bluuebunny/crossref_metadata_2025 huggingface dataset:
MicroRNA expression with “aging” in human aortic endothelial cells
Authors: Blimline Mark , Seals Douglas R. , Donato Anthony J. , Fleenor Bradley S. , Rippe Catarina
Published: June 2021

Vascular endothelial cell “aging” is associated with development of a senescent phenotype featuring oxidative stress, inflammation, increased extracellular matrix (ECM) and collagen deposition, and impaired angiogenesis. We hypothesized changes in micro‐RNAs (miR) known to modulate expression of proteins associated with these altered functions in human aortic endothelial cells (HAEC) with aging. HAEC aged to replicative senescence (45±1 population doublings (PDLs)) had greater % of cells stained for senescence‐associated β‐galactosidase (60±7% vs. 7±1%), expression of collagen I (+5.0±0.04‐fold, P<0.05) and vascular cell adhesion molecule‐1 (VCAM‐1, +3.6±0.8‐fold, P<0.05) protein (western blotting), and superoxide production (+2.7±0.3‐fold, P<0.001) (electron paramagnetic resonance) vs. early passage HAEC (28.0±0.4 PDLs). Consistent with these changes, senescent HAEC had lower expression of: collagen‐repressing miR‐29 (0.2±0.1 vs. 1.0±0.3 AU, P<0.05); miR‐21 (0.4±0.1 vs. 1.0±0.2 AU, P<0.05) and miR‐214 (0.3±0.1 vs.1.0±0.3 AU, P<0.05), two miRs that target PTEN, a tumor suppressor and cell cycle regulator; and miR‐126 (0.5±0.2 vs. 1.0±0.1 AU, P<0.05), which induces angiogenesis and down‐regulates VCAM‐1. In contrast, redox‐sensitive miR‐125 was greater in senescent HAEC (2.2±0.4 vs. 1.0±0.3 AU, P<0.05). Changes in miR may play an important role in development of a senescent phenotype in vascular endothelial cells and contribute to arterial endothelial dysfunction and increased risk of cardiovascular diseases with aging.

NIH AG013038, AG029337, HL007822, Swedish Research Council
Estrogen Modulation of Neuropeptide Y Receptor Signaling in Magnocellular Oxytocin Neurons of the Hypothalamus
Authors: Urban Janice H , Mitzey Ann M , Brownfield Mark S , Oblinger Monica M , Chura William E
Published: June 2021

Neuropeptide Y (NPY) plays an important role in the regulation of neuroendocrine secretions from the parvocellular and magnocellular groups of the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. Previously, we determined that NPY receptor expression is increased under conditions of hyperosmolality and elevated estrogen. These studies were designed to assess the role of estrogen receptors in regulating the expression, and response, of NPY Y1 receptors in cycling female rats. Y1 receptor immunoreactivity was increased in hypothalamic magnocellular neurons of proestrous female rats when compared with metestrous females. Challenge with the Y1/Y5 receptor agonist, [leu31pro34]NPY into the third ventricle significantly stimulated plasma oxytocin levels which suggested a correlation between receptor immunoreactivity and responsiveness. Since the magnocellular system contains primarily ERβ, we used cell culture with PC12 cell lines expressing either ERβ alone or ERα and β, to examine the contribution of ERβ to the regulation of NPY receptor expression. Overall, the results of these studies indicate that estrogen increases the expression of NPY Y1 receptors in the hypothalamus which is correlated with increased oxytocin secretion. The in vitro studies demonstrate that stimulation of ERβ increases the expression of NPY Y1 receptor immunoreactivity.
Apolipoprotein E Suppresses Atherosclerosis by Reducing Leukocyte Recruitment to Atheroma
Authors: Gaudreault Nathalie , Kumar Nikit , Possada Jessica , Raffai Robert
Published: June 2021

We investigated the anti‐atherosclerotic properties of apoE beyond its ability to lower plasma cholesterol level. We hypothesized that in the setting of dyslipidemia, apoE reduces the recruitment of leukocytes to atheroma.

Mice developing spontaneous dyslipidemia in the presence and absence of apoE expression were studied: Hypomorphic apoE mice deficient in low‐density lipoprotein receptor (Apoeh/hLdlr−/−) were compared to mice deficient in apoE and Ldlr (Apoe−/−Ldlr−/−). Despite similar plasma cholesterol levels (597.5±24.3 mg/dl versus 662.4±26.4 mg/dl, respectively), Apoeh/hLdlr−/− mice developed ~4 fold less oil red O and ~3 fold less macrophage‐positive aortic surface area than Apoe−/−Ldlr−/− mice by five months of age.

To examine the role of apoE on leukocyte recruitment, we performed adoptive transfer of fluorescently‐labeled leukocytes in both mouse models. Cross‐sections of the aortic root and isolated enface aortic arch were examined by confocal microscopy. Shoulder regions of aortic lesions accumulated two fold more fluorescent‐leukocytes in Apoe−/−Ldlr−/− than in Apoeh/hLdlr−/− mice. Moreover, the endothelium of Apoe−/−Ldlr−/− mice displayed elevated levels of PECAM and JAM‐A.

