bluuebunny/crossref_metadata_2025 huggingface dataset:
The association of Single Nucleotide Polymorphisms of PPARGC1B with AHR in asthmatic patients
Authors: Lee Shin‐Hwa , Park Ju Hyun , Chung Hun Soo , Uh Soo‐Taek , Kim Young Hoon , Park Jong‐Sook , Seo Ki‐Hyun , Na Joo Ok , Rhim Tai youn , Park Byung‐Lae , Park Choon‐Sik , Shin Hyung Doo , Chung Il‐Yup
Published: June 2021

PPARGC1B is a co‐activator for ER, PPAR, and GR, which are involved in asthma development. We investigated the genetic association of PPARGC1B polymorphisms with the risk of asthma and subphenotypes and the functional effect of these polymorphisms on the expression of PPARGC1B. Through direct DNA sequencing for PPARGC1B on chromosome 5q33 of 24 Koreans, we identified 18 polymorphisms. Among them, we choose 7 SNPs for large‐scale genotyping in Korean considering the location of SNPs, MAF and LD. In spite of the lack of association of SNPs with the risk of asthma, we found that −427C>T on promoter and +102525G>A on exon 5 of PPARGC1B were associated with airway hyperreactivity in asthmatics. LogPC20 values of C allele homozygotes on −427C>T was higher than of T allele homozygotes (p=0.004) and A allele homozygotes on +102525G>A was higher than of others (p=0.0001). Using Luciferase reporter system and Gel shift assay, we revealed that −427C type caused higher promoter activity than −427T type. In addition, we identified that B cells possessing −427C presented higher mRNA expression than that having −427T. These results suggest that the polymorphisms of PPARGC1B may affect on the promoter activity followed by development of airway hyperreactivity and the analysis for genotypes on PPARGC1B may be a genetic marker for severity of asthma. This study was supported by a grant of the Korean Health 21 R&D project.
Site Prediction for Computer Aided Functional Annotation of Structural Genomics Proteins
Authors: Hildebrand David GC , Ondrechen Mary Jo
Published: June 2021

Structural genomics (SG) projects are reporting new 3D structures of proteins at an increasing rate. Many of these new structures have novel features and some are novel folds. There are currently about 6000 such SG structures in the PDB; most of them are annotated as putative, hypothetical, or of unknown function. The determination of the function of a protein from its 3D structure has proved to be more difficult than anticipated. Here we report on recent efforts to accelerate the functional characterization of SG proteins using THEMATICS, a simple, fast computational predictor of functional sites. THEMATICS requires only the 3D structure of the query protein as input and thus applies to novel folds and remote sequences. It is shown that THEMATICS is effective for the most difficult cases, including proteins where ligand binding is coupled to a conformational change. The physicochemical basis for the success of THEMATICS predictions is discussed. Correlations of THEMATICS metrics with experimental kinetic data are studied. Examples of predictions for SG proteins are given. It is shown how THEMATICS predictions can be used to guide the experimental characterization of SG protein function, even in the absence of sequence similarity.
Mictotubules and actin differentially influence remodeling of connexin43 gap junctions
Authors: Hunter Andrew , Gourdie Robert
Published: June 2021

Regulation of Cx43 gap junction (GJ) size and organization is poorly understood. Previously we showed that fusion of GFP to the C‐terminus of Cx43 leads to the formation of aberrantly large GJs. Cx43 GJs are resistant to Triton detergent extraction, yet Cx43‐GFP GJs are largely Triton‐soluble. Interestingly, Triton‐insoluble Cx43‐GFP localizes predominately to plaque edges—the site of GJ growth—suggesting that GJ edges are stabilized by cytoskeletal interactions that influence GJ size. Fluorescence labeling revealed minimal interaction of actin filaments with Triton‐insoluble Cx43‐GFP. In contrast, plaques composed of native Cx43 were extensively colocalized with actin polymer. However, Cx43‐GFP plaques appear to acquire more microtubule contacts than native Cx43 GJs. Live cell imaging showed GJs containing a mix of Cx43‐GFP and native Cx43 are more dynamic than plaques comprised solely of Cx43‐GFP. Inhibition of either actin polymerization or Cx43 interaction with the actin binding protein ZO‐1 suppressed the dynamics of mixed Cx43 GJs. These results suggest that Cx43 C‐terminal elements, including the PDZ binding domain, determine cytoskeletal interactions at GJ edges, with ZO‐1‐mediated actin connections promoting active GJ remodeling, whereas microtubule contacts confer GJ stability and growth. Supported by NIH grants HL07260, K12GM081265, HL56728, HL082802.
Mitochondrial glycogen synthase kinase‐3beta: an investigation into its role in metabolism and cell fate
Authors: Clodfelder‐Miller Buffie J , King Taj D , Bijur Gautam N
Published: June 2021

Glycogen synthase kinase‐3ß (GSK3ß) facilitates apoptosis signaling and also regulates cell metabolism. GSK3ß is mostly in the cytosol, but an active pool of GSK3ß is present in the mitochondria. The present study focuses on the unknown actions of GSK3ß in mitochondria. A novel strategy was employed using a lentivirus gene delivery to express an inducible mitochondrial‐targeted, active GSK3ß (mS9AGSK3ß) in neuroblastoma cells. Immunofluorescence microscopy confirmed that mS9AGSK3ß was specifically localized in mitochondria. Increased expression of mS9AGSK3ß by itself did not activate apoptosis. However, increased expression of mS9AGSK3ß significantly enhanced apoptosis and cell death when cells were cultured under conditions of reduced glycolysis. It was also found that mS9AGSK3ß expression greatly facilitated apoptosis induced by the oxidative phosphorylation complex I inhibitors MPP and rotenone, and inhibition of GSK3? activity with lithium blocked apoptosis. Thus, increased GSK3ß activity in the mitochondria is deleterious under conditions that increase mitochondrial function and during mitochondrial stress.

