bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationNIH/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 InformationNational 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 InformationThis research is supported by NIH grant R01DK100595‐01 and NIH grant R01DK091277‐03.
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 InformationASBMB 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 InformationNational 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 InformationNational 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 InformationNational 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.Support or Funding InformationASBMB 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.