bluuebunny/crossref_metadata_2025 huggingface dataset:
Effector function and efficacy of CD8+ T cells activated in the absence of IFN‐γ
Authors: Nelson Michelle H. , Chu Chin‐Fun , Milligan Gregg N.
Published: June 2021

We examined the influence of early IFN‐γ exposure on CD8+ T cell effector function. Immunization of C57BL/6 mice with a thymidine kinase deficient herpes simplex virus type 2 (HSV‐2 TK‐) elicited CD8+ T cells secreting mainly IFN‐γ and TNF‐α (Tc1), whereas CD8+ T cells from immunized IFN‐γ−/−mice secreted mainly TNF‐α with a lesser population secreting type 2 cytokines (Tc2). To facilitate studies of the effector function of these cells we cultured OT‐I CD8+ T cells in conditions that generated Tc1 or Tc2. Like CD8+ T cells from IFN‐γ−/−mice, Tc2 secreted type 2 cytokines, IL‐4, IL‐5 and IL‐10 with a distinct population producing IFN‐γ and TNF‐α. Tc1 produced both IFN‐γ and TNF‐α, like B6 CD8+ T cells. Tc1 and Tc2 expressed similar surface activation and differentiation markers. Tc2 exhibited delayed proliferation and reduced CTL activity in vivo, when compared to Tc1. These differences correlated with a delay in clearance of an OVA expressing HSV‐2 strain by Tc2 recipients, relative to Tc1 recipients. Despite the ability to secrete regulatory cytokines, such as IL‐10, co‐transfer of Tc2 with Tc1 did not interfere with the Tc1‐mediated virus clearance. Together these results suggest that early exposure to IFN‐γnot only imprints type 1 cytokine secretion, but also affects proliferation and cytolysis. Supported by NIH AI42815 and AI054444. MHN supported by a Sealy Center for Vaccine Development Predoctoral Fellowship.
Chemical Sympathectomy Increases Sensitivity to Ocular HSV‐1 Infection
Authors: Templeton Amanda , Carr Daniel
Published: June 2021

We addressed the importance of sympathetic/parasympathetic innervation via chemical sympathectomy using the compound 6‐hydroxydopamine (6OHDA) in mice ocularly infected with herpes simplex virus type 1 (HSV‐1). The treatment of mice with 6OHDA (80 mg/kg, days ‐2, ‐1, and at the time of infection) was found to effectively eliminate or significantly reduce tyrosine‐hydroxylase‐staining nerves innervating the cornea. However, 6OHDA treatment had no impact on virus replication in the cornea or iris of mice at any time point evaluated post infection (pi). In contrast to the results found in the eye, 6OHDA treatment resulted in a time‐dependent increase in virus recovered in the trigeminal ganglia (TG) and brain stem (BS) of mice which was associated with increased mortality in comparison to vehicle‐treated controls. Ironically, there were no changes in leukocyte (myeloid and lymphoid) recruitment to the cornea, TG, BS, or draining lymph nodes (mandibular and deep cervical) of 6OHDA‐treated mice in comparison to controls during acute infection (day 3‐day 7 pi). Moreover, there was no change in the development or activity of CTLs in the draining lymph nodes or infected tissue of 6OHDA‐treated mice. Currently, measurement of cytokine levels in the nervous system is underway to determine if levels offer an explanation for the increased sensitivity to infection in the 6OHDA‐treated mice.
Liver NK cells play an inhibitory role in impairing antiviral CD8+ T cell effector function
Authors: Lukens John R. , Dolina Joseph , Hahn Young S.
Published: June 2021

NK cells play a pivotal role in innate immunity to viral infection and linking to adaptive immunity. The liver is a tolerogenic site and often succumbs to persistent viral infection. To determine whether NK cells are responsible for influencing CD8+ T cell responses in the liver, we examined intrahepatic CD8+ T cell responses in mice following infection with recombinant adenovirus expressing LacZ antigen (Ad‐LacZ). Two different routes of Ad‐LacZ infection were employed which allowed for delivery of antigen into the liver or secondary lymphoid organs preferentially. Notably, CD8+ T cells primed in the liver exhibit significantly impaired effector function with inhibition of IFN‐γ, TNF‐α production and failure of viral clearance. The impairment of CD8+ T cell effector function in the liver was associated with the increased level of PD‐1 expression. In contrast, the liver was able to sustain CD8+ T cell effector function when these CD8+ T cells were primed in secondary lymphoid organ. Surprisingly, the in vivo depletion of NK cells in Ad‐LacZ‐infected mice restored the impaired CD8+ T cell responses primed in the liver environment. These results suggest that liver NK cells are crucial for modulating CD8+ T cell effector activity in the liver.
T‐bet is required for survival and immunity to the intracellular pathogen Trypanosoma cruzi (T. cruzi)
Authors: Smeltz Ronald B , Lara Ana , Guo Siqi , Manque Patricio , Buck Gregory A
Published: June 2021

The T‐box transcription factor expressed in T cells (T‐bet) is a critical regulator of T cell effector functions, particularly interferon gamma (IFN‐γ) production. We investigated the role of T‐bet in immunity to T. cruzi, an intracellular pathogen that causes Chagas disease in humans, and which requires both CD4+ and CD8+ T cells for successful control of the parasite. T‐bet‐deficient mice (T‐bet KO) infected with T. cruzi exhibited elevated blood parasitemia, progressive weight loss, and significant mortality. Infection was accompanied by an increase in tissue parasitism, as well as liver inflammation in T‐bet KO mice. FACS analysis revealed that CD4+ and CD8+ T cell differentiation were altered in spleens of T‐bet KO mice. In addition, there was a significant delay in the kinetics and magnitude of CD8+ T cell expansion in the blood of T‐bet KO mice. Despite an increase in T cell‐derived IL‐4 production, T cells from infected T‐bet KO mice still produced significant quantities of IFN‐γ, and serum IFN‐γ levels were elevated in T‐bet KO mice. These data demonstrate that T‐bet is required for immunity to T. cruzi, and suggests that mechanisms other than a Th1 to Th2 switch may be responsible for the increased susceptibility of T‐bet KO mice to T. cruzi infection.
Farm Mechanization Early in Life is Associated with BMD and Bone Size Later in Life in a Farming Population
Authors: Arneson Lacie , Beare Tianna , Binkley Teresa , Specker Bonny
Published: June 2021

