bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sca‐1+PDGFRα− endothilial cell population maintains the ability of mouse mesenchymal stem cells in vitro
Authors: OGATA YUSUKE , MABUCHI YO , HORIIKE YUTA , YOSHIDA MAYU , SUTO ERIKO GRACE , SUZUKI NOBUHARU , AKAZAWA CHIHIRO 
Published: November 2023

BackgroundMesenchymal stem cells (MSCs) can renew themselves and differentiate into adipocyte, osteoblast, and chondrocyte. MSCs can be easily expanded to generate the large cell numbers required for transplantation. According to their stem cell ability, MSCs have been already used in clinical trials such as knee cartilage regeneration therapy. However, adherence culture condition on plastic dishes inevitably changes the surface marker expression and biological properties of the stem cells. Previous our report shows that the Sca‐1+/PDGFRα+ population is significantly enriched for mouse MSCs, and Sca‐1+/PDGFRα+ cells were located in the arterial perivascular space near the inner surface of the cortical bone (J Exp Med., 2009). Based in these findings, we established the new culture method using the purified MSCs and endothelial cells.Materials and MethodsFemurs, tibias and iliums from adult C57BL/6 wild‐type age of 6‐week were dissected and crushed. The crushed bones were collected and incubated for 1 hour in 20mL DMEM containing 0.2% collagenase. The digested cells were suspended in ice‐cold HBSS and the suspended cells were stained with antibodies for 30 minutes on ice. We sorted endothelial cells (Sca‐1+/PDGFRα− ) using cell sorter and analyzed the pattern of cell surface markers. In the same way, we sorted MSCs (Sca‐1+/PDGFRα+) from CAG‐EGFP transgenic mice and cocultured with wild‐type MSCs for 1 week.ResultsIn flow cytometric analysis, Sca‐1+/PDGFRα−expressed the endothelial cell markers such as CD31, CD34, CD133 and CD309 (VEGFR2), but did not that of blood cell markers (CD45 and Ter119). Sorted Sca‐1+/PDGFRα− cells higher expressed Pecam‐1, vWB, and Tie‐2. These data indicate that Sca‐1+/PDGFRα− population was enriched the endothelial cells. Next, we performed the co‐culture assay using Sca‐1+/PDGFRα−endothelial cells. MSCs (from EGFP mouse) kept spindle shape morphology and MSCs markers expressions (Sca‐1+/PDGFRα+) by the cocultivation with the endothelial cells. Furthermore, Ki‐67 (proliferation marker) was significantly higher co‐cultured condition compared to non‐cocultured.ConclusionThe results suggested that Sca‐1+/PDGFRα+ MSCs maintain their stem cell ability to culture with Sca‐1+/PDGFRα− endothelial cells. Further analysis of cell‐cell interaction between MSCs and Sca‐1+/PDGFRα− cells will develop new culturing method, which can prepare more suitable MSCs for regenerative therapy.
The Histone Tail of CPAR‐1, a CENP‐A Related Protein, is Cleaved by Separase at the Meiosis I Metaphase‐Anaphase Transition in C. elegans
Authors: Monen Joost , Hattersley Neil , Desai Arshad 
Published: November 2023

Centromeres are defined epigenetically in the majority of eukaryotes by the presence of chromatin containing the centromeric histone H3 variant CENP‐A. Most species have a single gene encoding a centromeric histone variant whereas C. elegans has two: HCP‐3 (also known as CeCENP‐A) and CPAR‐1. Prior RNAi replacement experiments showed that HCP‐3 is the functionally dominant isoform, consistent with CPAR‐1 not being detectable in embryos. GFP::CPAR‐1 is loaded onto meiotic chromosomes in diakinesis and is enriched on bivalents until meiosis I. Here we show that GFP::CPAR‐1 signal loss from chromosomes precisely coincides with homolog segregation during anaphase I. This loss of GFP::CPAR‐1 signal reflects proteolytic cleavage between GFP and the histone fold of CPAR‐1, as CPAR‐1::GFP, in which GFP is fused to the C‐terminus of CPAR‐1, does not exhibit any loss of GFP signal. A focused candidate screen implicated separase, the protease that initiates anaphase by cleaving the kleisin subunit of cohesin, in this cleavage reaction. Examination of the N‐terminal tail sequence of CPAR‐1 revealed a putative separase cleavage site and mutation of the signature residues in this site eliminated the cleavage reaction, as visualized by retention of GFP::CPAR‐1 signal on separating homologous chromosomes at the metaphase‐anaphase transition of meiosis I. Neither cleaved nor uncleavable CPAR‐1 were centromere‐localized in mitosis and instead localized throughout chromatin, indicating that centromere activity has not been retained in CPAR‐1. Although the functions of CPAR‐1 and of its separase‐dependent cleavage remain to be elucidated, this effort reveals a new substrate of separase and provides an in vivo biosensor to monitor separase activity at the onset of meiosis I anaphase.Support or Funding InformationJ.M. was supported in part by an Institutional Ruth L. Kirschstein National Research Service Award from the National Institute for General Medical Sciences to the UCSD Genetics Training Program (T32 GM008666). J.M. was supported in part by a grant from the Ramapo College Foundation and Faculty Development Fund. This work was supported by an NIH grant (GM074215) to A.D.
Regulation of HORMA domain proteins by the conserved remodeler Pch2/TRIP13
Authors: Corbett Kevin D , Ye Qiaozhen , Rosenberg Scott C , Su Tiffany Y 
Published: November 2023