Taken together these results indicate that apoE reduces atherosclerosis by suppressing the expression of cell‐adhesion molecules on the endothelium and in turn the recruitment of leukocytes to atheroma.
Increased systemic peroxynitrite levels and decreased expressions of lipolytic proteins in obese‐prone rats
Authors: Smith Brian , Ma Sheng Xing
Published: June 2021

Using the obese‐prone (OP) and obese‐resistant (OR) rat model, we studied the effects of diet induced obesity on peroxynitrite levels in various tissues/organs and expressions of lipolytic proteins in adipose tissue. After 8 weeks of high fat diet, animals were anesthetized and various tissues were harvested. Nitrotyrosine levels were analyzed by ELISA from the tissue homogenates. The protein levels of phosphorylated hormone‐sensitive lipase (phospho‐HSL) and perilipin, two key enzymes involve with lipolysis, in white adipose tissue were examined by Western blot analysis. Nitrotyrosine concentrations were 2–3 three times higher in all OP rat tissues we examined including skin, adipose tissue, skeletal muscle and small intestine. The protein level of phospho‐HSL in adipose tissue is as much as 20% lower in OP rats as compared to OR rats. As well, the protein levels of perliipin were also lower in OP rat adipose tissue as compared to OR rats. These results suggest a state of chronic systemic inflammation mediated by NOergic oxidant peroxynitrite may contribute to obesity development and attenuated lipolysis in OP rats. Supported by NIH (AT004620, and AT004504) and ADA 7‐07‐RA‐100 to S. Ma.
Aldosterone levels are inversely correlated to superoxide dismutase acitivity in the low birth weight newborn
Authors: Ojeda Norma Beatriz , Romero Damian Gaston , Dasinger John Henry , Alexander Barbara Thompson , Rhodes Phil
Published: June 2021

Low birth weight (LBW), is a risk factor for neonatal morbidity, mortality, and increased risk for cardiovascular (CV) disease. Epidemiological and experimental studies report increased aldosterone levels and oxidative stress as contributors to CV risk. Thus, the purpose of this study was to determine whether alterations in plasma aldosterone and activity of superoxide dismutase (SOD), an antioxidant, are associated to LBW. In a rodent model of pre‐term LBW induced by placental insufficiency Aldosterone levels were increased in LBW offspring (48±13 vs. 8±1 pg/ml/g, P<0.05; LBW vs. NBW, respectively). In a cohort of 39 newborn delivered at the University of Mississippi, pre‐term and term LBW newborns show increased aldosterone levels (354 ±110 vs. 197±11 pg/ml/k P<0.05; LBW vs. NBW, respectively); and reduced SOD activity (2±0.3 vs. 10±1 U/ml/k, P<0.05; LBW vs. NBW, respectively). A negative correlation was observed between aldosterone and birth weight in the animal model of LBW (Spearman r −0.9 P<0.05); and in human LBW newborns (Spearman r −0.4 P < 0.05). A negative correlation was observed between aldosterone levels and SOD activity in human LBW newborns (Spearman r −0.4 P < 0.05); and a positive correlation between SOD activity and birth weight (Spearman r −0.9 P < 0.05). Thus, studies from human and an animal model suggest parallel findings related to increased aldosterone and oxidative stress in LBW.
Cooperative role of NF‐κB signaling and PARP‐1 activity in the TNF‐induced inhibition of PHEX gene expression
Authors: Kiela Pawel R , Majewski Pawel M , Thurston Robert D , Ramalingam Rajalakshmy , Sabetisoofyani Arash , Ghishan Fayez K
Published: June 2021

Reduced bone mass is a common complication in chronic inflammatory diseases, although the mechanisms are not completely understood. Phex gene encodes an osteoblast Zn‐endopeptidase controlling bone mineralization and is inhibited by TNF in chemically‐induced colitis. Here, we confirm its downregulation in CD4+CD45RBhigh T‐cell transfer colitis, and delineate the molecular mechanism involved in TNF‐mediated inhibition of Phex transcription. TNF decreased expression of Phex in UMR106 cells and did not require de novo synthesis of a transrepressor. Transient transfections with Phex promoter constructs identified a region within ‐74nt containing weak putative NF‐κB binding sites. TNF induced recruitment of p65/p50 complex to two of those cis elements. Inhibition of NF‐κB signaling by a proteasome inhibitor increased the basal level of Phex transcription and abrogated the effects of TNF, while overexpression of p65 reduced promoter activity. Proteomic approaches identified poly(ADP‐rybose) polymerase (PARP‐1) constitutively interacting with Phex promoter, and TNF‐induced PARylation of p65 which potentiates its suppression of Phex transcription. TNF‐mediated downregulation of Phex was completely abrogated in PARP‐1 KO mice. NF‐κB signaling and PARP‐1 enzymatic activity are cooperatively involved in constitutive and inducible suppression of Phex.

Grant support: R37DK033209 (F.K.G.)
Integrated didactic and laboratory learning units in histology
Authors: Heidger Paul M. , Stone Justin A. , Dee Fred R. , Leaven Timothy C.
Published: June 2021

Medical and general dental histology at the University of Iowa are taught within the context of an organ systems course, with integration of structure and function serving as a guiding principle. Laboratory study utilizes both virtual and glass slides; pre‐laboratory tutorials precede formal, staffed laboratories, but many students choose virtual microscopy and individual home study as their primary laboratory learning mode. To integrate the learning of lecture and laboratory concepts, comprehensive study and review units have been developed that hyperlink lecture material, text assignments, and our Virtual Slidebox of Histology. The lecture outline for a given topic, complete with all illustrations and slides, is posted on the course web site. Hyperlinks are inserted directly within the lecture outlines to relevant text material in the licensed electronic version of one of the required texts (Junqueira, et al.), and to the appropriate annotated virtual slides in the Virtual Slidebox. Students have accepted enthusiastically this means of integrating didactic and laboratory study, as have faculty as a resource for reviewing concepts with students individually, or in small groups.
Interactive Learning Strategies for Moderately‐Sized Classrooms
Authors: Bruzzini Kristen Blake
Published: June 2021

Traditional classroom lecture experiences neither engage student interest nor foster student mastery and retention of course material. This research focuses on the use of interactive teaching strategies to engage student learning and understanding of Anatomy and Physiology (A&P). Activities occurred primarily in small groups and were designed to assess student understanding of core concepts without having to sacrifice content. These activities provided a key connection to learning using less time in lecture and more study time outside the classroom.
Expression of filamin‐A during tooth development
Authors: Miyamoto Ryoko , Yamada Aya , Iwamoto Tsutomu , Nakamura Takashi , Fukumoto Satoshi
Published: June 2021

