bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kir7.1 Disease Mutation in the Inner Pore is Critical for K+ Permeation
Authors: Beverley Katie Marie , Steffen Jack Thomas , Heyrman Joseph Andrew , Shahi Pawan Kumar , Pattnaik Bikash Ranjan 
Published: April 2020

BackgroundInwardly rectifying potassium (K+) channels (Kir) are critical for maintaining membrane potential and K+ homeostasis across many tissues. Mutations in the KCNJ13 gene that encodes for Kir7.1 protein, located in the retinal pigmented epithelium (RPE), contribute to the development of pediatric blindness. One such point mutation in the KCNJ13 gene c.458C>T changes Threonine (T) to Isoleucine (I) at amino acid position 153. T153 is located within the second transmembrane domain, lining the inner pore of the tetrameric protein.HypothesisWe hypothesized that T153I would not affect protein translation, assembly, or trafficking. Because of the specific location of T153 within the pore, the mutation might affect ion permeation through the channel.MethodsHEK293 cells in culture were transfected with either GFP tagged wildtype (WT) Kir7.1 or T153I plasmid DNA. Protein from these cells was isolated and used for Western blotting and Mass‐spectroscopy. Confocal live‐cell imaging, using Nikon‐C2, mapped the expression of GFP fusion protein. Hoescht marked the nucleus, and WGA‐Alexa 594 labeled the plasma membrane. Images were subjected to off‐line analysis with NIS Elements. Whole‐cell patch‐clamp electrophysiology provided insight into channel function. For ion permeation, we exploited K+ and Rubidium (Rb+) permeability of the channel and analyzed using the Clampfit program.ResultsThe T153I mutant protein size was identical to Kir7.1 WT protein. The specific protein band confirmed the Kir7.1 amino acid sequence. Live‐cell imaging indicated that both Kir7.1 and T153I are trafficked to the membrane. The IV plot for the Kir7.1 WT showed inward current measured at −150 mV with a mean amplitude of −943.49 ± 180.56 pA (n=6), but the T153I shows negligible inward current of −81.21 ± 20.29 pA (n=9, P=0.0051). Kir7.1 WT zero‐current potential was −62.21 ± 1.66 mV (n=6), however, the average T153I zero‐current potential was −5.98 ± 1.62 mV (n=9, P=6.2x10−12). Rb+ ion, known to permeate readily through Kir7.1 channel, showed current fold‐change of 4.95 ± 0.78 (n=6) in WT, while T153I showed current fold‐change of 2.60 ± 0.50 (n=9, P=0.033). P‐value <0.05 is regarded as significant.ConclusionThese data indicate that the T153I mutant channel is translated and trafficked to the membrane. However, our data indicate that T153I did not maintain WT zero‐current potential and decreased inward current compared to Kir7.1 WT. Surprisingly, T153I showed Rb+ permeability, albeit less compared to the WT channel. Since T153I is within the inner pore‐lining of the channel, it appears to constrict the channel to make it non‐permeable to K+, but Rb+ can pass through, resulting in a dysfunctional channel.Support or Funding InformationUniversity of Wisconsin‐Madison Endocrinology and Reproductive Physiology NICHD T32, NIH EY024995, NIH P30EY016665, NIH S10OD018221
Evaluation of In Vitro Models for Pyrrolizidine Alkaloid‐Induced Hepatocellular Injury
Authors: Ramos Kaydee Anne Garrote , Taylor Jonathan , Stamper Brendan D. 
Published: April 2020

