bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of isoflavone yields and profiles from hairy root‐induced kudzu genotypes
Authors: Mun Jonathan , Reppert Nelson Adam , Lachcik Pamela , Grannan Michael , Yousef Gad , Rogers Randy , Janle Elsa , Weaver Connie , Lila Mary Ann
Published: June 2021

Kudzu is a rich source of isoflavones, including puerarin which has positive effects on bone formation in vitro and in vivo, and may reduce bone loss associated with osteoporosis. Kudzu14C‐labeled isoflavones provide a means of studying their distribution and metabolic fate in vivo. In a recent pharmacokinetic trial, Sprague‐Dawley rats were gavaged using a crude extract containing P. lobata 14C‐isoflavones. Appreciable levels of radiolabel (0.42, 0.33, and 0.10% of the administered dose) were detected in femur, tibia, and vertebrae, respectively. In the current study, hairy roots of 4 kudzu selections, Pueraria montana var. lobata (USA), P. montana var. lobata (Japan), P. lobata (USA), and P. phaseoloides (Guatemala) were extracted with 80% methanol to determine isoflavone content. Extracts were evaluated by high‐performance liquid chromatography (HPLC) with puerarin, daidzin, daidzein, malonyl‐daidzin, genistin, genistein, and malonyl‐genistin standards. P. montana var. lobata (USA) contained the highest puerarin and total isoflavone content of the 4 selections, with concentrations of 53.4 and 112.4 mg/g dry extract respectively. This strain is under cultivation to enhance isoflavone yields and facilitate further radiolabel‐assisted pharmacokinetic studies.

Supported by NIH, 2 P50 AT000477–06.
Antioxidant Effects of Ethyl Acetate‐Soluble Fraction of Chrysanthemum coronarium
Authors: Kim Jong‐Sang , Seo Ji Yeon , Ha Young Ran , Lim Ji Sun , Lee Inae , Lim Soon Sung , Park Jung Han Yoon , Kong Tony An , Lee ChoongHwan
Published: June 2021

The present study was performed to purify phase 2 detoxifying enzyme inducer(s) from Chrysanthemum coronarium. Ethyl acetate‐soluble fraction was loaded on silica column and separated into seven subfractions. Among the subfractions of ethyl acetate‐soluble fraction, subfraction 3 was found to induce NQO1 activity the most strongly in murine hepatoma cells. Furthermore, subfraction 3 induced luciferase activities in HepG2‐C8 cells which were generated by transfecting ARE‐luciferase gene, suggesting ARE‐mediated phase 2 detoxifying enzyme induction. We also investigated whether the subfraction had effect on antioxidant/detoxyfying enzymes in whole animal system. We injected hyrogen peroxide and/or subfraction 3 to ICR male mice, and investigated the expression levels of antioxidant enzymes in tissues as well as FRAP assay. In conclusion, chromatographic subfraction 3 of ethyl acetate fraction of Chrysanthemum coronarium showed strong antioxidant potential and merits identification of active component(s).
PGK1 and p27/kip1 as Biomarkers for Intracellular Redox Status
Authors: Kim Jong‐Sang , Quintos Lesley , Lee In Ae , Lim Ji Sun , Seo Ji Yeon , Ha Young Ran , Kim Hak Ryul , Sung Mi Kyung
Published: June 2021

In this study we investigated the effect of delphinidin, a well‐known antioxidant present in pigmented fruits and vegetables, on the expression of not only PGK1 but also p27/kip1, one of the proteins induced in HT29 colon carcinoma cells treated with delphinidin, in whole animal model. ICR mice were injected with varying concentrations of hydrogen peroxide, delphinidin and both. Western blot analysis for the mouse large intestinal tissue showed that PGK1 expression was increased by hydrogen peroxide in a dose dependent manner but was attenuated by combined treatment with hydrogen peroxide and delphinidin. On the other hand, the expression of p27/kip1 was induced by both hydrogen peroxide and delphinidin in mouse organs. Plasma FRAP levels of delphinidin‐treated mice were decreased significantly as compared to those of hydrogen peroxide‐treated control group, maybe due to compensatory downregulation of endogenous antioxidants and/or relevant enzymes. In conclusion, our data suggest that the expression of PGK1 and p27/kip1 are sensitively responding to the presence of either oxidant or antioxidant.
HEPATOPROTECTIVE EFFECT OF SILICA HYDRIDE ON CARBON TETRACHLORIDE‐INDUCED HEPATIC INJURY IN RATS
Authors: Mendieta Jessica Elena , Correa José , Trujillo José , Rosales Martha
Published: June 2021

Reactive oxygen species (ROS) have been proven to cause numerous cellular anomalies, which cause oxidative stress. Taking into account that the most endogenous and exogenous antioxidants are not enough effective as reducing agents and as ROS selective scavengers, an anionic hydride organosiloxane, commonly named silica hydride (SiH−), was previously evaluated in vitro. However, none research had been done to prove its antioxidant and protective effects in vivo. For this reason, this work was aimed to demonstrate it suggesting its possible mechanism of action.

