bluuebunny/crossref_metadata_2025 huggingface dataset:
IP7K is kinetically well‐equipped for cell signaling but does not regulate CDK5 activity (1013.12)
Authors: Weaver Jeremy , Shears Stephen
Published: June 2021

Inositol pyrophosphates (PP‐InsPs) such as 1‐IP7 and IP8 serve as cellular signals that interface nutrient status with cellular growth and energy homeostasis; PP‐InsP turnover may impact bioenergetic diseases such as diabetes and cancer. In yeast, 1‐IP7 regulates phosphate response genes through its inhibition of the Pho85 cyclin‐kinase complex. Yet the relationship between cellular energy status and PP‐InsP synthesis is unclear. We characterized the ATP‐dependent kinase activity of human IP7K2. The IP7K2 Vmax (190 +/‐ 10 nmol/mg/min) is comparable to that of IP6K. Unlike IP6K, IP7K2 is not reversible in a physiologically‐relevant bioenergetic environment (i.e. [ATP]>[ADP]); the equilibrium point favors 80‐90% 1‐IP7 and IP8 accumulation. Furthermore, IP7K2 was insensitive to physiologically‐relevant changes to either [AMP], [ADP] or to [ATP] itself (since the ATP Km = 20‐40 µM). We further showed that regulation of yeast cyclin‐kinase by 1‐IP7 is not conserved for the homologous mammalian CDK5 complex. To interrogate potential signaling roles of IP7K2, we demonstrate in L6 skeletal myoblasts that either insulin, IGF or PDGF can each raise IP8 levels 2‐3 fold. Thus, we show independent of the bioenergetic context, IP7K2 is kinetically well‐equipped to participate in rapid cell‐signaling events and that cells place PP‐InsP synthesis under receptor regulation. Precedents suggest this is a valuable observation: demonstrations of receptor‐regulation of levels of IP3 ‐ and, later, PIP3 ‐ were critical in fermenting widespread interest in these molecules as cell‐signals.

Grant Funding Source: This work was supported by the NIEHS.
Phosphodiesterase expression and cyclic di‐GMP production in Streptomyces coelicolor (1013.22)
Authors: Geiger Robert , Tansey John , Bennett Jennifer
Published: June 2021

Streptomyces coelicolor is a pharmacologically important gram‐positive, filamentous, soil bacterium that displays a complex pattern of differentiation. The second messenger cyclic di‐GMP has been measured through the bacterium’s life‐cycle. We hypothesize that phosphodiesterases, which break down cyclic di‐GMP are differentially expressed to control developmental progression. We have constructed mutants that have a transposon insertion containing the gene for EGFP (enhanced green fluoresent protein) that disrupts a phosphodiesterase gene and forms a transcriptional fusion to the native phosphodiesterase promotor. EGFP was directly measured via ELISA and quantitated using fluorescence. Expression from the promotor for two phosphodiesterase genes varies between 36 and 96 hours. A plunge in EGFP expression is observed before the seventy‐two hour growth point which correlates with maximum sporulation in the wild type strain. In addition, cyclic di‐GMP production has been measured during the bacterium’s life cycle using HPLC‐MS/MS. The data shows differential production of the di nucleotide. These data provide further evidence that cyclic di‐GMP plays a role in the developmental progression of S. coelicolor.
Phosphorylation regulates association of PDE3A isoforms with distinct interactomes (1013.23)
Authors: Vandeput Fabrice , Szabo‐Fresnais Nicolas , Ahmad Faiyaz , Kho Changwon , Lee Ahyoung , Krall Judith , Hajjar Roger , Manganiello Vincent , Movsesian Matthew
Published: June 2021

Inhibitors of cyclic nucleotide phosphodiesterase PDE3A have inotropic actions in human myocardium, but their long‐term use increases mortality in patients with heart failure. We previously showed that two isoforms in cardiac myocytes, PDE3A1 and PDE3A2 ‐ whose amino‐acid sequences are identical except for a unique N‐terminal extension in PDE3A1 ‐ are selectively phosphorylated in response to the activation of different signaling pathways: PDE3A1 is phosphorylated at S312 by PKA and PDE3A2 is phosphorylated at S428 by PKC. Here we examined the effects of phosphorylation on protein‐protein interactions. Exposure of PDE3A1‐transfected HEK293 cells to isoproterenol (to activate PKA) and of PDE3A2‐transfected HEK293 cells to PMA (to activate PKC) led to shifts in their elution on gel‐filtration chromatography consistent with an increase in their interactions with other proteins. Two‐dimensional electrophoresis of co‐immunoprecipitated proteins indicates that the two isoforms bind to unique sets of proteins. These differences in the interactomes of PDE3A1 and PDE3A2 suggest that the two isoforms, which are indistinguishable with respect to catalytic activity and inhibitor sensitivity, may be selectively targeted by small molecules capable of disrupting their protein‐protein interactions and thereby blocking their recruitment to specific intracellular signaling complexes, with the possibility of eliciting inotropic actions without increasing mortality.
DNA methyltransferase knockout alters calcium homeostasis in HCT116 colon cancer cell line (1013.4)
Authors: Rovatti Jeffery , Wu Alexander , Kinney Shannon , Bose Diptiman
Published: June 2021

