bluuebunny/crossref_metadata_2025 huggingface dataset:
IL‐35 is a Novel Responsive Anti‐inflammatory Cytokine ‐ A New System of Categorizing Anti‐inflammatory Cytokines
Authors: Li Xinyuan , Mai Jietang , Virtue Anthony , Wang Hong , Yang Xiao-Feng
Published: June 2021

Interleukin‐35 (IL‐35), a recently discovered heterodimeric cytokine with anti‐inflammatory/immunosuppressive properties, has a central role in limiting the immune response in various disease models including colitis, arthritis and asthma. However, it remains unknown whether IL‐35 is different from other anti‐inflammatory cytokines such as IL‐10 and transforming growth factor (TGF)‐β in terms of inhibition of inflammation initiation or suppression of full‐blown inflammation. In this study, we examined the tissue expression profiles and regulatory mechanisms of IL‐35 in comparison to other anti‐inflammatory cytokines. Our results suggest that in contrast to TGF‐β, IL‐35 is not constitutively expressed in human tissues but is inducible in response to inflammatory stimuli. We also provide structural evidence suggesting that AU‐rich element (ARE) binding proteins and microRNAs target IL‐35 subunit transcripts, which are responsible for quick degradation of IL‐35. Furthermore, we propose a new system to categorize anti‐inflammatory cytokines into two groups: (1) the housekeeping cytokines, such as TGF‐β, inhibit the initiation of inflammation whereas (2) the responsive cytokines including IL‐35 suppress inflammation in full‐blown stage. Our in‐depth analysis of molecular events that regulate the production of IL‐35 and new categorization system of anti‐inflammatory cytokines are important for the design of new strategies of immune intervention.

This work was partially supported by the National Institutes of Health Grants HL094451 and HL108910 (XFY), HL67033, HL82774 and HL77288 (HW).
Direct G protein signalling mediates components of the vascular smooth muscle response to insulin‐like growth factor
Authors: Zahradka Peter , Perrault Raissa
Published: June 2021

Like many organs, the vasculature possesses an intrinsic repair ability that is critical for homeostasis. This ability is intimately linked to smooth muscle cells (SMCs) through their unique phenotypic plasticity, which allows them to proliferate and migrate in response to specific stimuli. However, phenotypic modulation is also the major underlying phenomenon behind pathogenic vascular disease and post‐surgery repair

Physiological control of the SMC response involves the presence of circulating factors, such as Insulin‐like Growth Factor‐ 1 (IGF‐1). IGF‐1 acts via its receptor, a member of the tyrosine kinase receptor family, but it may also require a G protein. Thus, the objective of this study was to investigate the structural aspects of IGR‐1R coupling to a G protein in SMCs, as well as the contribution of this pathway to the cellular response.

In primary SMCs, the IGF‐1R was found to interact with both Gαi and Gβ subunits and in the case of Gαi, this was a direct interaction. We found that IGF‐1‐induced activation of ERK1/2 was prevented with a Gβγ inhibitor, which also inhibited cell proliferation in response to IGF‐1. Specific cell‐penetrating Gαi inhibitor peptides were synthesized and these enabled identification of several unique downstream targets.

These findings support the conclusion that the responses of vascular SMCs to IGF‐1 are elicited at least in part through interaction with a G protein.

Source of research support: Canadian Institutes for Health Research
IGF1‐receptor signalling modulates diastolic cardiac function in aging hearts
Authors: Godecke Axel , Kessels Claudia , Peiseler Lena , Raupach Annika , Jacoby Christoph , Lindecke Antje , Koehrer Karl , Heger Jacqueline , Moellendorf Sarah
Published: June 2021

Insulin‐like growth factor receptor (IGF1R) signalling appears to play an important role in cardiac growth, contractile function, and the development of age related congestive heart failure. Here we address the question to what extent cardiomyocyte specific IGF1R signalling is essential for maintenance of the structural and functional integrity of the adult murine heart. We inactivated the IGF1R by a 4‐OHTX inducible Cre recombinase in cardiac myocytes of adult mice. Western blots and RT‐PCR demonstrated efficient inactivation of the IGF1R (iCMIGF1RKO)associated with reduced AKT and GSK3β‐phosphorylation. In adult iCMIGF1RKO mice(age 3 month)assessment of cardiac function by P/V catheter and MRI revealed no functional alterations. When the KO was induced in aged mice (11 month) diastolic cardiac function was more depressed in iCMIGF1RKO mice. To test if insulin signalling compensated for the loss of IGF1R, we inactivated β‐cells by STZ. The diabetes associated functional depression was similar in WT and iCMIGF1RKO mice. Analysis of the cardiac transcriptome on 44K microarrays did not reveal activation of overt adaptive processes.

Conclusion

Endogenous IGF1 receptor signalling is required for conservation of cardiac function of the aging but not the young heart.
Structural basis for type VI secretion effector recognition by a cognate immunity protein
Authors: Li Mo , Le Trong Isolde , Carl Mike A , Larson Eric T , Chou Seemay , De Leon Justin A , Dove Simon L , Stenkamp Ronald E , Mougous Joseph
Published: June 2021