Support by NARSAD, NIH‐NINDS, and the NIH Neuroscience Blueprint Core Grant NS57098.
ACTH/cAMP regulates the expression of acid ceramidase (ASAH1) in H295R human adrenocortical cells
Authors: Lucki Natasha Chrystman , Sewer Marion B
Published: June 2021

Acid ceramidase (ASAH1) is a key enzyme for sphingolipid metabolism that regulates the amount of ceramide, sphingosine, and sphingosine‐1‐phosphate within the cell. Based on our previous studies demonstrating that sphingosine is an endogenous ligand for the nuclear receptor steroidogenic factor‐1 (SF‐1), we examined the role of adrenocorticotropin hormone (ACTH) signaling in regulating the expression of ASAH1. Using H295R adrenocortical cells, we show that ACTH/cAMP increases mRNA and reporter gene activity of ASAH1. Using chromatin immunoprecipitation and reporter gene assays, we have identified the glucocorticoid receptor (GR) and cAMP‐responsive element binding protein (CREB) as important activators of this gene. We propose that ACTH signaling promotes the recruitment of GR and CREB to the ASAH1 promoter and that this increase in ASAH1 gene expression regulates ligand availability and SF‐1 target expression in the human adrenal cortex.
Regulation of CCT nuclear export and degradation by monoubiquitination
Authors: Chen Bill , Mallampalli Rama K
Published: June 2021

CTP:phosphocholine cytidylyltransferase (CCT), is an essential enzyme for phosphatidylcholine biosynthesis. CCT is also a nuclear enzyme that is proteolytically sensitive. We previously demonstrated that CCT availability is regulated by the ubiquitin‐proteasome pathway, and that tumor necrosis factor alpha (TNFalpha) induces CCT ubiquitination and degrades CCT (J. Biol. Chem. 275:2000). Herein, we demonstrate that CCT does not undergo polyubiquitination and degradation by the 26S proteasome, but rather the enzyme is monoubiquitinated, undergoes nuclear export, and subsequently is degraded in the endosome. The endosomal inhibitor ammonium chloride effectively blocked TNFalpha degradation of CCT. A CCT‐ubiquitin fusion protein that mimics monoubiquitinated CCT was found to accumulate in the endosome, indicating that monoubiquitination is critical for CCT trafficking and degradation. Mass spectrometry of the ubiquitinated CCT complex indicated thatCullin1, serves as a putative E3 ligase for CCT. Cullin1 recruits Skp1 and F‐box proteins to form an E3 complex, termed SCF. Both Cullin1 and Skp1 co‐immunoprecipitated with CCT in human lung epithelial cells. These results reveal the CCT monoubiquitination is a regulated process by distinct molecular factors that govern availability of this critical enzyme in vivo.
Microarray Analysis Reveals Hepatic Adaptive Responses to Excess Dietary Cholesterol
Authors: Ness Gene C. , Boone Lindsey R.
Published: June 2021

Microarray analysis was used to identify the alterations in hepatic gene expression in rats that occur in response to dietary cholesterol. Rats were fed normal chow or chow supplemented with 1% cholesterol for 3 days. RNA was prepared and reverse transcribed. It is well known that the rate of cholesterol biosynthesis is markedly down regulated by this treatment. Of the cholesterol biosynthetic enzymes, the largest decrease (7.9‐fold) was seen in squalene epoxidase expression. Lanosterol demethylase was decreased 3.5‐fold. The decrease in expression of these two genes is consistent with a build‐up an oxylanosterol‐the putative translational regulator of HMG‐CoA reductase. Several other cholesterol biosynthetic genes were decreased more than 2‐fold. These were 7‐dehydrocholesterol reductase, HMG‐CoA synthase, farnesyl diphosphate synthase, and acetoacetyl CoA synthase. HMG‐CoA reductase and mevalonate kinase were only decreased 2‐fold. HMG‐CoA reductase protein and activity are decreased by nearly 50‐fold. Hepatic LDL receptor expression was not significantly altered in response to feeding cholesterol. Unexpectedly, ABCG8 and ABCG5 were significantly decreased. However, cholesterol 7α hydroxylase was expression was increased 2.7‐fold. This suggests that in response to excess dietary cholesterol, the liver produces more bile acids to help keep cholesterol destined for secretion in solution rather than increasing the secretion of cholesterol directly by increasing expression of the ABCG8/5 transporters. (Supported in part by grant 04TSP‐03 from the Florida Department of Health.)
An intramolecular binding site for the myristoylated amino‐terminus of Gαi
Authors: Preininger Anita M. , Meier Scott M. , Funk Michael A. , Iverson Tina M. , Hamm Heidi E.
Published: June 2021

GPCRs act as guanine exchange factors for G‐proteins, catalyzing nucleotide release. The amino terminus of Gαi subunits is subject to reversible palmitoylation, as well as myristoylation, which is a permanent, co‐translational modification. Previous work has demonstrated that unmyristoylated Gαi proteins exhibit a disordered amino terminus in the absence of Gβγ, while myristoylated amino terminal residues are ordered even in the absence of Gβγ [Medkova, M. (2002) Biochemistry 41, 9963–9972; Preininger, A.M. (2003) Biochemistry 42, 7931–7941], suggesting an intramolecular binding site for the amino terminus of myristoylated Gαi subunits. In order to determine the location of this binding site, we utilized a combination of biophysical, biochemical and crystallographic methods. Using site‐directed‐mutagenesis combined with quenching studies, the extreme amino terminus undergoes activation‐dependent quenching by tryptophan residues within 15 Å of the extreme amino terminus, and this quenching occurs on the same timescale as that seen for nucleotide exchange. A crystal structure of a myristoylated Gαi protein demonstrates that myristoylation alters the conformation of Trp 258 (between the α‐3 helix and the β‐5 strand in the GTPase domain of Gαi proteins), and implicates this region as a participant in the intramolecular binding of the myristoylated amino terminus of Gαi proteins.

