bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of storage‐induced polymerization on the absorption and metabolism of fresh versus aged chokeberry juices in a rodent model
Authors: Graves Sarah Elizabeth , Howard Luke , Prior Ronald , Gilbert William , Devareddy Latha
Published: June 2021

Anthocyanins may be responsible for the health benefits of chokeberries, as they are present in large quantities in chokeberries and are potent antioxidant and anti‐inflammatory agents. Previous investigations on the bioavailability of anthocyanins illustrate that little of the ingested amount reaches systemic circulation. Furthermore, anthocyanins are known to undergo extensive polymerization during storage. Presently, little research has examined the effects of polymerization on the absorption and metabolism of anthocyanins. Therefore, the aim of this study was to investigate how storage‐induced polymerization affects the absorption and metabolism of anthocyanins in rats by comparison of fresh versus aged chokeberry juices. 75 eight week‐old male, Sprague‐Dawley rats were randomly assigned to receive one of the following treatments by gavage once daily for 7 days: water (CTRL), n=8; fresh chokeberry juice (FCB), n=32; and aged chokeberry juice (ACB), n=32. On day 7, rats from CTRL were sacrificed at 0.5h, and 8 rats from FCB and ACB were sacrificed at 0.5, 1, 2, or 4‐h post‐gavage for collection and analysis of blood and urine by HPLC/MS. Based on previous studies, we expect to find higher absorption and excretion of anthocyanins in the rats that received fresh chokeberry juice than in the rats that received aged chokeberry juice. Research support was provided by Arkansas Biosciences Institute.
Quantification of (−)‐epicatechin metabolites after ingestion of flavanol‐rich foods.
Authors: Actis-Goretta Lucas , Leveques Antoine , Renouf Mathieu , Rein Maarit , Williamson Gary , Dionisi Fabiola
Published: June 2021

Many previous studies have investigated the bioavailability of (−)‐ epicatechin. However, the majority of these reports have evaluated the total amount of (−)‐epicatechin absorbed by exposing the biological samples to glucuronidase and/or sulphatase enzymes. These methods do not provide any information about the (−)‐epicatechin metabolites that exist in the plasma. Therefore, our objective was to quantify the (−)‐epicatechin metabolites after flavanol‐rich foods consumption. At first, a new methodology for measuring (−)‐epicatechin metabolites using UPLC‐MS/MS was developed and validated. The recoveries for each metabolite were near one hundred percent while the limit of quantification was 10 nM. After that, plasma (−)‐epicatechin metabolites were evaluated after ingestion of chocolate by humans. Area under the curve, maximum concentration and Tmax of each metabolite was calculated. In conclusion, we were able to quantify the plasma (−)‐epicatechin metabolites after the consumption of a flavanol‐rich food. These data are helpful in understanding further elucidation of the bioactive compounds present in plasma after the ingestion of these highly consumed foods.
Determining functional fiber properties of berry pomaces via an anaerobic fermentation system
Authors: Goita Mfamara L , McCutcheon Kathleen Lee , Raggio Anne M , Finley John W
Published: June 2021

Berries such as cranberry, blueberry, black raspberry and grape contain bioactive carbohydrates, pigments and phenolic compounds that are shown to be beneficial in disease prevention. When berry fruits have been pressed for juice, solid by‐product pomaces from the skins, seeds, and pulp can be a good source of dietary fiber. A high intake of dietary fiber is linked to human health benefits including lowering the risk of cardiovascular, diabetes, obesity, and gastrointestinal related diseases. Also, fiber that is fermented by gut microflora produces short chain fatty acids (SCFA) that improve gut health and nutrient absorption. An in vitro fermentation system mimicking the digestion in the human gut was set up to determine the fermentability of various berry pomaces. Fecal samples from rats and humans were used as bacterial inoculum. The SCFA (acetate, propionate and butyrate) increased significantly over time of fermentation for all samples. Rat inoculum showed fermentation from cranberry pomace had the lowest pH, but the SCFA were similar for all. With the human inoculum, fermentation of black raspberry had the lowest pH and the highest acetate and butyrate production. To reduce variability, classification of the fecal microflora and chemical composition of the pomaces will help determine fermentability to select the best pomaces for use as functional fibers.
Influence of dietary methyl group donors on survival time after hemorrhage in an inbred rat strain
Authors: Rose Rajiv , Oh Taesung , Calderon Mariam , Klemcke Harold G
Published: June 2021

Our initial experiments with survival time after hemorrhage (STaH) in inbred rats revealed large intra‐strain differences in STaH, associated blood measures, and global DNA methylation levels. We hypothesized that feeding inbred rats post‐weaning diets varying in methyl group donors (MGDs; folic acid, choline, methionine and vitamin B12) starting at 4 weeks age for 9 weeks, may influence STaH and intra‐strain variability via altered DNA methylation patterns. To address this hypothesis, BN/Mcwi rats were randomly assigned to one of 4 diet groups: natural control (NC, n=13), synthetic control (SC, n=11), synthetic deficient (SD, n=6; 50–100% less MGDs than NC/SC) and synthetic supplemented (SS, n=11; 3–10 fold more MGDs than NC/SC). Each rat was bled to 50% of its blood volume and STaH observed up to 240 min. STaH was lower (p<0.01) in deficient rats (SD) compared to other diet groups: NC (110 ± 22 min), SC (141 ± 28 min), SD (27 ± 4 min), SS (166 ± 23 min). There were no differences in STaH between NC vs SC (p=0.84), SC vs SS (p=0.37), or NC vs SS (p=0.07). Percentages of survivors (ST=240 min) were: NC=23%, SC=36%, SD=0%, SS=45% (p=0.22). Coefficients of variation for STaH were not different among diets (p>0.25; NC=72.6%; SC=66.5%; SD=35.8%; SS=44.9%). Hence, SD diet decreased survival after hemorrhage, but dietary effects on DNA methylation patterns and other phenotypes are yet to be determined. (Funding by USAMRMC).
Vitamin K2 administration and bone mineral density in Senescence Accelerated Mice P‐6 (SAMP6)
Authors: Katsuyama Hironobu , Fushimi Shigeko , Yamane Kunikazu , Hinenoya Hajime , Akiyama Yuji , Tomita Masafumi , Okuyama Toshiko , Watanabe Yoko , Katsuyama Midori , Anh Le Ngoc , Saijoh Kiyofumi
Published: June 2021

The P6 strain of senescence‐accelerated mice (SAMP6) exhibit features of skeletal aging including reduced bone mineral density (BMD), so that various physiological active agents affecting BMD could be examined. In the present study, SAMP6 were used in order to clarify the effect of vitamin K2 on BMD.

