bluuebunny/crossref_metadata_2025 huggingface dataset:
Biomechanical and neurohumoral stimulation of neonatal rat ventricular myocytes induce focal adhesion kinase phosphorylation at S910
Authors: Chu Miensheng , Iyenar Rekha , Samarel Allen M
Published: June 2021

Biomechanical and neurohumoral stimulation of cardiomyocytes rapidly increase focal adhesion kinase (FAK) phosphorylation at tyrosine residues, but little is known about FAK phosphorylation at serine residues. Stimulation of cultured neonatal rat ventricular myocytes (NRVM) by 20% static stretch promoted a 4‐5 fold increase in FAK S910 phosphorylation, but not S722 phosphorylation, as revealed by Western blotting with phosphospecific antibodies. The neurohumoral agonists endothelin‐1, angiotensin II, and phenylephrine also stimulated FAK S910 phosphorylation. Direct activation of protein kinase C isoforms with phorbol 12‐myristate 13‐acetate (PMA) significantly increased FAK S910 phosphorylation, whereas the PKC inhibitor GF102903X, but not Gö6983 prevented ET‐1‐induced FAK S910 phosphorylation. These results suggest that both stimulation increase FAK S910 phosphorylation via novel, but not classical PKCs. Treatment with the MEK1 inhibitors U0126 or PD98059 also inhibited FAK S910 phosphorylation in response to stretch and ET‐1, whereas the Src inhibitor PP2 reduced FAK S910 phosphorylation induced by ET‐1, but not by stretch. Furthermore, inhibition of ILK by shRNA‐mediated knockdown prevented stretch‐induced FAK S910 phosphorylation. Thus, these data demonstrate that biomechanical and neurohumoral stimuli stimulate NRVM FAK S910 phosphorylation via different signaling pathways.
Vasoconstriction‐induced inward remodeling of isolated arterioles requires reactive oxygen species
Authors: Martinez‐Lemus Luis Arturo , Zhao Guiling
Published: June 2021

We previously demonstrated that inward remodeling induced by prolonged agonist‐induced vasoconstriction in isolated arterioles requires the activation of matrix metalloproteinases (MMPs). The vasoconstrictor agonists that induce inward remodeling also generate reactive oxygen species (ROS). Because ROS has been implicated in the activation of MMPs, we hypothesize that ROS‐generation participates in the vasoconstriction‐induce inward remodeling process in part by promoting MMP activation. Experiments were performed in isolated and pressurized (60 mmHg) rat cremaster arterioles. Results indicated that exposure to norepinephrine (NE, 10−5.5 M) plus angiotensin‐II (Ang‐II, 10−7 M) for 4 hours induced inward remodeling and increased the gelatinolytic activity of arterioles (5 fold vs. time controls, P<0.05). Incubation with apocynin or tempol prevented the development of inward remodeling and apocynin significantly reduced the gelatinolytic activity induced by NE and Ang‐II. These results: 1) indicate that MMPs, in particular the gelatinases, may play a role in the development of inward remodeling in the resistance vasculature; 2) indicate that ROS are required for the acute remodeling of the vascular wall; and 3) suggest that persistent vasoconstriction requires ROS‐dependent activation of MMPs to induce acute inward remodeling in resistance vessels. Funded by AHA.
Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF‐alpha and Pi
Authors: Villa‐Bellosta ricardo , Levi Moshe , Sorribas Victor
Published: June 2021

Pi transport by vascular smooth muscle cells (VSMC) has been proposed to play an important role in the pathogenesis of vascular calcification. In this study we have determined the correlation between calcification induced by Pi, PDGF‐BB, and TNF‐alpha and the expression of Pi transport in rat VSMC. These agents induced calcification and increased the expression of Cbfa1, Msx2, and Bmp2 RNAs in rat aortic VSMC, while Pi transport rate was not modified per milligram of protein. Only PDGF increased Pi transport when it was expressed per unit of DNA, as PDGF also increased total cell protein by 100 %, while DNA content was not modified. PDGF increased the expression of the Pi transporter, Pit‐1, but membrane protein biotinylation showed that Pit‐1 abundance was not modified in the cell surface. Immunofluorescence revealed that Pit‐1 is only slightly expressed at the cell membrane, but strongly expressed inside the cell, whose signal colocalizes with endoplasmic reticulum (ER) markers. PDGF increases Pit‐1 expression in the ER but not the plasma membrane.
Elevated fasting blood glucose levels depend on paternal history of diabetes in Wheaton College football players
Authors: Dozeman Michael Lee , Lawrence Abigail , Baybutt Richard
Published: June 2021

The purpose of this study was to examine the relationship between lean body mass and fasting blood glucose levels in 32 members of the Wheaton College football team. Procedure included a medical questionnaire, measuring fasting blood glucose levels, height, weight, and body composition. Regression analysis of the data showed no significant relationship between blood glucose levels and lean body mass, body weight, percent body fat, or fat mass. Interestingly, a relationship did emerge between fasting blood glucose levels and family history. Ten subjects had family history of diabetes; 7 maternal, 3 paternal. Those with maternal history had an average blood glucose level of 89 mg/dL; significantly lower than those with paternal history, who averaged 101 mg/dL. A multiple linear regression was calculated to predict subjects' fasting blood glucose levels based on their family history. A significant regression equation was found (p<.05) with R squared of .296. The equation for the subjects predicted blood glucose=86.464+.869(mom) +15.036(dad), where history is coded as a 1 if a subject has it, and 0 if they do not. History of the dad only was a significant predictor. Aldeen Grant from Wheaton College.
Chronic endometritis: How many plasma cells does it take to make the diagnosis?
Authors: Hartman Sarah K. , Symons Wendy A. , Yeh I‐Tien
Published: June 2021

3–10% of endometrial biopsies are said to show histologic evidence of chronic endometritis. In this study we correlate the # of plasma cells by H&E to the # of plasma cells by CD138 IHC in endometrial biopsies.