The AAA+ ATPase Pch2/TRIP13 is a key regulator of two related signaling protein families in eukaryotes, the meiotic HORMADs and the spindle checkpoint protein MAD2. These two families share a common domain, the HORMA domain, which can adopt two differently‐folded states: a signaling‐inactive “open” state and an active, peptide‐bound “closed” state. In the closed state, the C‐terminus of the HORMA domain, termed the “safety belt” region, wraps around a bound peptide to generate a topological linkage between the two proteins. To determine how Pch2/TRIP13 regulates HORMA domain protein function, we determined the crystal structures of Pch2/TRIP13 orthologs from C. elegans and H. sapiens. C. elegans PCH‐2 crystallizes in an asymmetric hexamer conformation, and H. sapiens TRIP13 crystallizes in a helical‐spiral conformation, with subunit‐subunit interfaces equivalent to those seen in C. elegans PCH‐2. Together, these structures reveal how ATP binding, hydrolysis, and release in the Pch2/TRIP13 hexamer power conformational changes that remodel substrate proteins. Further, we find that mammalian TRIP13, aided by the adapter protein p31(comet), converts MAD2 from its signaling‐active closed conformer to its inactive open conformer. We propose that TRIP13 partially unfolds the MAD2 C‐terminal safety belt region, allowing release of bound peptide and relaxation to the protein's open state. In the context of the spindle assembly checkpoint, TRIP13 and p31(comet) collaborate to recognize and disassemble mitotic checkpoint complexes, inactivating the checkpoint. A parallel HORMA protein complex disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination, where it acts on the meiotic HORMAD proteins.Support or Funding InformationThe authors acknowledge support from the NIH (R01GM104141), the March of Dimes Foundation, and the Ludwig Institute for Cancer Research.
Estrogen Receptor Alpha Utilizes TRP Channel 6 in Cancer Cell Growth
Authors: Rawlings Emily J , Rofelty Angela J , Schmitt John M 
Published: November 2023

The rapid signaling effects of the hormone estrogen (E2) are mediated through estrogen receptor alpha (ERα) that activates a Ca2+/calmodulin signaling pathway involving CaM KK, CaM KI, and ERK. Additionally, expression of transient receptor potential (TRP) channels, which are Ca2+ channels, may correlate with breast cancer cell growth. In this family of channels, TRPC6 has been shown to be expressed in MCF‐7 breast cancer cells and involved in ERK activation. Since both ERα and TRPC6 increase intracellular calcium levels, ERK activation, and cell growth our goal was to examine if ERα cooperates with TRPC6 to increase CaM Kinase and ERK phosphorylation as well as MCF‐7 cell growth. E2 treatment of MCF‐7 cells led to a significant rise in phosphorylation of CaM KI and its downstream target, ERK, and this effect was blocked by pretreatment with the ERa antagonist MPP. Treatment of MCF‐7 cells with E2 stimulated an increase in MCF‐7 cell growth that was blocked by pretreatment with MPP, but not by pretreatment with the ERb antagonist PHTPP. Interestingly, treatment of cells with the TRP channel inhibitor APB blocked E2 activation of ERK and cell growth. Immunoprecipitation (IP) of endogenous ERa pulled down TRPC6 as did the reciprocal IP from MCF‐7 cells. Confocal microscopy also revealed co‐localization of the proteins in MCF‐7 cells. Our data suggest E2 rapidly activates CaM KI and ERK via ERa and TRP channels and ERa may associate with TRPC6 to regulate breast cancer cell growth.Support or Funding InformationThe Paul K. and Evalyn E. C. Richter Memorial Fund.This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267.
Rab5‐Activating Protein 6 (RAP6) Activity in Adipogenesis
Authors: Chantarasinlapin Praew , Veisaga Maria‐Luisa , Huffman Fatma G. , Barbieri M. Alejandro 
Published: November 2023