Calcium (Ca2+) is a ubiquitous second messenger regulating a myriad of cellular processes including proliferation, metabolism, and cell death. Increased cytosolic Ca2+ activates apoptosis and is cytotoxic. Epigenetic modifications, such as DNA methylation, play a major role in transcriptional gene regulation. The link between epigenetics and Ca2+ signaling is not clear, in either normal or disease states, such as cancer. In order to study this relationship, we measured differences in Ca2+ handling between the colon cancer cell line (HCT116) and its genetic counterpart HCT116‐DKO (double knockout of DNA methyltransferases 1 and 3b). Incubation of HCT116 with thapsigarin (TG), a sarcoplasmic endoplasmic reticulum Ca‐ATPase (SERCA) pump inhibitor, resulted in a concentration‐dependent increase in cytotoxicity. Interestingly, HCT116‐DKO cells were clearly resistant to TG‐induced cytotoxicity. To explain the differences in cytotoxicity ER Ca2+ store capacity was measured in each cell line. Fluo‐4AM loaded cells were challenged with TG (5uM and 10uM). Addition of TG produced an increase in cytosolic Ca2+ in both cell lines. However, the amplitude of Ca2+ transient in the HCT116 was 30% higher than in DKO cells. This could be explained by altered levels of Ca2+ regulatory genes, such as ORAI, STIM, SERCA, and TRP. Initial analyses revealed increased ORAI2 expression in HCT116‐DKO as compared to HCT116 cells. These results indicate that epigenetic mechanisms may be responsible for conferring the resistance of HCT116‐DKO cells to TG mediated cytotoxicity, suggesting the possible role of DNA methylation in regulating calcium signaling events.
Potential of glycated proteins produced during aging of cheddar cheese to modulate fecal bacteria from obese mice ex vivo and protect against colon inflammation (1018.5)
Authors: Yuan Jialing , Noratto Katherine , Munske Gerhard , Pilla Paulina , Mohanty Indira , Alcantara Zapata Diana , Noratto Giuliana
Published: June 2021

Cheddar cheese aging promotes the production of glycated proteins with potential to improve intestinal health.

We investigated major changes on proteins of aged cheddar cheese (Cougar Gold) during ripening up to 24 months using HPLC/MS‐MS. The proteins effects on fecal bacteria from obese mice after in vitro intestinal digestion, and the effects of metabolites produced during fecal fermentation were evaluated. We further assessed the effects of metabolites from fecal fermentation on production of reactive oxygen species (ROS) in colon epithelial HT‐29 and the fibroblast CCD‐18Co cells challenged with E. Coli lipopolysaccharides (LPS) following standard analytical procedures.

Results demonstrated that cheese aging (6‐24 months) produced soluble glycosylated proteins of MW ~ 23 kDa mainly derived from α‐S1‐ and β‐casein. Cheese glycated proteins were resistant to in vitro gastrointestinal digestion and changed the relative abundances of fecal bacteria by increasing Bacteroidetes/Firmicutes ratio and Bifidobacterium compared to the casein fermented controls. These changes resembled relative abundances of bacteria in feces from lean mice control. Furthermore, metabolites produced during fecal fermentation protected both the HT‐29 and CCD‐18Co cells from LPS‐induced ROS. We are currently investigating the molecular targets of glycated proteins and metabolites produced after fecal fermentation that protect colon cells from inflammation. These results are relevant for gut health.
Comparison of corresponding commercially processed foods from store and restaurant for sodium, fat, sugar and potassium content in a nationwide sample (1019.11)
Authors: Ahuja Jaspreet , Pehrsson Pamela , Haytowitz David , Showell Bethany , Nickle Melissa , Wasswa‐Kintu Shirley , Cogswell Mary
Published: June 2021

Data comparing the nutrient content of commercially processed food items from restaurant and store are limited. Eleven highly consumed food items were sampled nationwide from the two sources ‐ restaurant and store (e.g., chicken nuggets, frozen and fast food), under the USDA’s National Food and Nutrient Analysis Program. Nutrient data are now available for 4 pairs. Two sample T‐tests and Mann‐Whitney U‐tests were used to compare corresponding foods. Store chicken nuggets were lower in sodium and total fat than fast food restaurants (559±98.8 vs. 594±241 mg/100 g, p=0.028 and 16.2±3.2 vs. 20.4±4.9 g/100 g, p=<0.0001, respectively), as were store thin crust cheese pizzas (471±45 vs. 742±349.9 mg/100 g, p<0.0001 and 11.1±1.3 vs. 14±4 g/100 g, p=<0.0001, respectively). Store chili with meat and beans were higher in sodium (449±21.7 vs. 381±38.3 mg/100 g, p=0.0003), but about a third lower in total fat and saturated fat than those obtained from restaurant. Store macaroni and cheeses were higher in sodium and potassium than restaurant (682±74.1 vs. 361±83.4 mg/100 g, p<0.0001 and 355±26.3 vs. 135±35.2 mg/100g, p<0.0001). These results do not indicate any consistent differences between the two sources. These studies will help our understanding of the food environment, strengthen national nutrition monitoring, and update the USDA National Nutrient Database for Standard Reference (SR), the most authoritative food composition database in the US.

Grant Funding Source: Funding Source: CDC‐USDA Agreement 60‐1235‐0‐185
Influence of editing free text responses on nutrient and food group estimates from the Automated Self‐Administered 24‐hour dietary recall (ASA24) (1022.7)
Authors: Potischman Nancy , Zimmerman Thea , Douglass Deirdre , Dixit‐Joshi Sujata , Kirkpatrick Sharon , Subar Amy , McNutt Suzanne , Coleman Laura , Alexander Gwen , Kushi Lawrence , Thompson Frances
Published: June 2021

OBJECTIVE: To assess the impact of editing data from the ASA24 on nutrient and food group estimates.

METHODS: About 900 adults from 3 integrated health care systems completed recalls in an ASA24 evaluation study. In using ASA24, respondents may enter free text in 2 contexts. “Other” is a response to questions about food detail (e.g., brand name), in which case text is collected but not used in assigning food codes. Text can be entered for “Unfound foods” when respondents cannot find a food. The system asks questions to identify the food type and assigns a food code. Free‐text responses were compared to system‐assigned food codes and edited when necessary. Nutrient and food group values for unedited and edited data were then compared.