The results of the present study demonstrate that the protective effect of SiH− is dose‐dependent. Pre‐treatment of Sprague‐Dawley rats with dosages of 180 and 200 mg/kg showed the better protection against CCl4‐induced hepatotoxicity. Lipid peroxidation and cytochrome P‐450 activity significantly decreased in relation to that of controls. In contrast, glutathione content, glutathione peroxidase and glutathione reductase activities significantly increased. The lower serum levels of alanine and aspartate amino transferases were restored. Histopathological studies confirm the protective effect of SiH−. In conclusion, this model in vivo and the different assays confirm that the mechanism of hepatoprotective action of SiH− may be due to its antioxidant and radical scavenger activities.
Polyphenols, Carotenoids, Tocopherols and Antioxidant Activity in Different Varieties of Angelica keiskei and Glycine max
Authors: Chun Hye‐Kyung , Li Lei , Aldini Giancarlo , Park Ki‐Moon , Cho Soo‐Muk , Chen Chun Yen Oliver , Blumberg Jeffrey B , Russell Robert M , Yeum Kyung‐Jin
Published: June 2021

The antioxidant content and activity as well as the stability of different varieties of Angelica keiskei (n = 3), and Glycine max (n = 5) were determined. Angelica keiskei, a dark green leafy vegetable, was lyophilized; and Glycine max, black soybeans, were roasted at 125°C for 2 min, milled to a powder and stored at room temperature, 4°C or −80°C for 4 mo. Carotenoids, tocopherols, and ascorbic acid, were analyzed by HPLC systems, total phenols by spectrophotometry, and antioxidant activity by fluorometry. Polyphenols in Angelica keiskei were further characterized by LC‐ESI‐MS/MS. A range in the content of lutein (205–265 μg/g dry wt), trans‐β‐carotene (103–130 μg/g dry wt), and total phenols (8.6–9.7 mg/g) was observed among three different varieties of Angelica keiskei. LC‐ESI‐MS/MS analysis identified that chlorogenic acid, chalcones and the glucosides of luteoline & quercetin as the major polyphenols in Angelica keiskei. Glycine max contained γ‐tocopherol and lutein at 82–133 and 6.5–14.6 μg/g dry wt, respectively. Major phytonutrients except for β‐carotene and antioxidant activity in lyophilized Angelica keiskei and Glycine max were stable up to 4 mo at the tested temperatures. Thus Angelica keiskei and Glycine max are stable sources of phytonutrients with their concentration dependent upon the variety.

[Supported in part by USDA #58‐1950‐7‐707, USA & BioGreen 21 Program (Code #20070301034009) RDA, Korea]
Genistein, daidzein, and equol increase quinone reductase activity and protein in Hepa‐1c1c7 cells
Authors: Froyen Erik , Steinberg Francene
Published: June 2021

Soy protein and isoflavones, genistein (G) and daidzein (D), are associated with protection against the risk of cancer. One postulated mechanism is modulation of phase I and II biotransformation enzymes. The objective of this study was to analyze phase II enzyme Quinone Reductase (QR) activity, protein, and RNA in Hepa‐1c1c7 cells treated with G, D, and equol (E), a daidzein metabolite. We hypothesize that G, D, and E increase QR enzyme activity, protein, and RNA via estrogen receptor beta binding and interacting with the QR antioxidant response element. Hepa‐1c1c7 cells were treated with DMSO (0.1% v/v), β‐naphthoflavone (positive control at 1 μM), and G, D, and E at concentrations of 1, 5, and 25 μM. After 24 hours of treatment (results expressed as treated/control ratios), G (1.32, 1.42, and 1.75), E (1.65, 1.58, and 1.65), and D (1.55 at 1 μM) increased QR activity compared to the DMSO treated cells (n = 3 per group) (P<0.05). After 48 hours, G (1.3 and 1.6), D (1.39 and 1.34), and E (1.46 and 1.55) at 5 and 25 μM concentrations increased QR activity (n = 12 per group) (P<0.05). Furthermore, G, D, and E increased QR protein in 25 μM treated cells. Future approaches include use of chromatin immunoprecipitation (ChIP) assay to determine the mechanism of isoflavone stimulated increase in QR expression. In conclusion, G, D, and E significantly increase QR activity and protein in Hepa‐1c1c7 cells.

Support: Departmental funds.
Infant diet differentially affects human electroencephalographic activities in the first year of life
Authors: Jing Hongkui , Pivik R.T. , Gilchrist Janet M. , Badger Thomas M.
Published: June 2021

The influence of infant diet (milk‐based formula [MF], soy‐based formula [SF], and breast milk [BF]) on brain EEG activities was studied in infants (20 males and 20 females per group) at 3, 6, 9, and 12 months of age. Power spectra were calculated in five frequency bands for scalp EEG signals recorded at 124 sites. ANOVA tests were applied to determine effects of diet, gender, age, brain area, and hemisphere. Diet significantly affected EEG spectral power. In Band 1 (0.1–3 Hz), the highest spectral power was observed at 9 months in BF infants in contrast to 6 months in MF and SF infants. In Band 2 (3–6 Hz), compared to MF and SF infants, BF infants had a smaller increase in EEG spectral power from 3 to 6 months. In Band 3 (6–9 Hz), 9‐month‐old MF infants had higher EEG spectral power in anterior brain areas and lower power in posterior areas relative to BF infants. In Band 4 (9–12 Hz), BF boys showed higher spectral power than BF girls in general, but girls in the MF and SF groups had higher spectral power than boys in those groups. This effect was also observed in Band 5 (12–30 Hz). In addition, EEG spectral power was higher in the left hemisphere relative to the right hemisphere in all infants. Based on these differences for gender, age, brain areas, and hemisphere, our results suggest that infant diet differentially modifies the development of brain physiological functions in the first year of life. (Supported by USDA CRIS 6251‐51000‐004‐01S)
Diet‐induced thermogenesis and post‐prandial substrate oxidation after soybean ingestion
Authors: Sakurai Masao , Iwashita Soh , Suyama Toru , Hayase Hideki , Morimoto Taketoshi , Hamada Koichiro
Published: June 2021

