bluuebunny/crossref_metadata_2025 huggingface dataset:
Reverse mode sodium‐calcium exchange (NCXrev) facilitated by co‐localization of the exchanger with TRPC channels contributes to Ca2+ influx and ER Ca2+ store refilling during agonist stimulation of endothelial cells
Authors: Liao Chiu‐Hsiang , van Breemen Cornelis , Braun Andrew P. , Cole William C.
Published: June 2021

The role of NCX as a mechanism for Ca2+ entry is receiving increased attention. NCX and TRPC channels were shown to co‐localize, facilitating NCXrev mediated Ca2+ influx in response to TRPC activation in over‐expression studies (Rosker C et al., JBC 279:13696 (2004)). Here, we employed patch clamp technique, Ca2+ fluorimetry and co‐immunoprecipitation to determine if: 1) NCXrev contributes to Ca2+ influx in response to agonist stimulation, and 2) TRPC and NCX are co‐localized in cultured endothelial cells (EA.Hy926). Stimulation with histamine increased steady‐state inward and outward current between −70 and +70 mV in K+‐free solution; KB‐R7943 inhibition of NCX selectively suppressed outward current, but SKF96365 inhibition of TRPC reduced both inward and outward currents. Histamine caused an increase in Fura‐2 cytosolic Ca2+ fluorescence and a reduction in ER Ca2+ D1ER fluorescence. SKF96365 and KB‐R7943 suppressed the increase in cytosolic [Ca2+] and enhanced the depletion of ER Ca2+ stores induced by histamine. Significantly, TRPC1, TRPC4 and caveolin‐1 immunoreactive proteins were detected in anti‐NCX1 immunoprecipitates of endothelial cell lysates. These findings suggest that co‐localization of NCX and TRPC channels in a signaling complex in endothelial cells may facilitate Ca2+ influx and ER Ca2+ store refilling via NCXrev during agonist stimulation. (funded by CIHR MOP‐13505, NSERC and AHFMR).
LH increases insl3 mRNA abundance in MA‐10 Leydig cells
Authors: Strong Mary Elizabeth , Burd Matthew Anderson , Peterson Daniel Gunnar
Published: June 2021

Leydig cells produce testosterone in response to luteinizing hormone (LH) via the cyclic adenosine monophosphate (cAMP)/protein kinase A pathway. Additionally, these cells are responsible for producing insulin‐like peptide 3 (INSL3), a peptide hormone that is essential for testicular descent. The insl3 promoter in Leydig cells can be activated by cAMP through the transcription factor Nur77. While the mechanism of LH action on testosterone production is well characterized, the effect of LH on insl3 abundance has yet to be shown directly. MA‐10 Leydig cells were treated with either 0 and 100 ng/mL LH for 3 and 6 hours in a 2×2 factorial design or 0 and 0.6 mM cAMP for 6 hours. Relative insl3 mRNA abundance was determined by quantitative reverse transcription PCR. While cAMP treatment failed to alter abundance of insl3 mRNA, treatment with 100ng/mL LH for 3 hours induced a significant increase in insl3 mRNA abundance. These data indicate that LH does increase insl3 mRNA abundance in MA‐10 cells. Research funded by California Polytechnic State Faculty Support Grant #SF0101.
Polymorphisms of the lamina maturation pathway and their association with the metabolic syndrome: the DESIR Prospective Study
Authors: Fontaine‐Bisson Benedicte , Fumeron Frederic , Marre Michel , Dutour Anne , Badens Catherine , Levy Nicolas , Tichet Jean , Tregouet David‐Alexandre , Balkau Beverly , Morange Pierre‐Emmanuel
Published: June 2021

Laminopathies are rare monogenic diseases, some of them exhibiting features of the metabolic syndrome. These diseases are mainly due to mutations in LMNA, encoding A‐type lamins. One LMNA polymorphism, rs4641, has been associated with the metabolic syndrome, but results have been controversial. We therefore investigated the effect of single nucleotide polymorphisms (SNPs) in the LMNA gene in combination with four other genes encoding enzymes influencing lamin post‐translational maturation on risk of metabolic syndrome (MS). Twenty‐three tagging SNPs characterizing the haplotypic variability of 5 genes (LMNA, ICMT, ZMPSTE24, FNTA, FNTB) were genotyped in 3916 French men and women who took part in the prospective DESIR study. Single‐locus and haplotype analyses were performed but did not detect any significant association with the risk of MS. No robust interaction between SNPs located in different genes on the risk of MS were neither identified. In conclusion, we did not observe any convincing evidence that common polymorphisms of the lamina pathway could modulate the risk of MS. The DESIR study was supported by INSERM‐CNAMTS.

Grant Funding Source: INSERM
Rab 11 is associated with Nectin 2 positive vesicles in Sertoli cells at newly forming adhesion junctions with early elongating spermatids in rat testis
Authors: Young J'Nelle Sarah , Vogl Wayne
Published: June 2021

Tubulobulbar complexes are podosome‐like structures that internalize intercellular junctions in the seminiferous epithelium. Near the ends of the complexes, enlarged swollen areas develop and separate away from the complexes forming double membrane vesicles. Although these vesicles are degraded by Sertoli cells, it also is possible that some of the junction molecules in the vesicles are sorted and are recycled to junctions assembling at other locations in the cell. In this study, we double label perfusion‐fixed and mechanically fragmented seminiferous epithelium of rat testis for Nectin 2 and Rab 11. The rabbit anti‐Nectin 2 antibody that reacts with rat protein was provided by Dr. Yoshimi Takai, Kobe University, Japan. As expected on the basis of data from studies in mice, Nectin 2 is found at adhesion junctions associated with late spermatids, and in vesicles at the ends of tubulobulbar complexes. Although Rab 11 is not associated with any of the Nectin 2 positive vesicles at tubulobulbar complexes, Rab 11 is associated with Nectin 2 positive vesicles at newly forming adhesion junctions attached to early elongating spermatids. Our data support the conclusion that some of the Nectin 2 being incorporated into newly forming adhesion junctions is from recycling pathways, possibly from junctions being internalized by tubulobulbar complexes. Supported by a NSERC discovery grant to AWV.

