bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone mineral density is affected by calcium nutritional status when assessed by ultrasound in school girls
Authors: Rio Maria E , Dupraz Hernán , Felipoff Ana Lia , Zago Liliana , Linari María , Piazza Norma
Published: June 2021

Grow process is a state of high bone turnover and an inadequate calcium nutritional status can lead to suboptimal peak bone mass and predispose to osteoporosis in adulthood. Previously, we have found that the a‐CTX marker of bone growth was highly affected by body Ca status. A similar study was carried out in relation to bone densitometry. For that, urine basal samples were obtained from 120 healthy girls, aged 5.8 to 9.2 years, attending public schools of Vicente Lopez (Bs. As) and over them Ca (AAS) and creatinine (Cr) (Wiener Lab®. Argentina) were determined to calculate Ca/ Cr ratio as marker of Ca status. Bone densitometry was assessed by ultrasound (Sahara Olohi. USA) and expressed as Quantitative Ultrasonic Index (QUI). The relation: QUI vs Ca/Cr divided in five ranges between < 0,10 and >0,15, fits a quadratic function so that QUI=92,828e0,3426x; R2=0,78; p<0,01. Results show a quite good no linear correlation presenting a flat area between Ca/Cr <0,07‐0.01 and then ahead a significant positive slope. As in the case of the evolution of the CTX bone marker previously studied, a Ca/Cc >0,01 was mandatory during bone growth to allow the age dependent increase in bone mineralization. Supported by UBCyT US.B/735. HD PhD Thesis. Fellow from UBA
Relationship among fatty liver, adipose tissue distribution and metabolic profile in moderately obese children
Authors: Jirillo Emilio , Chiloiro Marisa , Riezzo Giuseppe , Chiarappa Salvatore , Correale Mario , Guerra Vito , Amati Luigi , Noviello Marisa
Published: June 2021

We examined the relationship between moderate obesity and glucose metabolism, insulin sensitivity and suspected fatty liver.

Methods

z‐score body mass index( zBMI), caliper skinfold tickness, fasting blood glucose, insulin, HOMA score, lipid profile and transaminases (ALT, AST) were measured in 94 participants (mean age 9.7 ±2.2 years). Fatty liver and skinfold tickness were evaluated by ultrasound.

Results

The zBMI was 2.01 ± 0.39 (mean ± SD), and the duration of obesity was 4.3±3.03 years. Caliper and US skinfold thickness correlated for tricipital (r = 0.33; p= 0.003) and sovrailiac skinfold (r= 0.34; p=0.003). Fatty liver was diagnosed in 64% of children and it was positively related to anthropometric measurements. The three groups ‐ group 0 (normal US liver and normal transaminases); group 1 (US fatty liver and normal transaminases); group 2 (US fatty liver and elevated transaminases) showed difference in zBMI , insulin and HOMA parameters (Tukey test: zBMI group 1 vs group 0 and 2 vs group 0; serum insulin: group 2 vs group 1 and group 2 vs group 0; HOMA IR: group 2 vs group 1 and group 2 vs group 0).

Discussion

Moderately obese children with steatosis exhibited a clear increase of insulin and insulin resistance, indices of a future metabolic syndrome. Transaminases levels seems to be not adequate for the diagnosis of fatty liver.
Predictors of under‐ nutrition and overweight among children under age 5 years in India.
Authors: Chakraborty Priyanka , Anderson Alex Kojo
Published: June 2021

Objective

Although, undernutrition has prevailed in developing countries for years, prevalence of overweight among children has been on the rise in recent years. This study aims to assess the present trend of nutritional status of Indian children.

Method

Secondary data from the 2005‐2006 Demographic and Health Survey for India was examined. Secondary analysis was performed to identify trends of underweight and overweight of children under age 5.

Result

Stunting and wasting (HAZ, WHZ z scores <‐2.0) were widely prevalent among index children. While, 18% children were wasted, >38% children were stunted. At the same time, about 8% of urban and rural children were overweight (BMI z‐score >2.0). Analysis with respect to SES showed the difference of BMI‐for‐age between poorest and wealthiest households to be significantly different (mean= ‐0.52; 95% CI= ‐0.60 to ‐0.43; p<0.001). Multivariate regression analysis, (adjusted for birth weight) revealed that higher birth order and prolonged duration of breastfeeding had a negative impact on HAZ and BMI for age z‐scores of the index child. On the other hand maternal BMI was found to be positively associated with HAZ, WHZ and BMI‐for age z‐scores.

Conclusion

Further research and public health programs need to focus on these predisposing factors associated with nutritional status of under‐five children in India.

Grant Funding Source

Faculty Development Grant
Screening young children for lead poisoning in Bangladesh
Authors: Mitra Amal K. , Islam Manirul , Haque Akhlaque
Published: June 2021

To assess the risk of lead poisoning among children in Bangladesh, 345 children ages 6 mo‐12 y were screened for blood lead levels (BLL). The children were recruited from three locations: an urban industrial area (n = 105); a rural nonindustrial area (n = 183); and an urban nonindustrial area (n = 57). Possible sources of lead were identified, and GIS maps were constructed. BLLs were ≥10 μg/dL in all but one (99%) of 105 children in the industrial area. Industrial emissions and use of leaded gasoline were identified as possible sources of lead in that location. In the rural nonindustrial area, 14% of the children had BLLs ≥10 μg/dL. The possible source of lead in the rural area was from the use of agricultural pesticides. BLLs at the urban industrial area were significantly higher than those at the rural‐ and urban nonindustrial areas (24.58 ± 10.32, 7.24 ± 6.31, and 2.47 ± 3.32 μg/dL, respectively; p <.001). Weight‐for‐age z‐scores of the urban children were significantly poorer than that of the rural children (‐1.41 ± 1.88 vs. .20 ± 1.16, p <.001). Children with higher BLLs had poorer nutritional status (p =.05) compared to those with normal BLLs. Over 90% of the parents did not know that lead causes health problems. This study recommends programs for universal lead screening for preschool‐ and school‐aged children, and improving parents' awareness of lead poisoning. The study was supported by The University of Southern Mississippi.
Clustering of health and behavioral risk factors for cardiovascular disease in Alaska Native youth
Authors: Bersamin Andrea , Mohatt Gerald V , Zidenberg‐Cherr Sheri , Stern Judith S , Luick Bret R
Published: June 2021

