bluuebunny/crossref_metadata_2025 huggingface dataset:
Reconstituting human P‐glycoprotein (MDR1) into membrane nanodiscs
Authors: Farokhnia Roxana , Olivera Maisa C. , Okwuone Dakota D.D. , Jensen Madeline K. , Edgar Jennifer M. , Wise John G. , Vogel Pia D.
Published: June 2021

P‐glycoprotein (P‐gp) is a multidrug resistance protein found in the membrane of many cells. As an ATP powered pump, P‐gp exports toxins and other hydrophobic compounds outward across the plasma membrane. This trait becomes problematic when overexpression of P‐gp occurs in cancer cells and causes chemotherapeutics to be pumped out of the cancer cells, rendering therapy ineffective. For biochemical and/or biophysical assessment of P‐glycoprotein, the protein is removed from the membrane by detergents and purified into detergent micelles using affinity chromatography. Although P‐gp is active when dissolved into micelles, the detergent environment has been shown to affect its biological activity. To avoid this issue, native‐like membrane nanodiscs have been introduced and used successfully over the last several years. Nanodiscs typically contain the P‐glycoprotein surrounded by lipids and enclosed within membrane scaffold proteins (MSP) which create an amphipathic belt around the membrane disc for water solubility. This more native‐like environment within the nanodiscs allows access to both the nucleotide and drug binding domains of the inserted proteins. In order to effectively assemble proteins into nanodiscs, specific ratios of protein to MSP to lipid are needed. In our lab, different ratios of these components have been used to optimize the assembly of the mouse cysteine‐less variant of P‐gp (MDR3CL) into nanodiscs. For the human P‐gp (MDR1) preliminary assembly successes were achieved at a 1:10:500 ratio (P‐gp: MSP: phosphatidylcholine). However, the yield of P‐gp assembled into nanodiscs was very low (about 30%). Here we report our efforts to optimize the nanodisc assembly with the goal of increasing P‐gp reconstitution yield and specific ATP hydrolysis activity. We will use the nanodisc reconstituted P‐glycoprotein in our efforts to investigate efficacy and mechanism of inhibition of novel P‐glycoprotein inhibitors discovered in our lab.

Support or Funding Information

This work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU University Research Council, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Hydrogen Peroxide on Hydrolysis of Proteins
Authors: Ailes Isaiah , Tohidi David , Ngo Luke , Keenan Kelly
Published: June 2021

Hydrogen peroxide (H2O2) is the active ingredient in over‐the‐counter whitening strips for teeth. It has previously been shown to damage certain proteins by making them more susceptible to hydrolysis by proteases. The goal of this project was to characterize the hydrolysis of various proteins with H2O2. Albumin protein was mixed with different concentrations of H2O2 for one hour. The protease trypsin was added and portions were removed and immediately mixed with trichloroacetic acid to precipitate either unhydrolyzed proteins or proteins that were minimally hydrolyzed. After centrifugation, the supernatant, which contains small protein fragments that were released by hydrolysis, was collected and; the total amount was measured using the Lowry assay. The results indicate that even without the protease, there is significant hydrolysis with peroxide and none when the proteins were mixed with water. The amount of hydrolysis correlated with the amount of peroxide. In addition, the amount of hydrolysis in the presence of H2O2 with trypsin greatly exceeded that when only trypsin was present. It was important in this method to inactivate peroxide and efficacy of different metals were tested. This effect was further characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). These results suggest that hydrogen peroxide; even at the concentrations used in whitening strips, can damage protein by promoting hydrolysis. This work was supported by Research and Professional Development Grant from Stockton University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural insight into pre‐mRNA splicing catalyzed by the spliceosome
Authors: Zhao Rui , Li Xueni , Liu Shiheng , Zhou Hong
Published: June 2021

Pre‐mRNA splicing is catalyzed by the spliceosome, a huge protein‐RNA complex composed of the U1, U2, U4, U5, U6 snRNPs and many non‐snRNP related proteins. U1 snRNP plays a critical role in 5′ splice site recognition and is a frequent target of alternative splicing factors. These factors transiently associate with human U1 snRNP and are not amenable for structural studies, while their Saccharomyces cerevisiae (yeast) homologs are stable components of U1 snRNP. We have recently determined the structure of yeast U1 snRNP at 3.6Å resolution using cryoEM. The structure reveals common features as well as important differences from the human U1 snRNP. It provides atomic models of nearly all essential domains of U1 snRNA, all core proteins, and five auxiliary proteins. The structure offers a framework to integrate a wealth of existing genetic and biochemical data regarding the structure and function of yeast U1 snRNP and the mechanism of 5′ ss recognition. In addition, the yeast U1 snRNP structure and biochemical analyses based on the structure provided intriguing insight into the structure and function of these auxiliary human U1 snRNP proteins in alternative splicing in higher eukaryotes.

The spliceosome undergoes dramatic changes signified by the E, A, Pre‐B, B, Bact, B*, C, C*, P, and ILS complexes in a splicing cycle. CryoEM structures of B, Bact, C, C*, and ILS revealed mechanisms of 5′ ss recognition, branching, and intron release, but lacked information on 3′ ss recognition, exon ligation and release, all related to the post‐catalytic P complex. We have recently determined the cryoEM structure of the yeast P complex at 3.3Å resolution, revealing that the 3′ ss recognition is driven by the interaction between 3′ and 5′ ss, likely facilitated by a stem‐like structure formed in the intronic regions between the branch site and 3′ ss. The structure shows that a new protein(s) becomes stably associated with the core components of the P complex around Prp8 and Prp22, securing the 3′ exon and potentially regulating the activity of Prp22. The structure demonstrates that Prp22 binds 15–21 nucleotides downstream of the exon‐exon junction, enabling it to pull the 3′ intron‐exon and ligated exon in a 3′ to 5′ direction to achieve 3′ ss proofreading and exon release, respectively. By providing insights into the 3′ ss recognition, exon ligation, and the action of RNA helicase Prp22, the P complex structure fills a major gap in our understanding of the splicing cycle.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Studies of the Protein Degradation Complex
Authors: Zhao Gang , Li Guangtao , Zhou Xiaoke , Schindelin Hermann , Lennarz William J
Published: June 2021