We developed the further bone resorption model using water‐immersion restraint stress (WRS). Male SAMP6 were divided into three groups, i.e., control group, WRS (6 h/day, 5 times/wk) group, and WRS + vitamin K2 (30 mg/kg s.c.) group. SAMP6 were suffered from this stress between 6 to 10 weeks old. BMD was measured using computed tomography (CT) system for small animals, and bone turnover markers were also examined.

BMD of trabecular bone for WRS groups was significantly lower than that of control group, suggesting that WRS was effective to reduce BMD. However, no significant difference of BMD was observed between WRS group and WRS + vitamin K2 group. On the other hand, urinary excretion of CTX, bone resorption marker, was significantly higher in WRS + vitamin K2 group than in other groups. And total osteocalcin in serum was significantly higher in WRS group than in other groups. These results indicated that vitamin K2 could affect bone turnover. This study was supported in part by a Kakenhi (22500684), and a Research Project Grant (No. 22‐A‐59) from Kawasaki Medical School.
Effects of early growth restriction on development and insulin sensitivity in rats
Authors: Ebanks Erica , Lonnerdal Bo , Griffin Ian J.
Published: June 2021

There is increasing evidence that poor growth of preterm infants is a risk factor for poor long‐term development, while the effects of early postnatal growth restriction are not well known. We utilized a rat model to examine consequences of different patterns of postnatal growth and hypothesize that early growth failure leads to impaired development and insulin resistance. Rat pups were separated at birth into normal (N, n = 10) or restricted intake (R, n = 16) litters. At d11 R pups were re‐randomized into litters of 6 (RC; R/catch‐up), 10 (RN; R/normal) or 16 (RR; R/restricted). Pups were weaned at d21. Animals underwent developmental and behavioral tests, including open field maze, T‐Maze and passive avoidance. Insulin sensitivity was measured by i.p. insulin stress test (IPIST) and glucose tolerance test (IPGTT). By d10, N pups weighed 20% more than R pups (p<0.001). By d15, RC caught up to NN pups, RN showed partial catch‐up and RR showed no catch‐up. RR and RC groups showed less explorative behavior during developmental testing3. Further, there was evidence of learning in NN pups during T‐Maze and passive avoidance tests3. Though NN and RR were most insulin sensitive, all R groups were more glucose tolerant compared to NN. In rats, early postnatal growth restriction leads to abnormal development and insulin sensitivity. This “double hit” paradigm may contribute to poor developmental outcomes in preterms.

Grant Funding Source: UC Davis Department of Pediatrics
Severe heat stress affects the expression of Hsp90, GLUT4 and b0,+, in liver and leptin in adipose tissue of pigs
Authors: Garcia Hector , Grageola Fernando , Morales Adriana , Araiza Alfonso , Arce Nestor , Cervantes Miguel
Published: June 2021

Summer temperatures in northwestern México reach as high as 50°C. An experiment was conducted with 12 pigs (27.3 ± 1.92 kg, BW) to determine if severe heat stress modify the expression of Hsp90, GLUT4, AA transporter b0,+, and leptin, which participate in the regulation of nutrient metabolism and feed intake of animals. Treatments (T) were: T1, pigs housed at 22°C (Comfort); T2, pigs under heat stress (HS, 44°C). Pigs were fed a wheat‐soybean meal diet (16% CP, 0.95% Lys). At the end of the 21‐d trial, pigs were euthanized to collect liver and adipose tissue samples. Leptin expression was analyzed in adipose tissue; Hsp90, GLUT4, and b0,+ were analyzed in liver. The relative expression values (ratios of specific mRNA molecules:18S rRNA molecules) under Comfort or HS were: Hsp90, 0.025, 0.299; Glut4, 0.032, 0.070; b0,+, 2.983, 0.450; Leptin, 0.700, 0.066, respectively. Pigs under HS increased the expression of Hsp90 (P=0.004) and tended to increase GLUT4 (P=0.067) in liver, but reduced b0,+ (P=0.011) in liver and leptin (P=0.007) in adipose tissue. These data indicate that pigs exposed to severe heat stress modify the expression of proteins involved in regulation of feed intake and nutrient metabolism.
Anthropometric assessment of lactating mothers attending public clinics in the Guatemalan highlands in metropolitan Quetzaltenango
Authors: Oyesiku Linda , Doak Colleen M , Vossenaar Marieke , Solomons Noel W
Published: June 2021

Background

Early linear growth in Guatemala has historically been compromised, resulting in adults of short stature. A progressive maternal weight change from delivery through lactation has been described.

Objective

To assess maternal height and BMI in highland Guatemalan mothers at various periods of lactation.

Methods

Maternal weight and height were collected in 543 lactating mothers of infant and toddlers, with subsequent classification of mothers for low‐stature (relative to WHO/NCHS 1985 smoothed curve median) and for underweight (BMI <18.5 kg/m2), overweight (BMI 25–30 kg/m2) or obesity (BMI >30 kg/m2).