This work was supported by the National Institutes of Health (Grant 2R01 EY06062‐22A2).
Cloning and Expression of Epithelial Sodium Channels (ENaC) in Saccharomyces cerevisiae
Authors: Boswell Mikki Gage , Booth Rachell E
Published: June 2021

Epithelial Sodium Channels (ENaC) are integral membrane proteins which regulate sodium re‐absorption within cells. Current knowledge about the shoichiometry of these channels is limited; although a trimeric structure has recently been accepted. My research will aid in determining ENaC's structure by increasing the amount of expressed protein available for future studies. By cloning ENaC into Saccharomyces cerevisiae, we will be able to overcome the limitations of low protein expression which occur in traditional mammalian systems. For this study, the three ENaC genes were cloned into yeast vectors and transformed into S. cerevisiae cells. Expression conditions were optimized using time, sugar source, and various yeast strains. In all strains, induction times of approximately 4 hours resulted in maximum expression when raffinose was used as the sugar source, and expression of alpha ENaC in the yeast strain S1InsE4A resulted in a significant increase in protein yield. In this study, we have demonstrated that ENaC expression was achieved at the microgram level, which will provide adequate quantities for mass spectroscopy analysis of ENaC's post‐translational modifications and subunit associations. Funding for this project has been provided by the Department of Chemistry and Biochemistry Texas State University‐San Marcos, Welch Foundation, and Research Corporation.
Regulation of System xc‐ Expression and Localization in a Dopaminergic Cell Line
Authors: Wixson Matthew , Goltz Abby , Henderson Tara , Chase Leah A.
Published: June 2021

System xc− is a transport system that exchanges extracellular cystine for intracellular glutamate. The transporter plays a role in regulating intracellular cystine and cysteine levels, glutathione synthesis and maintains redox balance within the cell. Thus, the objective of this study was to examine how cellular redox state affects System xc− expression and cellular localization in dopaminergic cells. To manipulate cellular redox state, we exposed cells to either H2O2 or dopamine. We determined that exposure of PC12 cells to exogenous H2O2 (3 mM) or dopamine (10 mM) for 10 min results in an equivalent change in the intracellular H2O2 level in PC12 cells. Following either treatment, System xc− activity increases more than 2‐fold. Our preliminary data suggest that this increase is due to an increase in membrane localization of the transport system. Following oxidative insult, the glutathione levels in the cells are able to recover within one hour and the cells remain protected from cell death for at least 48 hours. Immunocytochemistry studies suggest that System xc− may be localized to a vesicles which may be important in the observed trafficking event. Thus, the regulated trafficking of System xc− may play a significant role in protecting dopaminergic cells form cell death. This research was supported by the Campbell Foundation, the NSF MRI and REU programs and the Biology and Chemistry Departments at Hope College.
A bimodality of Caveolin1 in regulation of TRPC1 function
Authors: Pani Biswaranjan , Singh Brij B
Published: June 2021

Caveolar microdomain constitutes a major cellular compartment that facilitates various signal transduction events, Ca2+ influx being one. TRPC1, a plasma membrane component of Ca2+ permeable channel is known to be activated by ER store depletion. However, its mode of activation remains illusive. In submandibular gland cells we show a dual role of Caveolin1 (Cav1) in regulating TRPC1 function. TRPC1 partitions into Cav1 enriched raft fractions as opposed to its non‐raft association in Cav1 knockout tissues, suggesting that Cav1 is required for TRPC1 membrane targeting. However, upon over expression, Cav1 reduces TRPC1 function. Activation of store operated calcium entry recruits TRPC1 to raft fractions, which is paralleled by its interaction with STIM1 and a subsequent reduction in its interaction with Cav1; suggesting STIM1 derepresses Cav1 suppression of TRPC1. Disruption of caveolae by sequestration of membrane cholesterol or silencing Cav1 significantly reduces raft association of TRPC1 and its interaction with STIM1, substantiating the requirement of a structured caveolar domain for activating TRPC1‐SOC. Hence, on one hand Cav1 is necessary for TRPC1 membrane targeting, whereas, on the other hand acts as a suppressor once over expressed. For a precise physiological relevance, this bimodality of Cav1 is further demonstrated in modulating TRPC1 mediated NFêB activation and cell proliferation.
Molecular analysis on growth factor and clock gene expression in the liver with streptozotocin‐induced diabetes
Authors: Won Jinyoung , Lee Sang‐kil , Park Sookyoung , Hong Yonggeun
Published: June 2021

Many biological systems are regulated by an intricate set of feedback loops that oscillate with a circadian rhythmicity of roughly 24 hr an increase in body temperature, heart rate, blood pressure, and cortisol secretion early in the day. Recent studies have shown amplitude of clock gene rhythm in heart and liver of streptozotocin (STZ) treated rats. Thus, our overall hypothesis is that diurnal variation in organ development and regulation of metabolism including growth and development during juvenile period of rat exists as a mechanism for anticipating and responding to the environment such as diabetic mellitus. In this study, we performed to disclose the expression pattern of clock genes including per1, per2, clock, bmal1, cry1 and cry2, and growth factors such as IGF (insulin like growth factors)‐I and ‐?, TGF (transforming growth factors)‐β1 on STZ‐induced diabetic rat. The expression of growth factors and clock gene at 1‐wk after inducing diabetes with STZ were clearly increased compared to control rats in the liver. In contrast, Expression pattern of these genes is very similar to 5‐wk DM rat after treating STZ. Thus we suggested that the reason upon the result described above was associated with insulin that is damaged to secretion of the insulin according to destruction of the pancreas beta cell by the STZ. [Partially supported by BioGreen21 Program (Code#20070401034006) and KRF‐2006‐E00344].
Inducing Cartilage Regeneration with Stem Cells for Treatment of Osteoarthritis Patients
Authors: Ferng Alice Shirong , Ferguson Christopher William , Szivek John A.
Published: June 2021

