bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of AT1, AT2 receptors, and a non‐AT1, non‐AT2 angiotensin II binding site in rat brain after endothelin‐1 induced ischemic stroke
Authors: Gonzalez Luz , Regenhardt Robert W , Haniff Aisha , Sumners Colin , Speth Robert C
Published: June 2021

New findings suggest that the activation of brain AT1 receptors decreases cerebral perfusion after an ischemic stroke while activation of brain AT2 receptors opposes those actions providing neuroprotective effects. Recent discoveries reveal the existence of a novel non‐AT1, non‐AT2 binding site for angiotensin II (Ang II) in the brain which may indicate additional effects of the brain angiotensin system after ischemic damage. To assess this, 5 rats were microinjected with 3 μl of 80 μM endothelin‐1 (ET‐1) to simulate middle cerebral artery occlusion in the right hemisphere of the forebrain. 24 hours later, the rats were sacrificed and the brains were removed and frozen. The brains were analyzed by observing the binding of 125I‐Sar1 Ile8 Ang II to the tissue by receptor autoradiography. Using quantitative densitometric analysis of the 125I‐Sar1 Ile8 Ang II binding (MCID) to the forebrain caudal to the ischemic region of the brain, no differences in AT1, AT2 or non‐AT1, non‐AT2 binding was observed between the hemispheres in either the density of the receptor binding or the area encompassed by each hemisphere. The results suggest that the ischemia does not alter the expression of angiotensin binding proteins in the region posterior to the stroke‐zone at 24 hours post‐ischemia. Supported by NHLBI HL‐096357 and American Heart Assoc.
Functional analysis of novel CLCNKB mutations in patients with Bartter syndrome
Authors: Keck Mathilde , L'Hoste Sébastien , Genete Mathieu , Grand Teddy , Vargas‐Poussou Rosa , Blanchard Anne , Lourdel Stéphane , Teulon Jacques
Published: June 2021

Bartter's syndrome is a hereditary renal tubulopathy characterized by salt‐wasting and hypokalaemic metabolic alkalosis, which can be due to loss‐of‐function mutations in genes encoding ClC‐Kb / Barttin chloride channel. Here, we investigated 7 novel CLCNKB mutations (L81P, V170M, G246R, R351P, G424R, R438H and L439P) and the A204T mutation (Waldegger et al. J. Biol. Chem., 2000, 275: 24527–33).

We studied WT and ClC‐Kb mutants in Xenopus laevis oocytes using two‐electrode voltage‐clamp for measuring ClC‐Kb currents and chemiluminescence for evaluating surface expression. Cellular expression was investigated in HEK cells using western‐blots. For the two latter purposes, we designed a ClC‐Kb construct carrying an extracellular FLAG epitope that does not alter ClC‐Kb currents.

No significant current was recorded with G246R, G424R, R438H and L439P mutants. L81P, V170M, A204T and R351P mutants produced 35–70% lower currents as compared to wild‐type. Accordingly, chemiluminescence showed that G246R, G424R, R438H and L439P were not addressed to the plasma membrane while surface expression of L81P, V170M, A204T and R351P was decreased by 30–85%. These results were confirmed by western‐blot on HEK cells, which indicated a defect in membrane targeting for all mutants.

We conclude that all these CLCNKB mutations are pathogenic and considerably alter membrane targeting of the channel.
High efficient transduction of renal tubular cells by Tyrosine‐mutant AAV2 sextuple vector
Authors: Qi Yanfei , Li QiuHong , Shenoy Vinayak , Jun JooYun , Verma Amrisha , Katovich Michael J , Raizada Mohan K
Published: June 2021

Gene therapy has distinct potential to treat kidney diseases; however efficient transduction of significant number of cells in the renal tissue by viral vectors has been difficult to accomplish. Previous studies have indicated that different serotypes of adeno‐associated virus (AAV) can transduce renal cells with variable and suboptimal efficiency. The availability of new and innovative mutants of AAV has led us to compare their efficacy in transducing rat kidney. Five types of AAV (AAV2 muttriple, AAV2 sextuple, AAV8 mut44, AAV8 mut733, and AAV9 mut446) carrying an enhanced green fluorescence protein (EGFP) reporter gene under the control of small chicken β‐actin (AAV‐CBA‐EGFP) were compared, which contain single or multiple point mutations in surface‐exposed capsid tyrosine residues. 1.5x1011 vector genome (vg) of AAV in 20μl was directly injected into the renal cortex at three different sites using a pressure microinjection technique. Three weeks after injection, the kidneys were collected and evaluated for EGFP expression. Among the various mutated AAV serotypes studied, only AAV2 sextuple showed a robust EGFP expression in significant renal tissue. About 45% of the renal tubular epithelial cells expressed EGFP. Our study shows that mutated AAV2 sextuple is an efficient gene transfer vector for renal tissue and may hold future therapeutic applications to treat kidney diseases.
AQP2 is Necessary for Vasopressin Mediated Filamentous Actin Depolymerization in Renal Epithelial Cells
Authors: Yui Naofumi , Lu Hua A. J. , Bouley Richard , Brown Dennis
Published: June 2021