Results

Mothers ranged in age from 15 to 48 y. The mean stature for the sample was 149.1±5.9 cm, with a median of 149.0 cm and a range, 136.0–169.0 cm. Women classified of Mayan ascent were significantly (p<0.001) shorter (146.9±5.1 cm) than others (150.3±5.9 cm). In terms of height percentiles for the adult reference, 423 (78%) of mothers were below the 5th percentile; 46 (8%) between the 5th and 10th percentile; and only 7 (1%) reached the median. Respective partition for underweight; normal weight; overweight; and obesity was: 4%; 49%; 37% and 10%. Variation in BMI with respect to the age of the offspring was not significant.

Conclusion

Highland Guatemalan women remain short, with no lactation‐duration effects on BMI.

Funded by the Nestlé Foundation and Sight and Life, Switzerland
Maternal and infant iron status in HIV‐infected Malawian women and their exclusively breastfed infants
Authors: Widen Elizabeth M , Bentley ME , Chasela C , Kayira D , Tegha G , Kourtis A , Jamieson D , van der Horst C , Allen L , Shahab-Ferdows S , Adair LS
Published: June 2021

Identifying infants at risk of early depletion of iron stores is essential to prevent postnatal iron deficiency. Maternal iron status may be a determinant of infant iron status, especially in the context of HIV where iron deficiency is common. We evaluated how iron status of HIV infected Malawian women related to iron status of their exclusively breastfed infants in the Breastfeeding, Antiretroviral and Nutrition Study. Participants were randomized to receive maternal lipid‐based nutrient supplements (LNS), providing 15 mg iron/d, or no LNS, and maternal, infant or no antiretroviral drugs. Infant iron status (Hb, log ferritin, log transferrin receptors TfR) at 2 (n=355) or 6 wk (n=165), and 24 wk (n=532) were regressed on corresponding maternal indicators, each corrected for inflammation (elevated C‐reactive protein and/or α‐1‐acid glycoprotein). Maternal Hb was marginally associated with infant Hb (6 wk β: 0.14, p=0.07; 24 wk β: 0.08, p=0.06). Maternal ferritin was related to infant ferritin only at 2 wk (β: 0.16, p<.01). Maternal TfR was associated with infant TfR at all periods (2 wk β: 0.14, 6 wk β: 0.30, 24 wk β: 0.21; all p<0.001). Maternal LNS predicted marginally lower maternal ferritin at 6 and 24 wk (p<0.1), but not maternal TfR, Hb or infant status. Given the observed maternal influence, optimizing maternal status at delivery and during lactation is important for reducing risk of iron depletion in infants.

Grant Funding Source : CDC(U48‐DP000059‐01); Bill & Melinda Gates Foundation(OPP53107); CPC(NICHD 5 R24 HD050924)
Clinical and Biological Significance of KISS1 Expression in Prostate Cancer
Authors: Wang Honghe , Jones Jacqueline , He Qinghua P. , Grizzle William E. , Welch Danny , Turner Timothy , Yates Clayton
Published: June 2021

For men in the United States, prostate cancer (PCa) is the most frequent malignancy and the second leading cause of cancer mortality. The metastatic spread of PCs is responsible for most PCa–related deaths. Although KISS1 functions as a metastasis‐suppressor in various cancers, its expression and functions in PCa development and progression remain undetermined. The goals of this study were to correlate the expressions of KISS1 in PCas with clinicopathological characteristics and to assess the biological relevance of KISS1 to the viability and motility of PCa cells. Strong KISS1 staining was detected in benign prostate tissues, but the staining was weaker in primary and metastatic PCas (p<0.001, t‐test). Furthermore, the low expression of KISS1 in PCas correlated with clinical stage (p<0.01) and with KISS1R expression (p<0.001). Over‐expression of full‐length KISS1 in low KISS1 expressing PC‐3M cells, but not KFMÄSS which lacks the secretion signal sequence, induced re‐sensitization of cells to anoikis, although it had no effect on cell proliferation or apoptosis. Over‐expression of KISS1 also suppressed steps in the metastatic cascade, including motility and invasiveness. Moreover, cells over‐expressing KISS1 demonstrated enhanced chemosensitivity. Collectively, our data suggest that KISS1 functions as a metastasis suppressor in PCas and may serve as a useful biomarker and a therapeutic target for PCas.
HLA‐A*7401 and HLA‐C*0401 ALLELIC DISEASE ASSOCIATION WITH PLASMA CELL NEOPLASIA
Authors: mannam venkat krishna r , Gilbert William , Green Taryn , Santos Mark , Goodin Joshua , Lewis Robert E , Cruse Julius M
Published: June 2021

The present investigation was designed to show the incidence of molecular Human Leukocytic Antigen (HLA) class I and class II allelic specificities in plasma cell neoplasia patients, identified by immunohistochemistry, flow cytometry, and high resolution molecular typing. Plasma cell myeloma is the second most common hematological malignancy (13%), comprising 1% of all cancers. Each year, 21000 new cases are diagnosed with a two‐fold prevalence in African Americans in contrast to Caucasians. Our hypothesis is that plasma cell neoplasia have a significant disease association with selected HLA class I and class II alleles. We found that in African American patients diagnosed with plasma cell myeloma (n=10), 50 % expressed HLA‐A*7401 (p= 0.002) and 60% were HLA‐C*0401 positive ( p= 0.004). In patients suspected to have plasma cell neoplasia ( controls n=50), 6% were HLA‐A*7401 positive and 14% expressed HLA‐C*0401. Our results suggest that persons expressing these alleles are more likely to develop plasma cell neoplasia than are those not expressing these alleles. African American patients diagnosed with plasma cell myeloma have significant associations with HLA‐A*7401 and HLA‐C*0401 alleles. Future studies will be directed to identification of HLA class II alleles associated with plasma cell neoplasia.
Assessment of dopaminergic involvement in cocaine‐induced conditioned taste aversions
Authors: Serafine Katherine Marie , Briscione Maria A , Rice Kenner C , Riley Anthony L
Published: June 2021