Increasing bone mineral density (BMD) during childhood through physical activity could possibly reduce osteoporosis later in life. Our hypothesis was that an active rural lifestyle, defined as low farm mechanization, during childhood could lead to increased BMD as opposed to a less active rural lifestyle defined as high mechanization. Level of farm mechanization prior to age 20 and bone data (distal radius by pQCT, and the spine and hip by DXA) on 330 individuals (173 men) aged 20–66 y were obtained. Bone differences among farm mechanization groups were compared stratifying by sex and controlling for covariates. Higher femoral neck bone area was observed among women with low compared to high mechanization and among women with moderate compared to high mechanization (both, p<0.05). These findings remained significant after controlling for covariates. Men from farms with low mechanization had greater cortical thickness than men from farms with moderate mechanization controlling for covariates (p=0.03). Men from farms with low mechanization also had greater hip T scores than men from moderate and high mechanization farms (0.59 ± 0.15, 0.12 ± 0.08 and 0.04 ± 0.20 respectively, p<0.01). A low farm mechanization level, or a “high” activity level, prior to 20 years of age is associated with a greater femoral neck bone area in women and greater cortical thickness and hip T scores in men in a rural farming population.
Soy protein diet suppresses COX‐2 induction by dextran sodium sulfate in CD‐1 mouse colon.
Authors: MacDonald Ruth S , Przybyszewski Joseph
Published: June 2021

Inflammatory bowel disease (IBD) affects nearly 1 million people per year and is associated with increased risk of colon cancer. Dextran sodium sulfate (DSS) is commonly used to induce mucosal tissue damage and an immune response in rodent colon that mimics IBD in humans. Cyclooxygenase‐2 (COX‐2) is typically associated with an inflammatory response and is an early marker of colon carcinogenesis. Activation of the wnt signaling pathway including release of β‐catenin, is associated with colon carcinogenesis. Dietary components, including soy proteins, may provide protection from inflammation induction. To define the role of dietary soy protein on DSS‐induced mucosal damage, female, CD‐1 mice were fed diets containing either 20% casein or soy protein. DSS (2%) was added to drinking water of half of the mice on each diet while the others received untreated water. Mice were euthanized 7 days later. Colon mucosa was collected into lysis buffer and frozen in liquid nitrogen. Extracts were processed for Western immunoblot using COX‐2 and β‐catenin antibodies. No change in body weight due to diet or DSS was observed. For mice fed the casein diet COX‐2 expression was increased 2.5 fold by DSS, whereas this effect was abrogated in mice fed soy protein. No differences in β‐catenin were observed. These results suggest soy protein may provide protection from inflammatory processes in colon leading to improvement in IBD.
Effect of dietary allyl isothiocyanate from Brassica vegetables on serum glutathione S‐transferase‐α concentration
Authors: Charron Craig S. , Clevidence Beverly A. , Milner John A. , Kramer Matthew , Baer David J. , Albaugh George , Davis Cindy D. , Ross Sharon A. , Seifried Harold , Kim Young S. , Emenaker Nancy , Novotny Janet A.
Published: June 2021

Allyl isothiocyanate (AITC) is a possible anticancer agent derived from foods including brown mustard and green cabbage. In a crossover study, 12 men and 34 women participated in 3 treatment phases, which consisted of an 11‐day basal diet supplemented as follows: 1) 11.4 mg AITC, 2) 100 g pureed cabbage + 30 g brown mustard (CM), 3) unsupplemented control. Subjects were selected by genotype for glutathione S‐transferase (GST) M1 and DNA repair enzyme gene XPD. Blood samples were collected on day 1 (baseline) and day 11, and outcome measures included COMET, 8‐oxo‐dG, F2‐isoprostanes, IL6, VEGF, GST activity, and GST‐α concentration. The mustard AITC content unexpectedly decreased over time. The response of log GST‐α concentration to treatment was proportional to baseline values, but the magnitude of the response was higher for subjects on the AITC treatment. Consumption of the CM treatment resulted in increased log GST‐α concentration independent of baseline value. Evaluation of other outcome measures and the effect of genotype on treatment response is underway. Funded by USDA/ARS and NIH/NCI.
A linkage disequilibrium block from the BRCA1 gene was associated with breast cancer in Korean population
Authors: Kim Kyoung‐Yeon , Lee Seung Ku , Kim Hyoun Geun , Kang HyunJun , Kim Jeong‐Geun , Oh Ah Reum , Pyun Jung A , Kim Min‐Ho , Kang Eun Joo , Won SeongSik , Kang Jason J. , Yang Song‐Ju , Noh Dong‐Young , Han Wonshik , Kwack KyuBum
Published: June 2021

Recently, the morbidity and mortality of breast cancer have markedly increased in Korea. Many studies were performed to find the genetic susceptibility genes that might be involved in oncogenesis and treatment of breast cancer in various ethnic populations. Among the susceptible genes, the BRCA1 and BRCA2 genes have been shown to be involved in hereditary breast cancer.

To identify whether the polymorphisms in these genes are associated with breast cancer, genetic association study was performed for tag single nucleotide polymorphisms (SNPs) located in the BRCA1 and BRCA2 genes with 346 breast cancer patients and 361 healthy controls in Korean ethnic group. We could not find any significant associations between SNPs in BRCA1 and BRCA2 genes and breast cancer. A single linkage disequilibrium (LD) block from the BRCA1 gene, however, was significantly associated with breast cancer. The haplotype (ht1) of this LD block was increased the risk of breast cancer (OR=0.584, p=0.039) and haplotype (ht2) revealed a protective effect (OR=1.272, p=0.027).

