bluuebunny/crossref_metadata_2025 huggingface dataset:
High resolution structure of the phosphatase VHZ explains unexpected substrate specificity, and suggests the presence of metavanadate at the active site
Authors: Kuznetsov Viacheslav I. , Johnson Sean J. , Hengge Alvan C.
Published: June 2021

VHZ, a recently discovered phosphatase expressed in most fetal tissues and associated with several types of cancer, had been classified as a dual specificity phosphatase (DUSP23). However, screening assays of VHZ with 360 phosphopeptides has revealed a number of peptide phosphotyrosine substrates, but no activity toward phosphothreonine or phosphoserine substrates. A 1.1 Å crystal structure of VHZ bound to vanadate shows that VHZ has a deep and narrow active site that is more characteristic of protein‐tyrosine phosphatases rather than dual‐specificity phosphatases. Together, these results suggest that VHZ's designation should be reconsidered. The vanadate species in the active site is unusual, with no discernable electron density observed between the VO3 unit and apical ligands. This structure suggests a stabilized metavanadate, a previously unobserved mode of vanadate inhibition. A mechanism is proposed explaining the strong potency of vanadate as an inhibitor of these phosphatases under conditions where non‐inhibitory polyvanadate species dominate in solution. (Funding from NIH GM47297)
Proteomic evaluation of a caveolae‐enriched fraction from variable beta3 integrin backgrounds
Authors: Liu Jinsha , Jin Lianji , Bush Jason A
Published: June 2021

Both integrins and caveolins are known tumor biomarkers for early and late‐stage cancers. Caveolin‐1 can function either as a tumor suppressor or promoter depending on tumor origin. Excess integrins are generally associated with cancer progression/angiogenesis but some integrin knockout mouse models have increased tumor burden. This dichotomy is compounded by data indicating that specific integrin subunits can bind caveolins. We hypothesize that the mechanism of integrin endocytosis/recycling is dictated by the combination of integrin subunits present on the cell surface and the interacting caveolin members. Our project focuses on cellular systems where the beta3 integrin subunit is overexpressed having a profound effect on cell functions. Using a combination of RT‐PCR, subcellular fractionation, immunoblotting, and proteomic workflows, our preliminary data indicate that 1) expression of caveolin isoforms changes in variable beta3 integrin backgrounds, 2) the contingent of proteins from the endosomal compartment changes with respect to beta3 integrin expression levels. Taken together, this data provides further understanding for molecular interactions associated with integrin recycling and has implication for cancer cell motility.
Urokinase plasminogen activator receptor induced non‐small cell lung cancer invasion and metastasis requires NHE1 transporter expression and transport activity
Authors: Provost Joseph J , Berthelsen Nicholas , Anderson Kristina , Wallert Mark
Published: June 2021

In this study we investigate the relationship between urokinase plasminogen activator (uPA)/uPA receptor (uPAR) signaling and Na+/H+ exchanger (NHE1). 10nM uPA increased the carcinogenic potential of three NSCLC cell lines. Including an increase in cell proliferation, the percentage of cells displaying stress fibers, and an increase in anchorage‐independent growth. NHE1 inhibitor EIPA blocked these effects. To further evaluate the role of uPA/uPAR and NHE1 in tumor progression we assessed signaling events using full‐length uPA and uPA amino terminal fragment (ATF). We found that both uPA and ATF increased stress fiber formation approximately 2 fold, while uPA increased MMP9 activity 5.44 fold compared to 2.81 fold for ATF. Using two cell lines, one with reduced NHE1 expression (NHE1 K/D) and one with reduced uPAR expression (uPAR K/D), neither uPA nor ATF could stimulate stress fiber formation or MMP9 activity in cells with dramatically decreased NHE1 or uPAR expression. Finally, using in vivo tumor formation studies showed that when mice were injected with H460 cells 80% of mice formed tumors. This was compared to 20% of H460 uPAR K/D injected mice forming tumors and 10% of NHE1 K/D injected mice forming tumors. These data demonstrate that uPA/uPAR‐mediated tumor progression and metastasis requires NHE1 and suggests a potential therapeutic approach to blocking cancer progression. NIH Grant 1 R15 HL074924‐01A1
Modulation of H2O2 production in vitro by low level magnetic fields
Authors: Martino Carlos F , Castello Pablo
Published: June 2021

Background

In this paper, we present data on the influence of low level magnetic fields (0.5 μT‐2 μT) on the modulation of hydrogen peroxide (H2O2) biogenesis in human fibrosarcoma cancer cell line HT1080, and bovine pulmonary artery endothelial cells (PAEC) compared to cells exposed to geomagnetic field (control; 45 μT‐60 μT).

Methods

The extracellular quantification of the production of H2O2 may be a direct measure of the production and decomposition of superoxide radical. H2O2 can freely pass through cell membranes and can be detected outside the cell. For the quantification of H2O2 we used a method that has been successfully applied before. This method involves the oxidation of Amplex Red by H2O2 in the presence of horseradish peroxidase (HRP). Tri‐axial Helmholtz coils established the control static magnetic field. Low level fields were implemented by a μ–metal cylinder. The background time‐varying magnetic field was measured by induction with a sensor comprised of a set of square perpendicular coils.

Results

Reduction of the Earth's magnetic field suppressed H2O2 production in cancer cells and PAEC. The addition of catalase and superoxide dismutase (SOD) mimetic MnTBAP inhibited the magnetic field effect on H2O2 production.

