bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhancing Production of Recombinant BMP‐2 in Mammalian Cell Culture
Authors: Zhou Aileen , Clokie Cameron ML , Peel Sean AF
Published: June 2021

Background

Bone morphogenetic proteins (BMPs) have been used successfully for bone repair; however recombinant BMPs are produced at relatively low yields in mammalian cells. Literature suggests the BMP pro‐domain may be involved in folding, stability, and secretion of the mature protein.

Objective

Investigate inhibition of pro‐domain cleavage on recombinant BMP‐2 production.

Methods

CHO cells transfected with the hBMP‐2 gene were cultured in the presence of various concentrations of the pro‐protein convertase inhibitor IND‐1 in 24 and 72hr multi‐well, and two‐month bioreactor cultures. Mature and proBMP‐2 secretion was measured by enzyme‐linked immunosorbent assay and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Cytotoxicity was determined by lactate dehydrogenase assay and cell viability counts.

Results

IND‐1 significantly enhanced secretion of both pro‐ and mature recombinant human BMP‐2 (rhBMP‐2) in short and long term cultures, without negative effects on cell growth or viability. The rhBMP‐2 was biologically active. ProBMP‐2 could be converted by enzymatic treatment into mature BMP‐2.

Conclusions

Addition of IND‐1 in cell culture medium increased rhBMP‐2 production in CHO‐BMP2 cells and is a simple, but effective way to enhance the production of active rhBMP‐2 in such production systems.

Grant

Oral Surgery Foundation.
Sphingosine kinase‐1 compartmentalization drives downstream metabolism of sphingosine‐1‐phosphate and upstream metabolism of ceramide biosynthesis
Authors: Wattenberg Binks , Siow Deanna , Berdyshev Evgeny , Pitson Stuart , Anderson Charles
Published: June 2021

Sphingosine kinase (SK) is a ubiquitous lipid signaling enzyme that produces the potent signaling lipid sphingosine‐1‐phosphate (S1P). SK has a dual function as a signaling enzyme and as a key regulator of sphingolipid metabolism. In this work we focus on the consequences of subcellular compartmentalization of SK on the metabolism of S1P. We find that the targeting of SK‐1 to either the endoplasmic reticulum or the plasma membrane promotes degradation of S1P by both a S1P lyase and phosphatases. Both sets of enzymes appear to contribute to this degradation equally. Surprisingly we find that association of SK to the endoplasmic reticulum promotes a dramatic increase in dihydro‐S1P. Dihydrosphingosine is the metabolic precursor for ceramide formation by the de novo pathway. Our data suggests that association of SK dwith the endoplasmic reticulum drains dihydrosphingosine from the ceramide synthetic pathway. Plasma membrane‐associated SK does not have this effect. Our data suggests that both the fate of S1P as a second messenger, and the metabolism of sphingosine metabolites is controlled by the subcellular localization of SK.
Molecular mechanisms of signal transduction by PAS sensor proteins
Authors: Ayers Rebecca A , Möglich Andreas , Moffat Keith
Published: June 2021

The modular nature of proteins containing PAS (Per‐ARNT‐Sim) or other sensor domains enables signaling networks to be diverse and poses an interesting question: how can sensor domains with largely conserved tertiary structures regulate effector domains with very diverse structures and functions? In the absence of high‐resolution structures of full‐length PAS proteins it is instructive to study the structures of isolated PAS sensor domains where a signaling event is recognized and transmitted. We present the structure of a heme‐PAS domain dimer from Bradyrhizobium japonicum (bjFixLH) in a new space group (P1) and at higher resolutions (1.5‐1.8 Å) than those previously obtained [Ayers RA and Moffat K (2008) Biochemistry 47, 12078‐86]. Interestingly, bjFixLH can form two different dimers in the same crystallization solution, where the monomers in one dimer are rotated ~175º relative to the second. This suggests that PAS monomers are plastic and that two quite distinct quaternary structures are closely similar in free energy. As in the light‐sensitive PAS domain YtvA‐LOV from Bacillus subtilis, bjFixLH undergoes signal‐induced quaternary structural changes where monomers rotate ~2º relative to each other. Signal‐induced quaternary structural changes accommodate the ability of PAS sensor domains to regulate a wide variety of effector domains since PAS and effector domains would not be required to interact with each other in a structure‐specific manner. Our results are relevant for the rational design of novel PAS signaling proteins [Möglich A, Ayers RA, and Moffat K (2009) J Mol Biol 385, 1433‐44].
Adult rats, but not adolescents, become tolerant to Δ9‐tetrahydrocannabinol in a hippocampal learning task
Authors: Moore Nicole L T , Wilson Wilkie A , Swartzwelder H Scott , Kuhn Cynthia M
Published: June 2021

Previous work in rats has shown that adolescent spatial learning in the Morris water maze task is more impaired by Δ9‐tetrahydrocannabinol (THC) than in adults, especially during the first three days of training. We hypothesized that development of tolerance in adults but not adolescents might mediate this difference. To investigate this hypothesis, we evaluated water maze performance and CB1 receptor function after brief repeated treatment with THC. Adolescents (PN28‐35) or adults (PN70‐75) were treated with THC (10 mg/kg) or vehicle for 5 days. Then they began water maze training, receiving vehicle or additional THC (10 mg/kg) during each day of training. To assess receptor function, adolescent and adult rats were treated with THC (10 mg/kg) or vehicle for 3 or 7 days, CB1 receptor function assessed by the ability of the agonist WIN55,212‐2 to stimulate GTPγS activation. Adults pre‐treated with THC were significantly less impaired by THC during training, whereas adolescents showed no effect of THC pre‐treatment. Hippocampal membrane GTPγS assays showed that adult CB1 receptors became more desensitized than adolescents. We conclude that greater THC‐induced adolescent learning impairment is due to lesser tolerance development to THC in adolescents.