The type VI secretion system (T6SS) has emerged as an important mediator of interbacterial interactions. A T6SS from Pseudomonas aeruginosa targets at least three toxic effectors, type VI secretion exported 1–3 (Tse1‐3), to recipient Gram‐negative bacterial cells. Tse2 is a potent cytoplasmic toxin that inhibits target cell proliferation through an unknown mechanism. When translocated into other bacterial cells, the protein provides a fitness advantage for P. aeruginosa. The type VI secretion immunity protein 2 (Tsi2) was found to protect against endogenous and intercellularly‐transferred Tse2. Here we showed that Tse2 delivered by the T6SS efficiently induces stasis, not death of recipient cells. Despite direct interaction of Tsi2 and Tse2 in the cytoplasm, we found Tsi2 is dispensable for targeting the toxin to the secretory apparatus. By mapping the results of genetic screening data onto our 1.0 Å X‐ray crystal structure of Tsi2, we discovered an acidic patch on the Tsi2 surface that mediates Tse2 interaction and immunity. Interestingly, our results indicated that Tsi2 shares structural similarity to the N‐terminal domain of the Vti1p SNARE protein and that these proteins interact with binding partners in a chemically and spatially overlapping motif. Our findings provided the first molecular insights into T6 effector secretion and immunity that distinguish the system from analogous cellular pathways.
Characterization of diverse internal binding sequences to PDZ domains by screening special random peptide library in yeast two‐hybrid system
Authors: Mu Yi , Hu Siqi , Wang Xiaorong , Gao Youhe
Published: June 2021

There are probably more ways of protein interaction than we currently realize. PDZ domains are flexible protein interaction modules that are considered mainly recognize C‐termini. However, the binding characteristics of PDZ domains to internal sequences have not been systematically studied. In this study, a nearly random octapeptide library with no stop codon designed in the insert was constructed in the yeast two‐hybrid vector. A total of twenty PDZ domains were used as baits to screen the library. They were either previously reported to bind internal sequences or had no known C‐terminal ligand. Among the twenty PDZ domains, eleven were found to bind internal sequences, including three PDZ domains that were not previously reported to bind internal sequences. Eight PDZ domains showed strong amino acids preferences; one of the consensuses was similar to its C‐terminal ligand, but the other consensuses were more diverse than those of common C‐terminal ligands. In addition, one PDZ domain with no strong consensus recognized extended hydrophobic peptides. The results suggest that PDZ domains binding abilities to internal sequences may have been underestimated. Moreover, this data can help us to find potential binding partners which are currently under validation.

This work is supported by grants from National Science Fund for Distinguished Young Scholar 30725009.
An expanded self‐antigen peptidome and proteome is carried by the human lymph as compared to the plasma
Authors: Clement Cristina Corina , Santambrogio Laura
Published: June 2021

The pre‐nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. Herein, we showed a major difference between lymph and plasma that could be visualized by FPLC and 2D gel in the amount of low molecular weight products corresponding to peptide fragments with MW<5000 Da. Analysis of more than 300 sequences identified self‐peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Moreover, an advanced proteomics analysis of the afferent pre‐nodal lymph in parallel to matched human plasma from healthy donors was performed using two proteomics strategies: 1D gel LC‐MS/MS analysis, and 2‐D‐DIGE coupled with mass spectrometry nanospray LC‐MS/MS. Scaffold 3.0, combined with Ingenuity Pathway analysis, highlights the proteomic functional signature of the prenodal lymph and plasma. The distinctive proteome of the human lymph is characterized by several products derived from the processing of extracellular matrix as well as cellular catabolism, metabolism and apoptosis. Whereas the distinctive plasma proteome is mostly represented by soluble molecule of the coagulation system and cell‐cell signaling factors.
Large scale analysis of synaptic phosphorylation and O‐GlcNAcylation reveals complex interplay between these post‐translational modifications
Authors: Trinidad Jonathan C , Barkan David T , Gulledge Brittany , Thalhammer Agnes , Sali Andrej , Schoepfer Ralf , Burlingame Alma L
Published: June 2021

O‐linked N‐acetylglucosamine (O‐GlcNAc) is a dynamic, reversible monosaccharide modifier of serine‐ and threonine‐residues on intracellular protein domains. Crosstalk between O‐GlcNAc and phosphorylation signaling mechanisms has been proposed. However, to date there has not been a large scale evaluation of these two PTMs at endogenous levels in any complex biological system. Here, we identified over 1,750 GlcNAcylation and16,500 phosphorylation sites from the same mouse brain synaptosomes.

As has previously been demonstrated for phosphorylation, we show that GlcNAcylation tends to occur in clusters on disordered regions of proteins. However, the distribution of GlcNAcylation sites is virtually random with respect to phosphorylation, suggesting that negative crosstalk at the structural level is not a general phenomenon. We frequently observed proteins extensively phosphorylated with minimal GlcNAcylation. However, proteins extensively GlcNAcylated were almost always phosphorylated to a similar or greater extent, indicating the process of GlcNAcylation appears functionally correlated to phosphorylation.
Genetic responses in a plant‐endophyte interplay
Authors: Robb Jane , Shittu Hakeem , Soman Kizhake V. , Kurosky Alexander , Nazar Ross N.
Published: June 2021

A plant can respond to the threat of a pathogen through resistance defenses or tolerance. Resistance has been widely studied in many host pathosystems but little is known about genetic events in tolerance. In this study a recently developed model for a tolerant tomato‐fungal (Verticillium dahliae) wilt pathosystem was used to examine changes in gene expression during both a tolerant interaction and in competition with a susceptible infection. Relative changes in mRNA levels were measured using a customized TVR (tomato‐Verticillium response) DNA chip and actual changes in cellular proteins were assessed using two dimensional gel electophoresis and MS‐based protein identification. Heat maps were used to identify responding groups of proteins and individual groups were examined further for common functions and other relationships including correspondence between mRNA and protein changes. In addition to elevated levels of defense gene responses to pathogen colonization, suppression or blocked responses were evident in mixed infections. These included some transcription factors and PR proteins such as class IV chitinases and beta glucanases known to target fungal spores and mycelium. Taken together the results reveal intriguing but complex molecular changes with potential agricultural benefits. Supported by NSERC (R.N.N. and J.R.), NIH, NHLBI (A.K.) and a Canadian Commonwealth Scholarship (H.O.S.).
Adiposity, but not chronological age, promotes accumulation of some old and damaged proteins
Authors: Grubina Rozalina , Klaus Katherine A. , Morse Dawn M. , Nair K. Sreekumaran
Published: June 2021