Filamentous actin (F‐actin) depolymerization is critical for vasopressin mediated AQP2 membrane accumulation. In this study, we compared the F‐actin polymerization state in MDCK and LLC‐PK1 cells with or without AQP2 transfection using a rhodamine‐phalloidin assay. In AQP2 expressing cells, the F‐actin level was significantly decreased 10% by vasopressin and forskolin (VP/FK); from 1.00 ± 0.02 to 0.90 ± 0.02 (n = 5, P < 0.001) at 60 min in AQP2 MDCK cells, and from 1.00±0.02 to 0.90±0.04 (n = 5, P < 0.001) at 20 min in AQP2 LLC‐PK1 cells. However, the F‐actin decrease induced by VP/FK was not observed in untransfected cells; from 1.00 ± 0.02 to 0.99 ± 0.03 (n = 5, P = 0.53) in MDCK cells, from 1.00±0.03 to 0.99±0.04 (n = 5, P = 0.59) in LLC‐PK1 cells. Furthermore, the extent of F‐actin depolymerization by VP/FK in AQP2 expressing cells was correlated with the AQP2 expression level. These results show that vasopressin mediated F‐actin depolymerization requires AQP2. This function of AQP2 probably involves interaction with regulatory components of the actin cytoskeleton, including, but not limited to, tropomyosin‐5b (Noda, Sasaki et al) and ERM proteins (Tamma, Valenti et al).
What do dietary K+ restriction and pregnancy share in common?
Authors: Crambert Gilles , Salhi Amel , Edwards Aurélie
Published: June 2021

Modern dietary habits are characterized by high‐Na+ and low‐K+ intakes, each of which has been correlated with a higher risk for hypertension. In this study, we examined whether long‐term variations in the intake of Na+ and K+ induce lasting changes in the plasma concentration of circulating steroids. We first developed a mathematical model of steroidogenesis in mice that predicted that mice increase their plasma progesterone levels specifically in response to K+ depletion. This prediction was confirmed by experimental measurements in both male mice and men. Further investigations revealed that progesterone regulates renal K+ handling by stimulating the expression of H,K‐ATPase type 2 (HKA2) under K+ restriction conditions. This new pathway linking progesterone, HKA2 expression and K+ retention was then investigated in the context of pregnancy, a condition in which progesterone is elevated and K+ retained. We showed that, during the late part of gestation in mice, the decrease in urinary K+ excretion is accompanied by overexpression of HKA2. Conversely, HKA2 knock‐out mice are unable to efficiently retain K+ during gestation. This occurs in parallel with a higher mortality rate during gestation in this strain compared to wild‐type mice. All together, our results suggest the existence of a hereto unknown regulatory process involving progesterone, its nuclear receptor, the HKA2 and renal K+ retention.
Morphological variability of the vascularisation of the polar renal parenchyma. A study on corrosion casts and dissections
Authors: Zahoi Delia Elena , Daescu Ecaterina , Enache Alexandra , Barsasteanu Florin , Pusztai Agneta Maria
Published: June 2021

A comparative analysis of the origin and number of the arterial sources of the two poles of the renal artery was performed on 100 corrosion pieces and 50 dissection pieces. The renal artery trunk developed two branches (anterior and posterior) in 86.7% of cases, three branches (anterior, posterior and superior/inferior) in 10.7% of cases, and four branches (anterior, posterior, superior and inferior) in 2.6% of cases. In 78% of cases, the superior pole had only one arterial source originating from: the anterior branch (54%), the posterior branch (14.67%) or the superior branch (9.33%). In 22% of cases, it had two arterial sources arising from the anterior and posterior branches (18%), the superior and posterior or the posterior and superior branches in equal share (2%). In 88.67% of cases, the inferior pole was supplied only by one segmental artery arising from the anterior branch (82.67%), the posterior branch (4%) and the inferior branch (2%). In 11.33% of cases, two arterial sources of the inferior pole were revealed: anterior and posterior. The study reveals the greater variability of the vascular sources of the superior pole. The anterior branch was the main source of both poles. When the inferior pole was supplied by the inferior or posterior branch, these were sole arterial sources. Such morphological aspects are useful in renal vascular surgery.
Mitochondrial gene variant contributing to coronary artery disease
Authors: Almontashiri Naif A. , Teng Allen C.T. , Chen Hsiao‐Huei , Cheng Brian L.M. , Alam Mohammad A. , Ta Matthew , Roberts Robert , Langer Thomas , McBride Heidi M. , Stewart Alexandre F.R.
Published: June 2021

Genome‐wide association studies (GWAS) have identified genetic variants that are associated with the risk for coronary artery disease (CAD), but the functional significance of these loci has remained obscure. We identified a genetic variant associated with CAD risk that changes the sequence of a mitochondrial protease called paraplegin (SPG7) and confirmed this association by meta‐analysis of 14 GWAS including 22,000 CAD cases and 60,000 controls. The variant changes an arginine residue at position 688 to a glutamine residue in the protease domain of SPG7. Most mutations in SPG7 cause a loss of protease function leading to spastic paraplegia. Although the Gln688 variant was considered benign in terms of spastic paraplegia, we have evidence that it is in fact a protease gain‐of‐function variant. We observed a strong correlation between the presence of the variant allele and the level of mature active SPG7 in peripheral lymphocytes, as well in primary cultures of human aortic smooth muscles. Consistent with increased SPG7 protease activity, increased production of cytochrome c oxidase subunits, ATP synthesis, cell proliferation and ROS production were seen in cells stably expressing this variant. Our data suggest that this gain of function may also be detrimental to mitochondrial function with aging. Our study is the first to link a mitochondrial matrix AAA protease variant to the risk of CAD.

