bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of PON2 and Proinflammatory Cytokine Genes in Rat Tissue Exposed to Combined oral Contraceptive Ethinylestradiol and Levonorgestrel
Authors: Wusu Adedoja Dorcas , Rotimi Solomin Oladapo , Saibu Gbemisola Morounke , Rotimi Oluwakemi Anuoluwapo , Giwa Rahman Ayodele
Published: November 2023

Paraoxonase (PON2) was identified as a genetic risk factor for cardiovascular disease (CVD) and usage of oral contraceptive (OC) is associated with increased cervical cancer and cardiovascular risk. PON2 protect against atherosclerosis development at the cellular level and this phenomenon could be related to their antioxidative properties. Therefore, the aim of the present study was to investigate the effect of OC on the expression of PON2, pro‐inflammatory cytokines interleukin one alpha (IL1α) and tumor necrosis factor alpha (TNFα) in the liver, kidney and brain of rats. Different dosage groups of eight female rats were treated with oral contraceptive (0.15mg levonorgestrel 0.03mg ethinylestradiol(A); 0.3mg levonorgestrel 0.06 mg ethinylestradiol (B) and 0.075 mg levonorgestrel 0.015 mg ethinylestradiol (C))/kg body‐weight(bw)). Two groups of eight rats were included in the study for a control group (D) and ≤0.1% DMSO (drug vehicle) group (E), which were not subject to drug administration for 21days. The levels of expression of the gene were assessed using quantitative reverse polymerase chain reaction technique. Combined oral contraceptive treatment produced a significant increase(p<0.001) in the level expression of renal IL1α and TNFα in all the groups compared to control in a dose‐dependent manner but has no significant effect on PON2.

Meanwhile, OC resulted in significantly (p<0.0001) reduced level of expression of hepatic IL1α with no significant effect on hepatic PON2 and TNFα level. In the brain, OC resulted in significantly (p<0.0001) reduced level of expression of TNFα in all dose groups and IL1α level at 0.015mg/bw. Although OC treatment did increase the expression of brain PON2 significantly (p<0.05) at the lowest dose. Therefore, pharmacological modulation of the expression of genes could constitute a useful approach for preventing atherosclerosis
Epidermal Growth Factor Substrate 8 is an essential partnering factor of the Forkhead box transcription factor FOXM1 in the regulation of cell proliferation in cervical cancer cells
Authors: Ngan Wan Ling , Yao Kwok‐Ming , Leung Wai Ying , Chan David Wai
Published: November 2023

The proliferation‐associated Forkhead box transcription factor FOXM1 (named Molecule of the Year in 2010) is increasingly recognized as marker and therapeutic target in cancers of multiple tissues of origin. FOXM1 plays critical role in maintaining the proliferation of cancer cells and its function is activated by mitogenic signals. In a yeast two‐hybrid screen for FOXM1‐interacting proteins, we identified Epidermal Growth Factor (EGF) Substrate 8 (EPS8), the phosphorylation of which is stimulated by EGF signaling. FOXM1 co‐localized with EPS8 at the G2/M phase of the cell cycle and both proteins can be reciprocally immunoprecipitated from lysates prepared from cervical cancer cells. Interestingly, the nuclear translocation of EPS8 was dramatically enhanced by treatment with the exportin 1 inhibitor leptomycin B, suggesting that EPS8 is actively regulated by nuclear export. Site‐directed mutagenesis defined a functional nuclear export signal within EPS8. To test the relevance of FOXM1‐EPS8 interaction in the regulation of FOXM1 function, we depleted EPS8 expression in cervical cancer cells by RNA interference. Importantly, depletion of EPS8 led to G2/M arrest and suppression of expression of FOXM1 and its known target genes including CCNB1 and CDK1. Moreover, chromatin immunoprecipitation analysis revealed recruitment of both FOXM1 and EPS8 to the promoters of CCNB1 and CDK1, consistent with EPS8 being a partnering factor of FOXM1 in transcriptional regulation. Taken together, our findings provide the first evidence to support a novel role of EPS8 in modulating the function of FOXM1, which is relevant to the development of cancer diagnostic strategy and targeted therapy.

Support or Funding Information

This work was supported by a grant from the Hong Kong Research Grants Council (17125214) awarded to Kwok‐Ming Yao
Effect of Covalent TAK1 Inhibitors on the Viability of Pancreatic, Renal and Colon Cancer Cells
Authors: Bernard Faviola , Singer William D. , Gurbani Deepak , Li Tan , Lim Sang Min , Gray Nathanael , Westover Kenneth
Published: November 2023

Many types of pancreatic, renal, and colon cancers are highly resistant to chemotherapy. A major contributor to this resistance is relatively high levels of TGF‐b‐activated kinase‐1 (TAK1) in the cancer cells, whose kinase activity leads to the suppression of pro‐apoptotic signaling pathways. Molecules which impede the binding of ATP to TAK1 have been developed as a strategy to overcome this resistance. They contain acrylamide groups that covalently attach to a cysteine in close proximity to the ATP‐binding site and irreversibly inhibit the activity of TAK1. Reversible analogs lacking the acrylamide group have been synthesized for comparison. We hypothesize that covalent inhibition of TAK1 will lead to loss of cell viability for selected cancer cell lines. The goal is to find cell lines where the covalent compound has significantly more activity than the non‐covalent counter‐part, suggesting that covalent TAK1 inhibition is responsible for the anti‐proliferative effects observed. We have examined the effect of three covalent‐ and non‐covalent‐binding pairs of inhibitors on the viability of six cell lines derived from these cancers. To test the viability we use the CellTiter‐Glow Assay, to determine the amount of ATP present in the cells. In many instances, the viability of these cell lines in culture was inhibited 50% – 100% compared to untreated controls following a 72‐hour exposure with 1 mM concentrations of the drugs. The covalent inhibitors were generally more effective than their non‐covalent analogs. One of the three pairs, Covalent SM1‐71, reduced viability of every cell line to a greater extent than the other two. The IC50 values for this effect were determined by dose‐response analysis, and the most potent covalent inhibitor often produced values lower than 100 nM. Future experiments will examine whether the reduction in vitality is a direct result of inhibition of the TAK1 signaling axis. Ultimately, these compounds may have potential as clinical candidates.
Role of ERK1/2 and p38 MAPKs in Tributyltin‐stimulated Interleukin 1 Beta Secretion and Production from Human Immune Cells.
Authors: Boules Mariam , Brown Shyretha , Whalen Margaret
Published: November 2023