ObjectivePyrrolizidine alkaloids (PAs) are toxic chemicals found in many herbal medicines, dietary supplements and even food sources such as honey, bread, and tea. Cases of drug‐induced liver injury due to herbal and dietary supplements are relatively common in Asia and are increasing in the United States. The literature suggests that PAs containing macrocyclic structure are more toxic than those with smaller sidechains. The goal of this project was to evaluate two PAs with macrocyclic structure (monocrotaline and retrorsine) and one PA will a smaller sidechain (retronecine) for their potential to elicit toxicity across two in vitro model systems.MethodsPA toxicity was measured by determining the concentration at which 50% of the cell population was killed (LC50). PAs were dissolved in DMSO and PA‐induced toxicity was measured after 48 hours in two in vitro models: TAMH (transforming growth factor‐α transgenic mouse hepatocytes) and THLE‐2 (transformed human liver epithelial‐2 cells). Statistical analysis of cell viability data was performed using SPSS (Probit regression) to obtain LC50 values.ResultsA decrease in cell viability was observed as PA concentration increased. Interestingly, both cell lines were relatively resistant to PA‐induced toxicity. In many cases only predicted LC50 values for each compound could be generated and are indicated by an asterisk (7.0mM*, 11mM*, and 30.mM* in TAMH for monocrotaline, retrorsine and retronecine, respectively; and 2.2mM, 3.2mM, and >100mM* in THLE‐2 for monocrotaline, retrorsine and retronecine, respectively. Based on the toxicologic parameters, monocrotaline and retrorsine were more toxic (lower LC50 values) than retronecine.ConclusionsWhile PAs have been associated with hepatotoxicity, limited toxicity was seen in both the TAMH and THLE‐2 cell lines. Predicted LC50 values were higher than expected, suggesting TAMH and THLE‐2 cells are not good models for studying PA‐induced liver toxicity. Retronecine, which contains a smaller sidechain, was found to be less toxic than the larger compounds. Future work is currently underway to identify biomarkers responsible for the observed resistance to PA‐induced toxicity as potential targets for preventing hepatocellular injury.Support or Funding InformationThis work was supported by the Pacific University Research Incentive Grant Program and an ASPET Summer Undergraduate Research Fellowship (SURF) award at Pacific University.
Examining the mechanism of the methionine E. coli ABC transporter MetNI
Authors: Foronda Matthew 
Published: April 2020

To maintain chemical homeostasis, cells need to transport substrates against their concentration gradients through the cellular membrane. In all life forms, ATP‐binding‐cassette (ABC) transporters utilize the energy of ATP binding and hydrolysis to drive this crucial process. When these transporters malfunction, diseases such as cystic fibrosis, Stargardt’s disease (vision loss), and drug‐resistant cancers can develop. Currently, the structure of a number of ABC transporters in different states has been solved, providing snapshots of the transporter in action. Functional analyses oftentimes reinforce structural studies, however in some cases the proposed models are in disagreement. Recent studies suggest that individual transporters may have multiple mechanisms for transport and that the mechanisms between transporters vary significantly.MetNI is an ABC transporter that imports methionine in bacterial cells. MetI is the transmembrane domain that passes through the cell’s lipid bilayer, and MetN is the site of ATP binding and hydrolysis. Assisting substrate arrival to MetNI is substrate‐binding protein MetQ. Our research focuses on determining the conditions necessary for the interaction of MetNI and MetQ to occur. Using fluorescence anisotropy and fluorescently labeled MetQ, we measure the change in anisotropy as the concentration of MetNI increases. From the anisotropy data we determine the binding dissociation constant of MetNI‐MetQ in the presence and absence of ATP, ADP, and methionine.
Dysregulation of Inositol Synthesis Affects Obesity and Causes Developmental Birth Defects
Authors: Rivera Maria J. , Nguyen LongThy , Eldon Elizabeth D. , Klig Lisa S. 
Published: April 2020

Myo‐inositol is a six‐carbon sugar alcohol that is a precursor of phosphatidylinositol (PI), a cell membrane phospholipid. Inositol is synthesized from glucose‐6‐phosphate by the product of the Drosophila melanogaster Inos gene, myo‐inositol‐3‐phosphate synthase (MIPSp). Abnormalities in myo‐inositol metabolism have been implicated in diseases and complications such as type 2 diabetes, polycystic ovary syndrome, and bipolar disorder. Obesity can be induced by a high sucrose diet in D. melanogaster third instar larvae. The hypothesis of this study was that obesity induced in D. melanogaster by high sucrose diet can be alleviated by inositol, either dietary or endogenously synthesized. A float buoyancy assay was used to assess obesity of larvae of three strains: a wild type strain (CS), a homozygous P‐element insertion strain with UASGAL4 5’ of the Inos gene (inosP‐UAS), and a heterozygous inosP‐UAS strain with GAL4 production driven by an actin promoter (inosP‐UAS ; Act5C‐Gal4). This last strain has up‐regulated inositol synthesis. Larval Inos transcript and MIPS protein expression levels in the three strains were confirmed by RT‐qPCR and Western blots. The inosP‐UAS larvae with GAL4 production driven by the actin promoter (inosP‐UAS ; Act5C‐Gal4) have the highest levels of Inos transcript and MIPS protein relative to the homozygous inosP‐UAS insertion larvae which have an intermediate level of expression, and the wild‐type CS with the lowest level of expression. In the float‐buoyancy assay, larvae with increased buoyancy are scored as ‘obese’. Provision of dietary inositol reduced the percentage of ‘obese’ wild‐type larvae from 74.75±1.94 to 63.95 ±1.70. Moreover, up‐regulation of inositol synthesis via inosP‐UAS ; Act5C‐Gal4 dramatically reduced the percentage of ‘obese’ larvae to 13.28±4.42. During these studies, it became apparent that dysregulation of inositol synthesis resulted in developmental abnormalities and birth defects. Attempts to grow inosP‐UAS ; Act5C‐Gal4 larvae to maturity (adult flies) resulted in a surprising outcome – nearly all flies died as pharate adults. Some structural alterations of wings, halteres, and legs were immediately apparent. The few flies that did eclose died within a few days. Scanning electron and light microscope imaging revealed that they all had malformed or missing proboscises. These data give us a better understanding of the importance of the regulation of inositol in obesity and development.Support or Funding InformationThis project is supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R25GM071638 and by the Frank Schatzlein Endowed Student Award.
Assembly the [2Fe‐2S] Cluster in the Cytochrome bc1 Complex from Saccharomyces cerevisiae
Authors: Briones Luis Alberto Sanchez , Pérez Alma Laura Díaz , Alcántar Lorena Martínez , García Jesús Campos 
Published: April 2020