Remodeling of the cytoskeleton is central to the modulation of cell shape and migration. Filamin‐A, encoded by the gene FLNA, is a widely expressed protein that regulates re‐organization of the actin cytoskeleton by interacting with integrins, transmembrane receptor complexes and second messengers. OPD1 belongs to a group of X‐linked skeletal dysplasias known as oto‐palato‐digital syndrome spectrum disorders that also include OPD2, Melnick‐Needles syndrome (MNS), and frontometaphyseal dysplasia (FMD). Recently, it has been demonstrated that mutations in the gene encoding the cytoskeletal protein filamin A (FLNA) are responsible for this group of clinically overlapping human syndromes. We identified the novel mutation in FLNA gene in MNS patient with tooth agenesis, root dysplasia and severe enamel hypoplasia. Mutation was observed in the rod domain (11th filamin repeat) of FLNA gene. In rodent tooth germ development, Filamin‐A was expressed in inner enamel epithelium and odontoblasts, but not other tooth cells. In cultured dental epithelial cells, filamin‐A localized under the cell surface and associated with F‐actin. These results indicate that filamin‐A is important for the organization of cytoskeleton and the differentiation of ameloblasts and odontoblasts.

Grant Funding Source: Ministry of Education, Culture, Sports, Science and Technology of Japan
Assessing joint vibration during spinal manipulation
Authors: Cramer Gregory D. , Ross Kim , Pocius Judy , Cantu Joe , Laptook Evelyn , Fergus Michael , Gregerson Doug , Selby Scott , Raju P. K.
Published: June 2021

This IRB‐approved study assessed the relationship between cavitation and zygapophysial (Z) joint gapping following spinal manipulation (adjusting) in 5 healthy volunteers. High signal MRI markers were used to accurately identify the T12, L3, and S1 spinous processes during scout views. Axial images of the L4/L5 and L5/S1 levels were obtained in the neutral supine position. Each subject was then positioned on the side, and accelerometers were placed over the marked spinous processes. Recording from the accelerometers was done from final positioning through adjusting. The subject was then scanned in side posture position (accelerometers removed). Using a digitizer, the greatest A‐P central Z joint articular surface distances were measured on the 1st and 2nd scans; the difference between the 2 measurement values was the gapping difference, GD; a positive value indicating an increase in gapping following the spinal adjustment. GD was compared between up‐side (adjusted), down‐side (non‐adjusted), cavitation, and non‐cavitation joints.

Results

GD was increased in Z joints that were adjusted [0.5 (SE 0.2) mm] vs. non‐adjusted [−0.2 (SE 0.2) mm], and vertebral segments with cavitation gapped more than no cavitation [0.8 (SE 0.4) vs. 0.4 (SE 0.2) mm].

Conclusions

A future clinical study is quite feasible. Forty subjects would be needed for appropriate power (0.80). Funding: NIH/NCCAM (#2R01AT000123).
The Effects of Exercise on Ventricular Wall Thickness and Fibrosis in Wild‐Type and Dystrophin‐Deficient Mice
Authors: Plochocki Jeffrey H , Costas Jeffrey M
Published: June 2021

Dystrophin is a cytoplasmic protein that connects muscle cells to the extracellular matrix. When absent, muscle degeneration and fibrosis occurs that resembles the histopathological presentation of Duchenne muscular dystrophy. In mice that lack dystrophin (Mdx mice), there is muscle degeneration and regeneration in the first few months of life, with fibrosis increasing after that. In this study, we evaluated the relationship between exercise and lateral ventricular wall thickness (LVT) in wild‐type and Mdx mice aged 11 weeks. Degree of cardiac fibrosis was assessed using a trichrome stain. Sedentary Mdx mice exhibited significantly larger LVT than wild‐type mice (P < 0.05). Exercise treated wild‐type mice had larger LVT than sedentary wild‐type mice on average, but this difference was not statistically significant. The LVT of sedentary Mdx mice was 28% larger than that of exercise treated Mdx mice (P < 0.05). Exercised treated Mdx mice were the only group that exhibited any noticeable fibrosis in the lateral aspect of the heart. Our findings suggest that exercise within the normal physiological range can accelerate degeneration and fibrosis of the lateral ventricular wall in Mdx mice.
Effects of swimming exercise and diet in tibias of rats: a biomechanical study
Authors: Okubo Rodrigo , Fernandes Cleverson Rodrigues , Frajacomo Fernando Tadeu Trevisan , Farnesi Ana Paula , Cardoso Vinicius Kannen , Falcai Maurício José , Marine Tassiana , Fazan Valéria Paula , Shimano Antônio Carlos , Volpon José Batista , Garcia Sérgio Britto
Published: June 2021

The purpose of this study was to evaluate the effects of swimming exercise in tibias of rats fed with different controlled diet. This study was approved by Ethical Committee in Animals Experiment. Ninety three‐month‐old male Wistar rats were randomly assigned to nine groups (n=10): (G1) normal diet control, (G2) hypo caloric diet control, (G3) hyper caloric diet control, (G4) normal diet 20‐minutes training, (G5) hypo caloric diet 20 minutes training, (G6) hyper caloric diet 20‐minutes training, (G7) normal diet 90‐minutes training, (G8) hypo caloric diet 90‐minutes training, e, (G9) hyper caloric diet 90‐minutes training. Hypo caloric diet is a 40% restriction of the total diet of the normal animals. Hyper caloric is 14% high‐fat diet. Normal diet is 4% fat. Physical exercise consisted of swimming on a large aquarium. Rats swam 5 days a week, 20 or 90 minutes, during 8 weeks. Tibias were dissected and tested in a universal testing machine. Data were analyzed using level of significance set at 5%. Data from our study suggest that bone resistance depends of the weight of animals acquired through diet. Swimming exercise programs did not alter mechanical properties of bones.

Grant Funding Source: FAPESP
Association Between Anatomical Measurements and Dynamic Knee Valgus in Healthy Women And Women With ACL Reconstruction
Authors: Rosario Martin G , Ortiz Alexis
Published: June 2021

Anthropometric measurements have been associated with anterior cruciate ligament (ACL) injury in women.