Altered protein homeostasis may explain the increased risk of degenerative and cardiometabolic disorders with aging. Here, we investigated the impact of chronological age and obesity on the accumulation (ratio of old to de novo synthesized proteins) of haptoglobin, fibrinogen, and apolipoprotein A‐1 (ApoA‐1) in 7 each of elderly lean (69.8 ± 5.0 years, 23.2 ± 1.8 kg/m2), elderly obese (72.6 ± 5.6 years, 30.6 ± 2.3 kg/m2), young lean (23.1 ± 3.2 years, 23.4 ± 1.9 kg/m2), and young obese (25.2 ± 2.4 years, 30.6 ± 2.4 kg/m2). Proteins were labeled with [13C6]‐phenylalanine, separated by 2‐dimentional gel electrophoresis, and identified by mass spectrometry. Insulin sensitivity was lower in the obese groups, while percent body fat was higher, and lean mass was lower, in the elderly. Haptoglobin accumulation was greater in the obese compared to lean (0.512 ± 0.229 vs. 0.272 ± 0.114; p = 0.008) across all ages, with no age effect. Fibrinogen trended toward higher accumulation in the obese compared to lean, with no age effect, but did not reach statistical significance (1.263 ± 2.108 vs. 0.598 ± 0.197; p = 0.094). ApoA‐1 accumulation showed no obesity or age variation. Associations remained unchanged when controlled for gender, age, and BMI. Thus, adiposity, and not chronological age, promotes protein accumulation, potentially contributing to degenerative and cardiometabolic disease risk. Supported by NIH grant RO1‐AG09531.
Molecular characterization of the 1‐Deoxy‐D‐xylulose‐5‐ phosphate Synthase (DXS) from marine bacterium, Kocuria gwangalliensis
Authors: kim young tae
Published: June 2021

In addition to the ubiquitous mevalonate pathway, Kocuria gwangalliensis utilizes the nonmevalonate pathway for isopentenyl diphosphate biosynthesis. 2‐C‐methyl‐D‐erythritol‐4‐phosphate synthase (MEP synthase) catalyzes the rearrangement of 1‐D‐deoxyxylulose‐ 5‐phosphate (DXP) to methylerythritol‐4‐ phosphate(MEP) as the first pathway‐specific reaction in the MEP biosynthetic pathway. In the present study, the gene coding for 1‐ Deoxy‐D‐xylulose‐5‐phosphate Synthase (DXS) was isolated from the marine bacterium, Kocuria gwangalliensis. The nucleotide sequence of DXS consisted of 2013 bp encoding 671 amino acids residues and turned out to be well conserved during evolution. DXS gene was subcloned into the pColdI expression vector which allows the expression of the recombinant DXS protein with C‐terminal fusion His‐tag and designated to pColdI‐DXS plasmid. The predicted size of recombinant DXS protein is approximately 73 kDa including a C‐terminal His‐Tag fusion. The resulting plasmid was transformed into the E. coli strain BL21(DE3) codon plus cell and the expression of the recombinant DXS protein was induced by the addition of IPTG at a final concentration of 0.1 mM. The expression patterns of recombinant DXS protein were analyzed by 10% SDS‐PAGE.
Kinetic characterization of binding among Helicobacter pylori nickel maturation enzyme accessory proteins
Authors: Hill Stephanie A. , Helms Katy A. , Brown Christopher C. , Benoit Stephane L. , Maier Robert J. , McMurry Jonathan L.
Published: June 2021

Helicobacter pylori causes peptic ulcers and gastric cancers in humans. Two nickel‐containing enzymes, the hydrogen uptake hydrogenase and urease, are required for colonization and persistence in the stomach. Hydrogenase and urease maturation require number of accessory proteins, HypABCDEF and UreEFGH, respectively. To investigate interrelationships between each set of accessory proteins, a kinetic survey of potential binding among HypA, HypB, UreE and UreG using optical biosensing was conducted. Interactions between HypA and UreE and UreE and UreG were observed and competition binding results show that UreG and HypA compete for binding to UreE. Full kinetic characterization of HypA‐UreE revealed complex binding that fit a two‐state model. UreG‐UreE binding exhibited fast‐on, fast off kinetics. Binding was faster and tighter in the presence of zinc than its absence, perhaps reflecting a change in oligomeric state but also significant kinetic differences. Experiments examining the stoichiometry and thermodynamics of binding will also be described. This work was supported by NIH grant GM080701 and a KSU Mentor‐Protégé Grant.
Functions of Monocyte Chemotactic Protein‐3 in Transgenic Mice Fed High‐fat Diet
Authors: An So Jung , Choi Myung-sook , Chae Chan Kyu , Oh Goo Taek , Park Yong Bok
Published: June 2021

In order to look into the function of the monocyte chemotactic protein‐3 (MCP‐3) in the atherogenesis, transgenic mice in which the MCP‐3 is over‐expressed in a specific tissue (vesicular endothelium) have been produced in the previous work, genotyped by a specific primer set designed from the MCP‐3 cDNA and fed high‐fat‐high‐cholesterol diet for 12 weeks to provide extreme atherogenic conditions. In the level of plasma lipids, the mice fed HFHC diet showed a higher level of total cholesterol and lower in the triglyceride level. On the other hand, the level of HDL‐cholesterol was surprisingly increased in the mice fed normal diet showing 72.6 in the HTR (HDL vs. total cholesterol ratio). Accordingly, the atherogenic index of the mice fed a normal diet was drastically lower than in the wild‐type mice. This was supported through a marked increase in the expression of Apo‐AI gene in the transgenic mice fed normal diet identified by microarray analysis. Activities of anti‐oxidant enzymes were also increased in the transgenic mice fed HFHC diet, indicating that there might be more oxidative damage to tissues of the mice fed HFHC diet leading to a progress toward the atherosclerosis. Supplementation of anti‐oxidants to the diet, naringin and hesperidin, was shown to lower the level of the activities of anti‐oxidant enzymes.
Hyperhomocysteinemia accelerates atherosclerosis and induces inflammatory monocyte differentiation in hyperglycemic mice
Authors: Fang Pu , Zhang Daqing , Jiang Xiaohua , Yang Xiaofeng , Wang Hong
Published: June 2021