Diet‐induced thermogenesis (DIT) is higher in protein than in fat or carbohydrate. However, higher protein intake in a mixed meal does not always result in higher DIT. In addition, the relation between the type of protein consumption and DIT is not clear. Therefore, we investigated DIT of soybean, which is rich in protein. Ten healthy male volunteers (29.2 yrs, BMI=23.7) participated in both soybean (Soy) and Wheat trials in blinded random order with at least one week of wash out period. Soy and Wheat were baked before serving. After an overnight fasting (>12 hrs), subjects ate test meal (29.3 KJ/kg) at 0900. Expired gas and blood samples were collected every hour from 0800 until 1500, and DIT, substrate oxidation, glucose, insulin and lipid profiles in the blood were determined. DIT was significantly higher in Soy than in Wheat (P<0.05). AUC in fat oxidation (OX) was higher and that in carbohydrate OX was lower in Soy (P<0.05). AUCs in plasma glucose and serum insulin level were remarkably lower in Soy than in Wheat (P<0.05). Serum free fatty acids and triglyceride were higher in Soy than in Wheat (P<0.05). These data suggested that soybean has a potential for enhancing DIT and post‐prandial fat oxidation. Soybean is expected to be an anti‐obesity food.
Egg white protein prevents hyperhomocysteinemia in folate‐deficient rats
Authors: Luchtel Rebecca A , Garrow Tim A , Schalinske Kevin L
Published: June 2021

Maintenance of homocysteine (hcy) homeostasis is regulated through folate‐independent and folate‐dependent remethylation, as well as irreversible transsulfuration. Folate deficiency perturbs hcy balance resulting in hyperhomocysteinemia, an independent risk factor for cardiovascular disease. Because of the marked difference in sulfur amino acid profile, we conducted a study to compare egg white and casein protein with respect to their ability to prevent hyperhomocysteinemia in folate‐deficient rats. Rats were fed 20% casein or egg white protein diets for 2 wk, followed by folate‐free diet treatment in half of the rats for an additional 4 wk. As expected, hyperhomocysteinemia was seen in folate‐deficient rats fed a casein diet. However, the folate‐deficient rats fed egg white protein exhibited hcy levels that were not statistically different from those rats receiving adequate folate. It appears that the effectiveness of egg white protein to prevent hyperhomocysteinemia was due to a 40% increase in hepatic betaine methyltransferase, the enzyme that catalyzes folate‐independent hcy remethylation. Taken together, these results indicate that dietary egg white protein has a significant impact on hcy metabolism and is extremely effective in the prevention of hyperhomocysteinemia (Support: Egg Nutrition Center, Iowa Egg Council).
Anemia and malnutrition is equal in children included and not included in the Oportunidades supplementation programme in Guanajuato Mexico
Authors: Delgado Luz María , García Olga Patricia , Caamaño María del Carmen , Rosado Jorge Luis
Published: June 2021

The objective of the present study was to compare nutritional status and anemia in children who have been part of the Oportunidades program in Mexico and consumed a fortified porridge (N=87) with similar children who have not been included in the program (N=89). The study was carried out in infants (35 ± 13 mo) and both groups were from the same rural communities in the state of Guanajuato. Weight and height were measured in all subjects, and a fasting blood sample was taken for the determination of hemoglobin. The results are shown in the table.

No differences were found between both groups in anemia or any of the growth variables. In conclusion, the inclusion of children in the Oportunidades national program including the intake of the fortified porridge to infants of these communities does not have a significant impact in the nutritional status, growth and anemia.
L‐arginine reverses Radiation‐induced immune dysfunction: the need for optimum treatment window
Authors: Shukla Jyoti , Chatterjee Saurabh , Thakur V.S. , Poduval T.B.
Published: June 2021

The aim of the present study was to investigate the protective efficacy of L‐arginine (LA), in mitigating gamma radiation‐induced injury in mice. Administration of LA at +2 h after 2‐Gy total body gamma radiation (TBI) was effective in preventing, the decrease in spleen size, weight and splenocyte number caused by TBI (Fig. 1a). LA administration at +2 h of TBI also prevented specific antibody response of splenic B cells to sheep red blood cells (Fig. 2). Splenic DNA fragmentation, which was significantly increased in TBI mice, was significantly reduced by the administration of LA at +2 h of TBI (Fig. 3). However, LA administered just before TBI did not reverse the radiation‐induced splenic damage. Serum nitrite levels in mice administered with LA just before TBI, were significantly higher, when compared to levels in mice, administered with LA at +2 h of TBI. The results suggest the importance of the correct therapeutic window of administering LA in mitigating the radiation induced host splenic immune dysfunction.
Morphometric Evaluation of Tongue Epithelium in Experimental Chromium Administration
Authors: Farinha Rodrigo , Rafael Ana I.V. , Falacho Rui , Alpoin Carmen , Cabrita A.M. Silvério
Published: June 2021

Recent reports alert to the presence of chromium in drinking water, mainly related with industry contamination. The hexavalent form is a carcinogenic for lab animals. In this study we make a morphometric evaluation of tongue epithelium in the experimental administration of chromium per os.

We used 20 animals, distributed into two groups: one kept with no manipulation (control) and one submitted to the administration of drinking water contaminated with chromium, 20 ppm (test). After 30 weeks all the animals were sacrificed and the necropsy was performed, collecting the tongue for histology. The histological slices were observed at three different levels and analyzed using Sigma Scan Pro®. Taking the epithelium area (EA) and the total area of the tongue at each level (TA), we calculated the ratio: EA/TA.

We observed in the test group some cases of focal increased thickness of the epithelium with expressive endophytic growth. No neoplastic lesions were observed. The calculated ratio didn't shown significant differences between the two groups, for a 95% confidence interval.