Evidence indicates that rural youth are more likely than their urban counterparts to participate in risky behaviors, many of which translate into a higher burden of cardiovascular disease (CVD). Alaska Native youth may be at particularly high risk. Our objective was to evaluate the clustering of health and behavioral risk factors for CVD in 315 Alaska Native youth and young adults (ages 14‐25 years) who participated in the Center for Alaska Native Health Research Study. The prevalence of health risk factors (overweight, hypertension and high HDL, low glucose, and high triglyceride concentration) was estimated using age and gender appropriate criteria. Behavioral risk factors (diet and smoking) were assessed using computer assisted 24‐hour recalls and standardized questionnaires. Clustering of risk factors was common. Approximately 75% of the sample had 0‐1 health risk factors; the remaining 25% had 2‐3 risk factors. The most prevalent risk factors were overweight (40.3%), low HDLs (26.3%) and high triglycerides (21.3%). More than 30% reported smoking regularly. Added sugars and saturated fat contributed substantially to energy intake (19% and 11%, respectively). The risk of CVD increases with each additional risk factor. To promote healthy aging among Alaska Native youth the development of ecologically appropriate CVD prevention programs is essential.

NIH (P20 RR16430)
RNAi‐Mediated Knockdown of DNMT3b in Hypermethylator Breast Cancer Cell Lines Leads to Normalization of Gene Expression Patterns
Authors: Roll J Devon , Rinkenbaugh Amanda L , Sandhu Rupninder , Rivenbark Ashley G , Coleman William B
Published: June 2021

A subset of breast cancer cell lines express a hypermethylation defect characterized by DNMT hyperactivity, overexpression of DNMT3b, and silencing of numerous genes. To investigate the role of DNMT3b in the hypermethylation defect, we examined the effect of RNAi‐mediated DNMT3b knockdown on expression of methylation‐sensitive genes in model hypermethylator cell lines: MDA‐MB‐453 and BT549. Western blot analysis confirmed reduction of DNMT3b in cells transfected with the DNMT3b RNAi targeting construct. RT‐PCR was used to analyze expression of six methylation‐sensitive genes (CEACAM6, CST6, ESR1, SCNN1A, GNA11, CDH1 and/or MUC1) that are methylated and exhibit diminished or no expression in parent cell lines compared to MCF12A. In MDA‐MB‐453 cells, all six genes were reexpressed or increased expression in response to DNMT3b knockdown. CEACAM6, CST6, ESR1, and GNA11 were expressed at normal levels, while MUC1 and SCNN1A were expressed at low but detectable levels. In BT549 cells, CEACAM6, CST6, ESR1, GNA11, and SCNN1A were reexpressed or increased expression in response to DNMT3b knockdown, whereas CDH1 was not expressed. These results strongly suggest that CEACAM6, CST6, ESR1, GNA11, SCNN1A, and MUC1 are direct methylation targets for DNMT3b. Further, these results suggest that DNMT3b overexpression governs the hypermethylator defect associated with some breast cancer cell lines.

Support: NIH CA78343
Loss of Nrf1 Function in the Brain Causes Age‐Dependent Neurodegeneration
Authors: Lee Candy Shih‐Chi , Chan Jefferson J
Published: June 2021

Neurodegenerative disease is an important public health issue as the aged population continues to increase. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis are caused by gradual loss of neurons. Although molecular mechanisms involved are not fully understood, accumulation of abnormal proteins, mitochondrial defects, and oxidative stress are common in many forms of neurodegenerative diseases. In order to cope with high levels of reactive oxygen species that are normally generated in the brain, antioxidant enzymes are transcriptionally activated in neurons to prevent oxidant damage. Members of CNC‐bZIP (Cap'n'Collar basic leucine zipper) transcription factor family have been implicated in regulating antioxidant gene expression through the antioxidant response element (ARE). Nrf1 is a CNC‐bZIP that is highly expressed in the adult brain, but its role in neurons is not known due to embryonic lethality in Nrf1 germ line mutant mice. Here, we generate and analyze Nrf1 brain conditional knockout mice by using Cre‐Lox system. Mice deficient of Nrf1 in the brain showed age dependent neurobehavioral abnormalities including abnormal leg‐clasping reflex, impaired rotarod performance and hyperactivity. Correspondingly, loss of Nrf1 in the mouse brain leads to age‐dependent brain atrophy as a result of apoptosis. Our studies here demonstrate a novel function of Nrf1 in protecting against neuronal apoptosis. Nrf1 Brain knockout mice may provide a model to study mechanisms of neurodegenerative diseases.
Aβ Oligomers Induce Neurotoxicity: Effects of HDACs and Nrf2 Modulation
Authors: Ebenezer Philip J. , Weidner Adam , LeVine Harry , Keller Jeffrey
Published: June 2021

The 40‐42 amino acid amyloid βpeptide (Aβ) contributes to one of the central neuropathologic features of Alzheimer's disease (AD). Recent evidence suggests that the oligomeric forms of Aβ may play a role in AD. The objective of the present study was to evaluate the morphological and neurochemical effects of oligomers in primary rat neuronal cells. Neuronal cells were exposed to different doses of soluble Aβ oligomers (0‐90 nM) and analyzed for multiple indices of neuronal homeostasis. Our results indicate that Aβ oligomers promote morphological alterations, neurotoxicity and oxidative stress. The effects of histone deacetylase inhibitors and modulators of the Nrf2 pathway were analyzed for their ability to inhibit the toxicity of Aβ oligomers. Our studies indicate a role for Aβ oligomers as mediators multiple aspects of AD pathogenesis and outline the effects of potentially novel therapeutic strategies as mediators of neuroprotection against Aβ oligomer toxicity.