Rab5‐Guanine Exchange Factor (GEF) has been speculated to involve in regulation of intracellular membrane trafficking and receptor signaling. The present study demonstrated that Rab5‐Activating Protein 6 (RAP6), a Rab5‐GEF containing a Vps9 domain, PH domain and Ras‐GAP domain, plays an important role in adipogenesis. Expression of RAP6:wild type blocked differentiation of cultured 3T3‐L1 preadipocytes. Consistently, expression of other Rab5‐GEFs (i.e., Rin1, Rabex‐5 and RinL) inhibited differentiation of 3T3‐L1 preadipocytes. In contrast, RNAi‐mediated depletion of RAP6 inhibited cell differentiation in 3T3‐L1 preadipocytes, suggesting that RAP6 activity is essential for adipogenic process. Furthermore, expression of RAP6:wild type, but not RNAi‐mediated depletion of RAP6, suppressed the expression of adipogenic‐specific transcriptional factors, PPARγ and C/EBPα. Interestingly, expression of RAP6 domains individually blocked cell differentiation in 3T3‐L1 preadipocytes. In addition, RAP6 mutants (i.e., D138A and Y1420F) that interfered with Rab5‐GEF activity poorly diminished 3T3‐L1 preadipocyte differentiation. The findings indicate that altering the level of active Rab5 by RAP6 influences the differentiation of 3T3‐L1 preadipocytes, suggesting RAP6 as one of key modulators in adipogenesis.Support or Funding InformationChulalongkorn University Doctoral Scholarship Program
CaM Kinase Co‐localization and Regulation of p53 in Breast Cancer Cells
Authors: Coblentz Cody , Geck Renee , Schmitt John M 
Published: November 2023

The Ca2+/calmodulin dependent kinases (CaM KK and CaM KI) have been implicated in several types of cancer including breast cancer. CaM KK and CaM KI are known to control several transcription factors. CaM KI has also been shown to bind and phosphorylate the cell cycle protein p27 to promote cell cycle progression. Interestingly, the tumor suppressor p53 contains a possible CaM KI phosphorylation site at S366. The aim of this study was to investigate CaM KI‐p53 association, CaM KI targeted phosphorylation (S366) of p53, and subsequent breast cancer growth in both MDA‐MB‐231 and MCF‐7 cells. GST pull‐downs, immunoprecipitation, as well as confocal microscopy of endogenous proteins showed that CaM KI associates with p53 phosphorylated at S366. Treatment of MCF‐7 and MDA‐MD‐231 cells with estrogen (E2) and carbachol respectively, increases the association of CaM KI‐p53 and S366 phosphorylation. siRNA knockout of CaM KI blocked E2 and carbachol‐induced S366 phosphorylation and constitutively active CaM KK increased p53 phosphorylation in breast cancer cells. Interestingly, both E2 and carbachol increased MCF‐7 and MDA‐MD‐231 cell growth respectively through CaM Kinase and p53. In summary, our data suggests that CaM KI binds and phosphorylates p53 in MCF‐7 and MDA cells, and that E2 and carbachol stimulate cell growth through a novel CaM KI‐p53 pathway.Support or Funding InformationThe Paul K. and Evalyn E. C. Richter Memorial Fund.This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267
FAM83G is a novel apoptosis inducer
Authors: Okada Shuichi , Yamada Masanobu 
Published: November 2023