Osteoarthritis is a progressive disease characterized by breakdown and loss of articular cartilage, which results in joint pain, stiffness, and limitations in activity and affects millions of people worldwide. Although artificial joint replacements are currently the gold standard for osteoarthritis patients, they can cause problems, such as bone loss, pain if the implant becomes loose, and a loss of proprioception.

Stem cells converted to cartilage cells (chondrocytes) can be used to induce cartilage regeneration. Stem cells isolated from adipose (fat) tissue can differentiate into chondrocytes under the appropriate conditions (Guilak et al. (2003). Cytotherapy 5(5):362–9). This project investigates the chondrocytic potentials of human stem cell lines in vitro isolated from: 1) adipose tissue, 2) bone marrow, and 3) umbilical cord blood. Growth factors, TGF‐β3 and BMP‐6, known to encourage chondrogenesis of stem cells have been used to treat these stem cell lines. Additionally, a pilot study with canine adipose‐derived stem cells is underway to determine how stem cells respond in vivo.
Effects of Media pH on VACM‐1/cul5 Protein Expression in T47D Cancer Cell Line
Authors: Pustelak Jessica
Published: June 2021

VACM‐1, a cul‐5 gene product is involved in the ubiquitin ligase complex formation and subsequent protein degradation. When expressed in T47D breast cancer cells, VACM‐1 inhibits growth by inhibiting MAPK phosphorylation (MAPK‐P) and decreasing nuclear estrogen receptors, ERα concentration. Factors that can regulate VACM‐1 protein concentration have not been identified. We have observed that changes in protocol from powder media to a purchased liquid media have led to an increase of endogenous VACM‐1 concentration in control T47D cells. The goal of this project was to examine whether changes in pH will affect VACM‐1 concentration and cell proliferation. Our work has shown that endogenous VACM‐1 protein expression in T47D cells is induced by changes in media pH. Immunocytochemistry data have verified the increased concentration of VACM‐1 in cells grown in liquid media (lower pH) when compared to the powder media (higher pH). The increase in VACM‐1 expression was directly correlated with the decrease in cell growth. These differences were no longer observed when culture media was adjusted to the same pH. These results support the hypothesis that changes in pH may affect VACM‐1 concentrations. This work was supported by a grant from NCI (R15CA104014) and by Dept of Chemistry.
Involvement of cGMP‐dependent protein kinase in colon cancer cells: analysis of cell proliferation, apoptosis and beta‐catenin expression
Authors: Sellak Hassan , Wu Songwei , Lincoln Thomas M.
Published: June 2021

Cyclic GMP‐dependent protein kinase (PKG) contributes to cell proliferation and differentiation as well as gene regulation. PKG expression is often reduced in cancer cell lines and its role in cell transformation is unknown. In this study, the effects of PKG‐Iß overexpression in cancer cell lines on proliferation, migration and the expression of a proto‐oncogene marker (beta‐catenin) was examined. PKG‐Iß expression in colonic cancer cells, Caco‐1 and Lovo‐1, was performed through stable transfection or lentiviral infection. Cells overexpressing PKG‐Iß proliferate at a higher rate compared to control cells harboring only empty vector, as analyzed by [3H]thymidine incorporation. Incubation with 8‐pCPT‐cGMP (10 μM) increased proliferation of PKG‐Iß‐overexpressing cells compared to the control. Overexpression of PKG‐Iß also had increased levels of apoptosis markers such as caspase 7 and caspase 9 compared to non‐transfected or non‐infected cells. Expression of beta‐catenin, a proto‐oncogene marker, was also analyzed in cells overexpressing PKG‐Iß. The results show that beta‐catenin expression was increased in PKG‐Iß‐overexpressing cells in both cytosolic and nuclear compartments compared to control cells. Taken together, these results suggest that PKG‐Iß may be involved in stimulating apoptosis of colon cancer lines but has no inhibitory effect on cell proliferation. PKG‐Iß may also regulate cell migration by altering the level of beta‐catenin.
Basal prostaglandin levels are required for PYK2 expression and Ang II‐dependent MAPK phosphorylation in intestinal epithelial cells
Authors: Slice Lee Warren , Wu Steven S. , Pham Hung , Vincenti Romina , Rozengurt Enrique
Published: June 2021

Objective

Ang II rapidly induces PYK2 phosphorylation on Tyr402 along with increased PG production in epithelial cells suggesting possible feedback regulation of Ang II signaling by PGs. We investigated the role of PGs in PYK2 signaling in cells over‐expressing 15‐hydroxyprostaglandin dehydrogenase (PGDH), an enzyme that inactivates PGs.

Methods

Intestinal epithelial cells (IEC‐18) were transduced using lentivirus to express green fluorescent protein (18.GFP) or PGDH (18.PGDH).