Supported by R01‐DA019346.
Molecular biomarker panels for assessment of high selenium status in rats
Authors: Sunde Roger Allan , Raines Anna M
Published: June 2021

Molecular biomarkers are mRNA transcripts that indicate the (nutrient) status of an organism or tissue. We recently examined 13 liver biomarkers from rats fed graded levels of selenium (Se) from 0–0.8 μg Se/g diet to study molecular biomarker panels for predicting Se status (Exp Biol Med 235:1046), and found that a 4‐selenoprotein mRNA biomarker panel predicted liver Se concentration with a correlation of 0.948, but rather poorly predicted Se status above 0.2 μg Se/g. To develop molecular biomarker panels that cover the full spectrum of Se status from deficient to toxic, we conducted microarray experiments in weanling rats fed up to 5 μg Se/g diet. Rats fed 5 μg Se/g diet had significantly altered expression of 1193 liver transcripts, whereas rats fed ≤2 μg Se/g diet had <10 transcripts significantly altered relative to Se‐adequate animals within an experiment. Multiple regression analysis starting with 6 transcripts significantly altered by 2 μg Se/g plus 6 transcripts significantly altered by Se‐deficiency identified an 11‐transcript biomarker panel (r=0.9988, P<10−6) that accounted for 99% of the variation in liver Se concentration over the full range from 0 to 5 μg Se/g diet, whereas conventional liver selenoenzyme activity biomarkers (Gpx1 plus Gpx4 activity) only predicted 57% of the variability, showing that molecular biomarker panels can accurately predict supernutritional and toxic Se status.
Megalin−/− mice excrete N‐terminal fragments of selenoprotein P (Sepp1) in urine
Authors: Kurokawa Suguru , McDonald W. Hayes , Hill Kristina E. , Burk Raymond F.
Published: June 2021

Sepp1 transports selenium from the liver to other tissues via the plasma. Apolipoprotein E receptor‐2 facilitates Sepp1 uptake by brain and testis. Megalin, a related receptor located on the apical membrane of renal proximal convoluted tubule (PCT) cells, binds glomerular filtrate proteins for endocytosis. Sepp1, detected by an antibody (9S4) to its selenium‐poor N‐terminal domain, is endocytosed by PCT cells in a megalin‐dependent manner. Because of its large size, full‐length Sepp1 would not be expected to appear in the glomerular filtrate. Megalin−/− mice were studied to assess the nature of Sepp1 in their urine. They were injected with 75SeO32−. Urine was analyzed by SDS/PAGE autoradiography. Several 75Se‐labeled bands appeared in the urine of megalin−/− mice but not megalin+/+ mice. One bound to an antibody to glutathione peroxidase‐3 (Gpx3). Several bound to 9S4 but one did not bind to either antibody. LC‐MS/MS identified one band as Gpx3 and all others as N‐terminal fragments of Sepp1, each containing only one selenocysteine. Also, knockout of megalin decreased kidney selenium and plasma Gpx activity. We postulate that the selenium‐rich C‐terminal domain of Sepp1 is removed in the body and the resulting N‐terminal fragments are filtered and taken up by PCT cells, supplying selenium for Gpx3 synthesis. Supported by NIH grant ES02497.
CFA and IFA can independently provoke myocarditis in rats
Authors: Morozova Marya P , Gavrilova Svetlana A , Knyazhentseva Alina K , Zemcova Ludmila V , Pogodina Larisa S , Chentsov Yury S
Published: June 2021

The aim of this research was producing model of autoimmune myocarditis induced by immunization with cardiac myosin and identification of acute stage of inflammation.

We shown that single subcutaneous injection of cardiac myosin (400 μg/kg) combined with incomplete Freund's adjuvant (CFA) or CFA alone led to inflammatory response in rat myocardium with maximal reaction on 14–21 days after immunization. It was earlier confirmed by test of antibody levels in blood serum, light and electron microscopy of heart sections, expression of inflammatory cytokines and iNOS by RT‐PCR methods and analysis of nitrite and nitrate levels in blood serum and myocardial homogenates on 2, 3, 4, 7, 14, 21 and 28 days after immunization. We substituted CFA by incomplete Freund's adjuvant (IFA) without Mycobacterium tuberculosis wall components and repeated experiment and all testes on 4, 14 and 21 days. Our data shown that immunization of rats with myosin mixed with IFA caused the inflammatory response in myocardium on 4 day with maximum was detected on 21 day. In IFA injected rats the inflammation was also detected, but had a slow onset and developed to 21 day's.

We examined two models of autoimmune myocarditis and shown that CFA and IFA can independently provoke inflammation in myocardium.
FGF‐2 Regulates Valve Interstitial Cell Repair Through TGF‐b/Smad Signaling
Authors: Han Li , Gotlieb Avrum I
Published: June 2021

Background

Transforming growth factor‐β (TGF‐β) and fibroblast growth factor‐2 (FGF‐2) both promote valve interstitial cell (VIC) repair. The relationship between TGF‐β and FGF‐2 in wound repair is not understood.

Methods

VIC confluent monolayers were wounded and treated with FGF‐2, FGF‐2 neutralizing antibody, TGF‐β neutralizing antibody and/or betaglycan antibody. Phosphorylated‐Smad2/3 (pSmad2/3) was localized at the wound edge (WE). Down‐regulation of pSmad2/3 protein expression was achieved via siRNA transfection.

Results

FGF‐2 treatment increased the number of VICs with nuclear pSmad2/3 staining at the WE compared to nontreated WE. Treatment with TGF‐β neutralizing antibody alone or with FGF‐2 present resulted in a similar decrease in nuclear pSmad2/3. Treatment with FGF‐2 neutralizing antibody alone or with FGF‐2 present showed a similar decrease in nuclear pSmad2/3; however the effect of FGF‐2 neutralizing antibody was less than that of TGF‐β neutralizing antibody. Incubation with betaglycan antibody inhibited FGF‐2‐mediated pSmad2/3 signaling. Down‐regulation of pSmad2/3 reduced the extent to which FGF‐2 promoted wound closure.