According to these results, the administration of chromium in the drinking water may be responsible for focal tongue morphologic changes with an endophytic proliferation of epithelial tissue.
Expression of BRCA1 and FAC1 in primary sporadic breast cancers.
Authors: White Kristen K. , Wells Wendy A. , Bowser Robert , Tsongalis Gregory J. , Coleman William B.
Published: June 2021

We have previously described a +ACA BRCA1 promoter polymorphism that creates a FAC1 transcriptional repressor binding site (AACAACAC), which may confer FAC1‐mediated transcriptional repression (depending on the BRCA1 promoter genotype). To explore the relationship between BRCA1 and FAC1 expression in primary breast cancers, we performed immunostaining on 69 luminal A and B subtype sporadic breast cancers (ER+, PR+, HER2+/−). Most tumors are positive for BRCA1 expression (40/69, 58%), while a subset express reduced BRCA1 levels (29/69, 42%). The majority of these tumors (64/69, 93%) are positive for FAC1 expression: 27/64 (42%) express cytoplasmic FAC1 (cFAC1), 23/64 (36%) express both cFAC1 and nuclear FAC1 (nFAC1) and 14/64 (22%) express nFAC1 only. Among tumors with reduced BRCA1 expression, 55% (16/29) are positive for nFAC1 expression (which represents 43% (16/37) of nFAC1 expressing tumors that have reduced BRCA1 protein expression). This data suggests that most primary breast tumors are positive for FAC1 expression, and that 58% of FAC1 expressing tumors have some degree of nuclear FAC1 expression. It is intriguing to speculate that nFAC1 may modify gene expression in breast tumors, resulting in repression of normal BRCA1 expression, and contributing to breast carcinogenesis.

Support: NIH CA78343, Komen Foundation BCTR0100575, Friends For an Earlier Breast Cancer Test.
Lectin glycosylation as a marker of thin gut inflammation
Authors: Guzyeyeva Gloria V.
Published: June 2021

Mucous maturation in human gastrointestinal system remains unexplored. It has been previously reported that changes in lectin receptors glycosylation might serve as histochemistry markers of thing gut inflammation. The aim of this study was to compare the binding pattern of biotinylated lectins in different subtypes of thin gut inflammation in the early childhood The study was performed on biopsy material of 120 patients with chronic thin gut inflammation and 30 healthy controls with different lectins: Peanut agglutinin (PNA), Soybean agglutinin (SBA), Wheat germ agglutinin (WGA), Lens culinaris (LcL), Laburnum anagyroides (LAL). Presence of N‐ and O‐glycans in the composition of proteins of plasmatic membranes of a thin gut was shown. Sialic acids were absent in the composition of alkaline phosphatase of mature mucous production. The expression of lectin‐binding glycoconjugates exhibited differences between certain types of chronic thin gut inflammation. WGA with affinity for GlcNAc and neuraminic acid labelled the cells with early thin gut inflammation but most intensely those of fibroblastic type. Staining with PNA and SBA, which are GalNAc specific, were strongly positive. Enhanced PNA reactivity reflected mainly cytoarchitectural pattern. These results documented the heterogeneous glycosylation pattern in different subtypes of thin gut inflammation and indicate the usefulness of lectins in the early evaluation of pluripotential differentiation of thin gut secretory cells.[Sponsored by NIH grants from Russian Federation].
Effects of Helicobacter typhlonius and H. rodentium Infections in IL‐10‐deficient Mice
Authors: Chichlowski Maciej , Sharp Julie M. , Vanderford Deborah A. , Myles Matthew H. , Hale Laura P.
Published: June 2021

Murine intestinal infections with Helicobacter typhlonius and/or H. rodentium are endemic within many research facilities. This study was designed to analyze effects of infection with these two Helicobacter species in the C57BL/6 IL‐10‐deficient (IL‐10−/−) mouse. Adult IL‐10−/− mice experimentally infected with H. rodentium and/or H. typhlonius developed inflammatory bowel disease (IBD), with the severity of inflammation at 11 weeks post‐infection being: non‐infected (no inflammation) < H. rodentium < H. typhlonius < mixed H. rodentium + H. typhlonius. Helicobacter DNA was detected in gastrointestinal and reproductive organs of infected mice by quantitative real‐time PCR. Infected mice also showed significant differences in pregnancy rates, number of pups born, and pups surviving to weaning compared with non‐infected controls. Treatment with 3 mg amoxicillin, 0.5 mg clarithromycin, 1 mg metronidazole, and 20 μg omeprazole per 5 g chow eradicated Helicobacter infection, ameliorated established IBD, and increased pregnancy rates and pup survival. Results demonstrate the pathogenicity of H. rodentium in a mono‐infection model and suggest a complex role of both Helicobacter and other intestinal microbiota in reproductive success and the development of IBD in susceptible hosts.

This work was supported by NIH grant # CA115480.
Variability among Macaca fascicularis in the innate immune response to Toll‐like receptor 4 (TLR4) agonists
Authors: Frevert Charles Wayne , Hukkanen Renee , Mongovin Steve , Larsen Kay , Agy Mike , Martin Thomas , Wurfel Mark
Published: June 2021

Despite being highly conserved, variability exists in the innate immune response to microbial pathogens. In man, individuals with distinct phenotypes are identified as high and low responders to TLR4 agonists. Whether similar variability exists among Macaca fascicularis is not known. Whole blood collected from 45 Indonesian‐origin monkeys was treated ex vivo with lipopolysaccharide (LPS) from Yersinia Pestis (YP), Pseudomonas aeruginosa, Salmonella minnesota, and Escherichia coli. Cytokines were measured at 4 h and the data organized in rank order ‐ lowest (1) to highest (45) ‐ for each cytokine and each TLR4 agonist. We identified high responders that consistently ranked in the top 20th percentile and low responders that consistently ranked in the bottom 20th percentile across all cytokines. In the entire group, cytokine production in response to TLR agonists varied by 10–100 fold. The range for TNFα in whole blood treated with YP LPS was 328 to 9,932 pg/ml. The high responders had 10‐fold more TNFα in plasma (5,300 +/−680 pg/ml) than low responders (520 +/−61 pg/ml) (p<0.0001). Similarly, high and low responders were identified for other TLR4 agonists and cytokines. These findings suggest that significantly different innate immune response phenotypes exist among M. fascicularis. Such variability may influence outcomes in studies of the systemic inflammatory response syndrome, sepsis, or vaccine trails.
TLR4 related to CXCR4 and CCR5 expression in HIV
Authors: Sanders Catherine Miller , Cruse Julius M , Lewis Robert E.
Published: June 2021