Grant Funding Source: NSERC
Eps15, but not AP‐2, is recruited and essential for the formation of enteropathogenic E. coli pedestals
Authors: Lin Ann , Benmerah Alexandre , Guttman Julian
Published: June 2021

Enteropathogenic Escherichia coli (EPEC) are extracellular pathogens that cause serious infantile diarrhea. The hallmark of EPEC pathogenesis is its ability to colonize the surface of gut epithelial cells and inject effector proteins to hijack sub‐cellular components, forming characteristic actin‐rich pedestals beneath the attached bacteria. Efficient formation of pedestals requires the phosphorylation of the EPEC effector protein Tir at Tyrosine 474 (Y474). Clathrin, a key component in receptor‐mediated endocytosis, was previously found to participate in pedestal formation. We examined the clathrin‐associated endocytic proteins AP‐2 and its binding partner, Eps15. We found that Eps15 is recruited to the pedestal together with clathrin while AP‐2 is not. Further analysis using GFP‐Eps15 mutants revealed that such recruitment does not require the AP‐2 binding domain at the Eps15 C‐terminus, confirming previous results that pedestal forms efficiently in the absence of AP‐2. RNA interference showed that pedestal formation is inhibited in the absence of Eps15, suggesting Eps15 is required to generate pedestals. Finally we showed that Eps15 recruitment to pedestals required Tir Y474 phosphorylation. These results reveal a novel internalization subversion strategy employed by extracellular pathogens, supporting the recent paradigm that endocytic proteins are important for EPEC‐mediated disease.

Grant Funding Source: CIHR
An animal model for simulating deep tissue injury for pressure ulcer research on spinal cord injury
Authors: Makhsous Mohsen , Lin Fang , Cichowski Andrew , Pandya Atek , Modi Mauli
Published: June 2021

Current animal models examining deep tissue injury (DTI) development as a mechanism for pressure ulcer (PU) formation are limited, as the created animal wounds do not usually reflect clinically observed tissue necrosis. This study aimed to establish a more clinically relevant rat PU model by including a SCI condition in the model and by examining the role of a simulated bony prominence in DTI formation. Tissue injury percentage of compressed tibialis anterior (TA) muscle from eight SCI (T9) rats was compared against eight neurologically intact control rats to examine PU development over the flat surface of the tibia. To examine DTI formation, five other rats were implanted with a hemispheric polypropylene bone mimic beneath the TA muscle and were allowed to heal prior to compression. The rats in the DTI model were then separated into three groups and were administered different levels of compression to determine the best experimental conditions for the model. Both MRI and light microscopy were used to analyze tissue necrosis due to compression, and histological data was processed and quantified using a custom Matlab code. Postoperative ultrasound images of the implant two weeks after surgery showed the implant was correctly oriented on the surface of the bone. Results from this experiment and subsequent histological analysis and MRI observation confirmed these models are successful in simulating a clinically relevant pressure‐induced deep tissue injury.
Angiotensin II signalling regulates skeletal muscle growth and myoblast chemotaxis
Authors: Johnston Adam , Baker Jeff , Bellamy Leeann , De Lisio Michael , Parise Gianni
Published: June 2021

The regulatory role of angiotensin II (Ang II) in mediating skeletal muscle hypertrophy is largely unknown. We examined the impact of Ang II on myoblast chemotaxis using transwell assays. Exogenous treatment of Ang II significantly increased the migratory capacity of both C2C12 and primary myoblasts by 30 and 43% respectively, while Ang II receptor knockout myoblasts (AT1a−/−) demonstrated a significant attenuation of basal migration (38% of control) and did not respond to exogenous Ang II treatment. Ang II appeared to increase migration by inducing MMP2 activity as gelatin zymography analysis of cell culture media and lysates revealed a ~40% increase in gelatinase activity and pharmacological inhibition of MMP2 abolished the Ang II mediated increase in myoblast migration. Ang II also appeared to signal in a paracrine fashion to induce migration as 4h of cyclic mechanical stretch of myoblasts enhanced migration of co‐cultured myoblasts. To test the involvement of Ang II signaling during skeletal muscle regeneration in vivo, control and At1a−/− mice were subjected to cardiotoxin injury. In comparison to controls, at 14 and 21 days following injury, At1a−/− mice demonstrated 34 and 25% reductions in cross sectional area respectively. Taken together, these results demonstrate that Ang II modulates skeletal muscle fibre growth by regulating myoblast chemotaxis through MMP2.
Using QuickTime videos of cone beam computed tomography of the mandible in the instruction of first year dental students
Authors: Smith Kyle D , Lanphier Terry F , Jergenson Margaret A , Edwards Paul C , Saini Tarnjit S , Norton Neil S
Published: June 2021

The mandible is a complex bony structure within the head. It either contains or has specific anatomical landmarks for several important nerves and vessels which provide innervation and vascular supply to teeth, bone, muscles, and surrounding soft tissues. As dental clinicians, it is important to have a thorough understanding of the anatomical structure and landmarks of the mandible. There are many procedures including anesthesia, restoring defective teeth, extraction of impacted third molars and placement of implants where the dentist must have a thorough knowledge of the osseous structure of the mandible. During the freshman year at Creighton University, first year dental students spend one semester in head and neck anatomy. This course is taught using both a traditional lecture format and cadaver laboratory dissection. We sought to utilize cone beam computed technology to offer 3‐dimensional anatomy as an adjunct to their education. A QuickTime video of the normal anatomy of the mandible using cone beam computed tomography images was created using axial, sagittal, and coronal planes. The video was distributed to the students as part of the regular course content. At the end of the course, students were provided a brief questionnaire to evaluate the resourcefulness of the video as an adjunct to their education.