Conclusion

FGF‐2 promotes in vitro VIC wound repair, at least in part, through the TGF‐β/Smad2/3 signaling pathway. Supported by the Heart and Stroke Foundation of Ontario (grant NA6204) and the Canadian Institutes for Health Research (grant 84228).
Fatty acid amide hydrolase is a key regulator of the endocannabinoid‐induced myocardial tissue injury
Authors: Horvath Bela , Mukhopadhyay Partha , Rajesh Mohanraj , Matsumoto Shingo , Saito Keita , Bátkai Sándor , Gao Yue R , Cravatt Benjamin F , Haskó György , Pacher Pál
Published: June 2021

Increased levels of endocannabinoids in various cardiovascular disorders (e.g. shock, cardiomyopathies, atherosclerosis) through the activation of CB1 cannabinoid receptors may promote cardiovascular dysfunction and tissue injury. We have investigated the role of the main endocannabinoid anandamide metabolizing enzyme (the fatty acid amide hydrolase; FAAH) in the myocardial injury induced by an important chemotherapeutic drug doxorubicin (DOX; known for its cardiotoxicity mediated by increased reactive oxygen and nitrogen species generation) using well‐established acute and chronic cardiomyopathy models in mice. The DOX‐induced myocardial oxidative/nitrative stress correlated with multiple cell death markers, which were enhanced in FAAH knockout mice exhibiting significantly increased DOX‐induced mortality and cardiac dysfunction compared to their wild type littermates. The effects of DOX in FAAH knockout mice were attenuated by CB1 receptor antagonists. Furthermore, anandamide induced enhanced cell death in human cardiomyocytes pretreated by FAAH antagonist, and enhanced ROS generation in inflammatory cells of FAAH knockouts. These results suggest that in pathological conditions associated with acute oxidative/nitrative stress FAAH plays a key role in controlling the tissue injury, which is, at least in part, mediated by the activation of CB1 receptors by endocannabinoids.

This study was supported by the Intramural Research Program of NIH/NIAAA (P.P.) and OTKA MB08‐A 80238 (B.H.)
Histomorphometric comparison of temporal and masseter muscles on an experimental malocclusion model
Authors: Gomes Marcolino , Farinha Rodrigo , Cabrita A.M. Silvério
Published: June 2021

Occlusal disarrangements are very often in the basis of the temporomandibular disfunction's sintomatology, and can course with muscular involvement. The aim of this study is to contribute to the understanding of pre‐morphologic changes in the masseter and temporal muscles in an experimental model of malocclusion, by morphometric analysis of muscular fibers areas, in transversal cuts.

20 male Wistar rats, 12 weeks old were distributed in 2 groups: the control (I), with the animals kept in the standard conditions and the test (II), in which we created a change of the normal occlusal contacts by adding fotopolimerized resin on the right upper teeth. After 2 weeks, all animals were sacrificed and the complete necropsy performed. Fractions of masseter and temporal muscles were collected for histopathological and morphometric analysis (using ImageJ).

The average area of muscle fibers on transversal section was bigger on masseter muscle of the left side, on group II. On the right side, we observed the opposite, with smaller average areas on group II.

The temporal muscle presented with an average area of muscle fibers much bigger on group II. These results suggest that both muscles are influenced by occlusal interferences, although the response is quite different between them. It was observed a difference of response on working and non‐working masseter muscles, too.
Nuclear localized GRP75 binds to retinoic acid receptors (RAR/RXR) and promotes retinoic acid‐induced neuronal differentiation of neuroblastoma
Authors: Shih Yu‐Yin , Pan Chien‐Yuan , Hsu Wen‐Ming , Liao Yung‐Feng
Published: June 2021

Neuroblastoma (NB) is the most common and deadly extracranial solid tumor in children. Retinoic acid (RA) has been used in clinic to treat NB by inducing neuronal differentiation of tumor cells. We previously found that glucose‐regulated protein 75 (GRP75) is correlated with the RA‐elicited neuronal differentiation of NB cells. In the present study, we further demonstrated that GRP75 can be translocated into nucleus and interact with retinoic acid receptor (RAR)α and retinoid X receptor (RXR)α to augment RA‐elicited neuronal differentiation. Down‐regulation of GRP75 diminishes the recruitment of RARα/RXRα to the retinoic acid response element (RARE) in the regulatory region of RA target gene promoters, resulting in a significant decrease in RA‐triggered gene expression. GRP75 also plays an essential role in stabilizing RARα/RXRα complexes in the presence of RA by reducing the proteasome‐mediated degradation of RARα and RXRα. Most intriguingly, the interaction between GRP75 and RARα/RXRα receptors was tightly associated with favorable outcome in NB patients and in a xenograft NB mouse model. Together, our present findings delineate a mechanism underlying the GRP75‐dependent regulation of RARα/RXRα‐mediated gene expression in the RA‐induced neuronal differentiation of NB, providing the basis for the development of novel therapeutics for NB.

Grant Funding Source: Academia Sinica
Changes in Apoptotic Mechanisms Following Penetrating Ballistic‐like Brain Injury
Authors: Cartagena Casandra M , Phillips Katie L , Tong Min , Tortella Frank C , Dave Jitendra R
Published: June 2021

