bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural basis for the regulation of endothelin‐1 mRNA stability by glyceraldehyde‐3‐phosphate dehydrogenase
Authors: Neal Jacob P. , Garcin Elsa D. 
Published: June 2021

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) plays a key role in vascular homeostasis by modulating levels of the vasoconstrictor endothelin‐1 (ET‐1). Elevated ET‐1 levels are associated with vascular diseases, and its expression is tightly regulated primarily via GAPDH‐mediated destabilization of its mRNA. Recent studies suggest GAPDH binding of adenine‐uridine rich elements in the 3′‐untranslated region of ET‐1 gene leads to decreased mRNA stability. However, structural and mechanistic details underlying the GAPDH‐mediated control of ET‐1 expression are lacking. We seek to probe the structural and mechanistic basis for the GAPDH‐induced destabilization of ET‐1 mRNA via electrophoretic mobility shift assay (EMSA) and x‐ray crystallography. To identify specific GAPDH binding sequences, we constructed short RNA transcripts of the putative mRNA binding region. Utilizing a modified EMSA with biotinylated RNA transcripts, we obtained association and dissociation constants. For transcripts yielding the greatest affinity for GAPDH, we obtained crystals. Ultimately, we aim to solve the crystal structures for these ET‐1 mRNA constructs with GAPDH. These studies will provide the first molecular basis for structure‐based drug design, leading to the development of novel molecules that specifically target ET‐1 mRNA.Funding: UMBC DRIF SRAIS (EG).
AKAP79 Controls β1‐Adrenergic Receptor Trafficking by Multiple Regulatory Mechanisms
Authors: li xin , Bahouth Suleiman 
Published: June 2021

Chronic activation of G protein‐coupled receptors promotes their endocytosis from cell membranes into intracellular endosomes. Efficient trafficking of these receptors back into the cell membrane is critical for receptor resensitization. Trafficking of many GPCR is dependent on cis‐acting domains and trans‐acting factors. Examples of cis‐acting domains in the β1‐adrenergic receptor (β1‐AR) are the type‐1 PDZ domain located in the carboxy‐terminus and serine at position 312 in the 3rd intra‐cellular loop of the β1‐AR., which is a substrate for phosphorylation by the cyclic AMP‐dependent protein kinase (PKA). In the β1‐AR, the PDZ type‐1 domain binds to a complex composed of synapse associated protein 97 (SAP97) and A‐kinase anchoring protein‐79 (AKAP79). The c‐terminus of AKAP79 in turn binds PKA, which phosphorylates Ser312 upon the activation of the β1‐AR signaling pathway.Preliminary studies showed that AKAP79 was involved in trafficking of the β1‐AR, but its precise mechanism was obscure. In this study, we determined that selective down regulation of AKAP79 inhibited the recycling of the WT β1‐AR in HEK‐293 cells and its restoration rescued the recycling of the agonist‐internalized β1‐AR. There are several functional domains in AKAP79 including PKA and PKC binding, membrane targeting/binding, SAP79 and calcineurin binding. We analyzed by confocal microscopy the effect of deleting each of these domains on the ability of the resulting AKAP79 mutant to rescue the recycling of the β1‐AR. Our data revealed that multiple domains of AKAP79 were required, indicating that the effect of AKAP79 on recycling of the β1‐AR occurred by multiple divergent mechanisms. Therefore membrane targeting of AKAP79 and its binding to other accessory protein were all required for AKAP79‐mediated regulation of β1‐AR trafficking. Funded by NIH HL‐085848.
Short‐hairpin RNA‐mediated silencing of NKCC1 in COS7 cells and its effect on cell volume
Authors: Alshahrani Saeed Ayed , Di Fulvio Mauricio 
Published: June 2021