RESULTS: Of 1,013 recalls collected, 440 recalls included 749 foods with free text responses. The ASA24 system assigned correct food codes 70% of the time. Edits were made to 225 foods (30% of foods reported using free text), affecting 188 recalls (18.5% of all recalls). This editing process required over 60 hours of specialized staff time. The mean nutrient and food group values before and after editing showed no significant differences.

DISCUSSION: These results allow researchers using ASA24 to weigh the benefits and costs of manually reviewing free text responses.
The impact of dairy consumption on circulating cholesterol levels is modulated by common single nucleotide polymorphisms in cholesterol synthesis‐ and transport‐related genes (1038.4)
Authors: Abdullah Mohammad , Cyr Audrey , Labonté Marie‐Ève , Lépine Marie‐Claude , Couture Patrick , Eck Peter , Lamarche Benoît , Jones Peter
Published: June 2021

Dairy consumption impacts circulating cholesterol levels, a traditional risk marker for cardiovascular health, however, whether genetics play a role in response to dairy has not been established. Our objective was to evaluate 24 candidate SNPs within 13 cholesterol synthesis‐ and transport‐associated genes in relation to the cholesterol metabolism response to an intake of conventional and commonly‐consumed dairy products in Canada. Normolipidemic adults (n=101) consumed 3 servings/d of dairy (1% fat milk, 1.5% fat yogurt, and 34% fat cheese) or energy‐matched control products for 28 d using a multicentre, randomized, free‐living, crossover design. Relative to the control diet, dairy intake was associated with an increase in plasma levels of total cholesterol (TC) and LDL‐C only in carriers of the cholesterol transport gene ABCG5 SNP rs6720173‐G/G (4.86±0.20 vs. 5.05±0.20 mmol/L, n=72, p=0.008) and (2.86±0.14 vs. 3.03±0.14 mmol/L, p=0.002), cholesterol synthesis gene SREBF2 SNP rs2228314‐G/G (4.98±0.16 vs. 5.21±0.16 mmol/L, n=50, p=0.02) and (3.06±0.15 vs. 3.24±0.15 mmol/L, p=0.03), and bile acid synthesis gene CYP7A1 SNP rs3808607‐G/T (5.00±0.16 vs. 5.21±0.16 mmol/L, n=53, p=0.04) and (2.93±0.14 vs. 3.11±0.14 mmol/L, p=0.01) genotypes, respectively. These findings accordingly suggest the existence of a gene‐diet interaction modulating the impact of dairy intake on circulating cholesterol levels.

Grant Funding Source: Supported by Dairy Farmers of Canada (DFC) and Agriculture and Agri‐Food Canada (AAFC)
Bridges and barriers: understanding the educational pathways of underrepresented minority students in STEM (104.2)
Authors: Eagan Kevin
Published: June 2021

This presentation draws from data and analyses of a national study regarding postsecondary pathways of students who started college with an interest in pursuing a bachelor’s degree in science, technology, engineering, and mathematics. The study utilizes both quantitative (e.g,. multilevel modeling) and qualitative analytic methods to understand the college experiences of STEM aspirants that contribute to their successful completion of a STEM bachelor’s degree and future enrollment in STEM graduate and professional programs. Findings indicate that the structure of introductory science courses are critical in facilitating or hindering undergraduate STEM students’ successful matriculation through their programs. More frequent incorporation of evidence‐based teaching practices significantly and positively contribute to students’ level of academic engagement in these courses. Over the longer term, students who participate in undergraduate research, identify faculty mentors, and participate in department‐related academic clubs have significantly better odds of completing a STEM degree and enrolling in STEM graduate and professional programs.
The Undergraduate Affiliates Network as a faculty network (104.3)
Authors: Benore Marilee
Published: June 2021

Faculty advisors of the ASBMB Undergraduate Affiliate Network clubs continuously cite the importance of the faculty networking opportunities during the year and at the national and regional meetings. The opportunity to engage in creative and stimulating discussion with others teaching courses, and getting feedback from colleagues on research, is frequently noted as valuable. Junior faculty welcome the mentoring and career advice. Financial support for activities such as outreach is valuable to both students and advisors. In this session we will discuss the UAN, networking, and how to participate.
Effect of vegetarian diets on zinc status: a systematic review and meta‐analysis of studies in humans (1043.1)
Authors: Samman Samir , Foster Meika
Published: June 2021

Plant‐based diets contain more folate, fibre, and phytochemicals than omnivorous diets, however some micronutrients, especially zinc, are poorly bioavailable. The findings of studies exploring the zinc intake and zinc status in populations that habitually consume vegetarian diets are inconsistent. This study aims to investigate the effects of plant‐based diets on dietary zinc intake and status in humans using systematic review and meta‐analysis techniques. Thirty‐four studies were included in the systematic review. Of these, 26 studies (reporting 48 comparisons) compared males and/or females consuming vegetarian with non‐vegetarian groups, and were included in meta‐analyses. Dietary zinc intakes and serum zinc concentrations were significantly lower (‐0.88 ± 0.15 mg/d, and ‐0.93 ± 0.27 μmol/L, P=0.001, respectively; mean ± SE) in populations that followed habitual vegetarian diets compared to non‐vegetarians. Secondary analyses showed greater impact of vegetarian diets on the zinc intake and status of females, vegetarians from developing countries, and vegans. Populations that habitually consume vegetarian diets have low zinc intakes and status. Dietary practices that increase zinc bioavailability, the consumption of foods fortified with zinc, or low dose supplementation are strategies that should be considered for improving the zinc status of vegetarians.
A new biomarker for quinoa intake (1044.25)
Authors: Ross Alastair , Savolainen Otto
Published: June 2021

Quinoa (Chenopodium quinoa) is a pseudocereal grain consumed widely in the Andean regions of South America, and now receiving greater attention in many countries as a gluten‐free grain with a balanced amino acid profile. To date there has been little work on quinoa composition, clinical effects, or association of quinoa intake with health outcomes. We identified a range of alkylresorcinols in quinoa using LC‐MS/MS and GC‐MS/MS, including odd numbered homologues C17:0 ‐ C25:0 identified in whole grain wheat, rye and barley. In addition, we have identified homologues C18:0, C22:0, and C24:0, to our knowledge the first time these have been reported in nature. As these even numbered homologues may be unique to quinoa they may serve as a biomarker of quinoa intake. To test this hypothesis, we measured alkylresorcinols in plasma before and after intake of quinoa and were able to measure alkylresorcinol homologues in plasma.