Following penetrating ballistic‐like brain injury (PBBI), we examined 84 genes known to be involved in apoptosis. Enzyme activity for Caspases 3, 8, and 9 was also examined. PCR arrays (n=2) were done using pooled RNA from sham or PBBI rats collected 4 h post‐injury. In death receptor mediated apoptosis the mean relative quantity (RQ) of RNA for tnf increased > 5 fold but did not reach significance (p=0.13). In contrast, RQs for fas and trail were significantly decreased (p<0.05). There were no significant changes in RQs for casp8 or casp3; however, significant increases in enzymatic activity were measured for both (p<0.001). In the anti‐apoptotic TNF‐R2 pathway we found significant increases in RQs for tnfr2 and flip (p<0.001), indicating that TNF may mediate both pro‐ and anti‐apoptotic pathways following this brain injury. For mitochondria mediated apoptosis RQ increased > 3 fold for the anti‐apoptotic gene birc 3 but did not reach significance (p=0.11). There were no changes in RQs for p53 or bcl2, but a significant decrease in bax RQ was measured (p<0.05), possibly due to compensatory mechanisms following changes in either quantity of Bax protein or translocation of Bax to mitochondria. There was no change in RQ for casp9, but enzymatic activity was significantly increased post‐PBBI (p<0.001). These data indicate that within 4 hours of a PBBI injury multiple mechanisms promoting and inhibiting apoptosis are at play.
Report of a cubilin deficient patient
Authors: Storm Tina , Emma Francesco , Verroust Pierre J. , Hertz Jens Michael , Nielsen Rikke , Christensen Erik Ilsø
Published: June 2021

Background

Cubilin is an important receptor involved in intestinal absorption of vitamin B12 and reabsorption of proteins from the glomerular ultrafiltrate. Murine studies furthermore suggest that both cubilin and the receptor partner amnionless are essential for normal embryonic development. Cubilin function is affected in Imerslund‐Gräsbeck syndrome (IGS) patients. IGS is a rare, autosomal recessive disorder characterized by a selective, intestinal, intrinsic factor‐vitamin B12 malabsorption and is often accompanied by an additional proteinuria.

Aim

To investigate the role of cubilin in renal protein handling in a patient with cubilin deficiency.

Results

A novel homozygous null mutation of the CUBN gene was identified in a conserved donor splice site in a male IGS patient. No cubilin was detected in a renal biopsy and an abnormal, cytoplasmic distribution of the receptor partner amnionless was also observed. Immunoblotting of patient urine, accordingly, showed selective tubular proteinuria with increased excretion of cubilin ligands.

Conclusions

This clearly demonstrates a central role of cubilin in handling of filtered proteins in the human kidney and demonstrates the interdependent relationship of cubilin and amnionless in man. This patient furthermore, presented with no additional developmental abnormalities suggesting that cubilin is not essential for human embryonic development.
Protein Chip Array Profiling of Molecular Variants of Hirudins Using Surface Enhanced Laser Desorption Ionization (SELDI) Technique
Authors: Paramatmuni Vijaya , Biljani Rahul , Litinas Evangelos , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Various molecular variants of natural hirudin are developed for pharmaceutical purposes. Most of these are molecular variants, their conjugates with PEG Hirudin and fragment conjugates. Desirudin and Refludin represent recombinant hirudin variants with minor structural differences. PEG Hirudin represents a polyethylene glycol conjugate. Angiomax represents a terminal decapeptide of hirudin which is complex with a tripeptide and linker polypeptide. Although these products are homogeneous, their immunogenic responses reportedly vary. The purpose of this investigation was to investigate the molecular profile of each of these agents utilizing the SELDI technique employing a gold chip. Desirudin showed a single peak at 6.97kda, where is Refludin showed a single peak at 6.98kda. PEG Hirudin showed a heterogeneous peak at 17.8kda Angiomax showed a major peak at 2.18kda and a minor peak at 2.39kda. All of these products were free of other minor components below 1kda. These results show that ProteinChip array can be used to determine the molecular weight profile of each of these proteins and to check the presence of other impurities such as peptide fragment and other contaminants.
Chronic Coxofemoral Luxation in a 13th Century Virginia Dog: Differentiation from Hip Dysplasia
Authors: Kelderhouse Charles , Blick Jeffry , Uhl Elizabeth W
Published: June 2021

The almost complete skeleton of a male dog with hypertrophic osteopathy and a chronically luxated right coxofemoral joint was one of over 100 canine skeletons excavated from Weyanoke Old Town, a Native American village site in what is now Virginia. Radiometric analysis indicated this dog lived between 1224 and 1296 AD. It was of medium size (46 cm tall, 12–13 kgs), and was an older individual. The right femoral head was dorsally luxated and articulated with an acetabular‐like structure composed of a rim of irregular osteophytes with eburnated bone at its center. The ventral aspect of the right femoral head was flattened and eburnated where it was in contact with the dorsal rim of the acetabulum. Hip dysplasia is common in modern dogs and is characterized by bilateral, gradual, post‐natal craniodorsal subluxation of the femoral heads, which results in erosion of the acetabulum and cranial flattening of the femoral head. This dog's hip lesions were most likely traumatic, as they were not bilateral, the dorsal acetabular rim was still intact, and there was ventral rather than cranial erosion of the femoral head. Animal paleopathology is not as well‐established as human paleopathology, but careful comparisons of lesions found in ancient animal remains with those in modern animals can provide important information about animal health both in the past and the present.
The Association between Insufficient Vitamin D Levels, Ethnicity and Diabetes Status
Authors: Shaban Lamya , Zarini Gustavo G , Huffman Fatma G
Published: June 2021

To examine the association in a cross‐sectional study of the differences in 25 hydroxyvitamin D [25(OH) D] levels across a tri‐ethnic population of Cuban (CA= 202), Haitian (HA=251) and African American (AA=227) with and without Type 2 Diabetes (T2D). Participants were recruited from multiple sources from Miami‐Dade and Broward Counties, Florida. Serum vitamin D levels were measured with enzyme immunoassay and dietary variables were collected using a food frequency questionnaire. Data were analyzed with chi square and analysis of variance (ANOVA). AA with and without T2D had significantly higher incidence of 25(OH) D insufficiency (<75 nmol/L) than HA (p= .021 and p= .034) and CA (p= .001 and p= .002) with and without T2D respectively. HA without T2D had significantly higher incidence of insufficient 25(OH) D (P=.001) than CA without T2D. HA with and without T2D had significantly higher proportions of inadequate vitamin D intake (p= .003 and p=.014) compared to AA with and without T2D. A significantly higher proportion of HA with T2D had inadequate vitamin D intake (p= .003) than the CA with T2D. The findings of this study indicate that ethnicity and diabetes status play a significant role on vitamin D status. A need for further exploration as to why these differences are presented is warranted. Funding for this research was provided through an NIH/NIDDK sponsored grant.
Lymphocytic Variant of Hypereosinophilic Syndrome: diagnostic features of a rare entity
Authors: SHAHAB ASIF SAIYED , ZULFIQAR MUHAMMAD IMRAN
Published: June 2021