Grant Funding Source: Departmental
Development of a synchronous online microscopic anatomy course using virtual microscopy
Authors: Barbeau Michele , Rogers Kem
Published: June 2021

The use of virtual microscopy has become a well established and accepted tool for microscopic anatomy instruction. This tool has been tested for its educational effectiveness and it has shown to be as effective as traditional microscopes.

The popularity of distance education (DE) courses continues to increase as more students become comfortable with this method of teaching and as more courses are offered. Using the Wimba Collaboration Suite videoconferencing software, we are combining the technologies of virtual microscopy with synchronous videoconferencing to create an online microscopic anatomy course. Our institution has offered a traditional comprehensive microscopic anatomy course for decades. This course includes both lectures and traditional laboratories using microscopes. With online technologies, it is possible to offer the same course, including the laboratory exercises, as a DE course. Synchronous online courses allow many of the benefits of face to face instruction where the instructor can ask questions to check for understanding or the class can have a discussion. The combination of technologies allows this course to be offered to a much wider audience, including participants that are unable to attend campus.

Grant Funding Source: Not available
Low cost, high impact video system for use in a gross anatomy laboratory
Authors: Goldman Evan Michael
Published: June 2021

Results

A customized wireless video system was developed for the gross anatomy lab at Philadelphia University. The system was designed to be extremely simple to set up and use. Minimal interaction with podiums, computers, or switches is required. The total price was kept extremely low ($1224) yet the system is high‐resolution and allows for live recording on digital media. The video system displays high‐resolution live video feeds on ten lab monitors with a setup time of less than one minute. With the system, instructors are able to relay details and images of noteworthy dissection techniques, structures, and pathologies to the entire class.

Conclusions

Viewing the dissections of multiple cadavers prepares students to understand the natural variations of human anatomy. Each cadaver also provides a glimpse into the person's medical history providing an opportunity to see, in situ, a variety of pathological states. For a variety of reasons students will often fail to see structures or interesting dissections on other group's cadavers within a lab. With the current video system, the instant availability of live video eliminates the barriers that had precluded students from viewing specimens throughout the room. This novel system within the anatomy laboratory now allows the instructor to provide interesting and pedagogically relevant content to all students simultaneously.
Use of CT Imaging to Enhance Learning of Gross Anatomy in Medical Curriculum
Authors: Nyauncho Clive P , Barlow John M , Christensen Kevin , Scoville Elizabeth A , Hassinger James P , Kumar Neha , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

The increasing dependence on radiologic diagnostic modalities in clinical practice has necessitated innovative teaching methods that introduce radiology into early medical curriculum. At Mayo Medical School cadaveric CT images are integrated into the first year gross anatomy course. Using post‐preservation, non‐contrast full body CT scans of their cadavers, medical students are able to correlate radiologic images to their dissection specimen. The AquariusNET thin client software used to view the images provides students numerous options for image manipulation including standard and oblique sagittal and coronal images and three‐dimensional renderings. Students are requested to record interesting findings from the imaging studies as part of an autopsy report. They are also provided with daily radiology learning objectives that require them to visualize specific anatomic structures. At the end of the course, students were comfortable accessing the cadaver CT data, correlating gross and CT anatomy and appreciating relationships of anatomic structures. They enjoyed interactions with radiology faculty and were more prepared to evaluate radiologic examinations.

Grant Funding Source: Department of Anatomy
Exercises to encourage analytical skills and enrich understanding in a pre‐baccalaureate anatomy course
Authors: Miller Sue Ann
Published: June 2021

General perception is that study of anatomy is just memorization of parts. Anatomists know that there is more to understanding than memorization, but the format of many courses rewards memorization to the extent that it perpetuates the myth while perhaps not encouraging important and fundamental skills. Exercises that require thoughtful analysis of information and reward understanding can help. Teaching dissection as a means to learning and understanding is basic. Connecting dissection to “butcher shop anatomy” or “popular anatomical terms in sports, history, or literature” connects understanding anatomy to common uses and gives students additional reasons to know about the parts. Cuts of meat from a grocery store encourage interpretation of sectional anatomy. Oral presentation helps students learn to “speak anatomy” with confidence and credibility. Short reports about eponymous anatomy provide history of anatomical enquiry. Readings that precede a course or build on experience late in a course and that are tied to class discussion can help establish appreciation for how anatomists think (First Cut. A Season in the Human Anatomy Lab, A.H. Carter) and consideration of related ethical issues (Stiff. The Curious Lives of Human Cadavers, M. Roach). I will present these and other enrichment exercises that students find useful in an anatomy course at a liberal arts college.
Effectiveness of a web‐based cross‐sectional anatomy learning tool (3D‐X) at improving students’ ability to interpret CT images
Authors: Dorosh Kyle , Bhattacharyya Siddhartha , Haase Peter
Published: June 2021

Background

Residency programs are demanding an increased knowledge of cross‐sectional anatomy from entering physicians studying radiological imaging. In response to medical schools decreasing the time allotted to teaching anatomy and increasing self‐directed computer‐based learning, a web‐based learning tool (3D‐X) was developed to facilitate the study of cross‐sectional anatomy. The effectiveness of 3D‐X was assessed.