Tributyltin (TBT) is an organotin compound that contaminates the environment due to its use as a biocide in a wide variety of applications. TBT has been found in human blood samples at levels as high as 265 nM. Previously, we have demonstrated that TBT interferes with the lytic function of human natural killer lymphocytes and alters the secretion of several pro‐inflammatory cytokines from lymphocytes, including interleukin 1 beta (IL‐1β). IL‐1β regulates cellular growth and immune responsiveness. Elevated levels of IL‐1β have been shown to increase the invasiveness of tumors. Our recent studies showed that TBT exposures of 5–25 nM (levels that are seen in human blood samples) caused increased secretion of IL‐1β from monocyte‐depleted peripheral blood mononuclear cells (MD‐PBMCs). In this study we examine the roles of the ERK1/2 and p38 MAPK pathways in TBT‐induced increases in IL‐1β secretion, protein production, and mRNA levels. MD‐PBMCs were treated with the MEK1/2 inhibitor PD98059 or the p38 inhibitor SB202190 for 1 h prior to exposure to TBT concentrations of 25, 10, and 5 nM for 24 h. Secretion of IL‐1β was measured using ELISA, intracellular protein levels were monitored using western blot, and mRNA expression was determined by RT‐qPCR. Results indicated that the TBT‐induced secretion of IL‐1β requires the ERK1/2 pathway. The increases in secretion are accompanied by increased protein levels in TBT‐exposed cells and this increase in protein production also utilized the ERK1/2 pathway. These data suggest that increased protein production accompanies the increases in secretion of IL‐1β seen in human immune cells exposed to TBT and these increases are dependent on MAPK pathway activation.

Support or Funding Information

Supported by NIH grant 5U54CA163066.
Effects of Pentachlorophenol (PCP) on Secretion of Interferon Gamma (IFNγ) from Human Immune Cells
Authors: Massawe Reda , Whalen Margaret
Published: November 2023

Pentachlorophenol ( PCP) is an environmental pollutant due to its use as an insecticide, fungicide, herbicide, and disinfectant. PCP has been found in human blood and urine samples. Exposure to PCP has been linked with cancers of the blood (lymphoma and myeloma) and kidney. Previous studies in our lab, have demonstrated that PCP interferes with the lytic function of human natural killer (NK) lymphocytes and reduces the levels of several cell surface proteins in NK cells. Interferon gamma (IFNγ) is an inflammatory cytokine that is produced by activated T cells and NK cells and to some extent by monocytes. IFNγ is a regulator of immune system function and inhibits viral infections. The objective of this study is to examine whether PCP is able to affect the secretion of IFNγ from several human immune cell preparations, which include: enriched NK cells; monocyte depleted (MD) peripheral blood mononuclear cells (PBMCs); and PBMCs. PBMCs were prepared by back flushing leukocyte filters followed by density gradient separation from red blood cells and granulocytes, with additional enrichment to yield MD‐PBMCs. Enriched NK cells were prepared using a rosetting procedure. IFNγ secretion was detected using an enzyme linked immunosorbent assay (ELISA). The results indicate that the secretion of IFNγ from all three types of cell preparations was altered when cells were exposed to concentrations of PCP ranging from 0.05 – 5 μM for 24 h, 48 h, and 6 days. The data showed that at least one concentration of PCP at one or more length of exposure significantly increased secretion of IFNγ from NK cells, MD‐PBMCs, and PBMCs in from all donors examined. These data suggest that exposure to PCP may significantly disrupt the immune regulation mediated by IFNγ. Additionally, the cell signaling pathways involved in the PCP‐induced increases in IFNγ secretion were examined.

Support or Funding Information

Supported by NIH grant 5U54CA163066.
Dibutyltin Exposures Alter Secretion of Interleukin 6 from Human Immune Cells
Authors: Brown Shyretha , Wilburn Wendy , Whalen Margaret
Published: November 2023

Dibutyltin (DBT) is an organotin compound that is used as a stabilizer in polyvinyl chloride (PVC) plastics (including pipes that distribute drinking water and bottles) and as a de‐worming agent in poultry. DBT is found in human blood samples, and DBT exposures alter the secretion of tumor necrosis factor alpha (TNFα) from lymphocytes. IL‐6 is an anti‐inflammatory agent, as well as a pro‐inflammatory mediator, that is produced by T lymphocytes and monocytes. It is responsible for immune response regulation as well as tissue repair and cellular growth. The aim of the current study is to determine whether exposure to DBT alters the secretion of IL‐6 from increasingly reconstituted preparations of human immune cells. We examined whether exposure to DBT concentrations of 0.05 to 5 μM after 24h, 48h, or 6 days alters IL‐6 secretion in a preparation of highly enriched human NK cells, a monocyte‐depleted preparation of human peripheral blood mononuclear cells (PBMCs) (MD‐PBMCs), PBMCs, granulocytes, and a preparation combining both PBMCs and granulocytes. The levels of IL‐6 were monitored using an enzyme‐linked immunosorbent assay (ELISA). The results indicate that DBT alters IL‐6 secretion from all cell preparations. The concentrations ranging from 0.5 μM to 5 μM caused diminished IL‐6 secretion, while the lowest concentration caused elevation of IL‐6 secretion. The concentrations and lengths of exposure to DBT that caused statistically significant increases in IL‐6 secretion from human immune cells varied from one donor to the next. Therefore, the data indicates that DBT‐induced alterations of IL‐6 secretion from immune cells may be a significant consequence of DBT exposures that may potentially affect immune competence.