Hypereosinophilic syndrome (HES) includes a spectrum of diseases with eosinophilia and tissue damage. Two idiopathic causes of HES are the myeloproliferative variant (M‐HES)and the lymphocytic variant (L‐HES). L‐HES is a rare entity. It is characterized by clonal population of phenotypically abnormal T‐ lymphocytes. Clinical manifestations vary but cutaneous involvement is common. In this case we report a patient diagnosed with L‐HES with no clinical symptoms on presentation.

A 56 year old male was initially being evaluated for leukocytosis with a differential showing 33% eosinophilia. Physical exam was unremarkable. The patient had an extensive workup to rule out secondary causes. Bone marrow biopsy revealed cellular marrow with eosinophilia (10% of cellularity). No evidence of myeloproliferative disorders. JAK2 mutation was negative as well as FISH for bcl‐abr gene and PDGFRA gene. Flow cytometry revealed an unusual population of CD4+, CD2+, CD3‐, and CD8‐ T‐lymphocytes.

HES is an extremely heterogeneous disorder. Diagnostic criteria includes persistent eosinophilia of 1.5 × 109 eosinophils/L of blood, lack of evidence for secondary causes and presumptive signs of organ involvement. The L‐HES is an uncommon variant. Patients usually present with dermatologic manifestations. GI symptoms and obstructive lung disease are also common. Endomyocardial fibrosis and myelofibrosis are rarely seen. Cytokine IL‐5 also plays a role in pathogenesis. The most prevalent T cell clone associated with LHES seems to be the CD3‐CD4+ clone. L‐HES has a much better prognosis than M‐HES but patients with the CD3‐CD4+ clone can progress to T cell lymphoma. Despite the relatively low mortality in patients with LHES, morbidity is significant. L‐HES is steroid responsive while M‐HES is not. It is crucial to differentiate between the variants because of the vastly different treatment regimens and thus disease course.
Gallbladder as a novel source of stem cells of the hepatobiliary system
Authors: Manohar Rohan Raoul , Guzik Lynda , Stolz Donna Beer , LaFramboise William , Lagasse Eric
Published: June 2021

Background

The identification and characterization of stem cells within the biliary system has great importance for basic biology and potential cell‐based therapy. Currently, there is a paucity of data regarding stem cells in the gallbladder. Recent data have shown that the intra‐hepatic and extra‐hepatic biliary systems develop from separate progenitors. Therefore, in the adult hepatobiliary system extra‐hepatic stem cells would be unique compared to intra‐hepatic bile duct stem cells.

Aim

To isolate and characterize stem cells from the adult mouse gallbladder and determine their uniqueness relative to intra‐hepatic bile duct (IHBD) cells.

Methods

Gallbladder cells were expanded in vitro on feeders. Limiting Dilution Analyses and Index sorts were performed to evaluate candidate stem cells markers. A matrigel‐based assay was used to examine lineage commitment of the gallbladder cells.

Conclusions

We identify CD49f as a gallbladder stem cell marker. Gallbladder stem cells exhibit clonogenic self‐renewal and morphogenesis in vitro, expand and form structures in vivo short term. Primary gallbladder and IHBD cells exhibit different phenotypic profiles by flow cytometry. Microarray analyses on gallbladder stem cells and IHBD cells in vitro indicate different genomic expression patterns. These data underscore the uniqueness of gallbladder stem cells.

This work was in part supported by the Commonwealth of Pennsylvania and by the NIH grant R01 DK085711.
ER Stress‐Induced SREBP‐2 Activation Contributes to Lipid Accumulation in Tubular Nephrotoxicity
Authors: Lhotak Sarka , Sood Sudesh , Ingram Alistair , Austin Richard
Published: June 2021

Disruption of protein folding in the endoplasmic reticulum (ER) leads to ER stress and activation of the unfolded protein response (UPR). Tubular damage caused by nephrotoxic drugs is associated with ER stress. Since ER stress is known to induce lipid dysregulation through the activation of sterol regulatory element binding proteins (SREBPs), we hypothesized that lipid accumulation observed in proximal tubules affected by nephrotoxic drugs is a consequence of ER stress‐induced SREBP activation. Mice injected with tunicamycin display vacuolization and ER stress in proximal tubules. The vacuoles contain lipids that co‐localize with increased SREBP‐2 expression and markers of ER stress. In kidney biopsies from cyclosporine A (CsA)‐treated patients we show by immunohistochemistry the co‐localization of SREBP‐2 and ER stress markers in the damaged tubules. Treatment of HK2 renal cells with tunicamycin, thapsigargin or CsA caused UPR activation and increased lipid content. SREBP‐2 activation was shown by the enhanced sterol‐regulatory element‐GFP fluorescence, increased mRNA of downstream genes, and the presence of the mature form of SREBP‐2 on Western blot. These outcomes were blocked by AEBSF, an inhibitor of SREBP‐2 processing. We conclude that lipid accumulation in diseased kidney is associated with SREBP‐2 activation by ER stress. Supported by Heart and Stroke Foundation grant NA6024.
NAD+ Treatment Can Attenuate the Oxidative Stress‐Induced, PARP‐Mediated ATP Depletion in PC12 Cells
Authors: Chen Heyu , Chen Chen , Xia Weiliang , Ying Weihai
Published: June 2021