According to the premise of LD and previous reports, we hypothesized that any causative or protective variants contribute to or against breast cancer may exist in this LD block. In this presentation, possible causative variations in ht 1 and ht 2 will be discussed.
Copyright Issues: a University Lawyer's Perspective
Authors: Hood William
Published: June 2021

Technological changes have caused heightened awareness of Copyright laws on university campuses. Additionally, while the legal factors of the bedrock principle of “fair‐use” still apply, newer copyright law such as the Digital Millennium Copyright Act for web content and the TEACH Act for digitally transmitted education provide additional scenery – both opportunities and challenges – to the legal landscape. A university legal department must provide guidance to faculty for compliance with copyright laws, without significantly comprising the academic mission of the university. In that regard, the legal departments at the Medical University of South Carolina have taken steps recently to develop additional copyright policies; to enhance faculty awareness of the latitude provided in the laws; to enhance awareness of the limitations; and to develop practical solutions for some of the challenges encountered. Electronic and live resources to aid compliance with copyright restrictions have been developed and provided, and training presentations have been well received.
Copyright Issues: A Faculty Member's Perspective
Authors: Bacro Thierry R H
Published: June 2021

As universities across the US become increasingly concerned about copyright infringements, faculty who have traditionally drawn rather freely from existing resources are being asked to review how they use copyrighted materials in teaching. In order to improve copyright compliance, the Medical University of South Carolina has adopted a university‐wide policy guiding the faculty in terms of US Copyright Law and has promoted strategies to help with its implementation. The strategies included organizing several educational sessions by both copyright lawyers and faculty members to promote these changes. The lawyers’ presentations focused on the legal texts whereas the faculty presentations discussed short and long term strategies about how to practically implement these recommendations in daily teaching activities. An MUSC website was created to provide support for these endeavors. Specifics about MUSC copyright resources and strategies will be shared with the audience during this talk.
Exposure of cultured murine brain cells to both lithium and estrogen results in altered NR1 and GSK‐3 beta mRNA expression
Authors: Valdes James Jason , Weeks Ophelia Inez
Published: June 2021

Lithium, traditionally used to treat bipolar disorder, evidentially is not simply a psychotropic drug. The literature shows that lithium contributes to activation of CREB by inhibiting the negative regulator GSK‐3β, a kinase involved in multiple signaling pathways. CREB is a transcription factor that binds to DNA and, upon activation via phosphorylation, regulates gene expressions that are involved in learning, memory, and neuronal protection/survival. Using this information, we have inferred that, since lithium specifically inhibits GSK‐3β, an enzyme that weakens CREB's transcriptional properties, GSK‐3β mRNA expression should be altered when exposed to lithium. Additionally, the literature suggests that estrogens have effects on CREB similar to those of lithium, however, via promoting positive regulators of CREB that affect NMDA receptor‐mediated calcium influx and promote CREB activation. We became interested in determining the extent to which estrogen and lithium, together, affect mRNA of NR1, a critical subunit of NMDA receptors, and mRNA of GSK‐3β, in areas of the brain involved in learning and memory processing. We treated primary mixed brain cell cultures harvested separately from the hippocampus and cortical regions of C57BL6 mouse embryos (E18.5) with both lithium and estrogen, isolated total RNA from these cultures for RT‐PCR, and detected unique changes in NR1 and GSK‐3β mRNA expression. We anticipate that this study will contribute data to the literature useful in developing palliative therapies using lithium as a treatment for cognitive decline. Supported in part by NIH/NICHD, through the EARDA Program.
Cross‐talk between Angiotensin II and IGF‐1‐induced connexin 43 expression in human saphenous vein smooth muscle cells involving αvβ3 integrin clustering and activation of focal adhesion kinase
Authors: Aggarwal Himanshu , Jia Guanghong , Cheng Gang , Agrawal Devendra K.
Published: June 2021

Vascular restenosis following coronary artery bypass grafts is a major clinical complication due to intimal hyperplasia in venous conduits. However, the precise underlying mechanisms of intimal hyperplasia are still unclear. We have recently reported that increased expression of connexin43 (Cx43) is involved in the proliferation of vascular smooth muscle cells (VSMCs) in human saphenous vein (SV). In this study, we investigated the signaling transduction pathway involved in Cx43 expression and SV SMC proliferation. Angiotensin‐II (AT‐II, 100ng/ml) increased AT‐1R mRNA expression and IGF‐1 (100ng/ml) upregulated IGF‐1R mRNA expression in SV SMCs. Interestingly, AT‐1R expression was also increased by IGF‐1 treatment and IGF‐1R expression was increased by AT‐II treatment. Both AT‐II and IGF‐1 significantly induced the expression of αvβ3 integrin and downstream activation of FAK in SV SMCs. AT‐II and IGF‐1 also significantly induced the expression of Cx43 in SV SMCs. The effect of AT‐II and IGF‐1 on Cx43 expression was attenuated by AT‐1 receptor antagonist, Losartan, IGF‐1R blocking antibody and αvβ3 integrin antibody. These data demonstrate a cross‐talk between IGF‐1R and AT‐1 in AT‐II and IGF‐1‐induced Cx43 expression in SV SMCs involving αvβ3 integrin clustering and downstream activation of FAK signaling pathway.

(Supported by a grant from the NIH R01HL07349)
Differential Bone Marrow Stem Cell Mobilization by G‐CSF Injection or Arterial Ligation in Baboons
Authors: Shi Qiang , Wang Xingli , VandeBerg John L.
Published: June 2021

In the current study, we investigated whether G‐CSF‐mobilized BMSCs share the same subtype profiles as the ischemia‐induced repair in nonhuman primate baboons.