This work was supported by a grant from the Alzheimer's Association and NIA (P01AG005119) to Dr. Jeffrey Keller.
A new phosphatase that modulates the C. elegans Insulin/IGF‐1 signaling pathway
Authors: Tissenbaum Heidi A , Padmanabhan Srivatsan , Mukhopadhyay Arnab , Narasimhan Sridevi
Published: June 2021

The prevalence of Type II diabetes is rapidly expanding in the US. Two factors that contribute to this are the incidence of obese individuals (strong correlation between obesity and Type II diabetes) as well as the increased average age of the population. As individuals age, they become more likely to develop Type II diabetes. The overall goal of our studies is to uncover the molecular mechanism that explains the connection between Insulin/IGF‐1 signaling, the aging process and fat storage, so that Type II diabetes and can be better controlled in humans. We use the nematode, C. elegans, as our model system since mutations in the genes in the Insulin/IGF‐1 signaling pathway lead to changes in both fat storage and longevity and the pathway is highly conserved between humans and C. elegans. The major regulatory control of IIS is through phosphorylation of its components by serine/threonine‐specific protein kinases. In an RNAi screen for serine/threonine protein phosphatases that counter‐balance the effect of the kinases in the IIS pathway, we identified pptr‐1, a B56 regulatory subunit of the PP2A holoenzyme. Modulation of pptr‐1 affects phenotypes associated with the IIS pathway including life span and fat storage. We show that PPTR‐1 functions by regulating worm AKT‐1 phosphorylation. In C. elegans, this modulation ultimately leads to changes in sub‐cellular localization and the transcriptional activity of the forkhead transcription factor DAF‐16. This study reveals a c role for the B56 regulatory subunit in modulating Insulin/IGF‐1 signaling through the dephosphorylation of AKT and thereby has widespread implications in diabetes research and cancer.
Epoxyeicosatrienoic acids (EET) contribute to flow‐induced dilation in mice mesenteric resistance artery
Authors: Varadarajan Sudhahar , Falck John R , Imig John D
Published: June 2021

Resistance arteries dilate in response to flow in an endothelial dependent manner and flow‐induced dilation (FID) has been demonstrated to be impaired in patients with cardiovascular disease. We investigated the contribution of endothelial vasodilator pathways to FID in mice mesenteric resistance arteries. Mesenteric resistance arteries pressurized to 65 mmHg averaged 179 ± 2 μm in diameter and were preconstricted with phenylephrine prior to increasing flow to 52 ± 1 μl/min (low flow) or 304 ± 1 μl/min (high flow). FID over a 5‐minute period averaged 101 ± 5 μm ( min and 138 ± 11 μm ( min in response to low flow and high flow, respectively. Inhibition of the nitric oxide (NO) and cyclooxygenase (COX) pathways with L‐NAME and indomethacin decreased the response to low flow by 57% and high flow by 50%. An EET antagonist, 14,15‐EEZE decreased the mesenteric resistance artery response to low flow by 46% and high flow by 34%. A combination of 14,15‐EEZE, L‐NAME & indomethacin further decreased FID (low, 73%; high, 70%). This decrease in FID was similar to that when L‐NAME, indomethacin and the non‐selective K+ channel inhibitor, tetraethyl ammonium were administered in combination. These data indicate that EET activation of K+ channels is responsible for the NO and COX independent FID in mice mesenteric resistance arteries.
New NO donor inhibits contraction of smooth muscle cell through activation of guanylyl‐cyclase and sarcoplasmic reticulum Ca+2‐ATPase (SERCA).
Authors: Bendhack Lusiane Maria , Pereira Amanda de Carvalho , Biazzotto Juliana C. , Da Silva Roberto S.
Published: June 2021

This study aimed to investigate the pathways involved in the vasodilatation induced by the new NO donor cis‐[Ru(bpy)2(py)(NO2)](PF6)3 (PY) synthesized in our laboratory. Vascular reactivity to PY was studied in denuded rat aorta pre‐contracted with phenylephrine (Phe) or 60mM KCl. Concentration‐effect curves for PY were constructed in the presence or in the absence of the selective inhibitor of soluble guanylyl‐ cyclase (sGC) (ODQ), superoxide scavenger (Tiron) and SERCA inhibitor (Thapsigargin, TG). It was also constructed concentration‐effect curves for CaCl2 under stimulation with 100nM Phe with or without PY. NO was released by the compound only in presence of the aortic tissue as detected by amperometry with a selective sensor. The NO donor reached the maximum aortic rings relaxation (ME) in 210 seconds. PY induced complete relaxation of the aortic rings pre‐contracted with Phe or KCl in a concentration‐dependent way but the ME and potency (pD2) values were lower in KCl than in Phe‐contracted aorta. These responses were inhibited by ODQ and TG. The contractions to increased concentrations of extracellular Ca2+ were abolished by the 3 uM PY. The effect of PY was blocked by ODQ or TG. Our results show that the NO released from PY in the tissue induces relaxation due to the activation of the enzymes sGC and SERCA. It also inhibits Ca2+ influx and consequent contractile response. Financial Support: Fapesp and CNPq.
Role of NADPH oxidase in A3 adenosine receptor‐mediated contraction using knockout mouse aorta
Authors: Ansari Habib R. , Nadeem Ahmed , Roush Kevin P. , Tilley Stephen L. , Mustafa S. Jamal
Published: June 2021