NAD+ is a critical factor in multiple biological functions. Poly(ADP‐ribose) polymerase‐1 (PARP‐1), a major NAD+‐consuming enzyme, plays a key role in oxidative stress‐induced cell death. Our previous study has indicated that NAD+ administration can greatly decrease ischemic brain injury, while the mechanisms underlying the effect remain unclear. In this study we tested our hypothesis that NAD+ treatment can attenuate the oxidative stress‐induced, PARP‐mediated ATP depletion, which may underlie the protective effects of NAD+ on brain ischemia. Our study shows that H2O2 treatment can significantly decrease intracellular ATP levels in PC12 cells, which was largely prevented by treatment of NAD+. In contrast, nicotinamide ‐‐‐ the degradation product of NAD+, produce weaker protection against the ATP depletion compared with NAD+, suggesting that NAD+ does not produce its effect on the ATP levels by producing nicotinamide. We also found that PARP inhibitors can prevent the H2O2‐induced ATP depletion, suggesting that NAD+ treatment can prevent the oxidative stress‐induced, PARP‐mediated ATP depletion in PC 12 cells. We also found that neither pyruvate nor antioxidants can attenuate the ATP depletion, arguing against the possibility that NAD+ restores the ATP levels by enhancing antioxidantion capacity of the cells or by increasing glycolysis. Collectively, our study has suggested that NAD+ treatment can restore the ATP levels in cells exposed to oxidative stress, which may at least partially underlie the protective effects of NAD+ on ischemic brain damage. (Supported by a Key Research Grant of Shanghai Municipal Scientific Committee #08JC1415400, and a Shanghai Engineering Center Grant of Equipment and Technology of Physical Therapy for Major Diseases)..
Dopamine transporter recruits α‐synuclein to the plasma membrane and cotrafficks
Authors: Goodwin Jeffrey Shawn , Patel Tina , Khoshbouei Habibeh
Published: June 2021

The dopamine transporter (DAT) and α‐synuclein are both expressed in dopaminergic neurons, and it is suggested they form a heteromeric complex in cells. Despite the involvement of α‐synuclein in PD and related α‐synucleinopathies, both its normal function and the mechanism of accumulation in neurons in disease states remain unclear. The primary structure of α‐synuclein suggests it can dynamically associate with plasma membrane and may regulate membrane proteins such as DAT. In the current study we used fluorescently tagged DAT and α‐synuclein to investigate the interaction of these two proteins in living cells using confocal microscopy. We found that DAT interacts with α‐synuclein and recruits it to the plasma membrane. Importantly the level of α‐synuclein recruitment to the plasma membrane directly correlated with surface DAT levels. Moreover, our preliminary data suggest that two populations of DAT exist: one that is colocalized with α‐synuclein and another that is not colocalized with α‐synuclein. Our preliminary findings suggest that the DAT colocalized with α‐synuclein, co‐traffics with this protein in the presence of 10 μM METH. Surprisingly, we found that α‐synuclein does not co‐traffic with DAT in response to PMA. Continued studies will examine these two DAT populations and whether they are differentially regulated by α‐synuclein.
Levadopa Activates Apoptosis Signaling Kinase 1 and Promotes Apoptosis in a Neuronal Model: Implications for the Treatment of Parkinson's Disease
Authors: Sabens Elizabeth A. , Mieyal John J.
Published: June 2021

Oxidative stress is implicated in the etiology of Parkinson's disease (PD), the second most common neurodegenerative disease. PD is treated with chronic administration of Levodopa (L‐DOPA), and typically increasing doses are used during progression of the disease. Paradoxically L‐DOPA is a pro‐oxidant and induces cell death in cellular models of PD through disruption of sulfhydryl homeostasisinvolving loss of the thiol‐disulfide oxidoreductase function of the glutaredoxin (Grx1) and thioredoxin (Trx1) enzyme systems (Sabens et al. (2010), Biochemistry 49, 2715‐24). Considering this loss of both Grx1 and Trx1 activities upon L‐DOPA treatment, we sought to elucidate the mechanism(s) of L‐DOPA induced apoptosis. In other contexts both the NFκB (nuclear factor κB) pathway and the ASK1 (apoptosis signaling kinase 1) pathway have been shown to be regulated by both Grx1 and Trx1, and both pathways have been implicated in cell death signaling in model systems of PD. Using SHSY5Y cells as model dopaminergic neurons we found that NFκB activity was not altered by L‐DOPA treatment. In contrast, ASK1 was activated with L‐DOPA treatment as indicated by phosphorylation of its downstream mitogen activated protein kinases (MAPK), p38 and JNK. Chemical inhibition of either p38 or JNK provided protection from L‐DOPA induced apoptosis. Moreover, direct knockdown of ASK1 protected from L‐DOPA induced neuronal cell death. These results identify ASK1 as the main pro‐apoptotic pathway activated in response to L‐DOPA treatment, implicating it as a potential target for adjunct therapy in PD. This work was supported in part by NIH Grant PO1 AG 15885 (J.J.M.) and a Department of Veterans Affairs Merit Review Grant (J.J.M.) and NIH Training Grants T32 GM008803 (E.A.S) and T32 DK007319 (E.A.S.).
3D‐based analyses of pharmacological modulation of zebrafish locomotion
Authors: Cachat Jonathan , Reihl Russell , Cosgrove Jessica , Gaikwad Siddharth , Green Jeremy , Hart Peter , Vollmer Karoly , Utterback Eli , Kyzar Evan , Rhymes Kate , Hook Molly , Chang Katie , Stewart Adam , Newman Alan , Allain Alex , Pham Mimi , Kalueff Allan
Published: June 2021

