bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction sites on H2Dd for Ly49A‐dependent natural killer (NK) cell licensing
Authors: Choi Taewoong , Poursine‐Laurent Jennifer , Matsumoto Naoki , Yokoyama Wayne M
Published: June 2021

NK cells become functionally competent through the interaction of NK cell inhibitory receptors with their cognate self‐MHC ligands, a process termed “licensing.” Licensed NK cells express self‐MHC‐specific receptors and can be triggered through their activation receptors to produce cytokines. Structural studies indicated that the Ly49A inhibitory receptor interacts with two sites on its H2Dd ligand. Site 2 encompasses the α1/α2/α3 domains of the H2Dd heavy chain and β2‐microglobulin (β2m), and is the functional binding site for the Ly49A in effector inhibition. However, it is currently unknown whether this same site is involved in Ly49A‐dependent licensing. Herein, we produced transgenic C57BL/6 mice expressing wild type, or mutant H2Dd molecules with site 1 or site 2 point mutations and studied whether Ly49A+ NK cells are licensed. Our data from these transgenic mice and human β2m‐transgenic, murine β2m‐deficient mice indicate that site 2 is critical in Ly49A‐dependent NK cell licensing. Thus, NK cell licensing through Ly49A involves interactions with its MHC ligand that are similar to those involved in effector inhibition.

Supported by the Howard Hughes Medical Institute and grants from the NIH.
The role of traditional foods for infants and young children in the Peruvian Amazon
Authors: Roche Marion Leslie , Creed‐Kanashiro Hilary M , Tuesta Irma , Kuhnlein Harriet V
Published: June 2021

Rationale. To understand the role of traditional foods in dietary quality and child growth, Awajún infant and young child feeding practices and nutritional status must be established. Methods. Research took place in 6 Awajún communities, Cenepa, Peru. Mothers completed repeat dietary recalls and infant feeding histories for their children (n= 32, 0–25 mo). Adequacy of complementary foods was compared to WHO guidelines. Anthropometry was measured to estimate nutritional status. Results. Based on dietary data some infants received adequate nutrition from breastfeeding and complementary foods; however, feeding practices varied. Complementary feeding from 12–23 months was nearer nutrient intake recommendations than for 6–11 months. Purchased or donated foods were not as nutrient dense as traditional foods. Overall, 39.4% of the children were stunted (HAZ <−2), with mean HAZ −1.71 ± SD 1.41. Nutritional status declined with increasing age. Conclusions. Several mothers practiced exclusive breastfeeding and appropriate complementary feeding with Awajún traditional foods; however, given the range of feeding practices, introduction of market foods and inadequate nutritional status of children, health promotion for infant and young child feeding is warranted.

Funding: Canadian Institutes for Health Research (CIHR), Institutes of Aboriginal Peoples Health (IAPH) & Population and Public Health (IPPH)
Lessons learned while assessing food insecurity, acculturation, and dietary intake among Somali refugees in Lewiston, Maine
Authors: Dharod Jigna , Croom Jamar Eliah , Sady Chris , Morell Dale
Published: June 2021

A survey designed to assess nutrition needs was pilot tested among Somali refugees in Lewiston, Maine using 24 face‐to‐face interviews. Inclusion criteria for the survey were: 1) Somali mother; 2) main meal preparer of the household; 3) Lewiston resident. Bilingual Somali women, hired as field workers (FW), were trained to administer the survey. During the trainings, it was found that FW had difficulty reading their native language and following research protocols due to cultural differences. The pilot survey revealed that average household size was 6, while 38% of the participants reported monthly income < $1,000. Of those (83%) participating in the Food Stamp Program, 60% reported their benefit lasting for 15 days or less. Most families experienced food insecurity (71%), and 25% reported using food pantries and soup kitchens. When testing for food insecurity using the Cornell questionnaire, many participants did not understand the concept of a “balanced meal” or “variety.” To explain the concept of “variety” and a “balanced meal,” FW provided examples to participants using a standard Somali meal. Future research with this population must take into account cultural differences; allow adequate time to train field workers on research protocols; and use culturally appropriate methods to assess food insecurity. The study is supported by the USDA‐FNS Food Stamp Program funds.
Relation of Parental Feeding Practices to Overweight in 3‐ and 4‐year‐old Children
Authors: Baker Eric R. , Kennedy Tay S. , Hubbs‐Tait Laura
Published: June 2021