Methods

100 endometrial biopsies were selected, excluding specimens with limited material, cancer and menstrual phase. H&Es were reviewed, and the # of plasma cells/10 hpfs were recorded. Additional sections were stained with CD138 antibody (clone B‐A38; Cell Marque, Rocklin, CA). Hot spots of CD138+ cells were recorded as the #/10 hpfs. Histologic findings near the CD138+ cells were also recorded.

Results

Plasma cells were identified in 29 cases by H&E alone (ave. 2.1, range 1–12) and 90 cases were + for plasma cells by CD138 (ave. 13.9 plasma cells, range 1–190). Of the 29 cases with plasma cells on H&E, CD138 highlighted an average of 24.3, with a minimum of 1. 71 biopsies lacked plasma cells on initial H&E review, but averaged 7.7 plasma cells by CD138. Often, the endometrial stroma contains scattered lymphocytes or mild edema, but lacked the spindled stroma typically associated with chronic endometritis.

Conclusions

By careful exam on H&E, >1 plasma cells were seen in 29% vs. 90% of endometrial bxs by CD138. These cases include diagnoses of gland‐stromal dyssynchrony, disordered proliferative endometria, and endometrial hyperplasias. The presence of CD138+ plasma cells may not equate to chronic endometritis.
Alpha2‐Adrenoceptor Blockade Accelerates the Neurogenic, Neurotrophic, and Behavioral Effects of Chronic Antidepressant Treatment
Authors: Yanpallewar Sudhirkumar , Fernandes Kimberly , Marathe Swananda , Vadodaria Krishna , Jhaveri Dhanisha , Rommelfanger Karen , Ladiwala Uma , Jha Shanker , Muthig Verena , Hein Lutz , Bartlett Perry , Weinshenker David , Vaidya Vidita
Published: June 2021

Enhanced adult hippocampal neurogenesis and increased trophic factor expression mediate the behavioral effects of antidepressants. We investigated the influence of alpha2‐adrenoceptors on adult hippocampal neurogenesis. We found that alpha2 agonists selectively decrease adult hippocampal progenitor proliferation. These effects persist in Dbh−/− mice lacking norepinephrine, suggesting an action through alpha2‐heteroceptors. Adult hippocampal progenitors express all the alpha2‐adrenoceptor subtypes, and decreased neurosphere frequency and BrdU incorporation indicate direct effects of Alpha2‐adrenoceptor stimulation on progenitors. Furthermore, coadministration of the alpha2 antagonist yohimbine with the antidepressant imipramine accelerates effects on hippocampal progenitor proliferation, the maturation of newborn neurons, and the increase in expression of BDNF and VEGF implicated in the neurogenic and behavioral effects of antidepressants. Finally, short‐duration (7 d) yohimbine and imipramine treatment results in robust behavioral responses in the novelty suppressed feeding test, which normally requires 3 weeks of treatment with classical antidepressants. Our results highlight importance of alpha2‐adrenoceptors as targets for faster acting antidepressants.
NAD+ treatment can prevent multiple rotenone‐induced apoptotic changes of PC12 cells
Authors: Hong Yunyi , Shi Shengtao , Shao Jiaxiang , Ma Yingxin , Xia Weiliang , Ying Weihai
Published: June 2021

NAD+ plays critical roles in various biological functions. A number of studies have also indicated that NAD+ treatment can prevent the necrosis of primary neurons and astrocytes, which are induced by multiple insults such as oxidative stress. NAD+ administration has also been shown to decrease ischemic brain damage. However, there, there has been little information regarding the effects of NAD+ treatment on apoptotic changes, which are critical pathological changes in such neurological diseases as brain ischemia. In this study we determined the effects of NAD+ on multiple rotenone‐induced pathological changes by applying flow cytometry‐based Annexin V assay, TUNEL say, Hoechst staining and immunostaining of apoptosis‐inducing factor (AIF). We found that NAD+ treatment, but not nicotinamide, can markedly prevent all of these apoptotic changes, suggesting that NAD+ can prevent cell apoptosis by mechanisms that are independent of its degradation product nicotinamide. Our study using propidium staining assay and lactate dehydrogenase (LDH) assay also showed that NAD+ treatment can block rotenone‐induced cell necrosis. We further found that NAD+ treatment can prevent rotenone‐induced ATP depletion, which could in part underlie the protective effects of NAD+. In summary, our study has provided first evidence that NAD+ treatment can prevent multiple rotenone‐induced apoptotic changes, in addition to its reported preventive effects on cell necrosis. Because mitochondrial impairments belong to a key pathological change in multiple neurological diseases, our study has suggested that NAD+ may be used to prevent mitochondrial impairment‐induced cell death (Supported by a Key Research Grant of Shanghai Municipal Scientific Committee #08JC1415400 (to W. Y.)).
Activity assessment of organic cation/carnitine transporters in A549 alveolar epithelial cells
Authors: Salomon Johanna Jessica , Yahara Tohru , Hosoya Ken‐ichi , Ehrhardt Carsten
Published: June 2021