In the present investigation, flow cytometric techniques were utilized to evaluate 20 cases of HIV for CXCR4, CCR5, and TLR4 expression in CD4 positive T cells, CD8 positive T cells, regulatory T cells, and Th17 cells, and mean fluorescence intensities (MFI) were determined. RT‐PCR was utilized to measure gene expression of CXCR4, CCR5, and TLR4. CD4+ T cells positively expressed CXCR4 in 16 cases, CCR5 in all 20, and TLR4 in 17. CD8+ T cells were positive for CXCR4 expression in 15 cases, CCR5 in all cases, and TLR4 in 18. Regulatory T cells positively expressed CXCR4 and CCR5 in 12 cases, and TLR4 in 11 cases. Th17 cells were positive for CXCR4 expression in 19 cases and TLR4 in 17. When compared to stage of disease, receptor expression by all cell types increased greatly as infection progressed from acute to chronic phase, but decreased only slightly as infection progressed from chronic phase to AIDS. Results reveal a positive correlation between CXCR4, CCR5, and TLR4 expression which may indicate that TLR4 also serves as a co‐receptor for HIV. In addition, these data suggest that TLR4 up‐regulation is not limited to gram‐negative bacterial infection nor is expression limited to myeloid cells.
Expression of neuregulin‐1 and ErbB‐receptors mRNAs is modulated by denervation and reinnervation in adult rat skeletal muscles
Authors: Geuna Stefano , Nicolino Silvia , Raimondo Stefania , Tos Pierluigi , Perroteau Isabelle
Published: June 2021

The Neuregulin/ErbB signaling pathway plays and important role in the neuromuscular system. The aim of the present study was to investigate by immunohistochemistry, RT‐PCR and Western blotting analyses, in an in vivo model of adult skeletal muscle denervation/reinnervation, if the expression of Neuregulin‐1 (NRG1) and ErbB receptors is regulated by the innervation condition.

Results showed that denervation of flexor digitorum muscles of the rat forelimb by median nerve transection led to a significant up‐regulation of the α, but not β, isoform of NRG1, as well as of ErbB2, ErbB3 and ErbB4‐cyt1 isoform. Reinnervation, i.e. the arrival of regenerated axons after median nerve surgical reconstruction, induced a prompt down‐regulation of all messengers of the NRG1/ErbB system. Altogether, our results describe the variations of the NRG1/ErbB system in skeletal muscles and suggest the existence of a NRG1‐mediated autocrine and/or paracrine trophic loop that is activated after muscle denervation. This myotrophic loop can represent a promising therapeutic target for the prevention of various muscle diseases.

Financial support: Italian M.U.R. (PRIN2005 fund) and Regione Piemonte (CIPE fund).
Arsenic signals through g‐proteins and NADPH Oxidase (NOX) to promote mouse liver sinusoidal endothelial cell capillarization
Authors: Straub Adam Carl , Beer‐Stolz Donna , Clark Katherine A , Barchowsky Aaron
Published: June 2021

Arsenic is a global health hazard that may cause disease in over 100 million people world‐wide. Liver disease including portal hypertension, non‐cirrhotic portal fibrosis, and vascular shunting and oxidative injury are associated drinking water with elevated arsenic levels. Liver sinusoidal endothelial cell (LSEC) capillarization with loss of fenestrations and increased surface expression of PECAM‐1 is an early event in these diseases. Drinking water containing 10 ppb or more of arsenic promotes LSEC capillarization in mice through unknown mechanisms. Therefore, the hypothesis that arsenic signals through NOX to promote LSEC capillarization was tested in primary mouse LSEC cultures. Ex Vivo arsenic exposures (0.5–5 μM) for 8 h recapitulated in vivo effects on capillarization. The ex vivo exposures increased superoxide generation that was prevented by pre‐incubating cells with the scavenger tempol or a selective NOX2 inhibitory peptide, gp91‐tat. Both tempol and gp91‐tat prevented arsenic‐stimulated capillarization. Finally, inhibition of either Gai or Rac1 GTPase also blocked arsenic‐stimulated oxidant generation and capillarization. These data are first demonstrating that environmental levels of arsenic stimulate LSEC capillarization through a g‐protein coupled, NOX‐mediated signaling cascade. Sponsored by EPA STAR grant FP‐91654201 and NIEHS grant R01ES013781.
New role of Human myofibroblasts on neovascularisation during wound healing
Authors: Mayrand Dominique , Cordier Benoît , Blanchette Samuel , Lopez‐Vallé Carlos A. , Roy Michel , Genest Hervé , Moulin Véronique
Published: June 2021

Myofibroblasts are involved in healing mechanism and on tumor development. In both cases, these processes are carried out in synergy with revascularization ensuring the survival of the tissue. We used a three‐dimensional model to investigate the role of myofibroblasts on neovascularization in a context of wound healing. The angiogenic development was analyzed in a fibrin‐based matrix after 14 days via a co‐culture of human skin microvascular endothelial cells (MVEC) and normal skin wound myofibroblasts (Wmyo) isolated from the same patient. Human skin fibroblasts (Fb) was used as control. In our culture conditions, we observed a significant increase of capillary‐like structure number and length when MVEC were co‐cultured with Wmyo instead of Fb. In the same way, we used myofibroblasts (Hmyo) and microvascular endothelial cells (MVECH) isolated from human hypertrophic scars. A positive effect of Hmyo addition on the angiogenic process was also observed but to a lesser extent than with Wmyo. The MVEC and MVECH had similar responses in the presence of dermal cells. From these results, we hypothesize that myofibroblasts could have a more important role on neovascularization than expected on normal wound healing but also in hypertrophic scarring characterized by a large number of myofibroblasts and an extensive microvascular network. This study was supported by CIHR and a scholarship from FRSQ (VM).
Stem cell mobilization and vasculogenesis stimulated by lactate and oxygen
Authors: Milovanova Tatyana N. , Sorokina Elena M. , Bhopale Veena M. , Moore Jonni S. , Hunt Thomas K. , Velazquez Omaida C. , Thom Stephen R.
Published: June 2021

