissue following myocardial infarction. These protective properties may be due to a role of H2S in control of mitochondrial oxidation, in addition to its ties to the localization and signaling of Protein Kinase C. Recent studies have indicated an involvement of H2S in the localization of certain isoforms of Protein Kinase C to the cellular membrane (epsilon and eta). In particular, PKC‐ε knockout has been found to be deleterious to cardiac recovery and also has probable involvement in the process of mitochondrial respiration. We are investigating the molecular basis of H2S induced translocation of PKC isoforms to the cellular membrane. One possibility of H2S involvement in PKC‐ε localization is through a diacylglycerol (DAG) synthesis response. DAG is known to be the main activator of PKC, and it is possible that H2S serves as a signaling molecule to initiate DAG synthesis. The molecular basis of PKC recruitment to cellular membranes by H2S signaling will be presented.
Posttranslational regulation of mitochondrial glycerol‐3‐phosphate acyltransferase by Protein Kinase C in rat hepatocytes
Authors: Pandey Atul , Zhang Man , Haldar Dipak
Published: June 2021

Glycerol‐3‐phosphate acyltransferase catalyses the first step in glycerolipid biosynthesis. The mitochondrialisoform (mtGPAT) is mainly expressed in liver cells where it is involved in fatty acid metabolism. Analysis of the cDNA of mtGPAT indicates 10 putative Protein Kinase C (PKC) phosphorylation sites. In the current investigation, we present evidence that PKC regulates mtGPAT in rat hepatocyte cells, BRL‐3A. When isolated mitochondria were treated with PKC, the activity was stimulated by approximately 50%. mtGPAT was identified by Western blot, or when the mitochondria or whole cells were incubated with γ‐32P‐ATP, by autoradiography. The activation of endogenous BRL‐3A cells with the cell permeable activator PMA (0.1 μM) also gave a stimulation of 50% suggesting a role of PKC in the regulation of mtGPAT. The mtGPAT activity was inhibited when the whole cells were treated with PKC specific inhibitor, Gö6976 (10 μM), and H‐7 (10 μM) which inhibits both Protein Kinase A and PKC suggesting the role of PKC in regulating mtGPAT. Given the important role of PKC in cellular neoplastic transformation, carcinogenesis and tumor cell invasion, the current study further explores the potential role that PKC might be playing in hepatic fatty acid metabolism by regulating mtGPAT by direct phosphorylation‐dephosphorylation mechanism. Supported by NIH grant GM‐57643.
Oxidative and nitrosative stress in human placenta in Preeclampsia
Authors: Campos‐Arroyo Ana Gabriela , Trejo‐Mora Iván , Pamatz‐Martínez Antonio Jesús , Maya‐Ramírez Luis Octavio , Clemente‐Guerrero Mónica , Saavedra‐Molina Alfredo , Manzo‐Avalos Salvador
Published: June 2021

Preeclampsia (PE) is a pregnancy specific disorder characterized by hypertension, proteinuria and edema after 20 weeks of gestation. There is a profound effect on the fetus, often resulting in intrauterine growth retardation or perinatal death. Oxidative stress is the biggest contributor to endothelial dysfunction in PE. It causes an increased sensitivity to constriction of blood vessel endothelial affecting endothelium‐dependent vasodilatation. In addition, several studies suggest that nitric oxide (NO) levels increase leading to increased protein nitration, which may lead to the development of PE. The increased oxidative and nitrosative damages are due in part to the decreased expression and activity of antioxidant systems. The aim of this work was to determine the status of oxidative and nitrosative stress in human placenta in PE. In homogenates of umbilical cordon, vein, artery and cotyledons of human preeclamptic placenta we determinate the oxidative stress measuring the levels of lipoperoxidation, and nitrosative stress by measuring the concentrations of NO, profiles of nitrosylation, and the levels of the nitric oxide synthases (NOS). The data obtained shown a significant increase in the levels of TBARS and NO, and a decrease in the levels of nitrosylation and NOS's in preeclamptic placenta.
The Prediction of The Risk for Hypertension in Carrier of Set of Alleles of Gene Encoding Methylene Tetrahydrofolate Reductase
Authors: Hemimi Neveen Salah , Oda Naglaa Rafaat , Hemedi Samira Ali , Shaafie Ishtiyaq Ahmad
Published: June 2021

Background

MTHFR is a regulatory enzyme of homocysteine metabolism. The genetic polymorphism of the MTHFR gene has been reported to be associated with elevated plasma homocysteine in individual with low folic acid intake.

Aim

To investigate the influence of the C677T and A1298C polymorphisms of MTHFR gene on the development of hypertension together with the study of other metabolic risk factors for hypertension.

Material& Method

66 normotensive individual and 60 hypertensive patients were enrolled in the study. The C677T and A1298C polymorphisms of MTHFR were identified by RFLP‐PCR of peripheral blood samples. Analysis of data was done by using SPSS program.