In eukaryotes, the endoplasmic reticulum associated degradation (ERAD) pathway plays an important role in the degradation of misfolded glycoproteins. Using functional and biochemical techniques we identified a higher order protein complex consisting of two ER membrane proteins, Derlin1 and gp78, and the cytoplasmic proteins p97/VCP, peptide:N‐glycanase (PNGase) and HR23. This complex may assist the retro‐translocation process and subsequently escort misfolded glycoproteins to the proteasome for degradation. The multifunctional AAA‐ATPase p97 is believed to extract ERAD substrates through the retrotranslocon and, by association with various cofactors, recruit and process the ubiquitinated substrates. One of the p97 binding proteins is the cytosolic PNGase, which hydrolytically removes the N‐linked glycan chains from denatured glycoproteins. Recently, we found a novel binding motif in the C‐terminal of mouse p97, which mediates the binding of p97 to PNGase and other substrate‐processing cofactors. The crystal structure of the N‐terminal domain of mouse PNGase in complex with this motif demonstrates the pivotal role of the penultimate tyrosine residue of p97 in its interaction with PNGase.
Mechanism of inhibition of glyceraldehyde‐3‐phosphate dehydrogenase by glucose
Authors: Subramaniam Ram , Easterday Ashton , Major Amanda , Vu Andrew
Published: June 2021

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that converts glyceraldehyde‐3‐phosphate to 1,3 bisphospho glycerate. In tissue extracts from diabetic mice and cytosolic extracts from RAW264.7 cells grown under hyperglycemic conditions (10–25 mM glucose), we found that the activity of GAPDH is decreased in comparison to control. We also found that the amount of GAPDH protein expressed is up‐regulated as a result of hyperglycemia and diabetes. In order to understand the mechanisms by which GAPDH activity is inhibited by glucose, we studied the effect of glucose on chicken muscle GAPDH. Reaction of GAPDH with glucose for 48 hours decreased the activity of GAPDH by 50% compared to control enzyme. No conformational differences were observed in glucose‐reacted GAPDH compared to native enzyme (based on fluorescence and circular dichroism spectroscopy). Kinetic data indicates that the KM for GAPDH reacted with or without glucose does not change, whereas the VMAX is decreased at high concentrations of glucose (>20 mM). Based on these results we concluded that glucose inhibits GAPDH by a non‐competitive mechanism, by binding to a site on the enzyme which is different from the substrate binding site. Our findings indicate a novel mechanism for glucose‐mediated inhibition of GAPDH activity.
Allosteric modulation of mGluR4 as a novel therapeutic direction for the treatment of Parkinson's disease
Authors: Niswender Colleen Marie
Published: June 2021

We have performed a series of anatomical, cellular, molecular, and behavioral studies that suggest that agonists of the metabotropic glutamate receptor mGluR4 could provide a novel approach to treatment of Parkinson's disease by actions at a key synapse in the basal ganglia motor circuit. Unfortunately, the glutamate binding site is highly conserved across mGluR subtypes, making it difficult to develop highly selective agonists of this receptor. Also, known glutamate site agonists do not possess properties needed to allow them to be useful as drug leads. We have now identified novel compounds that act at allosteric sites on mGluR4 to potentiate mGluR4 activity. These compounds have no effect on mGluR4 when added alone but induce progressive leftward shifts in the agonist concentration‐response curve for activation of this receptor. Interestingly, a representative compound potentiates mGlu4‐mediated inhibition of transmission at the striato‐pallidal synapse and induces therapeutic effects in rodent models of Parkinson's disease that are similar to those of the mGluR4 agonist, L‐AP4. A recent HTS campaign yielded over 400 novel mGluR4 potentiators belonging to multiple structural classes, many of which exhibit submicromolar potencies. We are now making significant progress in using technology‐enabled medicinal chemistry approaches to optimize these compounds as useful in vivo tools and as potential drug leads. These exciting findings suggest that allosteric potentiators of mGluR4 may provide a viable approach for selectively activating this receptor for treatment of Parkinson's disease. Supported by NIH, NIMH, and the Michael J. Fox Foundation.
Liver X receptor activation in cultured rat neonatal cardiomyocytes suppresses lipopolysaccaride‐induced inflammatory responses
Authors: Ricks Ernest , Yin Ran , Li Yuquan , Yang Qinglin
Published: June 2021

Elevated inflammation responses are a main feature for heart failure. Therapies of anti‐inflammation may have beneficial effect in preventing further progression of heart failure. LXR is a member of the nuclear receptor super‐family and is generally involved in cholesterol metabolism. LXR has two isoforms, LXRα and LXRβ. In macrophages, activation of LXRs has been shown to suppress lipopolysaccaride (LPS)‐induced inflammatory genes expression, suggesting that LXRs play a protective role against inflammation. It is unclear if LXR activation exerts similar anti‐inflammation effect in cardiomyocytes. To investigate this, we assess how pretreatment of a LXR synthetic ligand T1317 influences LPS‐induced inflammation responses in cultured rat neonatal cardiomyocytes. We confirmed that both LXR subtypes were expressed in cardiomyocytes and T1317 treatment induced more LXR expression by activating LXR. LPS treated cardiomyocytes showed markedly increased Tumor necrosis factor α(TNFα) and interleukine 6 (IL6) transcripts than those treated with DMSO or T0901317 alone. Pre‐treatment with T0901317 (2, 5 and 10 μM) for 2 and 24 hours revealed various degrees of suppression on LPS induced IL‐6 and TNF‐α expression in cultured cardiomyocytes (n=6, P<0.05). Therefore, we conclude that LXR activation play a protective role in the heart by suppressing inflammatory responses.
Temporal eicosanoid profiling of the inflammatory response to infection by the Lyme disease bacterium
Authors: Buczynski Matthew W. , Blaho Victoria A. , Brown Charles R. , Dennis Edward A.
Published: June 2021