This investigation assessed the effects of lactate and hyperoxia on recruitment and differentiation of stem/progenitor cells (SPCs). Within 18 hours after Matrigel plugs were implanted subcutaneously in mice, over one million CD34+ SPCs were recruited. Including a lactate source in Matrigel increased SPCs by 3.6‐fold at 18 hours and 5.8‐fold at 5 days. Lactate accelerated CD34+ cell differentiation based on surface marker expression, development of intricate vascular channels, and a higher fraction in the S and G2‐M phases of the cell cycle. If mice were intermittently exposed to hyperbaric oxygen, there were increased numbers of CD34+ cells in blood and Matrigel, accelerated differentiation and more complex vascular channels. Combining a lactate source and hyperoxia had an additive effect on CD34+ cells. If the Matrigel contained oxamate, a lactic dehydrogenase inhibitor; or antibodies that neutralize vascular endothelial growth factor or stromal derived growth factor, CD34+ cell recruitment and differentiation were inhibited. Hypoxia inducible factor‐1α (HIF‐1) was elevated in CD34+ cells from lactate‐supplemented versus un‐supplemented Matrigel. Hyperbaric oxygenation caused significant elevations of HIF‐1 in CD34+ cells in bone marrow, blood and Matrigel.

Conclusion: SPCs influence their own homing response by metabolizing lactate and generating growth factors to mediate progressive SPCs mobilization, recruitment and differentiation into vascular channels [N00014‐06‐1‐0363].
Accelerated Atherosclerosis in Low Density Lipoprotein Receptor Deficient Mice Lacking the Membrane Complement Regulator CD59
Authors: Yun Sheng , Leung Viola , Botto Marina , Boyle Joseph , Haskard Dorian
Published: June 2021

Background: CD59 is a membrane glycoprotein that inhibits terminal complement pathway C5b‐9 assembly. Here, we tested the hypothesis that CD59 limits the proinflammatory effects of complement activation in atherosclerosis.

Methods and Results: CD59 gene targeted mice (CD59a−/−) mice were crossed with Ldlr−/− mice to give double knock‐outs (DKO), and studied aged 22 weeks. Immunocytochemical staining showed CD59 on endothelial cells, vascular smooth muscle cells (VSMC) and macrophages in Ldlr−/− mice, with absent staining in DKO. DKO had more extensive en face Sudan IV staining of thoraco‐abdominal aorta than Ldlr−/− single knock‐outs, both following a normal chow (6.51 + 0.36% vs 2.63 + 0.56%, p<0.0001) or a high fat diet (17.05 + 2.15% vs 7.69 + 1.17%, p<0.02). Accelerated lesion formation in DKO mice was associated with significantly increased complexity of lesions in the aortic root, as assessed by increased VSMC number and fibrous caps development. The acceleration of atherosclerosis in DKO mice was associated with significantly greater deposition of C5b‐9, both following a chow or a high fat diet.

Conclusion: The acceleration of lesion formation in the absence of CD59 provides definitive evidence for complement activation in the Ldlr−/− mouse model and underlines the importance of CD59 in protection from the proatherogenic effects of the terminal complement pathway.

Funded by the British Heart Foundation
Brief hypercholesterolemia induces athero‐protective pathways on the patho‐susceptible aortic side endothelium of swine aortic valves
Authors: Guerraty Marie A , Grant Gregory R , Karanian John W , Chiesa Oscar A , Pritchard Wiiliam F , Davies Peter F
Published: June 2021

The aortic valve of humans and swine is preferentially susceptible to calcific sclerosis on the aortic side of the leaflets. Side‐specific endothelial gene expression in normal valves revealed a balance between pro‐pathologic and protective phenotypes on the aortic side (Simmons et al. 2005). We studied the effect of a modest exposure to hypercholesterolemia on the balance of endothelial phenotypes in a side‐specific manner. Eleven adult male swine were fed a normal chow diet (NC; n=6) or an isocaloric high cholesterol diet (HC; n=5) for 2 weeks. Transcript profiles of aortic (A) and ventricular (V) sides of the aortic valves of NC and HC animals were compared. The patho‐susceptible A side showed a markedly greater number of differentially expressed genes than the patho‐protected V side (1325 vs 87; false discovery rate 25%) indicating higher sensitivity to the risk factor on the susceptible side. These phenotypic differences were mirrored at the tissue level with early pathological changes confined to the A side of the leaflets of the HC animals. Pathway analysis showed that the HC treatment decreased TNF and NFkB pathway‐associated gene expression on the A side of the valve relative to the V side, and that a PPARgamma program was activated by the HC treatment on the A side. Together, these findings suggest a risk factor‐driven upregulation of athero‐protective pathways on the susceptible aortic side. NHLBI support.
Decay‐Accelerating Factor plays a critical atheroprotective role in Low Density Lipoprotein deficient (ldlr−/−) mice
Authors: Leung Viola WY. , Yun Sheng , Mason Justin , Song Wenchao , Botto Marina , Boyle Joseph , Haskard Dorian
Published: June 2021

Background: The GPI‐anchored membrane glycoprotein Decay‐Accelerating Factor (DAF, CD55) inhibits complement activation by downregulating C3 and C5 convertases. We asked whether DAF × LDLR double knock‐out (DKO) mice are protected from atherosclerosis.

