bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell Adhesion and Fibrosis Affect the Mechanical and Electrical Coupling Of Cardiomyocytes in Human Atrial Fibrillation
Authors: Bodi Ilona , Jessen Walter , D'souza Karen , Koch Sheryl , Akhter Shahab , Wolf Randall , Schwartz Arnold , Aronow Bruce
Published: June 2021

Background: Atrial fibrillation (AF) is the most common sustained arrhythmia. Long term AF leads to extensive structural remodeling with progressive contractile and electrophysiological dysfunction and system‐wide sequelae. We compared gene expression profiles of left atrial appendages from patients suffering from AF to normal atrial tissue from patients without history of AF.

Methods and Results: The study consisted of three groups of patients; those with no history of AF in sinus rhythm (SR) and patients undergoing cardiac Mini‐Maze surgery for paroxysmal AF (PAF) and chronic AF (CAF). Gene expression analysis was carried out using Affymetrix microarrays and confirmed by western‐blot. Relative to SR controls, PAF and CAF samples showed statistically significant increases in the expression of genes associated with TGF‐β signaling and focal adhesion, including BMP6, CTGF, DPT, FN1, FST, JAM2, POSTN, SMAD7 and TNFAIP6. Increased expression of POSTN has also been implicated in myocyte hypertrophy in human ventricle during end‐stage heart failure. Dysregulated expression of TRDN, HDAC9, MYL2, TPM3 and TNNI3, significantly associated with calcium signaling, was only identified in patients with CAF. A key biomarker of inflammation, IFNG, was significantly upregulated between PAF and CAF. Conclusions: Our results strongly support the hypothesis that therapeutic measures designed to limit the extent of TGF‐β signaling and fibrosis such as the use of the ACEI losartan which induces regression of severe myocardial fibrosis, may be highly effective at limiting long‐term electromechanical dysfunction.
The functional analysis for the association of ALOX5 polymorphisms with asthma
Authors: Chang Hun‐Soo , Park Se‐Min , Park Jong Sook , Jang An‐Soo , Park Sung Woo , Park Byung Lae , Shin Hyoung Doo , Park Choon‐Sik
Published: June 2021

The leukotrienes constitute a group of arachidonic acid‐derived compounds suggesting important roles in inflammation and immediate hypersensitivity. ALOX5 is a key enzyme required for the production of the cysteinyl‐leukotrienes.

We genotyped five SNPs among these polymorphisms in a Korean population. The ratio of major allele homozygote of −754C>G and +21757G>A, which were located on promoter region and intron 3 of ALOX5, respectively, were higher in asthmatics than those in normal subjects (p = 0.001). In haplotype analysis, the frequency of ht1 were significantly higher in asthmatics (p = 0.001), and that of ht5 were lower in asthmatics (p = 0.007). Using reporter assay and Real time‐PCR, we revealed that genotypes on −754C>G affected the promoter activity followed by mRNA expression. Furthermore, we found genotypes on +21757G>A affected on the production of a novel alternatively spliced variant of ALOX5 resulting protein which lack lipoxygenase domain. And we revealed that genotypes on +21757 G>A affected by mRNA expression.

Our results suggest that the ALOX5 polymorphisms may affect on the gene expression and/or alternative splicing of ALOX5 and are associated with the development of asthma.

* This study was supported by a grant of the Korean Health 21 R&D project, Ministry of Health & Welfare, Republic of Korea (01‐PJ10‐PG6‐01GN14‐0003).
Plasminogen trafficks through the regulated secretory pathway in hippocampal neurons
Authors: Chavarha Mariya , Jacobs Conor , McAllister Kevin , Scalletar Bethe A , Lochner Janis E
Published: June 2021

Plasminogen has emerged as a key player in learning and memory and neurodegeneration in the hippocampus, and yet little is known about its localization or secretory trafficking to synaptic sites in hippocampal neurons. To address these issues, we constructed a fluorescent chimeric protein, plasminogen‐mCherry, to facilitate a study of the packaging, transport, and synaptic localization of plasminogen in cultured hippocampal neurons. Our results indicate that the plasminogen chimera localizes to regions of the soma typically occupied by the Golgi and to spherical organelles that are distributed throughout the soma and processes. We find 82% co‐localization of puncta containing plasminogen chimeras with markers for dense‐core granules (DCGs), suggesting that secretion of plasminogen from hippocampal neurons is regulated. Plasminogen chimeras were found, co‐localized with the neuromodulatory protein pro‐brain derived neurotrophic factor (proBDNF), within DCGs at post‐synaptic sites. These results demonstrate that plasminogen is co‐packaged with proBDNF and traffics through the regulated secretory pathway of hippocampal neurons to post‐synaptic sites where it is strategically positioned to undergo co‐release with other neuromodulatory proteins. This work was supported by National Institutes of Health grants 2 R15 NS40425‐02 and 2 R15 GM061539‐02.
Down‐regulation of the mitochondrial pathway for de novo fatty acid synthesis compromises lipoylation pathways in human embryonic kidney cells
Authors: Feng Dejiang , Smith Stuart
Published: June 2021

Recent studies have demonstrated the presence of freestanding enzymes in the mitochondrial matrix that constitute a pathway for de novo biosynthesis of fatty acids. One of the major products, octanoyl‐ACP, is utilized as a precursor for lipoyl moieties that are essential for functioning of several key mitochondrial enzymes. The objective of this study was to assess the importance of this pathway to mitochondrial integrity by down‐regulating formation of the ACP component. HEK293 cells were transfected with siRNA targeting mitochondrial ACP. Knock‐down efficiency was monitored by quantitative real‐time RT‐PCR and protein lipoylation was assayed by Western blotting, using lipoic acid antibodies. Preliminary results show that exposure to siRNA reduced ACP mRNA levels by 95%, significantly retarded cell growth and lowered lipoylation of the E2 subunits of both pyruvate and oxoglutarate dehydrogenases by 90%. Thus the de novo synthesis of fatty acids in the mitochondrial matrix appears crucial for maintenance of mitochondrial integrity. Supported by NIH grant GM06717
Role of MGA2 in regulation of lipid metabolism in Saccharomyces cerevisiae
Authors: Rice Caitlin , Heneveld Katherine , Vankempen Rachel , Cooke Matthew , Treloar Natalie , Stukey Joseph , McDonough Virginia
Published: June 2021