Cardiovascular events are increased in patients with diabetes mellitus (DM) and hyperhomocysteinemia (HHcy). However, the underlying mechanism is unclear. In this study, we examined the role and mechanisms of HHcy on atherosclerosis under hyperglycemic condition. We established an atherosclerosis‐susceptible mouse model with both severe HHcy and hyperglycemia in which the mouse Cystathionine β‐Synthase (Cbs) and Apolipoprotein E (ApoE) genes are deficient and an inducible human CBS transgene is introduced (Tg‐hCBS ApoE−/− Cbs−/− mice). HHcy aggravated metabolic abnormalities in the hyperglycemic mice, including increased urine secretion, increased water consumption, increased blood glucose level and decreased body weight. Increased atherosclerotic lesions were also found in the hyperglycemic HHcy mice. Furthermore, HHcy increased inflammatory Ly6Chi MC in peripheral blood, spleen and bone marrow of the hyperglycemic mice. This may be due to hypomethylation since plasma level of Hcy is negatively associated with SAM/SAH ratio in mice. In cultured mouse primary splenocytes, L‐Hcy with D‐glucose induced Ly6C+ subset. These Ly6C+ MCs exhibited increased TNF‐α production, confirming their inflammatory properties. We concluded that HHcy promotes differentiation of inflammatory MC subset and contributes to atherosclerotic lesions in hyperglycemia. This study is supported by a NIH grant.
Acid Ceramidase Represses Steroidogenic Factor 1‐dependent Gene Transcription by Binding to the Receptor in the Nucleus of H295R Human Adrenocortical Cells
Authors: Lucki Natasha C , Li Donghui , Bandyopadhyay Sibali , Wang Elaine , Merrill Al H , Sewer Marion B
Published: June 2021

Adrenocorticotropin (ACTH) regulates glucocorticoid biosynthesis primarily through the activation of a cAMP/PKA‐dependent pathway to promote the interaction of transcription factors and coactivator proteins with the promoter of steroidogenic genes. The nuclear receptor steroidogenic factor 1 (SF‐1) is essential for steroidogenic gene transcription. We have shown that sphingosine (SPH) is a ligand for SF‐1 and that suppression of acid ceramidase (ASAH1) expression, an enzyme that produces SPH, increases the transcription of multiple steroidogenic genes. Given that SF‐1 is predominantly expressed in the nucleus, we sought to define the molecular mechanisms by which ASAH1 regulates SF‐1 function. We show that ASAH1 is localized in the nuclei of H295R human adrenocortical cells and ACTH signaling promotes nuclear sphingolipid metabolism in an ASAH1‐dependent manner. Significantly, ASAH1 suppresses SF‐1 activity by directly interacting with the receptor through an LXXLL nuclear receptor‐binding motif. Furthermore, ASAH1 is recruited to the promoter of various SF‐1‐target genes and co‐localizes with the receptor on the same promoter region of the CYP17A1 and steroidogenic acute regulatory protein (StAR) genes. Taken together, we establish ASAH1 as a novel coregulatory protein that represses SF‐1 function by directly binding to the receptor on the promoter of SF‐1‐target genes.
A Copper Complex, ghn‐12, as a sensitization of DNA to UVA offers potential for a novel photochemotherapy
Authors: Lai Feng-Jie , Liang Cheng-Hsuan , Cheng Ching-Li , Lin Rrong-Kai , Chang Nan-Shan , Lin Chu-Chieh
Published: June 2021

Photochemotherapy is a modern therapy modality, based upon the application of a photosensitizing agent onto the tissue followed by illumination with light sources, which is a proven therapeutic strategy for a number of non‐malignant hyperproliferative skin diseases and various cancers. The aim of the present study is to investigate a new synthesis copper complex (ghn‐12, Chemical Formula: C53H39ClCuN8O5) as a potent photocytotoxic agent. Photoinduced DNA cleavage activity, cell cytotoxicity, cell cycle distribution and apoptosis assay are used to investigate the effect of ghn‐12 on therapeutic applications. Ghn‐12 induced supercoiled pUC19 DNA cleavage from supercoiled to nicked circular form at a low‐power monochromatic UV‐A light of 365 nm. Flow cytometry analysis revealed that photoinduced ghn‐12 sensitized squamous cell carcinoma (SCC15) cells to arrest in the G0/G1 and S‐G2/M phases of the cell cycle with a concomitantly significant increase in the sub‐G1 cell population, indicating cell death by apoptosis. This photoinduced apoptosis process was accompanied by activation of caspase‐3 expression and FITC Annexin V staining after SCC15 cells treated with ghn‐12. Our data suggest that a new photosensitizing compound, ghn‐12, exhibiting light‐induced cleavage of double‐stranded DNA and causing apoptosis of squamous cell carcinoma. Ghn‐12 is a potential therapeutic reagent. Research support by grant from the National Science Council, Taiwan Grant NSC99‐2314‐B‐384 ‐002 ‐MY3.
The effect of benzosuberene analogues on endothelial cell morphology and tube formation
Authors: Tidmore Justin Keith , Strecker Tracy E. , Odutola Samuel O. , Charlton-Sevcik Amanda K. , Tanpure Rajendra P. , George Clinton S. , Sriram Madhavi , Chaplin David J. , Pinney Kevin G. , Trawick Mary Lynn
Published: June 2021