Although cocaine induces conditioned taste aversions (CTA), little is known about the mechanisms underlying this effect. It has been suggested that cocaine's actions as a nonselective monoamine transport inhibitor may be mediating this suppression. Given the role of dopamine (DA) in cocaine reward, this neurotransmitter system is of particular interest. The present experiments used direct pharmacological antagonism (with the DA antagonist haloperidol) to determine a role, if any, of DA in the induction of CTAs by cocaine. Following the determination of behaviorally active doses of haloperidol with no aversive effects on their own (Experiment 1), animals were given 1 mg/kg haloperidol prior to various doses of cocaine in a taste aversion procedure (Experiment 2). Under these conditions, haloperidol blocked cocaine‐induced CTAs (at the 18 and 32 mg/kg doses). These results suggest that DA has a role in cocaine‐induced CTAs. These data are discussed in the context of previous work demonstrating roles for both norepinephrine and serotonin in cocaine‐induced taste aversion learning as well as current research investigating DA transporter inhibitors and cocaine within the cross‐drug preexposure preparation.
The omega and omega‐1 monohydroxyl metabolites of the abused K2/Spice synthetic cannabinoids JWH‐018 and JWH‐ 073 bind with high affinity and act as agonists at human cannabinoid 2 receptors (hCB2s)
Authors: Brents Lisa K , Rajasekaran Maheswari , Franks Lirit , Moran Jeffery H , Prather Paul L
Published: June 2021

JWH‐018 and JWH‐073 are synthetic cannabinoids present in K2 or Spice, drugs of abuse marketed as incense. These compounds produce psychoactive effects similar to marijuana, but can also result in death and severe adverse effects including hypertension, tachycardia, agitation, anxiety and hallucinations. Recently, we reported that mono‐hydroxylated metabolites of JWH‐018 and JWH‐073 retain high affinity and exhibit a range of activity at cannabinoid 1 receptors (CB1Rs), potentially contributing to the toxicological consequence of K2 use. Since toxicity of these compounds could also result from action at CB2Rs, this study examined the affinity and intrinsic activity of JWH‐018, JWH‐073 and several phase I monohydroxylated metabolites for hCB2Rs stably transfected in CHO cells. Competition binding experiments and functional studies examining CB2R‐mediated G‐protein activation and inhibition of adenylyl cyclase activity indicated that several metabolites retain nanomolar affinity for hCB2Rs and exhibit partial to full CB2R‐agonist activity. Importantly, this included the omega and omega‐1 monohydroxylated metabolites of JWH‐018 and JWH‐073, which are major metabolites excreted in human urine as glucuronide conjugates. Retention of high CB2R affinity and activity post‐metabolism may prolong CB2R activation, resulting in immune system dysregulation.

APHL Award (JHM); 1UL1RR029884 (JHM, PLP)
Anti‐hypernociceptive property of the methanolic extract of stems of Kielmeyera rugosa in mice
Authors: Quintans-Junior Lucindo Jose , Melo Monica , Oliveira Makson , Santos Marcio RV , Araujo Adriano AS , Costa Emmanoel V , Anjos Charlene S , Jesus Aline M , Nogueira Paulo CL , Moraes Valeria RS
Published: June 2021

The present study examined the anti‐hypernociceptive effect of the methanolic extract of stems of Kielmeyera rugosa (MEKR) in mice. For assess this effect were realized models of mechanic hypernociception induced by carrageenan (CG) and substances which mediate the various reactions involved CG, such as TNF‐α, prostaglandin (PGE2) and dopamine (DA). The mice were pretreated with vehicle (saline + Tween 80 0.2%; p.o.) or MEKR (100, 200 and 400 mg/kg, p.o.). Thirty minutes after the treatments, were injected in region subplantar of the rear right paw: 20 μL of CG (300 μg/paw), PGE2 (100 ng/paw), DA (30 μg/paw) or TNF‐α (100 pg/paw). The hypernociception was assessed in times of 0.5, 1, 2 and 3 hours after the injection of CG, TNF‐α, PGE2 and DA, with digital Von Frey. It was observed that MEKR was able to maintain the baseline nociceptive threshold in all tests. The results suggest anti‐hypernociceptive property of MEKR, probably by a mechanism inhibition of production of cytokines, which activate various reactions connected with inflammation, reducing thereby hipernociception.

Financial support: FAPITEC/SE, CAPES, CNPq (Brazil).
Increased Astrocyte Thrombospondin‐4 Expression in Dorsal Spinal Cord Correlates with Neuropathic Pain States
Authors: Yu Yanhui Peter , Li Kang-Wu , Chen Xiao-Gu , Luo Z. David
Published: June 2021

Thrombospondin‐4 (TSP4), a member of a family of secreted extracellular matrix proteins, has previously been shown to contribute to certain neuropathic pain states. Blockage of TSP4 expression or actions by intrathecal antisense oligos or neutralizing antibodies, respectively, led to a reversal of behavioral hypersensitivity in rat neuropathic pain models. In spinal nerve ligated (SNL) mice with TSP4 null expression, behavioral hypersensitivity was significantly diminished compared with that in SNL WT mice. However, the cellular location of injury‐induced TSP4 expression in spinal cord is not known. Using immunohistochemical detections in sham and SNL TSP4‐eGFP mice, in which green fluorescent protein (eGFP) expression is driven by the TSP4 promoter, we examined the expression level of eGFP and its co‐localization with different cellular markers in the dorsal spinal cord. SNL injury induced an astrocyte specific increase of TSP4 production that correlates with the expression of behavioral hypersensitivity. Our data suggest that increased astrocyte TSP4 production in dorsal spinal cord plays an important role in the development of behavioral hypersensitive states after peripheral nerve injury.
Suppression of paclitaxel (PAC)‐induced neuropathic pain by hyperbaric oxygen (HBO2)
Authors: Zhang Yangmiao , Gibbons Carlee R. , Howlader Saad , Son Myngoc Chung , Yeon Jiwoong , Shirachi Donald Y , Quock Raymond M.
Published: June 2021