Methods and Results: We subjected 5 baboons to femoral artery ligation and 5 baboons to subcutaneous G‐CSF injection. Blood BMSCs were measured by surface antigens; functional differentiation to endothelial cells was assessed by colony forming capacity, expression of mature endothelial cell antigens and tube‐like formation. The blood CD34+ cells were spiked on day 3 post‐stimulation in both groups. While CD34+ cells released by artery ligation was 2‐fold lower than those by G‐CSF administration, significantly more CD133+/KDR+/CXCR4+/CD31+ cells were detected in baboons with artery ligation. After culture in endothelial growth medium, mononuclear cells from baboons with artery ligation formed more endothelial cell colonies (p < 0.05), formed more capillary‐like tubes (p < 0.05), expressed higher vWF and phagocytosed more Dil‐LDL (p < 0.05).

Conclusions: While G‐CSF and artery ligation can mobilize BMSCs capable of differentiating into endothelial cells, BMSCs mobilized by the artery ligation simulating in vivo ischemic attacks have higher potential for vascular differentiation. Our findings suggest that direct bone marrow aspiration may provide the pool of BMSCs capable of transforming into mature cardiovascular cells.
Physical interaction of CD36 with membrane associated proteins in mouse macrophage
Authors: Huang Wenxin , Febbraio Maria , Silverstein Roy
Published: June 2021

CD36 is an integral membrane protein of the class B scavenger receptor family. It is expressed on many cell types where it interacts with multiple different ligands that give rise to cell and tissue specific functions of CD36. CD36 plays an important role in atherosclerosis, thrombosis, malaria infection, inflammation, lipid metabolism and fatty acid uptake. Currently, very little is known as to how CD36 mediates these diverse functions. The strategy of our present work was to search, confirm and study CD36 interacting molecules. We used primary macrophages (from wild type and CD36 KO mice) to immunoprecipitate (IP) CD36 and interacting proteins, followed by SDS‐PAGE and mass spectrometry for identification of specific proteins. Then we used other methods including western blot, confocal microscopy and immunocytochemistry to validate these findings and to investigate the function of these proteins and how they may relate to CD36 functions. Thus far, we have identified 22 proteins that co‐immunoprecipitate with CD36. Some of these overlap with those previously reported in the literature. We have validated CD9 and flotillin‐2, and currently are pursuing further study of their relationship to CD36‐dependent functions.
Serotonergic involvement in the effects of chronic cold stress on reversal learning in the rats
Authors: Lapiz‐Bluhm M. Danet , Soto‐Pina Alexandra , Morilak David
Published: June 2021

Chronic stress is a risk factor and an antecedent to depression and to dysregulation of brain modulatory systems. Chronic intermittent cold (CIC) stress sensitized the increase in norepinephrine (NE) release in hippocampus and medial prefrontal cortex (mPFC) in response to acute stress (Mana & Grace, 1997. Neuroscience 81:1055). Cognitive performance in an attentional set‐shifting task (AST) can be enhanced by tonic NE transmission in mPFC (Lapiz & Morilak, 2006. Neuroscience 137:1039), and is disrupted by chronic unpredictable stress (Bondi et al. 2007. Neuropsychopharmacol. e‐pub). This study addressed the effects of CIC stress on performance of rats in the AST. Male Sprague‐Dawley rats exposed to a 4°C cold room (6 hr/day × 14 days) were tested in the AST. Analyses showed a significant effect of Stress (P = 0.043), Task (P < 0.0001) and a Stress × Task interaction (P = 0.001), with CIC‐stressed rats requiring more trials during reversal 1. The orbitofrontal cortex (OFC) has previously been implicated in reversal tasks, possibly modulated by serotonin (5‐HT). We showed that 5‐HT depletion with Para‐chlorophenylalanine (200 mg/kg/day, i.p.) induced a similar selective deficit in reversal, while the CIC‐induced impairment in reversal learning was attenuated by the 5‐HT reuptake inhibitor (SSRI) Citalopram (5 mg/kg, i.p.). These results indicate that the CIC stress‐induced impairment in cognitive flexibility may involve an alteration in 5‐HT function in OFC. These deficits may be relevant to the symptoms of neuropsychiatric disorders that respond to SSRI treatment, i.e., depression and anxiety disorders.

Support: NIMH grant MH72672 & NARSAD Award to MDLB
Peptide Ligands of Regulator of G‐Protein Signaling 4 (RGS4) Identified from a One‐Bead, One‐Compound LIbrary
Authors: Roof Rebecca A. , Clements Samuel , Roman David L. , Sobczyk‐Kojiro Katarzyna , Turbiak Anjanette , Mosberg Henry I. , Neubig Richard R.
Published: June 2021

RGS proteins accelerate the hydrolysis of GTP by the Gα subunit of the heterotrimeric G‐protein and therefore quicken the inactivation of G‐protein signaling. The distinct expression patterns and pathophysiologic regulation of RGS proteins suggest that inhibitors of their activity may have therapeutic potential.

The objective of this study is to identify peptide inhibitors of RGS4. We synthesized a focused One‐Bead, One‐Compound peptide library that retains structural features known to be necessary for the activity of a previously identified peptide. The library of 2.5 million peptides was screened and peptide‐beads with increased binding to a fluorescently labelled RGS4 were isolated. One of these hit peptides (9, Tyr‐Trp‐[Cys‐Lys‐Leu‐Cys]‐Lys‐NH2, S‐S) blocked the interaction between biotin‐RGS4 on avidin beads and a fluorescent Gαo with an IC50 of 28 μM in a Flow Cytometry Protein‐Protein Interaction Assay. It had RGS selectivity with the following order of inhibition RGS4>RGS8>RGS16≫RGS19>RGS7. Surprisingly, inhibition of RGS4 was irreversible. A methylene dithioether bridged peptide did not inhibit RGS4/Gαo interactions, suggesting that peptide 9 was forming a disulfide bond to RGS4. Future directions include determining which cysteine in the RGS is modified using mutagenesis and mass spectrometry. Supported by 5R01DA003910‐21 to HIM.
Regulator of G protein Signaling 4 (RGS4): A Novel Inhibitor of Breast Cancer Metastasis
Authors: TU Yaping , Xie Yan , Kirui Joseph K , Wolff Dennis W
Published: June 2021