ObjectiveParticipation of the Fe‐S‐IBG subsystem in the assembly of the [2Fe‐2S] cluster into the bc1 complex.MethodsMutants of S. cerevisiae: Δiba57, Δisa1, Δgrx5, and Δrip1 were grown in YPD medium at 30°C. Mitochondria were isolated by differential centrifugation according to Auchére et al. (2008). Respiratory complex and aconitase activities were evaluated according to Gomez et al. (2014). Mitochondria were treated with digitonin to evaluate the formation of respiratory supercomplexes by electrophoresis in Polyacrylamide Blue Native Gels (BN‐PAGE), and after 2‐dimensional SDS‐PAGE analysis according to Schagger et al. (2000). Detection of the Rip1p subunit was performed by Western blot using the anti‐Rip1p monoclonal antibody at 1:20,000 dilution and developed with anti‐mouse IgG HRP‐conjugate.AbstractThe iron‐sulfur clusters [Fe‐S] are inorganic cofactors contained in enzymes involved in multiple cellular processes. In eukaryotes, [Fe‐S] synthesis mainly occurs in the mitochondrial matrix by the biogenesis of Iron Sulfur Cluster system (ISC). This system involves two steps: the novo assembly of the [Fe–S] clusters on scaffold proteins, and the transference of the [Fe–S] cluster to target apo‐proteins. These steps involve the participation of several proteins, which perform specific reactions. The ISC subsystem Fe‐S‐IBG conformed by the Grx5p, Isa1p, Isa2p, and Iba57p proteins is essential for the synthesis of the [4Fe‐4S] clusters. Yeast mutants in the Fe‐S‐IBG subsystem showed deficiency in respiration and excessive ROS generation was found, similarly to the mutant Δrip1, which gene encodes for the Rieske subunit of cytochrome bc1. In this work, the activity of the cytochrome bc1 in mitochondria from the Δiba57,Δisa1, Δgrx5, and Δrip1 mutants strains were determined. Results showed that the cytochrome bc1 complex in mitochondria from the Δiba57,Δisa1, and Δrip1 mutants was dysfunctional; whereas in the Δgrx5, the cytochrome bc1 activity was partially decreased in comparison with the WT strain. BN‐PAGE analysis shows that the respiratory complex lost the capacity to form supercomplexes in mitochondria from the Δiba57 and Δisa1 mutants, Immunodetection assays showed that Rip1p was absent in the iba57Δ and Δisa1 mutants; whereas the Δgrx5 mutant shows an unaltered pattern profile of the supercomplexes bands in BN‐PAGE, without the loss of Rieske protein. In conclusion, results indicate that the Isa1p and Iba57p proteins are involved in the insertion of the [2Fe‐2S] cluster on the Rip1p apoprotein, suggesting that the Fe‐S‐IBG subsystem is not exclusive for the maturation/assembly of proteins with [4Fe‐4S] clusters, but also is involved in [2Fe‐2S] clusters assembly such as occurred in the Rieske subunit.
Characterization of skeletal muscle fiber types and electrocytes in the tail of the electric fish Apteronotus albifrons before and after regeneration following tail amputation.
Authors: Singh Anita Ler , Mills Kayley , Unguez Graciela 
Published: April 2020