The study objective was to evaluate correlates of overweight and at‐risk‐for overweight in low income children. Participants were 165 3‐ and 4‐year‐old children, 48% 3 year olds, attending Head Start in rural Oklahoma. Maternal ethnicity was 43.6% non‐white, 18.4% did not complete high school (or GED), 84.8% were receiving one or more forms of federal or state assistance. Twenty‐eight children (17.0%) were at‐risk‐for overweight (BMI ≥ 85th percentile and < 95th percentile) and twenty‐nine children (17.6%) were overweight (BMI ≥ 95th percentile). Correlations were computed between the overweight versus non‐overweight distinction and parental reports. Parents of overweight children reported greater concern about their child's being overweight, r = .159, p < .05, but were also more likely to tell their child, if they ate a particular food, they would be rewarded with dessert, r = .177, p < .05. They were more likely to perceive their child as more healthy than parents of non‐overweight children, r = .163, p < .05. Parents of overweight children were more likely to spank them, r = .155, p < .05, and parents of at‐risk‐for overweight and overweight children were more likely to be verbally harsh, r = .164, p < .05. In conclusion, parents’ feeding practices appear related to early unhealthy weight gains. Funding: USDA‐CREES Number OKL0 2605.
Development of web‐based nutrition education system for prevention and management of childhood obesity
Authors: Hyun Taisun H. , Yon Miyong , Han Young‐Hee , Park Chan , Yoo Kwan‐Hee
Published: June 2021

Increase in childhood obesity is a serious problem worldwide. To combat this issue, web‐based nutrition education may be effective due to easy access and availability to children. We developed a website consisting of 3 parts; 1) to self‐assess obesity index, dietary intake and behavior, energy consumption, food frequency, nutrition knowledge, and the stages of behavioral change. This site also provides messages on the assessment results and advice for improvements; 2) to keep follow‐up records for a user to establish dietary and exercise goals, and to maintain follow‐up records of dietary intake and exercise; and 3) to allow children to study by providing tailored information and games. For this entire system, a screening instrument (dietary behavior, nutrition knowledge and etc.) was developed and the reliability was tested. A database including nutrients of 500 common cooked foods was created to calculate dietary intake, and a database with energy expenditure of 165 physical activities was developed to calculate energy expenditure. Message source files designed for all potential feedback for each individual were also created. Our web‐based tailored nutrition education system can be used by a child alone or with parents or teachers who desire to control body weight through healthy eating habits and physical activities.

Research Support: Korea Research Foundation Grant (2005‐KRF‐204‐C00102)
Social‐class specific, prescriptive‐eating menus for Guatemalans: A Positive Deviance approach
Authors: Campos J Raquel , Solomons Noel W. , Vossenaar Marieke , Soto Maria J. , Medina Susana , Anderson Annie S.
Published: June 2021

Background:

Consuming diets closely guided by the 1997 World Cancer Research Fund tenets for cancer prevention could reduce tumor incidence.

Objectives:

To elaborate social‐class specific menu guides for a cancer‐risk reducing diet for Guatemalans using a Positive Deviance approach.

Methods:

A total of 873 Guatemalan participants from 3 SES groups were enrolled: rural (n=301); low‐income urban SES (n=298); and higher‐income urban SES (n=274). Subjects with inadequate diets and current smokers were excluded from analysis. Concordance with 14 selected WCRF/AICR individual guideline components, related to diet and lifestyle, were evaluated to select subjects' regimens to serve as models for elaborating the eating guides.

Results:

Among 873 surveyed, we identified a final total of 23 model finalists from the rural, 21 from the low‐income urban, and 15 from the high‐income urban classes, those with compliance with the highest number of selected WCRF/AICR guideline components. The food frequency records for 14 individuals per class, corrected in the remaining components, were the basis of the 42 single‐day menus to create a diet guide.

Conclusion:

Adherence to such culturally‐sensitive menu guides could contribute to reduction of cancer in Guatemala.