Lyme disease is caused by infection with the spirochete B. burgdorferi. If untreated, 60% of infected individuals will develop an arthritis characterized by the infiltration of macrophages and neutrophils into the infected joint. Eicosanoids play a critical role in recruiting innate immune cells during the development of arthritis; however, their role in the response to infection is poorly understood. To study the contribution of eicosanoids to infection response, we utilized a murine model of Lyme disease that recapitulates many of the pathological hallmarks of the human disease before spontaneously resolving. Susceptible C3H/HeJ mice were infected with B. burgdorferi, and joint tissue was removed at days 0, 3, 7, 10, 14, 17, 21, 24, 28 and 35 for histological and molecular analysis. Using LC‐MS/MS methodology that has the capability to identify and quantitate over 100 different eicosanoids, we detected over 30 different metabolites from COX, 5‐LO, 12‐LO, 15‐LO and CYP450s in the infected joints. Furthermore, we have identified temporal eicosanoid patterns that correlate with the pathological progression of infection. Application of this method may allow for the identification of lipid mediators with previously unappreciated functions in this as well as other disease models. This work was supported by the LIPID MAPS Large Scale Collaborative Grant GM069338, NIH AR052748 and 2T32DK007202‐32.
Identification and characterization of inhibitors of hsFATP‐dependent fatty acid uptake in humanized yeast and human Caco‐2 cells
Authors: Sandoval‐Alvarez Angel , Chokshi Aalap , Singer Diane , Black Paul N. , DiRusso Concetta C.
Published: June 2021

We have employed a live‐cell high‐throughput method to screen for compounds that inhibit FATP‐mediated fatty acid uptake. Over 100,000 compounds from 3 chemically diverse compound libraries were screened. After preliminary selection using yeast expressing hsFATP2, secondary screens were performed to eliminate compounds that were autofluorescent, quenched fluorescence of the fatty acid analogue C1‐BODIPY‐C12 used in the primary screen, and permeabilized or were otherwise toxic to yeast. The initial set of selected hits included 145 compounds. Each potential hit was secondarily screened for inhibition of C1‐BODIPY‐C12 import into human Caco2 cells and this reduced the subset of potential hits to 22 compounds. Among these, six compounds were chosen as representative of a unique structural class for further characterization. Sigmoid dose‐response curves for C1‐BODIPY‐C12 uptake were generated in Caco‐2 cells and EC50 values were within the lower μM range. The compounds were determined to be non‐toxic to Caco‐2 cells using the MTT cytotoxicity assay. Teer tests confirmed that the integrity of the epithelial cell barrier of the intestinal cell line was not disrupted. As expected for a transport inhibitor, treatment with compound suppressed incorporation of radiolabelled fatty acids into all classes of lipids without suppressing acyl‐CoA synthetase activity. Supported by DK07076 from the NIH (CD and PNB).
Sensitive method of detecting myosin phosphorylation: effects of angiotensin II on the renal afferent arteriole
Authors: Takeya Kosuke , Loutzenhiser Kathy , Walsh Michael P , Loutzenhiser Rodger
Published: June 2021

Phosphorylation of myosin light chain (LC20) plays a key role in smooth muscle regulation, and is governed by the relative activation of myosin light chain kinase and phosphatase. The role of these two pathways in the renal microcirculation is not known. Since pharmacologic probes are often not selective, a capability to measure LC20 phosphorylation in small resistance vessels is needed and current techniques have inadequate sensitivity. To address this issue, we developed a highly sensitive method using Phos‐tag PAGE (Mol Cell Proteomics 5:749, 2006). SDS was included during sample preparation and electrophoresis to enhance protein extraction and minimize loss due to nonspecific binding. Sensitivity of western blotting was increased by fixing proteins on the membrane and optimizing all conditions. Using isolated afferent arterioles, we were able to detect LC20 phosphorylation in as little as one afferent arteriole (<50 pg LC20). LC20 phosphorylation levels could be reproducibly measured in pooled samples of 3 vessels and concentration‐dependent increases in LC20 phosphorylation were seen in response to 1–100 nM angiotensin II. Parallel studies using fura‐2 demonstrated corresponding increases in intracellular [Ca2+]. This new method will facilitate studies into the physiologic roles of Ca2+ sensitization pathways in the renal microcirculation.
High‐throughput evaluation of Janus Kinase‐STAT and MAPK pathways in normal peripheral whole blood cells using 96 deep well plates by flow cytometry
Authors: Sisouvanthong Chad , Ernst David , Li Li , Wang Xiao , Chen Jie , Shih Jing Ping , Huang Joseph , Gao Guo‐jian
Published: June 2021