Uptake and release of reported substrates via organic cation/carnitine transporters (OCT/Ns) was studied in A549 monolayers in an attempt to investigate the role of OCT/Ns in alveolar cell function. The OCT/N probes, ASP+, [14C]‐TEA and [3H]‐acetylcarnitine (Ac‐Car) were used in the experiments. [3H]‐verapamil, generally considered a substrate for P‐gp/MDR1, was also studied. The effect of time, temperature and various pharmacological agents on uptake and release of the compounds was investigated. All compounds exhibited temperature‐sensitive and time‐ and concentration‐dependent uptake and release. The involvement of both non saturable and saturable uptake carriers was observed in the case of the OCT/N probes. Km values were 27.8 μM (ASP+), 744.4 μM (TEA) and 17.7 μM (Ac‐Car), respectively. Uptake was significantly inhibited by MPP+, Decynium 22 and amantadine. Verapamil uptake was found to be saturable, mediated by a single transporter (Km 57.9 μM) and not inhibited by organic cations. The efflux of ASP+ from A549 monolayers was also saturable (Km 32.9 μM). L‐carnitine and amantadine had no significant effect on ASP+ efflux. Our data suggest that OCT/Ns are involved in membrane transport of organic cations in the alveolar epithelium. In addition, OCTs, but not OCTNs contributed to ASP+ efflux from A549 monolayers.
Constitutive stimulation of vascular smooth muscle cells by Angiotensin II derived from an adenovirus encoding a fulin‐cleavable fusion membrane protein
Authors: Bourne Allison , Kimura Keita , Eguchi Kunie , Takaguri Akira , Elliott Katherin , Eguchi Satoru
Published: June 2021

Angiotensin II (AngII) plays critical roles in cardiovascular pathophysiology. Acute stimulation of cultured cells by synthetic AngII has been utilized to elucidate the signal transduction mechanisms of the peptide. However, such treatment may not mimic disease conditions where the circulating and/or tissue renin‐angiotensin systems are chronically upregulated to produce AngII consistently. To fill the gap between acute stimulation of cell cultures and chronic stimulation in vivo, and to obtain clinically relevant signaling information from cell cultures, we have made an adenovirus vector encoding a fulin‐cleavable AngII fusion protein. Acute stimulation of vascular smooth muscle cells (VSMCs) with synthetic AngII (100 nM) in cultured medium showed the peptide had a half life of less than 1 hour. In contrast infection of VSMCs with the AngII fusion virus showed a time and dose dependent production of AngII (peaking at 300 pM at 250 moi) in the medium as early as 2 days and up to 7 days post‐infection. Consistent with the peptides function in VSMCs, the AngII fusion virus increased protein content and cell volume, but not cell number and stimulated ERK1/2 activation and EGR‐1 induction. In conclusion, application of the AngII adenovirus to cultured cells will be useful to elucidate molecular and signaling mechanisms of cardiovascular diseases associated with enhanced AngII production.
Mechanisms of Neurotrophin Action on Human Airway Smooth Muscle
Authors: Abcejo Amard J , Sathish Venkatachalem , Aravamudan Bharathi , Thompson Michael A. , Pabelick Christina M. , Prakash Y.S.
Published: June 2021

Neurotrophins (NTs), which play an integral role in directing proper neuronal development and function, have been found in non‐neuronal tissue (including lung), but their role is still under investigation. We previously found NTs such as brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT3) and their receptors to be expressed in human airway smooth muscle (ASM). In this study, we hypothesized that NTs contribute to regulation of ASM function via regulation of mechanisms that control intracellular Ca2+ ([Ca2+]i). Human ASM cells isolated from lung samples incidental to patient surgery were incubated for 24 or 48 h in medium (control), 10 nM BDNF or NT3 in the presence or absence of inhibitors of signaling pathways such as MAP kinases (PD98059), PI3/Akt (Wortmannin) and NFκB (SN50). In fluo‐4 loaded cells, [Ca2+]i responses to ACh and histamine were significantly higher following BDNF and NT3 exposure: effects that were blunted by SN50 and PD98059, but not Wortmannin. Western analysis of cell lysates showed higher expressions of Ca2+ regulatory proteins such as Orai1, TRPC3, and SERCA with 1 or 10nM BDNF exposure, but decreased expression by NT3. These data show that NTs activate signaling pathways in human ASM that lead to enhanced [Ca2+]i responses, and could thus contribute to airway hyperresponsiveness. Supported by NIH grants HL088029, HL090595 and FAMRI.
Claudin‐4 promotes repair and barrier function in lung epithelia
Authors: Rokkam Deepti , Hirsch Jan , Ho Gwen , Griffin Chris , LaFemina Michael J , Frank James A
Published: June 2021

Claudin‐4 is a transmembrane protein important in paracellular sealing at tight junctions. Recent studies have found that claudin‐4 is specifically induced during epithelial repair and higher claudin‐4 expression levels are associated with intact barrier function in injured human lungs. In this study, claudin‐4 expression and trafficking were examined in primary rat alveolar type 2 cells and human bronchial epithelial cells in a scratch wound migration assay. To determine the role of claudin‐4 in migration, cell morphology, actin distribution and focal adhesions were examined. To discover claudin‐4 binding partners, coimmunoprecipitates were evaluated by mass spectrometry. Induction of claudin‐4 during repair was conserved in alveolar and bronchial epithelia and knockdown of claudin‐4 slowed migration. EGFR and JNK signaling were required for claudin‐4 expression. During repair, claudin‐4 was trafficked to the leading edge of migrating cells where it appeared to influence actin nucleation and lamellipodia formation. Novel protein‐protein interactions with claudin‐4 may provide clues to the role of this protein in collective migration. The combined effects of claudin‐4 on tight junction sealing and epithelial repair potentially explain the positive correlation between claudin‐4 expression levels and epithelial barrier function in human lungs.

