bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Nat5/San, a novel protein expressing both N‐alpha and N‐epsilon acetyltransferase activity
Authors: Evjenth Rune Henrik , Hole Kristine , Karlsen Odd Andre , Ziegler Mathias , Arnesen Thomas , Lillehaug Johan R
Published: June 2021

Protein acetylation is among the most common protein modifications in eukaryotic cells. There are two types of acetylation; N‐epsilon and N‐alpha which are performed by distinct types of enzymes. N‐epsilon acetylation is performed by histone acetyltransferases (HAT) targeting epsilon‐amino group of lysines within histone proteins, a central modification in regulating gene transcription.

Protein N‐terminal acetylation is performed by different N‐terminal acetyltransferase complexes (NAT). In yeast, three different NAT complexes are identified. One of them; NatA, is composed of two main subunits; Ard1p (catalytic subunit) and Nat1p (auxiliary subunit). Nat5p is a third subunit physically associated with NatA, but with unknown functions.

We wanted to investigate the substrates of hNat5. Purified recombinant hNat5 and oligopeptide substrates were mixed and separated with reverse phase HPLC to study hNat5 acetylation activity. We found that hNat5 prefers substrates with N‐termini Met‐Leu‐Xxx‐Pro. In addition, mass spectrometry analysis of hNat5 incubated with acetyl CoA, demonstrated that hNat5 has autoacetylation activity. Autoacetylation and acetylation of other subset of substrates indicate activity similar to HAT.

In conclusion, we here present a novel human acetyltransferase, hNat5 with a dual activity targeting both N‐terminal (NAT activity) and lysine ‐ amino groups (HAT activity).
Functional role of domains in the Snf2 subunit of the SWI/SNF complex outside the catalytic domain
Authors: Sen Payel , Ghosh Sujana , Pugh Franklin , Blaine Bartholomew
Published: June 2021

SWI/SNF is the classical ATP dependent chromatin remodeler that restructures nucleosomes by virtue of its DNA and nucleosome stimulated ATPase activity. In this context, much attention has been devoted to the biochemical and genetic analyses of the ATPase‐helicase domain of the catalytic subunit Snf2. The catalytic contribution of the regions outside this domain has been explored to a limited extent, mostly focusing on the C terminal bromodomain. In this study, we use bioinformatic tools to identify putative domains in Snf2 (N, QLQ, spacer and AT), selectively delete them and characterize the domain deleted complexes both in vitro and in vivo. Our results indicate that complex integrity is preserved in the absence of these domains. Deletion of N terminus and the double AT hook show no difference in activity in vitro although these deletions have a snf2 null global gene expression pattern. In contrast, "QLQ" and "spacer" deletions are catalytically defective both in vitro and in vivo. In vitro, they show poor ATP hydrolysis and remodeling capacity. In vivo, they closely mimic snf2 null phenotype. Site directed DNA and histone crosslinking studies identify subtle conformational changes in presence of these deletions that may perturb proper nucleosomal contacts critical for remodeling. In summary, regions outside the ATPase‐helicase domain of Snf2 contribute positively to the catalytic function of the holocomplex.
Insights into the substrate specificity of INO80 remodeling
Authors: Udugama Maheshi Imalka , Sabri Abdella , Bartholomew Blaine
Published: June 2021

INO80, a multisubunit SWI2/SNF2 superfamily member has recently gained attention because of its involvement in transcription regulation and DNA repair. Not much is known about its substrate specificity and remodeling mechanism and how it differs in comparison to more characterized complexes such as SWI/SNF or ISWI. In this study we focused on identifying its optimal substrate, extranucleosomal DNA dependence, and histone tail requirement. High resolution site directed photocrosslinking and mapping of histone‐DNA contacts showed that INO80 remodeled end‐positioned nucleosomes towards the center, but it was incapable of remodeling centrally positioned nucleosomes. The length of extra nucleosomal DNA is found to play an important role in nucleosome binding as well as remodeling by INO80. Kd values of INO80‐nucleosome complexes with varying lengths of extranucleosomal DNA available on one side of the nucleosome revealed that INO80 preferentially bound to nucleosomes =53bp of DNA. Similarly, INO80 remodeling on mononucleosomal substrates with different linker lengths showed that it required at least 53bp of extranucleosomal DNA on one side of the nucleosomes for it to remodel. INO80 remodeling on short arrays further revealed that it remodels nucleosomes away from DNA ends and it tends to reorganize a short array of tri‐nucleosomes spaced with 50bp of linker to an array spaced with 30bp linker DNA in between the nucleosomes. Although N‐terminal histone tails are known to interact with various chromatin remodelers, INO80 did not require any of the histone tails for its nucleosome binding and sliding activities.
Successful inhibition of Dnmt1 expression in bovine using RNA interference.
Authors: Sosa Hector Eduardo , LaMarre Jonathan , King Allan
Published: June 2021

Epigenetic modifications, such as methylation are essential for normal mammalian development. Dnmt1 is the predominant and most active methylase, which is responsible for copying methylation patterns after replication. During early development, Dnmt1 is highly regulated to support passive demethylation and normal reprogramming. In somatic cells, Dnmt1 is localized in the nucleus and its introduction into cloned embryos is believed to be the main limiting factor for proper reprogramming of the donor genome. The aim of this study was to transiently suppress Dnmt1 by RNA interference (RNAi) to investigate the role of Dnmt1 during early development and its effect on nuclear transfer technology. RNA and protein extracts were collected from fibroblasts treated with control or Dnmt1 RNAi. Real time RT‐PCR revealed a 93.4% reduction of Dnmt1 transcript levels. Consistently, Western blot analysis showed a protein decrease of up to 98.8%. Gene expression of housekeeping genes, GAPD, ACTB and lamin A/C, were not affected after RNAi treatment however, expression of HPRT1, H19, MMP1 and SCYA genes was increased. This is the first report to demonstrate a specific and highly effective knockdown of bovine Dnmt1 by RNAi at both mRNA and protein levels. Dnmt1 RNAi could be a useful tool to better understand epigenetic reprogramming events as well as syndromes related to abnormal methylation patterns. (Funding by CONACyT & NSERC)
To Degrade or Not to Degrade, That is the Question
Authors: Brooks Jeanne Kaye , Greer Susanna F.
Published: June 2021