Support or Funding Information

Supported by NIH grant 5U54CA163066
Polyisoprenylated cysteinyl amide inhibitors (PCAIs) block innate and VEGF‐induced angiogenesis
Authors: Nkembo Augustine T , Ntantie Elizabeth , Poku Rosemary A , Salako Olufisayo , Amissah Felix , Lamango Nazarius Saah
Published: November 2023

Angiogenesis is essential for solid tumor growth, therapeutic resistance and metastasis, the latest accounting for 90% of cancer deaths. Although angiogenesis is essential for the malignant transformations in solid tumors and therefore an attractive target, few drugs are available that block tumor angiogenesis. The focus has been to block signaling by receptor tyrosine kinases (RTKs), such as for VEGF, whose activations abrogate apoptosis and promote angiogenesis. The PCAIs were designed to modulate aberrant polyisoprenylated small G‐proteins such as mutant Ras whose constitutive activation promote RTKs activities, and cancer progression and insensitivity to drugs targeting the RTKs. We hypothesized that if the PCAIs disrupt the monomeric G‐protein activities, then they will effectively block angiogenesis and potentially be more effective than the monoclonal antibody and kinase inhibitor drugs that target the RTKs. In the study we determined the effects of PCAIs on human umbilical vein endothelial cells (HUVEC) tube formation, cell viability, cell migration and invasion. The effects of the PCAIs were also tested in vivo using the chick chorioallantoic membrane (CAM) assay model. The PCAIs, NSL‐BA‐040 and NSL‐BA‐055 inhibited HUVEC tube formation in a concentration dependent manner, effectively blocking tube formation but not killing cells at 0.5 μM. Also at 0.5 μM, NSL‐BA‐040 inhibited by 75% VEGF‐induced HUVEC migration and invasion in scratched and Transwell assays. These concentrations are approximately 4‐ to 15‐fold lower than the EC50 values against the viability of the pancreatic cancer cells, MIAPaCa‐2, Panc‐1 and BxPC‐3 with EC50 values ranging from 1.8 to 7.5 μM. Compared to the EC50 values of 8 to more than 50 μM against the viability of normal human embryonic kidney cells (HEK‐293), the concentrations that block angiogenesis were 16‐ to over 100‐fold lower. In the CAM assay, we observed a proportional decrease in the number of branched arterioles with respect to treatment with 0 – 10 μM NSL‐BA‐040. At 2 μM, arteriole branching was completely abolished and 10 μM induced the capillaries to rupture. Given that the PCAIs block angiogenesis at concentrations that spare normal cells and that angiogenesis is essential for tumor growth but otherwise limited to wound healing, feeding fat cells and uterine wall repair in adults, it is conceivable that these compounds can be developed into safer therapeutic agents for cancers and loss of vision due to retinal neovascularization.

Support or Funding Information

NIH/NIMHD G12 MD007582 and NIH/NCI/SC1CA190505
G‐protein Gamma Subunits As Novel Regulators of G‐protein Signaling
Authors: Choudhury Shilpa , Torres Matthew
Published: November 2023

G protein signaling systems (GPS), comprised of G protein coupled receptors (GPCRs), heterotrimeric G proteins (Gαβγ) and downstream effectors, are responsible for regulating a wide range of physiological processes including neurotransmitter and hormone signaling. Although the structure and function of GPS proteins are well known, evidence of post‐translational modifications (PTMs) that regulate signal transduction continue to emerge. Using a custom PTM informatics approach (Structural Analysis of PTM Hotspots; SAPH‐ire), we identified the N‐terminal unstructured tail of G protein gamma subunits (Gγ‐Nt) as a phospho‐regulatory element conserved between yeast and humans. Here, we used budding yeast, which harbors a single canonical GPS, to test the hypothesis that phosphorylation of Gγ‐Nt regulates signaling in vivo. We find that Gγ‐Nt in yeast (Ste18‐Nt) is hyper‐phosphorylated upon GPCR activation and in a cell‐cycle dependent manner, both of which events are independent of each other and suggestive of multi‐kinase phosphorylation. A systematic genetic screen revealed the MAPK, Fus3 (orthologous to MAPK‐Erk1), as the primary kinase responsible for GPCR‐dependent phosphorylation. Furthermore, analysis of G protein‐dependent signaling in yeast harboring Ste18‐Nt phosphorylation site mutations suggest that phosphorylation of Gγ N‐terminal tails may be important for regulating the kinetics of G protein pathway activation. Taken together, we have identified the unstructured N‐terminal tail of Gγ subunits as a target of MAPK‐dependent feedback phosphorylation and G protein signal regulation. We further conclude that using SAPH‐ire for computational prediction of biological function can be effective for identifying novel PTM regulatory elements in protein families. Finally, these studies can serve as a model to elucidate the regulatory role of Gγ‐Nt phosphorylation in mammalian G protein signaling systems.