Methodology

Undergraduate anatomy students (n=82) were randomly assigned to two groups. Each group participated in a 50‐minute learning session prior to identifying anatomical structures in CT images of the abdomen. Group 1 (control) spent the learning session studying the structures of the abdomen from prosection in an anatomy lab. Group 2 spent the first half of the lab session studying from a prosection in the anatomy lab and the second half in the computer lab using 3D‐X.

Results

A two‐tailed independent t‐test revealed a significant difference in test scores (mean% ± SD) between group 1 (58.4 ± 2.50, n=41) and group 2 (72.1 ± 2.44, n=41). Qualitative analysis revealed that students found the tool useful when presented as an adjunct to traditional gross anatomy.

Conclusion

This study demonstrates that 3D‐X, when presented in addition to traditional gross anatomy, can improve students’ ability to identify anatomical structures in CT images, as well as improving anatomical knowledge of the abdomen.

Grant Funding Source: N/A
Mentoring Students from Undergrad Through Tenure
Authors: Blatch Sydella
Published: June 2021

We often think of mentoring as an informal way to help students we encounter. Mentoring efforts usually enhance the professional development of mentees. However, as concerns about the strength and diversity of the scientific workforce grow, mentoring could play a larger role in fostering all scientists. This could be when (1) mentoring is institutionalized to benefit more people, especially under‐represented persons and (2) when mentoring specifically targets factors contributing to attrition. This talk will illustrate this by describing an institutionalized mentoring program for undergraduate and graduate students in the sciences at a large research‐intensive university. The goal of the program is to improve the professional development and academic experiences of students with professional development and the creation of a community. Graduate students are paired with minority undergraduates based on shared academic and career interests. Students communicate one‐on‐one and all participants gather a few times a semester for professional development seminars designed for scientists. Potential programs with similar approaches for mentoring postdocs and junior faculty will also be discussed.
Retinoic Acid Enhances Natriuretic Peptide Receptor‐A gene Transcription: Role of Additive Effect of Retinoic Acid Receptor‐α and Ets‐1
Authors: Kumar Prerna , Bolden Gevoni , Pandey Kailash N
Published: June 2021

Activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) by cardiac hormones atrial and brain natriuretic peptides produces the second messenger cGMP, which activates downstream signaling and biological effects of NPRA including vasorelaxation, anti‐mitogenic, and anti‐hypertrophic effects. The objective of the present study was to gain insight into the signaling mechanism of all‐trans retinoic acid (ATRA) in the regulation of Npr1 gene (coding for GC‐A/NPRA) transcription in cultured mouse mesangial cells. Cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and ITS (insulin,transferrin, and sodium selenite) and were transiently transfected using Lipofectamine. The results showed that ATRA significantly increased Npr1 mRNA, NPRA protein expression, and intracellular accumulation of cGMP in a dose‐dependent manner. ATRA caused a 7‐fold increase in luciferase activity from the region −365 to +55 base pairs of the Npr1 promoter. ATRA‐dependent Npr1 gene transcription was effectively inhibited by Ro 41‐5253, a specific antagonist for RARα. ATRA enhanced in vivo binding of Ets‐1 to Npr1 promoter as confirmed by ChIP assay. Moreover, sequential ChIP assay showed simultaneous presence of RARα and Ets‐1 in the Npr1 promoter containing Ets‐1 binding sites. Collectively, our results show that retinoic acid induces Npr1 gene transcription and expression via RARα and Ets‐1 transcription factors. The findings of the present studies will have important implications in prevention of high blood pressure and cardiac remodeling.
Transcriptional regulation of the lysyl oxidase gene by the hypoxia‐inducible factor 1 (HIF1)
Authors: Li Wande , Zhao Yinzhi , Gao Song , Toselli Paul
Published: June 2021

To elucidate transcriptional regulation of lysyl oxidase (LO), a key enzyme for crosslinking collagen and elastin in the extracellular matrix, we cloned the 5'‐flanking region (3,979 nt) of the rat LO gene into the pGL3‐Basic vector upstream of the luciferase gene. 5'‐Deletion assays showed the maximal promoter activity obtained from the 804 base pair fragment upstream of ATG. There are at least 4 hypoxia‐response element (HRE) consensus sequences (ACGTG) existing in this LO promoter region. Treatment of RFL6 lung fibroblasts with cobalt (Co) chloride (100 μM) mimic the hypoxia conditions enhanced LO promoter activities. Dominant negative HIF 1α inhibited LO promoter activities stimulated by Co. Electrophoretic mobility shift, oligonucleotide competition and in vitro translation of HIF 1α assays indicated that only one HRE mapped at −387/−383 relative to the ATG was functionally active among 4 consensuses. Site‐directed mutation of this HRE significantly decreased the LO promoter‐directed expression of the reporter gene. Thus, HIF‐1 activation of the HRE plays a critical role in regulation of LO gene transcription (Supported by NIH grant R01‐ES11340 and Philip Morris ERP).
PC4 is a coactivator for basal and TSA‐induced Luteinizing Hormone Receptor (LHR) transcription
Authors: Liao Mingjuan , Zhang Ying , Dufau Maria
Published: June 2021