The ubiquitin‐proteasome pathway plays vital roles in multiple processes including protein turnover and transcription regulation. Control of each of these pathways relies on the formation of ubiquitin‐tagged proteins via a series of enzymatic reactions resulting in the addition of ubiquitin molecules to lysine residues within target proteins. The fate of the ubiquitinated protein is determined by both the number of ubiquitin molecules and the site to which they are added. Proteins that are mono‐ubiquitinated have been shown to be involved in chromatin remodeling, DNA repair, and transcriptional activation, while polyubiquitinated proteins are targeted to the 26S proteasome for degradation. Major histocompatibility complex class II (MHC‐II) molecules are a vital part of the adaptive immune response and are responsible for presenting exogenous antigens to CD4+ T cells. The class II transactivator (CIITA) is the master regulator of MHC‐II transcription and has been shown to have increased transactivity in the presence of monoubiquitin. Our study highlights the impact of CIITA ubiquitination on the expression of MHC‐II genes by identifying the E3 ligase responsible for CIITA ubiquitination. This research will improve our understanding of the regulation of activation and half‐life of CIITA and of the transcriptional regulation of MHC‐II expression. Research supported by The American Cancer Society.
Unique tuning of two cofactors in a cryptochrome framework
Authors: Hitomi Kenichi , Arvai Andrew S , Getzoff Elizabeth D
Published: June 2021

We have determined the 1.75 Åresolution crystallographic structure of Synechocystis cryptochrome with both cofactors bound. This protein product from the Sll1629 gene of Synechocystis sp. PCC6803 belongs to the blue light photoreceptor cryptochrome/ photolyase family and binds both flavin adenine dinucleotide (FAD) and N5,N10‐methenyl‐5,6,7,8 tetrahydrofolate (MTHF). For many eukaryotic proteins of the cryptochrome/photolyase family, MTHF has been implicated by spectroscopic studies as a second "antenna" cofactor, in addition to the redox‐active FAD cofactor. In contrast, recent structural studies of prokaryotic proteins from this family have indicated alternative options for the second cofactor: 8‐hydroxy‐5‐deazaflavin, flavin mononucleotide (FMN) or another FAD. The cofactor‐binding sites in our Synechocystis protein structure are not only perfectly matched with those of plant homolog, but also well conserved among the cryptochrome DASH gene cluster, which has bacterial, plant and animal members. The binding of MTHF in our Synechocystis protein structure is more constrained by protein interactions than is MTHF binding in E. coli photolyase. Structural and mutational analyses indicate that both cofactors are regulated differently in the DASH cluster proteins than in the bacterial photolyase.
Adenovirus type 36 reduces fatty acid oxidation by increasing Cidec/FSP27
Authors: Wang Zhong Q , Zhang Xian H , Yu Yongmei , Cefalu william T
Published: June 2021

It is well documented that adenovirus type 36 infection (Ad‐36) results in obesity. However, the molecular mechanism of Ad‐36 cause obesity remains unknown. Recent studies suggest that Cell death‐inducing DNA fragmentation factor‐alpha‐like effectors (CIDE) family proteins play important roles in modulating energy homeostasis, aging and the development of metabolic diseases such as obesity and diabetes. Cidec/FSP27 is one of Cide family identified as a novel lipid droplet‐associated protein in adipocytes.

We sought to investigate the effect of Ad‐36 infection on fatty acid oxidation, Cidec/FSP27, PGC 1a, LPL, UCP3 and ACC protein abundance in primary cultured human skeletal muscle cells. Muscle cells were infected with various doses (1.9 ‐ 7.6 PFU/cells) of Ad‐36 and adenovirus type 2 (Ad‐2) with non‐lipogenesis as negative control as well as uninfected control.

Our data show that Ad‐36 significantly reduces fatty acid oxidation with does dependant manner (100±7%, 62±9%, 56±11%, and 43±5% in control, 1.9, 3.8, 7.6 doses of Ad‐36, P<0.01 and P<0.001 respectively), but Ad‐2 had little effect on fatty acid oxidation (P>0.05). Ad‐36 at dose of 3.8 PFU/cell substantially increased Cidec/FSP27, ACC abundance, slightly decreased LPL and increased PCG 1a proteins comparison with control (3.37±0.4, 2.02±0.21, 0.74±0.04 and 1.82 ±0.47 fold respectively). Ad‐2 only slightly increased these proteins (1.27±0.13, 1.28 ±0.24, 1.07±0.07 and 1.26±0.14 fold of control, p>0.05 respectively).