Alkylresorcinols, in food previously only thought to be present in wheat, rye and barley, are also present in quinoa, and unique alkylresorcinol homologues may serve as specific biomarkers of this pseudocereal.
Epithelial‐mesenchymal cross talk in cancer behavior (1047.1)
Authors: Szabo Pavol , Smetana Karel , Dvorankova Barbora , Kolar Michal , Strnad Hynek
Published: June 2021

Epithelial‐mesenchymal interaction is important in the morphogenesis, development, wound healing and also in cancer behavior. It is dependent on the range of cytokines, chemokines, growth, proliferating and transforming factors produced by both the counterpartners. Tumor stroma contains, except the mesenchyme, also blood vessels, and the inflammatory cells. We focused our research on most abundant cell component of cancer stroma on cancer‐associated fibroblasts (CAF). They are producers of extracellular components, which are necessary to formation bioactive cancer microenvironment and are able to influence the biology of tumor predominantly the differentiation status of tumor cells and their migratory potential. We have isolated CAF from malignant tumors (squamous cell carcinoma, basal cell carcinoma (BCCF), melanoma, and skin metastasis of breast cancer) and have shown that these CAF are able to influence the differentiation status of co‐cultured cells from normal squamous or breast cancer epithelium. The results were compared with control experiments using normal human dermal fibroblasts, 3T3 mouse fibroblasts, and 3T3 fibroblasts influenced by the fibroblasts prepared from the basal cell carcinoma. Our results demonstrated that expression of luminal marker keratin 8 was influenced only by CAFs prepared from any tested tumors. In contrast, all tested types of fibroblasts showed a strong stimulatory effect on the expression of basal/myoepithelial marker keratin 14. Since keratin 14 is a marker of basal myoepithelial cells and keratin 8 is a marker of luminal cells, these double‐positive cells can be considered for precursor cells with properties close to stem cells. Their presence in clinical samples indeed signals very poor prognosis in cancer‐suffering patiens. In conclusion, our data indicate that CAFs are able to influence the phenotype of a breast cancer cell line and this effect is based on a tumor type‐unspecific mechanism.
Biomarkers for Western diet accentuated cellular damage in benzo(a)pyrene‐induced colon cancer (1052.2)
Authors: Harris Kelly , Pulliam Stephanie , Niaz Mohammad , Okoro Emmanuel , Guo Zhongmao , Washington Mary , Adunyah Samuel , Ramesh Aramandla
Published: June 2021

The role of fat content in diet has been linked to the development of several types of cancers. We have investigated the effect of dietary fat type on benzo(a)pyrene (an environmental toxicant)‐induced colon cancer in an adult male rat model, the Polyposis In the Rat Colon (PIRC) kindred type. Groups of PIRC rats (n = 5) were fed with AIN‐76A regular diet (RD) or Western diet (WD) and these rats also received 25,50 and 100 µg B(a)P/kg body wt., daily via oral gavage for a period of 60 days. At the end of exposure period, rats were sacrificed; blood samples were collected and concentrations of cholesterol, triglycerides, glucose, insulin, adiponectin, reactive oxygen species (ROS) and oxidative damage to DNA were measured. Colon tissues were scored for tumors, and preserved in 10% formalin for observation of pathological changes. Rats that received WD + B(a)P registered increased levels of cholesterol,triglycerides and insulin in comparison to rats that received RD + B(a)P and also controls. Levels of glucose stayed the same between WD and RD for 25, and 50 µg B(a)P/kg body wt., but showed a significant difference (p < 0.001) at 100 µg B(a)P/kg body wt. with WD group showing high concentrations. The concentrations of adiponectin did not vary much between WD and RD groups for all the B(a)P doses used. Maximal levels of ROS and oxidative DNA damage in blood were noticed in WD + B(a)P rats compared to RD + B(a)P rats and controls. Additionally, the DNA damage was found to be B(a)P dose‐dependent. The colon tumor counts were more in B(a)P + WD rats compared to their B(a)P + RD counterparts, and also exhibited a B(a)P dose‐response relationship, with 100 µg B(a)P/kg registering greater counts. Adenomas with high grade dysplasia were prominent in B(a)P + WD rats compared to B(a)P + RD rats. Taken together, our findings revealed that WD potentiates the development of colon tumors induced by B(a)P through proinflammatory action, characterized by an increased generation of ROS and oxidative damage.