Sponsored by WCRF and AICR
THE INFLUENCE OF DOCOSAHEXAENOIC ACID (DHA) ON RAT OVULATION
Authors: Broughton Kenneth Shane , Hahn Brittney L , Ross Erica Christine , Barber‐Heidal Kimberly
Published: June 2021

Pregnant rat dams were fed diets enriched in DHA (0.3g/100g diet), EPA (0.3), arachidonic acid (0.3) or mixed EPA and DHA (0.3/.3 or 0.15/0.15). The diet influence on tissue composition, ovulation and ovarian prostaglandin synthesis was assessed in female offspring at 26 days of age following ovulation induction. EPA alone or in concert with DHA at 0.3g resulted in the lowest ovarian long‐chain n‐6 PUFA incorporation. This was offset by elevated LA tissue incorporation when compared to DHA alone. When DHA was fed alone, tissue long‐chain n‐6 increased exceeding that seen when ARA was fed, though not differing from control (0.15g LA/100g diet).

With DHA feeding alone, total PGE increased almost 2‐fold over control while neither differed from the other treatments. The increased PGE was induced by marked elevation in PGE3 with unaltered PGE2 release. PGE2 remained unchanged in all dietary treatments while PGE3 increased in response to DHA alone. Total PGF did not differ from control in any dietary treatment though PGF2α in response to ARA was significantly (P<0.05) greater than that seen with DHA. DHA at the 0.3 level, alone or with EPA, resulted in elevated PGF3α when compared to control or to all of the experimental diets. Consumption of the experimental diets did not alter ovulation from control though the EPA only diet yielded a higher ovulation when compared to the D/E .15 diet.
Iron chelation in PC12 cells alters the mRNA expression of monoamine‐related proteins
Authors: Unger Erica Lynn , Hao Lei , Gonzalez Eduardo G , Beard John L
Published: June 2021

Iron deficiency in brain tissue induces a number of genomic alterations, some of which persist into adulthood despite iron repletion (Clardy et al 2006). To investigate potential transcriptional events underlying changes in neurochemistry and neurophysiology during acute iron deprivation, we used microarray gene analyses with follow‐up RT‐PCR to identify candidate pathways affected by iron chelation in PC12 cells. Desferrioxamine (200 mM) treatment for 24 hr resulted in 322 significantly upregulated and 313 downregulated genes. Slc6 (norepinephrine transporter) and Slc3 (anion transporters) families of genes were reduced more than 3 fold in iron‐chelated cells. RT PCR verified this and also showed 3‐fold decrease in dopamine transporter mRNA. A pathway oriented analysis showed genes involved in dopamine transporter trafficking and dopamine receptor signaling were significantly up‐ or down‐regulated including glycogen synthase kinase 3β, protein phosphatase 2B, ubiquitin 2c and regulator of G protein signaling 2. Transcription factors related to neuron development (Nurr1, Nur77 and Phox2a) were also affected. These studies indicate acute iron deprivation alters the gene expression of key proteins involved in the biology of monoamines and will allow us to begin to target key molecular pathways underlying iron deficiency‐induced changes in behavior and neurophysiology.
Hop Extract Meta060 Attenuated TNFalpha Induced Inflammation and Endothelial‐Monocyte Interaction
Authors: Konda Veera Reddy , Desai Anuradha , Hall Amy , Bland Jeffrey , Tripp Matthew
Published: June 2021

TNFα induced endothelial activation and vascular inflammation plays a critical role in atherogenesis. We have investigated the effects of Meta060, a modified hop extract, on TNFα activated endothelial inflammation and endothelial‐monocyte interaction. We found that Meta060 or the NF‐κB inhibitor parthenolide inhibited TNFα activated HAEC‐monocyte interactions. Meta060 also inhibited TNFα activated endothelial biomarkers such as MCP‐1, VCAM‐1 and E‐Selectin in HAEC cells. We compared Meta060 with clopidogrel, a known inhibitor of platelet and monocyte activation. Treatment of THP‐1 cells with clopidogrel inhibited the monocyte‐HAEC interactions, but pretreatment of HAEC cells had no effect. These results suggest that clopidogrel inhibited only monocyte mediated interactions. Conversely, Meta060 inhibited both endothelial and monocyte cells independently, resulting in a more effective reduction of adhesion. Meta060 also inhibited a key inflammatory marker, MCP‐1 in THP‐1 cells. We evaluated inhibitory effects of Meta060 on inflammatory kinases in cell free assays. Meta060 dose dependently inhibited several kinases. These kinases mediate multiple signaling pathways, such as PKC, MAPK and NF‐κB. Taken together, this data suggests that Meta060 may be a novel therapeutic to treat inflammation, an important component of atherogenesis (Supported by Metagenics Inc.).
Structure‐based JAZ ligand‐J1 and its analogues show strong cytotoxic effects on glioma cells in vitro ‐ A new class of glioma therapy drugs?
Authors: Qiu Jingxin , Yang Mingli , May W. Stratford , Ostrov David A. , Keith Robertson D. , Harrison Jeffrey K. , Yachnis Anthony T.
Published: June 2021