Na+K+2Cl− cotransporters (NKCCs) effect the electroneutral movement of Na+, K+ and 2Cl− across the plasma membrane. Under physiological conditions NKCCs function as active, Na+‐dependent, Cl− uptake transporters. Two NKCC isoforms, encoded by two different genes have been identified: NKCC1 and NKCC2. NKCC1 is a ubiquitously expressed gene whereas NKCC2 is considered to be exclusive of the kidney. We describe for the first time a viable knock‐down strategy to silence endogenous NKCC1 expression in a widely used, easy to transfect cell line. NKCC1 mRNA expression in COS7 cells was eliminated transfecting these cells with a poly‐cistronic vector encoding for GFP and an shRNA directed against the third exon of human NKCC1. Stable transfectants were selected by continuous incubation in the presence of puromycin. Cells stably expressing GFP and resistant to the drug were selected and cloned. NKCC1 expression was followed by Western bolt, immunofluorescence and reverse transcription (RT) coupled to the polymerase chain reaction (PCR). The impact of NKCC1 depletion on cell volume was followed morphometrically. We found that cells lacking NKCC1 are shrunken suggesting that NKCC1 is the sole regulator of cell volume in these cells. This new cell system will open the possibility to study the functional, pharmacological and molecular properties of candidate genes involved in volume regulation.This work was supported by funds awarded to MDiF by the American Diabetes Association (Grant JF‐1001730).
REGULATION OF CANNABINOID RECEPTOR SIGNALING BY RGS (REGULATORS OF G‐PROTEIN SIGNALING) PROTEINS: A NEW PARADIGM FOR MODULATING CB1 AND CB2 RECEPTOR‐MEDIATED SIGNALOSOME
Authors: Mukhopadhyay Somnath , Jha Shalini , Poltoratsky Vladimir , LaRoche Genevieve , GiGuere Patrick , Oestreich Emily , Siderovski David 
Published: June 2021

CB1 and CB2 cannabinoid receptors belong to Gi protein‐coupled receptor superfamily. RGS proteins are classically known for turning off Gi and Gq‐coupled receptor signaling. CB1 and CB2 cannabinoid receptors are known to elicit a plethora of cellular responses acting through different G‐proteins. However, till date almost nothing is known about RGS‐mediated regulation of cannabinoid receptor signal transduction. In the current study we attempt to define the role of RGS‐proteins in the regulation of CB1 and CB2 receptor signaling in neuronal and endothelial cells respectively.In the current study using real‐time PCR we first screened for native expression of individual RGS proteins in CB1 and CB2 receptor expressing neuronal and endothelial cells lines respectively. Once we found the expression profile of RGS proteins in the specific cell lines we selectively knock down (using SiRNA ) individual RGS proteins in these cell lines. We then measured nitric oxide production following CB1 or CB2 receptor activation in these RGS‐knock down cell lines to determine the specificity of RGS proteins for intercepting CB1 vs. CB2 receptor signaling. Our initial findings showed that a) different RGS proteins are coupled to CB1 and CB receptor signaling and b) RGS proteins can differentially regulate ( inhibit or activate) CB1 and/or CB2 receptor‐mediated nitric oxide production. These results suggest that RGS proteins play a pivotal role in the regulation of cannabinoid receptor signalosomes.
LETM1 is an essential component of mitochondrial calcium uptake and regulation of cellular bioenergetics
Authors: Doonan Patrick John , Mallilankaraman Karthik , Chandramoorthy Harish , Madesh Muniswamy 
Published: June 2021