Grant Funding Source: Supported by 5RO1CA142845‐04,5T32HL007735‐19,5R25GM059994‐11,Southern Regional Education Board
Academia‐industry alliances for biomedical innovation ő what can we do better? (1053.1)
Authors: Seymour E
Published: June 2021

Research conducted at academic medical centers significantly contributes to discovery, development, or repurposing of drugs and biologics. Industry partnerships can be a vital component to the initiation and translational success of such research. The financial benefits to both parties are considerable, but industry directly and indirectly assumes additional risk due to less control over the research program and from increased safety reporting burden to regulatory authorities. As a result, industry “external R&D” divisions are increasing their selection scrutiny and regulatory oversight of these partnerships. Academic investigators can improve opportunities for success by adopting Good Laboratory Practice in alliance with industry requirements, but these practices are uncommon in academic labs. In addition, with the support of industry, investigators can submit their own Investigational New Drug application (IND) to FDA to begin clinical research. This IND should be in electronic Common Technical (eCTD) format to align with industry practice. Finally, safety reporting should comply with industry needs, but industry should resource the investigator to help them meet their corporate regulatory requirements. Academia‐industry alliances are an increasingly important source of biomedical innovation, but should be carefully vetted to maximize benefit and minimize risk.
Methyl salicylate lactoside inhibits inflammatory response on fibroblast‐like synoviocytes and collagen‐induced arthritis by inhibiting NF‐κB activation (1056.2)
Authors: Zhang Tiantai , Xin Wenyu , Huang Chao , Du Guanhua
Published: June 2021

Methyl salicylate 2‐O‐β‐D‐lactoside (MSL), whose chemical structure is correlated with salicylic acid, is a natural product derivative isolated from a traditional Chinese herb. The aim is to investigate the therapeutic effect of MSL in collagen‐induced arthritis (CIA) mouse and explore its underlying mechanism. DBA/1 mice CIA model was adopted to evaluate the anti‐arthritis effect of MSL. Fibroblast‐like synoviocytes (FLS) from RA patients were stimulated by TNF‐α to determine the production of proinflammatory cytokines, immunofluorescence staining, and proteins expression.

The results showed that MSL significantly prevented the progression of arthritis in CIA mice without gastric mucosa toxicity. Administration of MSL inhibited the production of TNF‐α and IL‐1β, and the phosphorylation of IKKβ, IκBα, and p65 in joint tissue of CIA mice. MSL also suppressed the IL‐1β and IL‐6 production, and release of NO and PGE2 in TNF‐α‐induced FLS. Accordingly, the expression of iNOS, COX‐1, and COX‐2 were also reduced by MSL. We further demonstrated that MSL inhibited the phosphorylation of IKKβ, IκBα, and p65, blocked the nuclear translocation of NF‐κB in TNF‐α‐induced FLS. MSL inhibits the development of arthritis in CIA mice and without gastric mucosa toxicity. The underlying mechanism may be related to suppress the NF‐κB activation, and non‐selective inhibit the COX. Therefore, MSL has great potential to be a novel natural product agent for prevention and/or treatment of RA.
Anti‐inflammatory effect of IMMP in a metabolic syndrome model (1056.3)
Authors: Gomez‐Solis Antonieta , Reyes‐Esparza Jorge , Garcia‐Vazquez Francisco , Alvarez‐Ayala Elizabeth , Farfan‐Morales Eduardo , Rodriguez‐Fragoso Lourdes
Published: June 2021

The metabolic syndrome (MetS) is a prothrombotic, proinflammatory chronic state, that affects more than a third of the US and Mexican populations. The administration of IMMP (immunomodulator metallopeptide) in a model of liver damage modified markers of liver damage and decreases the degree of inflammation with decrement of IL‐6 levels. Therefore, the aim of this project was to evaluate the anti‐inflammatory effect of IMMP in a metabolic syndrome model. Zucker fa/fa and Zucker Lean rats were used to develop this protocol experimental. We formed 4 groups with 5 rats each: 1) Lean rats as control 2) Lean + IMMP 3) fa/fa rats as damage control and 4) fa/fa + IMMP. PBS was administered to the control groups and IMMP (50ng/Kg) to the treated group 3 times per week for 8 weeks. After of treatments were evaluated cytokine (IL‐6, TNF, IL‐1 β, IFN‐ γ and IL‐10), adipokines (leptin and adiponectin) and a chemokine (MCP‐1) in serum by ELISA and also were performed H&E histological staining in liver. The results showed a decrease inflammatory state with decreasing of levels of proinflammatory markers (IL‐6, TNF, IL‐1 β and IFN‐ γ, MCP‐1 and leptin) and increasing adiponectin levels in serum in fa/fa + IMMP group. In addition, we could see an improvement in the liver of fa/fa rats treated with the IMMP, decreasing the degree of steatosis in these rats. The IMMP had anti‐inflammatory effect and improved of damage liver in fa/fa rats.
New analogs of 3‐pyridyl ether as potential new ligands with high affinity and efficacy at selective nAChRs (1059.10)
Authors: Ogunjirin Adebowale , Fortunak Joseph , Brown LaVerne , Xiao Yingxian , Davila‐Garcia Martha
Published: June 2021

The development of subtype‐selective ligands for nAChRs holds high potential for better, more targeted therapeutic management of conditions where nicotinic acetylcholine receptors are implicated, including brain and peripheral neuropathies. Here it is proposed that subtype selectivity for a particular nAChR may be achieved by small structural modifications of a ligand, including a change in the distance between the electronegative elements of the compound containing the nicotinic pharmacophore. In order to test this hypothesis, analogs of A‐84543 (compound 1) were designed and synthesized as potentially new nAChR subtype‐selective ligands. A‐84543 and four analogues (compounds 2‐5) were characterized in rat brain tissues. All synthesized analogs of A‐84543 were partially characterized and their binding affinities and subtype‐selectivity and function were determined using stably transfected cell lines expressing different nAChR subtypes. Of all the analogs, only compound 1 and 2 were shown to have higher selectivity for β2‐containing, compared to β4‐containing nAChRs, with a selectivity ratio between α3β4 and α4β2 of 1,350 and 530 fold, respectively. Finally, A‐84543 was shown to be a partial agonist at α3β4 nAChRs (EC50 = 160 ± 40 nM, Emax of 45%), while compound 2 was a full agonist and had higher potency (EC50 = 24 ± 1 nM, Emax of 100%), compared to 100 µM nicotine. Based on its higher efficacy and good selectivity ratio, compound 2 can be useful as a pharmacological tool for β2‐containing as well as α3β4*‐containing nAChRs receptors if selective pharmacological tools are used to protect or mask the other receptors. From our results we conclude that compound 2, has good potential as a lead compound to produce more selective ligands.