Methods and Results: Immunostaining showed expression of DAF by endothelial cells, macrophages and vascular smooth muscle cells in ldlr−/− but not in DKO. At 22 weeks on a chow diet, DKO mice had more en face Sudan IV staining of thoracoabdominal aorta than LDLR single knock‐outs (4.66 ± 0.63% vs 2.85 ± 0.34%, mean ± SEM, p<0.05). Accelerated lesion formation in DKO mice was also evident in aortic roots, with lesions being larger (76.16 ± 9.53 × 103 μm2 vs. 29.42 ± 3.83 × 103 μm2, mean ± SEM, p<0.0005) and more complex (vascular smooth muscle cell content 13.86 ± 5.82 % vs. 2.23 ± 1.54 %, p<0.0005). DKO mice had increased aortic root C3d staining (% lesion positive for C3d 11.5 ± 1.3% vs 3.0 ± 0.2%, p < 0.001), and higher levels of IL1β and TNFα (p<0.01 each).

Conclusion: Our data indicate that DAF plays a critical role in regulating C3 activation in the arterial wall. Effective complement regulation enables the protective actions of the classical complement pathway on debris clearance to occur whilst reducing deleterious proinflammatory downstream effects of C3 activation.

Funded by the British Heart Foundation
Alpha 1A‐Adrenergic Receptor Signaling Promotes Antidepressant‐Like Behavior and Increased Anxiety in the Mouse
Authors: Handel Evelyn M , Jensen Kelly A , Talbot Jeffery N , Rorabaugh Boyd R
Published: June 2021

Norepinephrine plays a role in the regulation of antidepressant behavior. In the present study, we used mice expressing constitutively active mutant α1A‐adrenergic receptors (CAM α1A‐ARs) to examine the effects of α1A‐AR signaling on behavioral depression and anxiety. CAM α1A‐AR mice exhibited significantly less immobility than nontransgenic mice in the tail suspension test (TST) indicating that α1A‐AR signaling promotes antidepressant‐like behavior. WAY100635 (5‐HT1A receptor antagonist) potentiated the antidepressant‐like effect of CAM α1A‐AR expression but had no effect on nontransgenic mice. Distance traveled in the open field did not differ between CAM α1A‐AR mice and nontransgenic mice confirming that decreased immobility in the TST was not caused by differences in overall motor activity. However, CAM α1A‐AR mice spent significantly less time than nontransgenic mice in the center of the open field suggesting that α1A‐AR signaling is anxiogenic. We used the elevated plus maze to confirm the anxiogenic effect of α1A‐AR signaling. CAM α1A‐AR mice spent significantly more time than nontransgenic mice in the enclosed arms of the maze and had fewer entries than nontransgenic mice into the open arms. Our data indicate that α1A‐AR signaling promotes antidepressant‐like behavior and is anxiogenic. (This study was supported by a Bower, Bennet, & Bennet Endowed Research Chair Award from Ohio Northern University).
Differential effects of histamine‐3 receptor activation on the behavioral‐stimulant effects of methamphetamine and cocaine in mice and squirrel monkeys
Authors: Banks Matthew L , Manvich Daniel F , Bauzo Rayna M , Howell Leonard L
Published: June 2021

Histamine‐3 receptors (H3R) are G‐protein (Gi/o) coupled receptors primarily expressed in the CNS and can function as either auto‐ or heteroreceptors regulating the release of histamine or other neurotransmitters, such as dopamine. Furthermore, there are high densities of H3R located in striatal regions of mice and monkeys. The aim of the present study was to examine the role of H3R in the behavioral‐stimulant effects of methamphetamine (METH) and cocaine in both mice and monkeys. Mice were pretreated with the H3R agonist, Imetit, 30 min. before cocaine or METH administration and locomotor activity was assessed for 90 min. Squirrel monkeys were pretreated with Imetit 30 min. before cocaine or METH administration and tested on a fixed‐interval operant schedule of stimulus avoidance. Imetit decreased the stimulant effects of METH in both mice and monkeys. In contrast, Imetit did not attenuate the stimulant effects of cocaine in mice, but did in monkeys. Overall, these results suggest an inhibitory role of H3R on the stimulant effects of METH with species differences present in the role of H3R on the stimulant effects of cocaine.
Food restriction alters pramipexole‐induced yawning, hypothermia, and locomotor activity in rats: Evidence for sensitization of D2, but not D3 receptor‐mediated effects
Authors: Collins Gregory Thomas , Calinski Diane M , Woods James H
Published: June 2021

It has been well established that food restriction enhances sensitivity to the reinforcing effects of drugs of abuse, and that food restriction alters a variety of behavioral and pharmacological responses to dopaminergic agonists. Evidence from behavioral and molecular studies suggests that augmented dopaminergic responses observed in food‐restricted animals result from a sensitization of the D2 receptor, however, little is known about how food restriction affects the D3 receptor. These studies were aimed at defining the effects of food restriction on D2 and D3 receptor function by assessing pramipexole(PPX)‐induce yawning, penile erection (PE), hypothermia, and locomotor activity in free‐fed and food‐restricted rats. Food restriction resulted in a suppressed level of PPX‐induced yawning, a sensitized hypothermic response, and an enhanced locomotor response to PPX; there was no effect on PPX‐induced PE. In food‐restricted rats, the D3‐selective antagonist, PG01037, inhibited PPX‐induced yawning and PE while the D2‐selective antagonist, L‐741,626, restored PPX‐induced yawning to free‐fed levels. These results suggest that food restriction sensitized rats to the D2‐mediated effects of PPX while having no effect on the D3‐mediated effects of PPX.