This study suggests that Ad‐36 promotes Cidec/FSP27 and ACC protein expression, and decreases LPL abundance. Ad‐36 reduces fatty acid oxidation and increases lipid accumulation in the muscle cells may be mediated by elevated Cidec/FSP27 protein.
Human PON2 S311C polymorphism impairs airway epithelia 3OC12‐HSL inactivation and alters PON2 glycosylation
Authors: Stoltz David Anthony , Ozer Egon , Recker Thomas , Estin Mira , Yang Xia , Gansemer Nicholas , Lusis Aldons , Shih Diana , Zabner Joseph
Published: June 2021

Airway epithelial cells can degrade the P. aeruginosa quorum sensing molecule 3OC12‐HSL and inactivation of 3OC12‐HSL is mediated by an enzyme termed paraoxonase (PON). Polymorphisms have been described for PONs including a serine (S) to cysteine (C) amino acid substitution at position 311 in PON2. Human airway epithelia (HAE) homozygous for C/C at 311 have an impaired ability to inactivate 3OC12‐HSL compared to HAE homozygous for S/S. Western blot analysis for PON2 showed similar levels of PON2 protein between PON2 311C and 311S. However, while PON2 311C lysates had only one protein band at approximately 39 kDa, PON2 311S cell lysates demonstrated an additional lower molecular weight band with higher electrophoretic mobility. After N‐linked deglycosylation with PNGase F there was no difference between the appearance of the PON2 band between 311C and S. Site‐directed mutageneis of the 4 putative N‐linked glycosylation sites in PON2 showed that PON2 311C and the larger immunoreactive band in PON2 311S undergo N‐linked glycosylation at Asn residues 254 and 323, while the lower immunoreactive band in PON2 311S is only glycosylated at Asn 254. We also found that glycosylation at Asn residue 323 is required for PON2 lactonase function. In summary, we found that airway epithelia homozygous for PON2 311C have an impaired ability to inactivate 3OC12‐HSL and that an altered glycosylation pattern is observed compared to PON2 311S.
Design and signaling mechanism of light‐regulated histidine kinases
Authors: Möglich Andreas , Ayers Rebecca A , Moffat Keith
Published: June 2021

Signal transduction proteins are organized into sensor (input) domains that perceive a signal and, in response, regulate the biological activity of effector (output) domains. We reprogrammed the input signal specificity of a normally oxygen‐sensitive, light‐inert histidine kinase by replacing its chemosensor domain by a light‐oxygen‐voltage (LOV) photosensor domain. Illumination of the resultant fusion kinase YF1 reduced net kinase activity by ~1000‐fold in vitro. YF1 also controls gene expression in a light‐dependent manner in vivo. Signals are transmitted from the LOV sensor domain to the histidine kinase domain via a 40‐60º rotational movement within an α‐helical coiled coil linker; light is acting as a rotary switch. These signaling principles are broadly applicable to domains linked by α‐helices, and to both chemo‐ and photosensors. Conserved sequence motifs guide the rational design of light‐regulated variants of histidine kinases and other proteins.

Funded by NIH grant GM036452 to K.M.
Study of the activity of Estrogen Response Elements present on Pax‐5 promoter and their potential role on regulating Pax‐5 expression in breast cancer
Authors: Faye Mame Daro , Ouellette Rodney
Published: June 2021

Pax‐5 is a transcription factor playing a crucial role in B lymphopoïesis and its deregulation has been associated to certain malignancies, namely non‐Hodgkin lymphomas and acute lymphoblastic leukemia. Although Pax‐5 is mainly known to be expressed in B cells, there is a body of preliminary data generated in our laboratory suggesting its expression in some breast cancer cell lines and primary breast tumors.

In an attempt to explain Pax‐5 expression in these breast cancer cases, we analyzed its promoter and found a number of Estrogen Receptor Elements (EREs) and Estrogen Related Receptors sites (ERRs), suggesting a role for estrogen receptors in Pax‐5 expression in breast cancer. Therefore, electrophoretic mobility shift assays (EMSA) were performed to assess the binding capacity of estrogen receptors ERα and ERβ on those predicted sites, which were also cloned in the Luciferase reporter vector pGL3‐Promoter to evaluate the receptors activity by dual luciferase reporter assays.

We report here the specific binding of estrogen receptors on some of the EREs located on Pax‐5 promoter and their differential activation under 17β‐estradiol stimulation in MCF‐7 cells. These results suggest a role for estrogen receptors in the expression of the oncogene Pax‐5 in breast cancer and suggest a mechanism that contributes to breast carcinogenesis.

This work has been supported by an ''Atlantic Innovation Fund'' grant.
Modulation of Cell Growth and Gene Expression in LNCaP prostate cancer cells by 1,25 dihydroxyvitamin D3 (1,25(OH)2D3)
Authors: Wang Wei‐Lin Winnie , Russell Nicholas , Chittur Sridar , Ensslin Courtney , Mordan‐McCombs Sarah , Welsh JoEllen , Tenniswood Martin
Published: June 2021

Epidemiological evidence suggests that vitamin D may be a potent chemopreventive agent for prostate cancer. It has been proposed that vitamin D can be used as a combination therapy with anti‐androgens for the treatment of early stage and locally invasive disease. In LNCaP cells, bicalutamide induces the rapid degradation of the androgen receptor (AR) and apoptosis in the presence or absence of testosterone. In the absence of testosterone, 1,25(OH)2D3 has little or no effect on either parameter. However the effect of 1,25(OH)2D3 is significantly altered in the presence of androgens. The AR is stabilized and cell death is abrogated. These results suggest that vitamin D mediated signaling interacts with AR mediated cell survival pathways, and reduces the efficacy of anti‐androgen therapy in hormone replete prostate cancer patients. Using genome‐wide microarray analysis, we have identified a set of genes that are differentially regulated by both vitamin D and testosterone. Most genes involved in cell cycle progression and apoptosis are not affected by both steroids. However, genes involved in the maintenance of intracellular calcium levels, such as TRPV6 are affected and may further influence the activity of various Ca2+ responsive genes, including ANXA2 and STK17B. These suggest a novel calcium‐related anti‐apoptotic pathway that is unique to prostate cancer cells.
Development of a High Throughput Flow Cytometry Protein Interaction Assay to Identify Inhibitors of G Protein‐Coupled Receptor Kinase 2
Authors: Thal David Michael , Tesmer Valerie M. , Tesmer John J.G.
Published: June 2021