Support or Funding Information

National Institute of Health (NIH) Funds
O‐GlcNAc Regulates CD4+ T Cell Differentiation
Authors: Machacek Miranda , Slawson Chad , Fields Patrick
Published: November 2023

In a normal immune response, antigen presenting cells (APC) engage the T cell receptor on CD4+ T cells, stimulating T cell proliferation and inducing T cell differentiation in conjunction with the cytokines secreted by APC. CD4+ T cells differentiate into effectors which secrete cytokines able to specifically neutralize different classes of pathogens. Pro‐inflammatory Th1 and Th17 effector cells are known to increase in situations of altered metabolism, such as type 1 and 2 diabetes and inflammatory bowel disease. A clear mechanism linking metabolic changes with pro‐inflammatory T cells is lacking. We hypothesize that elevated levels of O‐linked β‐N‐acetylglucosamine (O‐GlcNAc), a post‐translational modification of nuclear and cytoplasmic proteins, promotes pro‐inflammatory CD4+ differentiation. Since production of O‐GlcNAc involves input from carbohydrate, amino acid, fatty acid, and nucleic acid metabolism, the modification acts as a general sensor of a cell's nutritional status. To investigate the potential pro‐inflammatory role of O‐GlcNAc, we cultured mouse CD4+ T cells isolated from spleens with Thiamet‐G (TMG), a selective inhibitor of the enzyme that removes O‐GlcNAc (O‐GlcNAcase, OGA), before activation of the T cell receptor by plate‐bound anti‐CD3 and anti‐CD28. ELISA analysis of cytokines revealed a significant 95% increase in IL‐17A, the eponymous Th17 cytokine, in cells with TMG treatment compared to control cells that were cultured under permissive conditions in which differentiation of all effectors is equally possible. RT‐qPCR analysis showed significant increases in transcripts of IL‐17A and IFNγ, the main cytokine secreted by Th1 effectors. Taken together, these results suggest elevated O‐GlcNAc promotes the differentiation of pro‐inflammatory Th17, Th1, and/or Th1‐like Th17 cells, which are particularly pathogenic in autoimmunity. We then looked for changes in STAT3 and STAT4 activation, since these STATs are necessary for the transcription of Th17 and Th1 lineage defining programs, respectively. Phosphorylated STAT3 and STAT4, the transcriptionally active form of STATs, are increased in TMG‐treated cells within 10 minutes of activation of the T cell receptor. The relatively short time frame of this increase in phospho‐STAT levels after T cell receptor activation strongly implies that these changes are occurring as a result of T cell receptor‐induced kinase activity rather than through cytokine signaling via a JAK/STAT pathway. Taken together, our preliminary data suggest that elevated O‐GlcNAc levels prime pro‐inflammatory Th17 and Th1 CD4+ T cell differentiation by increasing activated STAT3 and STAT4 in a manner dependent on T cell receptor activation. Further study into the molecular mechanism of how O‐GlcNAcylation promotes pro‐inflammatory CD4+ T cells may provide insight into how nutritional excess affects inflammatory disease progression.

Support or Funding Information

This research is supported by NIH grant R01DK100595‐01 and NIH grant R01DK091277‐03.
A novel compound PT‐262 inhibits Oct4 and Nanog on the survivin blockage and apoptosis induction in human lung cancer
Authors: Chen Yan‐Ting , Li Jing‐Yi , Pan Pei‐Ying , Chen Chin‐Piao , Chao Jui‐I
Published: November 2023

PT‐262 (7‐chloro‐6‐piperidin‐1‐yl‐quinoline‐5, 8‐dione) is a novel synthetic compound, which derived from 6, 7‐dichloro‐quinoline‐5, 8‐diones that shows anticancer activities. However, the inhibition of cancer stem cells (CSC) and tumor growth in lung cancer by PT‐262 is still unclear. Here, we show that PT‐262 effectively inhibits the cancer stem proteins of Oct4 and Nanog, which are transcription factors in regulating CSC malignancy. Overexpressing Oct and Nanog in A549 lung cancer cells induced CSC‐like cell line, which we called A549‐ON, displayed drug resistance to chemotherapeutics. Interestingly, PT‐262 elicited higher cell death in the A549‐ON than the parental A549 cells. PT‐262 inhibited the protein expression of Oct4 and Nanog associated with survivin suppression and apoptotic induction. Moreover, A549‐ON cells expressed higher survivin protein levels. Transfection of Oct4 vector in A549 cells increased survivin protein expression. Besides, PT‐262 inhibited the anchorage‐independent ability and the xenografted lung tumor growth of SCID mice. Together, these results demonstrate that PT‐262 has ability to inhibit CSC malignancy and tumorigenesis in lung cancer.
Effects of Gryllus bimaculatus (Gb) fractions on alcoholic hepatitis
Authors: Ahn Mi Young , Hwang Jae Sam , Yun Eun Young
Published: November 2023