Results

Ang II induced PYK2 Tyr402 phosphorylation in IEC‐18 and 18.GFP cells. Both cell lines produced low basal levels of PGs that were increased by stimulation with Ang II or EGF. These agonists induced ERK phophorylation, which was blocked by AG1478. PG levels were undetectable in 18.PGDH cells and did not increase with stimulation. PYK2 expression was absent in 18.PGDH cells. Ang II did not induce ERK phosphorylation. Conversely, EGF induced ERK phosphorylation. Ang II‐dependent ERK phosphorylation was rescued by expression of wt PYK2 but not by an inactive PYK2 mutant. Reduction of PYK2 expression by siRNA in IEC‐18 cells correlated with a decrease in Ang II‐dependent ERK phosphorylation.

Conclusions

Basal PG levels are required to maintain PYK2 expression in IEC‐18 cells. In turn, PYK2 plays a critical role in mediating ERK signaling by Ang II. Supported by RO1 DK061485
Defining the role of the sugar transporter Sts1 in sexual development of the mushroom Schizophyllum commune
Authors: Kasper Dionna M. , Hickernell Thomas R. , Horton J. Stephen
Published: June 2021

Mushroom development in the fungus Schizophyllum commune is normally the result of a sexual interaction between two individuals differing at what are termed the A and B mating type loci. sts1 is a putative sugar transporter gene also implicated in the regulation of mushroom development. Null (Δ) mutant strains lacking functional copies of sts1 displayed severely attenuated mushroom production. When Δsts1 strains were outcrossed, some progeny displayed a “flat” phenotype, suggestive of an inappropriately‐activated B mating type pathway. Activation of this pathway normally requires the mating of two haploid individuals with different B mating type specificities. Specific B loci encode both a G‐protein coupled receptor (GPCR) and small lipopeptide pheromones that do not normally interact within self. We have investigated a possible link between the B mating type pathway and sts1 by outcrossing our Δsts1 strain with a compatible strain containing an incomplete B mating type locus. Only progeny with an intact B mating locus inherited from the sts1 parent were found to display a “flat” phenotype. This result suggests that the activation of the B pathway by Δsts1 is occurring at the level of the interaction between the GPCR and its “self” pheromones. Supported by a Union College IEF Grant to D.M.K.
The effect of apolipoprotein E and hypercholesterolemia on lipoproteins and amyloid metabolism
Authors: ONG Qi‐Rui , LIM Mei‐Li , CHUA Ching‐Ching , WONG Boon‐Seng
Published: June 2021

Apolipoprotein E (ApoE) is a major component of lipoprotein and has an important role in cholesterol homeostasis. ApoE is highly expressed in liver and brain. Altered apoE expression is associated with defective low‐density lipoprotein receptor (LDL‐R) and may cause hypercholesterolemia, a prominent feature in atherosclerosis and a growing risk factor for Alzheimer's disease (AD). Indeed, there is accumulating evidence demonstrating a pathogenic connection between atherosclerosis and AD. Animal studies showed that ApoE is involved in amyloid catabolism but the lack of ApoE can dramatically reduces amyloid‐beta (Aβ) deposition. In addition, ApoE also has anti‐atherogenic function and played an important role in the atherosclerotic process. However, the mechanisms underlying these contrasting properties in the liver and brain remained an enigma. In this study, we will present ApoE contribution to the biochemical profile of high‐density lipoprotein (HDL), LDL, triglycerides and cholesterol in control and ApoE‐null mice fed on a high‐fat diet. We will show that hypercholesterolemia affects Aβ production via modulating the expression of several enzymes involved in Aβ metabolism.
Neuroprotective effect of estrogen on hypoglycemic injury in hypothalamic cells
Authors: Loera Melinda , Chakraborty Tandra R.
Published: June 2021

Glucose is the main metabolic fuel for the neuronal cells. In the event of low glucose the regions of the brain like the cortex, hippocampus and hypothalamus become vulnerable to injury. The projects aims to find the effect of hypoglycemia on hypothalamic cells and explore estrogen as a potent neuroprotective agent. Hypothalamic cells HT39 (both express ERalpha and ERbeta) grown in the presence and absence of glucose. Cell death was analyzed at various time points using parameters like cell count, 3‐(4,5‐Dimethyl‐2‐thiazolyl)‐2,5‐diphrnyltetrazolium bromide (MTT) assay and Alamar blue. In addition, calculated amounts of beta‐estradiol (100nM) were used in order to access the neuroprotective effect of estrogen. Preliminary results usind Alamar Blue showed that within 24 hours, there is a ∼17–20% decrease in the cell proliferation when exposed to hypoglycemic insults. However, if exogenous estrogen is present in the same hypoglycemic media, there is a ∼10–12% increase in cell proliferation at that same 24 hour time point. Trypan Blue cell counting analysis revealed a similar trend after 24 hours: ∼14% decrease cell viability under hypoglycemic conditions, while in the presence of estrogen, cell viability increases ∼5%. These observations lead us to believe that estrogen has a neuroprotective effect on hypoglycemic shock in hypothalamic cells.
Beta‐arrestin‐dependent localization of actin‐turnover proteins downstream of Protease‐Activated Receptor‐2
Authors: Zoudilova Maria , Ge Lan , DeFea Kathryn
Published: June 2021

Beta‐arrestins are multifunctional adaptor proteins that mediate 7‐trans‐membrane (7TM)‐receptor signal desensitization and internalization, scaffolding of signaling molecules to specific cellular microdomains including those traditionally associated with chemotaxis. Protease‐activated‐receptor‐2 (PAR‐2) is a 7TM receptor that employs scaffolding function of beta‐arrestins for directed cell migration. Previously, we have shown that PAR‐2 elicits specific responses by differential activation of two pathways: classic Gαq/Ca2+ and beta‐arrestin, both of which required for chemotaxis. Cell motility requires localized generation of free barbed ends, which can be achieved through regulation of the cofilin pathway. We show that localization cofilin, its phosphotase Chronophin (CIN), and its inhibitory kinase LIMK to the leading edge requires beta‐arrestins. Furthermore, beta‐arrestins, CIN and cofilin are essential for initial pseudopod extension in response to PAR‐2 activation. Both cofilin and CIN are found at the sites of newly generated barbed ends. Beta‐arrestin‐dependent localization of signaling molecules has significant implications for understanding of PAR‐2 induced chemotaxis during cancer metastasis, inflammation and wound healing. This work was supported by R01GM066151 (KAD)
Atrial Natriuretic Peptide (ANP) and Islet Beta‐Cell Growth
Authors: You Hui , Laychock Suzanne G
Published: June 2021