HBO2 produces relief of acute pain (Chung et al., J. Pain 11:847, 2010). Recent studies have shown that HBO2 can also relieve chronic pain (Thompson et al., Neurosci. Res. 66:279, 2010; Li et al., Anesth. Analg. 113:626, 2011). The aim of this study was to ascertain whether relief of neuropathic pain could be enhanced by increasing the number of HBO2 treatments. Male Sprague Dawley rats were injected i.p. with either vehicle or PAC (1.0 mg/kg) on days 0, 2, 4 and 6 (cumulative dose 4 mg/kg). Twenty‐four hr after the last injection, rats were treated with 60‐min room air or HBO2 at 3.5 ATA for either one or four days. Mechanical thresholds were assessed with an electronic von Frey anesthesiometer. PAC‐treated rats showed an allodynia that persisted for more than 30 days after the initial injection. Rats that underwent a single 60‐min HBO2 treatment at 3.5 ATA on day 7 exhibited a recovery of the threshold after 3 weeks. However, rats that underwent four 60‐min HBO2 treatments from days 7 thru 10 recovered approximately 7 days earlier than the rats with a single treatment. It is concluded that HBO2 treatment is capable of causing dose‐dependent relief of PAC‐induced neuropathic pain. (This research was supported by NIH Grant GM‐77153 and the Allen I. White Distinguished Professorship.)
Biphenols block calcium entry in response to activation of the M3 muscarinic receptor
Authors: Huang Yue-Wern , Martin Alexis G. , Chao Po-Kuan , Wang Hsiu-Jen , Martin Adam L. , Shannon Erica K. , Reichard Rhett A. , Chan Ming-Huan , Aronstam Robert S.
Published: June 2021

Cell‐permeable biphenols, such as honokiol, present in herbal teas, inhibit autonomic responses. The effects of biphenols on calcium signaling activated by human M3 muscarinic receptors was studied in Chinese hamster ovary (CHO) cells. Receptor binding was determined by radiolabelled ligand binding; intracellular calcium concentrations were determined using a fura‐2 ratiometric imaging protocol; cytotoxicity was determined using a dye reduction assay. Activation of M3 muscarinic receptors with carbachol induced a biphasic increase in [Ca2+]i: an initial, IP3‐ mediated release of Ca2+ from endoplasmic reticulum stores followed by a sustained phase of steady Ca2+ entry (i.e., store operated calcium entry, SOCE). Biphneols had potent (EC50 ≈ 5 μM) inhibitory effects on SOCE that was induced by the activation of the M3 receptor. SOCE was also examined by acute exposure to thapsigarin (2 μM) in a calcium‐free medium, followed by reintroduction of calcium. Again, SOCE was severely depressed by biphenols. The effect of honokiol on SOCE was specific, rapid and partially reversible, and was seen at concentrations not associated with cytotoxicity, inhibition of IP3 receptor‐mediated calcium release, or depletion of ER calcium stores. Moreover, biphenols did not disrupt the ligand binding properties of M3 receptors. An inhibition of SOCE probably contributes to biphenol disruption of parasympathetic functions.
Dynamic Formation of a Ternary PTH receptor−Arrestin−Gβγ Complex required for sustained Signaling
Authors: Wehbi Vanessa Leila , Stevenson Hilary Paige , Feinstein Timothy Nathan , Romero Guillermo , Calero Guillermo , Vilardaga Jean-Pierre
Published: June 2021

Arrestins and heterotrimeric G proteins (Gαβγ) regulate G‐protein‐coupled receptors (GPCRs) signaling and trafficking. Arrestin binding to an activated GPCR terminates receptor and G protein coupling, and promotes receptor internalization. The binding of the β‐arrestins and G‐proteins on agonist‐bound GPCRs is thought to be mutually exclusive. Here we show that β‐arrestins prolong rather that desensitize parathyroid hormone (PTH) receptor (PTHR) signaling. By using optical approaches (confocal microscopy, FRAP, TIRF, fluorescence correlation spectroscopy, and FRET) in live cells in real time we found that PTHR forms a ternary complex with β‐arrestin1/2 and Gβγ subunits in response to PTH stimulation. Additionally, we found that the rapid (t1/2 < 60 s) assembly/disassembly dynamics formation of a receptor microdomain that contains PTH, PTHR, arrestin and Gβγ subunits increases the levels of G‐protein activation and cAMP accumulation in magnitude and duration. These data contradicted established tenets of the regulation of GPCR signaling and raise the emerging concept that the formation of a long‐lived GPCR‐Gβγ‐arrestin ternary complex contributes to prolonged receptor signaling by mechanisms that presumably permit multiple rounds of GαS subunit coupling and activation.
Membrane microdomains and cAMP compartmentation
Authors: Agarwal Shailesh , Caldwell Kacie , Owens Ashley , Singer Cherie , Nikolaev Viacheslav , Lohse Martin , Harvey Robert
Published: June 2021

The role of different membrane domains in the compartmentation of cAMP signaling was investigated using the FRET‐based Epac2‐ camps biosensor. The MyrPalm sequence from Lyn kinase and the CAAX sequence from Rho GTPase were used to target this probe to lipid raft (Epac2‐MyrPalm) or non‐lipid raft (Epac2‐CAAX) membrane domains in HEK293 cells. FRET responses were used to monitor relative changes in cAMP activity. Stimulating cAMP production by maximally activating prostaglandin or beta‐adrenergic receptors elicited similar sized responses in cells expressing Epac2‐MyrPalm. Yet, the maximal responses to receptor activation were significantly smaller in cells expressing Epac2‐CAAX. Differences in the magnitude of the responses detected by the two biosensors could reflect phosphodiesterase (PDE)‐dependent compartmentation of receptor‐mediated cAMP signaling. However, those differences remained even after PDE activity was inhibited. Alternatively, Epac2‐CAAX could be sensing higher basal levels of cAMP, reducing the apparent magnitude of the maximal response detected upon subsequent receptor activation due to saturation of the probe. Consistent with this idea, inhibition of basal adenylyl cyclase activity with MDL 12330A produced an inhibitory response in cells expressing Epac2‐CAAX, but not Epac2‐MyrPalm. These results indicate that there are diffusionally‐restricted pools of cAMP associated with different microdomains of the plasma membrane under basal conditions.
Effects of Fresh and Aged Traffic‐Related Particles on Breathing Pattern, Cellular Responses and Oxidative Stress
Authors: Diaz Edgar A. , Chung Yeonseung , Lamoureux Denise P , Papapostolou Vasileios , Lawrence Joy , Long Mark s , Sato Rodrigo , Joao Oliveira , Godleski John J
Published: June 2021

Background

Air pollution is a mixture of gaseous and particulate pollutants emitted from multiple sources. During transport in the atmosphere, emissions undergo photochemical reactions that may render them more harmful.