KGP18 is a tubulin‐binding, benzosuberene‐based small molecule with remarkable cytotoxicity against selected human cancer cell lines (BMC 2008 16, 8161–8171). Its structure together with its ability to inhibit tubulin polymerization into microtubules in vitro suggest that it may act as a vascular disrupting agent (VDA). An adequate blood supply is required for tumor growth and metastasis. VDAs represent a promising new class of anti‐cancer agents that function by selectively disrupting the tumor vasculature, thus starving the tumor of nutrients and oxygen. Tubulin‐binding VDAs inhibit microtubule assembly in the activated endothelial cells, lining the tumor vasculature, leading to cytoskeletal reorganization, increased vascular permeability, cell rounding and detachment, and vessel disruption. The lead benzosuberene analogue (KGP18) and several analogues were assessed for their ability to disrupt microtubules, and cause a rearrangement of F‐actin in human umbilical vein endothelial cells (HUVECs) in cell culture. The disruption of pre‐established tubules formed by HUVECs on Matrigel™ by these benzosuberene analogues was also evaluated. (Supported by Oxigene Inc., the NIH, and CPRIT).
“LEFT HEMICRANIUM: CRANIAL NERVES” AN ANATOMICAL MODEL IN WAX BY TRAMOND (ORFILA MUSEUM, PARIS): STUDY AND RECONSTRUCTION BY PHOTOGRAPHY IN ROTATION
Authors: Le Floch-Prigent Patrice Pierre , PARAVEY S.
Published: June 2021

An anatomical model in wax by Tramond (middle of the 19th century) represented the cranial nerves of a left hemicranium. The aim of the study was to verify its anatomical veracity; to realize a tri‐dimensional vizualisation by computer; and finally to digitize them. It belonged to the Orfila museum (Paris). It represented the last cranial nerves specially the facial nerve and its branches. To perform the photographic rotation every 5° along 360°, we used a special device made of two identical, superimposed, marble disks linked by a ball bearing. A numerical camera and the Quick Time Virtual Reality software were used. 72 pictures were shot. This wax was realized with a great morphological accuracy from a true cranium as a support for the cranial nerves. The Orfila museum was closed and evacuated on december 2009.
SCFFbxo45 controls cytokinesis through ubiquitin‐mediated proteolysis of GEF‐H1
Authors: chen xiaofei , Ahn Joon-Young , Szankasi Philippe , Chung Fuzon , Basrur enkatesha , Lim Megan S. , Miller Ann L. , Pagano Michele , Elenitoba-Johnson Kojo S. J.
Published: June 2021

The rho guanine nucleotide exchange factor H1 (GEF‐H1) is a critical regulator of cytokinesis, but the requirement and mechanism for its timely destruction for proper execution of cytokinesis are unknown. Here we show that GEF‐H1 is regulated by the evolutionarily conserved E3 ligase SCFFbxo45. Fbxo45 promotes GEF‐H1 ubiquitylation upon PLK1‐mediated phosphorylation of GEF‐H1 on serine 644, and this event is necessary for GEF‐H1 degradation during mitosis. Fbxo45 silencing causes stabilization and abnormal cellular distribution of GEF‐H1 with diffuse RhoA hyperactivation. A GEF‐H1 mutant that is unable to bind Fbxo45 is stabilized in mitosis, and results in an uncoordinated hyperactive membrane phenotype, with abnormal aberrant cytokinesis leading to cell death or multinucleation. Our studies provide evidence that limiting activation of the RhoAGTPase via Fbxo45‐mediated degradation of the GEF‐H1 activator is essential for proper execution of cytokinesis. This work was supported by NIH grants R01 DE119249 and R01 CA136905 to KSJ E‐J, R01 CA140806 to MSL.
Magic Marker Mystery: Why Does Antibody AC133 So Specifically Recognize Stem Cells?
Authors: Cooper Douglas N. W. , Buselli Monica , Castellanos Elizabeth , Enerio Shiena T. , Murti Akikta D. , Reed Kimberli D. , Wong Alyssa M.
Published: June 2021

Highly specific markers that distinguish stem from other cell types are critical tools for isolating stem cells, assaying their purity and functional status, and potentially targeting cancer stem cells for destruction. One of the most widely used markers for normal and cancer stem cells is a plasma membrane protein of unknown function, CD133. However, most antibodies to CD133 also recognize some non‐stem cell types. By contrast, the monoclonal antibody, AC133, seems to only recognize a subset of CD133 uniquely expressed on stem cells. Such specificity suggests a functionally role for this epitope in stem cell biology. Our aim has been to identify what epitope variation on CD133 makes AC133 so precise in identifying stem cells: e.g. splice variation in the CD133 protein sequence, post‐translational modification, or alternative conformation. Suspecting that tyrosine sulfation, a modification increasingly recognized on cell surface and extracellular proteins, might be involved, we tested the effect of sodium chlorate, a competitive inhibitor of sulfation, on CD133 expression in culture cells. 30mM chlorate blocked CD133 recognition by both AC133 and a specific anti‐sulfotyrosine antibody, indicating that tyrosine sulfation is a critical feature of the epitope recognized by AC133. We are now seeking other features of this epitope, its location on CD133, and its significance for stem cell physiology.
Styloid process elongation or stylohyoid ligament calcification is not necessarily a predictor of Eagle's Syndrome
Authors: Hessel Kara Marie , Heller Aaron , Wright Barth
Published: June 2021