Atrial natriuretic peptide (ANP) is a stimulus for particulate guanylyl cyclase and cyclic GMP (cGMP) formation through natriuretic peptide receptors A and B. Natriuretic peptide receptor subtypes A, B, and C mRNAs were identified by RT‐PCR in rat pancreatic islets of Langerhans. Culture of isolated rat islets at 5.5 mM glucose with ANP (1 M) for 7 days resulted in increased [3H]thymidine incorporation that was 200±36% of control (P<0.05). Rat insulinoma INS‐1e cell [3H]thymidine incorporation were similarly stimulated by ANP to 163±18% (P<0.05) of control. 8‐Bromo‐cGMP (0.5 mM) also stimulated islet [3H]thymidine incorporation to 207±15% of control (P<0.001). The expression of cyclin D2 mRNA was also increased by ANP. The total protein content of INS‐1e cells did not change during ANP stimulation. The total insulin content of pancreatic islets did not change during 7 day culture with ANP. However, the islet insulin secretory response to 11 mM glucose was markedly inhibited (74±7%) (P<0.01) by ANP after 7 day culture (but not a 3 hour pretreatment). Thus, ANP stimulates a mitogenic response in islets and INS‐1 cells that can be mimicked by cGMP. Long‐term ANP treatment also blunts the β‐cell insulin secretory response to glucose, but does not significantly alter total insulin content.
Cardioprotection in iNOS transgenic mice is independent of mitochondrial biogenesis.
Authors: Keith Rachel , Hill Bradford , Conklin Daniel J , Bolli Roberto , Bhatnagar Aruni
Published: June 2021

Inducible nitric oxide synthase (iNOS) is a known mediator of the late phase ischemic preconditioning (PC) against myocardial infarction. We have found that cardiac‐specific iNOS transgenic mice are protected from ischemic injury. It is also known that NO donors trigger PC by increasing iNOS and that they stimulate mitochondrial biogenesis. We hypothesize that protection by iNOS is linked to an increase in mitochondrial biogenesis. Mitochondrial density was examined by transmission microscopy of left papillary muscle from iNOS‐TG or wild type (WT) mice. No significant difference in mitochondrial count or size distribution was observed. Citrate synthase activity was not significantly different between iNOS‐TG and WT hearts. There was also no significant difference in the ratio of the mitochondrial DNA to the genomic DNA of iNOS‐TG or WT mice when compared cytochrome b primers with GAPDH using quantitative PCR or PCR. No difference in the levels of mitochondrial proteins cytochrome c, CcO‐IV was observed between iNOS‐TG and WT hearts. Addition of NO donors decreased ΔΨ and prevented permeability transition in mitochondria isolated from either WT or iNOS‐TG hearts. We conclude that overexpression of iNOS does not induce mitochondrial biogenesis and that cardioprotection due to iNOS may be related directly to the ability of NO to prevent mitochondrial permeability transition and subsequent activation of cell death. This work was supported by NIH grants HL5547 (to A.B) and HL78825 (to R.B.)
A role for Drak2 in the germinal center reaction and the antibody response
Authors: Al‐Qahtani Ahmed , Xu Jack , Zan Hong , Walsh Craig , Casali Paolo
Published: June 2021

DAP‐related apoptotic kinase‐2 (Drak2), a DAP family member, is highly expressed in B and T lymphocytes in the human and the mouse. To determine whether Drak2 plays a role in B cell activation and differentiation, we analyzed germinal center (GC) development in drak2−/− mice immunized with the T‐dependent conjugated hapten NP‐CGG. In drak2−/− mice, spleen GCs were normal in size and morphology, but their number was reduced by three to five‐fold as compared to wild type littermates. This was not due to a defect in B cell proliferation, as BrdU uptake in vivo and CFSE staining in vitro of B cells was comparable in Drak2‐deficient and wild type B cells. Nevertheless, the proportion of apoptotic GC B and T cells in drak2−/− mice was significantly higher than that in wild type littermates as shown by7‐AAD and TUNEL staining experiments. As a result, the generation of high affinity IgG antibodies was impaired in drak2−/− mice. Enforced expression of bcl‐xl in T cells of these mice increased the number of the GCs and rescued the high affinity IgG response to NP‐CGG. This was consistent with the findings that the somatic hypermutation and class switch DNA recombination machineries were inherently normal in drak2−/− mice. Thus, our findings suggest a novel role of Drak2 in regulating the GC reaction and the response to TD antigens.