Earlier we have reported that A3 adenosine receptor (A3AR) mediated endothelial‐contraction is via COX‐1 pathway in mouse aorta (Ansari et al Am J Physiol Heart Circ Physiol. 2007, 293:H3448‐55). In this study, we investigated signaling pathways coupled to A3AR activation using, Cl‐IBMECA in A3KO mouse aorta with apocynin (NADPH oxidase inhibitor) and PD98059 (p42/p44 MAP kinase inhibitor). We found that; a) treatment of aortic rings with Cl‐IBMECA resulted in NADPH oxidase subunits (gp91 & p47 phox) activation, p42/p44 MAP kinase phosphorylation and contraction, and b) preincubation of tissues with apocynin, PEG‐superoxide dismutase+PEG‐catalase and PD98059 inhibited A3AR‐induced aortic contraction significantly (p<.05). Cl‐IBMECA increased the ROS generation by 150±15% over control in WT which was inhibited by apocynin. In WT tissues, gp91 and p47 phox expressions were increased by 163±45% and 183±36%, respectively over control by Cl‐IBMECA which was blocked by apocynin. Cl‐IBMECA also increased p42/p44 MAP kinase phosphorylation by 116±42% over control and it was inhibited by PD98059 in WT. We conclude that Cl‐IBMECA leads to NADPH oxidase activation, ROS generation, p42/p44 MAP kinase phosphorylation which may play a role in A3AR‐mediated aortic contraction. Supported by HL‐027339, HL094447 and HL07180.
Zebrafish as a model to study cardiovascular functions
Authors: Horvat Christine M , Behler James H , AbouAlaiwi Wissam A , Mell Blair M , Williams Frederick E , Nauli Surya M
Published: June 2021

Background

Zebrafish have been used as a model for studying vertebrate development and genetics, including the cardiovascular system. Our project aims to monitor heart contraction and cytosolic calcium using a Fura‐2 probe to monitor calcium oscillations with regard to heart contraction.

Methods and Results

Using high resolution DIC and calcium imaging microscopy, we show that dopamine and verapamil alter calcium signals and muscle contraction in 6‐14 day old anesthetized zebrafish. Dopamine at 100 μg/mL increases heart rate. On the other hand, 40 μg/mL of verapamil decreases heart contraction, but it does not decreases heart rate. Interestingly, no significant difference in calcium in the heart is observed in the zebrafish treated with dopamine or verapamil.

Conclusion

Our preliminary data indicates that dopamine alters heart rate independent from cytosolic calcium. Furthermore, verapamil decreases heart contraction independent from the changes in cytosolic calcium. We propose here that zebrafish can be used as a model to study cardiovascular functions.
How matrix metalloproteinases regulate signaling in cancer
Authors: Werb Zena
Published: June 2021

Matrix metalloproteinases (MMPs) have many functions in the cellular microenvironment through cleavage of proteins leading to their activation, inactivation or removal. As a result broad‐spectrum inhibitors of these enzymes were unsuccessful in clinical trials in cancer patients. We have examined the nature of protumorigenic and antitumorigenic targets and functions of MMPs. Our data indicate that MMP9, MMP13 and MMP14 dependent remodeling of the collagen scaffold regulates angiogenesis, tumor cell invasion and migration and inflammatory cell infiltration. The outcomes vary with oncogenes, tumor type and location. MMPs increase VEGF bioavailability and also increase production of antiangiogenic extracellular matrix fragments.
IL‐8 release triggered by proteinase‐activated receptor‐2 stimulation involves both MEK/ERK and PI3 kinase/Akt pathways in gastrointestinal epithelial cells
Authors: Sekiguchi Fumiko , Tanaka Yusuke , Hong Hao , Kawabata Atsufumi
Published: June 2021

In the gastrointestinal (GI) system, proteinase‐activated receptor‐2 (PAR2) is widely expressed and involved in inflammatory responses. In the present study, we examined and characterized release of interleukin‐8 (IL‐8), a pro‐inflammatory cytokine, following PAR2 activation in human colorectal cancer‐derived HCT‐15 cells and human gastric adenocarcinoma‐derived MKN‐45 cells. A PAR2‐activating peptide (PAR2‐AP), but not a PAR2‐inactive scrambled peptide or a PAR1‐AP, induced IL‐8 release in these GI epithelial cells in 24 h. The PAR2‐triggered IL‐8 release was suppressed by inhibitors of MEK (U0126) or PI3 kinase (LY294002), but not by inhibitors of JNK (SP600125), p38 MAP kinase (SB206580), tyrosine kinase (genistein), EGF receptor tyrosine kinase (PD153035) or NF‐κB (PDTC). PAR2 stimulation actually induced phosphorylation of ERK and Akt. U0126 suppressed the phosphorylation of ERK, but not Akt, and LY294002 suppressed the phosphorylation of Akt, but not ERK. These results suggest that, in the GI epithelial cells, activation of PAR2 triggers release of IL‐8 via two independent pathways, MEK/ERK and PI3 kinase/Akt.
Rapid vascular effects of estrogen and aldosterone on MAP kinase activation: A role for GPR30.
Authors: Gros Robert , Ding QingMing , Chorazyczewski Jozef , Feldman Ross D
Published: June 2021

Recent studies have suggested that for EST, the rapid and non‐genomic effects may in part be mediated through the GPR30 receptor. However, the role of GPR30 in VSMC MAP kinase regulation is unclear. Therefore, we examined the role of GPR30, MR and ERa in mediating the rapid effects of EST and ALDO on ERK activation via western blotting.

In native VSMCs maintained in primary culture, short‐term exposure to ALDO stimulated ERK activation (146±7% of control), whereas EST inhibited of ERK phosphorylation (62±5% of control). Both GPR30 and MR gene transfer enhanced the effect of ALDO to stimulate ERK phosphorylation (GPR30:172±10% of control; MR: 156±6% of control). Interestingly, GPR30 gene transfer reversed the effect of EST from ERK inhibition to ERK activation (149±11% of control), whereas gene transfer of ERa further inhibited ERK activation (45±6% of control). Additionally, in freshly isolated aortic tissue, which express higher endogenous levels of GPR30 (as compared to cultured VSMCs), EST exposure mediated ERK activation‐ consistent with a GPR30‐predominant effect.