The temporal styloid process (TSP) has close anatomical relationships with the internal jugular vein, internal carotid artery, as well as the facial, glossopharyngeal, vagus, and hypoglossal nerves. Elongation of the TSP or calcification of the stylohyoid ligament (SL), may lead to compression of the surrounding structures, resulting in symptoms typical of Eagle's syndrome. These symptoms include dysphagia, odynophagia, unilateral face and neck pain, transient ischemic accident, and cerebrovascular accident. The average TSP is approximately 2.5 cm in length, with a length greater than 3 cm being considered elongated. This study discusses two cadaveric specimens with TSP elongation or SL calcification, neither of which displayed symptoms of Eagle's syndrome in life. The relatives of each specimen were contacted to determine whether symptoms were present. Case one, a 71‐year‐old male, was found to have bilateral calcification of the SL (right 88.18 mm, left 57.21 mm). The complete absence of symptoms was potentially due to the mobility of a pseudojoint attaching the calcified SL to the TSP. Case two, a 74‐year‐old male, displayed a right unilateral elongation of the TSP (48.74 mm). These two cases suggest that the degree of TSP elongation or SL calcification does not necessarily predict which patients will develop Eagle's syndrome.
Unraveling the role of exit tunnel residues of histone deacetylase‐8 in catalysis
Authors: Khanna Ruchi , Singh Raushan K. , mandal Tanmay , Cook Gregory , Srivastava D.K.
Published: June 2021

Histone Deacetylases (HDACs) are an important class of enzymes which are involved in epigenetic regulations and they have been identified as high priority targets for treatment of cancers. The structural data reveals that apart from the active site pocket, HDAC‐8 harbors an internal cavity through which the acetate product dissociates from the enzyme. To probe the role of the amino acid residues at the “exit port” of the above cavity, we performed site directed mutagenesis of Y18, Y20 and H42 to Ala. Although these mutations did not influence the binding affinity of the substrate, they drastically impaired the catalytic rate of HDAC‐ 8. In case of Y18A mutation, the magnitude of activation by our recently identified activator (TM‐2‐51) was far more pronounced than the wild‐type enzyme. Our circular dichroism studies indicated a change in the secondary structure of the enzyme in case of Y18A mutation (but not in Y20A and H42A mutations) which was manifested in an increased thermal stability of the mutant. Our experimental data reveals that there is a long range communication between the exit tunnel residues and the active site pocket of HDAC‐8, and such communication presumably regulates the overall catalytic feature of the enzyme

Research supported by NIH grants CA113746 and CA132034 to DKS and NIH COBRE grant NCRR‐P20‐RR15566 to GC.
Characterizing the Binding of Enzyme Inhibitors at the Molecular and Ensemble Levels
Authors: Cao Samantha Midung , Pham Benjamin , Radcliffe Amethyst , La Phuc , Wang Richard , An Yi , Sorin Eric J.
Published: June 2021

Recent studies have shown that butyrylcholinesterase (BuChE) activity is increased in patients suffering from Alzheimer's disease (AD) and is one of the main enzyme targets necessary to treat Alzheimer's symptoms. As BuChE is a key enzyme that catalyzes the hydrolysis of the neurotransmitter acetylcholine, increased control over inhibition of this enzyme is highly sought. We employ molecular dynamics (MD) simulations to study the physics of the inhibitor‐enzyme complex and the chemical characteristics necessary to effectively target BuChE. One family of inhibitors is the dialkyl phenyl phosphates (DAPPs), which have been shown to specifically target BuChE. We studied the interactions and binding affinities of a number of DAPP's docked in the active site of BuChE. Using the Folding@Home Distributed Computing Network, 8,000 all‐atom molecular dynamics simulations were collected, including the enzyme sans inhibitor, which is used to evaluate the mechanism and energetics of ligand‐binding, as well as the equilibrium dynamics of the enzyme‐substrate complex.

This work was made possible by the worldwide Folding@Home volunteers who contributed invaluable processor time; Women & Philanthropy, Kenneth L. Marsi, and James L. Jensen Student Scholarships; and a Research Corporation Cottrell College Science Award.
A Single Residue With Dramatic Impact On Substrate‐ Induced Spin‐State Change In P450BM‐3
Authors: Haines Donovan C. , Hegde Amita , Chen Baozhi , Tomchick Diana , Zhao Weiqiang , Bondlela Muralidhar , Humphreys John M. , Mullin David A. , Machius Mischa , Peterson Julian A.
Published: June 2021

Cytochrome P450 enzymes are important in Phase I xenobiotic metabolism, hormone metabolism, and the synthesis of numerous natural products. The bacterial enzyme P450BM‐3 has long served as a convenient model of P450 structure and function. As with many other P450s, P450BM‐3 undergoes a change in the spin‐state of the heme iron upon substrate binding. This change from the resting six‐coordinate low‐spin state to the five‐coordinate highspin state may help conserve electrons in the absence of oxidizable substrate, as the low‐spin state is harder to reduce. We have found that mutation of an active site alanine (A328) to valine produces an enzyme with greatly improved substrate binding, spin‐state conversion, and turnover rate. Mutation of the same residue to serine, however, results in greatly impaired spin‐state conversion and impaired substrate binding. Even more intriguing, the effect of the A328S mutant on Vmax depends on the type of substrate examine, with fatty acid oxidation being highly impaired but N‐acyl amino acid oxidation rate being enhanced ~60%. These results give us new insights into how protein conformational change couples substrate binding to spin‐state change. This research was supported in part by research grants GM43479 and GM50858 from the NIH (JAP) and X‐011 from the Robert A. Welch Foundation (DCH).
AN ENBLARGED DISMANTABLE ANATOMICAL MODEL OF EAR IN PAPIER MACHE BY AUZOUX, OBSERVATION AND PHOTOGRAPHICAL TRI‐DIMENSIONAL RECONSTRUCTION
Authors: Le Floch-Prigent Patrice Pierre , GILLOT Jean Bernard , UHL Jean François
Published: June 2021