Regulation of mitochondrial Ca2+‐dependent cellular bioenergetics has been implicated in a variety of pathophysiological settings including neurodegeneration, myocardial infarction and immunity. Recently, LETM1 was proposed as a mitochondrial Ca2+/H+ antiporter; however characterization of the functional role of LETM1‐mediated Ca2+ transfer has yet to be studied. Here we demonstrate that LETM1 knockdown significantly impairs mitochondrial Ca2+ uptake and results in bioenergetic collapse. We observed that mitochondrial Ca2+ uptake is impaired in shRNA‐mediated LETM1 knockdown and ΔEF hand LETM1 and D676AD688KLETM1 overexpression cells. Knockdown of LETM1 promotes LC3 positive multilamellar vesicle formation, indicative of autophagy induction. Furthermore, cellular bioenergetic parameters including ATP and oxygen consumption were reduced. In contrast, cellular NADH and mitochondrial membrane potential were unaltered in both control and shRNA LETM1 cells. Interestingly, knockdown of LETM1 by siRNA in primary neonatal cardiomyocytes resulted in a remarkable change in mitochondrial morphology and reduced Ca2+ uptake. We also observed that LETM1 knockdown results in increased ROS production. Our results represent the first time that LETM1 has been linked to cellular bioenergetics through regulation of mitochondrial Ca2+.Supported by NIH R01 HL086699, HL086699‐01A2S1, 1S10RR027327‐01
Infant and young child feeding in Mexico: National Health and Nutrition Survey 2006 and changes from 1999
Authors: de Cosío Teresita González , Escobar Leticia , González Luz Dinorah , Reyes Horacio , Rivera Juan 
Published: June 2021

We present evidence of infant & young child feeding practices (IYCFP) in Mexico using 2006‐WHO indicators (WHO‐I). Data from the last 2 national nutrition surveys (1999 & 2006) were used.Subjectswomen aged 12–49 y & their < 2 y child; n=2953 for 2006 & 3191 for 1999. Median lactation duration (MLD) and % of WHO‐I estimated with status‐quo method. Age of introduction (AI) to 7 food groups (FG) estimated (Kaplan‐Meier method). IYCFP analyzed by region, urban/rural location, socioeconomic & characteristics.ResultsMLD increased: 9.7–10.4 mo from 1999–2006, but decreased in indigenous populations: 17.4–14.6 mo. Proportion of exclusively breastfed infants <6 mo was 20% in 1999 & 22.3% in 2006; 66.2% of children 6–23 mo consumed Fe rich foods in 2006. Most lactation indicators decreased in the rural and indigenous groups from 1999–2006. Children in the Program OPORTUNIDADES had better IYCFP overall than population not enrolled. AI of the 7 FG: water 3 mo; non‐human milk, cereals & legumes, & animal sources 5 mo; non‐nutritive liquids, fruits & vegetables 6 mo; nutritive liquids 8 mo. Results show the poor lactation performance in Mexico, as well as untimely IYCFP. It is necessary to design programs aimed at protecting and promoting human lactation and adequate complementary feeding, especially for the poorest and indigenous groups. Research supported by Center on Nutrition and Health, National Institute of Public Health.
Liver Morphologic Changes Induced by 1‐naphthyl methylcarbamate
Authors: Costa Eduardo Leitão , Cavalcanti Karen , Costa Miguel , Pereira Felipe , Souza Ivone , Cabrita Silvério 
Published: June 2021

The chemical 1‐naphthyl methylcarbamate (1NC) belongs to a class of pesticides known as carbamates. This chemical substance can be produced by hydrolysis of the carbamate ester group with consequent degradation of the hydrolyzed fragments. It is moderately soluble in water, but has a good solubility in a large spectrum of organic solvents.The objective of this study was to evaluate the liver morphologic changes associated with the ingestion 1‐naphthyl methylcarbamate. One group of ten male Wistar rats, 8 weeks old, was submitted to the administration of 1NC 60mg/liter in drinking water during 8 weeks. Another group of ten male Wistar rats, 8 weeks old maintained during 8 weeks with no manipulation was used as a control group.All animals were sacrificed at the end of an 8 weeks period and a complete necropsy was performed. The slides of liver, stained by HE, were evaluated for morphologic lesions. In the group with 1‐naphthyl methylcarbamate some morphologic lesions were found, such as macrovesicular steatosis, some focus of stase with dilatation of sinusoids and small locals of necrosis. Mitotic figures were observed frequently. However, we did not observe focal hemorrhages, fibrosis, but only rare cases of cloudy swelling. In the control there was no significant lesions.These results suggest that an acute 1NC intoxication can induce minimal liver morphologic changes.
Dexamethasone induces apoptosis of osteoblasts in culture by interference in cell adhesion complex
Authors: Naves Marcelo Andery , Paterno Josne Carla , Oliveira-Sales Elisabeth B , Caparbo Valéria F , Pereira Rosa R , Teixeira Vicente Paulo Castro 
Published: June 2021

