bluuebunny/crossref_metadata_2025 huggingface dataset:
The influence of inflammation on the human visceral adipose tissue secretome
Authors: Vonk Roel J. , Szalowska Ewa , Weening Desiree , de Vries Marcel , Hoek Annemieke , Roelofsen Han
Published: June 2021

Chronic inflammation of obese adipose tissue is involved in the development of insulin resistance and type 2 diabetes, probably via altered secretion of adipokines and free fatty acids. To determine how inflammation changes the adipose tissue secretome, adipose tissue was challenged with LPS, a potent immune activator. Secretome changes were analyzed by a quantitative proteomics approach: Comparison of Isotope Labeled Amino acid Incorporation Rates (CILAIR). CILAIR compares incorporation rates of 13C‐labeled lysine in secreted proteins between conditions (Mol Cell Proteomics 2009, 8:316).

Methods

Human visceral adipose tissue was divided over six dishes. 13C‐Lys containing medium was added and to three dishes also LPS (100 μg/ml). Dishes were incubated for 24 hrs. Media were collected and processed separately, involving SDS‐PAGE fractionation, in‐gel digestion of excised bands and LC‐MS/MS. Database searching with ProteinPilot (Applied Biosystems) provided identifications and C13/C12 ratios.

Results

37 proteins were significantly changed (p<0.05) in expression of which 19 were up‐regulated by LPS and 19 were down‐regulated. Examples of up‐regulated proteins are: Plasminogen activator inhibitor 2, Growth‐regulated alpha protein and Interleukin‐6. In addition, 12 proteins were only expressed in the inflammatory condition. Examples are: Tumor necrosis factor‐α and C‐C motif chemokine 2 and 20.

Conclusion

These results indicate that inflammation profoundly changes the adipose tissue secretome. Secretion of several inflammatory cytokines and chemokines was up‐regulated as well as proteins involved in cell adhesion of attracted immune cells. These findings may have implications for the regulation of whole body insulin sensitivity and energy metabolism.
Impaired Energy Metabolism in Airway Epithelial Cells Exposed to the Industrial Toxicant Phosgene
Authors: Martens Margaret E , Olivera Dorian S
Published: June 2021

Phosgene is a common, highly toxic industrial gas that causes latent non‐cardiogenic pulmonary edema and is a model for chemically induced lung injury. Its toxicity arises from HCl release and acylation of proteins in pulmonary alveoli leading to breakdown of the blood:air barrier. We have developed an in vitro, air‐liquid interface exposure model in which airway epithelial cultures on Transwell inserts are combined with a Vitrocell exposure apparatus. Exposure to 0–64 ppm phosgene for 8 min caused dose‐dependent decreases in transepithelial electrical resistance, a measure of epithelial barrier integrity, and cell viability. Neither recovered at > 8 ppm. In view of the loss of cell viability, we examined the effects on energy metabolism. At 64 ppm phosgene, the rate of [14C]glutamine oxidation to [14C]O2 was inhibited by 72 ± 8% at 6 hrs and 68 ± 8% at 24 hrs after exposure (p < 0.05). However, 8 ppm only inhibited the rate by 23 ± 17% at 6 hrs and 16 ± 7% at 24 hrs (NS). Data for 6 vs 24 hrs did not differ. Preliminary assays of glycolysis showed no inhibition by phosgene. These data suggest that inhibition of aerobic metabolism may play a key role in the loss of cell viability and barrier integrity following phosgene exposure, in vitro. Supported by an Interagency Agreement from NIAID. The opinions, interpretations, conclusions, and recommendations are those of the author and are not necessarily endorsed by the US Army or the DoD.
Differential Gene Expression of Cutaneous Cells Upon Exposure to Staphylococcal Enterotoxin B (SEB) Superantigen
Authors: Srinivasan Seshamalini , Miller Stacy‐Ann , Shupp Jeffrey W , Crosby Margaret C , Hammamieh Rasha , Jett Marti
Published: June 2021

Staphylococcus aureus, a gram‐positive bacterium initiates lesions through the production of pyrogenic toxins termed Staphylococcal enterotoxins (SEA‐J), and toxic shock syndrome toxin‐1 (TSST‐1) causing staphylococcal food poisoning and toxic shock syndrome. SEs are prototypical superantigens (sAgs), which trigger massive V‐beta specific T cell proliferation without the need for internalization, processing, or presentation resulting in elevated serum cytokine levels. The presence and the detectability of these sAgs in the sera of burn victims is not clearly understood. We have detected circulating SEA, SEB and TSST‐1 toxins among 20% of the burn victims. In this study, we aim to elucidate the chronology of intoxication along with in vitro studies to better understand the potential effects of this SEB toxin on skin cells. Primary cultures of human keratinocytes, melanocytes, dermal vascular endothelial cells and dermal fibrobalsts were grown and treated with varying doses of SEB for different lengths of time. RNA was extracted by trizol method and oligonucleotide microarray was performed. Feature extraction and statistical analysis show that genes encoding negative acute phase response proteins, oxidative stress response, NFκB signalling, Anti‐apoptosis were highly upregulated and genes responsible for death receptor signalling, endothelin‐1 signalling, growth hormone signalling were significantly downregulated. The objective of this study is to elucidate the pathogenesis of SEB intoxication in skin cells and thereby help with new treatment strategies in wound healing.