The aim of the study was to determine the accuracy of a 19th century anatomical model of ear by Auzoux (a french manufacturer). It measured 63cm in length; 43cm in width; 37cm in height. The disassembled parts were studied on the morphological aspect in its whole and after removing of its components: external ear (43×23×15cm), tympanic drum and middle ear ossicles; cochleo‐vestibular apparatus. The main dimensions of each part were recorded. The arterial vascularisation of the three components of the ear and its sensitive and sensorial nervous system were established on the model which represented the external carotid artery with its terminal bifurcation, several collateral branches specially the posterior auricular artery at the two extremities of the carotid canal. The cochleo‐vestibular and facial nerve as well as other elements of small caliber were exactly figured and labelled by a number. The middle ear components were disassembled. The ossicles of the middle ear and a dried, peritoneal tympanic membrane were dismountable as a whole. This kind of rare model can be dated at the earliest 1835.
Expression of PPARα, β, and γ in the Hartley guinea pig model of primary osteoarthritis
Authors: El Mansouri Fatima Ezzahra , Nebbaki Salwa Sarah , Zayed Nadia , Benderdour Mohamed , Martel-Pelletier Johanne , Pelletier Jean-Pierre , Fahmi Hassan
Published: June 2021

Background

Peroxisome proliferator‐activated receptors (PPARs) are members of the nuclear receptor superfamily (PPARα, PPARβ/δ and PPARγ). Little is known about the pattern of PPAR expression during the progression of osteoarthritis (OA).

Objective

To investigate the expression of PPARα, β, and γ in cartilage over the course of OA in the spontaneous Hartley guinea pig model.

Methods

Hartley guinea pigs were sacrificed at 2, 4, 8, and 12 month‐old of age. Cartilage was obtained from the central portion of the medial tibial plateau. Cartilage degradation was evaluated histologically using the Osteoarthritis Research Society International (OARSI) guidelines. The expression of PPARα, β and γ was analyzed by immunohistochemistry.

Results

PPARα, β and γ, were detected in medial tibial plateaus from control animals. There was no significant changes in the levels of PPARα and PPARβ over the course of OA. In contrast, PPARγ expression decreased during the progression of OA. Correlation analysis revealed a negative correlation between PPARγ levels and histological score of OA.

Conclusion

Expression of PPARγ in cartilage decreased during OA. These data suggest that loss of PPARγ expression in cartilage may contribute to the pathogenesis of OA.
Absence of selenoprotein p but not selenocysteine lyase results in neurological dysfunction in mice
Authors: Raman Arjun , Pitts Matthew , Seyedali Ali , Hashimoto Ann , Seale Lucia , Bellinger Frederick , Berry Marla
Published: June 2021

Selenium (Se) is a trace element that is important for the mammalian central nervous system (CNS). Selenoprotein P (Sepp1) is a Se‐rich protein that is important for brain Se supply, and is thought to supply Se via the enzyme selenocysteine lyase (Scly). Sepp1‐deficient mice develop neurological symptoms of spasticity, and have deficient synaptic plasticity and spatial learning.

In this study, male and female transgenic mice lacking Sepp1 or Scly on variable Se diets were evaluated using behavioral and biochemical paradigms for neurological dysfunction. Male mice lacking Sepp1 appear worse than age‐matched female mice, suggesting that males have a higher demand for Sepp1 and Se in the CNS than females. Surprisingly, no behavioral impairment was observed in mice lacking Scly unless they were restricted with a low Se diet. Se deficiency uncovered a mild learning disruption in Scly knockout mice without causing motor deficits. Scly‐deficient mice fed a low Se diet display reduced expression and activity of selenoproteins in brain. These results suggest that Sepp1 is more critical than Scly for CNS function and maintenance of brain Se, but that both gene products are physiologically relevant during dietary Se deficiency.

This research was supported by NIH grants RR003061, DK047320, RR016467 and DA027318.
Increase of Hormone‐sensitive Lipase Activity by Subfaction of Green Pepper Water Extracts in 3T3‐L1 Cells
Authors: Kim Kyoungkon , Lee Jaesung , Heo Jeongwon , Baek Jongmi , Kim Taehyuk , Kim Daejung , Kim Taewoo , Kim Cheonan , Choe Myeon , Ochir Sarangowa , Kanazawa Tsutomu
Published: June 2021

It has been well elucidated that excessive body weight could related to various diseases, for example cardiovascular diseases, diabetes mellitus.. We have investigated water extract of green pepper stimulating hormene‐sensitive lipase activity in adipose tissue. In order to find effective compound from green pepper, we subfractionated water extract of green pepper by using columns packed with sephadex LH20 and MCI CHP‐20. Subfractions were checked by UPLC and hormene‐sensitive lipase activity was also investigated.
Pannexin 3, a gap junction protein, regulates proliferation through AMPK pathway in odontoblasts
Authors: Iwamoto Tsutomu , Ishikawa Masaki , Yoshizaki Keigo , Nakamura Takashi , Yamada Aya , Yamada Yoshihiko , Fukumoto Satoshi
Published: June 2021

Pannexin 3 (Panx3), a member of the pannexin family of gap junction proteins, regulates chondrocyte and osteoblast differentiation. However, its expression and physiological functions in tooth development remain unclear. Our objective was to elucidate the expression and identification of molecular mechanisms of Panx3 functions in tooth development. In situ hybridization demonstrated that Panx3 mRNA was preferentially expressed in preodontoblasts. To analyze the role of Panx3 in odontoblast proliferation and differentiation, we used the odontogenic cell line mDP. We found that Panx3 expression was induced in differentiating mDP cells, and overexpression of Panx3 inhibited mDP cell proliferation and promoted cell differentiation into odontoblasts. We also found that Panx3 overexpression promoted the release of ATP into the extracellular space and induced the phosphorylation of AMP activated protein kinase (AMPK), a highly conserved energy sensor in all eukaryotic cells. Further, the activated AMPK pathway inhibited mDP cell proliferation and induced p21 expression. Taken together, our results suggest that Panx3 reduces intracellular ATP levels through a hemichannel, which results in inhibition of odontoblast proliferation though the AMPK/p21 pathway.