Adhesive interactions play a critical role in cell biology, influencing vital processes from proliferation to cell death. The objective of this study was to analyse the effect of DEX (dexamethasone) on the biology of osteoblasts and its influence on the expression of ILK and β1 integrin. Primary human osteoblast culture were treated with dexamethasone in 10−9M (physiological) and 10−6M (pharmacological) doses of DEX for 24 and 48 hours. Cell viability (MTT assay), adhesion (crystal violet assay), apoptosis (flow cytometry), expression and immunolocalization of β1‐integrin and ILK (western blot and immunofluorescence) were analyzed. It was observed that cell viability and adhesion were reduced in the cultures evaluated. In comparison with the control cultures, there was slightly less apoptosis in the cultures exposed to the physiological dose and considerably more apoptosis in those exposed to the pharmacological dose. In all treated cultures, protein expression of ILK was slightly higher than in the control cultures, whereas that of β1 integrin was significantly lower. Both proteins under study were co‐localized at the cell periphery in all cultures. Our results suggest that DEX causes osteoblast anoikis, probably due to decreased β1 integrin expression, which might have had a direct influence upon ILK, reducing its activation and preventing it from playing its characteristic anti‐apoptotic role.Supported by FAPESP, CAPES
Disorder in functional phenotype of alveolar macrophages in influence of risk factors for chronic obstructive pulmonary disease (COLD): age‐ and genetic predisposition
Authors: Malyshev Igor Yu , Lyamina Svetlana V , Vedenikin Timofey Yu , Malysheva Elena V 
Published: June 2021

The defining role of tobacco smoking as a risk factor of COPD is of no doubt. However, there are still some unanswered questions. Do genetic determination and age‐related changes of macrophage's phenotype contribute to the development of lung pathology? Does alveolar macrophageˈs phenotype change with tobacco smoking? We modeled COPD in mice of different genetic strains (C57 and BALB/c) by tobacco smoke and studied phenotypes of alveolar macrophages. High production of NO is an indication of M1 phenotype; low production of NO after stimulation with LPS is an indication of M2 phenotype. It was shown that 1) there is a genetic predisposition to the influence of tobacco smoke, associated with proinflammatory M1 phenotype of macrophages; 2) aging promotes macrophages phenotype transformation towards antiinflammatory M2 which is more expressed in macrophages of mice with initially genetically determined M1 phenotype (C57), and 3) inhalation of tobacco smoke enhances age‐dependent transformation of macrophages aside M2 phenotype, that is also more expressed in macrophages of mice with genetically determined M1 phenotype (C57).Grant #x00039A; No#x0003A0;811 Federal Special Program of Russia 2009–2013.
Arterial stiffness and hypertension in diet‐induced obese mice
Authors: Seta Francesca , Shiang Tina , Weisbrod Robert , Chauhan Pratibha , Bajpai Saumendra , Reinhart-King Cynthia , Cohen Richard 
Published: June 2021