We extend our heartfelt thanks to Washington Hospital ct Res Fund and Fire Fighters Burn res for their support
Exploratory Microarray Analysis Provides Genetic Insights into Nutrition and Nutrition Plus Strength Training Interventions in Latino Adolescents
Authors: Gao Michael C , Croymans Daniel M , Allayee Hooman , Goran Michael I , Roberts Christian K
Published: June 2021

Diet and exercise are critical for preventing metabolic diseases such as type 2 diabetes, but the underlying gene expression changes associated with metabolic adaptations are unclear. We previously implemented a 16‐week high‐fiber low‐sugar nutrition education program (N, n=16) or that plus 2 d/wk of strength training (N+ST, n=12) in Latino adolescents, and explored the intervention effects on transcriptional profiles. We measured mRNA levels pre‐ and post‐intervention using Illumina's Expression BeadChip in white blood cells. We selected genes which were changed in N or N+ST (p<0.05, fold change >1.41, unchanged in control group) and then used Ingenuity Pathway Analysis to determine functional networks (p<0.001) and their representation in established canonical pathways (p<0.01). For N, we found 14 changed functional networks. Carbohydrate metabolism, lipid metabolism, and small molecule biochemistry were 3 of the top 5 pathways noted. For N+ST we found 33; cell‐to‐cell signaling and interaction, cell growth and proliferation, cellular movement, and cell development were 4 of the top 5. In addition, we found 8 canonical pathways for N, and 5 for N+ST. Results show relevant mRNA level changes after N and N+ST and their functional groupings broadly relate to the type of intervention. Further investigation of these genes may provide valuable insights into therapeutic mechanisms.
Eukaryotic DNA Replication Fidelity
Authors: Kunkel Thomas A.
Published: June 2021

Many studies over the past half century have led to an appreciation of the enormous complexity of the cellular machinery required to replicate DNA with the accuracy needed to maintain genetic information over many generations, balanced by the need for mutations to arise on which selection can act to enhance fitness. This complexity is partly based on the need to repair, and sometimes replicate, potentially lethal and mutagenic lesions in DNA generated by endogenous and exogenous environmental stress. This talk will briefly consider the fidelity with which undamaged and damaged DNA is replicated by the many DNA polymerases now known to exist. Polymerase error rates can vary more than a million‐fold, depending on the polymerase, the composition and location of the mismatch and the ability to proofread. This variation offers the potential to modulate cellular point mutation over a remarkable range, with consequences that can be deleterious or beneficial. Recent work in budding yeast will then consider the fidelity of leading and lagging strand DNA replication, and several variables that influence the efficiency with which the mismatch repair machinery corrects replication errors.
Identification of novel target genes for the prevention of inflammatory monocytes adhesion to senescent endothelial cells
Authors: Boo Yong Chool , Mun Gyeong In
Published: June 2021

The mechanism for enhanced atherosclerosis with increasing age remains controversial. This study addressed this issue at the cellular level, by searching for genes uniquely expressed in senescent endothelial cells and functionally involved in inflammatory leukocytes adhesion, a critical initiation step of atherogenesis. Senescent human umbilical vein endothelial cells prepared by continuous sub‐culturing in vitro showed higher binding affinity for monocytes (THP‐1 cells, human acute monocytic leukemia cell line) compared with young cells. Comparing gene expression profiles between young and senescent endothelial cells by the cDNA microarray method, CD44 was identified as one of the ‘senescence‐induced cell adhesion genes’ whose expression was up‐regulated in senescent cells and whose gene ontology annotation indicated their role in cell adhesion. The enhanced gene expression of CD44 in senescent endothelial cells was verified at the mRNA and protein levels. Adhesion of monocytes to senescent endothelial cells was significantly reduced when endothelial cells were pretreated with CD44 antibody or small interfering RNA, supporting the critical role of CD44 in the inflammatory event. CD44 and other ‘senescence‐induced cell adhesion genes’ identified in this study may provide the novel targets for the prevention of inflammatory leukocytes adhesion leading to the development atherosclerosis.
Knocking down Interferon Induced Transmembrane protein 1 (IFITM1) Inhibited Cell Proliferation and Invasion in Glioma Cells
Authors: Lin Marie , Yu Fang , Ng Samuel , Kung Hsiang Fu
Published: June 2021

Here we investigated the role of Interferon Induced Transmembrane Protein 1 (IFITM1) in glioma carcinogenesis. We determined the expression levels of IFITM1 in different glioma cell lines, examined its role in cell proliferation/cell migration & invasion, and the corresponding molecular mechanism in glioma cells. Our results showed that IFITM1 mRNA level was elevated in four of five widely used glioma cell lines and positively correlated with their tumorigenic abilities. Suppression of IFITM1 by siIFITM1 inhibited the proliferation of U‐373 MG and U‐87 MG glioma cells lines in a time dependent manner. The growth inhibition effect of siIFITM1 was mediated not by apoptosis but G1 to S phase cell cycle arrest, which are mediated via decreased G1/S‐specific cylin‐D1, cyclin‐dependent kinase 2 (CDK2) and increased cyclin‐dependent kinase inhibitor 1B (p27kip1). Furthermore, IFITM1 knockdown significantly inhibit the migration and invasion of U‐373 MG and U‐87 MG cells, which was mediated by decreased MMP9 expression and activity. Taken together, these results suggested IFITM1 might be a potential candidate oncogene in glioma.
Suv39H1 Recruitment by Sp1 Causes a Decline in 12(S)‐Lipoxygenase Expression
Authors: Chuang Jian‐Ying , Chang Wen‐Chang , Hung Jan‐Jong
Published: June 2021