Conclusion

Because most cancer cells undergo increased oxidative stress and ROS production due to excessive metabolic requirements to support an increased rate of cell division, it is reasonable to consider investigating a therapeutic that might target cancer cells through a free radical mechanism. Whether radio frequency exposure can influence growth rates and H2O2 production remains open and we propose to investigate it in the future. Modulating ROS production by magnetic fields may open new venues of biomedical research and therapeutic strategies.
Gene engineering, purification, crystallization and preliminary X‐ray diffraction of cytochrome P450 p‐coumarate‐3‐hydroxylase (C3H), the Arabidopsis membrane protein
Authors: Kim Young Ha , Kwon TaeWoo , Yang Hee Jung , Son BeomSeok , Youn BuHyun
Published: June 2021

Cytochrome P450s (P450s) are the most versatile biological catalysts in plants; however, because the structure of the P450s has not been fully established, their broad substrate specificity has been limitedly discussed. p‐coumarate‐3‐hydroxylase (C3H) is an essential enzyme for the biosynthesis of phenolic natural products in plants, but all attempts to express and purify C3H, have failed. In this research, we developed a bacterial expression of Arabidopsis C3H by combinational mutagenesis and purified C3H as a catalytically active form. The modified C3H could be purified in the absence of detergent, and crystallized in two forms (orthorhombic and trigonal space group) under different conditions. X‐ray diffraction was processed to a 4.0 Å resolution (first type crystal) and a 3.8 Å resolution (second type crystal). Although the diffraction results of C3Hmod crystals are not enough to determine crystallographic structure due to low resolution, the simplicity and rapidity of this technology are competitive advantages in comparison with other methods, and may contribute to structural analyses of other membrane proteins including P450s family.
CD45 is a Major Receptor Involved in Galectin‐8 Signaling of Preaparesis in HL‐60 cells
Authors: Gourdine Jean-Philippe Francois , Stowell Sean R , Cummings Richard D
Published: June 2021

The mechanisms of leukocyte turnover during an inflammatory response and its resolution are poorly understood. We recently showed that upon interactions with Galectin‐8 (Gal‐8), a 34 kilodalton heterodimeric tandem‐repeat galectin expressed by endothelial and epithelial cells, activated leukocytes express phosphatidylserine (PS) on their surface in the absence of apoptosis or DNA fragmentation. This can lead to phagocytosis of the cells in a process termed preaparesis, which fundamentally differs from that induced by factors signaling apoptosis. To identify the surface receptors involved in Gal‐8 signaling we took a proteomic approach to identify Gal‐8‐binding glycoproteins from human HL‐60 cells, a promyelocytic leukemic cell line with many signaling pathways in common to human neutrophils. Our previous studies showed that glycans containing poly‐N‐acetyllactosamine (poly‐N‐LacNAc) on HL‐60 cells were important for signaling. One major glycoprotein identified as a Gal‐8‐binding receptor on HL‐60 cells is CD45, a membrane glycoprotein that bears poly‐N‐LacNAc. We further characterized CD45/Gal‐8 interaction, by Western‐Blot, flow cytometry and confocal microscopy. Inhibition of CD45 cytoplasmic phosphatase activity reduced Gal‐8 induced PS exposure indicating a possible role of CD45 in Gal‐8 signaling preaparesis in human leukocytes.

This work was supported by National Institutes of Health Grant HL085607 to R.D.C.
Acid sphingomyelinase, syntaxin 4 and nitric oxide: how regulation of exo‐endocytic pathways is implicated in tumor chemotherapy
Authors: Perrotta Cristiana , Assi Emma , Bizzozero Laura , Cazzato Denise , Clementi Emilio
Published: June 2021

Acid sphingomyelinase (A‐SMase) plays an important role in apoptosis and its activation involves the exocytosis from intracellular compartments to the plasma membrane. One of the players in the apoptotic event is nitric oxide (NO) that inhibits apoptosis through the generation of cyclic GMP (cGMP) and the ensuing inhibition of A‐SMase. Here we demonstrate the mechanism of this regulation and the possible implications.

We found that A‐SMase is localized in lysosomes and in early/recycling endosomes, where it co‐localizes with syntaxin 4 (synt 4), a SNARE protein. After receptor activation A‐SMase translocates to the cell surface through an exocytic event in which synt 4 is required. In addition, we demonstrated that translocation of A‐SMase is required for its activation because the silencing of synt 4 inhibited A‐SMase activation. Synt 4 is the target of NO that acts via generation of cGMP and activation of G‐kinase, inducing phosphorylation of synt 4 on serine 78 and triggering proteasome‐dependent degradation of synt 4. Of importance, we found that this is a relevant mechanism in chemoresistance since NO produced by tumor‐associated macrophages protects glioma cells from cisplatin‐induced apoptosis. The identification of the interaction among ASMase, synt 4 and NO reveals an important mechanism of action of NO relevant in cancer and indicates synt‐4 as a novel target for antitumour strategies.
Signaling Mechanisms of Apoptosis Resistance in Lymphoid Cells Exposed to Hyperosmotic Stress
Authors: Scoltock Alyson Bell , Bortner Carl , Cidlowski John
Published: June 2021

Hyperosmotic stress is well known to induce apoptosis in T‐lymphocytes. We have now generated a lymphocyte cell line (OS 4–15) that is highly resistant to hyperosmotic stress when compared to the parental, S49 mouse t‐cell lymphoma cells. These cells are also resistant to other stimuli that trigger the intrinsic apoptotic pathway but not to activators of the extrinsic pathway. To delineate the mechanism of resistance in the OS 4–15 cells we studied the expression of the apoptosis proteins Bim, Bax, Bcl‐2, Bclx‐L, and survivin as well as caspases 8, 9 and 3. Little difference in the levels of these proteins was detected when compared to the parental cells. Since the OS 4–15 cells retain the ability to undergo apoptosis, we evaluated the initial signaling pathways activated by hyperosmotic stress in these cells. Hyperosmotic treatment resulted in p38 and JNK activation to a similar extent in both cell types, with no activation of ERK. In the parental cells, AKT was rapidly inactivated by hyperosmotic stress however AKT activity remained elevated in the OS 4–15 cells. Interestingly, inhibition of AKT activation sensitized the OS 4–15 cells to osmotic stress‐induced apoptosis. These data suggest that increased activation of AKT is at least partially responsible for the resistance to intrinsically induced apoptosis observed in the OS 4–15 cells.
How do infants acquire creatine?
Authors: Brosnan Margaret E , Edison Erica E , Brosnan John T
Published: June 2021