BACKGROUNDArterial stiffness (AS) may contribute to increased blood pressure (BP) during aging and obesity and is an independent predictor of cardiovascular complications.AIMSTo elucidate the molecular mechanisms of arterial stiffness and its relationship to the development of hypertension in a model of diet‐induced obesity.METHODS AND RESULTSC57BL/6J mice fed high fat‐high sucrose diet (HFHS), showed increased fat mass, body weight and insulin resistance compared to normal diet (ND). Pulse wave velocity (PWV), the speed of pressure waveform propagation along the aorta used as index of AS, increased by 2.5‐fold after 2 mos HFHS and remained elevated thereafter while BP, measured by radiotelemetry, increased only after 8 mos HFHS. Microalbuminuria, an index of renal dysfunction, developed after 4 mos HFHS. Aortic wall thickness and renin increased after 2 mos HFHS suggesting that renin‐angiotensin‐aldosterone system (RAAS) may contribute to AS in advance of hypertension. Stiffness modulus of aortic extracellular basal membrane, determined in vitro, was increased at 8 mos HFHS.CONCLUSIONSIn diet‐induced obese mice, AS precedes the development of hypertension and kidney dysfunction. Our results suggest that RAAS may contribute to structural changes in aortic smooth muscle, as evidenced by aortic wall thickening and extracellular basal membrane stiffening, causing AS.
Analysis of Lindane and Metabolites by HPLC‐UV‐Vis and MALDI‐TOF
Authors: Naidu Naga V , Smith-Baker Charlotte , Yakubu Momoh A 
Published: June 2021

Lindane (hexachlorocyclohexane) is a persistent environmental toxicant and a known neurotoxicant. High levels of Lindane have been detected in postmortem human brain from Parkinson disease patients. Metabolism of Lindane produces several metabolites which are still imperfectly identified. Therefore, easy, fast and sensitive method to detect Lindane is highly desirable. We determined Lindane and its metabolites in urine by HPLC‐UV‐Vis and characterized it with MALDI‐TOF. Rats received Lindane (17.6 mg/Kg) or vehicle orally for 4 wk and urine collected for analysis. Lindane and metabolites were extracted and analyzed by HPLC followed by MALDI‐TOF characterization. HPLC spectrum of standard Lindane peaked at 6.0 min. and urine samples (n=6) peaked at 6.10–6.30 min. Control urine did not show any peak corresponding to Lindane (6.0–6.30 min). MALDI‐TOF analysis of standard Lindane showed a peak corresponding to 293 m/z whereas the urine samples showed major peaks at 292–293 m/z corresponding to Lindane along with minor peaks at 259, 269, 277, and 318 m/z corresponding to Lindane metabolites: ‐ chlorobenzene, ‐chlorophenol, ‐hexacholorohexane, and other derivatives. These results show that HPLC is sensitive for detecting Lindane and its metabolites and MALDI‐TOF can identify specific metabolites of Lindane in urine by matching spectrum peaks generated to the molecular weights of the metabolites. Thus, HPLC‐UV‐Vis‐MALDI‐TOF analysis can be a reliable, non‐invasive method of detecting lindane exposure and burden.Supported by NHLBI (HL‐70669 and HL03674)
Nanoliposomes restore endothelial function of human adipose arterioles exposed to AL amyloidosis light chain proteins
Authors: Truran Seth , Weissig Volkmar , Franco Daniel A , Maltagliati Anthony , Burciu Camelia , Liu James , Migrino Raymond Q 
Published: June 2021

AL amyloidosis light chain proteins (LC) induce endothelial dysfunction and tissue injury. Nanoliposomes (NL) are <100 nM phospholipid vesicles that could bind amyloid proteins. We tested whether NL can protect against LC induced endothelial dysfunction.MethodsNL from cholesterol, sphingomyelin (1:1) and 5% phosphatidic acid were prepared by probe sonication. Subcutaneous adipose arterioles from patients without amyloidosis/vascular disease were cannulated. Baseline dilator response was measured (acetylcholine 10−9–10−4M and papaverine 10−4M, n=11). A second response followed 1 hour exposure to LC (20 μg/mL from AL patients’ urine, n=6) or LC+NL (1:1 mass, n=5). LC (20 μg) were also mixed with NL at 1:1, 1:5 and 1:10 mass ratios, centrifuged and the supernatant tested by Western blot using antibody to LC.ResultsLC caused impaired dilation to acetylcholine (10−4M: 49±6% vs. 89±4% control, p=0.003); the papaverine response was not different. NL restored dilator response to acetylcholine (10− 4M: 94±3%, p=NS vs. control, p<0.001 vs. LC). Western blot showed progressive reduction of soluble LC with increasing doses of NL suggesting physical sequestration by NL.ConclusionsNL protected human adipose arterioles against LC induced endothelial dysfunction, possibly through sequestration of LC. This proof of concept finding supports exploring use of NL to treat AL amyloid tissue injury.
THE EFFECT OF OMEGA‐3 SUPPLEMENTATION IN HEARTS OF AGED RATS
Authors: Lennon-Edwards Shannon , Schellhardt Taylor , Kuczmarski James 
Published: June 2021