Sp1 is an important transcription factor that regulates gene expression. Several modifications such as phosphorylation, acetylation, glycosylation, ubiquitination, and sumoylation have been found in Sp1, and these affect its transcriptional activity. In this study, we found that Sp1 can directly interact with the methyltransferase Suv39H1 and that it is also modified by methylation. In addition, to inhibit the lynsine‐specific demethylase 1 with pargyline or Suv39H1 overexpression would increase the Sp1 methylation signal. In the early stages of mitosis, the Suv39H1 protein level increases and co‐localizes with Sp1. Sp1 plays the role of an anchor protein, recruiting Suv39H1 to the chromatin region in order to methylate histone H3, which leads to chromosome packaging during mitosis. In addition, the increase in Sp1 methylation represses the expression of Sp1 target gene, 12(S)‐Lipoxygenase. Taken together, these data indicate that Suv39H1 could be recruited to the genomic region by Sp1 to affect the Sp1‐denpendent gene expression through modulation of the post‐translational modification of Sp1 and histone H3 by methylation.
Pausing vs. Poising of RNA Polymerase II on Rapidly Induced Genes
Authors: Adamik Juraj , Wang Kent Z.Q. , Auron Philip E.
Published: June 2021

Evaluation of kinetic site occupancy for Pol II bound to human IL1B and TNF genes, as well as the state of CTD Ser5 and Ser2 phosphorylation and NELF and P‐TEFb recruitment following LPS induction, reveals differences between these two rapidly induced human monocyte genes. Pol II is primarily transiently recruited to TNF, whereas it is constitutively pre‐recruited and poised on IL1B. For IL1B, NELF recruitment and P‐TEFb‐dependent release following stimulation is similar to that observed for classical downstream pausing, and continues hours after initial induction. In contrast, TNF recruitment of Pol II is rapidly lost after 30 minutes. Both of these genes differ from a GAPDH control, which reveals constitutive Pol II recruitment, as has been reported by others. These data argue for two novel mechanisms regulating rapidly induced genes that differ from that associated with classical polymerase poising/pausing at TSS‐proximal downstream sites. TNF utilizes de novo recruitment followed by rapid shut‐down, whereas IL1B employs poised polymerase and subsequent continuous expression. The differences between TNF and IL1B are also observed to be manifested at the level of polymerase run‐on and mRNA synthesis, suggesting important distinctions between the kinetic expression of these two immediate‐early inducible genes.
Manipulation of ribosomes by viral RNAs
Authors: Kieft Jeffrey Scott
Published: June 2021

Viruses employ a variety of strategies to manipulate the basic molecular machinery of their hosts and to coordinate important events during infection. In many cases, virally encoded RNAs are at the heart of these strategies. One example of this are in viral internal ribosome entry sites (IRES) RNAs, which recruit, position, and activate the host cell's translation machinery to the viral RNA. The correct functioning of IRESs is critical for the successful infection of these viruses, which include hepatitis C virus, polio, HIV‐1, and others. In addition, IRESs are used by some cellular mRNAs and are thus are a potentially important mechanism of post‐transcriptional gene regulation. The intergenic region (IGR) IRESs of the Dicistroviridae viruses use the most streamlined IRES mechanism identified to date, binding directly to the ribosome and manipulating it into initiating translation of the viral message. As such, the IGR IRESs have been dubbed “molecular hijackers.” We are using the IGR IRESs as a model to understand some of the basic molecular tenets of IRES RNA structure, IRES‐ribosome interactions and IRES function, as well as to understand the basic workings of the ribosome that are potentially important for post‐transcription regulation, in general. We have developed a detailed model for the mechanism of the IGR IRESs using results from cryo‐EM, X‐ray crystallography, and other structural, functional, and biophysical methods. I will present new data from both structural and functional studies that test our mechanistic model and also suggest new ideas for how these RNA‐based molecular hijackers work.
RNA Transport in the Cytoplasm: How to Get There from Here
Authors: Mowry Kimberly
Published: June 2021

RNA localization is a widely conserved mechanism for generating polarized gene expression. We have studied the localization of mRNA in the frog oocyte as a model to gain mechanistic insight into how RNA molecules can be targeted to specific regions of the cell cytoplasm to generate spatially restricted gene expression. A major focus of our studies is Vg1 mRNA, which is transported to the vegetal hemisphere cytoplasm and encodes a secreted grown factor whose restricted expression is required for proper embryonic patterning. Our recent studies have focused on tracing the molecular pathway for cytoplasmic RNA localization, from the initiating events in the nucleus to motor‐driven RNP transport in the cytoplasm. Our results indicate that specific molecular motors transport RNP cargoes in discrete regions of the oocyte cytoplasm.
The Role of Glycosylation in the Efficacy of Cyclooxygenase‐2 Inhibitors
Authors: Graham Kamara Whitney , Sevigny Mary B
Published: June 2021

Cyclooxygenase‐2 (COX‐2) is an enzyme that catalyzes the rate‐limiting step in the prostanoid synthesis pathway, which plays an important role in a variety of physiological and pathophysiological processes including inflammation and cancer. COX‐2 exists as two major glycoforms of 72 and 74kDa, the latter resulting from an additional oligosaccharide chain at amino acid residue Asn580. The purpose of this study is to determine if this additional glycosylation affects the inhibitory ability of various COX‐2 inhibitors. COS‐1 cells were transiently transfected with either the wild‐type or Asn580‐mutant COX‐2 gene. Subsets of both cell groups were treated with various concentrations of either Aspirin, Flurbiprofen, Ibuprofen, or Celecoxib. After addition of the COX‐2 substrate arachidonic acid to inhibitor‐treated and untreated (control) cells, media was collected and subjected to an ELISA which measured levels of the downstream product prostaglandin E2. Results indicate that, at low concentrations, all of the inhibitors seem to have a greater effect on the 70/72 kDa glycoform. However, the effects of aspirin and flurbiprofen were less profound. This indicates that glycosylation of COX‐2 at Asn580 influences the efficacy of COX‐2 inhibitors.
A Robust and Convenient Fluorimetric Assay for Measuring Acetylcholinesterase Activity and Screening Acetylcholinesterase Inhibitors
Authors: Liao Jinfang , Zhao Qin , Diwu Zhenjun
Published: June 2021