In most groups of electric fish, cells that make up the electric organ (EO), or electrocytes, derive from skeletal muscle cells that suppress many muscle properties. This muscle‐to‐electrocyte conversion is repeated during tail regeneration in adult fish after tail amputation. One of few exceptions to the adult muscle‐derived EO is the Apteronotus albifrons. In A. albifrons, the first electrocytes that form during the larval stage do derive from muscle. However, an EO that is solely composed of the terminal endings of electromotor neurons subsequently replaces this larval EO. We hypothesize that following tail amputation, regeneration of the adult electric organ includes the process of formation of the myogenically derived EO as it occurs during larval stage of development.Adult fish were obtained commercially and housed individually in 15 to 20‐gallon aerated aquaria maintained at 25–28°C and fed three times weekly. Ten fish were anesthetized using 2‐phenoxy ethanol (1:1,500 in tank water), and the distal segment of the tail was transected at the caudal end of the anal fin, then returned to their tanks and monitored until they fully recovered from anesthesia. The cut control tail segments were mounted on cork, frozen in liquid nitrogen‐cooled isopentane, and stored at −80°C. These fish were re‐anesthetized 2 weeks after amputation and their regeneration blastema were removed and frozen as done with control tails. Longitudinal and cross‐sections (20μm‐thick) were mounted on glass slides and processed histologically for analysis of general cell type morphology. Immunolabeling studies used antibodies against markers known to detect different muscle fiber types, axon projections, myogenic stem cells, and nerve endings. These approaches will allow us to determine the muscle fiber type composition of the tail region that contains the adult EO in A. albifrons and the fiber type transition that occurs during regeneration. Our preliminary data shows that 1) type I and type II adult muscle fibers surround the EO with type II being more predominant in number, and 2) muscle proliferation is abundant along the entire periphery of the regeration blastema. Further immunolabeling studies are underway to complete testing our hypothesis.Support or Funding InformationFunded in part by the NIH MARC Program Grant #GM07667‐41
Overexpression of catalase targeted to mitochondria improves neurovascular coupling responses in aged mice.
Authors: Csiszar Anna , Yabluchanskiy Andriy , Ungvari Anna , Ungvari Zoltan , Tarantini Stefano 
Published: April 2020

Moment‐to‐moment adjustment of cerebral blood flow (CBF) to neuronal activity via the homeostatic mechanism known as neurovascular coupling (NVC) has an essential role in maintenance of normal brain function. In advanced age cerebromicrovascular endothelial dysfunction impairs NVC responses, which contribute to age‐related cognitive decline. Recently, we have shown that pharmacological treatments that attenuate mitochondrial production of reactive oxygen species (ROS) provide significant neurovascular protection, improving NVC responses in aged mice. Transgenic mice that overexpress human catalase localized to the mitochondria (mCAT) are protected from age‐related mitochondrial oxidative stress and exhibit a longevity phenotype associated with resistance to several age‐related pathologies. The present study was designed to test the hypothesis that mitochondria‐targeted overexpression of catalase also confers protection against age‐related impairment of NVC responses. To achieve this goal, NVC responses were assessed in aged (24 months old) mCAT mice and compared with those in age‐matched wild‐type mice and young control mice by measuring CBF responses (laser speckle contrast imaging) evoked by contralateral whisker stimulation. We found that mitochondrial overexpression of catalase resulted in improved NVC in aged mice due to preserved NO‐mediated (L‐NAME inhibitable) component of the response. Thus, our present and previous findings demonstrate that interventions that boost mitochondrial antioxidative defenses confer significant cerebromicrovascular protective effects, which preserve NVC responses in aged mice. Our findings provide additional proof‐of‐concept for the potential use of mitochondria‐targeted antioxidants as therapy for prevention of vascular cognitive impairment associated with aging.Support or Funding InformationThis work was supported by grants the Oklahoma Center for the Advancement of Science and Technology, the Oklahoma Shared Clinical and Translational Resources (OSCTR) program funded by the National Institute of General Medical Sciences (GM104938), the NIA‐supported Geroscience Training Program in Oklahoma (T32AG052363), the Oklahoma Nathan Shock Center (P30AG050911), the Cellular and Molecular GeroScience CoBRE (1P20GM125528, sub#5337), and the NIGMS supported Center of Biomedical Research Excellence (CoBRE).
Two‐pore channel 2 (TPC2) Role in Melanoma
Authors: Hanbashi Ali , D'Amore Antonella , Parrington John 
Published: April 2020