(Supported by CIHR)
Allogenic Demineralized Dentin Matrix, An Ideal Bone Graft
Authors: Bakhshalian Neema , Hooshmand Shirin , Campbell Sara Chelland , Chai Sheau Ching , Arjmandi Bahram H
Published: June 2021

The objective of this study was to evaluate the osteopromotive property of allogenic demineralized dentin matrix. There are different kinds of grafts and biomaterials being used for bone regeneration that are neither cost effective nor free of complications. Therefore, it would be ideal to utilize teeth that are extracted from other individuals (due to orthodontic treatments or wisdom teeth extractions, which are routinely discarded) to make an appropriate bone regenerative material, allogenic DDM. We hypothesized that allogenic DDM has bone regenerative properties. To test this hypothesis 30 New Zealand White rabbits were used. The rabbits were divided into four groups and sacrificed after 15, 30, 60, and 90 days. The thickness of bone specimens were measured before utilizing for micro‐CT. Blood samples were collected from all rabbits at each time point. Average bone thickness was increased by 54% in experimental group and was significantly higher at all time points. Moreover, microstructural properties showed superior bone quality in experimental group. White blood cell count was numerically higher in control rabbits compared to experimental rabbits ruling out any inflammation or infection due to allogenic DDM. The results of the present study indicate that allogenic DDM significantly improves both bone quantity and quality through its osteoinductive and osteoconductive properties.
The influence of dietary lipids on mitochondrial hydrogen peroxide production in skeletal muscle of calorie restricted mice
Authors: Chen Yana , Hagopian Kevork , Ramsey Jon
Published: June 2021

Calorie restriction (CR) has been shown to reduce reactive oxygen species (ROS) production, attenuate oxidative damage and retard aging in a variety of species. Interestingly, CR has been reported to alter membrane composition towards saturation. This has led to a theory that changes in membrane saturation are central to the actions of CR. As a first step towards testing this theory, C57BL/6 mice were assigned to 4 dietary groups (control and three CR groups) and fed AIN‐93G diets at either 95% (control) or 60% (CR) of ad libitum. To manipulate membrane composition, the primary dietary fats for the CR groups were soybean oil (also used in the control diet), fish oil or lard. The diets were fed for 1 month and then mitochondria from hindlimb skeletal muscle were isolated for measures of mitochondrial lipid composition, proton leak, and H2O2 production. Results indicate that changes in mitochondrial lipids in the CR mice reflected the dietary lipid sources. Mitochondrial H2O2 production was decreased (P<0.02) in all three CR groups compared to the control group regardless of the substrate used for respiration. However, there was no evidence of difference when comparisons were between CR groups. The results of this study indicate that an increased degree of saturation in mitochondrial membranes is not required for reduced ROS production with CR in muscle. (Supported by NIH RO1 AG028125)
Morphological considerations regarding renal microvascularisation
Authors: Daescu Ecaterina , Enache Alexandra , Zahoi Delia Elena , Taban Sorina , Alexa Aurora , Motoc Andrei
Published: June 2021

The study is an immunohistochemical evaluation of the microvascularisation of the human kidney. The study group consisted of 45 post‐autopsy human renal pieces from adults with no renal affections. The common usual histopathological processing and the HE, PAS and Gomori trichrome staining allowed the morphological evaluation of the preparations. Immunohistochemical staining with anti‐CD31 and anti‐CD34 monoclonal antibodies were performed for all the cases included in the study. Vasa vasorum (VV) were revealed in interlobe, arcuate and interlobular vessels, and perivascular capillary luxuriant plexuses were identified mainly at the arterial adventitial level (arterial vasorum plexuses). Larger veins have internal VV that originate from the venous luminal area, have a centrifugal course, are directed to the periadventitial tissues and are apparently connected to microvascular structures derived from the peritubular capillary network. External microvascular elements and the aspect of the capillaries suggest vasorum neovascularisation at vein level. The presence of muscle arterioles and endothelial tubes disposed symmetrically to venous lumina can give birth to extrinsic vascular sphincters, which can slow down the venous flow and reduce protection of the renal parenchyma against ischemia. The degree of VV in the media and intima of an artery correlates with the risk of ischemic lesion.
Use of Angle‐Resolved Surface Plasmon Resonance Imaging (SPRi) for the Characterization of Protein Binding Dynamics
Authors: Golden Mary S. , Whitty Adrian , Georgiadis Rosina M.
Published: June 2021

Protein‐protein interactions are essential at almost every level of cellular function. Although structures of various protein complexes have been characterized, the mechanisms involved in protein binding events and the transient dynamics of complex formation are not fully understood. Here we present an improved angle‐resolved surface plasmon resonance imaging (SPRi) methodology that allows real time multi‐array kinetic and thermodynamic analysis of protein binding events on surfaces. Using Tumor Necrosis Factor alpha (TNFα), a homotrimeric protein, as a test system we have developed surface fabrication techniques including multi‐channel microfluidic delivery systems and patterning capabilities, and have employed various surface immobilization strategies to investigate the influences of density, orientation, and heterogeneity of surface immobilized TNFα on protein binding efficiency and kinetics. A unique multi‐wavelength SPRi approach is utilized to simultaneously determine dielectric constants and thicknesses of TNFα layers, allowing characterization of the oligomeric structure of the immobilized trimer. Results showed that TNFα activity for known binding partner TNFR2, as well as TNFα subunit dissociation dynamics, were highly dependent on the selected immobilization conditions. This work was supported by NIGMS and Boston University, Department of Chemistry.
Effect of protein blend vs whey protein ingestion on muscle protein synthesis following resistance exercise
Authors: Reidy Paul , Walker Dillon K , Dickinson Jared M , Gundermann David M , Drummond Micah J , Timmerman Kyle L , Fry Chris S , Cope Mark , Mukherkea Ratna , Volpi Elena , Rasmussen Blake B
Published: June 2021

Resistance exercise followed by the ingestion of amino acids or protein causes an additive stimulation of muscle protein synthesis (MPS). Proteins such as whey are digested at a fast rate whereas soy and casein are digested at an intermediate and slow rate, respectively. To determine if ingestion of a protein blend (soy and dairy protein) extends the post‐exercise MPS response compared to whey protein we conducted a double‐blind, randomized clinical trial in 20 young adults before and after ingestion of ~20g of the protein blend or whey (N=10 per group) given 1h after a bout of high‐intensity leg resistance exercise. We utilized stable isotopic methods to measure the mixed muscle protein fractional synthetic rate (FSR) and western blotting to examine mTORC1 signaling. Muscle biopsies (vastus lateralis) were collected at baseline and at two time points during post‐exercise (Early: 1–3h & Late: 3–5h post‐ex). We report differences between the two beverages as Protein A and B (data will be unblinded at the EB meeting). FSR increased by ~40–70% in both groups during Early and remained elevated into Late. Protein A and B also activated mTORC1 signaling to a similar extent following exercise, yet Protein B tended to be greater. We conclude, from these preliminary data that following resistance exercise, the ingestion of a protein blend enhances mTORC1 signaling and MPS similarly to that of whey protein.