Acetylcholinesterase (AchE) is one of the most crucial enzymes for nerve responses and neurological functions. AChE inhibitors are the key drugs for management of Alzheimer's disease and myasthenia gravis. Although AchE is a proven drug development target, the existing technologies for screening AChE inhibitors are a compromise between throughput, sensitivity and physiological relevance. We have developed a robust fluorimetric Thiolite Green–based assay for measuring AchE activities and screening AchE inhibitors. This new fluorimetric method is much more sensitive for detecting AChE activity than the existing colorimetric methods that is generally based on DNTB. The assay is based on the fluorogenic reaction of Thiolite Green with thiocholine. This specific fluorogenic reaction can be conveniently used to quantify thiocholine produced from the AChE–induced hydrolysis of acetylthiocholine. The fluorescence intensity of Thiolite Green‐thiocholine adduct is proportional to the formation of thiocholine, therefore the AChE activity. The Assay provides a continuous and ultrasensitive fluorimetric assay to detect as little as 0.1 mU/mL AChE, which is 100 times more sensitive than the DNTB colorimetric method. Its signal can be easily read by a fluorescence microplate reader with Ex/Em = 490/520 nm.
Overexpression of recombinant human procathepsin D in E. coli
Authors: Keefer Ryan , McConnell Matthew , Ye Alice , Chmielowski Jennifer , Berryman Laura , Wen Lisa , McConnell Rose
Published: June 2021

Cathepsin D is an aspartyl lysosomal protease that catalyzes protein cleavage. It is involved in the process of tumor invasion and metastasis. The cathepsin D enzyme has been considered as a potential target for cancer therapy. The objective of this research is to prepare functional recombinant cathepsin D enzyme which will be used in the development of new cathepsin D inhibitors. Human procathepsin D cDNA, purchased from OpenBiosystems, was amplified by PCR and sub‐cloned into XhoI site of pET15b. The procathepsin D‐pET15b construct was then transformed into JM109. The recombinant plasmid was isolated from the JM109 and transformed into BL21(DE3)pLysS cells for expression. Expression of recombinant procathepsin D protein in the transformed BL21(DE3)pLysS cells was induced with IPTG. The level of expression was analyzed by SDS‐PAGE. High level expression (greater than 50% of total cellular proteins) of the His‐tagged procathepsin D fusion protein was achieved when cells were grown in terrific broth. The ability to produce procathepsin D enzyme will permit a drug discovery program for development of newer effective inhibitors of cathepsin D in the near future.

Supported by National Cancer Institute at NIH Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1)
Targeting parasite FAR proteins in an attempt to restore host mucosal immunity during helminth infections
Authors: Maetzold Erin Charleen , Bath Jennifer
Published: June 2021

Hp‐FAR‐1 is a fatty acid and retinol binding protein that is secreted by the mouse parasite H. polygyrus. Previous groups in our lab have identified that the protein is secreted during each of the stages of the parasite's lifecycle. The corresponding gene, hp‐far‐1 was cloned into the pET‐30a vector and the expressed recombinant protein was purified on a HIS‐trap column and eluted in 500mM imidazole buffer. We demonstrate the binding capabilities and dissociation constants for both fatty acids and retinol with the purified recombinant protein. Hp‐FAR‐1's ability to bind retinol is of particular interest, given that retinol is the ligand for the retinoid X receptor, the transcriptional factor which controls the gene encoding IL‐4. IL‐4 deficiency has been demonstrated to contribute greatly to the severity of helminth infections. We are currently studying the effects of oral all trans‐retinoic acid (ATRA) treatment on H. polygyrus infected Balb/c mice. We hypothesize that treatment with ATRA will decrease the localized Th2 cell polarization, decrease parasitic worm burdens, and possibly (by preventing immunosuppression) correlate with improved immunity to protect against reinfection.
Possible Involvement of Manduca sexta Hemolymph Protease‐1 in Melanization
Authors: Suwanchaichinda Chansak , Kelo Lisha , Kanost Michael R
Published: June 2021

Melanization is a complex innate immune response in insects, involving multiple proteases in the prophenoloxidase (proPO) activation cascade. Hemolymph protease‐1 (HP1), a clip‐domain serine protease expressed in hemocytes, is found constitutively in the Manduca sexta hemolymph, yet its function is still unknown. RNAi techniques have been unsuccessful as a means to study the protease in vivo. As a result, we created a model by using the Drosophila S2 cell system to study the protease. The HP1 cDNA was cloned and incorporated into the S2 cellular genome. The cells were selected, and a stable cell line was established. Western blot analysis indicated that the expression of proHP1 in the cell culture was highly efficient. When the stable cells were treated with dsRNA against HP1, the expression of HP1 was blocked. To test whether HP1 might influence the activation of hemolymph proPO, the stable HP1 S2 cells treated with or without dsRNA were cultured as a monolayer. After induction of HP1 gene expression of the stable cells, M. sexta plasma was added to the culture. We found that the culture media of the cells expressing the HP1 protein turned dark‐colored, with an increase of PO activity, whereas the culture media of the cells in which the HP1 gene was silenced by RNAi remained unchanged. This result suggests that HP1 may function in the melanization/PO activation cascade. (supported by NIH grant no. GM41247)
Synthetic Method for the Development of Monorhamnolipids
Authors: Coss Clifford Scott , Polt Robin , Pemberton Jeanne , Maier Raina
Published: June 2021