This work is supported by NIH grants AI 45011, AI 60573 and AR 40908.
Positive selection‐associated skin‐homing of fetal thymic gamma/delta T cells
Authors: Jin Yan , Xia Mingcan , Saylor Christina , Xiong Na
Published: June 2021

Murine skin‐specific intraepithelial γδ T cells (sIELs) are prototypic non‐lymphoid tissue specific “innate lymphocytes”. Nearly all sIELs express identical Vγ3/Vδ1 γδ T cell receptors (TCR) and originate from the fetal thymus. Previously, we found that positive selection of γδ T cells in the fetal thymus is associated with a unique expression profile of chemokine receptors, such as upregulation of potentially important skin‐homing receptor CCR10, suggesting that a unique selection process of the fetal thymic γδ T cells determines their homing properties and peripheral localization. Confirming this, we now generated CCR10 knockout mice and found that development of γδ T cells in the skin, but not in other tissues, were severely impaired in these mice. Furthermore, we found that positively selected fetal thymic γδ T cells developed into sIELs without requirement of further peripheral TCR/ligand interaction, demonstrating that the central selection process of γδ T cells in the fetal thymus is sufficient to direct their migration and expansion in the skin. These findings may serve a paradigm for understanding development of other tissue‐specific “innate lymphocyte”, including γδ, NK, CD8αα, mucosal associated invariant T cells and B1 B cells, a group of important but poorly understood immune cells.
The roles of recombination signal sequence efficiency and location within the TCR β locus on the relative frequencies with which V β gene segments participate in V(D)J recombination
Authors: Cowell Lindsay Grey , Devlin Blythe , Hsieh Chia‐San , Harley Susan , Li Jie , Louise markert Mary
Published: June 2021

The different T cell receptor (TCR) β variable (Vβ) gene segments do not appear in the naïve TCR repertoire with equal frequencies. To identify the relative roles of recombination signal (RS) sequence efficiency and location within the TCRβ locus in determining the bias in Vβ gene segment utilization, we are studying TCR diversification after thymus transplantation in athymic infants with complete DiGeorge anomaly. Thymus transplantation reconstitutes T cells, and the course of T cell development allows study of TCR diversification. We sequenced mRNA from peripheral blood T cells sampled from two patients at multiple times after thymus transplantation. For each of six Vβ gene segments, we estimated the diversity of rearrangements from the number of unique rearrangements utilizing that gene segment and the number of identical sequences for each unique rearrangement. The RS recombination efficiency was predicted using the RS Information Content (RIC) score computed using a statistical model of mouse RS. We find that Vβ gene segment diversity varies significantly in the early repertoire indicating differences in the relative frequencies of recombination. Neither RS efficiency nor distance from the D‐J cluster solely determine the observed differences, rather the two factors appear to interact, jointly influencing recombination frequencies.

This work is supported by an NIH grant to MLM and a BWF award to LGC.
Regulation of CD4 T Cell Differentiation by CD44
Authors: Guan Hongbing , Nagarkatti Prakash , Nagarkatti Mitzi
Published: June 2021

CD4 T cells can polarize to Th1, Th2 and newly emerged Th17 lineage. The involvement of cytokines and signaling pathways that trigger such polarization are largely known. However, cells and molecules regulating the differentiation are not identified, especially in consideration of primary versus secondary immune response. In the current study, we found that CD44 plays an important role in CD4 T cell differentiation. By comparing immune responses to SRBC and OVA in CD44‐deficient and CD44‐sufficient mice, we observed that CD44 deficiency triggers a decreased Th1 immunity and an increased Th2 response. The decreased Th1 response correlated with a decrease in many of the components including macrophage migration, delayed‐type hypersensitivity, IgG2a production, and SRBC‐induced hemagglutination. However, there was an increase in IgG1 production. CD44‐deficient mice also had a down‐regulated expression of T‐bet and Th1‐cytokines but an enhanced expression of GATA3 and Th2‐cytokines. The skewed immunity occurred in either primary or secondary immune response. The decreased serum IL‐17 production, splenic Th17 population and dendritic cell functions in CD44‐deficient mice may contribute towards a shift from Th1 to Th2 cell polarization.

(This work was supported in part by NIH grants: AI053703, ES09098, AI058300, DA016545, HL058641 and P01AT003961).
Aging down‐regulates the transcription factor E2A, activation‐induced cytidine deaminase and Ig class switch in human B cells
Authors: Blomberg Bonnie B , Frasca Daniela , Landin Ana Marie , Riley Richard L
Published: June 2021

The capacity to class switch the Ig heavy chain is critical to the effectiveness of humoral immune responses in mice and humans. We have previously shown in mice that the E2A‐encoded transcription factor E47 is down‐regulated in old splenic B cells. This leads to a reduction in the activation‐induced cytidine deaminase (AID), which is known to induce class switch recombination (CSR) and Ig somatic hypermutation (SHM). We have extended our study here to investigate whether aging also affects antibody production, E47 and AID expression in B cells isolated from the peripheral blood (PB) of human subjects (18–86 years). Our results obtained with activated CD19+ B cells show that the expression of E47, AID and Igγ1 circle transcripts progressively decrease with age. We also show an age‐related decline in the percentage and numbers of switch memory B cells, no change in the percentage but a decline in the numbers of IgM memory B cells and an increase in the percentage but not in the numbers of naive B cells, consistent with our data on the decrease seen in CSR in vitro. In addition, we suggest that the subsets of B cells able to class switch in culture are the naïve and the IgM memory populatio, depending on the stimulus. Our results give possible molecular mechanisms for an intrinsic decrease in B cell function and the humoral immune response in aging.