The Saccharomyces cerevisiae MGA2 gene encodes a protein that is an important regulator of lipid metabolism in the cell. Mga2p plays a major role in unsaturated fatty acid production through its regulation of the transcription and mRNA stability of OLE1, whose product is the Δ‐9 fatty acid desaturase. We have found that mga2Δ strains also contain elevated relative amounts of squalene, a sterol pathway intermediate, when compared to wild‐type cells. A possible explanation for the higher squalene levels detected in mga2Δ cells is lower expression of the ERG1 gene. ERG1 encodes squalene epoxidase, which catalyzes the conversion of squalene to squalene epoxide. To explore whether MGA2 plays a role in regulating ERG1 expression, we first examined effects on ERG1 transcription using a promoter‐lacZ reporter gene. We report here that in addition to MGA2's role in regulation of unsaturated fatty acids, MGA2 is required for full basal expression of ERG1. In contrast to OLE1, we found that ERG1 expression was not repressed by unsaturated fatty acids. However, ERG1 expression was elevated in response to miconazole, an inhibitor of sterol synthesis in yeast
Why does a defect in choline kinase beta cause muscular dystrophy in mice?
Authors: Wu Gengshu , Vance Dennis
Published: June 2021

Muscular dystrophies (MD) are a group of more than 30 genetic diseases characterized by progressive weakness and degeneration of the skeletal muscles. There is no specific treatment to stop or reverse any form of MD due to limited knowledge. We recently uncovered a natural mutation (rmd) in the Chkb gene that resulted in a complete loss of choline kinase (CK) activity in skeletal muscle, and the mice developed severe and progressive hindlimb MD. It is the first demonstration of a defect in a phospholipid biosynthetic enzyme causing MD. Why does muscular dystrophy occur? We observed decreased phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio both in hindlimb muscle and in mitochondria associated with sarcolemmal integrity and megamitochondria formation. Decreased PC level is due to three factors: down‐regulated CTP:phosphocholine cytidylyltransferase (CT) activity, the rate‐limiting enzyme for PC biosynthesis; increased PC turnover due to increased phospholipase activity, and a limited source of phosphocholine in Chkb−/– mice. In vivo experiments further demonstrated that the incorporation ability of 3H‐choline into PC was significantly decreased in affected muscle. Decreased regeneration ability of Chkb−/–mice may also contribute to muscular dystrophy. In affected muscle there was increased expression of myostatin and decreased proliferating cell nuclear antigen (PCNA).

This research was supported by a grant from Alberta Heritage Foundation for Medical Research and Canadian Institutes of Health Research.
On the differential lipolytic capabilities of heart kidney, testis and spleen for young adult guinea pig. A chromatographic study
Authors: Helmy Fatma M. , White Sanese , Amiri Sohrab , Amiri Rohina , Saliu Aminat , Juracka Amal
Published: June 2021

We have recently reported the preferential deacylation of cardiolipin (CL) in a variety of mammalian myocardia and pectoral muscle of some birds by endogenous phospholipases (PLA1 and/or PLA2) during in vitro incubation at pH 7.4 and 38°C for 60 minutes of whole tissue homogenate (as source of phospholipids and phospholipases) with production of monolysocardiolipin (MLCL) and concurrent reduction of CL. In contrast, whole tissue homogenate of rat spleen treated similarly selectively deacylated ethanolamine plasmalogen (PE) producing lyso alkenyl PE.

In the present study, we conducted in vitro incubation of whole tissue homogenate of guinea pig heart, kidney, testis and spleen at pH 7.4 and 38°C for 60 minutes. Subsequent TLC (thin layer chromatography) analysis revealed: a) a noticeable high level of MLCL and a very low level of CL in control testis, b) guinea pig heart and kidney revealed a high level of CL and no MLCL was detected. MLCL was only produced (in both heart and kidney) subsequent to in vitro incubation, c) very low level of CL was shown in control sample of guinea pig spleen and no MLCL was produced, d) guinea pig heart has both ethanolamine (PE) and choline plasmalogens (PC). PE plasmalogen is the only alkenyl species present in guinea pig kidney, testes and spleen.

These data raised some questions pertaining to the lipolytic capabilities of diverse tissues and will be discussed in details.
Down‐regulation of mannosylphospho dolichol synthase (DPMS) in tunicamycin treated capillary endothelial cells
Authors: Banerjee Dipak K , Martinez Juan A , Banerjee Aditi , Baksi Krishna
Published: June 2021