In the present study, several Gb fraction samples derived from ethanol extracts of Gb crickets, such as Gb butanol fraction, Gb ethylacetate fraction, Gb chloroform fraction, and Gb hexane fraction (100 mg/kg each), were examined for their liver protective effects against alcoholic hepatitis in SD rats. We used silymarin 40 mg/kg as a positive control. Animals in each group were intraperitoneally given glycosaminoglycan (5 mg/kg or 10 mg/kg), followed by treatment of each fraction sample. One hour later, 1.5 mL of 30% ethanol was orally administered to all groups. The above procedure was repeated for consecutive 3 days. Then, animals were selected to collect their blood samples for blood chemistry analysis, and the following results were obtained using plasma samples harvested: The serum alcohol concentration decreased by 0.08%, when treated with PBS only. However, the serum levels of alcohol in groups treated with Gb etylacetate fraction, Gb chloroform fraction, and hexane fraction decreased by 0.06%, 0.02% and 0.01%, respectively. The loss of alcohol concentrations was observed in the groups treated with 5 mg/kg and 10mg/kg of glycosaminoglycan derived from Gb, and the levels of alanine transaminase (ALT) also decreased. Serum ethanol concentrations decreased in the Gb ethylacetate 100 mg/kg group, and anti‐alcoholic hepatitis effects through inhibition of hyaluronate secretion were also noted. In the chloroform fraction group, anti‐alcohol accumulation effects in the body were found through the loss of plasma concentrations of ethanol, hyaluronate, alkaline phosphatase, and ALT. Serum alcohol levels were reduced in the Gb hexane fraction group. When each of Gb glycosaminoglycan at a dose of 5 or 10 mg/kg was administered to rats for consecutive 3 days, four serum parameters (ethanol, alkaline phoshosphatase, AST, and ALT) showed significant losses in alcohol absorption. The results suggest that Glycosaminoglycan may improve alcoholic hepatitis. We could also confirm the decreased levels of creatinine, hyaluronate and free fatty acid in the blood.

Support or Funding Information

ASBMB member
Chlorinated Lipids are Potential Mediators of Microcirculatory Dysfunction During Sepsis
Authors: Ford David A , Korthuis Ron J , Hotchkiss Richard S , Albert Carolyn J , McHowat Jane
Published: November 2023

Plasmalogen phospholipids contain a vinyl ether bond that is targeted by hypochlorous acid (HOCl) produced by myeloperoxidase activity in activated leukocytes. The product of plasmalogen oxidation by HOCl is 2‐chlorofatty aldehyde (2‐ClFALD), which in turn can be oxidized to 2‐chlorofatty acid (2‐ClFA). Previous studies have shown these chlorinated lipids are produced by activated human neutrophils. Our multi‐PI group is testing the hypothesis that these chlorinated lipids are mediators of endothelial dysfunction during sepsis. Initial investigations showed that plasma 2‐ClFA levels were significantly greater in septic patients in comparison to control healthy humans. Similarly, both plasma and cecum levels of 2‐ClFA were elevated in rats subjected to cecal ligation and puncture protocols compared to rats undergoing sham surgeries. Using intravital microscopy, superfusion of the mesenteric microcirculation with physiological levels of the 2‐ClFA molecular species, 2‐chloropalmitic acid, resulted in significant increases in leukocyte rolling and adhesion compared to both vehicle superfusion as well as superfusion with equimolar concentrations of the non‐chlorinated fatty acid, palmitic acid. The mechanism for leukocyte rolling and adhesion likely is mediated through increased surface expression of adhesion molecules since human endothelial cells treated with either 2‐chloropalmitic acid or 2‐chloropalmitaldehyde had significantly increased surface expression of P‐selectin, E‐selectin, VCAM‐1, and ICAM‐1 in comparisons to endothelial cells treated with either vehicle or non‐chlorinated fatty acid or fatty aldehyde (palmitic acid and palmitaldehyde, respectively). Further studies with human microvessel endothelial cells from both lung and mesentery demonstrated that in comparison to palmitic acid and palmitaldehyde both 2‐chloropalmitic acid and 2‐chloropalmitaldehyde reduced endothelial barrier function. These in vivo and in vitro studies support our hypothesis that chlorinated lipids alter endothelial function in the microcirculation. Furthermore, the robust production of chlorinated lipids at the level of the neutrophil/endothelial interface in the microcirculation during sepsis suggests that these lipids potentially mediate endothelial dysfunction during sepsis leading to multi‐organ failure.

Support or Funding Information

National Institutes of Health Grant GM115553
Production of Novel Glutathione Adducts of Halogenated Fatty Aldehydes in Inflammatory Animal Models
Authors: Duerr Mark A , Aurora Rajeev , Korthuis Ron J , Ford David A
Published: November 2023

α‐Halofatty aldehydes (α‐haloFALD) are liberated from plasmalogen pools found in most plasma membranes following attack by either hypochlorous acid (HOCl) or hypobromous acid (HOBr), which are enzymatically produced from hydrogen peroxide by myeloperoxidase and eosinophil peroxidase activity, respectively. Myeloperoxidase is released from the granulocytes of neutrophils, monocytes, and some tissue macrophages while eosinophil peroxidase is released from eosinophils. The exact role of α‐haloFALD in inflammatory pathologies is not entirely known, but the production of both α‐chlorofatty aldehydes (ClFALD) and α‐bromofatty aldehydes (BrFALD) by activated neutrophils and eosinophils has been previously established. The presence of a halogen at the alpha position of the aldehyde likely makes the alpha carbon susceptible to nucleophilic attack by cellular nucleophiles such as glutathione (GSH). In this study, the reactivity of the sixteen carbon ClFALD (α‐chlorohexadecanal) and BrFALD (α‐bromohexadecanal) with GSH is compared ‐‐in vitro and in primary human neutrophils and eosinophils. Both the chlorinated and brominated aldehydes are shown to be reactive with GSH and yield the same reaction product, however the brominated α‐haloFALD appears to be more reactive with GSH as the α‐bromine likely promotes a faster substitution reaction at the α‐carbon than the α‐chlorinated fatty aldehyde. The novel peptidoaldehyde products of both the chlorinated and brominated fatty aldehyde reaction with GSH (FALD‐GSH) have also been shown to be elevated in the plasma of both a rat cecal ligation puncture (CLP) sepsis model and the K/BxN inflammatory arthritis mouse model. Together, this study compares the reaction of ClFALD and BrFALD with GSH in vitro and in primary human neutrophils and eosinophils, and demonstrates the production of the resulting peptidoaldehydes, FALD‐GSH, in animal models of sepsis and inflammation‐induced arthritis. This novel class of peptidoaldehydes may prove to be a valuable biomarker of inflammatory pathologies, and future studies will investigate their role as mediators of disease.