A growing infant acquires creatine (CR) from mother's milk (or infant formula) and/or de novo synthesis. We have measured CR and guanidinoacetate (GAA) content of mother's milk and of a number of commonly used infant formulas. We used these data to estimate CR intake by breast‐fed and formula‐fed infants and to compare these estimates to the infants’ daily requirements for CR. Our data reveal that diet is a relatively minor source of CR in breast‐fed infants; therefore, the great bulk of new CR must be synthesized by the infant. The GAA concentration in milk and formulas is extremely low. The situation with formula‐fed infants is more complex. The CR levels in cows’ milk‐based formulas are higher than in mothers’ milk so that the burden of CR synthesis is less in infants fed these formulas than in breast‐fed infants. Soy‐based formulas, however, contain essentially no CR; infants fed these formulas must synthesize all of their CR endogenously. These data have implications for infants suffering from CR‐deficiency syndromes as well as for the burden that CR synthesis places on infants’ glycine, arginine, and methionine metabolism (the amino acids required for CR synthesis). Funded by CIHR
Does Digital Photography Improve the Accuracy of Diet Records in the Elderly?
Authors: Brown Alisa , Hakkak Reza , Gonzales Dana , Wolfe Robert , Hays Nicholas
Published: June 2021

Utilizing digital photography alongside food records could prove to be a feasible way to refine the accuracy of food records in the elderly population. We determined whether the accuracy of 3‐day food records is improved by use of an accompanying digital photographic food record. Three day diet records were entered into the Nutrition Data System for Research (NDSR) both with and without the accompanying digital photography. We used total estimated energy expenditure (TEE), as assessed by the DRI's to determine overall accuracy of each method. Bland Altman regression, paired t‐tests and ratio of energy intake to total energy expenditure was used to determine variance between the methodologies. Our results shows that the digital photographic record helped to refine 157 items during data entry. Overall energy intake to TEE ratio was similar, indicating a high level of agreement between each method. Mean energy and protein intake did not significantly differ between the methodologies (p=0.75 and 0.73). We concluded that while accuracy was improved through the use of accompanying digital photographs, this did not significantly correct the tendency of underreporting. Given the qualitative improvement in food record data obtained using this technique, further research is needed to determine optimal use of digital photography.

Supported by Dept. Dietetics and Nutrition and NIH to RW.
Association of body fat indicators with lipid concentrations in US children aged 6–11
Authors: Masters Melissa , Stanek-Krogstrand Kaye , Albrecht Julie A
Published: June 2021

Childhood obesity is a major health issue in the US. The associations of body fat indicators were compared with lipid concentrations in a representative sample of US children.

Data from 1819 participants aged 6–11 y from the 2001–2004 NHANES were assigned to specific groups according to gender (male/female) and BMI (normal/overweight/obese) and total cholesterol and HDL cholesterol were compared. Prevalences of adverse total cholesterol (>200 g/dL) and HDL cholesterol (<35mg/dL) in this population were determined according to BMI categories. Associations between three indicators of body fat (BMI, subscapular skinfold, triceps skinfold) and lipid concentrations were assessed.

For children 6–11 y, 8.6% ± 0.9% had high total cholesterol and 5.0 ± 0.9% had low HDL cholesterol. Prevalence of adverse total and HDL cholesterol in obese US children was greater than normal weight participants. In multiple linear regressions adjusted for age, gender, survey period, and race‐ethnicity, HDL cholesterol levels were significantly inversely associated with BMI (p<0.001), subscapular skinfold (p<0.001), and triceps skinfold (p<0.001). Total cholesterol levels were significantly associated with subscapular skinfold (p=0.03) and triceps skinfold (p=0.007).

High adiposity and adverse lipid concentrations were significantly associated in children aged 6–11 y.

Grant Funding Source: NA
Consuming a lentil‐based sports nutrition bar affects metabolic and performance measures during endurance exercise
Authors: Zello Gordon Alexander , Jochim Jennifer T. , Rooke Julianne , Chilibeck Philip D.
Published: June 2021

Low glycemic index (GI) carbohydrates (CHO) may be superior to high GI CHO if consumed before exercise by providing a prolonged energy source and sparing muscle CHO. We compared pre‐exercise feeding with: 1) a low GI lentil‐based sports nutrition bar; 2) a commercially‐available moderate GI sports nutrition bar with a similar macronutrient content; and 3) diet jelly (placebo) on metabolism and performance during endurance cycling. Using a randomized, counterbalanced, cross‐over design, endurance athletes (n=6) consumed 1.5 g/kg available CHO from a lentil bar, the commercially‐available bar, or consumed placebo 1h before endurance cycling (75min, 65% of max aerobic power), followed by a 7km time trial. We also compared post‐exercise consumption of these bars on next‐day exercise performance as an assessment of recovery. Each feeding condition was separated by 1wk. Respiratory exchange ratio (RER) during the last 10 minutes of the 75min cycling trial was significantly different between conditions with control (0.82 SD 0.01) < lentil bar (0.87 SD 0.04) < commercially‐available bar (0.89 SD 0.03), respectively (p<0.05) indicating more fat oxidation relative to CHO oxidation late in exercise after consumption of the lentil bar compared to the commercially‐available bar. Time trial performance was significantly improved after consumption of the lentil bar (587 SD 42 s) and the commercially available bar (584 SD 61 s) compared to control (618 SD 72 s) (p<0.05). The next‐day recovery time trial performance was better in the lentil bar condition (547 SD 23 s) compared to the commercially‐available bar (568 SD 29 s) and control (566 SD 34 s) (p<0.05). A low GI lentil‐based sports bar offers a metabolic advantage during endurance exercise (i.e. lower RER) and enhances recovery following exercise compared to a moderate GI sports nutrition bar.
Evaluation of monoamine oxidase‐B (MAO‐B) inhibitory activity of different plant sources
Authors: Deiab Shihab E , Mazzio Elizabeth A , Soliman Karam F.A.
Published: June 2021