Two‐pore channels (TPC), a recently described class of NAADP‐ and PI(3,5)P2–sensitive calcium‐permeable cation channels in the endolysosomal system of cells have a crucial rule in cellular functions and have been linked to various type of diseases including cancer. Here, TPC2 protein stable silencing in melanoma cancer cell lines was developed using CRISPR/Cas9 genome editing technology. TPC2 knockout (KO) in B16 murine melanoma cancer cell was associated with higher viability and migration rates with higher melanin accumulation inside the cells compared to wild type controls. TPC2 KO CHL1 human melanoma cancer cells showed an increased level of proliferation with reduced migration capacity. Both KO cell lines had lower invasion rates and lacked capacity for angiogenesis compared to wild type controls. Proteomic analysis was used to identify novel targets which were affected by genetic modification of TPC2. Thus, the findings of the present study indicated that CRISPR/Cas9 model is an ideal and cost‐effective tool to study the role of TPC2 in melanoma cancer cells which can be further used as a target to understand the biological basis of this disease.Support or Funding InformationRoyal Embassy of Saudi Arabia Cultural Bureau
Chronic oral NaHCO3 treatment and high tubular flow promote insulin resistance in rats
Authors: Mannon Elinor , O'Connor Paul 
Published: April 2020

Diabetes is the leading cause of chronic kidney disease (CKD), and worsening insulin resistance likely contributes to CKD progression. Several small clinical trials have indicated that sodium bicarbonate (NaHCO3) slows the decline in GFR in CKD. Chronic ingestion of alkali like NaHCO3 can lead to potassium (K+) wasting by the kidney. While K+ wasting has been linked to the dysregulation of insulin metabolism, no studies have examined the metabolic impacts of chronic NaHCO3. We hypothesized that chronic NaHCO3 would promote K+ wasting and worsen insulin resistance in rats. In order to test our hypothesis, 16 male, 12 wk old Sprague Dawley (SD) rats underwent four weeks of high‐fructose (60%) diet (HFruD) prior to a baseline insulin tolerance test (ITT). Treatment with 0.1M NaHCO3 (Bicarb; n=8) or 0.1M NaCl (Vehicle; n=8) was initiated in drinking water after the baseline ITT and continued along with fructose feeding for another 3 weeks. ITTs were performed again following 1 week and 3 weeks of either bicarb or vehicle drinking. A similar protocol was performed using 18 male, 12 wk old SD rats, with the exception that these animals were maintained on normal rat chow, and following a baseline ITT, were placed on either Bicarb (n=6), Vehicle (n=6), or Tap H2O (n=6). At the conclusion of the protocol, animals were sacrificed, and visceral and subcutaneous fat pads and the spleen and kidneys harvested. In HFruD rats, there were no significant differences in the magnitude of the fall in blood glucose (area under the curve (AUC)) in response to insulin (0.75U/kg) at baseline (Veh 4229 ± 1625 vs Bicarb 3624 ± 1093; unpaired t‐test, p=0.76) or following 1 week of treatment (Veh 7187 ± 1613 vs Bicarb 5151 ± 1121; unpaired t‐test, p=0.32). However, following 3 weeks of treatment, animals that received bicarb demonstrated a significant decrease in the response to insulin, when compared to vehicle treated animals (Veh 5170 ± 690.0 vs Bicarb 1902 ± 846.0; unpaired t‐test, p=0.001). In naïve animals, there were no significant differences in the response to insulin at baseline (Veh 2337 ± 396.2 vs Bicarb 3651 ± 690.7 vs Tap H2O 3349 ± 544.2; One‐way ANOVA, p=0.25) or following three weeks of treatment (Veh 1773 ± 559.5 vs Bicarb 1745 ± 338.3 vs Tap H2O 1320 ± 215.7; One‐way ANOVA, p=0.67). In conclusion, our data show that prolonged NaHCO3 ingestion can significantly impair the response to insulin. As this effect was only present in HFruD fed rats, we speculate that high tubular flow may act synergistically with NaHCO3 to increase K+ wasting or that underlying metabolic dysfunction may be required to observe impairments in insulin sensitivity. As both underlying metabolic dysfunction and high single tubular flow due to reduced nephron mass are present in many CKD patients, these findings could have significant implications for CKD patients who are currently supplemented with NaHCO3. Such patients may require more vigilant blood glucose monitoring.Support or Funding InformationNIH PO1HL134604 and DK099548