Here, we integrated manual observations, computerized event‐based scoring and automated video analysis of zebrafish traces to generate three‐dimensional (3D) swim path reconstruction of general behavioral patterns, thereby improving the characterization and interpretation of complex, anxiety‐related adult zebrafish behavior. Drugs known to evoke a high‐anxiety state (caffeine, repeated morphine withdrawal) or an anxiolytic‐like effect (ethanol, morphine, fluoxetine, nicotine) wereused to detect multiple anxiety‐like behaviors and correlate them to those established manual observations. Additionally, we used this method to characterize the effects of lysergic acid diethylamide and 3,4‐methylenedioxymethamphetamine on zebrafish novelty‐evoked exploration. Our approach markedly enhances the power of zebrafish behavioral pharmacology, by providing innovative techniques for high‐throughput, multidimensional neurophenotyping of adult zebrafish behavior.
Affective stress applied in different periods of brain development induces changes on the expression of APP isoforms and β‐amyloid in the hippocampal formation of rats
Authors: Limonte Fabio Henrique , Pereira Marina Teixeira Ramalho , Iyomasa Melina Mizusaki , Rosa Maria Luiza Nunes Mamede
Published: June 2021

This study aimed at evaluating the changes induced by isolation in different periods of brain development on the expression of APP695, APP751/770 and Aβ in the hippocampal formation of rats. Isolation rearing (IR): Pups remained with their mothers (6/mother) until weaning (PND21) when were allocated for ten weeks to: 1) grouped, housed 4/cage; 2) isolated, housed individually. Social isolation (SI): Rats (140g) were allocated in the same conditions. Maternal separation (MS): Pups were either separated from their mothers for 180min or left undisturbed from PND1‐21 when were housed 4/cage for 5 weeks. 40‐μm brain sections were used for immunohistochemistry. The immunopositive cells (IC) were quantified in hippocampus and entorhinal cortex (EC). Data were compared by t‐test (p<0.05). MS induced a significant increase in APP695 IC in hilus (35%, p=0.028) while IR induced a significant decrease (41%, p=0.001). No change was induced by SI in hilus but a significant decrease (10%, p=0.02) was found in EC. APP751/770 IC were seen only in CA2 of the hippocampus where a significant decrease was induced only by IR (24%, p=0.005). For Aβ IC a significant decrease was induced by MS (32%, p=0.026) and IR (98%, p=0.001) in hilus. In contrast, a significant increase was found in CA2 (62%, p=0.001) in SI. The isoforms of APP are differently affected in specific areas of the hippocampal formation by affective stress applied in different periods of brain development. These alterations are not necessarily associated to the alterations on Aβ suggesting that several plasticity mechanisms may be triggered by the association of different stages of development and different kinds of affective stress. Financial support: FAPESP and FPA.
Strain Differences Determine the Response to Citalopram on Hippocampal Neurogenesis, Neurotrophin Mobilization and Behavior
Authors: Brookshire Bethany R , Lucki Irwin
Published: June 2021

The objective of this study is to examine strain differences on measures of antidepressant‐related behavior and hippocampal neurogenesis in response to chronic treatment with the selective serotonin reuptake inhibitor citalopram. MRL/MpJ mice, known for superior wound healing and resilience to stress, were compared with C57Bl/6J mice on responses to chronic infusion of the selective serotonin reuptake inhibitor citalopram (5 mg/kg/day for 21 days). The animals were then evaluated in the novelty‐induced hypophagia (NIH) test and examined for increased hippocampal cell proliferation after BrdU labeling by flow cytometry. Exposure to chronic citalopram significantly reduced the latency to approach food in a novel environment in the NIH paradigm in the MRL/MpJ mouse strain but had no effect on approach latency in C57Bl/6J mice. MRL/MpJ mice also showed increased cell proliferation and increased BDNF levels in response to citalopram treatment, while C57Bl/6J mice showed no significant change in cell proliferation or BDNF levels. Overall, MRL/MpJ mice showed a significant behavioral and neurobiological response to citalopram, in contrast to the C57Bl/6J strain. These results indicate that strain differences strongly impact responses to chronic citalopram. Future studies will determine the mechanisms of antidepressant action in these mice.

This research was supported by USPHS T32 MH14654 and R01 MH72832.
Regulation of the growth factor signaling modulator, sprouty 2, by PHDs and pVHL/E3 ligase
Authors: Anderson Kimberly Michelle , Ying Chunyi , Patel Tarun B
Published: June 2021

Sprouty (Spry) proteins modulate receptor tyrosine kinase signaling and inhibit cellular processes. While levels of Spry isoforms, especially Spry2, have been shown to influence the growth of tumors from the lung, liver, breast, and prostate, little is known about the interaction of Spry with proteins involved in the hypoxic response. Here, we demonstrate that HA‐Spry 2 levels are elevated after exposure to hypoxia (3% O2) and DMOG (200μM), conditions that are known to inhibit the activity of prolyl hydroxylases (PHDs). Spry2 interacts with PHDs 1–3 as well as von Hippel‐Lindau tumor suppressor protein (pVHL)—two key players in the activation of the hypoxic response. Overexpression of all PHDs under normoxic conditions (21%O2) resulted in decreased HA‐Spry2 levels in HeLa cells. Similar results were obtained when pVHL was overexpressed in HEK293T cells. Moreover, expression of shRNA against PHD1 and PHD3 or treatment with siRNA against pVHL resulted in increased levels of overexpressed HA‐Spry2. Knockdown of endogenous PHD1 in HEK293T cells caused a further reduction in the phosphorylation of ERK upon FGF stimulation when HA‐Spry 2 was present, confirming that Spry2/PHD interactions represent an alternate mechanism by which Spry2 function can be regulated.