Presented here is a cost‐effective and efficient synthesis and characterization of a variety of monorhamnolipids for the purpose of identification of biosurfactant properties. A complex mixture of mono‐ and dirhamnolipids can be extracted from the lipopolysaccharide (LPS) of the Gram‐negative bacterium, Pseudomonas aeruginosa, for the identification and understanding of the biosurfactants’ structural features, micellar activity, surface tensions and critical micelle concentrations (CMC). Figure 1 shows examples of a C10, C10‐mono‐ and diramnolipid. However, purification of this complex mixture is quite difficult and tedious and is never truly achieved. Therefore, surface tension and CMC measurements do not represent one pure rhamnolipid micellar system. Pure rhamnolipids can be achieved through synthesis and purification, thereby eliminating a need for extraction and the tedious purification process. Synthesis is achieved using fundamental carbohydrate and lipid chemistry, including an in depth study of rhamnoside glycosylation using a variety of Lewis acids. The final result is a general procedure for the cost‐effective, high‐yield production of monorhamnolipids varying in fatty‐acid chain length. This work is supported by a grant from the National Science Foundation (NSF).

Examples of a mono‐ and dirhamnolipid.

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Sequence and biochemical characterization of equilibrative nucleoside transporters from Crithidia fasciculata: seeking ligand binding residues
Authors: Arendt Cassandra S. , Beverley Stephen , Ullman Buddy
Published: June 2021

Eukaryotic cells acquire purines via de novo synthesis or salvage mechanisms. Unlike human cells, protozoan parasites from the Apicomplexan and Trypanosomatid lineages such as Plasmodium, Toxoplasma, Leishmania, Trypanosoma and Crithidia species lack de novo purine synthesis enzymes, and therefore rely on salvage of pre‐formed purine bases for growth and proliferation, making purine salvage an attractive drug target. A key step in purine salvage is transport of purine nucleobases and nucleosides into the cell, and each species encodes a unique repertoire of transporter proteins from the equilibrative nucleoside transporter (ENT) family, each with its own ligand preference. No detailed structural information for ENT proteins yet exists, but residues within four of the eleven transmembrane domains (TMs) appear to be important for ligand selectivity. Recently we have described the pivotal role of a lysine residue within TM4 of the Crithidia fasciculata purine nucleoside transporter CfNT2 in ligand discrimination. Here we describe the cloning of additional ENT genes from C. fasciculata using partial genomic sequencing and molecular techniques, and biochemical characterization of the encoded proteins. The role of sequence variations among the C. fasiculata ENTs in determining ligand preference may shed additional light on residues and regions of ENTs that contribute to ligand binding.
GILZ1: a key molecular determinant of intracellular signaling specificity governing epithelial sodium transport
Authors: Soundararajan Rama , Wang Jian , Melters Daniel , Shih I‐Chia , Pearce David
Published: June 2021

The kidney plays an important role in the long‐term regulation of sodium (Na+) balance, which is central to the maintenance of appropriate extracellular fluid volume and blood pressure. Renal Na+ reabsorption via the epithelial Na+ channel (ENaC) is dependent on the mineralocorticoid aldosterone. Aldosterone‐mediated Na+ transport includes the function of the PI3K‐dependent kinase SGK1, and a small leucine‐zipper protein called GILZ1, which negatively regulates the Raf‐MEK‐ERK1/2 MAPK pathway. Our studies suggest that GILZ1 and SGK1 demonstrate functional synergy in the modulation of epithelial Na+ transport, and point to a key role for GILZ1 as a scaffolding protein capable of harnessing pleiotropic signaling pathways to control Na+ transport. SGK1 is a short‐lived protein which is predominantly targeted to the ER to undergo rapid proteasome‐mediated degradation. GILZ1 redirects SGK1 away from the ER, and selectively recruits it to an ENaC‐regulatory complex (ERC), which includes Raf‐1 and the ubiquitin ligase Nedd4–2, the constitutively expressed inhibitory components of the ERC. Once in the ERC, GILZ1 and SGK1 (the aldosterone‐stimulated components of the ERC) synergistically stimulate ENaC surface expression and function by disinhibiting the inhibitory activities of Raf‐1 and Nedd4–2. This work is supported by NIH K01‐DK078679 (to RS) and R01‐DK51151 (to DP).
The role of Wip1 in regulating environmental stress‐induced cellular responses
Authors: Xia Yun , Liou Yih‐Cherng
Published: June 2021

The oncogenic phosphatase Wip1 acts as an important regulator in DNA damage pathway by dephosphorylating a spectrum of proteins including p53, p38, Chk1, Chk2, ATM and Mdm2. In this study we show that Wip1 negatively regulates the activation of MKK4‐JNK‐c‐Jun signaling during stress‐induced apoptosis. Loss of Wip1 function sensitizes mouse embryonic fibroblasts to stress‐induced apoptosis via the activation of both p38‐ATF2 and JNK‐c‐Jun signaling. Here we reveal that Wip1 has dual roles in alternatively regulating stress‐ and DNA damage‐induced apoptosis through p38/JNK MAPKs and p38/p53‐dependent pathways, respectively. Our studies also discover differential performances of Wip1 protein in response to different doses of genotoxicity. More importantly, the delicate regulation of Wip1 protein level is tightly related to different cell fates. Our results point to Wip1 as a general regulator of apoptosis, which further supports its role in tumorigenesis. Moreover, Wip1 may act as an indicator of cell fate upon different levels of DNA lesion.
NAD+ Treatment Induces Tumor Cell Necrosis by P2X7 Receptor‐Independent Pathways
Authors: Zhao Cuiping , Hong Yunyi , Han Jin , Xia Weiliang , Ying Weihai
Published: June 2021