When performing high‐throughput, flow cytometric screening of the patterns by which treated cells induce phosphorylated proteins involved in signaling pathways, tedious preparation of peripheral blood mononuclear cells (PBMC) was usually required. However, we have developed a Whole Blood Lyse/Fix Buffer System. This system allows for simultaneous red blood cell lysis and the fixation of leukocytes human whole blood. The cells can be stained for cell surface markers and intracellular cell signaling molecules and subsequently analyzed by flow cytometry. This eliminated the need for preparing PBMC. We have recently extended the application of this Whole Blood Lyse/Fix Buffer System to process cell samples in 96‐deep‐well plates. This method greatly facilitates the ease of preparing multiple human whole blood samples for cell signaling flow analysis. Whole blood stimulation, fixation, permeabilization, staining, and acquisition were done in deep well plates. Using deep well plates, we were able to evaluate the dose responses and time courses of STAT1, STAT3, STAT5, STAT6, Erk1/2, and P38 phosphorylation expressed by human whole blood cells that received various treatments. Inhibitory reagents, such as MEK and JAK inhibitors, were also applied to look at their differential effects on the phosphorylation status of intracellular signaling molecules that can be modified by the targeted kinases.
Immunomodulatory activities of arctiin isolated from a Fruit of Forsythia viridissima in macrophages
Authors: Kim Kyungjae , Lee Sungwon , Kim Hyunyul , Han Shinha , Kwon Jeonghak , Shin Seulmee , Kong Hyunseok , Yim Dongsool , Lee Chongkil
Published: June 2021

Arctiin isolated from Forsythia viridissim was reported to have anti‐inflammatory, and anti‐oxidant in vitro. There is lack of studies regarding the effects of arctiin on the immunological activities. We examined through macrophages for understanding the mechanism as to how arctiin inhibit inflammation. We investigated the effects of arctiin on the production of various kinds of cytokines such as nitric oxide (NO), tumor necrosis factor‐α (TNF‐α), Interleukine‐1β (IL‐1β), interleukine‐6 (IL‐6) and Prostaglandin E2 (PGE2) in primary and cultured macrophage with various doses of arctiin. Arctiin significantly decreased production of inflammatory cytokines in macrophages. Arctiin dose dependently decreased the expression levels mRNA of inducible NO synthase (iNOS), IL‐1β, IL‐6, and TNF‐α gene expression and western blot analysis also supported that arctiin decreased expression levels of their cytokines. In addition, Arctiin inhibit cyclooxygenase‐2 (COX‐2)‐dependent prostaglandin E2 (PGE2) production in a concentration‐dependent manner. The expression levels of costimulatory molecules such as B7‐1, B7‐2 and ICAM‐1 were suppressed by arctiin. These results showed that arctiin has immunomodulating effects in both innate and adaptive immunity. We suggest that arctiin could be a new suppressive immunomodulator.
Converting neural signals into short term immunological memory in macrophages
Authors: Parrish William R , Ochani M , Rosas‐Ballina M , Khalili H , Damle A , Gallowitsch‐Puerta M , Harris Y , Ochani K , Lin X , Miller E , Huston J , Czura C , Li W , Batliwalla F , Gregersen P , Tracey K J
Published: June 2021

Macrophage cytokine production is inhibited by cholinergic signals transmitted via the vagus nerve, an α7 nicotinic acetylcholine receptor (α7nAChR) dependent pathway termed the “cholinergic anti‐inflammatory pathway” (Nature 420:853–9, 2002). Here we addressed the hypothesis that this pathway is capable of providing short‐term memory by downregulating macrophage responses to endotoxin for up to 48 hr. Vagus nerve stimulation (VNS) (5V, 2 ms, 1Hz, 2.5 min) in rats 24 h prior to endotoxin (LPS) significantly reduced serum TNF (sham =767 pg/ml; VNS =358 pg/ml, p<0.01); this effect persisted for 48 h (sham TNF = 1327; VNS TNF = 824, p<0.05). The molecular mechanism is attributable to α7nAChR signaling, because VNS fails to reduce TNF levels in α7nAChR KO mice. This molecular memory is recapitulated in cultured human macrophages pulsed with acetylcholine (ACh) (60 min pulse 24 h prior to LPS). LPS‐induced TNF release was reduced 54% as compared with vehicle controls (p<0.05); pulsing also significantly suppressed activation of NF‐κB (74%). Ach‐pulsing significantly altered the macrophage transcriptome response to endotoxin, indicating that vagus nerve signals “train” macrophages as a short‐term (48 hr) memory response. Evidence that discrete neural signals can mediate short‐term memory in macrophages via α7nAChR has surprising implications for understanding innate immunity. Supported in part by NIGMS.
Expression of lymphotoxin‐αβ on antigen‐specific T cells is required for dendritic cell function
Authors: deLuca Leslie Summers , McCarthy Douglas D , Cosovic Bojana , Ward Lesley , Lo Calvin , Scheu Stefanie , Pfeffer Klaus , Gommerman Jennifer
Published: June 2021

The lymphotoxin (LT) pathway has been shown to be important in T cell responses and DC homeostasis/activation. LTαβ is a TNF family member upregulated on activated T cells and its receptor, LTβR, is constitutively expressed on DC. Elucidating the role of LTβR signaling in DC function is complicated by the expression of LTβR on both lymphoid stromal cells and DC. Here we have used two methods for inhibiting LTβR signaling. In the first we use an in vivo adoptive transfer system where the expression of the LTβR ligands is manipulated only on the Ag‐specific T cells that interact with and condition Ag‐bearing DC. Using this approach we demonstrate a novel requirement for the LTαβ for optimal DC conditioning during an immune response against protein antigen.