G protein‐coupled receptors (GPCRs) are key regulators of cell physiology, controlling processes that range from glucose homeostasis to contractility of the heart. Phosphorylation of these receptors by GPCR kinases (GRKs) is a major regulatory mechanism for the desensitization of activated receptors. Our lab is particularly interested in GRK2, which plays an essential role in the regulation of myocardial signaling. Small molecule inhibitors of GRK2 will not only provide a novel mechanism for understanding GRK2's regulatory mechanisms, but may ultimately serve as potential therapeutics for the treatment of heart disease. Our lab has utilized a high throughput flow cytometry protein interaction assay allowing us to study the binding interactions of GRK2. The first interaction we have studied is between GRK2 and the heterotrimeric G protein Gαq. In the presence of GDP·AlF4− GRK2 has 3.2 ± 1.7 nM binding affinity for Gαq. So far we have screened nearly 40,000 compounds obtaining Z' values ranging from 0.5‐0.9 in a 384‐well format. We are currently in the process of confirming the activity of approximately 20 compounds that showed activity in follow up dose‐response titrations. Preparations are also underway to screen GRK2 against a novel RNA aptamer that selectivity binds and inhibits the kinase activity of GRK2. Z' values for this assay also range from 0.5‐0.9. Supported by grants from the NIH and AHA.
AMP‐activated Protein kinase regulates FasL expression in Jurkat T lymphocytes via NF‐AT and AP‐1 dependent pathways
Authors: Lee Jung Yeon , Kang Insug
Published: June 2021

T cell activation by T cell receptor (TCR) engagement or its pharmacological mimics induces FasL expression to maintain T cell homeostasis by apoptosis of activated T cells. AMP‐activated protein kinase (AMPK), an important regulator of energy homeostasis, is known to be activated during T cell activation. In this study, we examined the roles and signaling mechanisms of AMPK in T cell receptor‐induced FasL expression. AMPK is activated at the downstream of Ca2+/CaMKK during T cell receptor activation. Inhibition of AMPK by pharmacological agent or small interfering RNA suppressed FasL expression as well as secretion of FasL protein in T cells stimulated with PMA plus ionomycin (PMA/Io) or with monoclonal anti‐CD3 plus anti‐CD28 (CD3/CD28). Consistently, inhibition of AMPK blocked promoter activity of FasL in Jurkat T cells. We found that inhibition of AMPK reduced transcriptional activation NF‐AT and AP‐1 in Jurkat T cells. Furthermore, we found that inhibition of AMPK blocked phosphorylation and activation of PKC‐theta, a critical molecule in T cell activation and FasL expression. Taken together, our results show that AMPK regulates T cell receptor‐induced FasL expression through activation of NF‐AT and AP‐1 pathways in Jurkat T cells and imply that AMPK may play a role in activation‐induced cell death during T cell activation.

This work was supported by grants from KOSEF and KRF of Korea Government.
Small G‐protein Activated Kinase Pak2 Promotes 3T3‐L1 Preadipocyte Differentiation
Authors: Ling Jun , Traugh Jolinda
Published: June 2021

Adipocyte differentiation plays an important role in many human diseases, including obesity, diabetes mellitus and hypertension. Pak2 has been studied to arrest cell cycle progression, phosphorylate c‐Myc and regulate stem cell differentiation. To study whether Pak2 regulates 3T3‐L1 cell differentiation, the changes in Pak2 were first analyzed during adipogenesis. It was found that the amounts of Pak2 were increased by 2‐3 folds as compared to the control from the day 2 to day 6 of induction, and then declined down to the levels of control. The level of Pak1 was constant during the same period of time. This result suggested that Pak2 could regulate 3T3‐L1 cell differentiation. With 3T3‐L1 cell lines stably transfected with Pak2, it was shown that over‐expression of WT‐Pak2 enhanced the progression of differentiation. The numbers of differentiated cells at day 4 were largely increased, but the final differentiated adipocytes were less than the control. In contrast, the kinase‐inactive mutant of Pak2 (K278R) completely abolished the differentiation in 3T3‐L1 cells, hence identifying that Pak2 is a key regulator of initiation of differentiation in the preadipocyte. Further analysis detected that WT Pak2 activated c‐Myc and PPARγ, but K/R did not support the elevated level of PPARγ at the intermediate stage, which accounted for one of mechanisms that Pak2 regulated 3T3‐L1 cell differentiation.
Get “Hooked” On Brugia malayi Asparaginyl tRNA Synthetase
Authors: Arnold Amanda , Schulman Amanda , Bach Charles , Bach Sam , Faught Andy , Haeflinger Sadie , Her Alex , Lillie Kristin , Martin Trevor , Noll Kristi , Peak Kate , Rice Collin , Rice Pat , Rogacheski Kaitlyn , Suggs Aaron , Weber Samantha , Vogt Gina , Kron Michael A
Published: June 2021

A partnership between the Brown Deer High School students participating in the MSOE SMART Team (Students Modeling A Research Topic) program and a scientist enabled the team to explore asparaginyl‐tRNA synthetase (AsnRS), a potential drug target to treat lymphatic filariasis, and to build a 3D physical model of the protein. Lymphatic filariasis results from mosquitoes transferring the nematode, Brugia malayi, to host lymph nodes, leading to swelling of affected limbs. AsnRS hooks asparagine to tRNA, used during protein synthesis. AsnRS is a member of the aminoacyl tRNA synthetase (AARS) family, a set of structurally heterogenous enzymes, specific for each amino acid. AARS are potential drug targets as they are essential for survival and are structurally different between species. AARS also functions as an immunosuppressant, blocking interleukin 8 receptors in humans. Current research for treatment targets parasitic AARS. If multiple functions could be mapped to the same region of the protein, a single drug could target these functions. Inhibition of the tRNA‐aminocylation function of AsnRS would prevent protein synthesis, thus causing death of the parasite. Preventing AsnRS from blocking interleukin 8 receptors, would act as an immunostimulant in humans. Further research on this family of enzymes could provide alternative therapies to treating parasitic diseases. Supported by a grant from NIH‐NCRR‐SEPA.
Physical Model of EGFR and Ras protein using RP‐Rasmol
Authors: Cox Justin , Ferguson Michael , Telscow Jenniffer , Whitehurst Chanelle , Stubbs Tamica , Colton Shannon , Herman Tim
Published: June 2021