NAD+ plays important roles in various biological processes. The aim of this was to determine the effects of NAD+ on the survival of cancer cells. We found that NAD+ treatment can dose‐dependently and time‐dependently induce the necrosis of various types of cancer cells without producing apoptosis. Our study has also excluded the possibilities that NAD+ induces the cell death through its degradation product nicotinamide, and that P2X7 receptors mediate the effects of NAD+ on cancer cells. Our study has provided evidence indicating NAD+ as a novel cancer cell‐killing agent, and suggested presence of novel NAD+‐dependent mechanisms for inducing cancer cell death. (Supported by a Key Research Grant of Shanghai Municipal Education Committee, by Shanghai Engineering Center Grant of Equipment and Technology of Physical Therapy for Major Diseases, and by a 973 Grant 2010CB834306)
Overexpression of human glycolipid transfer protein transcript (GLTP), but not novel splice variants GLTP_v1 or GLTP_v2, induces apoptosis
Authors: Zou Xianqiong , Gao Yongguang , Kamlekar Ravi Kanth , Pike Helen M. , Brown Rhoderick E.
Published: June 2021

Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer and serves as the conformational prototype defining the conserved GLTP superfamily. In humans, the single‐copy GLTP gene at 12q24.11 encodes the five‐exon GLTP transcript. Herein, two previously unknown GLTP splice variants are identified and functionally evaluated. Exon1 was the only exon common to the GLTP, GLTP_v1, and GLTP_v2 transcripts. In GLTP_v1, a new exon has been discovered within sequence previously designated GLTP intron2. The GLTP splice variants are highly tissue specific compared to the more ubiquitous GLTP transcript. Immunolocalization reveals distribution of exogenously‐expressed GLTP_i1 and GLTP_i2 throughout HeLa cells and, unlike GLTP, presence within nuclei. Exogenous GLTP expression induces S phase cell cycle arrest and apoptosis in HeLa cells. The pro‐apoptotic effect of GLTP occurs independently of glycolipid binding/transfer. In contrast, exogenous expression of GLTP_v1 and GLTP_v2 protects cells from apoptosis induced by hydrogen peroxide or etoposide without altering the cell cycle. Neither GLTP splice variant affects the expression or glycolipid transfer activity of GLTP. The data show for the first time that overexpression of GLTP, but not GLTP_v1 or GLTPv2, induces cell apoptosis, underscoring the biological diversity of the GLTP gene [Support: NIH GM45928, CA121493 & Hormel Fnd.]
Peroxiredoxin, a mitochondrial‐specific peroxidase, as a possible key regulator of the β‐adrenergic receptor anti‐apoptotic pathway
Authors: Slusser Andrea M. , Porter James E.
Published: June 2021

We previously reported a β‐adrenergic receptor (βAR) modulation of apoptosis requiring formation of a β‐arrestin/heat shock protein (HSP) 27 signaling complex for protection against program cell death. Our hypothesis is that a protective mechanism for this signalosome occurs by localized cellular trafficking of specific proteins. Non‐biased gene microarray profiling was used in epithelial (UROtsa) cells to identify gene products altered after co‐incubation with isoproterenol (ISO) and an apoptotic inducer, staurosporine (STS). Gene products found significantly changed when compared to STS alone included PRX, a mitochondrial peroxidase, FBXO28, a nuclear ubiquitin ligase and ZNF638, a pro‐apoptotic transcription factor. Quantitative real‐time PCR was used to validate changes of these gene products. There was increased transcription significantly different from STS only for PRX and FBXO in cells treated with ISO plus STS. Similar results were found for PRX using a neuronal cell model. Conversely, there was a significant decrease in ZNF638 transcription for UROtsa cells treated with ISO plus STS when compared to STS alone. All results corroborate observations from the gene microarray analysis. Higher PRX expression seen with concurrent ISO incubation is hypothesized as a compensatory mechanism for the increased O2− production initiated by STS. Supported by a SURF from ASPET and NIH grant P20RR016741.
Depletion of PIN1 increases APE1 activity in EMT6 murine mammary adenocarcinoma cells
Authors: Stevens Rachel L. , Schneider Ryan A. , Liu Tongzheng , Hoyt Dale G.
Published: June 2021

Base excision repair (BER) proteins are regulated by post‐translational modifications including phosphorylation. Protein Interacting with Never In Mitosis Gene A‐1 (PIN1) regulates the turnover and function of many phosphoproteins by facilitating the cis‐trans isomerization of phospho‐serine/threonine‐proline motifs. Consequently, we have examined the role of PIN1 in the regulation of BER proteins. Previously, we found that depletion of PIN1 with shRNA in EMT6 murine mammary xenograft tumors increased levels of the BER endonuclease, apurinic/apyrimidinic endonuclease 1 (APE1), compared with control tumors. Here we determined the effect of PIN1 depletion on APE1 expression, subcellular distribution, and activity in EMT6 cells in culture. Cells were transduced with lentivirus encoding small hairpin RNA targeting PIN1, or with an inactive control sequence. Stably transduced cells were selected with puromycin. Cells were lysed, and nuclei and cytosol were separated by centrifugation. Western blotting revealed no difference in total, nuclear, or cytosolic APE1 between the control and knockdown cells. However, APE1 activity, assessed by cleavage of a fluorescently tagged DNA oligomer containing an abasic site, was significantly higher in knockdown compared to control cells (p<0.05). The present results suggest that PIN1 can regulate APE1 function in EMT6 cancer cells.
Are estrogen and leptin the sole factors affecting the estrous cycle of ob/ob and ovariectomized mice?
Authors: Yong Jennifer Pui‐Wah , Ng Ka Yan , Chakraborty Tandra R.
Published: June 2021