In the second system, we have manipulated the expression of LTβR on DC using a mixed bone marrow chimera approach. Here we report a requirement for DC‐intrinsic LTβR signaling for the optimal upregulation of CD86. In addition, DC‐intrinsic LTβR signaling influences DC accumulation in the inflamed LN. Together these data identify a fundamental role for DC‐intrinsic LTβR signaling during T cell immune responses. Funding from CIHR (JLG and LSD) and MS Society of Canada (LSD) supported this research.
Capsaicin induced regression of established tumors is in consequence of enhanced antigen presentation and alteration of stromal microenvironment
Authors: Ghosh Amiya , Basu Sreyashi
Published: June 2021

Tumor microenvironment is crucial in preventing the immunological destruction of antigenic tumors. The phenomenon that intratumoral administration of Capsaicin (CP) into an established tumor results in retarded progression of the tumor led us to examine the potential function of CP in the tumor microenvironment. We have previously reported that CP causes apoptosis of tumor cells in vitro but a direct cytotoxic effect of CP responsible for tumor growth retardation seems unlikely as CP mediated immunotherapy is T cell mediated and tumor‐specific. In this report we confirm that CP injection specifically induces apoptosis in tumor cells in vivo. We show that CP induced apoptosis leads to increased sensitization of the surrounding stroma manifested by enhanced antigen presentation by stromal cells and thereby its destruction by tumor specific T‐cells. Most importantly we demonstrate that CP injection alters the tumor microenvironment with regards to tumor‐infiltrating T lymphocyte population as well as the cytokine milieu at the tumor site. CP treatment promotes antitumor immune response by blocking anti‐inflammatory cytokines and depletion of FoxP3+ suppressor T cells. Our study illustrates that CP mediated immunotherapy is attained by diverse immunological events initiated at the tumor environment.
Efficient delivery of vector DNA to dendritic cells using silica‐nanoparticles
Authors: Messmer Davorka , Huang Chien‐Tze , Seible Daniel , Yang Jian , Saenz Rebecca , Esener Sadik , Trogler William
Published: June 2021

Objective: Optimize plasmid DNA delivery to DCs using silica‐nanoparticles (NPs) for tumor immunotherapy.

Methods: Human monocyte‐derived DCs were generated from PBMCs by culturing adherent cells in GM‐CSF and IL‐4 for 5 to 7 days. SiO2 nanoparticles were generated using polymer beads as template. The surface was functionalized with amino silane or amino phosphoric acid and the polymer core was removed by calcination or organic solvents.

Results: We sought to determine the optimal parameters in terms of size and surface modification of silica‐NPs with regard to DNA adsorption. DNA could be adsorbed to silica‐NPs of different sizes and surface modifications including amine groups and poly‐L‐lysine (pLL) lead to increased DNA adsorption to the NPs. NPs of 45, 100, and 200 nm diameter were effectively taken up by DCs and no obvious preference was observed. Interestingly pLL‐modified NPs were not taken up by DCs. Confocal microscopy indicated that NPs were also present in the nucleus of the DC which showed high viability for at least 7 days. To determine whether the NPs can be used to deliver DNA into DCs and lead to gene expression of the transgene, immature DCs were exposed to NP‐DNA complexes containing gfp‐plasmid and analyzed for Gfp expression by flow cytometry. 45% of the DCs expressed GFP at 48h.

Conclusion: SiO2‐nanoparticles of 80 and 120 nm are more efficient than electroporation to introduce a transgene into DCs in vitro and possibly in vivo for DNA vaccines.
15‐deoxy‐Δ 12,14 ‐prostglandin J2 (15d‐PGJ2) increases oxidative stress and differentially upregulates hemeoxygenase‐1 (HO‐1) in malignant B cells
Authors: Bancos Simona , Bernstein Steven , Baglole Carolyn , Phipps Richard P.
Published: June 2021

Background: 15‐deoxy Δ‐ 12,14 ‐prostaglandin J2 (15d‐PGJ2) is an endogenous ligand of peroxisome proliferator‐activator receptorγ (PPARγ ). Treatment of some B cell lymphomas with 15d‐PGJ2 increases oxidative stress and induces apoptosis. HO‐1 is an oxidative stress‐inducible enzyme but its expression in human B cell lymphomas is unknown. We hypothesized that PPAR ligands induce HO‐1 in malignant B cells.

Methods: Ramos and Granta B cell lymphoma cells and 8226 multiple myeloma cells were treated with 15d‐PGJ2. HO‐1 protein expression was assessed by western blot. Markers of oxidative stress included mitochondrial superoxide production and generation of reactive oxygen species (ROS).

Results: Treatment of Ramos cells with 15d‐PGJ2 strongly increased ROS and mitochondrial superoxide production and significantly reduced cell viability. Granta and 8226 cells had a similar response to 15d‐PGJ2 treatment but they exhibited a concentration‐ and time‐dependent increase of HO‐1 expression. Interestingly, Ramos B cells did not up‐regulate HO‐1.

Conclusions: The PPARγ ligand 15d‐PGJ2 induces cell death in some B malignant cell lines and differentially regulates the induction of HO‐1. This suggests that malignant B cells have different anti‐oxidant defense mechanisms.

Research supported by DE011390, ES01247, The Leukemia and Lymphoma Society and the Lymphoma Research Foundation.
Differences in signaling properties of FcγRI and FcγRIIa in human monocytic cells
Authors: Mehta Payal , Wavreille Anne‐Sophie , Butchar Jonathan P. , Tridandapani Susheela
Published: June 2021