Grant Funding Source : Solae LLC
Challenges and progress towards universal salt iodization (USI) in Ethiopia
Authors: G/Egziabher Tafere , Girma Meron
Published: June 2021

The challenges and progress towards USI in Ethiopia were assessed in December, 2010. A qualitative study was conducted among ten key organizations and comments from the key informants are summarized. Salt iodization was first started in1998 and 80% of salt in Ethiopia was iodized. But due to the Ethio‐Eritrean war iodization was not maintained. To meet the needs of the Ethiopian population salt was imported for a short time but was replaced with local production of non‐iodized salt due to the high cost of importation. Local production of non‐iodized salt accounts for 90–95% of the salt supply in the country. Salt iodization was then revitalized in 2004. Accordingly small scale salt iodization was started in Afar region. Although commitment to solve the problem was stronger, lack of technical skills, harsh environmental conditions and instability of the region prevented large scale salt iodization. Hence increased levels of iodine deficiency were reported in a 2005 national survey. At the time of the study 3–5% of salt in the country was iodized (ICCIDD/UNICEF Standards) and the maximum iodization capacity could only meet 30–40% of salt needed by the country. In March, 2011, Ethiopia passed a law that prohibits processing, importing, and sale of non‐iodized salt for human consumption in Ethiopia. Although this is an important step in the right direction, more efforts are needed to successfully implement USI to combat IDD in Ethiopia. (Supported by Hawassa University, Ethiopia)
Dietary spray‐dried plasma attenuates inflammation caused by transport stress and increases pregnancy rate of mated female mice
Authors: Song Minho , Liu Yanhong , Lee Jeonjae , Che Tung M. , Soares-Almeida Juliana A. , Campbell Joy M. , Polo Javier , Crenshaw Joe D. , Chun Ju Lan , Seo Seongwon , Pettigrew James E.
Published: June 2021

Previous studies indicated that prolonged transport stress immediately after mating resulted in a low pregnancy rate (10%). Dietary inclusion of 8% spray‐dried plasma (SDP) consistently and markedly increased the pregnancy rate to 49%, and 1% SDP increased it to 35%. We hypothesized that SDP increases pregnancy rate of mated female mice by attenuating inflammation caused by transport stress. Mice (C57BL/6 strain) were shipped from Bar Harbor, ME to Urbana, IL on the day the vaginal plug was found [gestation day (GD) 1], arriving at the laboratory in IL on GD 3. Mice were randomly assigned to diets [0 (CON), 1, or 8% SDP]. Randomly selected mice were euthanized each day from GD 3 to 7 to collect blood and uterus to measure cytokines, Creactive protein (CRP), and cortisol by ELISA and to check pregnancy by inspection. Indicators of inflammation and stress were greatest soon after arrival on GD 3 or 4 (time, P < 0.05). SDP decreased (P < 0.05) uterine TNF‐α (45–49%) and IFN‐γ (18‐ 20%), and serum TNF‐α (27–41%), CRP (17–19%), and cortisol (8‐ 12%), compared with CON, but increased (P < 0.05) uterine TGF‐ β1 (15–16%). SDP markedly increased (P < 0.05) pregnancy rate (176–190%) compared with CON. No difference was found between 1% and 8% SDP. These data support the hypothesis that SDP increases pregnancy rate of mated female mice under these conditions by attenuating inflammation.

Grant Funding Source: APC, Inc., Ankeny, IA
A global partnership to improve nutrition medicine in Vietnam
Authors: Lenders Carine M , Henry Elizabeth ,   AFINS group
Published: June 2021

Background

Although the nutrition status in Vietnam has improved dramatically this past decade, malnutrition is still prevalent and studies in the hospital setting are lacking. The Vietnamese government recently urged hospitals to start addressing nutrition.

Objective

To create a partnership between Boston University (BU), Bach Mai Hospital (BMH), the National Institute of Nutrition (NIN), and the Hanoi Medical University (HMU) to improve patient outcomes and health services in Vietnam.

Intervention

After an in‐depth assessment, the AFINS partners have developed a hospital‐centered integrative model of nutrition medicine.

Results

We found that the rate of acute undernutrition in a general hospital ranges between 30–45% based on age while overnutrition is rare. With support from AFINS partners, the academic and hospital leadership is creating new training opportunities and systems applicable to the Vietnamese context.

Conclusions

Using government guidelines and feedback, AFINS partners plan to develop strategies to further evaluate, sustain, and disseminate this model. Support from the Abbott Fund.
The behavior of mitochondria
Authors: Nunnari Jodi
Published: June 2021

Mitochondria are double membrane‐bounded organelles that perform a myriad of diverse and essential functions in cells. These functions are dependent on the collective intracellular behavior of the organelle. We have characterized key features of mitochondrial behaviors. Our work has addressed the physiological functions and mechanisms of mitochondrial division and fusion, which are important determinants of overall mitochondrial shape and distribution. We have uncovered contact sites that intimately link mitochondria with the ER and determined their roles in mitochondrial positioning and dynamics. We have also addressed the fundamental question of how mitochondrial membranes are sub‐compartmentalized to reveal how the complex internal architecture of the organelle is generated. One of our current challenges is to determine how mitochondrial behaviors are integrated with one another and physiologically regulated within cells and organisms.
Antagonism of the hypothermic effects of nicotinic receptor ligands in mice
Authors: Rodriguez Jesse , Cunningham Colin , Carroll F Ivy , McMahon Lance Richard
Published: June 2021