Overall, these data support the hypothesis that the vascular expression of GPR30 plays an important role in mediating the common and rapid vascular effects of ALDO and EST. Further, the net effect of EST to regulate ERK activation is dependent on the balance between ERα‐mediated signaling and GPR30‐mediated signaling in VSMCs.
The Adenosine A2A Receptor Modulates TRPV1 Expression and Function by Inhibiting Nerve Growth Factor (NGF) Signaling in Peripheral Neurons
Authors: Mukherjea Debashree , Jajoo Sarvesh , Puntambekar Preeti , Ramkumar Vickram
Published: June 2021

NGF is implicated in sensitization of nociceptive neurons and mediation of inflammatory pain. One mechanism underlying this process is activation and induction of transient receptor potential vanilloid 1 (TRPV1) channel via generation of reactive oxygen species (ROS) and activation of p38 mitogen activated protein kinase (MAPK) pathway. Previous studies from our laboratory showed that NGF regulates the expression of the adenosine A2A receptor (A2AR) through activation of nuclear factor (NF)‐κ B via the p75 receptor. In this study, we determined whether activation of the A2AR could in turn modulate NGF signaling and TRPV1 channel expression in rat pheochromocytoma (PC12) and dorsal root ganglion (DRG) cells, known to express both of these receptors and are responsive to NGF. We observed that the A2AR agonist, CGS21680 (1 μM) reduced NGF mediated ROS generation (via the NADPH oxidase system) in both DRG and PC12 cultures and reduced p38 MAPK activation. Furthermore, CGS21680 reduced the induction of TRPV1 expression by NGF. These results suggest that the A2AR could serve as an endogenous regulator of NGF signaling and thereby modulates the thermal/inflammatory pain pathways controlled by TRPV1. Disruption of such an endogenous regulator would be expected to exacerbate the pain response by removing this inhibition of NGF signaling. This study was supported by an Excellence in Academic Medicine grant from SIU School of Medicine.
Ex vivo and in vivo changes in function of the serotonin 2C receptor due to RNA editing
Authors: Canal Clinton Ernest , Watt Erin Elizabeth , Sanders‐Bush Elaine
Published: June 2021

In vitro, RNA editing of the serotonin 2C receptor (5HT2CR) alters its signal transduction efficiency. Fully edited (VGV) 5HT2CR's show low constitutive and agonist‐stimulated activity, whereas non‐edited (INI) 5HT2CR's exhibit the converse pattern. To test whether 5HT2CR editing alters its function ex vivo, 125‐I DOI and 3‐H mesulergine autoradiography was performed on brain sections from mice solely expressing either the INI or VGV receptors. Similar to in vitro observations, VGV receptors exhibited decreased, whereas INI receptors exhibited increased high affinity G‐protein coupling relative to wild‐type (WT) receptors. In addition, relative to WT mice, INI expressing mice showed greater increases in plasma corticosterone following treatment with the 5HT2CR agonist, MK‐212. INI expressing mice did not show alterations in expression of glucocorticoid receptor, suggesting that changes in the stress circuit did not account for the differences in 5HT2CR agonist‐induced corticosterone release. These data show that editing of the 5HT2CR alters its function in vivo.

Supported by: NIH T32‐MH065215 (CEC) and R01‐MH34007 (ESB)
Beyond Prozac: Generation and characterization of SSRI Insensitive Transgenic Mice
Authors: Thompson Brent J, , Jessen Tammy , Henry L. Keith , Gamble Karen L. , Chisnell Peter J. , McMahon Douglas G. , Blakely Randy D.
Published: June 2021

Altered activity of the serotonin transporter (SERT) transporter has been hypothesized to contribute to major depression, anxiety, obsessive compulsive disorder, and autism. A principal component of this theory is the belief that SERTs are the primary target for selective serotonin reuptake inhibitor (SSRI)‐class antidepressants, as well as many tricyclic antidepressants (TCA's). Cross‐species analysis of SERT has identified a substitution (I172M) that, in vitro, results in a dramatic loss of affinity for cocaine, several SSRI's and TCA's with no appreciable effect on 5HT transport (Henry, L.K., et al., 2006). We have introduced the I172M substitution into the SERT locus of 129S6 mice by homologous recombination. As predicted this substitution has no impact on 5HT transport and these animals exhibit no overt behavioral phenotype. Behavioral studies revealed a reduction in responsiveness to citalopram in the tail suspension test, as well as reduced responsiveness to fluoxetine and citalopram in the forced swim test. Ex vivo analysis of forebrain synaptosomal 5HT transport from mice carrying this substitution demonstrate ~1,000 fold shift in citalopram potency, ~12 fold shift in fluoxetine potency and ~32 fold shift in cocaine potency. In contrast, paroxetine displayed less than a 2 fold shift in potency. These findings are complemented by ex vivo midbrain slice recordings of serotonergic neurons in the dorsal raphe, which show a significant shift in SSRIs induced autoinhibition of neuronal firing. Ongoing efforts aim to define the full impact of the I172M mutation on in vivo and ex vivo SERT pharmacology as well as the effect this loss of SSRI potency has on SSRI induced behavior.

Grant Funding Source

NARSAD Distinguished Investigator Award, NIDA Award (DA07390) and The Silvio Conte Center for Neuroscience Research at Vanderbilt University (MH078028)
An SNP in the Human GRK3 Gene Promoter‐Luciferase Construct Eliminates Hormone‐Induced Activation of GRK3 Transcription
Authors: Eikenburg Douglas , Salim Samina , Taneja Manish
Published: June 2021

Epinephrine (EPI) and corticotrophin releasing factor (CRF) selectively increase GRK3 expression by an ERK1/2‐dependent activation of the transcription factors Sp‐1 and Ap‐2 in two neuronal cell lines, BE(2)‐C and CATH.a cells. Moreover, a single nucleotide polymorphism (SNP; ‐382G/A) in the promoter region of the human GRK3 gene reportedly decreases GRK3 expression, and increases the risk for bipolar disorder (BPD). In this study we generated plasmids to express a WT human GRK3 promoter (1.3kb, ‐1597 to ‐297)‐luciferase reporter construct containing the predicted Sp‐1 and Ap‐2 binding sites and a second construct containing the SNP. BE(2)‐C cells and CATH.a cells were transfected to express each construct and the effects of EPI and CRF on luciferase reporter activity, respectively, were studied. EPI/CRF treatment (1‐8h) increased WT GRK3 promoter driven luciferase activity; this was prevented by the MEK1/2 inhibitor, U1026. EPI/CRF treatment (1‐8h) failed to stimulate SNP‐GRK3 promoter driven luciferase activity. These results demonstrate that our WT GRK3 promoter construct accurately models regulation of endogenous GRK3 gene expression and that the SNP in the GRK3 promoter eliminates the ability of the stress hormones EPI/CRF to stimulate ERK‐dependent GRK3 expression. This mechanism may contribute to the reduced GRK3 levels observed in cells from BPD patients with the SNP. (Supported by NARSAD)
Roles of Rab1 GTPase and dimerization in the cell surface transport of angiotensin II type 2 receptor
Authors: Zhang Xiaoping , Dupre Denis , Feng Ying‐Hong , Hebert Terence E , Wu Guangyu
Published: June 2021