Fcγ receptors are involved in mediating a variety of effector functions such as phagocytosis of IgG‐coated particles and the release of pro‐inflammatory mediators. Given the importance of FcγR function in autoimmune diseases and antibody‐based therapies for cancer, our goal is to understand the mechanism of action of these receptors. Two classes of FcγR exist. The activating receptors include FcγRI, FcγRIIa and FcγRIII whereas FcγRIIb is an inhibitory receptor. The activating receptors are associated with an Immunoreceptor Tyrosine‐based Activation Motif (ITAM). Interestingly, our experiments show that activation of FcγRI but not FcγRIIa leads to cytokine production. Further, blocking of FcγRI, but not FcγRIIa, activity leads to decreased killing of antibody‐coated tumor cells suggesting a primary role for FcγRI in cancer therapy. We speculated that these differences may be due to differential interaction of downstream signaling proteins with the γ‐chain versus FcγRIIa ITAMs. To test this notion we performed peptide pull‐downs and surface plasmon resonance analyses with synthetic peptides designed to mimic the ITAMs of the γ‐chain or FcγRIIa. Our data indicate that inhibitory phosphatases associate significantly better with FcγRIIa ITAM. We are currently investigating which of the two ITAM tyrosines is responsible for this association.
Breastfeeding Education and Support Trial for Obese Women
Authors: Chapman Donna J , Bermudez‐Millan Angela , Wetzel Katherine , Damio Grace , Kyer Nan , Young Sara , Pérez‐Escamilla Rafael
Published: June 2021

The objective of this randomized trial is to evaluate the efficacy of a specialized breastfeeding (BF) peer counseling intervention promoting exclusive breastfeeding among obese women. In this study, 206 obese, low‐income, pregnant women who are considering BF are being recruited from the prenatal clinic of a Baby‐Friendly Hospital. They are randomized to receive either specialized peer counseling or usual care. The intervention consists of 3 prenatal home visits, daily visits during hospitalization and up to 11 postpartum (pp) home visits. Breast pumps are available at no charge. The specialized peer counselors work under the supervision of an IBCLC and receive 40 hours of classroom training, plus 3 months of supervised clinical experience. To date, 59 participants have been recruited, with a mean BMI of 34.1 ± 5.5 kg/m2. Thirty‐four participants (intervention n = 20, control n = 14) have delivered their infants. All postpartum participants have initiated BF and 100% of those followed to 2 weeks pp (n=15) continued to BF. Our preliminary findings suggest that in a supportive environment, obese women are able to successfully establish breastfeeding, with or without a specialized peer counselor. Funded by the Donaghue Foundation and NIH EXPORT grant #P20MD001765.
Characteristic changes in plasma phosphatidylcholine species in humans depleted of choline
Authors: Costa Kerry‐Ann da , Miller Jameson , Giddings Morgan C. , Cui Zheng , Zeisel Steven H.
Published: June 2021

Phosphatidylcholine (PtdCho) is an essential membrane phospholipid synthesized from choline via the CDP‐choline pathway. PtdCho is also formed via sequential methylation of phosphatidylethanolamine (PE) using PE methyltransferase (PEMT). In the latter case, choline contributes its methyl group to PtdCho via betaine. PtdCho produced from the CDP‐choline pathway mainly contains medium chain, saturated fatty acid species such as 16:0/18:0, while those from the PEMT pathway preferentially have long chain polyunsaturated species such as 22:6. We investigated whether there are changes in PtdCho species that are characteristic of choline deficiency. Adult men were fed a diet containing 550 mg choline/d for 10 d. They were then fed a choline deficient diet for up to 42 d. Choline was returned to the diet of subjects who developed clinical symptoms of deficiency. At the end of each phase, plasma was collected and analyzed using gas chromatography/mass spectrometry. Changes in PtdCho mass 758 (16:0/18:2) and 760 (16:0/18:1) appear to be a signature of choline status in that the higher the ratio, the less likely the subject will deplete on a low choline diet. Principal component analysis identified other variations in PtdCho profiles that correlate with clinical choline status. Thus, PtdCho profiling may be a useful indicator of choline status.

This research is supported by grants from NIH‐ DK55865, DK56350 and RR00046.
Effects of Marginal Vitamin B6 Deficiency on Plasma Fatty Acid Profiles in Healthy Men and Women
Authors: Zhao Mei , Lamers Yvonne , Stacpoole Peter W , Gregory Jesse F
Published: June 2021

The interaction between vitamin B6 (VB6) and lipid metabolism is not well understood. Previous studies indicated that linoleic acid (LA) levels increased and arachidonic acid (AA) levels decreased in different tissues of VB6‐deficient rats, suggesting an altered n‐6 pathway of fatty acid (FA) synthesis. Our study aimed to determine the effect of marginal VB6 deficiency on plasma FA in human subjects. Thirteen healthy subjects (7 men and 6 women, aged 20–39 yr) received a 2‐d adequate diet followed by a 28‐d VB6 restricted diet (0.5 mg/d) to reach a marginal VB6 deficiency (plasma pyridoxal‐5′‐phosphate 20–30 nM). Fasting blood samples were collected after 2‐d of adequate diet and after 28‐d of VB6 restricted diet. Plasma total FA profiles were analyzed by gas chromatography/flame ionization detection. After 28‐d of VB6 restriction, AA concentration decreased significantly from (mean ± SD) 533 ± 72 nM to 479 ± 59 nM (p=0.004). The conversion indices of AA/LA also significantly decreased from 0.23 ± 0.04 to 0.20 ± 0.04 (p<0.001). There was a slight but non‐significant increase in plasma LA concentration. However, we did not observe any significant alteration in n‐3 fatty acid concentrations. In conclusion, marginal VB6 deficiency is associated with alterations in the distribution of n‐6 fatty acids in plasma lipids in healthy men and women. Supported by NIH grant DK072398 and GCRC grant M01‐RR00082.
Consequences of high‐fat diets on body growth and bone mineral density, in growing rats
Authors: Friedman Silvia Maria , Macri Elisa Vanesa , Rodriguez Patricia Noemi , Juiz Natalia , Suarez Cristina Graciela , Chaves Macarena Maria Conzalez , Ponce Graciela , Zeni Susana Noemi , LifShitz Fima
Published: June 2021

Objective: To investigate in growing rats, the effect of high‐fat diets with different n‐6/n‐3 fatty acids ratios, on body growth and parameters of bone densitometry. Weaning male Wistar rats (n=50) were fed ad libitum one of the following diets for 8 wk: control (C; fat=7% w/w) (AIN‐93G) or a high‐fat diet (20% w/w): soybean oil (SO), corn‐oil (CO), linseed oil (LO) or beef‐tallow (BT). Body weights (W) and lengths (L) were recorded every 4 days. W and L gain velocities were calculated (g W/100g W rat and L/10cm L rat, respectively). After 8 wk, rats were sacrificed. Total skeleton bone mineral density (BMD) and bone mineral content (BMC) were evaluated by DXA using a Lunar DPX‐L. Spine BMD was determined. Groups were analyzed as: a) BT vs. SO, CO and LO; b) SO, CO and LO; c) C vs. SO.