In humans, MAO‐B (EC 1.4.3.4) is located in neurons and glia where it is believed to contribute to age associated neurological disease and the generation of free radicals The purpose of the current study is to assess the ability of variety of plant sources in inhibiting MAO‐B aiming to identify natural products that can be used in the prevention or in retarding the progression of Parkinson's disease. In this study, enzyme activity of human MAO B‐ derived from insect cells infected with recombinant baculovirus was evaluated by monitoring production of peroxide upon the addition of the substrate benzylamine ± treatment (0.2–.83mg/ml final concentrations). Results obtained indicate that plant extracts having the greatest potency included: Humulus Lupulus, Gentian Root, Green Tea, Bakuchi Seed, Turmeric, Curcuma longa rhizome, Skull Cap, Amur Cork Tree, Spearmint, Wintergreen, Peppermint, Artemisia annual herb, Lady's Mantle, Psoralea corylifolia fruit, Glycyrrhiza uralensis Mai Dianthus superbus herb, Rosemary, Sage, Cilantro, Marjoram, Comfrey, Perilla frutescens leaf. Polygonum tinctorium levis, Isatis tinctoria and fruit extracts derived from orange, raspberry, strawberry and acai. The results of this study indicate that plant extracts examined which effectively inhibits MAO‐B may be promising for use in the management of patients with Parkinson's disease (Supported by a NIH grant RR 03020)
Effect of ketone treatment and glycolysis inhibition in brain cancer cells (U87MG) and rat primary cultured neurons exposed to hyperbaric oxygen and amyloid beta
Authors: Bennett Angela , Ari Csilla , Kesl Shannon , Luke Jaimie , Diamond David , Dean Jay , D'Agostino Dominic
Published: June 2021

Mild ketosis from caloric restriction or the ketogenic diet confers a therapeutic effect against a wide range of pathologies, including brain cancer and Alzheimer's disease. Based on previous work, we hypothesized that supplemental ketones, glycolysis inhibition (lonidamine) and hyperbaric oxygen (HBO) would decrease viability of U87 MG cells. Furthermore, we hypothesized that ketones would prevent oxidative stress in primary neurons associated with amyloid beta (Ab) toxicity and HBO. LDH assays were used to measure cell viability in response to ketones (1–10mM), lonidamine (glycolysis inhibitor) and HBO (3atm, 60 min.) Rat primary neurons were treated with 10nM Aβ42 for 24 hours (with and without HBO) and superoxide anion production was measured with dihydroethidium (DHE). Lonidamine and HBO increased U87 MG cell death in cultures exposed to acute drug treatment (1hr). Ketone treatment in primary neurons decreased superoxide anion production by 20.5% and 36.2% in normobaric treatment (NBO) and HBO, respectively. Ketone treatment in Aβ42 treated neurons reduced superoxide by 23% and 16.5% in NBO and HBO, respectively. We conclude that glycolysis inhibition decreases viability in U87 MG cells and that supplemental ketones reduce superoxide production in primary rat neurons exposed to HBO and Aβ. These observations support a therapeutic effect of ketones in brain cancer and Alzheimer's disease.
The Increase in Flow‐Mediated Vasodilation Induced by Grape Polyphenols is Positively Correlated with Increased Expression of Inducible Nitric Oxide (iNOS)
Authors: Barona Jacqueline , Lee Jiyoung , Park Youngki , Fernandez Maria Luz
Published: June 2021

Twenty four men (30–70 y) with metabolic syndrome (MetS) participated in a double blind study and were randomly allocated to either a freeze‐dried grape polyphenol powder (GRAPE) or a placebo for 30 d. After a 3‐wk washout, they followed the alternate treatment. Participants abstained from consuming polyphenol‐rich foods. Flow‐mediated vasodilation (FMD), Inflammatory markers tumor necrosis factor (TNF)‐a, interleukin 6 (IL‐6), IL‐8, and the anti‐inflammatory IL‐10 were measured in plasma as well as 8‐ isoprostanes in urine. Additionally, mRNA expression of IL‐6, IL‐ 1b, IL‐10, the antioxidant enzymes (SOD1&2, GPx1&4), the endothelial (eNOS) and inducible (iNOS) isoforms of nitric oxide synthase, and NADPH oxidase (NOX2) were measured in peripheral blood mononuclear cells isolated at both periods. FDM was increased during the GRAPE period (p<0.0001) while inflammatory or antioxidant markers were not different (p>0.05) between periods. However, iNOS expression changes were correlated with changes in absolute (mm) FMD response (r=0.514, p< 0.025) and with changes in SOD2 expression (r=0.642, p<0.01). These results suggest that the beneficial effects of GRAPE on FMD are positively associated with NO production in men with MetS. However, 4 wk of GRAPE supplementation were not enough to detect changes in anti‐inflammatory or anti‐oxidant gene expression [Supported by The California Table Commission]
Examining the Anti‐inflammatory Properties of Blueberry Polyphenols Using MC3T3‐E1 Cells
Authors: Elam Marcus L , Zhang Jiyao , Feresin Rafaela , Hooshmand Shirin , Arjmandi Bahram H
Published: June 2021