Results

Allele frequency for T allele of C677T polymorphism was higher in the hypertensive group compared to the normotensive group. The carriers of XT genotype (CT and TT) were at high risk of developing hypertension (OR= 1.8; 95% CI = 0.8–3.9) when compared with the carrier of CC genotype. Similarly carries of XC genotype (AC& CC) of A1298C were at high risk of developing hypertension (OR=1.5; 95%CI=0.6–4.1) when compared with AA genotype. The risk for hypertension was increased when the individual was carrier of both alleles (OR= 1.9; 95%CI= 0.6–5.8).

Conclusion

T and C allele of C677T and A1298C polymorphisms of MTHFR are possible genetic risk factors for hypertension.

Source of Fund: Dubai Medical College
Acetylbritannilactone is a potent synergist of celecoxib to inhibit cyclooxygenase‐2 expression and to induce apoptosis in human breast cancer cells
Authors: Liu Bin , Wen Jin‐kun , Wang Jun‐jie , Zhang Yan‐ping , Zhang Di‐qun , Han Mei
Published: June 2021

The use of cyclooxygenase‐2 (COX‐2) inhibitors is associated with a significant decrease in breast cancer risk. However, recent studies show that the long‐term use might be limited due to undesired side effects. Our previous study has demonstrated that acetylbritannilactone (ABL), isolated from Chinese herb Inula Britannica L, inhibit COX‐2 expression via blockade of nuclear factor‐kappa B (NF‐κB) activation. Here, the effects of ABL and celecoxib as single and their combination were examined in breast cancer cells, and CalcuSyn software was used to assess synergistic inhibition. Celecoxib (5 μM) and ABL (25–50 μM) synergistically inhibited cell growth and induced cell apoptosis. The results showed that ABL augmented celecoxib inhibition of COX‐2 expression and prostaglandin E2 synthesis. To confirm the mechanism by which ABL and celecoxib synergistically attenuate COX‐2 expression. Furthermore, the promoter activity of COX‐2 gene was analyzed by chromatin immunoprecipitation and DNA affinity protein‐binding assays. We found that the combination of ABL with celecoxib at lower concertration resulted in reduction of binding of CREB‐1, ATF‐2 and c‐fos to the COX‐2 promoter, and then decreased activity of promoter reporter gene. In addition, inhibition of Akt and p38 signaling pathways was required for the synergism, as a constitutively active Akt and p38 abrogated the reduction of COX‐2 transcription and protected cells against apoptosis induced by the combination. Taken together, the combination of ABL with celecoxib elicited significantly greater inhibition of cell growth and more apoptosis, compared with either drug alone. Thus, ABL is expected to be a potent synergist of celecoxib for treatment of breast cancer.
Directing human pluripotent stem cells toward the skeletal muscle lineage
Authors: Tran Tuan , Peterson Cory , Pyle April D.
Published: June 2021

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) hold great promise as a potential source of cells for use in therapeutic medicine due to their ability to differentiate into cells from each germ layer and continuously self‐renew. Despite this potential, there remains great variation in both methodology and efficiency of generating mesodermal lineages from either hESCs or hiPSCs. Of particular difficulty has been the development of reliable protocols to efficiently differentiate skeletal muscle lineages. Although CD73+ mesenchymal cells have been obtained through sequential culturing and cell sorting methods, skeletal muscle cell yield was low and the protocol requires media for neuronal growth. Therefore we are currently establishing an alternative approach for directing differentiation of hESCs and hiPSCs toward the muscle lineage. To this end, when embryoid bodies (EB) derived from hESCs and hiPSCs were differentiated after 24 days, maximal enrichment for early stage or progenitor muscle markers was achieved compared to those differentiated up to 30 days. Subsequently, dissociated cells from EBs at each time point were sorted for either NCAM or M‐Cadherin and expanded in standard culture conditions for promoting muscle cell growth. Both cell types were confirmed to express Pax3 via RT‐PCR. Establishment of standard protocols for the reliable generation of muscle progenitor cells from PSCs will be important in future experiments for assaying the differentiation potential of Duchenne Muscular Dystrophy (DMD) patient‐specific hiPSCs as well as for identifying the muscle cell type(s) needed for efficacious cell therapy.
Investigation of migrating neural cells during neurulation in zebrafish
Authors: Allette Yohance M , Jaychandran Pradeepa , Brewster Rachel
Published: June 2021

Our Central Nervous System is formed from an embryonic precursor tissue known as the neural tube. Birth defects caused by improper closure of this tube during a process known as neurulation are frequent in humans. Our laboratory uses the zebrafish as a model system to investigate the etiology of these defects, since mechanisms of neurulation appears to be conserved across vertebrates. We hypothesize that a specific midline‐derived signal may direct the movement of neural precursors. The goal of my project is to determine whether Neogenin mediates polarized cell migration during neurulation, by verifying that Neogenin is expressed in neural tissue at the time of neurulation using in situ hybridization. I am investigating whether Neogenin is required for mediating polarized cell movement during neurulation, by disrupting Neogenin function using translation‐blocking morpholinos. Defects in cell movement will be scored by using in situ to visualize the position of lateral neural cells during convergence. This research will increase our understanding of molecular mechanisms that regulate neurulation, and provide putative candidate genes implicated in the etiology of human neural tube birth defects.