Funded by NIH/NHLBI HL88440, HL88440S2
Alveolar acid transiently permeabilizes the alveolar epithelium in mouse lungs
Authors: Lindert Jens , Bhattacharya Jahar
Published: June 2021

Acid aspiration causes acute lung injury (ALI). However, acid interactions with the alveolar epithelial (AE) membrane are not understood. To understand acid interactions with intact alveoli, we set up isolated, perfused mouse lungs held at pulmonary artery, left atrial and alveolar pressures of 10, 3 and 5 cmH2O, respectively. Then we imaged intact alveoli by real‐time fluorescence microscopy. To view the alveolar cytosol, we microinfused alveoli with BCECF‐AM, that intra‐cellularly converts to the membrane‐impermeable fluorophore, BCECF. Then we imaged pH‐independent fluorescence (440 nm excitation). Alveolar microinfusion of HCl (pH 1.5) decreased AE fluorescence by 48±7 % of initial in 5 min (mean±SE, n=3). However subsequently, despite continued HCl infusion for 15 min, the AE fluorescence held steady. Further, after reloading BCECF to initial levels of AE fluorescence, a second HCl infusion given 20 min after the first, failed to decrease AE fluorescence (p<.05). We conclude, first contact with acid immediately permeabilized the AE membrane. However, the membrane spontaneously resealed and resisted further acid‐induced damage. We report a novel protective mechanism in ALI in which the cell spontaneously repairs chemically‐induced membrane damage. (Support: HL69514, 78645).
Undergraduate student‐led cadaveric dissection: an interdisciplinary approach to connecting pathological findings to health conditions
Authors: Drennan Rachel , Goatley Lee , Muff Jaclyn , Scavuzzo Raghela , Young Whitney , Zidon Terese
Published: June 2021

A cadaveric anatomical dissection was completed by five Westminster College undergraduate students with the objective to analyze anatomical abnormalities in order to compile a complete report of pathological findings. Abnormalities were identified through observation, histological preparation, and examination of aberrant tissues that may indicate possible health conditions at time of death. Preliminary pathological findings include enlargement of the thyroid gland (length=6.8 cm, width=4.6 cm), abnormal growth on the exterior surface of the superior vena cava (length=3.1 cm, width=1.6 cm), bronchopulmonary lymph nodes with anthracotic pigmentation, a papillary process present on the liver, numerous dense polyps on the descending colon, sigmoid colon and rectum, and uterine irregularities. Further histological analysis of the listed abnormalities was performed to identify the severity of disease(s) at the time of death. In addition to dissection, students documented personal experiences through journaling and participated in educational seminars to enhance their understanding and appreciation for human anatomy.

Grant Funding Source: Westminster College
Male‐male combat drives bite force evolution in the absence of mastication
Authors: Jones Katrina Elizabeth , Ruff Christopher B
Published: June 2021

Bite force production is often cited as a key influence on the anatomical evolution of the mammal jaw. However, most research focuses on mastication and food processing as the driving force for the evolution of large bite forces in terrestrial mammals. Pinnipeds (seals, sea lions and walruses) have lost the ability to masticate as an adaptation to marine feeding and instead use their jaws for a wide range of other behavior, including prey capture and male‐male combat in certain polygynous species. This study investigates the influence of male fighting on the jaw morphology of a mammal whose postcrania are entirely specialized for swimming. We measured cortical thickness of males and females of two polygynous (n=14) and three monogamous (n=19) species, from a wide taxonomic range. Section modulus of the corpus was calculated using a hollow beam model and bite force estimated by scaling by jaw length. We find that polygynous species that exhibit male combat (harem‐defense polygyny) produce significantly larger theoretical bite forces than females of the same species and males and females of monogamous species, including those with different feeding specializations. This indicates that male‐male combat is the most important influence on bite force evolution in pinnipeds and is a major contributor to variation in mandibular anatomy.

Grant Funding Source: n/a
Muscles Underlying the Elevation of the Hyolaryngeal Complex
Authors: Pearson William G. , Langmore Susan E , Yu Lou B , Zumwalt Ann C
Published: June 2021

Introduction

A critical event in the pharyngeal phase of swallowing is the elevation of the hyolaryngeal complex opening the upper esophageal sphincter. Current swallowing theory assigns this function to the suprahyoid (mylohyoid, geniohyoid, digastrics, and stylohyoid) and thyrohyoid muscles. The role of the long pharyngeal muscles (stylopharyngeus, palatopharyngeus, and salpingopharyngeus) that insert into the thyroid cartilage and distal pharynx is uninvestigated in humans. This study uses a cadaver model to document the structural properties of muscles to analyze their potential for elevating the hyolaryngeal complex.

Methods

Measurements to determine physiological cross‐sectional areas (PCSAs) of suprahyoid, thyrohyoid and long pharyngeal muscles were taken from hemisected head and neck formalin‐fixed cadaver specimens (n = 14). Coordinates of muscle attachment sites and PCSAs were used to calculate unit force vectors representing the relative maximum tetanic tension muscles in the superior direction. Force vectors of muscle samples were compared by an analysis of variance to evaluate their potential for force in elevating the hyolaryngeal complex.

Results

An ANOVA with Tukey HSD post hoc analysis of unit force vectors in the superior direction showed the following results: suprahyoid (1.54±0.34 cm2) > long pharyngeal (.74±0.20 cm2) (p<.01), and long pharyngeal > thyrohyoid (.49±0.17 cm2) (p<.05).

Discussion

The morphology of these muscles suggests that while the suprahyoid muscles have the most potential for force in elevating the hyolaryngeal complex, the long pharyngeal muscles contribute significantly more to UES opening than the thyrohyoid. If verified by physiological data, this data would add to the current understanding of swallowing.