The LHR has an essential role in sexual development and reproductive function, and its transcription is subject to several modes of regulation and multiple effectors. In this study we investigated PC4 coactivator function in the control of LHR transcription. In MCF‐7 and JAR cells, PC4 knockdown with siRNA significantly reduced the basal LHR promoter activity. While over‐expression of PC4 alone had no effect on the LHR promoter activity, it synergistically enhanced Sp1‐ but not Sp3‐mediated LHR transcriptional activity and GST‐pull down showed that PC4 interacted with Sp1. Moreover, DNA affinity precipitation assay revealed the association of PC4 with Sp1/Sp3 binding sites of the LHR gene promoter. This association was markedly increased by dephosphorylation of PC4 with treatment of calf intestinal phosphatase, suggesting a role of phosphorylation in the modulation of PC4/Sp1 interaction. Furthermore, PC4 was found to be involved in the activation of LHR gene expression by TSA in MCF‐7 cells, since the induced LHR transcription/expression was substantially reduced by >90% after knockdown of PC4 protein. Chromatin immunoprecipitation assay further showed significant occupancy of PC4 in the LHR promoter. Taken together, these studies have demonstrated another layer of LHR modulation whereby PC4 acts as a coactivator of Sp1 to contribute to the basal and TSA‐induced derepression of LHR transcription.
Molecular mechanisms of a disease susceptibility variant of SIRT1: Genotoxic stress‐induced, CTCF‐dependent activation of SIRT1 gene expression
Authors: Song Xiaoyuan , Cho Miook , Han Jeehae , Hazuda Helen , Duggirala Ravindranath , Blangero John , Rosenfeld Michael G. , Suh Yousin
Published: June 2021

SIRT1 is an evolutionarily conserved protein deacetylase that modulates life span and stress resistance in model organisms. While increased expression of SIRT1 has been shown to provide protection from various diseases and aging effects in many invertebrate and vertebrate organ systems, surprisingly, the link between SIRT1 and human diseases and aging is not well understood. Here, we report identification of functional sequence variants in the human SIRT1 promoter that are associated with myocardial infarction. These variants have led to the identification of a pathway through which genotoxic stress results in demethylation of a DNA binding site for CCCTC‐binding factor (CTCF) in the promoter of the SIRT1 gene. We show that the wild‐type SIRT1 gene is a stress‐induced transcriptional target of CTCF, while a variant SIRT1 gene promoter is not bound by CTCF in response to genotoxic stress and hence SIRT1 gene expression does not increase with stress. We hypothesize that changes in DNA methylation of the wild‐type SIRT1 promoter may be due to recruitment of the Activation‐Induced cytidine Deaminase (AID) and concurrent dismissal of polycomb repressor complexes in response to genotoxic stress. This initial example of stress‐induced binding of CTCF suggests the possibility that regulated demethylation of CTCF response elements represents a more widely‐used strategy for activation of gene expression.
Role of STAT6 in regulation of Matrix Metalloproteinase‐3
Authors: Borghaei Ruth , Chambers Mariah , Gunsolly Chad , Gorski Grzegorz , Evans Michel
Published: June 2021

Previously we showed that IL‐4 inhibits the IL‐1 induction of MMP‐3 in human fibroblasts. Since STAT6 is a transcription factor prominently involved in IL‐4 transcriptional responses, experiments were designed to determine its role in IL‐4 inhibition of MMP‐3 expression. Chemical activation of STAT6 using chromeceptin caused a super‐induction of MMP‐3 mRNA in IL‐1 stimulated fibroblasts, but IL‐4 was still able to suppress MMP‐3 expression in its presence. Chromeceptin also increased MMP‐3 transcription in transfected COS‐1 cells. Chemical inhibition of STAT6 using pifithrin‐á caused a dose‐dependent inhibition of IL‐1 induced MMP‐3 expression, which was not due to inhibition of p53, since nutlin, an activator of p53, had no effect. Interestingly, overexpression of STAT6 in transiently transfected COS‐1 cells was able to increase transcription from the MMP‐3 promoter, but only when it contained the 6T version of the 5T/6T polymorphism at −1610. Experiments are currently underway to determine whether the effects of STAT6 on MMP‐3 expression are mediated through interaction with NF‐êB binding to the polymorphic SIRE site. This work is supported by grant R15DE16277 from the NIDCR to R.C. Borghaei.
Tungsten Carbide‐cobalt Nanoparticles Induce Reactive Oxygen Species, AKT, ERK, AP‐1, NF‐κB, VEGF, and Angiogenesis
Authors: Ding Min , Zhao Jinshun , Bowman Linda , Castranova Vince , Jiang Bing‐Hua
Published: June 2021

Powder mixtures of tungsten carbide and metallic cobalt (WC‐Co) are widely used in various products. Nanoparticles are engineered structures with at least one dimension of 100 nanometers or smaller. WC‐Co is known to be associated with lung injury and diseases. However, the cellular effects of WC‐Co remain to be elucidated. In this study, we showed that WC‐Co nanoparticles at 5 ug/cm2 induced reactive oxygen species (ROS) production which activated AKT and ERK signal pathways in a lung epithelial cell line. ROS played a key role in WC‐Co induced transcriptional activation of AP‐1, NF‐kB,VEGF, AKT and ERK1/2; while the fine size of WC‐Co particles at the same concentration did not have much effect. However, at higher concentrations, fine‐size WC‐Co particles also induced ROS production, AKT and ERK1/2 activation. WC‐Co nanoparticles also affected the cells to induce angiogenesis. These results identify multiple signaling molecules that are induced by WC‐Co nanoparticle treatment, and elucidate the potential molecular mechanism of their regulation in the cells. This information may be useful for preventing potential damage from WC‐Co particle exposure in the future.
Roles of the yeast Ccr4‐Not complex in regulating transcription
Authors: Dutta Arnob , Kruk Jennifer A , Gilmour David S , Reese Joseph C
Published: June 2021