Hypoxia/ischemia and oxidative stress has been shown to be involved in glioma tumorigenesis. The Jaz gene encodes a zinc finger protein that can respond to oxidative stress with increased expression both in vitro and in vivo. We have previously reported that JAZ exhibits a tumor grade‐dependent increased expression in gliomas with the highest levels in GBM and shows increased cytoplasm‐nuclear translocation adjacent to pseudopalisading necrosis. We hypothesized that JAZ plays a role in glioma tumorigenesis. Targeting JAZ with a molecular docking approach, we identified one small molecule compound named “J1” that exhibited strong dose‐ and time‐dependent glioma cytotoxic effects in vitro (human U87MG, U118MG, A172, and mouse GL261 glioma cell lines) with GI50 around 25microM. Several known J1 analogues also displayed differential glioma killing effects. Flow cytometry showed that J1 led to cell cycle arrest. Quantitative proteomic analysis revealed that, in both U87MG and U118MG glioma cells, J1 treatment led to a significant decrease of PLOD2 (procollagen hydroxylase 2), which is known to be induced by hypoxia, associated with cancer progression, and was reported to be increased in pseudopalisading GBM cells. These results suggest that JAZ ligand J1 and its analogues have the potential to become a new class of chemotherapy drugs for glioma treatment.
Alcian Blue and Pyronine Y histochemical stains of paraffin embedded sections provide multi‐parameter morphologic assessment in experimental models of pulmonary disease
Authors: Meyerholz David K. , Rodgers Janice R. , Castilow Elaine M. , Varga Steve M.
Published: June 2021

Pulmonary researchers often assess morphologic parameters to determine disease progression or response to novel therapeutics. Detection of morphologic parameters by special stains is essential for assessment. We show novel application of Alcian Blue and Pyronine Y histochemical stains in experimental models of lung disease to provide multi‐parameter tissue assessment. The Alcian Blue provided intrinsic counterstaining through pale blue staining of nuclei. Acidic mucins of airway epithelia or submucosal glands were stained deep blue (sialylated mucins), red (sulfated mucins), or purple (sialylated/sulfated mucins). Elastic fibers stained red within the walls of pulmonary arteries, connecting airways, alveolar septa and subpleural space. Mast cells were readily detected in select species by red granular cytoplasmic staining. Mucin alterations were readily detected in a respiratory syncytial virus hypersensitivity model and elastin loss in a model of bronchiectasis. Experimental models of respiratory disease which had representative staining included: inbred mice, rat, ferret, cat, dog, sheep and pig. This histochemical stain assesses multiple morphologic parameters often required by pulmonary investigators, is cost effective, simple to perform, and reduces the needs for repeated serial sectioning. This work is supported by the Department of Pathology (University of Iowa) and the NIH.
Morphine induced tolerance to mouse intestinal but not colonic transit and constipation
Authors: Gabra Bichoy H , Ross Gracious R , Akbarali Hamid I , Dewey William L
Published: June 2021

The objectives of the present studies were to compare the development of tolerance to antinociception, constipation, intestinal and colonic transit in the mouse. Eight‐ (morphine pellet) and 45‐ (morphine pellet plus supplemental injections) fold antinociceptive tolerance to morphine were developed by standard techniques. Defecation was measured daily as the number of fecal boli and weight of feces. In both tolerant groups defecation was significantly reduced at 24h, 48h and 72h, compared to placebo‐pelleted mice, indicating that tolerance did not develop to morphine‐induced constipation. The effects of a 10 mg/kg dose of morphine on antinociception, intestinal and colonic transit were determined 72h following pellet‐implantation. Acute morphine reduced intestinal transit of a charcoal meal administered orally by 50% in placebo‐pelleted mice but did not produce a similar reduction in animals chronically exposed to morphine indicating the development of tolerance. The time for the expulsion of a glass bead inserted 3 cm proximal to the anus was used to measure colonic transit. Acute morphine significantly reduced the bead expulsion time in both placebo‐ and morphine‐pelleted mice indicating no tolerance. These findings confirm that chronic morphine produces tolerance to antinociception and intestinal transit but not to colonic transit or defecation. Supported by DK46367, DA01647, DA020836
Novel anti‐methamphetamine (METH) monoclonal antibodies (mAbs) substantially alter METH disposition and METH‐induced behavioral effects
Authors: Laurenzana Elizabeth M , Gentry W. Brooks , Hendrickson Howard P. , Owens S. Michael
Published: June 2021