PURPOSE

Assess the association between anthropometric measures of the lower extremities and dynamic knee valgus in women with and without ACL reconstruction.

METHODS

Twenty‐nine women (non‐injured=15, ACL=14) participated in this investigation. Anthropometric measures included: height, weight, inter‐ASIS distance, ASIS‐to‐trochanter distance, leg length, knee width, and ankle width. Dynamic knee valgus was measured with a three‐dimensional motion capture system (120Hz) as the distance between markers placed at the lateral femoral epicondyles during the landing phase of a double‐legged 60‐cm height drop jump. T‐tests were used to compare anthropometric measures between groups. Multiple linear regressions were used to examine the association between anthropometric variables and valgus.

RESULTS

All measurements except knee width were statistically significant different (p>.05) between groups with the ACL group exhibiting greater values. Regression analyses showed no statistically significant association for the control group (r2=.51; p>.05), whilst the association for the ACL group was statistically significant (r2=.89; p=.007).

CONCLUSIONS

It appears that anthropometric measures are predisposing factors to dynamic knee valgus that in turn are directly related to ACL injury.

Grant Funding Source: NIH‐Clinical Research Education and Career Development in Minority Institution R25RR017589
The Anabolic Role of Cannabinoid Receptor in Bone
Authors: Del Carpio‐Cano Fabiola Elizabeth , Marcu Jahan , Popoff Christopher , Pixley Robin , Abood Mary , Popoff Steven , Safadi Fayez
Published: June 2021

Endocannabinoids and their G protein‐coupled cannabinoid receptors 1 (CB1) and 2 (CB2) are present in the skeleton. CB2 is expressed in osteoblast (OB), though CB1 is debatable. We have begun a characterization of the skeletal phenotype in CB1/CB2 double receptor knockout (CB1/2 dKO) mice, We also utilized specific CB1&2 receptor antagonists to examine their effects, either individually or in combination, on OB differentiation. We conducted micro‐CT analysis of femurs from 12 week‐old CB1/2 dKO mice demonstrating a significant reduction in cortical and trabecular bone volume and thickness, when compared to wild‐type mice. To examine the functional significance of CB1&2 receptors on OB differentiation, we established primary OB cultures utilizing newborn wild‐type calvaria. Alkaline phosphatase (ALP) activity was assessed to evaluate the effects of CB receptor antagonists on OB differentiation. Treatment with the CB1 or CB2 antagonist caused a significant, dose‐dependent inhibition of ALP activity. In cultures treated with both antagonists, the inhibition of ALP was more pronounced compared to either antagonist alone. Our results suggest that CB1 & CB2 are functional receptors during OB differentiation and in regulation of bone mass. Additional studies are warranted to evaluate intracellular signaling subsequent to ligand activation of CB1&2 in OB

Grant Funding Source: NIH‐NIAMS AR048892
Maternal expression of atrial natriuretic peptide and the fetal programming of cardiac hypertrophy
Authors: Armstrong David WJ , Tse M Yat , Wong Philip G , Pang Stephen C.
Published: June 2021

Introduction

The natriuretic peptide system (NPS) is a key regulator of cardiovascular homeostasis. Mice lacking atrial natriuretic peptide (ANP) display chronic hypertension with salt‐sensitivity and cardiac hypertrophy (CH). The effect of maternal ANP expression on the development of CH in offspring is unknown.

Objective

To determine the effect of maternal ANP genotype on the development of CH in ANP heterozygous (ANP+/−) offspring born to either ANP wild‐type (+/+) mothers (ANP+/−WT offspring) or ANP knockout (−/−) mothers (ANP+/−KO offspring).

Methods

9‐week old male ANP+/−WT and ANP+/−KO mice were treated with normal salt (NS; 0.8% NaCl) or high salt (HS; 8.0% NaCl) diet for 5 weeks. Mice were euthanized, organs were weighed, and left ventricular (LV) gene expression was measured using real‐time qPCR.

Results

LV‐to‐body weight (LV/BW) ratio did not differ between ANP+/−WT and ANP+/−KO fed NS. HS increased LV/BW ratio in ANP+/−WT but not in ANP+/−KO. Gene expression of corin, iNOS, ACE, AT1a, sGC‐α1 and ‐β1 were increased, and NPR‐C was decreased in ANP+/−WT, and unaltered in ANP+/−KO.

Conclusions

Maternal ANP ablation appears to protect against the development of CH in the ANP+/− mouse. Compensatory changes in the ANP−/− mother during development may confer salt‐resistance in the ANP+/− mouse later in life, the mechanism of which remains to be elucidated. (Support: Heart & Stroke Foundation of Ontario)

Grant Funding Source: Heart and Stroke Foundation of Ontario
Primer delivery by T7 gene 4 proteins
Authors: Zhu Bin , Lee Seung‐Joo , Richardson Charles C.
Published: June 2021

Gene 4 protein (gp4) encoded by bacteriophage T7 contains a C‐terminal helicase domain and a N‐terminal primase domain. After synthesis of the tetraribonucleotide primers gp4 must then transfer them to the polymerase for use as primers to initiate DNA synthesis. A truncated version of the full‐length 63‐kDa gp4, the 56‐kDa gp4, lacks the N‐terminal Cys4 zinc‐binding motif that is important in the recognition of primase sites in the DNA. We find that the 56‐kDa gp4 is defective in primer synthesis but delivers a wider range of primers to initiate DNA synthesis compared to the 63‐kDa gp4. This property of the 56‐kDa gp4 is potentially significant in the initiation of T7 DNA replication. We have identified DNA primases that are defective in primer delivery by screening 56‐kDa gp4s containing mutations for their ability to support the growth of T7 phage lacking gene 4 but containing a suppressor that allows it to grow in the presence of the 56‐kDa gp4. We find that Trp69 of the 56‐kDa gp4 is critical for primer delivery. Substitution of lysine for Trp69 yields 56‐kDa gp4‐W69K and 63‐kDa gp4‐W69K that are defective in primer delivery to the polymerase with other functions unaffected. DNA primase harboring lysine at position 69 fails to stabilize the primer on DNA as analyzed by surface plasmon resonance.
The role of Toll‐like receptor 3 in apoptosis of bewo cells hepatitis B virus induced
Authors: Wang Suping , Li Xiaoyan , Song Xiuxia , Feng Liping , Qu Yiqun
Published: June 2021

Objective

The aim of this study was to explore whether HBVcan replicate in bewo cells, whether HBV can induce apoptosis of bewo cells, and the role TLR3 may play in apoptosis induced by HBV.