The MSOE SMART (Students Modeling A Research Topic) Team Program establishes partnerships between students and research mentors and enables teams to explore the nature of select proteins. The Waddell SMART Team researched and designed physical models using 3D printing technology of two proteins involved in cell division regulation, the Epidermal Growth Factor Receptor (EGFR), and Ras. EGFR, a tyrosine kinase receptor, has an extracellular ligand binding domain that leads to activation of the intracellular kinase signaling pathway. Once activated, EGFR phosphorylates a GRB2/SOS complex that binds to Ras, a regulatory transduction trans‐membrane protein. Ras has a phosphate binding loop that binds to GTP, providing energy for the reaction. Once activated, Ras undergoes a conformational change and signals cell division. Cancer results from uncontrolled cell growth. In some forms of breast cancer, EGFR is overexpressed, leading to tumor formation. A new drug, Lapatinib, may inhibit this excessive cell proliferation by blocking the ATP binding domain of the tyrosine kinase. In many types of cancer, Ras has been mutated, preventing hydrolysis of GTP, and the transduction pathway remains active, signaling for continued cell division, which may lead to tumor formation. Understanding the structure and function of these proteins may lead to further drug development. Supported by a grant from NIH‐NCRR‐SEPA.
Effects of the Scorpion Toxin (BmP09) on Muscle Cells in the Tobacco Hawkmoth
Authors: Gerner Emily , Gerner Zach , Kim Que , Speagle Josh , Vangala Sai , Shankar Darshan , Thompson Louise , Witten Jane
Published: June 2021

A partnership between the Brookfield Central High School students participating in the MSOE SMART Team (Students Modeling a Research Topic) program and a researcher enabled the team to explore the structure and function of a potassium channel bound to a toxin and to build a 3D physical model of the protein. In the larvae of the Tobacco Hawkmoth, the Tobacco Hornworm, the muscle under study is a posture muscle that has a slow recovery (repolarization) rate, and is classified as "slow‐twitch". Metamorphosis transforms this muscle into a flight muscle requiring a fast repolarization rate, becoming a "fast‐twitch" muscle. The repolarization rate of these muscle cells is directly linked to the muscle's rate of contraction. An explosive increase in the number of the Ca2+‐gated K+ channels (BK) in these muscles corresponds to these metamorphic changes. The Chinese scorpion toxin BmP09, from Buthus martensi Karsch, will be used to identify the BK channel as responsible for the change from posture to flight muscle. This toxin binds specifically to the BK channel, preventing the channel from removing K+ from the cell, slowing repolarization. Binding the toxin to flight muscle, repolarization rates can be studied to determine if the BK channel is involved in the transformation from posture to flight muscle. Supported by a grant from NIH‐NCRR‐SEPA.
EST‐based shRNA library screen identifies a critical role for the cholesterol biosynthetic pathway in beta2‐adrenergic receptor degradation
Authors: Pan Hui , Nabhan Joseph F , Lu Quan
Published: June 2021

This study describes the development of a novel expressed sequence tag (EST)‐based short hairpin RNA (shRNA) library and its application in screening for genes regulating the agonist‐induced degradation of the beta2‐adrenergic receptor (beta2‐AR). We established an efficient procedure for making a large number of shRNA expressing clones from the cDNA fragments. Using this procedure, we made a lentiviral library which could express about 600,000 individual shRNA based on a collection of approximate 40,000 human ESTs. We transduced the shRNA library into a human cell line stably expressing an epitope‐tagged beta2‐AR. We used fluorescence‐activated cell sorting to repeatedly enrich cells that retain more beta2‐AR on the cell surface after treatment with the receptor agonist isoproterenol. We have isolated and sub‐cloned the shRNAs expressing constructs from the enriched cell population. Interestingly, several clones target common cellular pathways related to lipid metabolism. One of these clones targeted the farnesyl diphosphate synthase (FDPS), a key enzyme in cholesterol and sterol biosynthesis. We found that a chemical inhibitor of FDPS, alendronate, and the cholesterol depleting reagent, methyl‐beta‐cyclodextrin, both had inhibitory effects on beta2‐AR degradation. Our results thus reveal a novel role for the cholesterol biosynthetic pathway in regulating beta2‐AR degradation.
A Role for Retinoblastoma Tumor Suppressor Protein and p21‐activated Kinase as Regulators of Cell‐to‐Cell Adhesion
Authors: SOSA BERNADETTE , VAZQUEZ VIVIANA , SANTIAGO PEDRO
Published: June 2021