Hormones like leptin and estrogen have been suggested to increase energy expenditure and modulate estrous cycle. The aim of this study was to investigate the stages of estrous cycle in mouse models with contrasting leptin and estrogen levels. Estrous cycle of Wild type (WT), WT ovariectomized (OVX), ob/ob and ob/ob OVX was observed by obtaining vaginal smear and staining with Papanicolaou (PAP) procedure. Quantitative analysis showed cellular morphology and predominance of cellular content across the estrous cycle. Microscopical and quantitative counts showed a 4–5 day regular cycle in WT mouse, WT OVX were acyclic, infertile and hardly showed any cells, ob/ob and ob/ob OVX were both acyclic, infertile and were at persistent estrous or persistent metaestrous phase. Enzyme‐Immunometric Assay results showed that leptin levels were about 3 fold higher in WTOVX than WT mouse (p<0.0001) with beta‐estradiol values being negligible in WTOVX. The beta‐estradiol level for ob/ob and WT were similar. These results suggest that although leptin and estrogen play an important role in the estrous cycle, they are not the sole factor influencing the cycle. It is probable that in the absence of leptin and decrease in sex steroid hormones with increase in acyclicity, the central nervous system probably interprets the circulation as absence of energy stores and shuts down reproduction indicated by the changes in the estrous cycle.
Contributions of familial mutation E46K, phosphorylation, protein domains, and alanine‐76 to α‐synuclein toxicity in yeasts
Authors: Fiske Michael , DebBurman Shubhik
Published: June 2021

Parkinson's disease (PD) is an incurable neurodegenerative disorder linked to the misfolded protein α‐synuclein, which is often phosphorylated and membrane‐lipid associated within the Lewy bodies in the dying substantia nigra neurons. Exactly how α‐synuclein phosphorylation, aggregation, and membrane association contribute to toxicity is unresolved, as is the mechanism of toxicity linked with newer familial mutant E46K. We recently created two yeast models that recapitulate α‐synuclein properties: budding yeast for membrane association and fission yeast for aggregation. Here, first, we report that E46K resembled wildtype and A53T α‐synuclein by associating with the plasma membrane in budding yeast and forming aggregates in fission yeast. Next, we demonstrate that serine phosphorylation promoted α‐synuclein aggregation, and also, unexpectedly, membrane association. In fission yeast, S87A & S129A mutants redistributed to diverse intracellular compartments, while in budding yeast both mutants reduced plasma membrane localization. Lastly, we provide evidence that the domains N and NAC (and alanine‐76 within NAC), but not domain C, contributed to membrane association in budding yeast. Furthermore, in fission yeast, A76E was less aggregated. Thus, both yeast models of α‐synuclein further illuminate mechanisms that underlie familial and sporadic PD. (Supported by NSF‐MRI, NSF‐CCLI & NIH R15)
Presence of calcium binding proteins in the echinoderm nervous system and their preferential distribution to the connective tissue plexi
Authors: Lázaro‐Peña María I. , Diaz‐Balzac Carlos A. , González Carlos I. , García‐Arrarás José E.
Published: June 2021

Echinoderms have been considered an enigmatic group of deuterostome animals. One of their most interesting characteristics, referred to as mutable connective tissue, is the ability of the non‐cellular connective tissue (CT) to modulate its stiffness. These changes in the CT are known to be under nervous system control. Here we investigate the association of nerve cell and fiber populations with the CT components of the sea cucumber Holothuria glaberrima. Antibodies made against intracellular calcium‐binding proteins with EF‐hand motifs, namely, rat Calbindin‐D28k (CB) and Parvalbumin (PV) and human Calretinin (CR), labeled cells and fibers within the echinoderm nervous system. The neural specificity was verified by co‐labeling with the neural‐specific marker RN1. Immunoblotting with anti‐CB and anti‐PV showed that these markers recognized an antigen of 32 kDa in homogenates of the radial nerve cord. Bioinformatic analyses of the possible antigens being recognized by these markers showed that a CR homolog, similar to the CB‐32 previously identified in other invertebrates, is present in the echinoderms. Our findings suggest that the CB‐32 present in invertebrates is the homolog of CR, while CB‐D28k is a paralog. The immunoreactivity of these markers in the CT component provides the first evidence for a candidate protein that could be playing a direct role in the control of mutability of the CT in echinoderms.
Maternal iron deficiency: effects on fatty acid and eicosanoid metabolism and spatial memory in the adult guinea pig offspring
Authors: LeBlanc Caroline P , Surette Marc E , Fiset Sylvain , Rioux France M
Published: June 2021