Methods

PCR was used to detect HBV cccDNA expression of bewo cells cocultured with HBV positive serum. Flow cytometry, tarnsefarse‐mediated dUTP nick end‐labeling were used to detect apoptosis of bewo cells. Flow cytometry was conducted to measure TLR3 protein.

Results

HBV cccDNA can be detected in bewo cells co‐cultured with HBV DNA positive serum for 48 hours. when bewo cells were cocultured with 100μl HBV serum for 24h or 48h, early apoptotic rates, total apoptotic rates and apoptotic indexes were all statistically higher than that in the separate cotemporaneous group (P < 0.001). After PolyI:C prestimulation, early apoptotic rates and total apoptotic rates of the bewo cells co‐cultured with HBV serum for 24h or 48h were significantly decreased(P < 0.05); TLR3 protein's mean fluorescence intensity was statistically higher than that in no‐PolyI:C stimulated group(P=0.02).

Conclusion

Bewo cells which hepatitis B virus can replicate in may be a good model for HBV infection experiments. TLR3 may mediate the apoptosis of bewo induced by HBV.The activation of TLR3 by PolyI: C may inhibit apoptosis of bewo induced by HBV.
Effect of fatty acids on translational control of leukocytes infected with HIV‐1
Authors: Luchessi Augusto Ducati , Cambiaghi Tavane David , Curi Rui
Published: June 2021

During a virus infection, host cells suffer a general inhibition of protein synthesis. Initially, the viral RNAs activate the protein double‐stranded RNA‐dependent protein kinase (PKR). Active PKR phosphorylates the eukaryotic translation initiation factor 2 (EIF2) and impairs the general protein synthesis. However, the Tat protein of the HIV‐1 virus inhibits EIF2 phosphorylation and so the synthesis of the viral proteins occurs freely. Tat is phosphorylated by PKR and activates the elongation of viral RNAs. Results from our group indicate that the palmitic acid (PA) stimulates EIF2 phosphorylation in Jurkat cells probably by a process involving the protein PKR‐like endoplasmatic reticulum kinase (PERK). The PERK protein phosphorylates EIF2 in response to endoplasmic reticulum stress. Under a controlled pulse of PA the phosphorylation of EIF2 is reversible and does not induce a significant toxicity. Indeed, the pulse of PA impairs the cell general protein synthesis as reveled by polysome profile assay. In addition, evidence has been obtained that PA can modulate HIV‐1 infection.
The fructose 2,6‐bisphosphate cycle bioswitch and metabolic coordination
Authors: Okar David Anthony
Published: June 2021

Objective

Provide a comprehensive mechanistic description of metabolic regulation that can account for the full spectrum of positive synergistic effects observed while investigating the modulation of hepatic glycolysis as a therapy for diabetes‐associated hyperglycemia.

Method

Search pertinent literature for examples of metabolic coordination including those observed for hepatic fructose 2,6‐bisphosphate (F‐2,6‐P2 and 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (PFK‐2/FBP‐2) and identify common elements, thereby highlighting mechanisms that coordinate metabolism within the liver and in relation to fuel homeostasis.

Results

The most obvious regulatory elements common to metabolic coordination are chemical cycles, protein:protein interactions, and translocation between sub‐cellular locations. All of these constitute biological switches and some coordinate across levels of biological organization.

Conclusions

The PFK‐2/FBP‐2 dimer is a bioswitch that along with F‐2,6‐P2 primarily modulates the committing step to glycolysis and coordinately regulates multiple aspects of fuel metabolism at the molecular, cellular, tissue/organ, and systemic levels of biological organization. Funding: VA.
Subcellular localization of Saccharomyces cerevisiae glutamate dehydrogenase isoenzymes
Authors: Petersen Max , Trotter Pamela J.
Published: June 2021

Reductive amination of 2‐oxoglutarate by glutamate dehydrogenase (GDH) is a primary pathway for glutamate synthesis in Saccharomyces cerevisiae. There are three GDH isoenzymes: NADP‐dependent Gdh1p and Gdh3p and NAD‐dependent Gdh2p. Knowing the subcellular localization of the GDH enzymes is vital to understanding the role of compartmentation in glutamate homeostasis. Previous studies suggested cytosolic, nuclear, and/or mitochondrial localization, but the precise location of the enzymes remains unclear. Wild‐type and mutant (gdh3Δ) cells were grown on lactate to induce mitochondrial biogenesis, spheroplasted, and homogenized. Then, crude mitochondrial and extramitochondrial fractions were isolated by sedimentation. Analysis of cytochrome c oxidase as a mitochondrial marker and hexokinase as a cytosolic marker revealed limited cross‐fractional contamination. Measurement of NADP‐dependent and NAD‐dependent GDH activity conclusively demonstrated extramitochondrial localization of Gdh1p and Gdh2p enzyme activity, respectively. Comparison of data from wild‐type as compared to gdh3Δ cells implies extramitochondrial localization for Gdh3p. These data indicate that in S. cerevisiae 2‐oxoglutarate generated within the mitochondria must be transported out for conversion to glutamate by GDHs. (Supported by NIH grant GM069372 [P. J. T.] and a gift from the Grilly family [M.C.P.]).
GSK3β: a possible therapeutic target in Cardiac Autonomic Dysfunction in a mouse model for Type I Diabetes
Authors: Zhang Yali , Picard Kristen , Georgescu Serban , Vaickus Celestia , Park Ho‐Jin , Galper Jonas
Published: June 2021