Supported by grants DA20669 & F013771 through NIDA
Effect of Pb‐acetate on neurobehavior and learning in the kitten
Authors: Andicochea Chad T. , Martin John R.
Published: June 2021

Pb‐induced learning and behavioral deficits, and hypo‐spiny neurons of the motor cortex, cerebellum, and hippocampus, do not occur in rats until blood‐lead levels (BLLs) reach at least 21 μg/dL. In contrast, elevated aggression and poor intelligence scores occur in children with BLLs lower than 10 μg/dL. Similarly, Pb‐exposed kittens show increased aggression with BLLs less than 10 μg/dL, which may be associated with Pb‐induced hyper‐spiny neurons of the motor cortex, hippocampus and cerebellum. These results suggest that Pb ingestion by kittens will result in learning and behavioral deficits similar to those observed in lead‐burdened children. To test this, kittens were treated (20 mg/kg/day, lead acetate or equal volume distilled water via esophageal intubation) from post natal day 1 to 7. At 8‐ and 10‐weeks of age, the kittens were tested in a reversal t‐maze, an open field, and a free fall test. Eight‐week‐old Pb‐treated kittens showed a delay in learning as demonstrated by a significant higher number of incorrect arm choices in the t‐maze that returned to control levels when the kittens reached 10‐weeks of age. No differences occurred between treatment groups at 8‐ and 10‐weeks of age in the open field or free fall tests. These results show the kitten to be a viable model for Pb toxicity studies in that it better simulates the effects of Pb toxicity found in children. Supported by an Institutional Graduate Program Grant.
G Protein‐Coupled Receptor Kinase 4γ Interacts with Inactive Gαs and Gα13 without Competing with Gβγ
Authors: Andresen Bradley T. , Keever Lindsay B. , Jones John E. , Linetsky Mikhail D.
Published: June 2021

G protein‐coupled receptors (GPCRs) are regulated by multiple families of kinases including G protein‐coupled receptor kinases (GRKs). GRK4 has been shown to be constitutively active towards GPCRs, and polymorphisms of GRK4γ are linked to, and experimentally can cause, hypertension. To better understand GRK4γ we examined the interactions between GRK4γ and Gα and Gβ subunits of heterotrimeric G proteins. Because GRK4 has been shown to inhibit Gαs‐coupled GPCR signaling and does not contain the PH domain found in GRK2, we hypothesized that GRK4γ would interact with active Gαs, but not Gβ. We examined GRK4γ and G protein interactions through co‐immunoprecipitation of GRK4γ with inactive and active Gα proteins and Gβ. Surprisingly, GRK4γ preferentially interacts with inactive Gαs along with Gβ to a greater extent than active Gαs. GRK4γ also interacts with inactive Gα13 along with Gβ to a lesser extent than Gαs. These results are in contrast to GRK2, which interacts with active Gαq, but not inactive Gαq. Functional studies demonstrate that wild‐type GRK4γ, but not kinase‐dead GRK4γ, ablates isoproterenol‐mediated cAMP production indicating that the kinase domain is responsible for GPCR regulation. This evidence suggests that the binding to inactive Gαs and Gβ may explain the constitutive activity of GRK4γ towards Gαs coupled receptors. Support provided by the American Health Assistance Foundation.
Hydrogen peroxide (H2O2) increased the expression of Gqα in vascular smooth muscle cells: role of growth factor receptors
Authors: Descorbeth Magda , Anand‐Srivastava Madhu B.
Published: June 2021

We have recently shown that high glucose increased the expressions of Gqα which was attributed to the enhanced levels of H2O2. The present studies were undertaken to investigate if H2O2 can mimic the effect of high glucose on the expression of Gqα and‐mediated signaling in A10 VSMC. Treatment of A10 VSMC with H2O2 for 4 h increased the levels of Gqα protein in a concentration‐dependent manner. A maximal increase of ~50% in the levels of Gqα proteins was observed at 50μM of H2O2. Since H2O2 transactivates growth factor receptors, we also investigated the implication of growth factor receptors in H2O2‐mediated increase in Gqα levels by using specifics inhibitors of growth factor receptors such as AG1024 for IGF‐R or AG1295 for PDGF‐R. Pretreatment of cells with growth factor receptor inhibitors resulted in the restoration of H2O2‐induced enhanced expression of Gqα proteins. In addition, PD‐98059, an inhibitor of mitogen‐activated protein kinase (MAPK), and wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (PI3‐K), decreased H2O2–induced enhanced levels of Gqα. On the other hand, the enhanced activity of MAPK induced by H2O2 was also inhibited by growth factor receptors inhibitors. These results suggest that H2O2‐mediated up‐regulation of Gqα expression in VSMC occurs through the transactivation of growth factor receptor‐induced activation of the MAPK/PI3K pathways. (Supported by Quebec Heart Foundation)
Selective regulation of G‐protein signaling pathways by AGS3
Authors: Oner Sukru Sadik , Blumer Joe B. , Lanier Stephen M.
Published: June 2021

Activator of G Protein Signaling‐3 (AGS3) contains 4 G‐protein regulatory (GPR) motifs each of which can serve as a docking site for Giα‐GDP free of Gβγ. AGS3 and related GPR proteins provide unexpected regulatory mechanisms for G‐protein signaling systems, which are involved in diverse various cellular functions. The mechanisms by which these proteins integrate into various G‐protein signaling pathways involving cell surface receptors are not fully understood. As an initial approach to address this issue, we optimized conditions for siRNA‐mediated knockdown of endogenous AGS3 in the neuronal cell line NG‐10815 and examined the consequences of this reduction in AGS3 on α2‐adrenergic receptor (α2‐AR) signaling. AGS3 protein was reduced by greater than 95% following AGS3 siRNA transfection without any change in the levels of Giα3, the related GPR protein AGS5/LGN or basal or forskolin‐induced increases in cAMP. α2‐AR mediated inhibition of cAMP production was also not altered by AGS3 knockdown. However, α2‐AR‐mediated increases in the phosphorylation of ERK1/2 were reduced by ∼40% following siRNA mediated knockdown of AGS3, without any change in the total ERK1/2 protein. These data suggest a role for AGS3 in the selective regulation of signaling pathways.