The physiological function of angiotensin II (Ang II) is mediated through Ang II type 1 (AT1R) and type 2 (AT2R) receptors. Our previous studies indicate that AT1R plasma membrane expression is regulated by Rab and Sar1 GTPases and the C‐terminal F(X)6LL motif. However, the molecular mechanisms of the export trafficking of AT2R remain poorly defined. Here we report that cell‐surface expression of AT2R was markedly augmented by Rab1 and attenuated by dominant negative Rab1 mutants (S25N and N124I) and siRNA‐mediated Rab1 depletion. The Rab1 mutants also inhibited the overall expression of AT2R, but not AT1R, likely via promoting ubiquitin‐mediated degradation of ER‐accumulated AT2R. Consistently, AT2R‐mediated inhibition of EGF‐activated ERK1/2 was significantly reduced by the Rab1 mutants, indicating that endogenous Rab1 controls the cell surface targeting and signaling of AT2R. Furthermore, ER‐export deficient AT1R mutant, in which the F(X)6LL motif were mutated, formed both homodimers and heterodimers with AT2R, which blocked AT2R trafficking to the cell surface, suggesting constitutive dimerization of both receptors in the ER and an important role of dimerization in ER export. These data demonstrate that nascent AT2R transport from the ER to its functional destination is a highly regulated process and its signaling can be manipulated by modifying its biosynthesis (R01GM076167).
Elucidating the molecular mechanisms controlling GPR54 expression
Authors: DeFino Mia C. , Wacker Jennifer L. , Lyssand John S , Wang Edith , Stella Nephi , Hague Chris
Published: June 2021

Increasing evidence suggests that the G‐protein coupled receptor GPR54, together with its endogenous peptide ligand kisspeptin (KP), play pivotal roles in the initiation of puberty and reproduction. For example, previous studies report blood plasma concentration of kisspeptin increases by ~9000 fold during pregnancy, and is accompanied by an immediate return to pre‐pregnancy levels postpartum, suggesting that expression of KiSS‐1, the gene encoding KP, is tightly controlled. Thus far, studies have implicated SP1 and estrogen as important regulators of KiSS‐1 gene expression (Mitchell et al, 2007). However, no research to date has examined the mechanisms of GPR54 regulation at the DNA, RNA or protein level. Hence identification of specific factors that regulate GPR54 expression at the DNA and protein level will provide critical information about the GPR54‐KP signaling system in reproduction. In our studies, we have utilized luciferase reporter assays and inducible expression vectors to characterize the mechanisms regulating GPR54 expression, and propose that sex steroids play a critical role in this key physiological process.

Supported by NIH 5 T32 GM07750.
Erythrocyte (RBC)‐Released ATP and Vascular Control: When it Works and What if it Does Not?
Authors: Ellis Christopher G. , Goldman Daniel , Sprague Randy S. , Ford David A. , Stephenson Alan H. , Ellsworth Mary L.
Published: June 2021

Substantial evidence suggests that RBCs contribute to matching of oxygen (O2) supply with demand via release of small amounts of ATP in regions of increased O2 need, enhancing blood flow appropriately. We simulated this system using a computational model incorporating network geometry and conducted signaling to predict microvascular flow responses to changes in tissue O2. To test this innovative model, we used Zucker Diabetic Fatty (ZDF) rats at 7 weeks (pre‐diabetes; high insulin, normal glucose) and at 12 weeks (type II diabetes [DMII]; normal insulin, high glucose). In pre‐diabetes, capillary O2 supply was decreased without a decrease in capillary density. RBCs exposed to pre‐diabetic insulin levels (1 nM) did not release ATP in response to reduced O2 and isolated arterioles perfused with insulin treated RBCs did not dilate in response to low extraluminal O2. Finally, RBCs of rats and humans with DMII did not release ATP when exposed to low O2 and isolated arterioles perfused with RBCs of humans with DMII did not dilate in response to low O2. These findings in the intact microcirculation and isolated vessels are consistent with predictions of the computational model. This systems biology approach provides novel insights into the mechanisms by which O2 supply is matched with O2 demand and will enhance our understanding of the causes of peripheral vascular complications in pre‐ and type II diabetes. (NIH R33 HL089094)
Effect of erythrocyte aggregation and flow rate on temporal variation of cell‐free layer width in arterioles
Authors: Ong Peng Kai , Namgung BumSeok , Johnson Paul C , Kim Sangho
Published: June 2021

The cell‐free layer (CFL) is a dynamic feature of microcirculatory blood flow and its variability is commonly described in terms of standard deviation (SD). However, CFL width in an arteriole follows a non‐Gaussian distribution imposed by asymmetrical nature of its physical environment, which implies that more detailed statistical analyses are required to better interpret CFL characteristics. In this study, rat cremaster muscle was used to visualize arteriolar flows and erythrocyte aggregability was raised by dextran infusion to levels seen in normal human blood. The effects of aggregation and flow reduction on CFL characteristics in arterioles (ID 20‐50 μm) were investigated. A positive relation was found between mean CFL widths and their corresponding SD and was predominantly enhanced by flow reduction. Both normalized mean and SD of CFL width increased significantly at low pseudoshear rates (17.1 ± 6.1 s−1), especially in the presence of aggregation. The variability of the CFL width was further analyzed in terms of its frequency and magnitude of deviations from its mean value, extending either towards the vessel wall or into the erythrocyte core. There was no significant effect of either flow condition or aggregation on the frequency of CFL deviations, whereas the magnitude of deviations significantly increased after flow reduction and dextran infusion (P < 0.05), especially into the erythrocyte core.
Revisiting cell flux vs bulk flow: bifurcation angle and Re.
Authors: Frame Mary D , Lehman Lisa , Mencher Eric
Published: June 2021