The exact mechanism of switching to a new angiogenic phenotype during tumor growth is currently unknown. But, we have observed a direct relationship between the dolichol cycle and the cell cycle dynamics during in vitro angiogenesis. A non‐transformed capillary endothelial cell line when exposed to 2mM 8Br‐cAMP the cell proliferation enhanced ∼70% with one‐half of the cell population in the S phase. A significant increase in the lipid‐linked oligosaccharide (LLO) biosynthesis and turnover, and the expression of cell surface N‐glycans was also observed. Mannosylphospho dolichol synthase (DPMS) activity was also upregulated. The objective of the present study is to evaluate the status of the DPMS during anti‐angiogenic response of tunicamycin. To test this hypothesis we have examined the DPMS activity in tunicamycin treated cells as a function of time. The DPMS activity in cells has been depleted well before the induction of apoptosis. Examination of DPMS expression by Western blotting indicated down regulation of DPMS by 70–80% within 3 hours of tunicamycin treatment. Furthermore, there was no trace of DPMS after 12 hours of tunicamycin treatment. We, therefore, conclude that DPMS is an angiogenesis sensor. Supported by the grants NIH U54‐CA096297 and the Susan G. Komen Breast Cancer Foundation BCTR5820 (DKB), and NIH/NCRR/RCMI grant G12‐RR03035 (KB).
Computational identification of novel peripheral protein‐membrane interactions
Authors: Kufareva Irina , Dancea Felician , Kiran Mahadev Ravi , Polonskaya Zinaida , Overduin Michael , Abagyan Ruben
Published: June 2021

Soluble proteins are recruited to membranes in many cellular processes. The information about the ability of a protein to transiently interact with the bilayer often helps understanding its function. However, obtaining such information experimentally is costly and complicated.

In this study, we present a fast computational method for detecting potential peripheral membrane proteins and their individual membrane‐inserting residues. The method relies on 3D structure alone and does not refer to homology with known membrane‐targeting modules. It is based on the analysis of protein surface curvature and statistical membrane propensities of the chemical groups.

Using the method, we screened a set of 521 medium‐size protein domains with known X‐ray structures and residue chemical shift assignments. We identified four novel transient membrane binders: plant acetyltransferase AT1G, bacterial methionine sulfoxide reductase, human ARF‐1, and human von Willebrand factor. Bilayer interaction of these proteins was assessed by monitoring chemical shift changes in their 1H,15N HSQC spectra upon micelle addition. Based on the obtained per‐residue data, membrane docking position of each domain was determined by rigid‐body Monte Carlo optimization in a membrane/solvent boundary map. Analysis of the obtained positions proved the validity of our prediction.

This work was supported by NIH grant 5‐R01‐GM071872‐02.
Inactivation of G‐protein coupled receptor 48 impairs definitive erythropoiesis at midgestation through downregulation of ATF4 pathway
Authors: Luo Weijia , Song Huiping , Luo Jian , Weng Jingsheng , li Dali , Liu Mingyao
Published: June 2021

G protein‐coupled receptors (GPCRs), one of the most versatile groups of cell surface receptors, can recognize specific ligands from neural, hormonal, and paracrine organs and regulate cell growth, proliferation and differentiation. Gpr48/LGR4 is a recently identified orphan GPCR with unknown functions. To reveal the functions of Gpr48 in vivo, we generated Gpr48−/− mice and found that Gpr48−/− fetuses displayed transient anemia during midgestation and abnormal definitive erythropoiesis. The dramatic decrease of definitive erythroid precursors (Ter119pos population) in Gpr48−/− fetal liver at E13.5 was confirmed by histological analysis and blood smear assays. Real‐time PCR analyses showed that in Gpr48−/− mice both adult hemoglobin α and β chains were decreased while embryonic hemoglobin chains (ξ, βH1 and Єy) were increased, providing another evidence for the impairment of definitive erythropoiesis. Proliferation but not apoptosis was deficient in Gpr48−/−fetal liver, associated with the down‐regulation of c‐Myc and cyclin D1 expression levels. Furthermore, ATF4, a key transcription factor in erythropoiesis, was downregulated in Gpr48−/− fetal livers during midgestation stage through cAMP‐PKA‐CREB pathway, suggesting that Gpr48 regulated definitive erthropoiesis through ATF4‐mediated definitive erythropoiesis.
The RGS homology domains of D‐AKAP2 regulate the endocytic recycling compartment through complexes with Rab4 and Rab11
Authors: Eggers Christopher , Taylor Susan S.
Published: June 2021

Although polymorphisms in D‐AKAP2 (AKAP10) have been linked to morbidity and mortality, heart disease, and breast cancer, the biological function of the protein has remained unknown. In addition to known PKA and PDZ binding motifs at the C‐terminus, D‐AKAP2 contains two Regulator of G‐protein signaling Homology (RH) domains with no known interacting partners. We have shown that the RH domains can localize the protein to the endocytic recycling compartment. Co‐expression of D‐AKAP2 with GFP‐tagged Rab4 or Rab11, but not Rab5 or Rab7, causes recruitment of D‐AKAP2 from the cytosol and enlargement of endosomal structures that are accessible to endocytosed transferrin. Flag‐tagged D‐AKAP2 can co‐immunoprecipitate the wild‐type and GTP‐locked mutants of Rab4 and Rab11, but not GDP‐locked mutants or Rab5. A yeast two‐hybrid screen identified the gamma subunit of the AP‐1 clathrin adaptor protein complex as an interacting partner, and we have confirmed this with co‐immunoprecipitation and in vitro GST pull‐down studies. Receptor trafficking experiments with overexpressed and knocked‐down D‐AKAP2 suggest that it may act as a Rab effector regulating the recycling of membrane proteins.
M3‐Muscarinic Receptor Activation of ERK and Cell Growth Requires Calcium/Calmodulin‐dependent Protein Kinases in MCF‐7 Cells
Authors: Abell Ellen , Wagner Andrea , Schmitt John Michael
Published: June 2021