Results: a) BT achieved the lowest W, total skeleton BMC, BMC/W and spine BMD (P<0.0001); b) No significant differences were observed on anthropometric measurements, total skeleton BMC, BMD, BMC/W or spine BMD, but spine BMD on SO showed a tendency to a diminution (P=0.064); c) Anthropometric measurements, total skeleton BMC and BMD were similar but SO showed reductions of 10% on total BMC/W and spine BMD.

Conclusion: The combination of adequate amounts of dietary fat and n‐6/n‐3 fatty acids ratio protects bone health and maintains body growth. Awarded by UBACyT O 003‐4.
Validity of stage of change tools targeting total fat, fruit and vegetable intake, and exercise in a cardiac rehabilitation program
Authors: Froriep Franziska L. , Steinberg Francene M.
Published: June 2021

The objective of this study was to investigate the utility of 3 previously validated stage of change (SOC) questionnaires among patients beginning a cardiac rehabilitation program at the University of California Davis Medical Center. SOC for fruit and vegetable intake increase (SOC F&V) and for exercise (SOC Ex) were each assessed with one question, and SOC for total fat intake reduction (SOC Fat) with 10 questions. Actual fruit and vegetable (F&V) and fat intake were assessed by a 4‐day food record and physical fitness by total achieved metabolic equivalents during a treadmill stress test. Test‐retest reliability (2 week interval, n=15) was reasonably good when considering that most variability came from participants moving up one level in SOC. Convergent/criterion validity was good for SOC Fat (p=0.023, n=52) and SOC Ex (p=0.040, n=80), but poor for SOC F&V (p=0.086, n=51). SOC F&V had no association with F/V intake standardized to total kcals, but a stronger association with F&V intake standardized to body weight (p=0.016), suggesting that people may overestimate their F&V intake to a different extent based on body size. The findings indicate good validity for SOC Fat and SOC Ex. However, staging for SOC F&V in this population may be more accurate with a multi‐item questionnaire. Support: Departmental funds
Additive impairment of beta‐adrenoceptor‐mediated renal vasodilation by nicotine and cyclosporine
Authors: El‐gowilly Sahar M , Ghazal Abdel‐Rheem M , Goher Eman Y , El‐Mas Mahmoud M
Published: June 2021

We tested the hypothesis that nicotine aggravates the deleterious effect of the immunosuppressant drug cyclosporine (CSA) on β‐adrenoceptor‐mediated renovascular control. The effect of nicotine alone or combined with CSA on isoprenaline vasodilation in phenylephrine‐preconstricted perfused rat kidneys was assessed. Bolus isoprenaline (0.25 μmol) caused 35.4±4.0% reduction in the phenylephrine‐induced tone, which was attenuated by propranolol and tetraethylammonium (TEA), potentiated by hexamethonium and diclophenac, and abolished in KCl‐preconstricted tissues. NG‐nitro‐L‐arginine (L‐NNA), methylene blue, CHAPS, nifedipine, or SQ22,536 had no effect on isoprenaline vasodilation. Nicotine infusion caused 30.2±3.6% reduction in isoprenaline vasodilation and this effect was potentiated when CSA was concomitantly infused with nicotine. The effect of nicotine on isoprenaline vasodilation was reduced by hexamethonium and potentiated by L‐NNA, methylene blue, CHAPS, or nifedipine. The CSA exacerbation of nicotine‐isoprenaline interaction was abolished by propranolol, L‐NNA, methylene blue, CHAPS, L‐arginine, TEA, or nifedipine. It is concluded that nicotine and CSA produce additive impairment of β‐adrenoceptor‐mediated renovascular control and implicated the endothelial NO/K+ pathway in the interaction.

Supported by Faculty of Pharmacy, Univ. of Alexandria, Egypt.
DIFFERENTIAL EXPRESSION OF PRO‐ AND ANTI‐APOPTOTIC GENES UNDER THE INFLUENCE OF OXIDATIVE STRESS DURING CCL4‐MEDIATED LIVER INJURY IN MICE
Authors: Patel Chirag , Bhatt Shruti , Ray Sidhartha D.
Published: June 2021

Although past few decades made substantial progress in the understanding of the mechanisms of the liver injury, it has not been possible to design non‐hepatotoxic drugs or identify ‘effective’&’clinically‐safe’ antidotes. This is because our comprehension of mechanisms of cell injury and cell death at the molecular level is incomplete. From a mechanistic standpoint, CCL4‐liver injury has emerged as a prominent model. Therefore, this study investigated modulation of a series of key death regulatory genes, such as, Bcl‐2, Bcl‐xl, Bax, Bad, & p53 during the progression of liver injury. Female ICR mice (28–35g) were gavaged with toxic doses of CCL4 (1 ml/kg) in corn oil, and sacrificed 0, 6, 12, 18, 24 h later. Blood was collected for serum chemistry and livers for measurement of oxidative stress (MDA, SOD, NO2+NO3) and western blot analysis. CCL4‐mediated liver injury was confirmed by increases in ALT activity (U/L in CON‐57±10, 6hr‐196±41, 18hr‐20635±1678). Analysis of the liver biochemistry showed increased lipid peroxidation, nitric oxide production, genomic DNA fragmentation and SOD activity. Expression of both pro‐ and anti‐apoptotic genes (Bax, Bad, Bcl‐2, Bcl‐xl) including p53 and Cyt c release were significantly elevated at 18 & 24 hr. In conclusion, this study undoubtedly extends our understanding of some of the complex interplay between cell‐death regulating genes during CCL4‐liver injury.
The Effects of Aging on Microcirculatory Oxygen Delivery (QO2) in Contracting Rat Spinotrapezius Muscle
Authors: Copp Steven Wesley , Ferreira Leonardo F , Herspring Kyle F , Musch Timothy I , Poole David C
Published: June 2021