Support or Funding Information

National Institutes of Health Grant GM115553 and MAD is an American Heart Association Pre‐Doctoral Fellow (14PRE20380048)
Characterization of 2‐Chlorofatty Acid Signaling Pathways
Authors: Hartman Celine , Cho Thomas Y , McHowat Jane , Ford David A
Published: November 2023

Monocytes and macrophages are key inflammatory cell mediators in the response to infection and tissue injury. Monocytes and macrophages produce a respiratory burst, resulting in the release of reactive oxygen species. Upon activation of these leukocytes, myeloperoxidase (MPO) is released. MPO catalyzes the conversion of hydrogen peroxide into hypochlorous acid (HOCl). HOCl then is able to target the vinyl ether bond of plasmalogens, an abundant class of phospholipids. This results in the production of a novel class of chlorinated lipids, including 2‐chlorofatty aldehyde (2‐ClFALD) and its oxidation product, 2‐chlorofatty acid (2‐ClFA). 2‐Chlorofatty acids have been shown to accumulate in activated human neutrophils and monocytes, as well as the plasma of rats subjected to lipopolysaccharide treatment or the cecal ligation and puncture sepsis model. The present study is designed to identify signaling mechanisms mediated by 2‐ClFA. Initial studies used dynamic mass redistribution (DMR) assays, which is a label‐free functional cell‐based technology that measures whole cell response to ligands. DMR analyses demonstrated that the 2‐ClFA, 2‐chloropalmitic acid, selectively stimulates THP‐1 cells and not HEK293 cells. Furthermore, the dose‐response for 2‐chloropalmitic acid was striking compared to the lack of response to equimolar concentrations of the non‐chlorinated fatty acid, palmitic acid. CCAAT/enhancer‐binding protein homologous protein (CHOP) mRNA increases selectively in response to 2‐chloropalmitic acid compared to palmitic acid in both RAW 264.7 and THP‐1 cells. CHOP mRNA expression in RAW 264.7 cells is inhibited by Bisindolylmaleimide, SB203580, and U0126 indicating 2‐ClFA elicited CHOP expression is PKC, p38 and MEK1/2 dependent. Thus, THP‐1 and RAW 264.7 cells are ideal cells to use for expression analysis and DMR to further elucidate the mechanisms and receptor by which 2‐ClFA elicits cell responses.

Support or Funding Information

National Institutes of Health Grant GM115553
Microarray analysis and anti‐hyperglycemia effect of Gryllus bimaculatus (GB, a type of cricket) extract in rats on high fat diet
Authors: Ahn Mi Young , Hwang Jae Sam , Yun Eun Young
Published: November 2023

Wistar rats fed a high‐fat diet (HFD) were orally administered with phosphate buffered saline (PBS, control), GB (100 mg/kg or 200 mg/kg), Provastatin or Isaria sinclairii (IS) extract, which is reported to have fat‐reducing effects, for either 1 or 2 months. GB's sero‐biochemial, hematological and anti‐oxidizing hepato‐cellular biomarker levels were evaluated to determine their anti‐lipidemic, anti‐inflammatory, and anti‐coagulant effects after 1 month of GB treatments in HFD (fat 60%) Wistar rats. The abdominal and epididymidal fat weights were measured and the composition of fatty acid was analyzed by GC/MS. The high fat dieted Wistar rats treated with GB showed increases in the unsaturated fatty acids (FA) ratio, especially with single (mono) FA. However, there were decreases in saturated fatty acids. Microarray analyses were performed with a rat 28K cDNA clone set array to identify the gene‐expression profiles for the GB exposed high fat dieted Wistar rat. The weight and fatty acid composition of abdominal fat and epididymidal fat, total cholesterol, LDL‐cholesterol, and triglyceride in GB treated rats were at lower levels than those of the control group. The anti‐oxidant hepato‐cellular biomarker levels, protein carbonyl content and malondialdehyde concentrationin GB treated rats were significantly decreased. Compared to the control, the GB treated rat group (treated at a dose of 100 and 200 mg/kg), had 190 up‐regulated genes including Gpm6a, Tmem14a and Fasin, with 235 down‐regulated genes including Cc121b, Glycan1 and Tcrb. The data suggest that Fasin‐related fatty acid synthesis and adipose differentiation related protein (Adfp), which is related to obesity, were upregulated by GB treatment, indicating their potential as therapeutic markers for anti‐atherosclerosis or inflammation.