The cellular pathway centered on the retinoblastoma (Rb) tumor suppressor protein, is inactivated in most human cancers. Rb itself is inactivated with a frequency of over 90% in a subset of human tumors such as retinoblastomas, osteosarcomas, and small cell lung carcinomas. In this study we investigated a novel role for the retinoblastoma as a regulator of cell‐to‐cell adhesion. Immunoblot and immunoflourescence analyses showed that Rb‐deficient cells have dramatically reduced levels of adherens junction proteins, and that these are abnormally localized in the cell. Rb deletion also resulted in overexpression of Rac1, a small Rho GTPase, and also elevated levels of Rac1' effector the p21‐activated protein kinase (PAK1) were observed at both the mRNA and protein levels. Our results suggest a molecular mechanism by which Rb promotes adherens junction assembly by blocking the activity of Rac1 and PAK1. A dual role for Rb as both a cell cycle repressor and as inducer of adherens junction assembly raises the exciting prospect that, at least in some tumor types, inactivating Rb could play a significant role not only during initial tumorigenesis by providing targeted cells with a replicative advantage over normal cells, but also during later stages by disrupting cell‐to‐cell adhesion with consequent metastasis.Support by: MBRS‐RISE Program (NIH‐NIGMS #1R25GM082406)
Role of ζ‐Crystallin and Other AUBPs in Bcl‐2 Over‐Expression Occurring in B‐cell CLLs
Authors: di Gesualdo Federico , Donnini Martino , Witort Ewa , Papucci Laura , Specogna Rebecca , Cianchi Fabio , Lulli Matteo , Capaccioli Sergio , Lapucci Andrea
Published: June 2021

We previously identified in the 3'UTR of bcl‐2 mRNA an adenine + uracil‐rich element (ARE) that

regulates bcl‐2 expression at the post‐transcriptional level by interacting with specific AU‐Binding

Proteins (AUBPs). In most solid tumours and leukemias, bcl‐2 is overexpressed without any obvious gene rearrangement or mutation, suggesting that it could depend on impaired ARE/AUBP interaction based post‐transcriptional control. Investigating this possibility, we have recently disclosed a new bcl‐2 AUBP, ζ‐crystallin, which increased binding to the ARE of bcl‐2 mRNA resulted in bcl‐2 over‐expression in T‐cells of Acute Lymphocytic Leukemias (ALLs) patients as compared to T‐cells from healthy donors. Here, we also show that ζ‐crystallin and Bcl‐2 protein levels are strictly co‐related in B‐cell Chronic Lymphocytic Leukemias (CLLs) and demonstrate that this is consequent to bcl‐2 mRNA stabilization by ζ‐crystallin, in a circuit involving shifting of other bcl‐2 AUBPs. Our results clearly indicate that ζ‐crystallin overproduction contributes to bcl‐2 over‐expression and thus to the onset of human B‐cell CLLs.
Immortalization of human small airway epithelial cells with characteristics of bronchioalveolar stem cells
Authors: Gao Boning , Huang Chunxian , Sullivan James P , spinola Monica , Raso Maria Gabriela , Wistuba Ignacio , Shay Jerry , Minna John D
Published: June 2021

Most of our current understanding of stem cells in the adult lung is based on mouse models while information in the human lung remains poorly defined. A major advance would be the ability to reproducibly grow human lung stem or progenitor cells to allow their systematic study and genetic manipulation. Toward this goal, we have worked out conditions to routinely immortalized (NB = 9) human small airway epithelial cells (HSAECs) from peripheral lung using Cdk4 and hTERT expression vectors in these cells. As an example strain HSAEC1 grows in serum free SAGM medium (Clonetics), forms colonies in liquid media efficiently, but does not form colonies in soft agar or tumors in SCID/NOD mice. HSAEC1 expresses lung basal/stem cell markers (aldehyde dehydrogenase, p63, Cytokeratin14 and 17, and is resistant to 1 mM naphthalene) but very low or undetectable Clara cell, Type1 and Type2 cell markers in SAGM, which are then expressed when grown in Matrigel culture or in serum. Finally, the cells can be genetically manipulated toward oncogenesis (with KRASV12, and or missense p53). We conclude, that we have prepared multiple, immortalized strains of human peripheral lung epithelial cells with many characteristics of lung stem or progenitor cells that are experimentally tractable for studying lung differentiation and tumorigenesis.

This work is supported by NCI SPORE P50CA70907, and NASA/DOE NSCOR NNJ05HD36G, DE‐AI02‐05ER64068
THE ABILITY OF NEONATAL RAT LUNGS TO RECOVER AFTER EXPOSURE TO HYPEROXIA
Authors: Totapally Abhinav S
Published: June 2021

Exogenous induction of hypercapnia is shown to improve pulmonary function during ventilator and endotoxin‐induced lung injury. These injuries, likewise hyperoxia‐induced lung injury, are partly mediated by neutrophils sequestration and production of reactive oxygen species. Therefore, we tested the hypothesis that hypercapnia may improve the morphological manifestations of prolonged hyperoxia in neonatal rats. One day‐old rats were randomly assigned for 96 hours of exposure to normoxia (21% O2), hyperoxia (>98% O2) and hypercapnic hyperoxia (95% O2 plus 5% CO2), (n=5/group). Pups were then euthanized, lungs cardiac lobe was dissected for histological preparations, and changes in the alveolar size (μ2) were measured, using a computerized image analyzer. The results showed characteristic manifestations of pulmonary oxygen toxicity, such as substantial hypercellularity in both hyperoxic groups, as compared to normoxia. Although the alveolar density (number of alveoli/mm2) was reduced, the alveolar size was increased by 21% (p>0.05) during hyperoxia, and by 60% during hypercapnic hyperoxia, as compared to normoxia (p<0.05; ANOVA followed by Tukey‐Kramer multiple comparisons test). These data suggest that exogenously induced hypercapnia does not improve the morphological manifestations of pulmonary oxygen toxicity.
Sex differences in anxiety after infusion of group I mGluR agonist within the basolateral amygdala
Authors: De Jesús‐Burgos María Inés , Pérez‐Acevedo Nivia L
Published: June 2021