Regulator of G protein signaling (RGS) proteins attenuate signaling of G protein‐coupled receptors associated with breast cancer metastasis. Using quantitative real‐time PCR, we found that RGS4 mRNA was selectively and significantly up‐regulated in highly metastatic breast cancer MDA‐MB‐231 and −436 cells. However, despite this high level of RGS4 transcription, RGS4 protein was barely detectable in these cells and additional studies determined that this was due to proteasome‐dependent degradation of RGS4 protein. Proteasome inhibitor‐induced blockade of endogenous RGS4 protein degradation attenuated the migration and invasion of theses cells in transwell assays. Similarly, transient expression of RGS4, but not other RGS proteins, in MDA‐MB‐231 cells also caused marked inhibition of these indices of tumor metastasis. Stable expression of RGS4, but not its function deficient N128A mutant, in MDA‐MB‐231 cells effectively attenuated cancer cell invasiveness both in vitro and in a mouse xenograft model. Other in vitro studies showed that RGS4 selectively inhibits Gi‐dependent activation of small G protein Rac, a key activator of breast cancer metastasis. Finally, immunohistochemistry studies of human breast carcinoma specimens indicated that RGS4 protein levels are down‐regulated in the cancer cells and that this down‐regulation is a relatively late event associated with the invasive phenotype.
Endogenous RGS proteins attenuate Gαi‐mediated Lysophosphatidic Acid signaling pathways in Ovarian Cancer Cells
Authors: Hurst Jillian Heyward , Brown April L. , Henkel Paul A. , Hooks Shelley B.
Published: June 2021

Lysophosphatidic acid (LPA) is the predominant growth factor driving the progression of ovarian cancer. Effects of LPA are mediated by cell surface G‐protein coupled receptors (GPCRs) that activate multiple heterotrimeric G‐proteins. G‐proteins are deactivated by Regulators of G‐protein Signaling (RGS) proteins. We hypothesized that RGS proteins may regulate G‐protein signaling pathways initiated by LPA in ovarian cancer cells. To determine the effect of endogenous RGS proteins on LPA signaling in ovarian cancer cells, we compared the activity of LPA in ovarian cancer cells expressing Gi subunit constructs that are either insensitive to RGS protein regulation (RGSi) or their RGS‐sensitive wild‐type (RGSwt) counterparts. LPA‐mediated inhibition of forskolin‐stimulated adenylyl cyclase activity was enhanced in cells expressing RGSi Gi proteins as compared to RGSwt Gi, suggesting that endogenous RGS proteins in ovarian cancer cells normally attenuate signaling by these G‐proteins. The enhanced Gi signaling reflected greater potency, efficacy, and duration of LPA effects in the absence of RGS regulation. Further, LPA‐stimulated ovarian cancer cell migration in a wound‐induced migration assay was enhanced in cells expressing Gai2 RGSi as compared to cells expressing Gai2 RGSwt. These data establish RGS proteins as important regulators of LPA signaling in ovarian cancer cells.
PI(3,4,5)P3 Potentiates Phospholipase C‐[beta] Activity
Authors: Zhang Yong , Kwon Sun Hyung , Vogel Walter , Filtz Theresa
Published: June 2021

Phospholipase C‐[beta](PLC‐[beta]) isozymes are key effectors in G protein‐coupled signaling pathways. Previously, we had shown that PLC‐[beta]1 and PLC‐[beta]3 bound immobilized PIP3. In this study, PIP3 was found to potentiate Ca2+‐stimulated PLC‐[beta] activities using an in vitro reconstitution assay. LY294002, a specific PI 3‐kinase inhibitor, significantly inhibited 10 minutes agonist‐stimulated total IP accumulation. Both LY294002 and wortmannin inhibited 90 seconds agonist‐stimulated IP3 accumulation in intact cells. Moreover, transfected p110CAAX, a constitutively activated PI 3‐Kinase catalytic subunit, increased 90 seconds oxytocin‐stimulated IP3 accumulation. Receptor‐ligand binding assays indicated that LY294002 did not affect G protein‐coupled receptors directly, suggesting a physiological role for PIP3 in directly potentiating PLC‐[beta] activity. When co‐expressed with p110CAAX, fluorescence‐tagged PLC‐[beta]3 was increasingly localized to the plasma membrane. Conversely, a greater proportion of PLC‐[beta]3 associated with cytosolic fraction following H9c2 cells treatment with LY294002. Additional observations suggest that the PH domain of PLC‐[beta]1 and ‐[beta]3 is not important for p110CAAX‐induced membrane association.

This work was supported by a grant from the NIH R01‐GM61244 (to T.M.F.).
The biosynthesis of secretory and membrane proteins
Authors: Hegde Ramanujan
Published: June 2021

Secretory and membrane proteins are essential to all intercellular and most intracellular communication, and their precise locations and abundances are tightly regulated to maintain normal organismal physiology. Indeed, the majority of current drugs target secreted and membrane proteins, underscoring their central role in human biology. Our laboratory is working to develop a molecular level understanding of the pathways of secretory and membrane protein biosynthesis and metabolism. We are especially interested in the regulatory machinery controlling protein entry and insertion into the endoplasmic reticulum (ER), the site where nearly all secreted and membrane proteins are first made. Biochemical approaches are being employed to purify, identify, and functionally reconstitute the machinery underlying these basic cellular pathways. In parallel, the physiologic importance of regulating the metabolism of secretory and membrane proteins are being analyzed in cellular and whole animal models. We anticipate that a greater understanding of these basic cellular pathways will provide insight into the various diseases caused by problems in protein localization, folding, and processing.
Identification of the site on p90 RSK1 that binds the catalytic subunit of PKA
Authors: Gao Xianlong , Chaturvedi Deepti , Patel Tarun
Published: June 2021