The highly conserved Ccr4‐Not complex has been ascribed many functions, from transcription regulation to mRNA decay. Initial studies described a role of this complex in preinitiation complex formation, but a number of studies have clearly shown Ccr4‐Not mediates deadenylation of mRNAs and protein ubiquitylation in the cytoplasm. It is still not clear how Ccr4‐Not regulates gene expression, and which of these functions are controlled directly by this complex. Our study demonstrates that Ccr4‐Not complex directly regulates PolII‐dependent transcription elongation. Using purified Ccr4‐Not complex and yeast PolII in an in vitro transcription system we have studied how this complex affects transcription elongation. Our studies show the complex binds directly to the elongation complex, which is partially dependent on the emerging transcript, and crosslinking studies show it also makes contacts with the transcript. Using transcription run on assays, we demonstrate that Ccr4‐Not affects the rate of PolII transcription and stimulates elongation through transcription blocks. Our in vitro results are substantiated by in vivo studies, showing that Ccr4‐Not co‐purifies with PolII and associates with the open reading frames of transcribing genes. Together these studies support a model by which the Ccr4‐Not complex directly associates with transcribing PolII and regulates elongation. Funded by NIH (GM58672).
The effects of flow on the activation of bovine prothrombin by prothrombinase at physiologically relevant shear rates
Authors: Haynes Laura M. , Dubief Yves , Orfeo Thomas , Mann Kenneth G.
Published: June 2021

The generation of thrombin by preassembled prothrombinase on phospholipid coated capillaries was studied under laminar flow at physiologic shear rates (100–1000 sec−1). When prothrombin (1.4μM) was perfused, thrombin levels reached a steady‐state that decreased with increasing shear rate; however, generation was independent of shear rate when corrected for the velocity of the effluent. The ratio of α‐thrombin to meizothrombin formed was 3:2 at shear rates of 250 and 500 sec−1. Kinetic constants determined at a shear rate of 250 sec−1 were in agreement with those obtained in closed systems, suggesting that the exchange between the bulk solution and the capillary wall region is limited by the competition between molecular diffusion of thrombin and flow convection. This results in the development of a diffusive boundary layer that spatially confines thrombin, yielding predicted concentrations of up to 1μM. The observation of extensive thrombin feedback cleavage of the phospholipid binding domain from prothrombin and meizothrombin is consistent with such high concentrations of thrombin. A flow transport model is presented for thrombin generation that estimates the development of the thrombin layer. Supported by NIH HL46703 and 5T32HL007594.
Activity Studies of Epulopiscium Type B 1,3‐β‐glucanase
Authors: Lajeunesse Karah Lynn , Piefer Andrew John
Published: June 2021

The activity of recombinantly expressed and purified Epulopiscium 1,3‐β‐glucanase was characterized in terms of optimal temperature, pH, and β‐glucan substrate specificity. Epulopiscium is a bacterial symbiont of surgeonfish that may express and secrete a putative endo 1,3‐β‐glucanase to convert β‐glucans in seaweed to simple carbohydrates, providing an energy source for both the bacteria and host. To study enzyme function, the active site of the gene encoding 1,3‐β‐glucanase was cloned into a pET24b vector, expressed in E. coli, and purified using Ni‐NTA chromatography. Purified enzyme was incubated with buffer and carbohydrate. Enzyme reactions were stopped by the addition of DNSA reagent to quantify liberated reducing sugars. The results for average specific activity at 37oC, 45oC, 60oC, and 75oC were 90 uM/min/mg, 110 uM/min/mg, 91 uM/min/mg, and 29 uM/min/mg respectively. Interestingly, these results suggest that the enzyme has optimal activity at 45oC, above the physiological temperature of the host. As expected, the enzyme has specificity for 1,3‐β‐glucans, as exhibited by the degradation of laminarin. Substrate specificity will be examined further by employing β‐glucan substrates with mixed linkages, and the optimal pH for activity will be found by varying the reaction pH. This research is supported through funding from a Faculty Research Grant and the Hartwick College Department of Chemistry.
Measuring Readily Exchangeable Zinc in Escherichia coli by Carbonic Anhydrase‐based Expressible Ratiometric Zinc Sensor
Authors: Wang Da , Hurst Tamiika K. , Fierke Carol A. , Thompson Richard B.
Published: June 2021

Zinc is essential in numerous cellular processes and its homeostasis is highly regulated in living organisms. Monitoring readily exchangeable zinc concentration in the cells under various conditions will provide insight into the regulation of zinc homeostasis. Here we engineered a series of genetically encoded fluorescence resonance energy transfer (FRET)‐based excitation ratiometric zinc sensors based on carbonic anhydrase (CA) that exhibit large signal increases in response to zinc. A fluorescent inhibitor of carbonic anhydrase, Dapoxyl sulfonamide (DPS), binds selectively to the active site zinc of CA, undergoing >100 fold increase in quantum yield. A red fluorescent protein (RFP) fused to the C‐terminus of CA functions as the FRET acceptor to zinc‐dependent DPS emission. The FRET signal normalized by the fluorescence from RFP is then calibrated in a series of chelated zinc buffers. These CA_RFP sensors were expressed in the E. coli strain BL21(DE3) in minimal medium and the readily exchangeable zinc concentration was measured at 30 ± 10 pM. The zinc concentration is not dependent on the sensor concentration or the growth stage but varies significantly (from 5 pM to > 300 pM) with changes in the zinc content in the medium. Furthermore, the cells’ ability to maintain low intracellular free zinc concentration is impaired during sugar starvation. This work was supported by National Institutes of Health (NIH) grant NIH/NIBIB RO1 EB003924.
Localization and Purification of Phagosome‐Associated Proteases in Tetrahymena thermophila
Authors: Kaur Tripta , Carreon Jay R. , Straus J. William
Published: June 2021