Grant Funding Source: n/a
Screening for additives that stabilize enzymes at higher temperatures
Authors: Coleman Jack , Dai Lijun , Patton Wayne F
Published: June 2021

In Molecular Biology, enzymes are often required that function at elevated temperatures. Enzymes produced by mesophilic organisms usually denature at elevated temperatures, followed by aggregate formation. We developed a rapid fluorescence‐based assay for assessing a range of parameters impacting the thermal stability of an enzyme. Overall, protein stability is monitored by a fluorogenic dye that selectively detects aggregated protein. Stability can be measured in the presence of different buffers, cryoprotectants and excipients. By systematically raising the temperature of the protein in solution, the temperature at which the protein aggregates (Tagg) can be determined. Using this method with Klenow DNA polymerase, we determined that trehalose significantly increases Tagg. The DNA polymerase activity of the enzyme is significantly enhanced at 50°C in the presence of trehalose under the same conditions. Low amounts of the detergents, Tween20® and Triton X‐100® (0.05%) decreased Tagg and also compromised enzyme activity, especially at elevated temperatures. T7 RNA polymerase was tested in a similar manner. Trehalose less significantly increased Tagg, and had a more modest effect on increasing enzyme activity at 42°C. The described assay facilitates screening for buffers and additives that structurally stabilize a protein of interest at elevated temperatures.

This work was supported by ENZO Life Sciences.
Biochemical and kinetic characterization of wild type and pathogenic mutant forms of human ETHE1
Authors: Kabil Omer , Banerjee Ruma
Published: June 2021

Hydrogen sulfide (H2S) is a recently described gaseous signaling molecule, which influences various cellular processes involving the central nervous system, cardiovascular system as well as mitochondrial energy metabolism. H2S is produced endogenously from the desulfhydration of cysteine and homocysteine by the enzymes of the transsulfuration pathway. Understanding the regulation of steady‐state H2S levels awaits biochemical characterization of the enzymes responsible for the oxidation and metabolic removal of H2S, sulfide quinone oxidoreductase, sulfur dioxygenase (ETHE1) and a sulfurtransferase. Mutations in the persulfide oxidizing enzymes, i.e. ETHE1, result in ethylmalonic encephalopathy, an autosomal recessive disorder. In this study, we report the biochemical characterization and kinetic properties of ETHE1 and describe the deficiencies associated with two patient mutations, T152I and D196N. Steady‐state kinetic analysis shows that T152I mutation results in 3‐fold lower activity, which is correlated with a 3‐fold lower iron content compared to wild‐type enzyme. The D196N mutation results in a 2‐fold higher Km for the substrate, glutathione persulfate (GSSH).
Initial Characterization of Human Dual Oxidase 2 (DUOX2) in a Baculovirus System
Authors: Meitzler Jennifer L. , Ortiz de Montellano Paul R.
Published: June 2021

As members of the NOX/DUOX family, the DUOX protein subfamily is responsible for hydrogen peroxide generation in human epithelial cells. The reactive oxygen species produced is utilized for cellular functions including innate immunity, hormone production, and signaling. Two highly conserved DUOX isoforms are encoded in the H. sapiens genome; hDUOX1 and hDUOX2. These membrane bound proteins have garnered much attention for their unique N‐terminal peroxidase‐like domain(s). Initial studies on the hDUOX1 peroxidase‐like domain have failed to demonstrate heme binding; whereas the C. elegans DUOX1 isoform co‐purifies with heme bound. To further characterize the heme‐binding potential and function of the hDUOX2 protein, both the full‐length protein and N‐terminal peroxidase domain have been expressed in a baculovirus system for investigation. Evaluation of these proteins will be presented. This work was partially supported by NIH Grant DK30297.
ANTIOXIDANTS DECREASE LIPOLYSIS AND LIPID SYNTHESIS IN HUMAN DIFFERENTIATED ADIPOCYTES
Authors: Krawczyk Sarah Anne , Deeney Jude T. , Corkey Barbara E.
Published: June 2021

OBJECTIVE

Reactive oxygen species (ROS) partially mediate the aberrant effects of chronic inflammatory states such as obesity. The goal of this research was to examine the effect of ROS on triglyceride synthesis and lipolysis by scavenging ROS with the antioxidant, diphenyliodonium (DPI).

METHODS

Human subcutaneous preadipocytes were differentiated for 12 days into adipocytes. Cells were then treated with DPI and labeled with tritiated oleate (3H‐FA) or 32P as a tracer to assess lipid synthesis, which was detected by thin layer chromatography (TLC). Glycerol release was measured using an enzyme‐linked assay. Protein was assessed using western blot.

RESULTS

There was a decrease in glycerol released into the media of cells treated with 10 μM DPI for either 1 or 4 hours, as compared to control cells under basal and forskolin‐stimulated conditions. In parallel, triglyceride (TG) synthesis was decreased with DPI treatment compared to control (for basal and stimulated conditions). A preliminary look at the PKA pathway by western blot showed no change in activation compared to control by DPI treatment.

CONCLUSIONS

Data indicate that both 1 and 4 hour treatment with DPI decreased basal lipolysis and concomitantly decreased neutral lipid synthesis. This may be due to decreased TG synthesis, decreased lipolysis, or a decrease in turnover.
Celecoxib preserves arachidonic acid‐induced cell degeneration: implications for the anticancer effects of NSAIDs and polyunsaturated fatty acids
Authors: AMISSAH FELIX , AYUK‐TAKEM LAMBERT T , ABONYO BARACK O. , LAMANGO NAZARIUS S.
Published: June 2021

Polyisoprenylated proteins mediate signaling, intracellular trafficking, cell proliferation, differentiation and apoptosis. Aberrant activities of such proteins as Ras and Rab families of monomeric G‐proteins are implicated in cancers. The polyisoprenylation pathway culminates in a reversible ester‐forming polyisoprenylated protein methyl transferase‐ and hydrolytic polyisoprenylated methylated protein methyl esterase (PMPMEase)‐ catalyzed reactions. Arachidonic acid (AA) was analyzed for its effects on various cancer cells and the cellular PMPMEase activity. COX‐2 metabolism of AA was explored to demonstrate the loss of inhibitory effect on PMPMEase activity in‐vitro which was restored in the presence of celecoxib. AA induced the degeneration of the cancer cells at physiologically relevant concentrations (EC50 from 1 – 24 μM). Cellular PMPMEase activity also followed a similar profile as the cell viability (IC50 6.6 – 21 μM). Addition of COX‐1 or COX‐2 to the assay mixture restored the lost PMPMEase activity in the lysates of the AA‐treated degenerating cells. Celecoxib maintained the inhibition of PMPMEase in the presence of COX‐2. These data suggest that AA, functioning through PMPMEase inhibition, may be a physiological regulator of cell growth. The results further elucidate the association between COX‐2 expression in tumors and the chemopreventive effects of PUFAs and NSAIDs.