The eight members of FAM83 family are mostly uncharacterized proteins. FAM83G is present across vertebrates and a missense mutation in dogs resulted in hereditary footpad hyperkeratosis and a truncation in mice was reported to be responsible for the wooly mutation (wly). Through a serine phosphorylation proteomic screen we uncovered that overexpressed FAM83G significantly reduced cell number and this effect was primarily due to activation of apoptotic programmed cell death, characterized by increased DNA fragmentation, mitochondrial release of cytochrome C into the cytosol and changes in the cleavage patterns of PARP, caspase 3 and caspase 7. Surprisingly, this did not require the addition of any stress inducers but simply occurred upon over expression of FAM83G.The ability of FAM83G to activate apoptosis in the absence any stressor requires the phosphorylation of S356 as phosphorylation of this site occurred in FAM83G over expressing cells and mutation of this residue to alanine protected against apoptosis and cell death. Although we have not directly identified the kinase(s) responsible the most likely candidates are PKD1/PKCmu. Moreover, PKD1/PKCmu have been shown to directly phosphorylation S82 of HSP27 that protects cells from apoptosis. In this regard, expression of wild type FAM83G suppressed S82‐HSP27 phosphorylation whereas expression of the S356A‐FAM83G mutant had no effect on S82‐HSP27 phosphorylation. We hypothesize that FAM3G and HSP27 may compete for substrate recognition by PKD1/PKCmu would account for the effects of phosphorylation site peptide to mimic the effect of wild type FAM83G. Further studies are therefore needed to determine whether FAM83G functions as a direct antagonist of PKD1/PKCmu mediated HSP27 phosphorylation.Support or Funding InformationNone
Protective roles of Sep15 against cataract formation
Authors: Yin Na , Huang Kaixun 
Published: November 2023

Sep15, a 15 KDa selenoprotein, is believed to involve in reduction or isomerization of disulfide bonds and the quality control of glycoprotein folding. However, most of its function remains unclear, especially its roles in the related diseases. For instance, it was reported that Sep15 knockout (KO) mice developed a prominent nuclear cataract in the early of age, but the protective role of Sep15 against cataract formation remains unaddressed. Here, we are trying to share our recent work aimed to unveil the underling mechanism of Sep15 deficiency in cataract development. In our recently published work, we showed that knockdown of Sep15 gene increased human lens epithelial cells’ susceptibility to Tm‐induced stress, and the protective role of Sep15 in human lens epithelial cells was operated via inhibiting oxidative stress. It suggests an interesting perspective to the relevance of Sep15 and cataract formation, i.e. regulating oxidative stress. To follow up, we investigated the effect of Sep15 gene knockdown on differentiation of human lens epithelial cells, since the differentiation of lens epithelial cells to fiber cells is critical to lens transparency. The results showed that Tm‐induced oxidative stress suppressed the expression of β‐crystallin as a lens fiber structural protein, and reduction of Sep15 expression exhibited the similar effect. These results suggested that Sep15 might exert roles on lens fiber cell differentiation by regulating oxidative stress.Support or Funding InformationThis work was supported by grants from the National Natural Science Foundation of China (Project No. 21271077).
Pentachlorophenol and Dichlorodiphenyltrichloroethane Alter Secretion of Interleukin 1 &[Beta]eta (IL‐1β) from Human Immune Cells
Authors: Martin Tamara J , Whalen Margaret 
Published: November 2023

Pentachlorophenol (PCP) and Dichlorodiphenyltrichloroethane (DDT) are environmental contaminants found in human blood. PCP has been used in fungicides, insecticides, herbicides, and antifouling paint. 4, 4′‐Dichlorodiphenyltrichloroethane (4, 4′‐DDT), has been banned in the U.S. however, it is still used in other countries as a pesticide to kill insects. A number of cancers are associated with exposures to these contaminants. Previous studies in our lab show that PCP and DDT inhibit the lytic function of highly enriched human natural killer (NK) lymphocytes and decrease the expression of several surface proteins on NK cells. Interleukin 1‐βeta (IL‐1β) is a cytokine produced by lymphocytes and monocytes. It is involved in stimulating cell proliferation, tissue repair, and present during the inflammatory response to injury. An increase or decrease in the level of Il‐1β may lead to immune dysfunction and increased potential for tumor development and invasiveness. The objective of this study was to examine if exposure of human immune cells to different concentrations of PCP and DDT alters the secretion of IL‐1β. Immune cells included Peripheral Blood mononuclear cells (PBMCS), monocyte‐depleted (MD) PBMCs, and enriched NK cells. Cells were exposed to concentrations of PCP ranging from 5 μM to 0.05 μM and DDT concentrations of 2.5 μM to 0.025 μM for 24 h, 48 h and 6 days. Secreted IL1‐β levels were measured using ELISA (enzyme‐linked immunosorbent assay). Results showed that PCP and DDT induced alterations in IL‐1β secretion from each of the human immune cell preparations. NK cells, MD‐PBMCs and PBMCs all showed increased secretion of IL‐1β after at least one length of exposure to at least one concentration of PCP. The concentrations and lengths of exposure at which these increases occurred varied among the donors. With DDT, all donors showed an increase in IL‐1β secretion after exposure to at least one concentration of DDT in all types of cell preparations. These results suggest that PCP or DDT‐induced alterations in IL‐1β secretion may have the potential to cause immune system dysregulation.Support or Funding InformationSupported by NIH grant 5U54CA163066
Tributyltin Alters Secretion of Interleukin 6 (IL‐6) From Human Immune Cells
Authors: Wilburn Wendy JoAnne , Brown Shyretha , Whalen Margaret 
Published: November 2023