Diabetic Autonomic Neuropathy (DAN) is characterized by parasympathetic dysfunction of the heart. Parasympathetic stimulation of the heart involves Ach binding to M2, and activation of the G protein‐coupled K+ channel, (GIRK1)2/(GIRK4)2. The transcription factor, SREBP1 is unregulated by insulin. Akt and GSK3β are important mediators of the effect of insulin. In type 1 diabetic mice, cardiac parasympathetic function was markedly decreased in response to carbacol, furthermore, the expressions of GIRKs, SREBP1, p‐Akt and p‐GSK3β in the atrium were decreased which were reversed to levels in WT by insulin and GSK3β inhibitor, LiCl. Insulin treatment of atrial myocytes increased GIRK4 expression and p‐Akt, which were inhibited by the PI3K inhibitor LY294002. MyrAkt induced the expression of SREBP1 and GIRK4. SREBP1 stimulated GIRK4 promoter activity in a dose‐dependent manner, and DN SREBP1 reversed the insulin effect on GIRK4 protein. In addition, insulin increased nuclear SREBP level, which was inhibited by LY. DA GSK3β decreased the expression of SREBP1 and GIRK4, while DN GSK3β increased. Our results suggest that increased parasympathetic response by insulin in type 1 diabetic heart involves activation of Akt, inhibition of GSK3β, and increased expression of SREBP and GIRKs. The role of Akt and GSK3β in this pathway may offer therapeutic targets for the treatment of DAN.
OASIS, an endoplasmic reticulum stress transducer, is involved in bone formation
Authors: Murakami Tomohiko , Nishimura Riko , Yoneda Toshiyuki , Ikegawa Shiro , Imaizumi Kazunori
Published: June 2021

OASIS (Old Astrocyte Specifically Induced Substance) is a bZIP transcription factor of the CREB/ATF family with a transmembrane domain. OASIS is cleaved at the membrane in response to endoplasmic reticulum (ER) stress. Its cleaved cytoplasmic domain translocates into the nucleus where it activates the transcription of target genes. OASIS is selectively expressed in some types of cells such as astrocytes and osteoblasts. Previously, we demonstrated that OASIS activates the transcription of the gene for type I collagen, Col1a1, and contributes to the secretion of bone matrix proteins in osteoblasts. OASIS−/− mice exhibit severe osteopenia and growth retardation. The osteopenia resulted from a decrease in type I collagen in the bone matrix and a dysfunction of osteoblasts, which show abnormal expansion of the ER with the accumulation of bone matrix proteins. To examine whether the phenotypes of OASIS−/− mice are rescued by OASIS transgene in osteoblasts, we generated transgenic mice that express exogenous OASIS specifically in osteoblasts (obOASIS mice). Introduction of exogenous OASIS in osteoblasts rescued severe osteopenia observed in OASIS−/− mice. In contrast, that could not improve the growth retardation in OASIS−/− mice. These results indicated that the growth retardation observed in OASIS−/− mice could be caused by other mechanisms different from osteoblast dysfunction.
Knockdown of Lysosomal Cystine Transporter Causes the Fanconi Syndrome in Primary Renal Cells
Authors: Taub Mary , Springate James E , Cutuli Facundo
Published: June 2021

The autosomal recessive disorder cystinosis is due to a defect in cystinosin, a lysosomal cystine transporter. The Fanconi Syndrome emerges in the kidney in this disorder, due to reduced reabsorption of phosphate (Pi) and other solutes by the renal proximal tubule (RPT). The hypothesis was examined that in RPT cells the defect in cystinosin not only causes a reduction in the activity of apical Na+ solute/cotransporters, but also results in increased apoptosis. In order to examine this hypothesis, siRNA was used to knock‐down cystinosin in primary rabbit RPT cells. Na+ dependent Pi and alpha methyl‐D‐glucoside uptake was reduced by more than 60% following a 3‐day incubation with cystinosin siRNA (and a 80% knock‐down of cystinosin) vs. scrambled (scr) controls. Na+ independent 3‐0‐methyl‐D‐glucoside uptake was unaffected. The sensitivity of primary RPT cells to apoptotic stimuli was examined. TNFα caused a 10.8 +/− 0.7 fold increase in caspase 3 activity in RPTs treated with cystinosin siRNA (2.4 fold higher than scr controls), and 20 μM cisplatin resulted in the death of 31 +/− 3% primary RPTs treated with cystinosin siRNA after 12 hr, a value 2.2 fold higher than in scr controls. Cystinosin siRNA also reduced cell growth by 45 +/− 10%. Further studies are in progress to determine whether these alterations can be explained by a change in the cellular redox state caused by reduced transport of cystine from lysosomes.
The antidiabetic metformin is toxic to liver mitochondria of Zucker Diabetic Fatty (ZDF) rats: a cellular metabolomic study with 13C‐lactate and carbon 13 NMR
Authors: Baverel Gabriel , Pinteur Claudie , Martin Guy , Gauthier Catherine
Published: June 2021

Metformin decreases hyperglycemia in diabetic subjects and animals. This effect results in part from the inhibition of hepatic gluconeogenesis. The objective of this study was to investigate in vitro the effect of metformin on hepatic lactate gluconeogenesis by using an innovative approach.

Precision‐cut liver slices from adult male ZDF rats were incubated with variously 13C‐labeled lactates (5 mM) or with 5 mM unlabeled lactate plus 25 mM 13C‐bicarbonate. Substrate removal and product formation were measured by enzymatic and 13C‐NMR methods.

In slices from fasted lean ZDF rats, metformin dose‐dependently inhibited 13C‐glucose synthesis from [2‐13C]‐lactate (IC50 2.3 mM). The labeling patterns of the glucoses synthesized indicated that lactate gluconeogenesis involved not only the passage of carbons through pyruvate carboxylase and the reversible equilibration of oxaloacetate with fumarate but also the entire tricarboxylic acid cycle.