Grant Funding Source: This research was supported in part by NIH grant R24MH06762701 and 5U54NS039407 to MDG.
Organization of cytochrome P450 system components in the endoplasmic reticulum (1064.8)
Authors: Park Ji Won , Reed James , Cawley George , Eyer Marilyn , Backes Wayne
Published: June 2021

Cytochromes P450 (P450s) are membrane‐bound proteins mainly found in the endoplasmic reticulum (ER). The ER membrane is heterogeneous, containing ordered (OD) and disordered domains (DD). These OD are more tightly packed and not readily solubilized with non‐ionic detergent, and are called detergent‐resistant membranes. Recently, our lab showed that CYP1A2 and CPR localize in OD. In this study, we examined the organization of other P450s in rabbit liver microsomes by Brij98 treatment and sucrose gradient centrifugation. Interestingly, CYP2E1 localized in DD and CYP2B4 was equally distributed between the OD and DD. Cholesterol depletion disturbed the distribution of OD‐localized CYP1A2, CYP2B4 and CPR. However, CYP2E1 localization was cholesterol independent. Lastly, to test if P450 localization is a property of the P450, P450 was incorporated into reconstituted systems, consisting of 1) phosphatidylcholine (PC) and 2) lipids mimicking the ER, forming both OD and DD. In PC vesicles both CYP2B4 and CYP2E1 proteins localized in late DD fractions. However, in ER mimicking vesicles, CYP2B4 was found in both OD and DD, whereas CYP2E1 distributed exclusively into DD, consistent with microsomal data. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and the distribution is dependent on the specific P450 being examined. Supported by R01 ES004344 and P42 ES013648
Nitric oxide attenuates the enhanced expression of Giα proteins in vascular smooth muscle cells from spontaneously hypertensive rats: molecular mechanisms (1065.15)
Authors: Sarkar Oli , Paul Ravinder , Anand‐Srivastava Madhu B.
Published: June 2021

We have previously shown that the nitric oxide donor, SNAP, decreased the expression of Giα proteins and associated functions in A10 vascular smooth muscle cells. The present study was undertaken to investigate if SNAP can also decrease the expression of Giα proteins and associated signaling in aortic vascular smooth muscle cells (VSMC) from 12‐week‐old Wistar‐Kyoto (WKY) and Spontaneously Hypertensive rats (SHR) and to further explore the underlying mechanism(s). Treatment of VSMC with SNAP (100 µM) for 24h decreased the expression of Giα‐2, ‐3 proteins. This decrease was subverted by inhibiting peroxynitrite with wither Mn (III) tetrakis (4‐benzoic acid) porphyrin (MnTBAP) or uric acid, and by inhibiting MEK with PD98059. However, inhibition of guanylate cyclase by 1H‐(1, 2, 4) oxadiazolo (4, 3‐a) quinoxalin‐1‐one (ODQ) was unable to restore the SNAP‐induced decrease in Giα‐2, ‐3. In addition, the enhanced activity of NADPH oxidase and protein levels of its subunits (Nox‐4, p47phox and p22phox) were decreased by SNAP. The decreased expression of NADPH oxidase subunits was restored towards control levels by MnTBAP. Furthermore, SNAP treatment decreased the enhanced levels of O2‐, TBARS and protein carbonyl in SHR to control levels. SNAP also attenuated the increased phospholyration of PDGFR, EGFR and IGF‐1R and c‐Src. These results suggest that SNAP decreased the enhanced expression of Giα proteins in VSMC from SHR by attenuating the increased oxidative stress, growth factor receptor activation and ensuing ERK1/2 signaling and not by a cGMP‐dependent mechanism.

Grant Funding Source: CIHR
Phospholipase C‐epsilon links G‐protein coupled receptor activation to inflammatory responses in astrocytes (1066.5)
Authors: Dusaban Stephanie , Purcell Nicole , Kunkel Maya , Smrcka Alan , Heller Brown Joan
Published: June 2021

Neuroinflammation underlies many diseases of the central nervous system (CNS) and the role of astrocytes in this process is increasingly recognized. Thrombin and the lysophospholipids, LPA and S1P are generated during injury and can activate G‐protein coupled receptors (GPCRs) on astrocytes. We hypothesized that GPCRs that couple to RhoA induce inflammatory gene expression in astrocytes through phospholipase C‐epsilon (PLCε). Using wild‐type (WT) and PLCε (KO) astrocytes, we recently demonstrated that thrombin, LPA, and S1P signal through PLCε to mediate sustained PKD activation and subsequently activation of NFκB to induce expression of inflammatory genes including COX‐2, IL‐1β, and IL‐6 (Dusaban, SS et al. PNAS, 2013). We are currently investigating the molecular mechanism by which PLCε mediates sustained PKD activation and downstream inflammatory responses. Preliminary data using Golgi targeted PKD FRET reporters suggests that PLCε mediates sustained activation of PKD at the Golgi. We also find that disrupting the Golgi with brefeldin A inhibits both PKD activation and COX‐2 expression. We further determined that COX‐2 gene expression was induced through PLCε following in vitro wounding and in vivo cortical stab wound injury. In light of growing evidence for a role of glial cells and S1P receptors in neuroinflammation, we investigated the S1P receptor subtypes coupled to PLCε and its downstream targets. Taking advantage of receptor KO astrocytes and receptor knockdown, we determined that coupling occurs primarily through the S1P3 receptor. Future work is aimed at identifying other GPCRs that signal through PLCε to mediate neuroinflammatory responses.