Phagosome maturation involves sequential introduction of oxidases, hydrolases and proteases that degrade encapsulated materials. In the ciliate Tetrahymena thermophila, we found that protease activity is associated with small vesicle‐like structures at the phagosome surface and occasionally in the lumen. Our current focus is to localize and purify phagosome associated cysteine proteases from T. thermophila using fluorescent and affinity‐tagged conjugates of an irreversible inhibitor DCG‐04 (lys(Boc)‐6‐aminohexanoic acid‐tyr‐leu‐ethyl (2S, 3S)‐oxirane‐2, 3‐dicarboxylate after Greenbaum et al, 2000). Two irreversible active‐site directed fluorescent cysteine protease inhibitors (6 FAM SE DCG‐04 and BODIPY 588/616‐DCG‐04) were synthesized using a combination of solution phase and standard F‐moc solid phase peptide chemistry. The products were confirmed by MALDI‐TOF mass spectrometry and purified by reverse phase HPLC. The binding of 6 FAM SE DCG‐04 to formaldehyde fixed, detergent permeabilized cells was assessed by fluorescence microscopy in the presence and absence of a 100‐fold excess of E‐64, a competing irreversible cysteine protease inhibitor. There was extensive punctate binding of the fluorescent inhibitor only in the absence of E‐64. 6 FAM SE DCG‐04 was taken up by live cells and following fixation, the fluor was observed to co‐localize with a fluorogenic protease substrate. Biotinylated DCG‐04 was used to isolate inhibited cysteine proteases by affinity chromatography. One‐dimensional electrophoresis revealed at least 21 distinct proteins ranging in size from about 18 ‐ 200 kDa. Future efforts will focus on proteomic analysis of these isolated proteins.
Signal transduction on the ribosome:timing EF‐Tu release from aminoacyl‐tRNA
Authors: Liu Wei , Poruri Kiran , Mandecki Wlodeck , Goldman Yale E. , Cooperman Barry S.
Published: June 2021

The QSY9 derivative of E348C‐EF‐Tu (EF‐TuQ348) is fully functional and is an efficient quencher of Cy3 fluorescence within the ternary complex made with Phe‐tRNAPhe(Cy3). Incubation of this ternary complex with a ribosomal initiation complex leads to a large increase in Cy3 fluorescence as EF‐TuQ348is released from the ribosome and Phe‐tRNAPhe(Cy3) is accommodated within the ribosomal A‐site. The fluorescence increase depends upon prior EF‐Tu‐dependent GTP hydrolysis, and occurs at a rate that is very similar to that of dipeptide formation, which rapidly follows the accommodation step. We conclude that disruption of the tight binding between EF‐Tu and aminoacyl‐tRNA, which follows the GTPase activation step that is a consequence of cognate codonanticodon recognition at the decoding center, is rate‐limiting for both accommodation and dipeptide formation. Experiments are underway to further examine the movement of tRNA within the ribosome immediately following EF‐Tu release, and to compare the kinetics of EF‐Tu release with cognate vs. near‐cognate codon recognition. Supported by NIH grants GM‐071014 (to BSC) and HG‐004364 (to WM).
Regulation of cyclin E stability in Xenopus embryos
Authors: Brandt Yekaterina , Mitchell Therese , Wu Yuehan , Hartley Rebecca
Published: June 2021

Cyclin E associates with Cdk2 to mediate the G1/S cell cycle transition and is overexpressed in many cancers. We are studying how cyclin E is downregulated in Xenopus laevis embryos to ask if similar mechanisms are disrupted during tumorigenesis. In Xenopus embryos, cyclin E level is constant until its abrupt destabilization by an undefined mechanism after the12th cell cycle, corresponding to midblastula transition (MBT). Degradation of mammalian cyclin E is regulated by the ubiquitin proteasome system and is phosphorylation dependent. To examine the role of phosphorylation in cyclin E turnover in Xenopus, we generated a series of threonine/serine mutations. Western blot analysis showed that mutation of threonine 394 to alanine (T394A) and both T394A/S398A increased cyclin E stability. T75A mutation had only a minor effect. Immunofluorescence analysis showed that cyclin E re‐localized from the cytoplasm to the nucleus preceding degradation. When cyclin E nuclear import was inhibited, its stability was markedly increased after MBT. We conclude from these studies that cyclin E destruction is facilitated by phosphorylation and requires nuclear import. Treatment of embryos with a proteasome inhibitor caused progressive cytoplasmic accumulation of cyclin E after MBT, suggesting that it may be exported back to the cytoplasm for destruction by the proteasome. This work was supported by NIH grant R01CA095898 to RSH.