The objective of this study was to investigate the mechanisms by which total blueberry polyphenol extract (BPE) exerts its anti‐inflammatory function on MC3T3‐E1 preosteoblasts under inflammatory conditions. Cells were treated with BPE at concentrations of 0, 10, 100 μg/ml. After 24 h incubation, cells were stimulated with TNF‐α (10 ng/ml) and IL‐1 (10 ng/ml) individually or in combination for 24 and 48 hrs. Cells and supernatants were collected to assess NO production and for RNA extraction. Select gene expressions involved in bone cell differentiation were assessed using commercial cDNA array plates. BPE was able to suppress the increased NO production induced by either TNF‐α or IL‐1, suggesting the prevention of impaired osteoblastic differentiation. However, BPE had no significant effect on NO production when TNF‐α and IL‐1 were both present. At the molecular level, BPE upregulated RANKL and IL‐6 via the NF‐κB signaling pathway. In spite of this, BPE tended to increase gene expression of Runx2 and osterix, albeit not significantly. From these preliminary findings it can be suggested that BPE does not suppress bone turnover through osteoblast‐mediated activity unlike in vivo findings.

Grant Funding Source : Margaret A. Sittion Professorship
Lipid extracts from edible blue‐green algae reduce the production of pro‐inflammatory cytokines by inhibiting nuclear translocation of NF‐κB in RAW 264.7 macrophages
Authors: Ku Chai Siah , Lee Jiyoung
Published: June 2021

Chronic inflammation contributes to the pathogenesis of several metabolic diseases including atherosclerosis. We investigated anti‐inflammatory effects of lipid extract from edible blue‐green algae (BGA) such as Nostoc commune var. Sphaeroides Kützing (NO) and Spirulina Platensis (SP) in RAW 264.7 macrophages. The algal lipids were extracted into chloroform/methanol (2:1). Cells were pre‐treated with 100 μg/ml of the lipid extract for 12 hr and subsequently activated by 100 ng/ml lipopolysaccharide for 18 hr. Quantitative realtime PCR analysis showed that mRNA levels of pro‐inflammatory cytokines such as tumor necrosis factor α, interleukin 1β, and interleukin 6 are significantly repressed by NO and SP lipid extract. Cytokine array demonstrated that the secretion of pro‐inflammatory cytokines was also markedly reduced by both NO and SP lipid extract. The repressive effect was at least partly due to the inhibition of nuclear translocation of NF‐ κB. In addition, the repression of pro‐inflammatory cytokine expression by algal extracts was more potent than SN50, an inhibitor of NF‐κB nuclear translocation, suggesting that additional pathways may exist for the anti‐inflammatory effect of NO and SP lipid extract. In conclusion, BGA lipid extract contains anti‐inflammatory compounds that could be developed as a nutraceutical agent against inflammation‐related diseases.

Grant Funding Source: NIH R21AT005152
Prevalence of dyslipidemia and cardiovascular disease (CVD) risk factors among young Mexican college students
Authors: Vazquez-Vidal Itzel , Andrade Flavia C.D , Teran-Garcia Margarita , Aradillas-Garcia Celia , Vargas-Morales Juan Manuel
Published: June 2021

Our objective was to evaluate the prevalence of dyslipidemias and CVD risk factors among young Mexican college applicants. Fasting lipid profile (triglycerides (TG), total cholesterol (TC), HDL and calculated LDL) from 794 individuals 18–25 year‐old (56% females, BMI: 24.3±4.1 kg/m2) who applied to the UASLP in 2009. Only 41% of the population had a normal lipid profile. Fifty percent of the population had high‐LDL and 44% had low‐HDL. The most common lipid disorders were: low‐HDL (30%), high‐TC (9%), combined high‐TG and low‐HDL (7%) and combined high‐TC, high‐TG and low‐HDL (4%). Men had higher atherogenic profile ratios than women (all p<0.001): TC/HDL (41% vs. 31%), LDL/HDL (45% vs. 37%), and TG/HDL (55% vs. 41%). Low‐HDL and high‐LDL were the most common lipid disorders. These results suggest that young Mexican adults might be at risk for coronary heart events later in life. Sponsor by C09‐PIFI‐030606 (UASLP) and the University of Illinois Vice‐chancellor of Research, CHAD and USDA/NIFA Hatch Projects (#600108‐793000‐793323 and #600109‐698000‐698354).

Grant Funding Source: USDA/NIFA Hatch Projects
Enteric Microbiome (EMB) of Breastfed Infants (BFI) on Complementary Feeding (CF) Regimens with Different Iron (Fe) Exposure
Authors: Krebs Nancy F , Sherlock Laurie , Robertson Charles E , Westcott Jamie , Culbertson Diana , Feazel Leah , Frank Daniel
Published: June 2021

Background

The low bioavailability of electrolytic Fe in infant cereals results in high Fe exposure for gut organisms, which may alter the balance of microbiota in the developing intestine.

Objective

Compare the development and composition of the EMB among groups of healthy BFI on CF regimens that provided a range of Fe intake and excretion.

Methods

Healthy BFI were randomized at 5 mo to Fe‐fortified infant cereal (IFC, n=4), Fe+Zn‐fortified cereal (IZFC, n=6), or meat (M, n=4). All infants continued breastfeeding and received no formula. Stool samples were obtained monthly from 5–9 months. Bacterial profiles were analyzed by broad‐range PCR of 16S rRNA genes and phylogenetic sequence analysis.