In slices from fed diabetic ZDF rats, 2 mM metformin inhibited lactate consumption, glucose production, the synthesis of all the 13C‐labeled glucose carbons, the production of 13CO2 and reduced the cellular ATP level. By contrast, metformin dramatically increased lactic acid production and ketogenesis.

These results strongly suggest that a compound found to be toxic to liver mitochondria in vitro should not necessarily be withdrawn from drug development.
Renin‐angiotensin system involvement in the generation of reactive oxygen and nitrogen species in kidney mitochondria in hypertension
Authors: López‐Castillo Lilia , Godínez‐Hernández Daniel , Manzo‐Avalos Salvador , Urquiza‐Marín Héctor , Saavedra‐Molina Alfredo
Published: June 2021

In maintaining blood pressure (BP) various vasoactive substances are in equilibrium. Nitric oxide (NO) and angiotensin II (Ang ll) play a key role in controlling the BP. The NO and Ang II are regulated each other to maintain adequate Ca2+ concentrations within the cell. Hypertension (HP) is a consequence of alterations in these mechanisms. HP presents a polygenic and multifactorial etiology yet to be defined completely. Studies found an increase production of reactive oxygen species (ROS), which uncoupled the endothelial nitric oxide synthase (eNOS) activity. In this work we analyze the effect of HP induced by L‐NAME (competitive inhibitor of NOS), in normotensive rats, inhibition of Ang II synthesis with captopril (an inhibitor of angiotensin converting enzyme (ACE) in normotensive and hypertensive rats, ACE inhibition with captopril, measuring the levels of AT1 receptor expression in the kidney, as well as the different mitochondrial parameters such as NO synthesis (mtNO), oxygen consumption and H2O2 production. Renal damage results measured as creatinine, urea, proteinuria has no changes during the time of HP induction. Mitochondrial NO synthesis diminished 36% in the hypertensive rats group, while had no changes in the other groups. Results of state 3 and 4 of mitochondrial respiration were increased 1.5‐fold and 2‐fold, respectively in the hypertensive rats group, and no change were detected in the other groups. The results suggest that mtNO exerted a crucial effect on rat kidney mitochondria in hypertension.
Extending XMLPipeDB to create GenMAPP‐compatible gene databases for the analysis of DNA microarray data from human pathogens
Authors: Parks Kelly C , Hirning Andrew J , McDonald Kelia , Dionisio John David N. , Dahlquist Kam D
Published: June 2021

The GenMAPP suite is a powerful tool for viewing and analyzing DNA microarray data on biological pathways and performing Gene Ontology term over‐representation analysis. However, the use of GenMAPP and MAPPFinder is limited to those species for which a GenMAPP‐compatible Gene Database exists. Currently, these Gene Databases are based on Ensembl, which is limited to (mostly) animal species. Furthermore, the process of creating a Gene Database is sensitive to changes in flat file formats. To extend the use of GenMAPP for plants and microorganisms, and to create a method for database creation that was robust to changes in file formats, we created XMLPipeDB, an open source tool chain for building relational databases from XML sources. The tool chain reads an XSD and automatically generates an SQL schema, Java classes, and Hibernate mappings. It also provides functionality for database configuration, importing data, performing queries, and exporting GenMAPP‐compatible Gene Databases based on data from UniProt and Gene Ontology. We used XMLPipeDB to update the Gene Database for Saccharomyces cerevisiae and create new ones for Mycobacterium tuberculosis, Pseudomonas aerugenosa, and Staphylococcus aureus. We have used our new Gene Databases to analyze previously published DNA microarray data for each of these organisms, generating new findings that were not previously reported.
Gene Ontology term enrichment analysis of gene expression changes observed in the TRAMP mouse model of prostate cancer upon treatement with green tea catechins
Authors: Speicher Stephen Vincent , Dahlquist Kam D
Published: June 2021

The primary purpose of this study was to examine the effect of green tea on the global gene expression profile in the transgenic adenocarcinoma mouse prostate model for prostate cancer (TRAMP model). The mechanism by which green tea alters gene expression and affects an organism on the cellular level is relatively unknown. We obtained published DNA microarray data from McCarthy et al. (2007, Molecular Oncology 1:196–204). There were three groups of mice in this study: water‐fed wild type C57BL/6 mice (controls), water‐fed TRAMP mice (prostate cancer progressing), and green tea catechin‐fed TRAMP mice (prostate cancer delayed) that were sacrified at three different time points 12, 17, 24 weeks of age, with three biological replicates in each group. Labeled RNA from each mouse was hybridized to an Affymetrix Gene Chip. We reprocessed the data using the dChip software to determine differential gene expression. We then used the GenMAPP and MAPFinder programs to perform Gene Ontology (GO) term enrichment analysis to determine which GO terms were overrepresented in the data that were not reported in the original published work. We would like to gratefully acknowledge Timothy J. Yeatman, Saverio Bettuzzi, and Steve Enkemann for providing us with the complete microarray dataset.
Visualizing influenza hemagglutinin nanoanatomy using ProfileGrids: dissecting novel H1N1 features
Authors: Roca Alberto I
Published: June 2021

ProfileGrids have been introduced as a novel bioinformatic visualization method to analyze very large multiple sequence alignments (http://www.biomedcentral.com/1471‐2105/9/554). We apply this technique to the influenza hemagglutinin protein family to characterize all of the conserved and variable bioinformatic elements. We define a uniform ontological nomenclature of these elements to facilitate the discussion of nanoanatomical observations. Two hemagglutinin features that we present with respect to the recent swine flu (H1N1) outbreak are the distribution glycosylation motifs and the visualization of rare residues unique to human Mexico H1N1 hemagglutinin sequences. We also describe an update to the JProfileGrid Java software that now includes abilities suitable for large protein sequence databases such as Pfam and the NCBI Influenza Virus Sequence Database. [AIR supported by the Erasmo Foundation (grant TSC13702).]
PKC‐alpha mediated induction of miR‐101 in human hepatoma cells
Authors: Chiang Chao‐Wei , Huang Yi , Leong Ka‐Wai , Chen Shu‐Jen , Chen Hua‐Chien , Chou Chen‐Kung
Published: June 2021