Previously, we reported novel interactions between subunits of cyclic AMP‐dependent protein kinase (PKA) and ribosomal S6 kinase 1 (RSK1). The interactions are dependent upon the activation state of RSK1, with inactive RSK1 associating with the type I regulatory subunit (RI) of PKA whereas active RSK1 binds with the catalytic subunit of PKA (PKAc). Through these interactions, PKA modulates the subcellular localization of RSK1 and thereby its anti‐apoptotic effects. Here we report the identification of the regions of RSK1 that are responsible for the interaction with PKA. We expressed several polypeptides of full‐length RSK1 (aa 1–735) in Hela cells and performed immunoprecipitation with anti‐PKAc antibody. RSK1‐NT (aa 1–317), which contains the N‐terminus kinase domain (NTK) of RSK1, was not co‐precipitated with PKAc, whereas RSK1‐LCT (aa 318–735), which includes the linker region and C‐terminus kinase domain of RSK1, was co‐precipitated with PKAc. Subsequently, we tested RSK1‐NT+Linker (aa 1–418), RSK1‐CT (aa 418–735) and RSK1‐CTK (aa 418–671). RSK1‐CT, but not RSK1‐NT+Linker or RSK1‐CTK, was co‐precipitated with PKAc, indicating that the extreme C‐terminus of RSK1 (aa 671–735) is crucial for the interaction with PKAc.

Supported by NIH grant GM 079226
CaMKIV Regulates Survival/Proliferation of Neuroblastoma Cells
Authors: Feliciano David Matthew , Edelman Arthur M
Published: June 2021

CaMKIV is a multifunctional, predominantly nuclear CaM kinase abundantly expressed in the nervous system. Upon activation by Ca2+/calmodulin and CaMK kinase (CaMKK), CaMKIV phosphorylates transcriptional regulators, notably Ca2+/cAMP response element binding protein (CREB) to transduce rises in Ca2+i into changes in gene expression. We studied the role of CaMKIV in survival and proliferation of multipotent BE(2)C neuroblastoma cells. RNA interference of CaMKIV or CaMKK inhibition by STO‐609 produced cell cycle arrest, a reduction in proliferative rate and induction of markers of apoptosis (caspase‐3 and poly ADP‐ribose polymerase [PARP] cleavage). Transretinoic acid treatment led to upregulation of cellular markers of neuronal differentiation (doublecortin, peripherin, Bcl‐2) and to co‐ordinate reductions in CaMKIV mRNA and protein and of CREB phosphorylation. To identify downstream effectors of CaMKIV, protein expression patterns of control and CaMKIV RNAi cells were compared by two dimensional difference gel electrophoresis. Differentially expressed proteins identified by mass spectrometry and peptide mass fingerprint database searching include PARP‐6, Mitofusin‐1, and Heat shock protein 60. CaMKIV may promote the survival and proliferation of neuroblastoma cells by transcriptionally or post‐transciptionally maintaining the levels of anti‐apoptotic proteins.
The Structure of Human Cytochrome P450 2C19: Active site differences between P450s 2C19 and 2C9
Authors: Reynald Rosamund L. , Sansen Stefaan , Stout C. David , Johnson Eric F.
Published: June 2021

Our objective was to determine the structure of 2C19 and to identify structural features underlying the distinct substrate and inhibitor profiles of 2C19 and 2C9, which differ at 49 of 490 residues. Crystal structures of 2C19 and 2C9 complexed with structurally related inhibitors, DMB and DMI, respectively, were determined to 2.9 and 1.9 Angstrom resolution. DMI is the 3,5‐diiodophenyl analog of DMB (2‐methyl‐1‐benzofuran‐3‐yl)‐(4‐hydroxy‐3,5‐dimethylphenyl)methanone. The increased bulk of the iodine relative to the methyl moieties and the lower pKa of the phenol are thought to contribute to 1000‐fold difference in the Ki's exhibited by DMI for the two enzymes (Locuson et al. J. Med. Chem. 47: 6768 (2004)). The two enzymes exhibit conformation differences that confer substrate selectivity with relatively few amino acid differences contributing directly to substrate binding. The increased flexibility of the helix B‐C loop of 2C9 relative to 2C19 allows Arg108 to complement the negative charge of the phenol, whereas the neutral form of DMB is more readily accommodated by 2C19. These structures elucidate the contributions of structural flexibility and active site hydration in substrate binding. Furthermore, the availability of a structure for 2C19 will facilitate computational approaches for predictions of substrate and inhibitor binding this enzyme. Supported by USPHS grant NIH GM031001.
Identification of active site residues essential for the metabolism of phenacetin to acetaminophen in human 2A cytochromes P450
Authors: DeVore Natasha M , Smith Brian D. , Urban Michael J. , Nikaeen Naseem , Blake Linda C. , Scott Emily E.
Published: June 2021

Human cytochromes P450 from the 2A subfamily have differing abilities to metabolize carcinogens and procarcinogens, including the nicotine‐derived 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), aflatoxin B1, and 4‐aminobiphenyl. Recently, Fukami et al. established that phenacetin, a hepatocarcinogen and known substrate for the CYP1A family, is even more efficiently metabolized by human lung cytochrome P450 2A13 (CYP2A13), while the human liver cytochrome P450 2A6 (CYP2A6) has almost no activity despite being 94% identical in amino acid sequence. To determine the basis for substrate specificity between these two closely related human 2A enzymes, site directed mutagenesis was used to construct eleven CYP2A13 single point mutants with mutations at positions in or near the active site where CYP2A13 and CYP2A6 wild type enzymes differ. Phenacetin metabolism kinetics were determined for the wild type enzymes CYP2A13 and CYP2A6, and eleven CYP2A13 mutants. Two CYP2A13 mutants, A117V and L366I, each increase phenacetin metabolism activity greater than four‐fold. Four amino acid substitutions, S208I, F300I, A301G, and G369S, virtually abolished phenacetin metabolism in CYP2A13. The CYP2A6 enzyme is being modified by substitution of one or more 2A13 active site residues to determine those required to incorporate phenacetin metabolism into CYP2A6.