Objectives

The aim of this study was to investigate the toxicity of primary and secondary traffic particles.

Methods

Sprague‐Dawley rats were exposed to either: fresh primary particles from a tunnel plenum (P); secondary organic aerosol formed from photochemical oxidation of primary gases after filtration of primary particles (SOA); photochemically aged primary particles plus secondary organic aerosol (P + SOA); or filtered clean air. In each exposure, pollutant gases were removed using a non‐selective denuder.

Animals were exposed for 5 hours/day, with varying numbers of days of exposure. Outcomes included breathing pattern, in‐vivo chemiluminescence (IVCL), broncho‐alveolar lavage, and complete blood count.

Results

Mass concentration was ~50μg/m3. Respiratory data showed many changes with each exposure type. All produced decreases in tidal/minute volumes and inspiratory/expiratory flows. There were mild inflammatory changes in BAL, with increased neutrophils for SOA and P+SOA and lymphocytes for P and P+SOA, without changes in total protein, β‐NAG or IVCL.

Conclusions

All exposures produced changes compared to filtered air; overall toxicity, P+SOA>P>SOA.
Cyp‐epoxygenases mediate adenosine A2A receptor induced vascular relaxation via KATP channels
Authors: Ponnoth Dovenia , Nayeem Mohammed A , Kunduri Swati S , Tilley Stephen L , Ledent Catherine , Mustafa S Jamal
Published: June 2021

A2A adenosine receptor (AR) causes aortic relaxation through generation of epoxyeicosatrienoic acids (EETs) formed via Cyp‐epoxygenases (Nayeem et al, AJP 2008). We hypothesize that A2AAR‐mediated aortic relaxation occurs via EETs through opening of KATP channels in smooth muscle cells. Organ bath experiments were done with isolated aortas from A2AKO and wild type (WT) mice. Pinacidil (KATP opener; 10−6M) caused higher relaxation in WT aorta compared to A2AKO (48.09±5.23% vs. 25.41±2.37%; p<0.05). Cyp‐epoxygenase inhibitor MSPPOH (10−5M) caused significant decrease (p<0.05) in pinacidil response in WT (48.09±5.23% vs. 24.93±5.34%) and A2AKO (25.41±2.37% vs. 14.66±3.88%) aorta. Adenosine analog NECA (10−5M) caused significant relaxation in WT aorta (19.65±3.25%) compared to contraction (13.94±6.57%) in A2AKO, that was lowered by KATP blocker glibenclamide in WT (0.17±6.43%) while higher contraction was observed in KO (28.47±5.1%). Relaxation to A2A agonist CGS 21680 (10−6M) in WT (19.02±2.75%) changed to contraction (23.66±6.31%; p<0.05) with glibenclamide. Mitochondrial KATP opener diazoxide had no effect on precontracted tissues, while 5‐HD (mitochondrial KATP blocker, 100uM) had no effect on CGS 21680 response in WT and A2AKO aorta. These data suggest adenosine mediated aortic relaxation occurs via generation of EETs through sarcolemmal KATP channels. (HL027339, HL09444, HL07180)
Role of adaptor protein IQGAP1 in regulating endothelial permeability of lung vessels
Authors: Garcia Alexander Nathaniel , Komarova Yulia , Vogel Stephen M. , Gao Xiao-pei , Malik Asrar B.
Published: June 2021

IQGAP1 is a scaffold protein that associates with the endothelial adherens junction (AJs) protein beta‐catenin and with the active form of monomeric GTPase Cdc42 to play an important role in maintaining the integrity of AJs. Here we addressed the role of IQGAP1 interaction with AJs in regulating barrier permeability. Lungs of IQGAP1−/− mice displayed a 2‐ to 3‐fold greater lung vascular capillary coefficient, a measure of vessel permeability, after PAR‐1 activation compared to WT. PAR‐1 activation resulted in reversible association of IQGAP1 with AJs as demonstrated by immunofluorescence and co‐immunoprecipitation. The time course of IQGAP1 re‐association with AJs correlated with activation of Cdc42 and AJ re‐sealing and integrity. Measurements of transendothelial electrical resistance in endothelial cells treated with IQGAP1 siRNA showed reduction in the rate of barrier recovery. Lung endothelial cells isolated from IQGAP1−/− mice demonstrated low basal activity of Cdc42 and impaired formation of AJs. The expression of constitutively active Cdc42 in IQGAP1−/− lungs resulted in reduction of vascular permeability back to WT levels. Thus expression level of IQGAP1 through its ability to induce Cdc42 activity is a critical determinant of lung vascular permeability.
In vitro neuroprotective activity of phenibut
Authors: Huynh Daisy , Wai Chong Shun , Liang Anna , Maher Timothy J. , Pino-Figueroa Alejandro
Published: June 2021