We recently developed a novel series of anti‐METH mAbs against a METH‐like hapten. (designated MO10). MO10‐antigen immunization of mice resulted in two unique mAbs with high‐affinity binding to METH (mAb7F9, KD 1.8 nM), or METH (mAb4G9, KD 34 nM) and (+)amphetamine (AMP, KD 70 nM), with no cross‐reactivity against (−)isomers or endogenous neurotransmitters. The resulting mAbs were tested in rat pharmacokinetic and behavioral assays. For the pharmacokinetic studies, METH (5.6 mg/kg/day) was chronically administered via sc osmotic minipumps to male Sprague‐Dawley rats. At METH steady state, mAb was administered. Serum samples were collected for 2 wks. Both mAbs caused >60‐fold increases in METH serum concentrations compared to pre‐mAb levels, and increases were maintained throughout the study. mAb4G9 also significantly increased AMP serum levels. For behavioral studies, baseline locomotor effects of a 1 mg/kg METH iv dose were determined on day 1 and 4. On day 7, mAb was given 30 min after METH. mAb4G9 caused 54% and 55% reductions in distance traveled and rearing, while mAb7F9 resulted in 48% and 64% reductions, respectively. These data show that anti‐METH mAbs effectively alter METH pharmacokinetic and behavioral effects in preclinical models of human METH abuse (Supported by NIDA P01 DA14361 and R01DA11560).
A Positive Allosteric Modulator of mGluR5 Potentiates DHPG‐induced Long Term Depression at the Schaffer Collateral–CA1 Synapse of the Rat Hippocampus
Authors: Ayala Jennifer Edl , Chen Yelin , Conn P Jeffrey
Published: June 2021

Activation of group I metabotropic glutamate receptors (mGluR) has a variety of actions that lead to alterations in synaptic transmission in area CA1 of the hippocampus. One effect is the induction of long term depression (LTD) of synaptic transmission by the group I selective agonist DHPG (DHPG‐LTD). Exciting advances have been made in the discovery of novel selective positive allosteric modulators (PAM) of both mGluR1 and 5. VU‐29, a PAM of mGluR5, has been shown to selectively potentiate the effect of submaximal concentrations of glutamate with nanomolar potency in cell lines expressing mGluR5. We have previously shown that VU‐29 can enhance long term potentiation induced by subthreshold theta burst stimulation at the Schaffer collateral‐CA1 synapse of the rat hippocampus. Therefore, we chose to determine the effect of VU‐29 on DHPG‐LTD at this synapse. Field excitatory postsynaptic potentials were recorded from area CA1 in response to stimulation of the Schaffer collaterals. We have shown that pre‐incubation of slices with VU‐29 followed by brief application of S‐DHPG induces a more robust LTD than that caused by S‐DHPG alone. Additionally, the effect of VU‐29 can be blocked by the neutral allosteric modulator 5MPEP. Interestingly, a PAM selective for mGluR1, Ro 67‐4853, had no effect on DHPG‐LTD. This work was supported by grants from the NIMH and NINDS.
Intermittent Hypoxia Protects Brain from Ethanol Withdrawal: Role of Free Radicals
Authors: Jung Marianna E , Mallet Robert T , Downey H Fred , Simpkins James W
Published: June 2021