This work is supported by NIH AG‐17618, AG‐23717 (BBB) and AG025256 and AI064591 (RLR)
CD5 regulates B‐1a B‐cell expansion and selection
Authors: Sestero Christine M. , Singh Simer Preet , Axtell Robert , Raman Chander
Published: June 2021

The expression of CD5 is the defining phenotypic characteristic of the B‐1a population of B‐cells. However, the role of CD5 in the development and physiology of B‐ 1a B cells has remained controversial. Our laboratory recently discovered that a major role of CD5 is to regulate survival in activated T‐cells and this activity was mediated by the unique interaction of CD5 with CK2. To determine if this biological activity of CD5 extended to B‐1a B‐cells, we developed a CD5 knock‐in mouse (CD5ΔCK2BD) in which the ability of CD5 to bind and activate CK2 was abrogated. The absolute number of B‐1a B‐cells in the peritoneum and natural IgM in the serum of the CD5ΔCK2BD mouse was dramatically lower than that in the CD5 WT mouse. Furthermore, we observed selective reduction of VH11Vê9 anti‐PtC B‐cells in the CD5ΔCK2BD mouse, suggesting altered positive selection. The contraction of B‐1a B‐cells in the CD5CK2BD mouse was in part due to arrest of cells from entering into cell cycle. Interestingly, costimulation with TLR 4/9 agonists and IFN‐β led to levels of IL‐10 production from CD5ΔCK2BD B‐1 B cells that was absent if B‐1 B‐cells were obtained from CD5 WT or CD5−/− mice. Our findings represent the first in vivo evidence that CD5 contributes to the development and biology of B‐1a cells in mice. Supported by the NMSS and NIH.
Activated human CD25+B cells are able to induce cell cycle arrest and apoptosis in CD4+T‐Helper cells
Authors: Tretter Theresa , Venigalla Ram Kumar , Eckstein Volker , Saffrich Rainer , Hanns‐Martin Lorenz
Published: June 2021

B cells play an important role as APC. However several hints indicating an additional regulatory role remain to be elucidated

Methods: CD19+B and CD4+T cells were separated from human PBMC by MACS. B cells were prestimulated with SAC or αIgM/IgG, irradiated and cocultured with CD4+T cells and αCD3+IL‐2 or αCD3+αCD28. T cell proliferation was determined by 3H Thymidine incorporation or PKH‐26 and apoptosis by AnnexinV

Results: T cell proliferation and cytokine production (IL‐10, IFNg) under optimal stimulatory conditions was inhibited by more than 70% in presence of preactivated, FACS‐sorted, large CD25+B cells vs. small CD25‐B cells. Suppression was strictly dependent on cell‐contact and constant presence of B cells as determined by cell culture inserts and reconstitution of cell division after removal from B cells. Blocking IL‐2Rα/β on B cells abolished suppression by about 50%, as did decreasing concentrations of IL‐2, suggesting that B cell suppressor activity is determined by the amount of IL‐2 available. Further, B cells induced apoptosis in a subpopulation of T cells (37% vs 17% after 24h) independent from blocking the CD95‐CD95L pathway

Conclusions: Polyclonally activated CD25+B cells are able to costimulate or suppress T cell mediated responses, depending on activation status and environmental conditions. Future experiments deal with the mechanisms behind and their pathophysiological impact.
Expression of CD103 by Foxp3+ regulatory T cells is critical for protective immune regulation in schistosomiasis
Authors: Hesse Matthias , Wilson Mark S. , Mahamed Deeqa , Cheever Allen W. , Wynn Thomas A. , Bajracharya Siddhartha
Published: June 2021

In schistosomiasis, failure to establish a tightly controlled chronic immune response is detrimental for the host. The responsible regulatory mechanisms are still unknown. We reported the presence of CD4+Foxp3+regulatory T cells (naTreg) in S. mansoni‐egg‐induced hepatic granulomas. Although they can control acute egg‐induced inflammation independent of IL‐10, their role in chronic schistosomiasis has not been determined. We found dramatically increased expression of the activation marker CD103 by granulomatous naTregs during disease progression. When infected with S. mansoni, CD103‐deficient mice succumb during transition from acute to chronic disease. They are rescued by adoptive transfer of CD103‐competent naTregs. CD103 does not control migration of naTreg to hepatic granulomas in our model. Strikingly, activated granulomatous B cells express e‐cadherin (the ligand for CD103) and high levels of MHC‐II and B7.2. In vitro LPS‐activated B cells strongly stimulate effector CD4+ T cell proliferation. NaTregs from naïve or from schistosome‐infected donors inhibit this response. Blocking of CD103 ameliorates the suppressor capacity of naTregs from infected animals. We propose that interaction of CD103+naTregs with activated B cells in granulomas is essential for host protection in schistosomiasis. This is a novel functional model for the control of inflammation by naTregs.
Dendritic cells use endocytic pathway for cross‐priming Qa‐1‐restricted CD8αα+ Treg
Authors: Smith Trevor Robert Frank , Tang Xiaolei , Maricic Igor , Garcia Zacarias , Kumar Vipin
Published: June 2021

Here, for the first time, we describe the mechanism by which Qa‐1‐restricted TCRαβ+CD8αα+ Tregs are primed. CD8αα+ Treg are a novel subset of naturally occurring suppressor lymphocytes that control activated Vβ8.2+ CD4+ T cells mediating experimental autoimmune encephalomyelitis in H‐2u mice. CD8αα+ Treg recognize a peptide determinant from the conserved region of the Vβ8.2 chain. Here we show that dendritic cells pulsed with apoptotic Vβ8.2+ CD4+ T cells can crosspresent TCR‐derived peptides to Treg in vitro. Apoptotic T cells do not induce DC maturation. Toll‐like receptor agonists augment the DC's ability to prime the Treg. Using specific inhibitors of antigen presentation, we demonstrate the involvement of endosomal and proteasomal processing in the formation of Qa‐1/peptide complexes. DCs that have captured apoptotic T cells can also prime CD8αα+ Treg in vivo and prevent autoimmune disease. Collectively, our data suggest during the exhaustion phase following a productive immune response, apoptotic T cells are engulfed by the DCs, and upon appropriate activation by innate signals DCs can crossprime CD8αα+ Treg to mediate immune suppression. Supported by grants from the NIH, MSNRC to VK.