NIH GM76343
Upregulation of Adenosine A1 Receptor in Coronary Atherosclerosis in the Metabolic Syndrome and in the in Vitro Organ Culture Model of Coronary Atherosclerosis
Authors: Long Xin , Edwards Jason M , Neeb Zachary , Alloosh Mouhamad , Sturek Michael
Published: June 2021

Ossabaw miniature swine fed excess atherogenic diet develop metabolic syndrome (MetS) and coronary atherosclerosis similar to humans. Organ culture is a useful in vitro model of arterial injury/atherosclerosis. Our lab previously made the novel discovery that adenosine A1 receptor (A1R) signaling via ERK phosphorylation mediates smooth muscle cell proliferation in intact coronary rings from Ossabaw pigs. Thus, we hypothesized that A1R will be upregulated in atherosclerotic coronary artery of MetS Ossabaw pigs and also in the organ culture model. Coronary intimal thickness in MetS pigs (n=7) increased 2.4‐fold compared to control pigs (n=6) (p<0.05). A1R mRNA and protein increased 4‐fold and 3‐fold, respectively, in MetS compared to control pigs (p<0.05). A1R mRNA increased 5‐fold in the 2‐day organ cultured coronary compared to cold stored ones (p<0.05), and A1R protein increased 2‐fold in the 4‐day organ cultured coronary compared to cold stored. In an in vitro organ culture model of arterial injury, A1R stimulated ERK phosphorylation increased 8‐fold compared to non‐injured artery segments.

CONCLUSION: Upregulation of A1 receptor expression and ERK signaling may contribute to coronary atherosclerosis in MetS and in the in vitro organ culture model of arterial injury.
Resistin promotes macrophage foam cell formation in rats via dysregulation of scavenger receptors and ATP‐binding cassette transporters
Authors: Tsai Jin‐Yi , Lin Chun‐Yueh , Kou Yu Ru , Wu Yuh‐Lin , Ho Low‐Tone , Lee Tzong‐Shyuan
Published: June 2021

We studied the cellular mechanisms underlying the acceleration effect of resistin on macrophage foam cell formation. In vivo studies demonstrated that resistin levels were elevated in atherosclerotic aortas of apolipoprotein E deficient mice compared with wildtype counterparts. In vitro studies showed that resistin increased lipid accumulation in rat macrophages treated with oxidized low‐density lipoprotein (oxLDL), as compared to control cells. Resistin also increased levels of mRNA and proteins of scavenger receptor class A (SR‐A) and CD36 (two types of SRs for internalization of oxLDL) and decreased protein level, but not mRNA level, of ATP‐binding cassette transporter‐A1 (ABCA1; a type of cholesterol exporter). The up‐regulations of SR‐A and CD36 by resistin were due to activation of AP‐1 and PPARγ, respectively, as evidenced by increased nuclear levels of these two transcriptional factors and by their preventions after pharmacological inhibition of AP‐1 (curcumin or SP600125) or PPARγ (GW9662). The down‐regulation of ABCA1 by resistin resulted from a more rapid degradation of protein via proteasome pathway, as revealed by its abolition after pharmacological inhibition (calpeptin or MG‐132) of the pathway. In conclusion, resistin may promote foam cell formation via dsyregulation of SR‐A, CD36 and ABCA1. SR‐A and CD36 are up‐regulated through transcription regulation, whereas ABCA1 is down‐regulated through proteasome‐mediated protein degradation.
Mitochondrial ROS mediates HNE‐induced MMP‐2 production in VSMC via activation of Akt/NF‐κB signaling pathways
Authors: Seo Kyo Won , Lee Seung Jin , Yun Mi Ran , Bae Sun Sik , Kims Chi Dae
Published: June 2021

To investigate the role of HNE in the progression of atherosclerosis, we examined the HNE‐triggered signaling pathway and its role in MMP‐2 production in vascular smooth muscle cells (VSMC). VSMC stimulated with HNE produced enzymatically active MMP‐2 with the phosphorylation of Akt. The increase in promoter activity and production of MMP‐2 by HNE was attenuated by PI3K/Akt inhibitors, LY294002 and SH‐5, suggest a role of Akt signaling in transcriptional regulation of MMP‐2 production. Among the various transcription factors involved in MMP‐2 transcription in VSMC, the activity of NF‐κB activity, but not others including CREB, AP‐1 and c‐Jun, was significantly increased by HNE. The increased MMP‐2 promoter activity by HNE was also completely abolished by mutation of NF‐κB binding site in MMP‐2 promoter region, demonstrating the participation of Akt‐mediated activation of NF‐κB in HNE‐induced MMP‐2 transcription. Moreover, the phosphorylation of Akt by HNE were significantly attenuated not only by inhibitors for mitochondrial generation of ROS, rotenone and stigmatellin, but also by depletion of mitochondrial function in VSMC, indicating that mitochondrial ROS mediates Akt phosphorylation and subsequent MMP‐2 production. Based on these results, it is suggested that mitochondrial ROS enhances MMP‐2 production in HNE‐stimulated VSMC via activation of Akt‐dependent NF‐κB signaling pathway.
Biologic nanoparticles induce a sensitizing immune response in naïve rabbit
Authors: Schwartz Maria A K , Lieske John C , Hunter Larry W , Miller Virginia M
Published: June 2021