This study examined receptor mechanisms responsible for the hypothermic effects of smoking cessation aids (nicotine, cytisine, and varenicline) and three epibatidine analogs (RTI 36, RTI 76, and RTI 102) in male C57BL/6J mice. The rank order potency of drugs for producing hypothermia was RTI 36>nicotine>RTI 102=varenicline=cytisine>RTI 76. The maximum effect was similar among drugs, although RTI 36 produced somewhat greater hypothermia (−10 degrees C) than nicotine (−7.4 degrees C) and the other drugs. Mecamylamine (3.2 mg/kg) and dihydro‐β‐erythroidine (DHβE; 3.2 mg/kg) did not significantly alter temperature, but antagonized the hypothermic effects of RTI 36, nicotine, varenicline, and RTI 102. Mecamylamine produced greater antagonism than DHβE. Mecamylamine and DHβE produced little or no antagonism of the hypothermic effects of cytisine and RTI 76. To the extent that DHβE is more selective than mecamylamine for α4β2 receptors, these results suggest that hypothermic effects are mediated by α4β2 receptors and other nicotinic receptor subtypes. There are marked differences in the receptor mechanisms that mediate some of the in vivo effects of nicotine versus other smoking cessation aids (cytisine) and novel drugs (RTI 76), especially with regard to nicotinic (mecamylamine‐sensitive) versus non‐nicotinic mechanisms.
Pharmacological Actions of L‐cysteine on Isolated Bovine Irides
Authors: Stroud Destinee Denea , Kulkarni Madhura , Njie-Mbye Ya Fatou , Opere Catherine A. , Ohia Sunny E.
Published: June 2021

Purpose

To investigate the pharmacological actions of L‐cysteine, a substrate for hydrogen sulfide (H2S) production on isolated bovine irides.

Methods

Isolated bovine iris muscle strips were set up in organ baths containing oxygenated Krebs buffer (pH 7.4) at 37□ C. Changes in isometric tension were recorded via grass FT03 Force‐displacement transducers and analyzed using PolyView software. The relaxant action of L‐cysteine on basal and carbachol‐induced tone was studied in the presence or absence of enzyme inhibitors of the biosynthetic pathways for H2S and prostanoids.

Results

L‐cysteine (0.1 nM –100 μM) elicited a concentration‐dependent relaxation of basal and carbachol‐induced tone in bovine irides. In contrast, L‐cysteine had no relaxant effect on basal tone in porcine irides. Inhibition of cyclooxygenase with flurbiprofen (3 μM) had no significant effect on relaxation induced by L‐cysteine. The inhibitor of cystathionine β‐synthase, aminooxyacetic acid (30 μM) significantly (p<0.02) reduced the relaxation induced by L‐cysteine.

Conclusion

L‐cysteine can relax isolated bovine irides, an effect that is dependent on intramural biosynthesis of H2S. Prostanoids are not involved on this relaxant effect. Bovine irides appear to be more sensitive to the inhibitory action of L‐cysteine than its porcine counterpart, indicating that species differences exist in the response to this substrate.
Contrasting the application process and daily life of MD, MD/PhD, and PhD career tracks
Authors: Gutierrez-Hartmann Arthur
Published: June 2021

Almost 60% of college matriculants who plan to pursue a STEM (science, technology, engineering and math) major end up switching to other degrees or not graduating at all. But for those persistent enough to complete their undergraduate STEM degree, then the choice of the next career step becomes critical. Every fall, tens of thousands of college seniors and college graduates apply for a coveted 19,230 medical school slots, 610 MD/PhD slots, and ~8,000 biomedical PhD graduate school slots. While a strong academic performance, national test scores, and leadership/extra‐curricular activities help identify the top candidates, each of these career tracks require a previous substantive “internship”‐type experience and outstanding letters of recommendation. Moreover, the character traits that might best predict success are not measured by any of the above criteria. Thus, applicants are faced with making the optimal career choice between these highly related fields, and Programs are faced with identifying the very best candidates destined for the most productive and highest impact careers. In this presentation, I will contrast the application process and the daily life of the MD, MD/PhD, and PhD career tracks, with the goal to help clarify the best path for any individual considering these diverse, but related career trajectories.
Antioxidant, anti‐glycation and ROS scavenging activities of Plantago asiatica
Authors: Nam Mi-Hyun , Hong Chung-Oui , Son Da-Hee , Oh Jun-Gu , Lee Kwang-Won
Published: June 2021

Plantago asiatica (PA) is widely distributed in Korea, China and Japan. It has traditionally been used as a folk remedy for various health benefits such as cough treatment and liver protection. The objective of this study was to demonstrate the reduction of excessive intracellular reactive oxygen species (ROS) through antioxidants and the anti‐glycation activity of PA. Dried roots of PA were boiled in ethanol for three hours, evaporated and lyophilized with a freeze‐dryer. The freeze‐dried powder of PAE was dissolved in distilled water and the solution was loaded onto a column packed with Diaion HP‐20 resin, which had been equilibrated with distilled water. The column was eluted with 2 L of 25% ethanol. Twenty‐five percent ethanol‐containing fraction (PAE‐D) was collected, and then concentrated using rotary vacuum evaporator. The concentrate was freeze‐dried. The total polyphenol content, flavonoid content, FRAP value, and DPPH‐ SC50 of PAED were 231.7 μg gallic acid equivalents/mg dry matter (DM), 211.7 μg quercetin equivalents/mg DM, 3.23 mmol FeSO4/mg DM and 282.0 μg DM/mL, respectively. The effects of the PAE‐D on AGE fluorescence were dose‐dependent, reaching 71% inhibition at 1 mg/mL of extract. In addition ROS scavenging assay were performed to investigate the possibility of PAE‐D in improving endothelial dysfunction‐induced diabetic complications. Treatment of PAE‐D significantly lowered the fluorescent formation due to advanced glycation reaction.