Supported by NIH/NIGMS/SCORE (GM 08111‐35) & NIH/NCRR (G12 RR0 3020).
The effect of flavonoids on mitochondria activity and cellular metabolism
Authors: Nguyen Duong T. , Ng Alexander S. , White J. Brandon
Published: June 2021

Studies have described the beneficial effects of dietary polyphenols such as flavonoids on the reduction of the risk of chronic diseases. We and others have shown that flavonoids can induce cellular cytotoxicity in breast cancer cell lines. More recently, we have identified that some flavonoids stimulate ATP production in breast cancer cell lines. Our research has focused on characterizing flavonoid stimulatation of ATP production. Fourteen different flavonoids were screened at different concentrations and incubation times in MDA‐MB‐231 cells. Trypan blue exclusion assay was performed to measure cell viability. Glucose uptake by the cells was measured in both low and high glucose. ATP assays were conducted to measure ATP levels in the cells at different flavonoid concentrations and in both low and high glucose. Our preliminary results indicate that the four out of fourteen flavonoids stimulate ATP production in breast cancer cells (apigenin, genistein, kaempherol, and quercetin). However, the level of stimulation depends upon the glucose levels; more stimulation was seen in high glucose compared to low glucose. There is also a correlation between the amount of glucose and cell viability; in low glucose cells are protected from flavonoid cytotoxicity. Taken together, these results suggest that mitochondrial activity is required for flavonoid induced cellular cytotoxicity.
Cytotoxic effects of combinational therapy of ascorbic acid and 3PO on breast and non‐small cell lung cancer cells
Authors: Rinkinen Jacob R. , Davis Keith R.
Published: June 2021

Many malignant cancer cells exhibit a high glycolytic rate even in the presence of oxygen. The Warburg effect has lead to the selective targeting of glycolytic enzymes by many novel therapeutic agents. Mitochondrial respiratory injury and damaged oxidative stress mechanisms within the cell are postulated to contribute to this cancerous phenotype. Therapies that inhibit glycolysis and induce oxidative stress may be particularly effective. We used the glycolysis inhibitor 3PO and pro‐oxidant ascorbic acid in combination against MCF7 and H1299 cells to test this hypothesis. We observed a synergistic loss of cell viability when a combination of drugs was used against H1299 cells, which correlated with induction of DNA damage and apoptosis in H1299. Interestingly, MCF7 cells did not respond to the treatment with the same efficacy as H1299 cells, implying a stronger resistance to 3PO and ascorbic acid. Reactive oxygen species (ROS) levels in drug treated H1299 cells display an initial increase and a later diminished degree of ROS. Mitochondrial dysfunction was also assessed to see if apoptosis proceeded via the intrinsic apoptotic pathway in H1299 cells. Data suggests at higher doses of combination drug therapy, mitochondrial integrity is disrupted. Our results suggest that this combinational therapy against glycolysis and oxidative stress show synergistic, cytotoxic results in non‐small cell lung cancer. This research was supported by the Medicus Society and the U.S. Department of Defense USAMRMC/TATRC, Award W81XWH‐09‐2‐0022.
Catalysis of carbonic anhydrase IX and pH control in MDA‐MB‐231 breast cancer cells
Authors: Frost Susan Cooke , Li Ying , Tu Chingkuang , Wang Hai , Silverman David N
Published: June 2021

Carbonic anhydrase IX (CAIX) is a membrane‐bound glycoprotein whose expression is considered a marker of tumor hypoxia and associated with tumor acidification. Our goal was to determine if the catalytic properties of native CAIX in MDA‐MB‐231 breast cancer cells are equivalent to those previously described in vitro and to evaluate its catalytic role in pH control. CAIX, like other CA family members, catalyzes the reversible hydration of carbon dioxide: CO2 + H2O ↔ H+ + HCO3−. We directly analyzed CAIX activity by using 18O exchange between CO2 and water as measured by membrane inlet mass spectrometry (MIMS). Data from membrane ghosts showed that the kinetic constants of CAIX catalysis in the membrane environment are very similar to those measured for purified, recombinant, truncated forms, suggesting that CAIX is not affected by the proteoglycan domain or the membrane environment. In intact cells, hypoxia increases CAIX activity compared to normoxia which is associated with increased expression. In the dehydration direction, the catalytic activity of CAIX increases as pH is decreased from pH 9 to pH 6.5. This suggests that at the pH of the tumor microenvironment (pH 6.8), CAIX uses protons to generate CO2. Thus, CAIX may contribute to both the development and maintenance of the acidic microenvironment of hypoxic tumors and thus to the survival fitness of tumor cells. BC073020 (SCF), DK45035 (SCF), and GM25254 (DNS)
PLD has a role in the formation of clathrin‐coated vesicles and in the structure maintenance of Golgi apparatus in HSY cells
Authors: de Souza Lorena Brito , Jamur Maria Célia , Oliver Constance
Published: June 2021

Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. PLD has been implicated in many cellular functions, however, the function and intracellular localization of PLD varies according to cell type. The aim of this study was identify the intracellular distribution of the isoforms PLD1 and PLD2 and determine the role of PLD in the formation of clathrin‐coated vesicles (CCVs) from Golgi apparatus in duct cells of parotid gland. Treatment of cells with the primary alcohol 1‐butanol (1‐ButOH) inhibits the hydrolysis of phosphatidylcoline by PLD thereby suppressing phosphatidic acid (PA) production. Instead, phosphatidyl‐1‐butanol is formed by a transphosphatidylation reaction. In untreated HSY cells, there is a co‐localization of PLD with CCVs. When the cells were incubated with 1‐ButOH, clathrin‐coated vesicles, PLD1 and PLD2 are recruited to the perinuclear region. The decrease in PA levels after treatment with 1‐ButOH also results in an accumulation of lysosomes in the perinuclear region and affects the Golgi apparatus, increasing the volume of the Golgi saccules. Therefore, PLD is involved in the formation of Golgi associated CCVs as well as in the structural maintenance of the Golgi apparatus in HSY cells. FAPESP, CNPq, CAPES
Genetically encoded probes for phosphatidic acid
Authors: Vitale Nicolas , Kassas Nawal , Tryoen‐Toth Petra , Pelletan Leonardo , Bailly Yannick , Grant Nancy , Belmonte Silvia
Published: June 2021

During the last two decades, it has become evident that cellular lipids not only form bilayers to separate compartments, but also participate in vesicular trafficking and signal transduction. Phosphoinositides have received major attention at this respect, but other phospholipids such as phosphatidic acid (PA) are quickly emerging as signaling molecules. The spatiotemporal distribution of cellular PA appears tightly regulated by localized synthesis and quick degradation. Although it has been reported that PA plays key roles in key cellular functions, it remains unknown when and where PA is produced in the living cells. Hence we have decided to characterize biosensors that specifically bind to PA through relatively compact binding domains.

After protein expression and purification, their interaction with PA was characterized using PIP strips and liposomes assays. The results confirm that they have high specificity for PA, but we also found that chain length and saturation affected interaction with membranes. Finally we used these probes to visualize the dynamic of PA in various cell models and found that the various probes had also different affinity for PA in cells. In conclusion, the use of a large choice of probes may be required to adequately follow the complex dynamic of PA in various cell types, subcellular compartments and cellular processes.
Lipidomics of Aiptasia pallida and Symbiodinium: a model system for investigating the molecular basis of coral symbiosis
Authors: Garrett Teresa A , Hwang Janice , Schmeitzel John L. , Schwarz Jodi
Published: June 2021

Changing oceanic environmental factors disrupt the symbiotic relationship between cnidarians and a photosynthetic dinoflagellate symbiont, leading to expulsion of the symbiont from the cnidarian and endangering fragile coral ecosystems. The symbiosis between the anemone Aiptasia pallida and Symbiodinium is a model system for studying the molecular changes that occur during the establishment, maintenance and disruption of symbiosis. The symbiont resides in a specialized membrane component called the symbiosome membrane indicating dramatic changes to the lipidome of the anemone and symbiont. We have undertaken a comprehensive characterization of the lipid composition of the anemone, and symbiont at all stages of the symbiotic relationship. Lipids from the aposymbiotic symbiont, aposymbiotic anemone, and symbiotic anemone and symbiont were extracted and analyzed using liquid chromatography electrospray ionization mass spectrometry. Preliminary results based on exact mass and collision‐induced decomposition mass spectrometry reveal the presence of glycerophospholipids, phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine and interestingly, several molecular ions do not correspond to any known lipid molecular species. Lipidomic changes will be correlated to transcriptomic changes known to occur at the various stages of symbiosis.
New unphosphorylated peptide motif interacts with Grb7 SH2 domain
Authors: Zhang Dan , Ma Sucan , Gao Youhe
Published: June 2021

The Grb7 protein is found to be highly expressed in metastatic tumors. And the SH2 domain in Grb7 C‐terminal is reported to be mainly involved in Grb7 signal pathways. Currently, most reported motifs interacting with Grb7 SH2 domain are phosphotyrosine motifs. This domain can also bind dephosphorylated motifs steadily from degradation of phosphatases. By yeast two hybrid screening random peptide library, we have found some nonphosphorylated consensus sequences interacting with Grb7 SH2 domain. All positive peptides comprised a 22 amino acids sequence, containing 7 conserved ones at the two terminals and 15 random ones in the interval including S, T and P residues. This new way of interaction was not a common phenomenon, for we did not detect the interaction between these peptides and other SH2 domain proteins such as ABL, Grb2 and Grb14. Deleting assays in yeast two hybrid system showed that 7 conserved amino acids were necessary for interaction between Grb7 SH2 domain and nonphosphotyrosine peptides. To further confirm the interaction, we selected two peptides and a mutation which was short of 7 conserved sequences and co‐transfected into 293T cells with Grb7 SH2 domain. We found 7 conserved residues played essential roles in interacting with Grb7 SH2 domain in co‐IP assay. In further experiments, it is interested to see if those dephosphorylated peptides as drugs against cancer with high expression of Grb7 protein.
Biochemical and structural characterization of the activation of soluble Guanylate Cyclase
Authors: Seeger Franziska , Laffly Emmanuelle , Garcin Elsa D
Published: June 2021

Soluble Guanylate Cyclase (sGC) is a heterodimeric enzyme converting GTP to cyclic GMP. Each subunit contains an N‐terminal, heme‐containing regulatory domain (HNOX: Heme Nitric oxide OXygen), a dimerization PAS (Per/Arnt/Sim) and coiled‐coil (CC) domain, and a C‐terminal catalytic domain (GC). Controlling crucial physiological functions such as vasorelaxation, a low turnover of sGC causes cardiovascular pathologies. Binding of nitric oxide (NO) to HNOX maximally activates sGC and alleviates a presumed inhibitory effect of HNOX on GC. However, the isolated GC exhibits low activity excluding HNOX as the only brake on catalytic turnover.