Omega‐3 supplementation has been shown to be beneficial in reducing oxidative stress and inflammation. The purpose was to determine if 12 weeks of a high omega‐3 fatty acid diet would reduce age associated increases in myocardial oxidative stress and inflammation. 20 mature (M) (6 mo.) and 20 older (O) (15 mo.) SD rats were assigned to 2 diet groups: Control (CON) or Fish Oil (FO). Following the diet, animals were sacrificed and left ventricular heart tissue was harvested. Interleukin‐6 was significantly lower in MFO vs. OCON and OFO (26.58±0.67 vs. 37.77±3.85 & 37.68±2.23 pg/mg, respectively; p<0.05). Tumor necrosis factor‐alpha was not different among groups. Hydrogen peroxide production (H2O2) was reduced by FO treatment (p<0.01) but not significant in regards to age. However, OFO H2O2 trended towards significance vs. OCON (0.10±0.02 vs. 0.12±0.01 uM/mg ptn; p=0.09). A significant effect was seen for catalase with age (p<0.05), between MCON and OCON (3.04±0.38 vs. 4.49±0.36 U/mg ptn; p<0.05) and trended towards significance in OFO (3.04±0.38 vs. 4.25±0.36 U/mg ptn; p=0.1). There were no differences among groups for superoxide dismutase (p>0.05). These results suggest age‐associated increases in myocardial inflammation regardless of FO treatment. However, FO supplementation appears to increase some myocardial antioxidant activity and reduce H2O2.Supported by UD Research Foundation Grant
Ethyl pyruvate protects against myocardial ischemia/reperfusion injury via mitochondrial membrane potential dependent and anti‐apoptotic mechanisms
Authors: Chun Byeong Jo , Moon Jeong Mi , Lee Byeong Guk 
Published: June 2021

PurposeA role Ethyl pyruvate (EP) in ischemia/reperfusion (I/R) injury is not clear. We investigated whether EP could inhibit cardiac myocyte apoptosis and reduce mitochondrial injury in the ischemic/reperfused heart of the mice.MethodsMale C57BL/6 mice of 12–15 weeks of age were subjected to 25 min of ischemia followed by 45 min of reperfusion. A modified KH perfusion fluid was used, contained except where EP levels were varied. JC‐1 staining flow cytometry was used to examine mitochondrial membrane potential. We investigated mitochondrial structure, apoptosis rate and Bcl‐2, Bax, Caspase‐3 proteins. RT‐PCR was used to determine the expression of P38MAPK mRNA and JNK mRNA. Western blotting was used to measure the expression of P38MAPK, JNK and NFκB P65.ResultsOur results show that 4.0 mM EP group increased LVDP recovery rate and decreased mitochondrial ultrastructural damage as compared to other EP groups. The apoptosis rate and PCI of Bax, Caspase‐3 proteins in the 4.0 mM EP group were decreased, while the PCI of Bcl‐2 protein was increased. The expression of P38MAPK mRNA, JNK mRNA and protein of P38MAPK, JNK and NFκB P65 in free EP group was increased. The rate of loss Δψm cells in the 4.0 mM EP group was lower than in the other groups.ConclusionOur dates suggest that the EP plays an important role in protection of the myocardium against I/R injury and may represent a novel therapeutic target.