The goal of this experiment was to determine the relationship between volumetric flow and particle flux in a large scale model of the arteriolar microcirculation. The initial hypothesis was that percentage flux (F) vs. volumetric flow (Q) to each branch would be linear, independent of Reynolds number and bifurcation angle. Particle F distribution as a function of prescribed Q distribution was tested using a large scale model where glycerol was the suspending medium and particles were made of PDMS. The relative density and particle to tube diameter ratios mimicked the arteriolar microcirculation. Reynolds number (Re, feed, 0.06‐0.2), bifurcation angle (45‐135°) and the percentage Q to each branch (10‐50%) were varied to determine the relationship between flow and flux in T‐shaped bifurcations. Examining all groups, F vs. Q was clearly non‐linear. With 10% Q to the branch, F = 0%. For Q = 33%, F = 6.8±6.1% (mean±SD) and Q = 50%, F = 38±9.1%. Both angle and Re influence flux distribution, e.g., with 90° and Q = 50%, F increases with Re (R=0.53, p<0.05). For either the 45 or 135° and Q = 50%, this is not significant (R=0.08, p>0.05). Significant effects of angle on F were seen at lower Re, for all Q, e.g., for Re=0.06 and Q = 33%, F decreases with increasing angle (R=‐0.057, p<0.05). For higher Re, this was not significant (R=0.02, p>0.05). Thus, part of the non‐linear behavior between F and Q is related to angle and Re. NIH HL55492 AHA 0655908T.
Differential actions of Sphingosine‐1 Phosphate (S1P) receptors in the regulation of microvessel permeability in rat mesenteric venules
Authors: Zhang gengqian , Wang Qian , He Pingnian
Published: June 2021

Our previous study demonstrated that preperfusing microvessels with S1P prevented platelet‐activating factor (PAF)‐induced endothelial gap formation and the increases in hydraulic conductivity, Lp. In vitro studies indicated that three different S1P receptors (S1PRs) existed in vascular endothelium, and play different roles in regulating endothelial functions. Our present study aims to identify the S1PRs responsible for the protective effect of S1P on PAF‐stimulated microvessels. Lp was measured in individually perfused rat mesenteric venules. Endothelial gap formation was examined three‐dimensionally with confocal microscopy using fluorescence microspheres (100 nm) as markers. The roles of S1PRs in the S1P effect were investigated using S1PR1 and S1PR3 antagonist, VPC23019 and S1PR2 antagonist, JTE‐013. Neither of the antagonist alone changed baseline Lp. Preperfusing vessels with VPC23019 (10 μM) for 30 min following 20 min perfusion of S1P (1 μM) plus VPC23019 abolished the inhibitory effect of S1P on PAF‐induced endothelial gap formation and the Lp increases. The magnitude of endothelial gap formation and the peak increases in Lp were similar to those obtained in PAF‐stimulated vessels in the absence of S1P. In contrast, preperfusing microvessels with JTE‐013 (10 μM) for 30 min showed no effect on the inhibitory effect of S1P on PAF stimulated vessels. Our results indicated that S1PR1 and/or 3, not S1PR2, are responsible for the protective role of S1P in permeability of intact microvessels by preventing the formation of endothelial gaps. Supported by HL56237 and HL084338.
Vascular dysfunction in transgenic mice expressing a PPARγ interfering mutation specifically in vascular muscle: role of aortic rho kinase
Authors: Ketsawatsomkron Pimonrat , de Lange Willem J. , Pelham Christopher J. , Halabi Carmen M. , Faraci Frank M. , Sigmund Curt D.
Published: June 2021

PPARγ is a ligand activated transcription factor expressed in vascular muscle and endothelium. We previously reported that targeted expression of a dominant negative PPARγ mutation in vascular muscle (S‐P467L) of transgenic mice caused severe aortic dysfunction. This included impaired responses to acetylcholine (ACh), sodium nitroprusside (SNP) and cGMP. Using a pressurized myograph preparation in vitro we now show that the mesenteric artery (~190μm) exhibits impaired relaxation to 1 μM ACh (22±6% S‐P467 vs 74±5% WT), but a normal response to 10 μM SNP (89±9% S‐P467L vs 79±11% WT), suggesting the mechanisms regulating vascular function by PPARγ may differ among vascular beds. We also reported that aorta from S‐P467L mice exhibited augmented contraction to ET‐1, which was rho kinase dependent. NO is thought to be an endogenous inhibitor of rho kinase. We therefore tested the hypothesis that inhibition of rho kinase may restore the response to NO in thoracic aorta from S‐P467L mice. Vehicle‐treated S‐P467L aorta exhibited impaired relaxation to ACh (5±3% vs 71±1% in WT). Y27632 (3 μM) caused improved relaxation to ACh (40±3%) in aorta from S‐P467L, but had no effect in aorta from WT. A similar improvement in relaxation to SNP was also observed (75±6% Y27632 vs 38±5% vehicle) in S‐P467L. These data implicate increased activity of the rho kinase pathway in mediating impaired responses to NO in the aorta of S‐P467L mice.
Pharmacological vasodilation improves the anabolic response of muscle protein synthesis to insulin in elders
Authors: Lee Jessica L. , Timmerman Kyle L. , Fujita Satoshi , Dreyer Hans C. , Fry Christopher S. , Dhanani Shaheen , Rasmussen Blake B. , Volpi Elena
Published: June 2021

Loss of muscle with aging (sarcopenia) contributes to disability in elders. Aging is associated with an insulin resistance of muscle protein anabolism, which correlates with impaired insulin‐induced muscle vasodilation and nutrient delivery.