Iron deficiency is the most prevalent worldwide nutritional deficiency. Groups at risk in industrialized countries are infants and pregnant women. The goal of this study was to evaluate the long‐term consequences of maternal iron deficiency on the offspring's fatty acid and eicosanoid metabolism, behavior and spatial memory. Female guinea pigs consumed iron sufficient (IS) and iron deficient (ID) diets for 14 days before mating and throughout pregnancy and lactation. Iron restriction resulted in ID in pregnant females. On post‐natal day (PND) 9, all offspring (ID and IS) were weaned on the IS diet and at 42 days all offspring were iron replete. Locomotion was tested in pups on PND 24 and 40 and spatial memory from day 25 through 40. Pups from the ID group were significantly more active in the open‐field at both times of testing while spatial memory was comparable in both groups. On PND 42, dihomogammalinolenic 20:3(n‐6), docosapentaenoic 22:5(n‐3) and docosahexaenoic 22:6(n‐3) acid content was significantly higher in brain phospholipids of offspring born to ID dams. Prostaglandin E2 and F2α concentrations were also significantly higher in brains of offspring born to ID dams. This demonstrates that moderate iron deficiency during gestation and lactation results in alterations of brain fatty acid and eicosanoid metabolism and perturbation in behavior in adult offspring. Funded by CIHR and CRC.
Effect of iron status on lipid and glucose metabolism
Authors: Kim Jonghan , Buckett Peter D. , Liu Sihao , Li Yingxia , Cohen David E. , Lee Chih‐Hao , Wessling‐Resnick Marianne
Published: June 2021

Belgrade (b/b) rats have impaired divalent metal transporter‐1 (DMT1) activity and display iron‐loading anemia. To investigate the relationship between iron status and lipid/glucose metabolism, we characterized the metabolic phenotype of b/b rats. Fasting serum triglyceride (TG) levels were higher in homozygous b/b rats compared to heterozygous littermate (+/b) control rats at the age of 6–18 weeks. By 18 weeks of age, b/b rats also exhibited mild hyperglycemia. Although serum insulin level was reduced in b/b rats, its secretion was not affected; rather, b/b rats were insulin‐sensitive, suggesting that b/b rats have altered lipid metabolism and consequently, impaired glucose homeostasis. These alterations appear to be related to iron‐loading since anemic +/b rats fed an iron deficient diet showed normal serum TG. Neither hepatic TG levels nor lipogenic gene expression was altered in b/b rats. After injection with Triton WR1339, a lipoprotein lipase inhibitor, TG secretion kinetics were unchanged in b/b rats, suggesting that hypertriglyceridemia results from reduced TG clearance and not from enhanced TG secretion. The apoE/apoB lipoprotein ratio in b/b rats was 50% of that observed for +/b rats, further supporting the idea that b/b rats have slower plasma VLDL removal. Supported by NIH R90 DK071507 and R01 ES014638.

Grant Funding Source: NIH
Changes of erythropoietin concentrations in plasma and kidney induced by zinc deficiency in rats
Authors: Konomi Aki , Yokoi Katsuhiko
Published: June 2021

Some researchers reported that oral zinc (Zn) effectively cured iron refractory anemia. We reported before that Zn deficiency depressed plasma erythropoietin (EPO) concentration in rats. We propose a hypothesis that Zn deficiency depresses EPO production in kidney. To test the hypothesis, thirty 3‐week‐old male SD rats were assigned into 3 groups of 10 for the 4‐week study receiving AIN‐93G‐based EDTA‐treated casein diet: ad libitum fed control group (AIN‐93G), moderate Zn‐deficient group (4.5 mg Zn/kg diet) and pair‐fed group (AIN‐93G). Plasma and kidney (medulla and cortex) EPO concentrations were measured using ELISA kit. Difference between groups was statistically analyzed by Fisher's PLSD. P value less than 0.05 was considered significant. Plasma EPO concentration of Zn‐deficient group was significantly decreased compared with control group, and pair‐fed group was marginally decreased compared with control group (P=0.056). Kidney medulla EPO concentration was decreased in Zn‐deficient group compared with pair‐fed group. Kidney medulla EPO concentration in pair‐fed group was higher than that of control group but the difference was not significant. No significant difference was observed in kidney cortex EPO concentration. These results support the hypothesis that Zn deficiency impairs EPO production in kidney.

This work was supported by Japan Society for the Promotion of Science Young Scientists (Start‐up) (21800073).
Identification of Potential Biomarkers for Assessing Zinc Status
Authors: McCormick Nicholas , Kelleher Shannon
Published: June 2021

Marginal zinc (Zn) deficiency is presumed to be prevalent throughout the world. Currently, there is no reliable biomarker of Zn status. Although plasma Zn concentration is routinely used, its reliability is questionable due to tight homeostatic regulation. Herein, we utilized microarray analysis of monocytes isolated from mice fed diets differing in Zn content to identify potential biomarkers for marginal Zn deficiency in humans. Weanling male Swiss‐Webster mice were fed a diet differing only in Zn content (n=10 mice/diet) for 30 days: supplemented (180 mg Zn/kg diet), control (30 mg Zn/kg diet) and marginally low (15 mg Zn/kg diet). Weight gain, food intake, and serum Zn levels were not affected. Zn concentration in liver, small intestine, spleen, kidneys, and muscle were reduced in mice fed a Zn deficient diet. Microarray analysis revealed 433 genes whose mRNA expression differed significantly between diet groups. Of 127 annotated genes there was an effect of Zn intake on the expression of 24 genes (p<0.05) including RNA‐binding proteins, mitochondrial proteins, and protein kinases. In conclusion, our data suggest that these novel genes may be useful for establishing a gene signature for Zn nutrition, and studies are under way to validate their usefulness in assessing Zn status in human populations. Supported by Intramural Funds to SLK.