Phenibut, a derivative of gamma‐aminobutyric acid (GABA) is said to exhibit neurological properties. Pharmacological studies are necessary to understand its full potential as a therapeutic agent and its possible pathway in which it exerts its biological activities. B35 rat neuroblastoma cells were pretreated with various concentrations of phenibut (1 – 30 μM) and then subjected to hydrogen peroxide or bicuculline damage to evaluate the neuroprotective activity of phenibut. The results showed that phenibut protected the cells in a dose‐dependent manner and could have acted on GABA receptors. Fatty acid amide hydrolase (FAAH) inhibition and antioxidant assays were performed in order to characterize other possible mechanisms of action. The results showed that phenibut did not have antioxidant potential nor FAAH inhibitory activity. Further studies with GABA agonists and antagonists are necessary to better understand the mechanism of action of phenibut and its possible therapeutic applications.
Atomic force microscopy study of ECM‐integrin modulation of neuroplasticity in the hippocampal dentate granule cells in epilepsy
Authors: Wu Xin , Muthuchamy Mariappan , Reddy Doodipala Samba
Published: June 2021

Integrins as membrane receptors on neurons play a key role in the development of neuroplasticity in the brain. Integrin recruitment and relocalization, and extracellular matrix protein (ECM)‐integrin attachments have been reported to dynamically change their position during cell spreading and migration. In this study, we hypothesized that ECM‐integrin bond formation and cell elasticity (remodeling) are altered in epilepsy. We tested this premise in adult mouse hippocampal dentate granule cells (DGCs) in stage 5 hippocampal kindling model of epilepsy by using nanoscale atomic force microscopy (AFM) and immunocytochemistry. The α5β1‐ integrin staining intensity in DGCs from epilepsy mice was at least 10% stronger than in control. In AFM study, the unbinding force (25.6 ± 0.2 pN) and binding probability between ECM protein FN‐coated AFM probe and membrane integrins in epilepsy DGCs were 49% and 18% lower than control, respectively (p<0.05, n=10). FN‐integrin binding probability was significantly reduced by anti‐α5β1 integrin in control. Moreover, the cell elasticity, representing changes in cytoskeletal reorganization, was 66% increased in epilepsy. These results indicate that FN‐adhesive activities are functional specific and primarily bound to α5β1 integrin. Therefore, it is likely that dynamic changes in plasticity properties of DGCs contribute to epileptogenesis.
The role of p300‐HAT in promoter‐associated histone acetylation and regulation of FasL gene expression in ethanol treated CD4+ T lymphocytes
Authors: Ghare Smita , Patil Madhuvanti , Joshi-Barve Swati , McClain Craig , Barve Shirish
Published: June 2021

Alcohol abuse is known to induce immunosuppression by depleting CD4+ T cells. We have shown that ethanol decreases S‐adenosylmethionine levels and enhances activation induced cell death (AICD) in CD4+ T cells. The Fas‐FasL system plays a major role in AICD of T cells. Increased FasL gene expression has been seen in HIV infection and cancer. Histone acetylation maintained by histone acetyltransferases (HATs) and histone deacetylases (HDACs) are important for transcription. p300 is a transcriptional co‐activator with intrinsic HAT activity and is directly involved in chromatin remodeling. Accordingly, we investigated the immunotoxic effects of ethanol by examining FasL promoter histone modifications in the regulation of FasL gene expression in CD4+ T cells. FasL promoter ChIP analysis in ethanol treated T cells showed increase in histone H3 acetylation which is associated with active transcription. In correspondence with increased acetylation, ethanol was observed to enhance the recruitment of p300 and relevant transcription factors which correlated with increased FasL expression. Notably, garcinol, a specific p300‐ HAT inhibitor markedly reduced p300 targeting, histone acetylation and FasL gene expression. Overall these data identify the role of ethanol‐mediated p300 targeting and resultant histone acetylation as a critical mechanism for ethanol‐induced FasL mediated AICD in CD4+ T cells.
Changes in the expression and function of beta‐adrenergic receptors subtypes In the rat aorta during development
Authors: Lopez-Canales Oscar A , Vargas-Robles H , Arellano-Mendoza Monica , Lopez-Canales Jorge , Castillo-Hernandez Maria C , Guevara-Balcazar Gustavo , Escalante-Acosta Bruno
Published: June 2021

During his life the rat shows changes in the relaxation response mediated by beta adrenergic receptors (βAR). In order to provide additional information on the topic, we studied the changes that may occur in the expression, localization and in vitro function of each subtype βAR in the thoracic aorta of male Wistar rats of two age groups: 3 weeks (prepuberty) and 9 weeks (postpuberty). The results indicate that, after the ninth week, the pretreatment with ICI 118.551 (β2 selective inhibitor, 10‐7 M) but not with CGP 12177 (selective inhibitor of β1, 10‐7 M) inhibited the relaxation response in all groups. However, the inhibition was greater when both antagonists were administered simultaneously. In addition, BRL 37344 (selective β3 agonist) caused relaxation only in prepuberty rats rings, which was blocked by SR 59230A (antagonist β3, 10‐6 M). Consistent with previous data, there was an increase in the expression of β1 and β2 receptors and decreased the β3 from the ninth week rats. Moreover, β1 and β2 subtypes are mainly located in smooth muscle, whereas β3 are located mainly in the endothelium. In conclusion, β1 receptors do not appear to participate functionally by themselves at any age, the β3 only involved in prepuberty rats and β2 are involved in all groups but their affinity is likely to change from the ninth week. Interestingly, the combination of β1 and β2 antagonists produced a synergistic effect which deserves further investigation.
Critical Role of Transglutaminase 2 in Endothelial Cell Inflammation and Lung PMN Sequestration
Authors: Bijli Kaiser M , Kanter Bryce G , Xu Lei , Fazal Fabeha , Rahman Arshad
Published: June 2021

We addressed the role of transglutaminase2 (TG2), an enzyme that catalyzes protein crosslinking reactions, in causing endothelial cell (EC) inflammation. Exposure of EC to thrombin resulted in activation of the transcription factor NF‐κB and its target genes, vascular cell adhesion molecule‐1 (VCAM‐1) and interleukin‐6 (IL‐6). RNAi knockdown of TG2 inhibited thrombin‐induced NF‐κB activity and expression of VCAM‐1 and IL‐6. Analysis of NF‐κB pathway revealed that TG2 depletion had no effect on IκBα phosphorylation/degradation and NF‐κB DNA binding function. Interestingly, TG2 knockdown impaired thrombin‐induced phosphorylation of RelA/p65 at Ser536, a crucial event that confers transcriptional competency to the DNA‐bound NF‐κB. Thus, TG2 mediates EC inflammation by promoting the transcriptional activity of NF‐κB. Because thrombin is released in high amounts during sepsis and its concentration is elevated in plasma and lavage fluids of patients with Acute Respiratory Distress Syndrome (ARDS), we determined the in vivo relevance of TG2 in a mouse model of sepsis‐induced lung PMN recruitment. A marked reduction in ICAM‐1 expression and lung PMN sequestration was observed in TG2 knockout compared to wild type mice after intraperitoneal lipopolyschharides (LPS) challenge. Together, these results identify TG2 as an important mediator of lung vascular inflammation associated with sepsis.