Protein Kinase C (PKC) is a serine/threonine kinase that involved in controlling of many cellular processes such as cell proliferation and differentiation. We have shown previously that TPA (12‐O‐tetradecanoylphorbol 13‐acetate) induces cell cycle arrest in G0/G1 phase in human hepatoma HepG2 cells. In this study, we examined TPA regulated miRNA expression profile in human hepatoma HepG2 cells. Six miRNA were identified as putative tumor suppressor genes since there both are induced by TPA and are also down regulated in human hepatoma tissues. Among six miRNA, miR‐101 has been intensively studied recently. MiR101 targets two subunits of PRC2 complex, enhancer of zeste homolog 2 (EZH2) and EED, and was shown to play negative role in cell growth in human prostate, breast and liver cancers. We showed that TPA not only up regulated miR‐101 expression, but also reduced protein level of EZH2, EED and H3K27me3 in HepG2 cells. Using lenti‐virus‐mediated shRNA to knockdown endogenous PKC‐alpha expression, we observed that TPA induced growth arrest, elevation of miR‐101 and reduction of EZH2, EED and H3K27me3 proteins were all PKC‐alpha dependent. Therefore, we proposed that PKC‐alpha mediated miRNA expression may play an important role in carcinogenesis of human hepatoma.
Over Expression, Purification and Crystallization of the PTF1‐J and PTF‐L Complexes
Authors: Kohrs Alix , Stein Kelly , Zhang Mengqing , Shuller Lauren , Marshall Andrea , Christy Rachel , MacDonald Raymond , Coats Ward
Published: June 2021

Students in Biomedical Research Studies at Hillcrest High School in Dallas, Texas, participate in a ongoing research program at the University of Texas Southwestern Medical Center to study the structure and function of a mammalian transcription factor complex. Ptf1a is a transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a common E‐box binding protein (E12/47, HEB, or TCF12), Ptf1a and either Rbpj or Rbpjl. The Rbpj form of the complex (PTF1‐J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl‐form (PTF1‐L) is required for the formation of mature acinar cells. PTF1‐L is involved in an auto‐regulatory loop for the maintenance of transcription of both Ptf1a and Rbpjl genes. We have utilized PCR to clone full length and truncated forms of Ptf1a, HEB, E12/47, Rbpj and Rbpjl. These cDNAs have been subcloned into the bacterial expression vector pSMT3. The over expressed proteins will be purified and crystallized in order to perform structural determination of PTF1‐J and PTF1‐L complexes. Determination of the structures for the PTF1‐J and PTF1‐L complexes will elucidate the structural motifs required for the protein‐protein interactions found in the trimeric complex, as well as the mechanism and the structural determinants for the strong cooperative DNA binding of the complexes.
Determination of the Transcription Factor Binding Specificity of the Id Protein Family Members
Authors: Osborn Nicholas , Williams Sam , Fein Zachary , Degani Jesse , Mayberry Brett , Boardman Brandon , Haspel Steele , Johnson Parker , Guthrey Reggis , MacDonald Raymond , Coats Ward
Published: June 2021

Students in Biomedical Research Studies at Hillcrest High School in Dallas, Texas, participate in a research program at the University of Texas Southwestern Medical Center to study the structure and function of Id transcription factors. The DNA‐binding Id proteins play roles in cell differentiation, aging, growth, and apoptosis. Id proteins affect the balance between differentiation and cell growth by negatively regulating the basic‐helix‐loop‐helix (bHLH) transcription factors, and are found in nearly all mammals (including humans). Id proteins also bind to certain cell cycle regulatory proteins, such as pRB and Ets‐family factors, thus making Id proteins in some cases tumor promoters. The Id proteins have been linked to the processes in tumors that facilitate growth and blood vessel formation. The role of Id proteins suggests they are possible targets for treatment in human diseases. In the current study, we will over‐express, purify, crystallize, and perform structural determination of the Id family members complexed with known bHLH protein partners. We have utilized PCR to clone mouse cDNAs for Id2, Id3, and Id4 from mRNA. Determination of the Id protein structures will augment our basic understanding of these proteins and their role both in cell cycle regulation and in disease.
Cyclic AMP/PKA‐triggered secondary transcrption networks associated with growth arrest and apoptosis in S49 lymphoma cells
Authors: Wilderman Andrea C , Insel Paul A , Zambon Alex C
Published: June 2021

The second messenger cyclic AMP (cAMP) acts via protein kinase A (PKA) to induce growth arrest and apoptosis in murine S49 lymphoma cells by mechanisms that are poorly understood. Here, we devised a bioinformatic method to predict the transcription factors (TFs) and their target genes that are activated or repressed downstream of cAMP/PKA. Our bioinformatic method identifies patterns of transcriptional co‐regulation by clustering and predicts the TFs that regulate the clusters by comparative genomic analysis of the 5′ regulatory regions for enrichment of TF binding sites. We used this methodology to analyze gene expression patterns of S49 cells treated for 0, 2, 6, and 24 hrs with the PKA‐activating analog 8‐CPT‐cAMP and found a potential role for a number of TFs and their target genes. We validated alterations of TF activity by electrophoretic mobility shift assay (EMSA). Increases in CREB activity reflect its role as a transcription activator of cAMP/PKA while increases in PPARγ, NFkB, and FOXO activity are associated with transcriptional down‐regulation. The findings indicate the utility of bioinformatic methods for predicting secondary transcriptional networks from gene expression data and define candidate TFs that regulate cAMP/PKA targets. (Supported by grants from NIH and the Lymphoma and Leukemia Society)