The extracellular signal‐regulated protein kinase (ERK) signaling pathway is found in diverse cells throughout the human body. ERK activation has been implicated in breast cancer cell growth and proliferation. Studies have shown that ERK is activated by carbachol, a G Protein‐Coupled Receptor (GPCR) agonist, which increases intracellular calcium in MCF‐7 cells. The calcium/calmodulin‐dependent protein kinase (CaM K) family of proteins including CaM KK, CaM KI, and CaM KII can be activated by increased intracellular calcium. Our goal was to determine whether CaM Ks may be responsible for ERK activation and cell proliferation in carbachol‐treated MCF‐7 cells and evaluate which GPCR was responsible for these events. Carbachol treatment of MCF‐7 cells triggered ERK 1/2 phosphorylation within 5 minutes. Treatment with KN‐93, a general CaM Kinase inhibitor and the MEK inhibitor U0126 blocked ERK activation. Carbachol increased MCF‐7 cell growth nearly 4‐fold, an effect that was also dependent upon CaM Ks and MEK. Interestingly, CaM KK was responsible for ERK activation and cell growth. Pretreatment of MCF‐7 cells with 4‐DAMP, a selective M3 receptor antagonist, completely blocked carbachol's activation of ERK and cell growth. Taken together these results suggest that carbachol stimulated ERK phosphorylation and MCF‐7 cell growth by the M3 subtype GPCR receptor perhaps through CaM KK.
Intersectin (ITSN) regulates Ras activation of PI3K Class2β
Authors: O’Bryan John P. , Chen Jun‐Yu , Wong Katy A
Published: June 2021

Intersectin (ITSN) is an evolutionarily conserved multi‐domain scaffolding protein that regulates endocytosis as well as a number of signal proteins including Ras, Cbl, and PI3K. Although ITSN activates Ras on intracellular vesicles, this pool of Ras did not activate classical downstream pathways such as ERK or JNK MAPKs. We now report the identification of the downstream target for ITSN‐Ras. We discovered that ITSN interacts with and activates a novel PI3K isoform, PI3K Class 2β (PI3K‐C2β), which is necessary for survival of differentiating neurons. PI3K‐C2β possesses a Ras binding domain (RBD) suggesting a potential interaction with Ras. We now demonstrate that Ras interacts with PI3K‐C2β and ITSN. This interaction requires the RBD of PI3K‐C2β, and ITSN interaction with PI3K requires both the PRD and RBD of PI3K suggesting that Ras is necessary for ITSN‐PI3K interaction. In support of this model, we demonstrate that ITSN, Ras, and PI3K‐C2β are part of a trimolecular complex on vesicles. Finally, we demonstrate that dominant‐negative Ras inhibits ITSN activation of AKT. These data support a model whereby ITSN activates Ras on vesicles leading to recruitment and activation PI3K‐C2β to stimulate cell survival.
Intracellular sorting studies of wild‐type and truncated forms of the D1 dopaminergic receptor unravel a chaperone‐like effect of antipsychotic drugs
Authors: Sedaghat Keyvan , Tiberi Mario
Published: June 2021

The D1 dopaminergic receptor (D1R) is a seven‐transmembrane protein linked to stimulatory heterotrimeric GTP‐binding proteins. Here, we investigate the role of the cytoplasmic tail (CT) of D1R in regulating intracellular sorting properties of the receptor. We show that wild‐type (WT) D1R and its truncated and phosphorylation‐deficient form (Δ351) were sorted at the plasma membrane of transfected embryonic kidney 293 (HEK293) cells. Antibody feeding and co‐localization studies indicate that similar to WT, Δ351 internalizes through a clathrin‐, CT‐ and receptor phosphorylation‐independent pathway following dopamine (DA) exposure. However, in the absence of DA, Δ351 displays a strong intracellular staining unlike WT as assessed using immunofluorescence studies. Our confocal microscopy analysis shows that Δ351 co‐localizes with the endoplasmic reticulum (ER) markers, calnexin and calreticulin. Intriguingly, we observe an increase in the mature form of Δ351 and a complete rescue of ER‐retained Δ351 following an overnight treatment of transfected HEK293 cells with the inverse agonist flupentixol (Flu), an antipsychotic drug. These data strongly suggest that Flu displays a chaperone‐like effect on ER‐retained Δ351. Overall, our studies underpin a pivotal role of the CT of D1R in regulating the ontogeny and sorting to cell surface of this dopaminergic receptor subtype.

Supported by CIHR (MOP‐81341; MT).
Phenotypic Characterization of the 7H24 Mutant in O‐xylosyltransferase
Authors: Gentile Nicole Beth , Selva Erica , Fuentes Julio
Published: June 2021

Formation of morphogenetic gradients is a fundamental mechanism of developmental signaling. Establishment of the gradient dictates how tissues respond to extracellular signals to elicit appropriate cellular responses to a secreted ligand. Recently, we have identified a novel mutation in the Drosophila O‐xylosyltransferase (oxt) that encodes a key enzyme required for heparan and chondroitin sulfate (HS and CS) biosynthesis, named 7H24. In Drosophila, HS has been shown to be essential for Wingless (Wg)/Wnt, Hedgehog (Hh) and Decapentaplegic (Dpp)/Bone Morphogenic Protein (BMP) extracellular ligand movement and gradient formation. When HS is absent downstream signaling of these elemental pathways that drive the Drosophila and human developmental program is disrupted. The 7H24 mutation in oxt is a functional null and removal of maternal and zygotic oxt is embryonic lethal with terminal segment polarity cuticle phenotypes suggesting disruption of the Wg and/or Hh signaling pathway. Removal of zygotic oxt causes homozygous mutants to die as pharate adults with small rough eyes and thorax defects that cannot close from the pupal case. We find deficiencies in Wg, Hh and Dpp signaling in the mutant larval wing imaginal discs and evaluation of larval eye phenotypes suggests they do not arise because of defects in cell type specification.
What is the pathway by which estrogen promotes the neuroprotective effect on hypoglycemic injury in hypothalamic cell lines?
Authors: Chakraborty Tandra R. , Loera Melinda , Chakraborty Sanjoy
Published: June 2021