Supported by NIH grant GM073181
The aryl hydrocarbon receptor nuclear translocator‐interacting peptide 1 suppresses hypoxia inducible factor‐1 alpha signaling via an Arnt‐dependent mechanism
Authors: Wang Yu , Li Yanjie , Peng Depeng , Chan William K
Published: June 2021

The aryl hydrocarbon receptor nuclear translocator (Arnt) is a basic‐helix‐loop‐helix‐PAS protein which dimerizes with hypoxia inducible factor‐1α (HIF‐1α) under hypoxia in solid tumors. Hypoxia causes HIF‐1α protein accumulation; binding of the HIF‐1α::Arnt heterodimer to the corresponding enhancers upregulates the expression of genes that are involved in carcinogenesis and angiogenesis. Our objective is to explore whether interference of the Arnt function would suppress the HIF‐1α signaling. We utilized a novel peptide called Arnt‐interacting peptide 1 (Ainp1, 58 amino acids in length), which we had discovered from phage display screening, to address whether the HIF‐1α signaling could be suppressed by an Arnt‐mediated mechanism. Ainp1 suppressed the cobalt chloride‐dependent, HRE‐driven luciferase expression in Hep3B cells in a dose‐dependent manner. Ainp1 did not appear to bind HIF‐1α directly; however, it suppressed the interaction between Arnt and HIF‐1α. The GFP fusion of Ainp1 was located at both the cytoplasm and the nucleus of Hep3B cells. This GFP‐Ainp1 fusion suppressed the CoCl2‐dependent recruitment of Arnt to a HRE‐responsive promoter and decreased the nuclear Arnt content, but not the total Arnt content in Hep3B cells. We concluded that Ainp1 suppresses the HIF‐1α function by reducing the nuclear Arnt content. This work is supported by NIH (WKC, R01ES014050).
Activation of PLEKHG2 RhoGEF by selective G beta gamma isoforms released from multiple classes of G proteins
Authors: Runne Caitlin , Sun Zhizeng , Tang Xiao Yun , Chen Songhai
Published: June 2021

PLEKHG2 is a novel Gβγ‐activated Rho guanine nucleotide exchange factor (GEF) which catalyzes the activation of the RhoGTPase family of proteins. Overexpression of PLEKHG2 has been associated with enhanced cell transformation. Additionally, PLEKHG2 may mediate lysophosphatidic acid (LPA)‐induced cell spreading. However; the mechanism of PLEKHG2 regulation has yet to be elucidated. In this study we aim to identify how LPA stimulation of G proteins activates PLEKHG2. Using a luciferase‐based reporter assay, we found that PLEKHG2 activation via LPA is selectively mediated by Gβγ subunits released from G proteins, as activation is completely abolished by scavengers of Gβγ, GαT or the C‐terminal tail of G protein‐coupled receptor kinase 2. Interestingly, PLEKHG2 activation is only partially inhibited by Pertussis toxin, the Gαq minigene, or the Gα13 minigene that selectively uncouple the Gi/o, Gq/11, and G12/13 classes of G proteins. The combination of these three inhibitors has additive effect on reducing activation, indicating that activation of PLEKHG2 involves Gβγ subunits released from multiple classes of G proteins. By comparing the activation of PLEKHG2 by different Gβ isoforms, we further provided evidence that activation of PLEKHG2 is Gβ isoform specific. Taken together, our data indicate that PLEKHG2 is a RhoGEF activated by selective Gβγ isoforms released from multiple classes of G proteins.
Characterization of a Muscarinic M3 Receptor‐Associated Protein Complex
Authors: Kan Wei , Lanier Stephen M. , Smrcka Alan V.
Published: June 2021

A receptor and its cognate G protein may be pre‐coupled in the absence of agonist. Here, we address whether a) the muscarinic M3 receptor (M3R) is pre‐assembled with G proteins, and b) whether other G protein‐binding signaling molecules are present in this complex. We confirmed that M3R, a Gαq‐coupled receptor, stably co‐immunoprecipitated G proteins. Notably, phospholipase Cβ (PLCβ) was also present in this complex, suggesting that a stable complex of receptor‐G protein‐effector could be isolated. In agreement with previous findings that deleting most of third intracellular loop (ICL3) from full length M3R (M3RΔi3) permitted coupling to PLCβ, PLCβ was found to interact with M3RΔi3. This finding indicates that there are non‐ICL3 regions that bind PLCβ, either directly or indirectly through other proteins. A non‐receptor Gα guanine nucleotide exchange factor, Ric‐8, binds to and catalyzes nucleotide exchange on Gαq. We observed that Ric‐8 interacted with both full length M3R and M3RΔi3. This interaction may be dependent on Gαq and/or βγ. Furthermore, we demonstrated that GST fusion proteins constructed from M3R ICL3 directly bound G proteins and Ric‐8. We postulate that Ric‐8 and Gαq can pre‐couple PLCβ to M3R, independent of ICL3, and the multiprotein architecture built around M3R may serve to exert distinct signaling influences.
Role for FAK in proliferation, migration, and metastasis of EL4 lymphoma cells
Authors: Zhang Zhihong , Meier Kathryn E
Published: June 2021

Focal adhesion kinase (FAK) is a cytosolic protein tyrosine kinase that participates in adhesion‐mediated signaling. Our group previously established a panel of clonal cell lines derived from the EL4 mouse lymphoma line. These cell lines have been grouped into three phenotypes, only one of which forms aggressive tumors in an experimental metastasis model in syngenic mice. Numerous differences in mRNA and protein expression have been profiled between metastatic and non‐metastatic EL4 cells. One of the hallmarks of the metastatic phenotype in EL4 cells is expression of FAK. Therefore, current study examined the role of FAK in metastatic EL4 cells, using a siRNA knockdown approach. Incubation with siRNA against FAK was successful in reducing FAK protein levels in a metastatic EL4 cell line, as detected by immunoblotting. FAK knockdown significantly reduced cell proliferation in response to serum. In addition, cells with reduced FAK levels showed significantly reduced migration in response to EGF in a Boyden chamber assay. Treatment of metastatic EL4 cells with EGF enhanced FAK phosphorylation (Y397), as well as proliferation and migration. FAK knockdown blocked EGF‐induced activation of Erk. In summary, the results suggest that expression of FAK is critical for proliferation and migration of metastatic EL4 cells, and that FAK plays an important role in EGF response. Supported by NIH CA0944144.