Grant Funding Source: NIH
THE IMMUNOPROTEASOME, THE 20S PROTEASOME, AND THE PA28αβ PROTEASOME REGULATOR ARE OXIDATIVE STRESS‐ADAPTIVE PROTEOLYTIC COMPLEXES
Authors: Pickering Andrew Michael , Koop Alison L , Pimental Cheryl Teoh , Ermak Gennady , Grune Tilman , Davies Kelvin J A
Published: June 2021

We demonstrate the importance of various forms of the proteasome in adaptation to the oxidative stress of hydrogen peroxide (H2O2). Following an initial direct proteasome activation, 20S proteasome, immunoproteasome, and Pa28αβ regulator, all exhibited de novo synthesis during a 24hour period in murine embryonic fibroblasts. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome, and Pa28αβ synthesis. Additionally, a Pa28αβγ knockout mutant achieved only half the H2O2 induced adaptive increase in proteolytic capacity of wild‐type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated, for the first time, that immunoproteasome can selectively degrade oxidized proteins; in fact, immunoproteasome was more selective for oxidized proteins than was 20S proteasome. Importantly, siRNA knock‐down of 20S proteasome, immunoproteasome, or Pa28αβ regulator blocked 50–100% of the increase in cell division and cell survival, when H2O2 adapted cells were exposed to a, normally lethal, challenge concentration of H2O2. Our results reveal the previously unknown ability of immunoproteasome to degrade oxidized proteins, and the ability of Pa28αβ to enhance such degradation. Our results also demonstrate the importance of 20S proteasome, immunoproteasome, and Pa28αβ in overall adaptive responses to oxidative stress.
Stimulation of adrenergic receptors regulates proteasome function in cardiomyocytes
Authors: Ranek Mark John , Wang Xuejun
Published: June 2021

Ubiquitin‐proteasome system (UPS)‐mediated proteolysis plays a central role in protein quality control and degradation in the cell. UPS dysfunction was observed in various forms of heart disease and might play an important role in the development of congestive heart failure (CHF). However, very little is known about how UPS proteolytic function is regulated in the heart.

The sympathetic nervous system (SNS) activity is increased during CHF. As an initial step to investigate if and how the SNS regulates UPS function in the heart, we employed neonatal rat ventricular myocytes (NRVM) culture. To monitor UPS function, a validated UPS surrogate substrate (a modified Green Fluorescent Protein, GFPu) and a red fluorescent protein (RFP, not a specific substrate of the UPS) were co‐expressed in cultured NRVMs through adenoviral gene delivery

We found that both stimulation of the beta 1 receptor with dobutamine and stimulation of the alpha 1 receptor with phenylephrine significantly reduced the GFPu/RFP ratio, suggesting an enhancement of UPS function. We have also discovered that the phosphorylation status of the Rpt6 subunit of the 19S proteasome and the beta 5 subunit of the 20S proteasome was markedly altered by stimulating each of the receptors. These preliminary findings suggest that SNS may play an important role in regulating cardiac proteasomes. Funding: NIH HL085629, HL072166, and HL068936.
TEMPERATURE‐SENSITIVE, POST‐TRANSLATIONAL REGULATION OF PLANT OMEGA‐3 FATTY ACID DESATURASES IS MEDIATED BY THE ER‐ASSOCIATED DEGRADATION PATHWAY
Authors: Zhang Daiyuan , O'Quin Jami , Bourassa Linda , Foss Spencer , Shocky Jay , Gidda Satinder , Chapman Kent , Mullen Robert , Dyer John
Published: June 2021

In plants, the endoplasmic reticulum (ER)‐localized omega‐3 fatty acid desaturases (Fad3s) increase the production of polyunsaturated fatty acids at cooler temperatures, but the FAD3 genes themselves are typically not upregulated during this adaptive response. Here, we expressed two closely related plant FAD3 genes in yeast cells and found that their enzymes produced significantly different amounts of omega‐3 fatty acids. Moreover, protein amounts were determined primarily by differences in rates of protein turnover, rather than mRNA levels, indicating that the Fad3 proteins are differentially regulated at the post‐translational level. Domain‐swapping and mutagenesis experiments revealed that each Fad3 protein contained a degradation signal in their N‐terminal region and that the charge density of a PEST‐like sequence within this region was largely responsible for the differences in their protein half‐lives. The half‐lives of both Fad3 proteins were increased at cooler temperatures and their protein degradation required Cdc48 adaptor proteins, Doa1 and Shp1, as well as proteasomal activity. Collectively, these findings indicate that the steady‐state amount of Fad3 proteins (and thus Fad3 activity) is modulated in response to temperature through a combination of cis‐acting degradation signals and specific components of the ER‐associated degradation pathway.
FATP4 contributes as an enzyme to the insulin mediated fatty acid uptake of C2C12 muscle cells
Authors: Digel Margarete , Staffer Simone , Stremmel Wolfgang , Ehehalt Robert , Fuellekrug Joachim
Published: June 2021

The function of membrane proteins in long chain fatty acid transport is controversial. The acyl‐CoA synthetase FATP4 has been suggested to facilitate fatty acid uptake by its enzymatic activity or by direct transport across the plasma membrane. Here, we investigated the role of FATP4 in C2C12 muscle cells, a model system relevant for fatty acid metabolism. Stable expression of exogenous FATP4 resulted in a twofold higher fatty acyl‐CoA synthetase activity, and uptake of radiolabeled oleate was enhanced similarly. We report for the first time that insulin increases the acyl‐CoA synthetase activity within minutes. This short term regulation offers a novel mechanistic explanation for the concomitantly observed enhanced fatty acid uptake. Confocal microscopy showed that FATP4 was localized in an intracellular ER‐like pattern clearly distinct from the plasma membrane. Neither differentiation into myotubes nor insulin treatment changed the intracellular localization of FATP4. We conclude that FATP4 functions by its intrinsic enzymatic activity. This is in line with the concept that intracellular metabolism plays a significant role in the uptake of fatty acids.