Control of serum glucose level is critically important in reducing the complications of diabetes mellitus. Reports show that recurrent episodes of hypoglycemia can lead to morbidity. In the event of low glucose the regions of the brain like the cortex, hippocampus and hypothalamus become vulnerable to injury. Our preliminary work has shown that estrogen has a neuroprotective effect on hypoglycemic cells. Using parameters like cell count, 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphrnyltetrazolium bromide (MTT) and Alamar Blue assay, the cells show ∼20% decrease in neuronal cell poliferation when exposed to hypoglycemic shock for 24 hours was observed. However, when beta‐estradiol (100nM) was added to the cells in the absence of glucose under identical conditions there was a ∼10% increase above the control. Neuronal cell death could lead to apoptotic cell death causing activation of caspases. Analyses of caspases‐3 would confer the hypoglycemia‐induced cell death and would be done using immunocytochemical techniques. Further, GLUT1 and ERalpha would be analyzed in the neuronal cells in order to access the signaling pathway and the mechanism of action.
Effect of creatine on the processing of amyloid precursor protein isoforms and mitochondrial function.
Authors: Chong Pey Rou , Chua Li Min , Lee Chia Wen , Wong Boon Seng
Published: June 2021

Glucose hypo‐metabolism is an early feature in Alzheimer's disease (AD), preceding the onset of the clinical symptoms of dementia and the appearance of amyloidbeta (Aβ) peptide, a proteolytic product of amyloid precursor protein (APP). Studies showed that this metabolic deficit is associated with altered expression and activity of enzymes involved in mitochondrial energy production. Mitochondrial dysfunction can lead to ATP depletion and trigger apoptosis. Inhibition of energy metabolism can also alter APP processing. Hence, buffering intracellular energy levels could exert neuroprotective effects. One such buffering system is creatine kinase and its substrate creatine. However, it is unclear if energy buffering can modulate aberrant APP processing. In human brain, APP695 is the predominant APP isoforms and APP751 and APP770 are two other major APP transcripts. APP751 and APP770 isoforms contain a Kunitz protease inhibitory (KPI) motif that APP695 lacks, and APP770 possesses an additional OX2 domain that is absent in both APP695 and APP751. In AD‐afflicted human brain tissues, APP751 and APP770 expression were elevated. In this study using an immortalized APP‐null neuronal cell line expressing APP695, APP751 and APP77, we observed creatine is able to reduce the production of amyloidogenic derivatives and lessen the impairment of mitochondrial function in cells expressing the KPI‐containing APP isoforms. This neuroprotective effect is due in part to the activation of the insulin signalling pathway. Taken together, we speculate that creatine may have an important role in preventing energy dysfunction and possibly modulating the neurodegenerative cascade in AD.
Effect of genetic polymorphism on apolipoprotein E glycosylation and lipoproteins metabolism
Authors: Chua Ching Ching , ONG Qi‐Rui , WONG Boon‐Seng
Published: June 2021

Alzheimer's disease (AD) is the leading cause of age‐related dementia and apolipoprotein E (ApoE) is a well‐established genetic risk factor in sporadic AD. ApoE is highly expressed in liver and brain, and has an important role in cholesterol metabolism. In human, there are three alleles (E2, E3 and E4). ApoE4 increases disease susceptibility by binding amyloid‐β (Aβ) and preventing their clearance, leading to Aβ accumulation. ApoE3 is the most common allele but ApoE2 is found to reduce AD risk. ApoE is also associated with hypercholesterolemia, a risk factor for AD. However, the mechanisms underlying ApoE role in AD pathology is poorly understood. ApoE undergoes O‐linked glycosylation and various glycosylated isoforms exist. This diversity is partly due to differential sialylation but the effect and identity of this chemical modification on ApoE function and between ApoE isoforms is unknown. In this study, we will present the glycan profile on different ApoE isoforms. We will also demonstrate ApoE contribution to the biochemical profile of high‐density lipoprotein (HDL), LDL, triglycerides and cholesterol as well as their effect on the expression of several enzymes involved in Aβ metabolism.
Correlation of Estrous cycle with estrogen and leptin levels in ob/ob and Agouti mice
Authors: Rayani Nikita , Sachdev Aman , Chakraborty Tandra R.
Published: June 2021

Gonadal hormones like estrogen acts as a metabolic signal of energy sufficiency and also regulates the stage of reproductive cycle. Apart from hormonal changes, feeding circuits also affects the estrous cycle. Estrogen, a hormone secreted by the ovaries regulates reproductive cycle and has a negative effect on food intake. Leptin, a hormone secreted by the adipose tissue has an inhibitory effect on food intake. In this study we tried to analyze the estrous cycle in relation with leptin and estrogen levels in two mouse lines ob/ob and Ay/a, both of which are obese, hypometabolic and infertile but with contrasting leptin levels. To assess the progression of estrous cycle, vaginal smears was performed on female WT, ob/ob and Ay/a (n=4) mice. In WT mice at proestrous; nucleated cells were round to hexagonal shaped, estrous; cornified cells and metaestrous; leukocytes with many nucleated cells, diestrous; the cells were all scattered leukocytes. We found that both ob/ob and Ay/a mice were not cycling and were at a persistent estrous phase with cornified cells. The number of cornified cells however was not similar at all stages. Quantitization of beta‐estradiol showed both ob/ob and Ay/a compared to WT had same or higher b‐estradiol levels. Thus, this study leads us to believe the estrous cycle of these mouse models is not only dependent on estrogen levels but interplay of gonadal hormones and feeding circuits.
Cardiac Lineage Protein‐1 (CLP‐1) regulates the skeletal myogenic program through its association with MyoD and histone deacetylases (HDACs)
Authors: Galatioto Josephine , Mascareno Eduardo , Siddiqui M.A.Q.
Published: June 2021