Results

At 9 mo, mean Fe intake was 13 vs 3.7 mg/d for IFC+IZFC vs M, respectively. Sequence data identified significant differences in longitudinal development of the EMB by feeding group, including greater abundances of Clostridium Group XIVa (M > IFC & IZFC) and Bacteroidetes (IFC > IZFC & M). The M group also had higher species diversity.

Conclusions

This longitudinal study indicates that the composition of CF, including Fe exposure, influences the development of the infant enteric microbiota.

Supported by NIH K24DK083772, T32 DK007658‐20S, 1R21HG00596, M01‐RR00069; The Beef Checkoff, NCBA
Regulation of the cell surface traffic of α2‐adrenergic receptors by small GTPases
Authors: Zhu Shu , Li Chunman , Wu Guangyu
Published: June 2021

The molecular mechanisms responsible for the cell surface targeting of nascent G protein‐coupled receptors (GPCRS) remain poorly elucidated. Our previous studies have utilized α2B‐adrenergic receptor (AR) as a model to define the pathways that mediate forward transport of GPCRs by studying the function of small GTPases in the Rab and ARF/Sar1 subfamilies. In this study, we compared the small GTPases‐coordinated cell surface movement of three α2‐ARs and investigated the possible mechanisms. Transient expression of dominant‐negative mutants of Rab, ARF, and Sar1 clearly inhibited the cell surface transport of α2A‐AR and α2C‐AR. Although the cell surface expression of α2A‐AR was much more abundant than that of α2C‐AR, their responsiveness to the small GTPase mutants was similar. In contrast, the cell surface transport of α2B‐AR was inhibited by some, but not all the mutants. More interestingly, three α2‐ARs differentially interacted with some small GTPases and the interaction was identified to be mediated via the third intracellular loops and/or the C‐terminal tails of the receptors. These data have demonstrated that the cell surface traffic of three α2‐ARs is controlled by different mechanisms and their differential interactions with different small GTPases involved in distinct transport steps along the secretory pathway may play an important role.
Neurabin scaffolding of adenosine receptor and RGS4 regulates anti‐seizure effect of endogenous adenosine
Authors: Chen Yunjia , Liu Yin , Cottingham Christopher , McMahon Lori , Jiao Kai , Greengard Paul , Wang Qin
Published: June 2021

An increase in adenosine receptor responsiveness to endogenous adenosine would enhance neuroprotection while avoiding the confounding effects of exogenous ligands. Here we report novel regulation of adenosine‐evoked responses by a neural tissue‐specific protein, neurabin. Neurabin attenuated adenosine A1 receptor (A1R) signaling by assembling a complex between the A1R and the regulator of G protein signaling 4 (RGS4). Inactivation of the neurabin gene enhanced A1R signaling and promoted the protective effect of adenosine against excitotoxic seizure and neuronal death in mice. Furthermore, administration of a small molecule inhibitor of RGS4 significantly attenuated seizure severity in mice. The enhanced anti‐seizure and neuroprotective effect achieved by disruption of the A1R/neurabin/RGS4 complex is elicited by the on‐site and on‐demand release of endogenous adenosine, and does not require administration of A1R ligands. These data identify neurabin‐RGS4 as a novel tissue‐selective regulatory mechanism for fine‐tuning adenosine receptor function in the nervous system. Moreover, these findings implicate the A1R/neurabin/RGS4 complex as a valid therapeutic target for specifically manipulating the neuroprotective effects of endogenous adenosine. This research is supported by UAB faculty development grant (QW) and NIH grants MH081917 (QW) and DA10044 (PG).
Role of increased GABAergic synaptic transmission in morphine tolerance
Authors: Bobeck Erin N. , Suchland Katherine L. , Haseman Rachel , Ingram Susan L.
Published: June 2021

Repeated administration of morphine increases desensitization of mu‐opioid receptor (MOP) coupling to many cellular effectors and has long been thought to be a cellular correlate of antinociceptive tolerance. However, recent studies observed no evidence of MOP desensitization in presynaptic terminals, although a shift in potency to the right was observed in morphine pretreated rats. Since opioid disinhibition of PAG output neurons is thought to drive antinociception, these results suggest that a cellular mechanism other than receptor desensitization may account for decreased opioid potency following repeated administration of morphine. We determined that repeated morphine administration (5mg/kg twice a day for 2 days) increased GABA release (1.1 ± 0.2 Hz) compared to saline pretreatment (0.5 ± 0.1 Hz) without altering the amplitude cumulative distribution in the PAG. ME (1 μM) inhibited GABAergic mIPSCs by 49 ± 6% in slices from saline compared to 30 ± 8% in morphine pretreated slices. Superfusion of forskolin (20 μM) also increased mIPSC frequency in slices from both saline and morphine pretreated rats. Interestingly, forskolin superfusion in slices from saline pretreated rats was sufficient to significantly decrease ME‐induced inhibition of mIPSCs suggesting that increased release of GABA causes an “apparent” change in affinity of opioid agonists at MOPs. Supported by NIDA grant DA027625.
Regulation of human oligodendrocyte differentiation by muscarinic M3 receptor
Authors: Vedia Bansi H , Pol Suyog U , O'Bara Melanie A , Sim Fraser J
Published: June 2021