Grant Funding Source: GM 36927
The unexpected sphingolipid and cholesterol distribution in the plasma membrane (107.1)
Authors: Kraft Mary
Published: June 2021

Though the cellular abundances of sphingolipids and cholesterol are known to influence many biological processes, the organizations of these components within the plasma membrane are not well established. To address this issue, we have used a chemically specific and spatially well‐resolved imaging technique, high‐resolution secondary ion mass spectrometry (SIMS), to image the distributions of metabolically incorporated 15N‐sphingolipids and 18O‐cholesterol in the plasma membranes of intact mouse fibroblasts. Our data reveal sphingolipid domains in the plasma membrane. Contrary to current models, the cholesterol is not enriched in the sphingolipid domains, and is instead fairly evenly distributed within the plasma membrane. We have also used this approach to characterize the effects of cholesterol depletion and disruption of the cytoskeleton on the sphingolipid and cholesterol distributions in the plasma membranes of mouse fibroblasts. Our findings are inconsistent with the prevailing hypothesis that cohesive sphingolipid‐cholesterol interactions are responsible for plasma membrane organization. Instead, our research shows the cytoskeleton plays a major role in organizing the sphingolipids at the cell surface.
Role for DNA repair signaling in coronary artery stenosis (1071.9)
Authors: Meloche Jolyane , Pflieger Aude , Provencher Steeve , Bonnet Sébastien
Published: June 2021

Coronary artery stenosis is a vascular disease characterized by sustained inflammation and oxidative stress, leading to DNA damage. Despite these detrimental conditions, coronary artery smooth muscle cells (CoASMC) show increased proliferation and suppressed apoptosis leading to luminal narrowing. PARP‐1 is a critical enzyme acting as DNA damage sensor by promoting either DNA repair or apoptosis depending on the amount of damage. Recent studies demonstrated the implication of the glycogen synthase 3 (GSK3) enzyme, which when inhibited, favors DNA repair (promoting cell survival and proliferation) and resistance to apoptosis (by promoting mitochondrial hyperpolarization). Thus, we hypothesized that increased DNA damage in coronary artery of patients with stenosis promotes PARP‐1 activation and GSK3 inhibition. In freshly isolated human CoASMC issued from control or stenosed arteries, we measured DNA damage, PARP‐1 expression and phosphorylated GSK3 protein levels. We demonstrated that CoASMC from stenosed arteries exhibit increased DNA damage, enhanced PARP‐1 expression and GSK3 inhibition. These cells also present mitochondrial membrane hyperpolarization and consequently show increased proliferation and suppressed apoptosis. Our study suggests an important role of DNA damage signaling and metabolism dysfunction in coronary stenosis and opens the door to new avenues of investigation and treatment.

Grant Funding Source: Supported by CIHR grants
Impaired fasting blood glucose and circulating endothelial progenitor cell function (1072.4)
Authors: Bammert Tyler , Beckstrom Collin , Diehl Kyle , Kavlich Philip , Greiner Jared , Stauffer Brain , DeSouza Christopher
Published: June 2021

Impaired fasting blood glucose (IFG: 100‐125 mg/dL) is a risk factor for type 2 diabetes and confers cardiovascular risk. Functional impairment of circulating endothelial progenitor cells (EPCs) is linked to atherogenesis. We tested the hypothesis that IFG is associated with EPC dysfunction. Peripheral blood samples were collected from 22 adults (19 M/3 F): 10 normal fasting glucose (NFG; age: 57±3 yr; BMI: 29.7±1.4 kg/m2; glucose: 90±1 mg/dL) and 12 IFG (59±1 yr; 28.6±1.1 kg/m2; 104±1 mg/dL). Cells with phenotypic putative EPC characteristics were isolated from peripheral blood mononuclear cells and EPC clonogenic (colony forming unit [CFU] assay) and migratory (Boyden chamber) capacity as well as apoptotic susceptibility (intracellular active caspase‐3 concentrations) and angiogenic growth factor release (ELISA) were determined. EPC colony forming capacity was ~60% lower (p<0.05) in the IFG (10±3) vs NFG (23±6) group. Basal (0.31±0.1 vs 0.21±0.1 ng/mL) and staurosporine‐stimulated (2.9±0.3 vs 2.1±0.3 ng/mL) active caspase‐3 levels were markedly (~45%; p<0.05) higher in EPCs from IFG compared with NFG adults. There were no significant differences in EPC migration or release of angiogenic factors. These results suggest that impaired fasting glucose adversely affects the ability of EPCs to form colonies and resist apoptosis, potentially contributing to the increased cardiovascular risk with hyperglycemia.
The interplay between eNOS palmitoylation and phosphorylation: the triacsin C effect (1075.9)
Authors: Blakeman Nina , Weis Margaret
Published: June 2021

Decreased endothelial‐derived (NO) is central to the development of vascular disease. Earlier, we showed that triacsin C (TriC), a long chain fatty acyl‐CoA synthetase inhibitor, increased NO in cultured endothelial cells, and decreased endothelial nitric oxide synthase (eNOS) palmitoylation. Inhibiting eNOS palmitoylation suggests a decrease in NO production; however, eNOS regulation is complex. This leads us to hypothesize that the interplay between eNOS palmitoylation and phosphorylation regulates a phosphoryl switch increasing NO synthesis. After incubating bEnd.3 cells with TriC for 24 hours, we measured the phosphorylation status of key regulatory residues of eNOS. The mean relative density of each site (p‐eNOS/total eNOS) is summarized:

VehicleN=3
TriCN=3

p‐serine 1177
0.85 ± 0.07
0.39 ± 0.03
p< 0.0001

p‐serine 635
0.12 ± 0.02
0.10 ± 0.02
ns

p‐serine 617
0.04 ± 0.01
0.05 ± 0.01
ns

p‐threonine 495
0.60 ± 0.06
0.70 ± 0.09
ns

p‐serine 116
0.17 ± 0.03
0.07 ± 0.01
p<0.0157

Phosphorylation can mediate eNOS uncoupling and superoxide generation. To address this concern, we evaluated TriC’s effect to alter lipid peroxidation or induce the thioredoxin/thioredoxin reductase (Trx/TrxR) system. TriC significantly decreased thiobarbituric acid (TBA)‐malondialdehyde (MDA) adduct formation. Although not an indication of their activity level, no significant change to Trx/TrxR protein expression was seen.