Females are twice affected by anxiety‐related disorders than males. These differences might be due to the effects of gonadal hormones. Metabotropic glutamate receptors (mGluRs) have been implied in the pathophysiology of anxiety. We hypothesized that S‐3,5‐Dihydroxyphenylglycine (DHPG), a group I mGluRs agonist, within the basolateral amygdala (BLA) will modulate anxiety according to sex. Intact males were compared to ovariectomized female rats that contained empty implants (OVX) or implants containing estradiol (OVX‐EB). We used the Vogel Conflict Test (VCT) to analyze conflict‐based anxiety and the Cat Odor Test (COT) for innate anxiety behaviors. In the VCT, intact males displayed more punished licks than OVX female rats (p=0.05). DHPG infusion did not alter any VCT parameter in intact male, OVX and OVX‐EB female rats. In COT, intact males showed lesser number of flat back approached (FBA) and spent more time in contact with the cat odor stimuli than OVX female rats (p= 0.01 & 0.04). DHPG infusion decreased FBA (p=0.02) and stretch attended behaviors in OVX female rats only (p=0.03). In males, DHPG reduced the number of freezing behaviors (p=0.004). Our results indicate sex differences in anxiety‐related behaviors which are task‐dependent. Infusion of DHPG within BLA modulates innate anxiety behaviors in OVX female rats. We concluded that mGluRs might be a pharmacological alternative to modulate anxiety.
Wireless Neural Recording for Mice and Rats
Authors: Morizio James Charles , Go Vinson
Published: June 2021

The continuous monitoring of electrical brain activity with implanted electrodes is essential for understanding the neural substrates of many physiological and pathological brain functions. Although this is done in some patients before undergoing surgical treatment, such recordings are extremely difficult in experimental animals, particularly in small rodents. The wires from the implanted electrodes restrain and interfere with the natural behavior of the animals. Consequently, it has been impossible to correlate truly normal behavior with neuronal activity. In addition, it would be ideal if brain activity could be monitored while animals live in a natural and enriched environment. Because of the complexity of such environments with natural hiding places and burrows, recordings of brain activity with wires attached to the animal's skull are impossible. Triangle BioSystems Inc. (TBSI) has overcome these challenges with the design of a miniature, multi‐channel wireless recording system with selectable gain and bandpass filtering. This will give rise to greatly expanded experimental capabilities, and serve as a platform for the development of clinical neural prosthetic devices to treat a variety of ailments including: Alzheimer's disease, epilepsy, and Parkinson's disease.
The acute effects of methamphetamine on synaptic plasticity in the CA1 region of the mouse hippocampus
Authors: Swant Jarod , Chirwa Sanika , Khoshbouei Habibeh
Published: June 2021

Monoamines affect LTP in the CA1 region of the hippocampus. GBR12935 and cocaine, both which increase the concentration of synaptic dopamine (DA), have been shown to increase LTP (Swant and Wagner 2006; Thompson et al. 2005). D1/D5 DA receptor agonists also increase LTP (Otmahkova and Lisman 1996; Stramiello and Wagner 2008). Methamphetamine (METH) application results in an increase in the concentration of extracellular monoamines. Here we study the acute effects of methamphetamine on LTP in the CA1 region of the mouse hippocampus. In contrast to cocaine, 1 and 10 microM METH application decreased LTP. The application of METH at a concentration of 30 microM resulted in a profound decrease in LTP, along with an increase in baseline synaptic transmission. Thapsigargin, verapamil, or DL‐APV did not block the effects of METH on baseline synaptic transmission and LTP. However, pretreatment with D1/D5 DA receptor antagonist SCH23390 (5 microM) eliminated the effects of METH both on baseline synaptic transmission and on LTP. These unexpected results show that unlike cocaine, which increases LTP in the CA1, METH decreases LTP through a process which requires activation of D1/D5 DA receptors.
Prostaglandins contribute to endotoxin‐induced increase in lipid peroxidation via nitric oxide production during endotoxemia in rats.
Authors: Sahan‐Firat Seyhan , Korkmaz Belma , Cuez Tuba , Ayaz Lokman , Tamer Lulufer , Buharalioglu C Kemal , Sari A Nihal , Tunctan Bahar
Published: June 2021

Endotoxemic shock is a systemic inflammatory response that is associated with increased nitric oxide (NO) and vasodilator prostaglandin production by inducible NO synthase and inducible cyclooxygenase (COX‐2) which contribute to the fall in blood pressure, vascular hyporeactivity and multiple organ failure. We have previously demonstrated that NO is involved in endotoxin‐induced changes in lipid peroxidation during endotoxemia in rats. The purpose of this study was to investigate whether COX‐2‐derived prostaglandins contribute to increase in lipid peroxidation via NO production in endotoxemic rats. Endotoxin caused an increase in nitrite (an index of NO production) and malonedialdehyde (MDA; an index of lipid peroxidation) levels as well as myeloperoxidase (MPO; an index of neutorphil infiltration) activity in the lung, spleen and femoral artery. Endotoxin‐induced increase in nitrite and MDA levels in these tissues of endotoxin‐treated rats was prevented by COX‐2 inhibitor, NS‐398. NS‐398 also prevented the effect of endotoxin to increase in MPO activity in these tissues. The data suggest that prostaglandins derived from COX‐2 contribute to the endotoxin‐induced changes in lipid peroxidation via NO production during endotoxemia.This study was supported by Mersin University Research Foundation (2006‐3, B Tunctan).
Reliability of grading zygapophysial (Z) joint degeneration from MRIs of subjects with acute low back pain
Authors: Grieve Thomas Joseph , Cramer Gregory D , Cantu Joe A , McKinnis Ray A , Bogar William C , Miley Heather L , Heiser Rudolf N , Little Joshua W
Published: June 2021