Oligodendrocytes are destroyed in demyelinating diseases such as Multiple Sclerosis. In a genomic study to identify novel drug targets, we identified the muscarinic cholinergic M3 receptor as highly expressed by human oligodendrocyte progenitor cells (OPCs). Human OPCs expressing CD140a antigen were isolated from 19–22 week fetal brain using immunomagnetic sorting. We treated OPCs with oxotremorine, a specific agonist of muscarinic receptors, for 4 days and assessed cell fate and proliferation using immunocytochemistry. Oxotremorine treatment resulted in a dose‐dependent decrease in oligodendrocyte differentiation (1‐way ANOVA, p<0.01). The proportion of O4+ oligodendrocytes was significantly reduced from 16.0±2.4% at 0μM to 8.3±1.5% at 40μM oxotremorine (n=3, Tukey's post‐hoc p<0.05). The proportions of dividing cells (BrdU) and oligodendrocyte lineage cells (Olig2) were not significantly altered suggesting that the effect of oxotremorine was to directly block oligodendrocyte differentiation. As such, we have identified an operative receptor expressed by human OPCs that may be targeted to regulate oligodendroctye differentiation. This work was supported by NYSTEM contracts (C026413 and C026428).
Avocado extract induces chromosomal aberrations in cultured human lymphocytes
Authors: Kulkarni Paresh Prahlad , Paul Rajkumar , Ganesh Narayan
Published: June 2021

Avocado fruit is consumed in many parts of the world due to its high nutritional value, while extracts from avocado fruit and leaves have been shown to exhibit pharmacological activity against diabetes, hypertension, infections and cancer. We carried out a chromosomal aberration assay in metaphasic preparations of cultured human lymphocytes to evaluate the potential genotoxicity of 50% methanolic extracts of avocado fruits and leaves. Exposure of cells to both leaf and fruit extracts, led to a significant concentration‐dependent increase in chromosomal aberrations as compared to untreated control. The lymphocytes exposed to fruit extract exhibited lower frequency of all types of aberrations at equal concentrations as compared to those exposed to leaf extract. Acrocentric associations and premature centromeric division, which are thought to indicate chromosomal instability and are potential harbingers of non‐disjunction and translocation mutations, were the two most common abnormalities observed in both the exposed groups. In conclusion, the present study suggests that the extracts of both avocado fruit and leaves can potentially induce significant genomic instability and some genetic damage in human lymphocytes in vitro. However, it is essential to carry out in vivo studies before making a final remark on the genotoxicity of the plant extract. This work was funded by JNCHRC, Bhopal, India.
Induction of kidney endonucleases by DNase I: evidence of endonuclease network
Authors: Basnakian Alexei G. , Fahmi Tariq , Wang Xiaoying , Savenka Alena , Fite Todd , Stewart Anna G. , Apostolov Eugene O. , Shah Sudhir V.
Published: June 2021

Endonuclease‐mediated DNA fragmentation is both an immediate cause and a result of apoptosis and all other types of irreversible cell death during kidney injury. It is produced by nine enzymes: DNase I and 2 families, CAD and EndoG, which act simultaneously. However, regulatory links between these enzymes have not been established. We hypothesized that DNase I, the most active kidney endonuclease, may regulate other endonucleases. To test this hypothesis, rat kidney tubular epithelial NRK‐52E cells were transfected with rat DNase I gene or its inactive mutant in pECFP expression vector, while control cells were transfected with empty vector. Cells were collected and DNA and mRNA were isolated from the cells at varying time points. mRNA expression of all nine endonucleases was studied using real‐time RT‐PCR; DNA strand breaks endonuclease genes was determined by PCR; and protein expression of the enzymes was measured by Western and quantitative immunocytochemistry. Our data showed DNase I, but not its inactive mutant, induces all other endonucleases at different time periods after transfection, DNA breaks in endonuclease genes, and protein expression of several endonucleases. This is the first evidence of an endonuclease network, in which endonucleases are orchestrated by DNA‐degrading activity of DNase I. The study was supported by NIH and VA grants to A.G.B. and S.V.S.
Human adipose‐derived stem cells attenuate cigarette smoke induced bone marrow hypoplasia via secretion of anti‐inflammatory cytokine TSG‐6
Authors: Xie Jie , Schweitzer Kelly , Johnstone Brian , Cook Todd , Feng Dongni , Albrecht Marjorie , Gao Yong , Justice Matthew , Cooper Scott , Broxmeyer Hal , Petrache Irina , March Keith
Published: June 2021

Introduction

We have previously observed bone marrow hypoplasia in a murine model of chronic smoking, which was ameliorated by murine adipose‐derived stromal cells (ASC). This study was designed to test the hypothesis that ASC exert their marrow protective effects through key paracrine factors.

Methods

Mice (NSG) were exposed to cigarette smoke (CS) for 1 day to 6 months. Human ASC or ASC conditioned media were administered through intravenous (i.v.) or intraperitoneal (i.p.) injections. Expression of TSG‐6 in ASC were knocked down by siRNA and confirmed by real‐time PCR and ELISA. BM hematopoietic progenitor subsets were quantified by colony forming‐unit assays. Possible engrafted human ASC in mouse BM were examined by anti‐human nuclei staining.

Results

CS (6 mos) significantly reduced BM hematopoietic progenitors (vs. nonsmoker, 50.7%, p<0.01), which was restored by human ASC i.p. (110.6%, p>0.05) and i.v. (70.9%; p>0.05). Such damage occurred acutely after one‐day exposure (61.6 %, p<0.01), and could be ameliorated with either ASC (111.0%, p>0.05) or ASC conditioned media (105.7%, p>0.05). Knocking down TSG‐6 (>90%) abolished the protective effect of ASC. No human cells were detected in recipient mouse bone marrow.

Conclusions

The protective effects of ASC against smoking‐induced myelosuppression are mediated by paracrine factor TSG‐6 rather than cell engraftment or differentiation.