Advancing age alters both the structural and functional determinants of convective muscle oxygen (O2) flux. However, capillary red blood cell (RBC) hemodynamics have not been investigated during contractions in muscles of older animals.

Purpose:

To test the hypothesis that, during muscle contractions, old rats exhibit capillary RBC hemodynamics that constrain convective muscle O2 flux.

Methods:

The spinotrapezius muscle was observed via intravital microscopy in 8 old (O: 26–30 months) and 5 young (Y: 6–8 months) Fischer 344 x Brown Norway rats. Electrodes elicited 1‐Hz (6–8 volts) contractions for 3 minutes. Capillary RBC flux (FRBC) and the lineal density of flowing capillaries (LD) were measured at rest and during contractions. QO2m was calculated as FRBC x LD.

Results:

At rest, FRBC was elevated (Y: 23 ± 3, O: 34 ± 3 cells/s, P < 0.05) and LD was reduced (Y: 30 ± 1, O: 24 ± 1 capillaries/mm, P < 0.05) in O compared to Y rats, resulting in a similar QO2m (Y: 723 ± 153, O: 773 ± 86 cells/s/mm). However, during contractions QO2m was reduced in O muscles (Y: 1615 ± 472, O: 781 ± 120 cells/s/mm, P < 0.05). In contrast to Y, during contractions O muscles exhibited stoppage of flow in some capillaries where continuous flow was observed at rest.

Conclusion:

These data indicate that capillary convective O2 flux is severely impaired in muscles of older animals.
Prostacylin(PGI2) analogs stimulate receptor‐mediated increases in cAMP accumulation and ATP release from rabbit and human red blood cells(RBCs)
Authors: Sridharan Meera , Bowles Elizabeth , DuFaux Eileen , Adderley Shaquria , Hanson Madelyn , Stephenson Alan , Sprague Randy
Published: June 2021

Activation of the G proteins, Gs and Gi, stimulates increases in cAMP and ATP release from rabbit and human RBCs. Previously, we showed that the PGI2 analog, iloprost (ILO), stimulates increases in cAMP and ATP release from rabbit RBCs. Here we show that a structurally dissimilar analog, UT‐15C (UT), also produces concentration‐dependent increases in cAMP (n=4) and a 329±136% increase in ATP release (1 μM, n=9) from these cells. This effect is not limited to rabbit RBCs since ILO stimulated concentration‐dependent increases in cAMP (n=4) and a 328±92% increase in ATP release (1 μM, n=9) from human RBCs as well. To determine if these increases are receptor mediated, RBCs were pretreated with CAY10441 (10 μM), a PGI2 (IP) receptor antagonist. CAY10441 prevented ILO‐ and UT‐induced cAMP accumulation and ATP release. To ensure that responses were not the result of PGE2 (EP) receptor activation, RBCs were incubated with PGE2 (1 μM). PGE2 had no effect on cAMP or ATP release. Finally, using Western analysis, we determined that the IP receptor is present in rabbit and human RBC membranes. These results are consistent with the hypothesis that PGI2 analogs stimulate receptor‐mediated cAMP increases and ATP release from RBCs. This suggests a heretofore unrecognized mechanism by which these agents, when administered pharmacologically, can decrease vascular resistance. Funded by NIH, HL‐64180 and ADA, RA‐133.
Effects of angiotensin II on the constriction of goat cerebral arteries to endothelin‐1
Authors: Garcia‐Villalon Angel Luis , Salcedo Adely , Fernandez Nuria , Monge Luis , Narvaez Raul , Dieguez Godofredo
Published: June 2021

Angiotensin II (AII) may produce deleterious effects on blood vessels, including cerebral ones, and it is thought to be mediated by AT1 receptors; AT2 receptors may have protective effects. The modulatory role of AII on the vascular response to other vasoactive agents is less known. To examine the effects of AII on the cerebrovascular response to endothelin‐1 (ET‐1), 3 mm‐long segments from the middle cerebral artery and its branches were obtained from goats previously killed, and were mounted in organ baths for isometric tension recording. Under resting conditions, ET‐1 (10−11–10−7 M) produced concentration‐dependent contraction which was increased in the presence of a subthreshold concentration of AII (10−7 M). This increased contraction by AII was diminished in the presence of the inhibitor of AT1 receptors losartan (3×10−6 M), and augmented in the presence of the inhibitor of AT2 receptors PD123319 (3×10−6 M). Losartan or PD123319 alone did not modify the response to ET‐1. The relaxation induced by ADP (10−8–10−5 M) in the arteries precontracted with ET‐1 was similar in the absence and in the presence of AII. Therefore, AII by activating AT1 receptors may potentiate the cerebral vasoconstriction to ET‐1. AT2 receptors may not oppose to the cerebral vasoconstriction to ET‐1 but blockade of these receptors may augment the potentiating effect of AII on this vasoconstriction. (Supported in part by FMMM, FIS and MEyC)