Grant Funding Source : iPET, Korea
Roles of mitochondrial and lysosomal alterations in potentiation by tumor necrosis factor‐alpha of amiodaroneinduced cytotoxicity in vitro
Authors: Lu Jingtao , Roth Robert A. , Ganey Patricia E.
Published: June 2021

We demonstrated previously that tumor necrosis factor‐alpha (TNF) plays an important role in a rat model of amiodarone (AMD)‐ induced hepatotoxicity under inflammatory stress and that TNF potentiates the cytotoxicity of AMD in Hepa1c1c7 cells in vitro. In this study, we explored the roles of mitochondrial and lysosomal alterations in TNF's potentiation of AMD cytotoxicity. The mitochondrial membrane potential (MMP) in cells treated with AMD alone was increased about 2 fold at an early time (1h) and decreased later (6h) to half that of the untreated cells. TNF cotreatment attenuated the AMD‐induced MMP increase at the early time but enhanced the MMP decrease at the later time. AMD caused mitochondrial superoxide production, whole‐cell reactive oxygen species (ROS) generation and lipid peroxidation, and TNF coexposure enhanced these responses. α‐Tocopherol, a lipid‐soluble free radical scavenger, prevented AMD cytotoxicity and caused pronounced protection against AMD/TNF cotreatment. α‐Tocopherol completely abolished lipid peroxidation in both the AMD‐ and the AMD/TNF‐treated cells and significantly reduced cellular ROS without affecting the AMD‐induced mitochondrial superoxide generation. α‐Tocopherol did not affect AMD‐induced early MMP increase but preserved MMP at later times. AMD decreased the lysosomal pH gradient, an effect that was partially attenuated by α‐tocopherol. Cotreatment with bafilomycin A1, which abolishes the lysosomal pH gradient, significantly potentiated AMD cytotoxity. Deferoxamine, an iron chelator, attenuated the cytotoxicity caused by AMD alone or AMD/TNF cotreatment. In summary, mitochondrial and lysosomal alterations were observed after AMD/TNF cotreatment, and α‐tocopherol attenuated AMD‐ and AMD/TNF‐induced cytotoxicity by preventing these alterations. (NIH R01DK061315)
Effect of ACE inhibition in renin‐angiotensin and α1‐ adrenergic renal vascular renal systems
Authors: Castro-Moreno Patricia , López-Guerrero Juan Javier , Ibarra Maximiliano , Pardo Juan Pablo , Hernández-Muñoz Rolando , Villalobos-Molina Rafael
Published: June 2021

The kidney is an important organ in the pathogenesis of hypertension and its complications, by the overactivation of intrarenal renin‐angiotensin system (RAS) and noradrenergic system leading to tissue injury. We evaluated captopril treatment at short and long term on Ang‐ II and phenylephrine‐induced renal vascular contraction and the participation of AT1 receptor. Five weeks‐old WKY and SHR rats were treated with captopril at 30 mg/kg/day, in drinking water for 2 and 14 weeks and perfusion pressure on isolated kidney was measured. In WKY rats the systolic blood pressure was not affected by captopril and hypertension was absent in SHR. When kidney was stimulated with Ang II, surprisingly we observed an increase in vascular contraction and maximal effect in captopril‐treated rats, but no for the α1‐adrenergic agonist phenylephrine. Losartan, AT1 receptor antagonist blocked responses to Ang II in WKY but not in SHR. Our data show that treatment with captopril modifies the intrarenal vascular response via RAS and not by α1‐adrenergic receptor pathway. It is possible that a different receptors population between strains might occur. [Supported by PAPIIT IN224408 and CONACYT 47481 grants; PC‐M is a doctoral fellow from CONACYT, 164454].
4‐Amino‐2‐chlorophenol nephrotoxicity in vitro: alteration of cytotoxicity by antioxidants
Authors: Rankin Gary OˈNeal , Ferguson Travis , Baksi Sukanya , Anestis Dianne , Racine Christopher
Published: June 2021

4‐Amino‐2‐chlorophenol (4A2CP, 1.0 mM) induces nephrotoxicity in isolated rat renal cortical cells (IRCC). The purpose of this study was to examine potential metabolic pathways (cytochrome P450 [CYP], flavin mono‐oxygenase [FMO], and oxidation‐reduction) for bioactivation of 4A2CP. IRCC were obtained from male Fischer 344 rats and incubated in Krebs‐Henseleit buffer. IRCC (4 million cells/ml; 3 ml) were incubated with dimethyl sulfoxide (DMSO) or 4A2CP (1.0 mM) for 60 min with shaking. In some experiments, IRCC were pretreated with an FMO inhibitor [N‐octylamine (0.2 mM) or methimazole (1.0 mM)], CYP inhibitor [isoniazid (1.0 mM), ketoconazole (1.0 mM), or metyrapone (1.0 mM)], or an antioxidant [ascorbate (1.0 mM), glutathione (1.0 mM), N‐acetyl‐L‐cysteine (NAC, 2.0 mM), DPPD (0.05 mM), deferoximine (0.1 mM), or alpha‐tocopherol (1.0 mM or 2.0 mM)]. Cytotoxicity was assessed by measuring the release of lactate dehydrogenase (LDH). FMO or CYP inhibitors didn't decrease 4A2CP cytotoxicity. Only ascorbate, glutathione and NAC provided protection from 4A2CP toxicity. These results suggest that the mechanism by which 4A2CP induces toxicity does not involve activation by FMO's, CYP2E1, 2B or 3A4. Antioxidant protection suggests that a toxic metabolite is created via an auto‐oxidation process and/or oxidative stress plays a role in 4A2CP nephrotoxicity. (Supported by NIH Grant 5P20RR016477 to WV‐INBRE)
Novel insights into renal angiotensin metabolism using mass spectrometric imaging
Authors: Grobe Nadja , Weir Nathan M , Elased Khalid M , Cool David R , Yan Zhongyu , Morris Mariana
Published: June 2021