Research supported by American Heart Association
Exercise training ameliorates age‐related diastolic dysfunction: the role of coronary microvascular function
Authors: Chen Bei , Ravikumar Vignesh S. , Muller-Delp Judy M.
Published: June 2021

The risk for diastolic heart failure increases with advancing age. Regular physical exercise has been shown to prevent and/or ameliorate the progression of age‐related diastolic dysfunction; however, the underlying mechanisms have not been identified. We tested the hypothesis that adaptations of coronary microvascular function contribute to age‐ and exercise‐related changes in cardiac diastolic dysfunction. Young and old Fischer 344 male rats were randomly assigned into either a sedentary control group or an exercise‐trained group. Doppler analysis was used to evaluate ventricular diastolic function. Coronary arterioles were isolated from the left ventricular free wall to assess arteriolar contractile function. Vascular smooth muscle (VSM) proliferation was assessed by measuring wall:lumen ratio and by analysis of phosphohistone H3. Doppler analysis showed that age impaired cardiac diastolic function and late‐life exercise training improved diastolic function. Age impaired contractile function and induced localized VSM proliferation in coronary resistance arterioles. Exercise training reversed age‐related contractile dysfunction of coronary resistance arterioles and attenuated VSM cell proliferation. Our findings suggest that age and exercise affect cardiac function, in part, through alterations of VSM function of coronary resistance arterioles.
Increased formation of functional caveolae due to increased content of fibrinogen
Authors: Muradashvili Nino , Khundmiri Syed J. , Benton Richard L. , Lominadze David
Published: June 2021

Previously, we showed that elevated level of fibrinogen (Fg) increase endothelial cell (EC) layer permeability through formation of filamentous actin and affecting EC junction proteins. These changes led to an increase in mouse pial venular permeability. We hypothesized that Fg‐mediated increased EC layer permeability may also involve transcellular transport pathway. Formation of caveolae was observed in mouse brain cortical cryo‐sections obtained from wild‐type or matrix metalloproteinase‐9 gene knockout (MMP9−/−) mice after infusion of Fg (a total blood content of 4 mg/ml) or similar volume of PBS. Expression of caveolin‐1 and plasmalemma vesicle associated protein‐1 (PV‐1) were increased in mouse brain vessels after Fg infusion compared to those after PBS infusion. These effects were ameliorated in MMP9−/− mice. Expression of PV‐1 was increased by 15±4% and expression of occludin decreased by 3±1% in mouse pial vessels after infusion of Fg. At higher levels Fg increased formation of functional caveolae in mouse brain ECs (MBECs) as detected by caveolar uptake of albumin. Fg‐induced enhanced formation of caveolae in MBECs was mitigated by inhibition of MMP9 activity. These results suggest that high levels of Fg increase cerebrovascular permeability by altering both paracellular and transcellular pathways. It is still need to be defined which one of these pathways is most affected by elevated levels of Fg.
Fluorescence imaging reveals differences in mitochondrial function along the collecting duct
Authors: Courtneidge Holly Robyn , Crawford Carol , Hall Andrew M , Peppiatt-Wildman Claire M
Published: June 2021

The collecting duct (CD) consists of two different cell populations, principal cells (PCs) and intercalated cells (ICs), which have distinct solute transport functions. Transport and metabolism are closely coupled in the nephron, thus we hypothesised that intrinsic differences in PCs and ICs mitochondrial function might exist. Mitochondrial function can be measured in intact rat kidney slices using confocal and multiphoton microscopy (1,2); here we apply this approach to investigate our hypothesis.

PCs and ICs have distinct morphological differences, which enabled their identification during live imaging. Heterogeneity was observed in mitochondrial signals along the CD. Mitochondrial membrane potential, measured using tetramethylrodamine methyl ester (TMRM), was higher in PCs (34.45±3.08) than ICs (16.82±8.23) (n=3, p=0.009), and this signal was not altered by dye efflux inhibitors. Fluorescence signals from the endogenous fluorophores NADH and FAD+ (substrates for complexes I and II in the respiratory chain, respectively) were higher in ICs than in PCs (p=0.03 and p=0.056 respectively).

In summary, fluorescence imaging reveals intrinsic differences in mitochondrial function between PCs and ICs, which may be important in understanding (patho)physiology of the CD.

Research supported by the Medical Research Council
K+ homeostasis is maintained with knockdown of big‐conductance K+ channel in principal cells of connecting tubule/collecting duct
Authors: Rieg Timo , Lukowski Robert , Dominguez Jessica , Sharik Meaghen , Ruth Peter , Vallon Volker
Published: June 2021

Big‐conductance K+ (BK) channels are expressed in principal cells (PC) and intercalated cells of the kidney and contribute to K+ homeostasis and flow‐induced K+ secretion. To define the role of BK channels in PC, we generated mice with conditionally inactivated BK channels in PC of the connecting tubule/collecting duct (AQP2:Cre; BKloxloxCre). Cre negative BKloxlox mice served as controls (Con). On standard diet (Teklad 7001), Plasma Na+ (Con vs BKloxloxCre: 150±0.4 vs 150±0.4 mmol/l), K+ (4.4±0.1 vs 4.3±0.1 mmol/l), pH (7.4±0.04 vs 7.4±0.04), and hematocrit (49±0.4 vs 50±0.5 %) were not different between genotypes. Systolic blood pressure and heart rate, determined by tail cuff, were comparable between genotypes (Con: 113±2 mmHg and 623±16 min−1, BKloxloxCre: 116±2 mmHg and 647±13 min−1). Spontaneous urine collections showed similar urine osmolality in Con and BKloxloxCre (2041±87 vs 1950±85 mmol/kg). However, urinary K+/Na+ ratio was significantly higher in BKloxloxCre vs Con (1.49±0.07 vs. 1.17±0.07, P<0.05) due to lower urinary Na+/creatinine (crea) in BKloxloxCre vs Con (21.7±1.3 vs. 25.4±1.1, mmol/mmol P<0.05), while urinary K+/crea was not different (31.9±1.8 vs 29.6±1.6 mmol/mmol). Our data indicate that under control diet conditional knockdown of BK channels in PC of the connecting tubule/collecting duct reduces Na+ excretion while K+ homeostasis is maintained.