This pilot study was performed to assess grading Z joint degeneration from MRI scans. Using a grading system with subscales assessing articular cartilage erosion, subchondral sclerosis, and osteophytes/facet hypertrophy, an atlas showing examples of the full range of Z joint degeneration was constructed. Three radiologists were trained to use the grading system, graded left and right L4/L5 Z joint degeneration of 20 joints, then repeated grading after 30 days. Radiologists were blinded to the individual results and previous grading scores. Intra‐ and inter‐observer agreements were assessed using percent agreement and kappa. Results were most promising for the articular cartilage subscale, with intra‐observer agreements of 75% (k=0.71), 60% (0.50), and 35% (0.30), and agreements within one grade of 100%, 100%, and 95%; inter‐observer agreements were: Obs 1&2, 40% (k=0.46); Obs 1&3, 25% (0.23); Obs 2&3, 75% (0.62), and agreements within one grade of 100%, 95%, and 100%. For total grading scores, overall intra‐observer agreements were 75% (k=0.52), 55% (0.27), and 60% (0.42), and agreements within one grade were 100% for all observers; inter‐observer agreements were: Obs 1&2, 65% (k=0.38); Obs 1&3, 75% (0.35); Obs 2&3, 55% (0.42), and agreements within one grade were 100%, 95%, and 100%. The percent agreement shows the methods to be reliable. Support: NIH/NCCAM (Grant# 3R01 AT000123).
Differential augmentation of the anticoagulant responses by newly developed oral anti‐Xa and Anti‐IIa drugs in patients with liver disease
Authors: Masood Soofia , VanThiel David , George Magdalena , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

The liver plays a central roll in the regulation of hemostasis. Beside synthesis of coagulation factors, the liver is responsible for the metabolism of newly developed anticoagulant drug such as the Rivaroxaban (R), Apixaban (A) and Dabigatran (D). These drugs may have a direct effect by compromising the coagulation process. The purpose of this investigation is to determine the differential effects of R, A and D in liver disease patients (LDP). Citrated plasma samples from LDP (n=10‐20) were supplemented with graded amounts of R, A and D and saline. PT, aPTT, thrombin time (TT), Heptest clotting times and FXIII activity were determined. In addition, thrombin induced fibrinokinetics were measured. These agents produced a concentration dependent increase in the clot based assays with the exception of the Heptest in the range of 0.125‐1 µg/ml. In comparison to saline, the anti‐Xa and anti‐IIa agents produced a sizeable increase in the INR, aPTT and TT. D produced the most pronounced effects on all clotting assays, whereas R and A produced a modest increase. R decreased the Heptest. The fibrinokinetogram revealed mark differences among the different patients indicating the defects in fibrin polymerization. Supplementation of R, A and D resulted in a strong inhibition of fibrin formation which was most pronounced with D. These results clearly demonstrate that (LDP) suffer from a pre‐existing hemostatic imbalance with multiple factor differences.
Glybenclamide Exhibits Thromboxane Receptor‐Dependent Antiplatelet Effects
Authors: Ting Harold J , Khasawneh Fadi T
Published: June 2021

Background

Proper platelet function is vital as they are the main cellular agents of hemostasis. Conversely, inappropriate activation of platelets is featured in occlusive disease such myocardial infarction, and stroke. While the thromboxane A2 pathway of platelet activation has been identified as an attractive target for pharmacological intervention, no thromboxane receptor (TPR) antagonists are available for clinical use. Given the structural similarity between the antidiabetic sulfonylurea agent Glybenclamide (Glyb) and the TPR antagonist SQ29548, we hypothesize that Glyb exhibits antiplatelet effects via interaction with TPR.

Objective

Characterize the potential antiplatelet capacity of Glyb.

Methods

Isolate platelets from healthy donors who denied taking medication in the last 10 days and then investigate the effects of Glyb on platelet aggregation induced by the TPR agonist U46619, the thromboxane A2 precursor arachidonic acid (AA), ADP, as well as the protease‐activated receptor peptide (PAR4) using optical aggregometry.

Results

Glyb was found to dose‐dependently inhibit U46619/AA‐ (i.e., TPR‐) mediated platelet aggregation, but had no detectable effects on that triggered by ADP or PAR4.

Conclusion

Our findings suggest a therapeutic role for Glyb in thrombotic diseases via blockade of TPRs, and may lay the foundation for the design of therapeutic agents for selective targeting of TPR.
Linoleic acid inhibits PDGF‐induced migration in aortic smooth muscle cells: the role of tissue inhibitor of metalloproteinase
Authors: Cho Min Kyung , Lee Ji Won
Published: June 2021

Linoleic safflower seed oil reduces the development of diet‐induced atherosclerosis. However, the underlying mechanism of anti‐atherosclerotic effect remains unclear so far. The present study aims to investigate the effect of linoleic acid (LA) on PDGF‐induced migration of rat vascular smooth muscle cells (rSMC) and its molecular mechanism. Wound healing assay and transwell migration assay indicated that PDGF induced migration of rSMC relative to control. LA pretreatment reduced PDGF‐induced migration in a dose‐dependent manner. These results were consistent with that of DQ gelatin degradation. PDGF increased the phosphorylations of ERK and Akt. LA inhibited PDGF‐induced phosphorylation of Akt but not that of ERK. Moreover, LA significantly increased mRNA levels and protein expressions of tissue inhibitor of metalloproteinase (TIMP)‐1 and TIMP‐2. TIMP activity by reverse zymography was also increased by LA. We showed that TIMP‐1 induction by LA was regulated by JunB/AP‐1 as determined by ChIP analyses. The transfection of TIMP‐1 or TIMP2 siRNA reversed the inhibitory effect of LA on the PDGF induced migration. We conclude that LA effectively suppressed the PDGF‐induced rSMC migration via induction of TIMP1/2 suggesting that LA might potently prevent migration‐related disease such as restenosis and atherosclerosis.