A molecular imaging technique was established to monitor tissue specific processing of the vasoconstrictor peptide, angiotensin II (Ang II). Mouse kidney sections were incubated with Ang II followed by analysis with MALDI (matrix assisted laser desorption/ionization) imaging. The method allows for the spatial and molecular characterization of peptide processing. Using this technique, conversion of Ang II to Ang III mainly occurred in the renal medulla. Ang (1–7) was predominantly formed in renal cortex as was Ang (1–4), a tetrapeptide generated from Ang (1–7). These results were verified with enzyme assays using tissue punches of renal medulla and cortex. Ang III and Ang (1–4) formation were comparable in Ang converting enzyme 2 (ACE2) knockout (KO) and wild type (WT) mice. Ang (1–7) formation was lower in ACE2KO, but still present. Using recombinant enzymes, we determined that two other peptidases, prolyl carboxypeptidase (PCP) and prolyl endopeptidase (PEP), are capable of converting Ang II to Ang (1–7). Incubation of kidney sections with a PCP/PEP inhibitor reduced Ang (1–7) formation in WT and ACE2KO suggesting a contribution of PCP/PEP to ACE2 independent Ang (1–7) generation in kidney. This novel molecular imaging technique can be expanded to assess actions and catalytic specificity of other enzymes in mammalian tissues. Supported by NIH R01HL093567 and F32DK093226‐01.
Protective role of the Angiotensin AT2 Receptor in obesity‐induced renal inflammation
Authors: Dhande Isha Shirish , Ali Quaisar , Nag Sourashish , Khan Mohammed Azhar , Hussain Tahir
Published: June 2021

The Angiotensin AT2 receptor (AT2R) is up‐regulated in response to tissue injury and is implicated in tissue protection and repair. Recent studies have also suggested an anti‐inflammatory role for the AT2R. The present study was designed to investigate whether chronic activation of the AT2R offers renoprotection by attenuating early stage obesity‐induced inflammation in the kidney. For this purpose, 5 weeks old lean and pre‐hypertensive obese Zucker rats (OZR) were treated for 2 weeks with either vehicle, AT2R agonist C21 (1 μg/kg/day, i.p.), AT2R antagonist PD123319 (50 μg/kg/min, osmotic pump) or both PD and C21. OZR had higher plasma and renal cortical levels of cytokines (TNF‐α, IL‐6 and MCP‐1) which were lowered by C21 treatment. Interestingly, C21 treatment raised the levels of the anti‐inflammatory cytokine IL‐10 in the plasma and renal cortices of OZR, while there was a marked reduction in IL‐10 levels in the OZR PD group. Renal histology revealed mesangial matrix expansion and mild mesangial hypercellularity in the OZR vehicle and PD treated rats, while the glomeruli of the OZR C21 group were normal. Our results suggest an anti‐inflammatory role for the AT2R in obesity‐linked renal inflammation. Pharmacological activation of AT2R in the early stages of obesity‐induced renal injury may serve as a potential therapy to prevent progression of the disease. Supported by NIH R01 DK‐61578.
Novel stretchable electronics platform for simultaneous high‐density electrical and optical recordings from ex vivo hearts
Authors: Gutbrod Sarah R , Laughner Jacob I , Sulkin Matt S , Xu Lihzi , Bonifas Andrew P , Ying Ming , Yu Alice , Rogers John A , Efimov Igor R
Published: June 2021

Although there are protocols for studying high‐resolution cardiac propagation on the bench top, there is a great need to develop a comparable monitoring strategy on intact hearts in vivo. Novel, stretchable electrodes offer the ability to measure electrical data from the dynamic beating heart without interrupting mechanical contractions. We developed a mechanically compliant, transparent sock electrode for measuring surface electrograms from a beating Langendorff perfused rabbit heart. Using a matrix of nanomembrane transistors, submillimeter and submillisecond resolution can be obtained. The purpose of this study was to correlate simultaneous high‐density surface electrograms with optical mapping recordings in explanted heart preparations. Perfusion was maintained at a constant pressure of 60 mmHg and electrical recordings were collected prior to and after mechanical uncoupling with Blebbistatin in sinus rhythm, epicardially paced through an external stimulator and through the electrode array, and during pinacidil induced ventricular arrhythmias. Optical mapping was performed only after mechanical uncoupling. Preliminary results show that the propagation maps created from the array of electrodes contains adequate activation, repolarization and conduction velocity information of normal and aberrant rhythms, verified by the optical recordings.

supported by NIH Grant RO1 HL067322
Aging Aggravates Cardiac Dysfunction in Severe, but not in Mild, Pressure‐Overload
Authors: de Boer Martine , van Deel Elza D. , Hoeijmakers Jan H.J. , Duncker Dirk J.
Published: June 2021

The aging heart undergoes structural and functional alterations, that are reminiscent of changes observed in the failing heart, and which thus may increase the vulnerability of the aging heart to develop heart failure. To investigate the effect of aging on left ventricular (LV) hypertrophy and dysfunction we subjected mice at 3, 12 and 24 mo of age to 8 wk of pressure‐overload produced by transverse aortic constriction (TAC), either mild (mTAC) or severe (sTAC).