Tributyltin (TBT) is a widespread environmental contaminant due its use as a biocide (including as an antifungal agent). TBT has been found in human blood samples and has been shown to increase tumors in mammals. IL‐6 is both an anti‐inflammatory and pro‐inflammatory protein that is produced by T lymphocytes (as well as other cells). It regulates cell growth, tissue repair, and immune functions. Previous studies have shown that TBT alters the secretion of cytokines IL‐1β, TNFα, and INFγ from human immune cells. Due to the important role IL‐6 plays in immune responsiveness, it is important to understand whether exposures to TBT are able to disrupt its secretion. Natural killer (NK) cells, monocyte‐depleted (MD) human peripheral blood mononuclear cells (PBMCs), PBMCs, PBMC+granulocytes, and granulotcytes were exposed to TBT at concentrations of 0–200 nM and monitored for secretion of IL‐6 at 24 h, 48 h, and 6 days using enzyme‐linked immunosorbent assay (ELISA). After 24 h exposures to TBT at concentrations of 100 and 200 nM, NK cells, MD‐PBMCs, and PBMCs showed significant decreases in secretion of IL‐6 while PBMCs‐granulocytes exposed to 100 nM TBT showed increased secretion of IL‐6 compared to control. Additionally, PBMCs exposed to 2.5 nM TBT showed increased in IL‐6 secretion compared to controls. These results indicate that TBT is able to disrupt IL‐6 secretion from immune cell preparations and the nature of those disruptions are dependent on the type of immune cell that is being tested as well as the concentration of TBT.Support or Funding InformationSupported by NIH grant 5U54CA163066.
Structural analysis of PTM hotspots reveals large body of putative regulatory elements with predicted biological function in eukaryotic protein families
Authors: Torres Matthew , Dewhurst Henry , Choudhury Shilpa 
Published: November 2023

Post‐translational modifications (PTMs) regulate protein behavior through modulation of protein‐protein binding and recognition site interactions, allosteric regulation, and conformational switching. In order to explore the structure/function relationships of large‐scale PTM data, we developed SAPH‐ire (Structural Analysis of PTM Hotspots), which has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we proceed by applying SAPH‐ire to the comprehensive set of eukaryotic protein families containing experimental PTM and 3D structure data – capturing 1,864 protein families with 60,756 unique PTM sites organized into 40,493 PTM hotspots, of which 2,659 possess known biological function (data obtained from PhosphoSitePlus), analyzed across 51,000 crystalized molecular structures. PTM hotspots with a known biological function undergo up to 6‐fold enrichment using SAPH‐ire ranking methods. Moreover, we find that a high percentage of top‐ranked hotspots lacking functional evidence in our input data could be linked to biological function by in‐depth literature searches. In contrast, ~10% of PTM hotspots predicted to be biologically impactful have yet to be verified experimentally ‐ representing putative regulatory elements. We provide experimental evidence for one such element in which we discovered a previously un‐described feedback phosphorylation mechanism in GPCR signal transduction systems. Taken together, these results highlight the utility of SAPH‐ire as a predictive tool for the identification of biologically important PTMs and provide a model through which the structural topology of PTMs may be explored.Support or Funding InformationThis work was funded by the Georgia Tech School of Biology (M.T.) as well as the National Institutes of Health grant 4R00 GM094533‐03 (to M.T.).