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ASBMB member
An ER‐Golgi tether facilitates ceramide transfer out of the ER and alleviates ceramide toxicity
Authors: LIU LI‐KA , Toulmay Alexandre , Prinz Will
Published: November 2023

Ceramide is key intermediate for synthesis of sphingolipids, It is also an important second messenger in signal transduction. However, overproduction of ceramide triggers apoptosis and causes cell death. Our goal is to understand how cell adapt itself from ceramide toxicity stress. Ceramide is synthesized in ER, but is transferred to Golgi to form complex sphingolipids. Such lipid trafficking events can be divided into vesicular and non‐vesicular transport. Non‐vesicular transport usually occurs at Membrane contact sites (MCSs), which are regions where two organelles come into close proximity and are thought to bridge the gap between compartments during lipid transport. We aimed to investigate the role of MCSs in non‐vesicular ceramide transfer between the ER and Golgi. We screened for proteins could serve as potential tethers or facilitate non‐vesicular lipid transport. One promising candidate we identified was Nvj2. In yeast, ER‐to‐Golgi ceramide transfer is partially blocked in mutant with defect in vesicular transfer, such as sec18‐1ts. Ceramide transfer in this strain occurs only via non‐vesicular transport. Expression of Nvj2 resulted in increased non‐vesicular ceramide transfer in sec18‐1ts cells. Conversely, knockout of Nvj2 in sec18‐1ts cells exacerbated the block in ceramide transport. Importantly, Nvj2 is specifically localized at ER/medial‐Golgi junctions, closely juxtaposed to Golgi‐localized Aur1 (the enzyme required for the next step of ceramide conversion to sphingolipid), indicating that Nvj2 might be ER/medial‐Golgi tether that facilitate ceramide transfer by non‐vesicular manner.

We further show that Nvj2 alleviates ceramide toxicity through non‐vesicular transfer of ceramide out of the ER. orm1Δ orm2Δ cells are hypersensitive to ER stress due to its unusual ceramide accumulation. Vesicular transport pathway is also slowed down in the strain. We would like to investigate if Nvj2 can alleviate ceramide accumulation by exporting excess ceramide from ER to Golgi. Indeed, overexpression of Nvj2 rescues hypersensitivity of ER stress and also reduces the abnormally high level of unfolded protein response in orm1Δ orm2Δ. Accordingly, we found that Nvj2 alleviates ceramide accumulation in orm1Δ orm2Δ by facilitating ceramide transfer out from ER. Interestingly, we found that Nvj2‐GFP accumulates at ER‐medial‐Golgi contact sites during ER stress, suggesting that Nvj2 is actively recruited to these sites to expedite ceramide export, Taken together, We prefer a model that cells accelerate non‐vesicular transport of ceramide out from ER to adapt from ceramide accumulation stress.

Importantly, Nvj2 is conserved in higher eukaryotes and may share a similar function in ER‐to‐Golgi transport of ceramide, an intriguing possibility that warrants further investigation.
Phosphatidylserine Production at Membrane Contacts Sites Enhances Its Transport out of the Endoplasmic Reticulum
Authors: Kannan Muthukumar , Lahiri Sujoy , Liu Li‐Ka , Prinz William A
Published: November 2023

Mitochondrial membrane biogenesis requires phospholipid import into mitochondria. One lipid that is critical for mitochondrial function, phosphatidylethanolamine (PE), is synthesized in mitochondria from phosphatidylserine (PS) that is made in the endoplasmic reticulum (ER) and imported into mitochondria. PS transfer to mitochondria occurs at regions of close contact between ER and mitochondria, often called membrane contact sites (MCSs). We investigated the mechanism of ER to mitochondria PS transport. It has long been known that PS synthase activity is enriched in portions of ER that are associated with mitochondria, called mitochondria associated membranes (MAM). We wanted to know whether the enrichment of PS synthase in MAM is necessary for efficient PS transfer to mitochondria. To address this, we generated strains in which PS synthase was located in different portions of the ER. In cells lacking endogenous PS synthase, we expressed E. coli PS synthase (PssA) in the cytoplasm or fused to Sec63p (which is all over the ER) or Mmm1p, which is enriched at ER‐mitochondrial contacts. Remarkably, only Mmm1‐PssA restores PE in mitochondria to near wild‐type levels while Sec63‐PssA or cytoplasmic PssA did not. Thus, the enrichment of PS synthase in MAM promotes PS transport to mitochondria. This finding indicates that non‐vesicular PS transport is affected by where PS is synthesized in the ER and that PS synthesized outside of MAM is not efficiently transferred to mitochondria. Interestingly, PS synthase enrichment at ER‐endosomes contacts also promote PS transport to endosomes at these junctions.
HspA1A, a seventy‐kDa heat shock protein, binds to phosphoinositides
Authors: Kdeiss Brianna , McCallister Chelsea , Nikolaidis Nikolas
Published: November 2023

Seventy‐kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for maintaining cellular homeostasis. Apart from their indispensable functions in protein folding and refolding Hsp70s also localize at the plasma membrane and bind to specific lipids. The interaction of Hsp70s with lipids has direct physiological outcomes including lysosomal rescue, microautophagy, and promotion of cell apoptosis. We have recently characterized the binding properties of HspA1A, a major inducible Hsp70 in humans and mice, to several lipids, including phosphatidylserine and sulfatide. However, whether HspA1A binds to lipids essential for cell signaling, like phosphoinositides, remains unknown. Therefore, in this study, we characterized the interaction of HspA1A with several such lipids. We first established that HspA1A binds to monophosphorylated inositides with much higher affinity than to di‐ and triphosphorylated inositides. We also used high concentrations of potassium and found that HspA1A embeds in membranes when bound to specific lipids. We also determined that the nucleotide‐binding domain (NBD) of the protein binds to lipids quantitatively more than the substrate‐binding domain (SBD). However, for all lipids studied, the full‐length protein is necessary for binding. Furthermore, we used calcium and reaction buffers equilibrated at different pH values and determined that electrostatic interactions alone cannot fully explain the association of HspA1A with phosphoinositides. We then determined that lipid‐binding is inhibited by nucleotide‐binding, but it is not significantly reduced by protein‐substrate binding. Together, these results suggest that the interaction of HspA1A with phosphoinositides is specific, depends on the physicochemical properties of the lipid, and is mediated by multiple molecular forces and binding sites. Based on these data we propose a model according to which the NBD domain of HspA1A contains two lipid binding sites, the first one targets and docks the protein to the membrane, while the second one facilitates the embedding of the SBD into the membrane. These results uncover mechanistic details of the Hsp70‐lipid interactions and establish a framework of possible physiological functions of these interactions as they relate to chaperone mediated cell signaling and communication.
Acrolein Modification of Apolipoprotein A‐I Alters Protein Stability and Antimicrobial Activity
Authors: Hong Hea Jin , Weers Paul
Published: November 2023