Aging affected cardiac geometry and function in sham mice, demonstrated by increases in LV weight (~26%) and diameter (~8%) and by decreases in LVdP/dt at 40 mmHg (dP/dtP40) (~13%) and in dP/dtmin (~26%) in 24 mo versus 3 mo old mice (all p<0.05). mTAC produced compensated LV hypertrophy (30–40%) with minimal signs of LV dysfunction, which was similar in all age groups. sTAC resulted in ~75% LV hypertrophy, LV dilation (~20%), global dysfunction and pulmonary congestion in all age groups. However, aging aggravated the responses to sTAC, demonstrated by a further decrease in dP/dtP40 (~30%) and dP/dtmin (~30%) and elevation of lung fluid weight (~27%) and right ventricular weight (~31%) in 24 mo versus 3 mo old mice (all p<0.05). The TAC‐induced increase in expression of atrial natriuretic peptide (ANP) was also most pronounced in the older age groups. In conclusion, the effects of severe pressure‐overload on cardiac hypertrophy and function are aggravated by aging.
Short‐Term ACE Inhibition Produces Long‐Term Protection Against NOS Inhibitor‐Induced Cardiac Dysfunction
Authors: Biwer Lauren Alysse , Xu Haodong , Carroll Chad , Broderick Tom , Hale Taben Mary
Published: June 2021

ACE inhibition produces changes that persist following cessation of treatment, resulting in a reduced inflammatory, proliferative and fibrotic response to the nitric oxide synthase inhibitor L‐NAME. The present study examines the impact of this pre‐treatment on L‐NAME‐ induced cardiac dysfunction.

Young adult spontaneously hypertensive rats were treated with enalapril (E+L) or tap water (C, C+L) for 2 weeks followed by a 2‐week washout period. At this point, C+L and E+L rats were given L‐NAME for 10 days. Hearts were perfused via the Langendorff working heart method, mean arterial pressure was assessed via radiotelemetry, and collagen by hydroxyproline assay.

L‐NAME increased MAP by a similar magnitude in C+L and E+L. In the LV, prior enalapril treatment tended to decrease collagen content. L‐NAME caused statistically significant decreases in flow‐mediated functional parameters in C+L rats such as cardiac output (Con: 45±11.1mL/min, C+L: 36±11.3mL/min, E+L:47±7.4mL/min), stroke volume, and coronary flow.

Despite equivalent impact on arterial pressure and incidence of myocardial infarction, prior enalapril treatment resulted in preservation of cardiac function following L‐NAME. Understanding the mechanisms by which transient ACE inhibition protects against reductions in cardiac function in the absence of ongoing treatment may reveal novel targets for heart failure treatment.
Inflammation, but not oxidative stress or apoptosis, predominates in atherosclerosis‐associated endothelial dysfunction in juvenile Ossabaw pigs with metabolic syndrome
Authors: Cui Jian , Zhang Hanrui , Li Jun , Chen Jianping , Perfield James W , Rector Scott , Hill Michael A , Zhang Cuihua
Published: June 2021

Mechanisms underlying premature atherosclerosis‐associated endothelial dysfunction in childhood obesity have not been clearly established. We hypothesized that inflammation, but not oxidative stress or apoptosis, contributes to endothelial dysfunction in obese juvenile Ossabaw pigs. To test this hypothesis, female Ossabaw pigs at 8‐weeks of age were fed with either a normal diet (lean control) or a high‐fat/high‐fructose diet (obese) for 18‐weeks. Bradykinin‐induced endothelium‐dependent vasorelaxation of the left anterior descending artery (LAD) was impaired in obese pigs, but endothelium‐independent vasorelaxation to sodium nitroprusside was preserved. Incubating the LAD with the superoxide scavenger TEMPOL did not ameliorate endothelial dysfunction in obese pigs. Nitrotyrosine protein expression, p38 phosphorylation, p53 protein expression, and DNA degradation were not increased in LAD of obese pigs. However, expression of lectin‐like oxLDL receptor 1 (LOX‐1) and suppressor of cytokine signaling 1 (SOCS‐1), key regulators of atherogenesis, as well as the inflammatory cytokine tumor necrosis factor‐alpha (TNFa) were higher in LAD of obese pigs. The data support an increased inflammatory response, as opposed to oxidative stress or vascular apoptosis, being the major factor in impaired endothelial function in juvenile Ossabaw pigs with metabolic syndrome.
Endothelin‐1 Signaling in Coronary Arteriolar Constriction
Authors: Lu Guangrong , Hein Travis W. , Xu Xin , Kuo Lih
Published: June 2021

Endothelin‐1 (ET‐1) is a potent vasoconstrictor implicated in coronary heart diseases, but its signaling for vasoconstriction remains elusive. Herein, we probed the role of voltage‐gated Ca2+ channels (VGCCs), phospholipase C (PLC), myosin light chain kinase (MLCK), protein kinase C (PKC) and Rho kinase (ROCK) in the porcine coronary arteriolar response to a pathologic dose of ET‐1 (0.1 nM). Isolated coronary arterioles (~70 μm) constricted to ET‐1 (from 52±3% to 32±3% max. diameter) in a manner sensitive to ETA receptor blocker BQ123, zero‐extraluminal Ca2+, and PLC inhibitor U73122. Inhibiting VGCCs by nifedipine or MLCK by ML‐9 abolished basal tone but did not affect ET‐1 response. ROCK inhibitor H1152 blocked vasoconstrictions to ET‐1 but not PKC activator PDBu. Broad‐spectrum PKC inhibitor Gö6983 blocked vasoconstriction to PDBu but not ET‐1. The PKC‐activated CPI‐17 protein was undetectable in arterioles. Moreover, ET‐1 elicited phosphorylation of myosin‐binding subunit (MBS) in H1152‐sensitive manner. ROCK2, but not ROCK1, siRNA inhibited vasoconstriction to ET‐1. We conclude that VGCCs and MLCK contribute to the generation of basal tone rather than the constriction to ET‐1. The sequential activation of ETA receptors, PLC and the VGCC‐independent Ca2+ entry leads to vasoconstriction to ET‐1 via a pathway involved in ROCK2‐associated MBS phosphorylation independent of PKC signaling.