Sponsors: NIH (HL067424 and HL096907) and AHA SDG grant.
Identification of Voltage‐Activated Calcium Currents in Renal Afferent and Efferent Arterioles of the Rat
Authors: Smirnov Sergey V , Loutzenhiser Kathy , Loutzenhiser Rodger
Published: June 2021

Based on indirect methods, it is suggested that both L‐ and T‐type Ca channels mediate signaling in the renal afferent arteriole (AA) and that T‐type Ca channels are involved in signaling in the efferent arteriole (EA). Our study was initiated to characterize Ca currents in these vessels, directly, using patch clamp. Native myocytes were isolated from individually isolated rat AA and EA and from tail arteries. Inward currents were measured in physiologic 1.5 mmol/L Ca and 10 mmol/L Ba using the whole‐cell configuration. By exploiting known differences in activation and inactivation properties and sensitivities to nifedipine and kurtoxin, we could readily demonstrate the presence of both L‐ and T‐type Ca channels in myocytes from the rat tail artery. AA myocytes exhibited relatively large Ca current densities (−2.0±0.2 pA/pF), which increased 3.6 fold in Ba. These currents were blocked by nifedipine, but not by kurtoxin or mibefradil and did not exhibit the activation and inactivation characteristics of T‐type Ca channels, compared to tail artery myocytes. EA myocytes did not exhibit a marked voltage‐activated inward current in 1.5 mmol/L Ca. Thus, our findings support the physiologic role of L‐type Ca channels in the AA, but not EA, and do not support the premise that T‐type Ca channels are significantly present in either vessel. Supported by the Royal Society and the Alberta Heritage Foundation for Medical Research.
Exercise training during human obesity protects against impaired microvascular function after acute exertion by enhancing hydrogen peroxide‐mediated flow‐induced dilation
Authors: Bian Jing-Tan , Yue Lili , Franklin Nina C , Mazzone Theodore , Phillips Shane A
Published: June 2021

Our recent studies suggest that increased superoxide dismutase and endothelial nitric oxide synthase (eNOS) contribute to the protective effects of chronic exercise on nitric oxide (NO) and hydrogen peroxide (H2O2)‐mediated acetylcholine (AChD) dilation after acute exertion. The goal of this study was to determine the effect of exercise training on flow‐induced dilation (FID) and AChD in resistance arteries (RAs) of obese patients. We hypothesized that H2O2 mediates the protective effect of exercise training against impaired FID and AChD induced by acute exertion. A protocol of weight lifting (WL; 8 weeks) was applied to subjects (BMI: 30–35). RAs were dissected from gluteal fat biopsies and cannulated for measurements of intraluminal diameters. NO and H2O2 production was assessed by confocal microscopy. All subjects had similar BP responses to WL. The eNOS inhibitor L‐NAME blocked FID (75% ± 12%) and AChD (60% ± 8%) in sedentary subjects. In contrast, PEG‐catalase, a H2O2 scavenger, blocked FID (68% ± 11%) and AChD (61% ± 7%) after exercise training. Fluorescence studies showed reduced NO (0.70 ± 0.05 fold) and increased H2O2 (4.55 ± 0.83 fold) production in RA post‐WL compared to pre‐WL. We conclude that H2O2 contributes to the protective effect of exercise training against reduced NO‐mediated dilation after exposure to acute exertion‐associated with hypertension in obesity. NIH (K23HL085614, R01HL095701).
Distinct roles for talin‐1 and kindlin‐3 in LFA‐1‐dependent neutrophil rolling and arrest
Authors: Lefort Craig , Rossaint Jan , Moser Markus , Petrich Brian G. , Zarbock Alexander , Monkley Susan J. , Critchley David R. , Ginsberg Mark H. , Fassler Reinhard , Ley Klaus
Published: June 2021

β2 integrins, including LFA‐1 (αLβ2), play critical roles in leukocyte recruitment during inflammation by binding to ICAM‐1 expressed on the endothelium. The affinity of integrin for ligand is determined by its global conformation, regulated by the binding of proteins to the integrin cytoplasmic tails. At least three different integrin affinity states have been observed: low affinity with bent ectodomain, intermediate affinity with extended ectodomain, and high affinity with extended ectodomain and open headpiece. Intermediate affinity LFA‐1 contributes to neutrophil rolling interactions with the endothelium, whereas high affinity LFA‐1 mediates neutrophil arrest. In the current study, we determined the roles for two integrin activating proteins, talin‐1 and kindlin‐3, in neutrophil rolling and arrest. Using established in vivo (intravital microscopy of post‐capillary venules) and ex vivo (microfluidic flow chamber) models, we found that talin‐1 is necessary for both LFA‐1‐dependent neutrophil rolling and arrest. In contrast, kindlin‐3 was required only for neutrophil arrest and was dispensable for rolling mediated by intermediate affinity LFA‐1. We further corroborate our results with imaging and in vitro studies using antibodies that directly report β2 integrin conformation. These data indicate that talin‐1 is sufficient to induce LFA‐1 extension to an intermediate affinity state whereas kindlin‐3 is critical for the transition of LFA‐1 to a high affinity state.