Biologic nanoparticles (NPs) can be isolated from bovine serum, human kidney stones, and human arterial calcifications. In addition, it is possible to detect both antigen and antibody for NPs in human blood, suggesting that perhaps NPs represent a novel infectious agent participating in human disease. However, it is not known when and how persons might become exposed to NPs and if exposure produces sensitization to subsequent exposures. Experiments were designed to determine whether exposure to nanoparticles would induce an immune response in rabbits that can be detected by a delayed‐type hypersensitivity test. Rabbits were injected intravenously with bovine‐derived NPs (1ml of 1000NTU solution). Plasma was collected prior to injection and on days, 3, 7, 14, 21, 28, 35 post‐injection. At this time, NPs (1ml of 1000NTU solution) were injected subcutaneously in the ear of exposed and naïve rabbits. Areas of injection were observed daily and after three days, blood and ears were collected for analysis. Injection sites showed leukocyte infiltration only in ears of rabbits previously exposed to NPs. These data indicate that biologic NPs induce immune sensitization in rabbits.
Biological nanoparticles and platelet activation
Authors: Chu Kevin , Kaul Vivasvat , Owen Whyte G. , Jayachandran Muthuvel
Published: June 2021

Self‐replicating, self‐calcifying nanosized particles (NPs) can be isolated from mammalian blood and calcified human tissue. Intravenous injection of these biologic NPs into rabbits accelerates occlusion of endothelium‐denuded carotid arteries. Since platelet activation is an initiating step in arterial repair, experiments were designed to determine if biologic NPs alter platelet functions. Platelet rich plasma was prepared from human citrate anti‐coagulated blood. Platelet aggregation (light transmission) and ATP secretion were monitored in response to sTRAP (10 μM) prior to and following incubation with either control solution, biologic NPs (50, 150, 500 NTUs) or hydroxyapatite (HA). NPs increased light transmission and ATP secretion when added immediately to the platelet solution. Both rate and maximal aggregation induced by sTRAP were reduced in the presence of NPs. NPs increased stimulated release of ATP at moderate doses but only after 15 minutes of incubation. HA crystals reduced rather than augmented ATP secretion. These results suggest that NPs cause a shape change in platelets and formation of microaggregates under basal conditions. NPs selectively augment agonist‐stimulated ATP secretion in a dose and time dependent fashion. Therefore, NP activation of platelets could contribute to the increased vascular response to injury in vivo.
Role of CD40‐CD40L signaling in the extra‐intestinal thrombosis associated with colonic inflammation
Authors: Russell Janice , Stokes Karen Y. , Granger D. Neil
Published: June 2021

The CD40‐CD40L signaling pathway has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Although IBD is associated with enhanced thrombosis in extra‐intestinal tissues, the mechanisms that underlie this response remain undefined. The objective of this study was to evaluate the contributions of CD40‐CD40L in the enhancement by dextran sodium sulfate (DSS) of thrombus formation elicited in cremaster muscle microvessels. Wild type (WT), CD40‐deficient (CD40−/−) and CD40L‐deficient (CD40L−/−) mice were fed 3% DSS drinking water. On day 6, intravital microscopy was used to monitor thrombus formation in a light/dye‐induced endothelial injury model. DSS enhanced the rate of thrombus formation; decreasing the time of thrombus onset and the time to flow cessation in arterioles of WT mice, while thrombosis formation in venules was not significantly altered by DSS. In arterioles of CD40L−/− mice, the time to onset and flow cessation was significantly increased after DSS when compared to WT‐DSS. In contrast CD40−/− mice were not protected against the enhanced thrombosis formation. These findings suggest that CD40L contributes to the enhanced extra‐intestinal thrombus formation during experimental IBD, most likely through a non‐CD40‐dependent pathway. The CD40L receptors responsible for this accelerated thrombus formation remain to be identified (Supported by P01 DK43785).
EXPERIMENTAL STUDY ON DIFFERENTIATION OF BONE MARROW STROMA CELLS TO CARDIOMYOCYTES‐LIKE
Authors: Bai Xiaojuan , Zhou Ying , Wang Bo
Published: June 2021

Objective To investigate the ultrastructure and cell factor changes of BONE MARROW STROMA CELLS(BMSCs) after induction and differentiation into cardiomyocytes‐like, in order to provide theory foundation for the therapy of dilated cardiomyopathy(DCM) with BMSCs transplantation.

Method BMSCs were isolated and proliferated in vitro, after induction and differentiation by 5‐azacytidine(5‐aza), they were observed by electromicroscopy to see the ultrastructure changes; RT‐PCR were also used to check cardiac‐special factors including ANP, BNP, α‐MHC, β‐MHC, comparing with the cardiomyocytes in primary culture in vitro.

Result BMSCs were verified by immunohistochemistry that they differentiated into cardiomyocytes‐like; under electromicroscopy, glycogensome and myofibril can be seen in cell plasm, which was similar to cardiomyocyte in primary culture. RT‐PCR showed that BMSCs expressed cardiac‐specific factors, including ANP, BNP, α‐MHC, β‐MHC after induction and differentiation.

Conclusion After induction by 5‐aza, BMSCs were similar to cardiomyocytes in primary culture in ultrastructure and cardiac‐specific factors expression, which suggested that they can be used for the therapy of DCM with BMSCs transplantation.
Simulation of angiogenesis, remodeling and pruning in microvascular networks: Control of branching angles
Authors: Secomb Timothy W. , Alberding Jonathan P. , Hsu Richard , Pries Axel R.
Published: June 2021

In the microcirculation, angiogenesis, remodeling and pruning redistribute flow in response to varying demands. Here, a previously developed theoretical simulation of these processes, based on local vascular responses to pressure, shear stress, oxygen partial pressure and growth factor concentration, is extended to include effects of axial tension in vessel segments. The network is represented as a large number of short segments in two dimensions, connected at nodal points. The total tension in each segment is assumed to be proportional to the vessel circumference. Each nodal point is assumed to migrate through the surrounding tissue at a rate proportional to the resultant force generated by the vector sum of the axial tensions acting at that point. In the simulations, most bifurcations initially have a ‘T’ shape, resulting from sprouting angiogenesis from an existing segment or from coalescence of a sprout with an existing segment. However, the effect of tension‐induced migration is to convert these to a ‘Y’ shape. The resulting distribution of branching angles is similar to that observed in mesenteric microvascular networks of the rat. Supported by NIH HL034555.