Transcription in eukaryotic cells is regulated by the positive transcription elongation complex b (P‐TEFb), which appears to be dynamically partitioned between the active versus inactive states. In its active form, P‐TEFb contains cyclin T1 or T2 and cdk9 kinase which phosphorylates the CTD of RNA polymerase II resulting in elongation of the nascent transcripts to full‐length mRNAs. P‐TEFb in the inactive form contains the inhibitory protein CLP‐1/HEXIM1 and 7SK snRNA. CLP‐1 (Cardiac Lineage Protein‐1), the mouse ortholog of human HEXIM1, was cloned and the gene for CLP‐1 was knocked out in mice in our laboratory. In this study, we have begun to examine the role of CLP‐1 in skeletal muscle differentiation using the mouse C2C12 skeletal muscle cell line. We observed that CLP‐1 participates with both MyoD and HDACs which mediate P‐TEFb function. To further understand the role of CLP‐1 protein, we produced CLP‐1 overexpressing and CLP‐1 +/− C2C12 cells. Overexpression of CLP‐1 did not induce any prominent phenotype. On the other hand CLP‐1‐deficient cells were unable to differentiate into myotubes. Taken together, these data suggest that CLP‐1 plays a critical role in the genetic differentiation program of skeletal muscle cells. Supported by a grant from the National Institutes of Health (HL 07339‐M.A.Q. Siddiqui)
Cyclic AMP pathway and endocytic mechanisms in Trypanosomatids
Authors: Torres Héctor N. , Schoijet Alejandra C. , Docampo Roberto , Flawiá Mirtha M. , Alonso Guillermo D.
Published: June 2021

Several species of kinetoplastids cause major human infectious diseases. Trypanosoma cruzi is responsible of the fatal Chagas disease in South America, and Trypanosoma brucei is the agent of human sleeping sickness in Sub‐Saharan Africa.

In trypanosomatids, cAMP has been involved in the control of several processes such as proliferation, cell differentiation and osmoregulation. Recently, we have characterized two phosphodiesterases in T. cruzi: TcrPDEA1 and TcrPDEC2. TcrPDEC2 shows homology with PDE4 family and possess a N‐terminal FYVE domain. This domain participate in vesicular trafficking by targeting proteins containing them to membranes enriched in PI 3‐phosphate. After pre‐incubation with specific type‐4 PDE inhibitors, it was observed that parasites exposed to hypo‐osmotic stress, showed an increase in the volume recovery. Moreover, TcrPDEC2 localized close to the contractile vacuole/flagellar pocket. We also characterize a putative class III PI 3‐kinase. This enzyme, named TcVps34, is homologous to the S. cerevisiae Vps34p. By using PI3K inhibitors, it was observed that TcVps34 play a role in osmoregulation. In addition, epimastigotes over‐expressing TcVps34, showed functional and large contractile vacuoles, defects in vesicular acidification and receptor‐mediated endocytosis. Supported by CONICET, FONCYT, UBA‐Argentina and Ellison Med. Fund.‐U.S.A.
Modulation of adrenal steroidogenesis by c‐AMP specific phosphodiesterase 8
Authors: Tsai Li‐Chun Lisa , Shimizu‐Albergine Masami , Beavo Joseph A
Published: June 2021

Corticosterone is secreted by mouse adrenal zona fasciculata cells in response to the pituitary hormone adrenocorticotropin (ACTH) in a cAMP/PKA dependent manner. The cAMP/PKA signaling pathway dynamically promotes steroidogenesis by increasing substrate availability, phosphorylation of key enzymes, and transcription of steroidogenic enzymes. Intracellular levels of cAMP are controlled through its synthesis by adenylyl cyclases and its degradation by phosphodiesterases (PDEs). We now show that one of the most recently discovered phosphodiesterase, PDE8B, is highly enriched in adrenal fasiculata cells. Interestingly, PDE8's physiological functions remain largely unexplored due to the lack of specific pharmacological inhibitors. Our laboratory has previously shown that PDE8A is a main modulator of testosterone production in the testes. Here we report that PDE8B modulates adrenal steroid production. We observe that cultures of primary adrenocortical cells isolated from PDE8B knockout mice show elevated levels of basal adrenal steroids, and respond more promptly to ACTH stimulation than wild‐type cells. We also find elevated corticosterone levels in the urine of PDE8B knockout mice, presumably due to adrenal hypersensitivity in vivo. These findings suggest that PDE8B plays an important role in the regulation of adrenal steroidogenesis. Supported by NIH grant DK21723.
Evolution of allostery in the cyclic nucleotide binding module: A comparative genomics study
Authors: Kannan Natarajan , Wu Jian , Yooseph Shibu , Neuwald Andrew Frank , Venter J. Craig , Taylor Susan S
Published: June 2021

The cyclic nucleotide binding (CNB) domain regulates signaling pathways in both eukaryotes and prokaryotes. In this study, we analyze the evolutionary information embedded in genomic sequences to explore how the CNB domain elicits a cellular response upon binding to cAMP. Toward this end, we have identified and classified CNB domain sequences in the Global Ocean Sampling data and other protein databases. A systematic comparison of the identifed sequences reveals that the CNB domain recombine with a wide variety of functional domains to acheive functional specificity in signaling pathways. CNB domains have also undergone major sequence variation during evolution. In particular, the sequence motif that anchors the cAMP phosphate (termed the PBC motif) is striking different in some families. This variation may contribute to ligand specificity inasmuch as members of the prokaryotic cooA family, for example, harbor a CNB domain, that contains a non‐canonical PBC motif and that binds a heme ligand in the cAMP binding pocket. Statistical comparison of the functional constraints imposed on the canonical and non‐canonical PBC containing sequences reveals that a key arginine, which coordinates with the cAMP phosphate, has co‐evolved with a glycine in a distal loop that allosterically couples cAMP binding to distal regulatory sites. In conclusion, our analysis suggests that CNB domains have evolved as a scaffold to sense a wide variety of second messenger signals. Based on sequence, structural and biochemical data, we propose a mechanism for allosteric regulation by CNB domains.
Role of a PKA Interacting Protein, AKIP, in Cell Signaling and Apoptosis
Authors: Sastri Mira , King Charles , Taylor Susan S
Published: June 2021