To elucidate the activation mechanism and identify the domains required for maximum activity, we are studying truncated bovine sGC constructs. These include GC, CC‐GC, and PAS‐CC‐GC as well as the HNOX‐PAS and HNOX‐PAS‐CC heterodimers.

We report preliminary SAXS (Small Angle X‐ray Scattering) ab initio envelopes on HNOX‐containing constructs and present activity assays for the GC‐containing constructs.

Combinations of activity measurements, SAXS, and x‐ray crystallography have been used to elucidate the activation mechanism of sGC. These studies will provide a foundation for structure‐based drug design of novel sGC activators.

Work supported by American Heart Association Scientist Development Grant (E. Garcin) and NIH CBI Training Grant NIH T32 GM066706‐06.
HUMAN NEUROGLOBIN FUNCTIONS AS A REDOX REGULATED NITRITE REDUCTASE
Authors: Tiso Mauro , Tejero Jesus , Basu Swati , Azarov Ivan , Wang Xunde , Simplaceanu Virgil , Frizzell Sheila , Jayaraman Thottala , Geary Lisa , Shapiro Calli , Ho Chien , Shiva Sruti , Kim‐Shapiro Daniel B , Gladwin Mark T
Published: June 2021

Neuroglobin (Ngb) is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. Ngb shows a six‐coordinate heme iron with proximal and distal histidines in the heme pocket directly bound to the heme iron however coordination of the sixth ligand is reversible. Deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox sensitive surface thiols, cysteine 55 and 46, which regulate the open probability of heme pocket, nitrite binding and NO formation. Replacement of the distal histidine by leucine (H64L) leads to a stable five‐coordinated geometry; the Ngb H64L mutant reduces nitrite to NO approximately 2000‐times faster than the wild type, while mutation of either C55 or C46 to alanine stabilizes the six‐coordinate structure and slows the reaction. Using lentivirus expression systems we show that the nitrite‐reductase activity of Ngb inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six‐to‐five coordinate status of Ngb regulates intracellular hypoxic NO signaling pathways. These studies suggest that Ngb may function as a physiological oxidative stress sensor and a post‐translationally redox regulated nitrite reductase, that generates NO under six‐to‐five coordinate heme pocket control.
Vitamin A bioefficacy of traditional and provitamin A‐biofortified banana
Authors: Bresnahan Kara Allison , Arscott Sara , Tanumihardjo Sherry
Published: June 2021

The provitamin A biofortification of banana could provide a sustainable and cost effective method to ameliorate vitamin A deficiency in many tropical countries where banana is a staple food. However, matrix effects in banana may limit the absorption of vitamins, which could render biofortification efforts ineffective. We examined VA bioefficacy of traditional and high provitamin A biofortified bananas using Mongolian gerbils (n = 97). After feeding VA‐deficient diet for 8 weeks, customized banana diets were fed for 17 days in studies 1 and 2. Both studies utilized VA positive and negative controls, as well as a VA plus banana group. In study 1, which assessed traditional banana, only the unripe Cavendish group VA stores (16.6 ± 7.7 nmol/liver) differed from baseline (15.7 ± 7.7 nmol/liver), (P < 0.05). In study 2, which compared traditional and high provitamin A biofortified bananas, no groups differed from baseline VA stores. Both studies provide support for matrix effects negatively impacting provitamin A absorption because limited VA bioefficacy was observed regardless of provitamin A concentration. α‐Retinol levels, derived from α‐carotene, were significantly elevated in gerbils fed traditional ripe and biofortified banana. This is of interest because α‐retinol may have partial VA function. Biofortification efforts in this crop need further evaluation. (Support from HarvestPlus 8907.)
Adaptation to and Intake Patterns of Traditional Foods Made from Biofortified Orange Maize (Zea mays) in Rural Zambia Children
Authors: Nuss Emily Taylor , Arscott Sara A , Bresnahan Kara , Pixley Kevin V , Rocheford Torbert , Hotz Christine , Siamusantu Ward , Chileshe Justin , Tanumihardjo Sherry
Published: June 2021

Vitamin A deficiency (VAD) persistently plagues much of the world, propagating poor health outcomes related to reproduction, growth, sight, and immunity. Biofortification of staple crops is a novel strategy for combating VAD in high‐risk groups. Many African populations are proposed beneficiaries for provitamin‐A biofortified maize (Zea mays), often called “orange maize” due to distinct yellow/orange kernels. Its color challenges cultural acceptance by maize‐consuming communities that traditionally eat white varieties. This study explores adaptation to, and intake patterns of, traditional foods made with orange maize compared with white in rural Zambian children (n = 189) during a 3‐mo intervention. Subjects were fed maize porridge breakfast, maize nshima with relish lunch, and snack. Consumption of menu items, except snacks, increased significantly by week (P<0.0084). Orange porridge and nshima intakes equaled white from week 2 onward. During the trial, 3 orange maize meals were fed: 2009 freezer (O1), 2009 cold room (O2), 2010 fresh (O3). Subjects consumed uniform amounts of porridge and nshima prepared from O1 and O2, but intakes became significantly higher after O3 maize meal substitution (P<0.014 porridge; P<0.013 nshima). Results demonstrate quick adaptation to orange maize, preference for fresh, and an optimistic outlook for similar adaption patterns in other orange‐maize target countries.

Grant Funding Source: HarvestPlus 8217