(Supported by grants from the Ministry of Education, Science, and Culture of Japan and the Intramural Program of the NIDCR, NIH, USA)
CDK9 regulates hypotonicity‐induced nuclear export of NFAT5
Authors: KO Ben C.B. , Cheung Chris Y.K. , Wong Catherine , Yates John , Xu SongXiao , Huang Tingting
Published: June 2021

NFAT5, also known as Osmotic Response Element‐binding Protein (OREBP), or Tonicity‐responsive Enhancer‐binding Protein (TonEBP), is an osmo‐ sensitive transcription factor that mediates cellular adaptations to extracellular hypertonic stress. The subcellular localization and activity of NFAT5 are tightly regulated by extracellular tonicity. Earlier we showed that hypotoncity‐induced nuclear export of NFAT5 is regulated by the sequential phosphorylation of two serine residues (ser‐155 and ser‐158), and we also identified Casein Kinase 1 (CK1) as the ser‐158 kinase. In this study, using protein fractionations, in‐vitro kinase assay and mass spectrometric protein identification approaches, we identified Cyclin‐dependent Kinase 9 (CDK9) as a putative ser‐155 kinase. NFAT5 is co‐immunoprecipitated with CDK9 and its regulatory subunit cyclin T. Furthermore, CDK9 inhibitors and siRNAs significantly diminished NFAT5 nuclear export in response to hypotonicity.
CARDIAC MORPHOMETRIC CHANGES IN RENAL ISCHEMIA/REPERFUSION INDUCED CARDIAC HYPERTROPHY IN MICE
Authors: Kmit Fernanda Vieira , Trentin-Sonoda Mayra , Carneiro-Ramos Marcela Sorelli
Published: June 2021

Objectives

The aim of this study was to investigate if particular morphological parameters were altered in heart after renal ischemia/reperfusion.

Methods

C57bl/6J mice were subjected to unilateral 60 minutes kidney ischemia, followed by reperfusion of 5, 8, 12, 15 and 20 days. Levels of urea were measured to confirm renal failure. Image captures was taken to analyze morphometric parameters and Cavalieri estimator was performed to estimate heart tissue volume and renal tissue alterations.

Results

Urea levels were increased in groups 5, 15 and 20 days confirming the experimental model. The renal ischemia/reperfusion model promoted a decrease in left kidney weight/ body weight and left kidney length/ tibia length ratio in group 20 days reperfusion as expected. An increase in heart weight and heart weight/tibia length ratio after 15 days of reperfusion was observed compared to sham group (P<0.001). The cardiac hypertrophy was accompanied by a decrease on Left ventricle lumen area in 12, 15 and 20 days when compared to sham (P<0.001), suggesting a concentric hypertrophy. Moreover the ventricles weight showed increased in 15 days group (p<0.001).

Conclusion

First results indicate that renal ischemia/reperfusion model was able to induce several morphometric changes in heart such as concentric cardiac hypertrophy in groups studied.

Financial Support: FAPESP and UFABC.
STAP‐2 interacts with and modulates BCR‐ABL‐mediated tumorigenesis
Authors: Matsuda Tadashi , Sekine Yuichi , Ikeda Osamu , Muromoto Ryuta , Oritani Kenji
Published: June 2021

In chronic myeloid leukemia (CML), the BCR‐ABL fusion oncoprotein activates multiple pathways involved in cell survival, growth promotion and disease progression. In this report, we show that the signal transducing adaptor protein‐2 (STAP‐2) is involved in BCR‐ABL activity. We demonstrate that STAP‐2 binds to BCR‐ABL, and BCR‐ABL phosphorylates STAP‐2 Tyr250 and the phosphorylated STAP‐2 in turn up‐regulated BCR‐ABL phosphorylation, leading to enhanced activation of downstream signaling molecules including ERK, STAT5, BCL‐xL and BCL‐2. In addition, STAP‐2 interacts with BCR‐ABL to alter chemokine receptor expressions. The interaction between STAP‐2 and BCR‐ABL plays a crucial role in conferring a growth advantage and resistance to imatinib, a BCR‐ABL inhibitor, as well as tumor progression. Notably, mice injected with BCR‐ABL/STAP‐2‐ expressing Ba/F3 cells developed lymph node enlargement and hepatosplenomegaly. Moreover, suppression of STAP‐2 in K562 CML cells resulted in no tumor formation in mice. Our results demonstrate a critical contribution of STAP‐2 in BCR‐ABL activity, and suggest that STAP‐2 might be an important candidate for drug development for patients with CML.
Examination of S‐nitrosylation sites in human Thioredoxin protein
Authors: Alicea Ismael , Schreiter Eric R
Published: June 2021

S‐nitrosylation (SNO) is a posttranslational modification involving the chemical attachment of a nitric oxide moiety to specific cysteine sites of a protein. Although a variety of proteins with SNO sites have been studied, experimental or computational methods for predictions of such sites are still limited. To learn more about the molecular determinants of protein‐SNO formation, we used human thioredoxin protein (hTrx) as a model for SNO because it has two distinct SNO sites, cysteines 62 and 69, and is involved in several aspects of cellular SNO signaling. Briefly, we used available three‐dimensional structures of hTrx (PDB codes, 2IIY and 2HXK) to identify amino acids in the immediate chemical environment of Cys62 and Cys69 that may be important for SNO formation. We generated five variants (E6Q, D61K, D61N, D61V, and V65T,) by site‐directed mutagenesis and monitored S‐nitrosylation of each variant using the Saville‐Griess assay. We found that all the variants, with the exception of V65T, exhibited higher level SNO than the wild type hTrx (which contains five cysteines including the redox‐active Cys32‐Cys35 pair), but similar to the control having only Cys62. In contrast, replacement of valine 65 with threonine (V65T) significantly reduced stable SNO formation at Cys62, suggesting that it may play a direct role in the activation of cysteine 62 towards S‐nitrosylation in hTrx. Research supported by RISE (NIH).