A Kinase‐Interacting Protein/Breast Cancer Associated Gene 3 (AKIP/BCA3), a protein with three splice variants that is up‐regulated in breast cancers, was shown to bind the N‐terminus of the catalytic subunit of PKA (PKAc), and results in nuclear translocation of PKAc. AKIP/BCA3 is neddylated and can bind to and regulate NF‐κB‐dependent transcription. These findings suggest that AKIP/BCA3 functions as a molecular shuttle and direct regulator of transcription. Using a proteomics‐based approach, we identified Apoptosis Inducing Factor (AIF) and HSP‐70 as AKIP/BCA3 binding proteins. HSP‐70 has previously been shown bind and block nuclear translocation of AIF. Taken together, this suggests that AKIP/BCA3 acts a molecular shuttle for PKAc and AIF, but may be attenuated by HSP‐70. Our initial analysis of these protein:protein interactions revealed that PKAc, AIF, and HSP‐70 preferentially bind different splice variants of AKIP/BCA3. The two splice variants of AKIP/BCA3 up‐regulated in breast cancer could with pro‐survival proteins suggesting that an imbalance of AKIP/BCA3 import may significantly contribute to the pro‐survival signaling observed in breast cancer.
Following the Trail of Frank Netter, Master Anatomical Illustrator
Authors: Machado Carlos A. G.
Published: June 2021

For decades Dr. Frank Netter's medical illustrations have been admired and have taught medicine to health students, health professionals and the lay public all over the world. Dr. Netter's unique style, technique, talent, knowledge and remarkable body of work that comprises more than 6,000 illustrated plates, with over 20,000 individual images, is still unsurpassed by the most prestigious contemporary medical illustrators. One of the key factors that made his style so distinguished is the association of the appealing language and concepts of commercial and advertising illustration with the transmission of scientific knowledge. For 14 years, in my updating of the illustrations in the Netter Atlas of Human Anatomy as well as many other Netter publications, I have faced the challenging mission of continuing Dr. Netter's legacy, of following and understanding his concepts, and of reproducing his style by using his favorite techniques. This lecture analyzes the factors that influenced Dr. Netter's style and contributed to his success. It also shows similarities and differences between his and my professional training, styles, concepts and particular techniques.
Western blot and RTPCR analysis of phosphodiesterase (PDE4) expression in mouse leg muscle
Authors: Maglic Dejan , Bloom Timothy J
Published: June 2021

Duchenne muscular dystrophy, an X‐linked recessive disorder, is a progressive skeletal muscle disease caused by absence of the protein dystrophin. Previous work in this lab showed elevated cAMP phosphodiesterase (PDE) activity in muscle from 5 week‐old dystrophic mdx mice versus control 10SnJ mice, followed by a decline in PDE4 activity at fifteen weeks in mdx muscle. We hypothesize that PDE4 expression is specifically and differentially regulated in mdx muscle. Specific PDE4 isoforms in muscle were identified using western blots with commercial affinity‐purified antibodies. A custom made antibody against a conserved peptide in all PDE4 families was used to confirm the other antibodies. PDE4A and PDE4D were identified in 7 week and 8‐week‐old male 10SnJ hind leg muscles, respectively. The presence of PDE4C and PDE4B was not detected in 6 week and 7‐week‐old male 10SnJ hind leg muscles, respectively. To more specifically identify expressed isoforms, the presence of specific PDE4 mRNAs was assessed using RT‐PCR. This approach found expression of mRNA from all four PDE4 families, including PDE4A5, PDE4B2, and at least 2 isoforms of PDE4D. Primers for conserved PDE4C sequences also produced a sequence‐verified fragment. These results lay the groundwork for examining differences in the expression of PDE4 in mdx versus 10SnJ tissue. The project described was supported by NIH Grant Number 1R15AR050408‐01A2 from NIAMS.
Delineating Early Events in B1 B‐cell Development
Authors: Esplin Brandt L. , Welner Robert S. , Chen Xinrong , Kincade Paul W.
Published: June 2021

The recent description of CD19+CD45R/B220lo/neg B1‐specified progenitors (B1P) by Montecino‐Rodriguez and Dorshkind adds support for the notion that these specialized lymphocytes arise via a distinct lineage. However, the origin of B1P was not described and developmental relationships to conventional B2 cells were unclear. Therefore, RAG‐1+ early lymphoid progenitors (ELP) and Lin‐ Sca‐1+ c‐KitLo IL‐7Rα+ common lymphoid progenitors (CLP) were isolated from bone marrow of adult Rag1/GFP knock‐in reporter mice. Both categories of conventional lymphoid progenitors generated CD19+CD45R/B220lo/neg B1P cells in culture and when transplanted to sublethally irradiated recipients. Moreover, significant numbers of peritoneal CD11b+ CD5+ B1a and CD11b+ CD5‐ B1b cells were produced. B1 lineage development was also found to be IL‐7Rα‐dependent, and developmentally blocked in Btk−/−mice. Further study is needed to determine the principal differentiation route(s) utilized by B1 cells. However, our findings indicate at least some B1P can be produced in adult bone marrow from primitive B2 progenitors, suggesting a developmental relationship between these lineages.
Positive regulation of Itk PH domain function by soluble IP4 is required for thymocyte positive selection but dispensable for negative selection
Authors: Huang Yina Hsing , Grasis Juris A , Miller Andrew T , Xu Ruo , Soonthornvacharin Stephen , Andreotti Amy H , Tsoukas Constantine D , Cooke Michael P , Sauer Karsten
Published: June 2021