Treatment
Trx
TrxR

Vehicle
0.05 ± 0.01
0.05 ± 0.01

TriC
0.06 ± 0.01
0.06 ± 0.02

Treatment
MDA Concentration (ÂµM)

Vehicle
100.0 ± 6.1

TriC
50.9 ± 4.7 ****

****=p<0.0001; n=9 These results support our hypothesis that TriC regulates an eNOS phosphoryl switch increasing NO synthesis, without increasing oxidative stress.
Interaction between the Cl‐ conductance associated proteins bestrophins and TMEM16A? (1077.2)
Authors: Aalkjaer Christian , Dam Vibeke , Boedtkjer Donna , Matchkov Vladimir
Published: June 2021

We have previously demonstrated two Ca2+ activated Cl‐ conductances in smooth muscle cells (SMC). One which is cGMP dependent and Zn2+ sensitive (IClcGMP) and one which is not (ICl). We have also shown that IClcGMP, but not ICl, disappears after knock‐down of bestrophin3. We here tested the role of TMEM16A for the two currents. In vivo downregulation of TMEM16A in segments of rat mesenteric small arteries with siRNA resulted in disappearance of both IClcGMP and ICl. Downregulation of TMEM16A also led to decreased expression of bestrophin1, 2 and 3. Immunoprecipitation of TMEM16A revealed bestrophin3 in the precipitate, while immunopreciptation of bestrophin3 revealed TMEM16A in the precipitate. Close apposition of TMEM16A and bestrophin3 in SMC was confirmed with proximity ligation assay. We suggest that TMEM16A is the poreforming unit in SMC and that bestrophin(s) may be subunit(s) which modifies the biophysical and pharmacological characteristics of the Cl‐ conductance of TMEM16A, although the data does not rule out the possibility that bestrophins are poreforming subunits idependently from TMEM16A.

Grant Funding Source: Supported by The Danish Research Council
The effect of substrate elasticity on the adhesion and elasticity of vascular smooth muscle cells (1077.8)
Authors: Hong Zhongkui , Jin Minshan , Bunyak Filiz , Ersoy Ilker , Sun Zhe , Li Zhaohui , Palaniappan Kannappan , Meininger Gerald
Published: June 2021

In previous studies, we found vasoactive agonists induce coordinated changes in elasticity and integrin‐mediated adhesion to extracellular matrix (ECM) in primary vascular smooth muscle cells (VSMC) that were cultured on plastic/glass dishes. In this study, we hypothesized that the mechanical properties/stiffness of the substrate may play an important role in determining cellular elasticity and adhesion in VSMC. To simulate the in vivo mechanical environment of the ECM for VSMCs, a flexible polyacrylamide (PA) gel 2D substrate with tunable elasticity was used for cell attachment. A comparison of two substrate elasticities (7 and 30 kPa) were tested and collagen‐I was coated on the surface of PA gel to provide a biologically functional substrate to promote adhesion of VSMCs. An atomic force microscope (AFM) was used to measure VSMC elasticity and VSMC adhesion to FN coated AFM probes. Quantitative characterization of changes in cell morphology, adhesion, and elasticity were determined by custom‐developed software. Initial results showed a significant increase in cell elasticity/stiffness (~ +120%), adhesion force (~ +30%), and adhesion probability (~ +100%) of the VSMCs cultured on the stiffer (30 kPa) substrates compared with the cells cultured on softer substrates (7 kPa). These findings support the hypothesis that elasticity of substrate exerts an effect on the mechanical and adhesive properties of VSMCs.

Grant Funding Source: NIH P01HL095486
The role of the VEGF/eNOS signaling pathway in cardiovascular development: a novel target to advance cardiovascular regeneration (1078.11)
Authors: Gentile Carmine , dos Remedios Cristobal , Drake Christopher , Davies Michael
Published: June 2021

In vivo and in vitro models of murine and human endothelial cells (ECs) and cardiomyocytes (CMs) have been used to study the emerging roles played by the vascular endothelial growth factor (VEGF)/endothelial nitric oxide synthase (eNOS) signaling pathway in cardiovascular development. In vivo studies using mouse embryos showed that eNOS is first expressed by Flk‐1 (VEGF receptor 2)‐positive ECs of the endocardium earlier than in CMs. In vivo studies also showed that this occurs as early as at 8 days post‐coitum in mice, a developmental stage when both ECs and CMs are proliferating. Our in vitro studies using human umbilical vein endothelial cells (HUVECs) identified cyclin B1 as a novel molecular target of the VEGF/eNOS signaling pathway controlling EC proliferation. VEGF/eNOS‐mediated S‐nitrosylation of cyclin B1 in HUVECs occurs following formation of the “signalosome”, the multimolecular complex responsible for the S‐nitrosylation of several target proteins via HSP90. Pharmacological inhibition of HSP90 using 17‐AAG reduced the VEGF/eNOS‐mediated proliferative effects on human ECs. Taken together, our studies showed that the VEGF/eNOS signaling pathway via S‐nitrosylation of cyclin B1 represents a novel molecular target for cardiovascular regeneration in patients with cardiovascular disease. Current studies are focused on establishing the role of the VEGF/eNOS signaling pathway in CM development.

Grant Funding Source: Supported by Marcus Blackmores Fellowship and the Translational‐in‐Aid‐Grant (Bosch Institute)