We hypothesized that pharmacological restoration of muscle perfusion to youthful values during hyperinsulinemia would improve the response of muscle protein synthesis and anabolism in older subjects.

We measured blood flow (BF), anabolic signaling, and muscle protein synthesis (Rd) and breakdown (Ra) with stable isotope tracers at baseline and during insulin infusion in one leg in 12 healthy, non‐diabetic older subjects (71 ± 2 yrs) divided in two groups: CTRL (insulin only) and SNP (insulin + sodium nitroprusside to double blood flow above baseline).

Blood flow and phenylalanine delivery increased significantly (P<0.05) in SNP only. Muscle protein synthesis (nmol·min−1 ·100mL leg−1) increased in SNP (35 ± 4 to 102 ± 26), but not in CTRL (34 ± 10 to 45 ± 10), while breakdown did not change in either group. Akt/PKB phosphorylation increased in both groups but to a larger extent in SNP (P<0.05) and S6K1 phosphorylation tended to increase more in SNP (P = 0.068).

In conclusion, pharmacological restoration of muscle perfusion and nutrient delivery during hyperinsulinemia increased muscle protein synthesis in elders.

Funded by NIH R01 AG018311 and P30 AG024832
Exercise effectiveness for atrophic muscle in estrogen (E2) replete and ovariectomized (OVX) female rats
Authors: Ferreira J Andries , Hemmann Kaitlyn M , Brown Marybeth
Published: June 2021

Studies suggest that women who are E2 deficient do not respond to exercise as well as women who are E2 replete.

PURPOSE

determine if differential recovery occurs in atrophic skeletal muscle in OVX rats, with and without E2 and with and without exercise.

METHODS

78 female rats age 5 mos were randomly placed into 6 groups: 1) control, 2) Hindlimb unweighted 4 weeks (HLU), 3) recovery no E2, 4) recovery + E2, 5) exercise no E2, 6) exercise + E2. Recovery groups cage recovered or were exercised daily for 14 days after HLU.

RESULTS

HLU resulted in significant atrophy and loss of muscle force (p<0.05) in soleus, plantaris, gastrocnemius, tibialis anterior. Return of muscle mass toward control levels occurred in both cage ambulation but not both exercise groups. Exercise + E2 was more effective than exercise without E2. Representative muscle mass/body mass ratio data and Po are given for gastrocnemius (mean ± sem).

Exercise was effective for E2 supplemented rats but not for rats that were E2 deficient. Exercise without E2 rats had histological evidence of damage (central nuclei).

CONCLUSIONS

Exercise + E2 appears to be the most effective stimulus for the recovery of atrophic muscle, whereas exercise without E2 may be detrimental.

Support: NIH HD058834, Spinal Injuries Research Board, Research Council at Univ. of Missouri
Gastric leptin plays a role in sympathetic vasomotor control via a CCK1 receptor‐dependent mechanism
Authors: Sartor Daniela Maria , Verberne Anthony JM
Published: June 2021

Gastric‐derived leptin acts on vagal afferents and interacts with the gastrointestinal hormone cholecystokinin (CCK). This study aimed to examine the significance of these signals in cardiovascular regulation. The effect of CCK (4 μg/kg, i.v.) and close arterial (c.a.; coeliac) infusion of leptin (15 μg/kg) on (a) arterial pressure (AP) and heart rate (HR), (b) firing rate (FR) of presympathetic vasomotor rostroventrolateral medullary (RVLM) neurons, and (c) splanchnic/lumbar sympathetic nerve discharge (SSND/ LSND), was examined in anesthetised, male SD rats. Within 5 minutes, c.a. leptin reduced the FR of CCK‐inhibited RVLM neurons (‐28 + 5 %; P < 0.01; n=10) but not CCK‐insensitive/activated neurons (8 + 6 %; n=6), compared to saline controls (‐1 + 2 %; n=11); i.v. leptin did not affect CCK‐inhibited neurons (2 + 4 %; n=5). Leptin (c.a.) significantly decreased AP (‐13 + 3 mmHg; P < 0.05; n=16) compared to c.a. saline (‐4 + 1 mmHg; n=11) and i.v. leptin (3 + 2 mmHg; n=5) without affecting HR. The CCK1 receptor antagonist lorglumide (10 mg/kg; i.v.) abolished leptin's inhibitory effect on RVLM neurons (n=3, P < 0.05). In separate experiments c.a. leptin inhibited SSND (‐9 + 2 %; P < 0.05; n=7) compared to c.a. saline (2 + 3 %; n=5); LSND was unaffected. We conclude that leptin acting within the gut may exert selective sympathetic effects via a CCK1 receptor‐dependent mechanism that may involve the release of CCK.
Role of nicotinic receptors on carotid body function in developing rats.
Authors: Niane Lalah , Joseph Vincent , Bairam Aida
Published: June 2021

Acetylcholine is an excitatory neurotransmitter in adult carotid body playing a significant role in the ventilatory response to hypoxia. We used epibatidine (Epi, 5μg/Kg i.p) as agonist and hexamethonium (Hexa, 1mg/kg i.p) as antagonist of peripheral nicotinic receptors to determine the function of nicotinic receptors in developing rats. Breathing frequency (Fr), tidal volume (VT) and minute ventilation (VE) were measured using whole body plethysmography in normoxia (21%O2) and in response to hypoxia (20 min‐12%O2) in rat pups at 1, 4, 7, 12 and 21 postnatal days before and after a single injection of Epi or Hexa. In normoxia, Epi increased significantly Fr by 4%, 21%, 30%, 40%, and 43% for P1, P4, P7, P12 and P21 respectively, p for age<0.001. In hypoxia, Epi increased Fr by 15% for P4 and 30% for P12 while Hexa decreased Fr by 13% and 31% respectively, p for age<0.01. We conclude that the functional expression of cholinergic nicotinic receptor on hypoxic ventilatory response is age‐dependent, thus presumably playing a significant role in the maturation of the carotid body response to hypoxia. Founded PF‐RSR, IRSC and Sick Kids Foundation.