Membrane recruitment of Itk and PLCγ1 require interactions between their Pleckstrin homology (PH) domains and PIP3, the phospholipid product of PI‐3 kinase. Dual anchoring via SH2 and PH domains are required for optimal Itk‐mediated activation of PLCγ1 and the subsequent generation of IP3 and DAG. We have identified a novel mechanism in which conversion of IP3 into IP4 by ItpkB creates a physiologically important soluble ligand for the Itk PH domain. IP4 specifically promotes PH domain‐dependent recruitment of Itk but not PLCγ1 to PIP3 and is required for optimal Itk activation. PH‐domain mediated oligomerization of Itk leads us to propose a model in which IP4 binding to one Itk PH domain can allosterically increase the affinity of associated Itk PH domains for membrane PIP3. IP4‐mediated positive feedback of Itk activation is required for positive selection during T cell development. Dose dependent reduction of single positive thymocytes suggests that ItpkB limits the efficiency of T cell maturation. Interestingly, our data indicates that thymocyte negative selection is normal in the absence of ItpkB and suggests that ItpkB participates in distinguishing TCR‐dependent death or survival signals.
Separate upstream and downstream promoters control Dbeta2 transcription during T cell development
Authors: Sikes Michael L. , McMillan Ruth E.
Published: June 2021

V(D)J recombinational accessibility is regulated by transcriptional enhancers and promoters positioned within each gene locus. In Tcrb, rearrangement initiates at two D‐J cassettes, each of which is transcribed coincident with DJ rearrangement in early pro‐T cells. Consistent with a model of promoter‐facilitated recombination, assembly of the DJβ1 cassette is dependent on a promoter within the Dβ1 5’RS. Assembly of DJβ2 proceeds independent from that of DJβ1, albeit with less efficiency. To gain insight into the mechanisms that selectively alter D usage, we have defined transcriptional regulation at Dβ2. We find that both DJβ cassettes generate germline messages and DJ joints in murine CD44+CD25− DN1 cells, though promoter activity at DJβ2 is located between the D and J elements. Unexpectedly, loci that contain DJβ2 rearrangements redirect transcription to sites immediately 5’ of the new DJ joint. We find that 3’PDβ2 activity is dependent on two NFβB binding sites, while 5’PDβ2 activity is controlled by an array of factors including GATA‐3, Runx‐1, and USF‐1. The unique organization and timing of Dβ2 promoter activity is consistent with a model in which promoter placement selectively regulates the rearrangement potential of Dβ2 during Tcrb assembly. This work was supported by the Elsa U. Pardee Cancer Foundation (2005‐0069) and the North Carolina Agriculture Research Service (NC06711).
Stage‐dependent molecular changes in Notch signaling are critical for normal human T cell development
Authors: Taghon Tom , Van de Walle Inge , De Smet Greet , De Smedt Magda , Plum Jean
Published: June 2021

Notch signaling is one of the major drivers of T cell development. While the requirements for this pathway have been well established during mouse T cell development, its role in this developmental process in human is less clear. Here, using the OP9‐DL1 coculture system, we demonstrate that different levels of Notch activation, through different downstream molecular events, are required at the major developmental checkpoints of human T cell development. We show that these Notch driven events in human induce different developmental cell fate decisions compared to during mouse T cell development. Furthermore, through characterization of the β‐selection checkpoint in human T cell development, we demonstrate that Notch signaling is mainly required for proliferation of human thymocytes, but much less important for differentiation, in contrast to during similar stages of mouse T cell development. Thus, our results reveal stage‐dependent molecular changes in Notch signaling that are critical for normal human T cell development and fundamentally different compared to the mouse.

This work was supported by the Belgian Science Policy, Research Foundation Flanders (FWO), Institute for Innovation of Science and Technology (IWT) and Concerted Research Activities (GOA).
Functional and Genomic Profiling of Effector CD8 T Cell Subsets with Distinct Fates
Authors: Sarkar Surojit , Kalia Vandana , Haining W. Nicholas , Konieczny Bogumila T , Subramaniam Shruti , Ahmed Rafi
Published: June 2021

An important question in memory T cell differentiation is understanding the mechanisms that regulate the generation of terminal effector cells that die following antigen clearance versus effector cells that give rise to the long‐lived pool of memory CD8 T cells. Using killer cell lectin‐like receptor G1 as a marker to effectively distinguish terminal effectors from memory precursors, we found that despite their diverse cell fates both subsets possess remarkably similar gene expression profiles and function as equally potent direct ex vivo killer cells, with similar ability for inflammatory cytokine production and granzyme B expression. Interestingly, in addition to these conventional effector CTL characteristics memory precursors also produced IL‐2, a property typically ascribed to naïve or memory cells; thus presenting them as a novel effector subset. On the other hand, terminal effector cells represent a “late leaving” subset of cells that continues to perceive stimulation towards the tail end of antigen clearance, and exhibits an enrichment of the “effector cell signature”. Importantly, curtailing stimulation during the later stages of infection enhanced the generation of long‐lived memory cells. Collectively, these data define a model of memory differentiation wherein effector CD8 T cells that are driven further down the differentiation pathway by continued stimulation towards the later stages of infection become metabolically and functionally unfit for memory lineage. These studies strongly support the classical decreasing potential model of memory differentiation, and further demonstrate that “late leaver” effector cells contribute less towards the long‐lived pool of memory CD8 T cells.