Human apolipoprotein A‐I (apoA‐I) isa major component of high density lipoprotein and has an important role in cholesterolefflux. The protein also has antimicrobial properties, capable of neutralizing lipopolysaccharides (LPS) and destabilizing inner membranes of gram‐negative bacteria. Previous studies have shown that acrolein modifies the ɛ‐amino sidechains of lysine residues of apoA‐I. Acrolein is a highly reactive αβ unsaturated aldehyde and is generated mostly by cigarette smoking or during oxidation of lipids and amino acids. Acrolein‐modified apoA‐I has been detected in humana therosclerotic lesions and studies have shown that acrolein impairs lipid transport, leading to severe heart disease. The current study aims to investigate the effect of acrolein exposure on the antimicrobial activity of apoA‐I. The protein, containing 21 lysine side‐chains, was modified by acrolein and analyzed by SDS‐PAGE. At a 20‐fold molar ratio of acrolein to lysine, acrolein modification was evident by the appearance of oligomers caused by crosslinking. As the molar ratio of acrolein to protein increased, the intensity of the apoA‐Iband at 28 kDa decreased while heavily cross‐linked apoA‐I appeared with protein bands at 63, 98, and 126 kDa. Acrolein modification was further verified by western blot using an antibody (mab5F6) that specifically detects acrolein modified lysine residues in proteins. This confirmed the presence of acrolein‐modified lysine in the oligomers. To examine the structural changes of the modified protein, α‐helical content and the stability of the protein was measured using circular dichroism. The α‐helical content of acrolein modified apoA‐I was similar to unmodified protein (helical content of ~55%). However, the midpoint of guanidine‐induced denaturation increased from 0.97 (unmodified protein) to1.50 M (acrolein modified protein), indicating a significant increase in protein stability. This suggests that while modification did not alter the secondary structure, the tertiary structure was altered due to cross‐linking. To examine the binding of cross‐linked apoA‐I to phosphatidylglycerol, vesicles entrapped with calcein were used. Addition of apoA‐I to the vesicles resulted in calcein release as indicated by the increase in fluorescence intensity. The percentage of calcein released by apoA‐I decreased from 87.5 ± 2.3% to 4.7 ± 0.13% when the protein was modified by acrolein. This shows that acrolein‐modified apoA‐I binds phosphatidylglycerol less effectively, possibly due to loss of electrostatic interaction with the anionic vesicles or by the increase in protein stability caused by crosslinking.

Support or Funding Information

Research reported in this presentation was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC3GM089564.
Binding of lysine residues of apolipophorin III to phosphatidylglycerol membranes
Authors: Shah Kriti , Weers Paul M.M.
Published: November 2023

Apolipoproteins are the protein components of lipoproteins, which are responsible for transport of lipids to target tissues through the aqueous environment of the body. Apolipophorin III (apoLp‐III) is an abundant apolipoprotein of the hemolymph of insects. The 18 kDa protein is made of a bundle of five amphipathic α‐helices, which are the structural elements responsible for lipid binding. ApoLp‐III facilitates the lipoprotein mediated transport of diacylglycerols, which are released from fat body tissue for transport to the flight muscles during flight. Another novel function performed by apoLp‐III is modulating the cellular and humoral immune response. The protein up‐regulates expression of antimicrobial proteins, neutralizes lipopolysaccharides, and binds and deforms bacterial membranes. Lipopolysaccharides and phosphatidylglycerol (PG) are abundant components of the bacterial membrane, and our studies aim to better understand the antimicrobial properties of apoLp‐III of Locusta migratoria. This eventually may lead to improved strategies to treat bacterial infection and sepsis. ApoLp‐III contains 8 lysine residues, and the objective of this study was to characterize the ionic interactions between lysine residues of apoLp‐III and the negatively charged membrane components of gram‐negative bacteria, lipopolysaccharides and PG. To mimic bacterial membranes, PG vesicles with encapsulated calcein were prepared by extrusion. Protein‐induced membrane perturbation causes release of calcein which fluoresces intensively. When lysine side‐chains were neutralized by acetylation, binding to PG membranes was greatly reduced as indicated by the large reduction in calcein release from 80 ± 3.3% (unmodified apoLp‐III) to 21 ± 0.2% (acetylated apoLp‐III). In contrast, no effect on lipopolysaccharide binding was observed. To identify the lysine residues responsible for PG binding, site‐directed mutagenesis was used to change lysine into glutamine at position 52, 54, 63, 68 and 121. The proteins were expressed in bacteria and purified to homogeneity. The lysine variants were all able to release calcein, with values for most mutants around 70 to 80%. Since this is similar to the wild‐type protein, this indicates that these residues are not critical for PG binding. The remaining lysine residues at positions 143,145 and 161 were also changed into glutamine; the mutant proteins are currently expressed and purified. In addition, studies are underway to specifically acetylate the N‐terminal amino group by lowering the pH since the pKa for α amino groups is lower than the lysine ɛ‐amino group and hence making it a better nucleophile.