We have reported that intermittent hypoxia (IH) conditioning protected against behavioral signs of ethanol withdrawal (EW) in rats. Here, we tested the hypothesis that IH‐induced free radicals evoke neuroprotective adaptations against EW. Male rats consumed a 6.5% ethanol diet for 5 weeks, and then the diet was abruptly withdrawn to create EW stress (EW group). During the last 20 d of ethanol consumption, subsets of these rats received IH [repetitive (5–8 cycles/d), brief (5–10 min) periods of hypoxia (9.5–10% O2)] with (EW/IH + NAC) or without (EW/IH) daily ip injections (100 mg/kg) of the antioxidant N‐acetyl cysteine (NAC) to test whether blockade of IH‐induced free radical generation would blunt IH‐induced protection against EW insults. Table (means ± SEM, n = 5–6): EW rats showed severe EW signs, including tremor and rigidity, and excess cerebellar superoxide content vs. sham rats fed an isocaloric dextrin diet (*P < 0.05 vs. sham). IH mitigated EW signs and superoxide formation (†P < 0.05 vs. EW), but NAC treatment during the IH program abrogated neuroprotection during subsequent EW. These results suggest that IH‐induced free radicals activate endogenous defenses against EW insults. (Support: NIAAA/AA013864, NIAAA/AA015982, NCCAM/AT003598)
Proteomic analysis detects cytoskeletal‐related proteins that interact with the intracellular C1 domain of adenylyl cyclase 6
Authors: Thangavel Muthusamy , Liu Xiaoqiu , Ostrom Rennolds S.
Published: June 2021

Lipid rafts are specialized structures of the plasma membrane that have an altered lipid composition as well as links to the cytoskeleton. These structures are membrane domains expressing high concentrations of signaling proteins such as receptors, G‐proteins, and certain isoforms of adenylyl cyclase (AC). The mechanism by which AC6 is localized in lipid rafts or caveolae is unknown. We attempted to identify proteins interacting with the intracellular C1 domain of AC6 using proteomics. We expressed a protein corresponding to amino acids 306–692 of human AC6 in Cos‐7 cells then immunoprecipitated the expressed protein via a fused epitope and analyzed the samples by MALDI/TOF mass spectrometry. Of the many proteins identified, 26 of the proteins had high association values with the AC6 C1 domain. Cytoskeleton‐related proteins such as smooth muscle actin, tubulin, MAP2, MAP4, CDC42 and Rho‐GEF were found to interact with the bait protein. Immunoprecipitation studies confirm these interactions. While further studies are needed to characterize the exact relationship between AC6, lipid rafts and microtubules, our study contributes to growing evidence that cytoskeletal and lipid organization are functionally interdependent elements responsible for AC6 localization. Supported by grants from the NIH and the AHA.
Prostaglandin E2 (PGE2) stimulates the secretion of brain‐derived neurotrophic factor (BDNF) from cultured human microglia
Authors: Hutchinson Anthony , Chou Chih‐Ling , Israel Davelene , Xu Wei , Regan John W.
Published: June 2021

PGE2 is produced at high levels in the injured central nervous system (CNS), where it acts as a pro‐inflammatory mediator. The cellular actions of PGE2 are mediated by G protein signaling pathways activated by receptors termed EP1, EP2, EP3 and EP4. Recent animal studies indicate that PGE2 also regulates the induction of growth factors that could participate in tissue repair after CNS injury. To examine the potential regulation of these factors by PGE2 we obtained immortalized human microglial cells, which are the resident macrophages of the CNS. We analyzed PGE2‐treated cultures using reverse transcriptase‐PCR, protein microarrays, ELISA, immunoblotting, and assays for both cyclic AMP production and reporter gene activity. We show that cultured human microglia express the mRNA for the EP2 receptor, whereas the EP1, EP3 and EP4 mRNAs are not detected. Our data also demonstrate that PGE2 stimulation promotes the secretion of BDNF, a neurotrophin growth factor that supports the survival of neurons. This induction of BDNF occurs in a time‐ and dose‐dependent manner through a cyclic AMP/protein kinase A pathway activated by the EP2 receptor. These results suggest that endogenous PGE2, acting through the EP2 receptor in microglia, may play a role in recovery from CNS injury via induction of BDNF release. Supported by EY11291.
Regional Hypoxia In Ischemic Cremaster Muscle Microcirculation
Authors: Duggan William , Ashby Marc , Hui Pan , Damaser Margot S. , Siemionow Maria
Published: June 2021

Purpose: The purpose of our study was to test the hypothesis that ischemia of the cremaster muscle induces extensive hypoxia and significantly reduces blood flow of the microcirculation.

Methods: 12 Lewis rats (75–100g) divided into 4 experimental groups underwent cremaster muscle preparations for in vivo microcirculatory recordings. Group 1 acted as the control group, whilst groups 2, 3 and 4 underwent 2, 4 and 6 hours of ischemia respectively by clamping the cremaster pedicle. After ischemia hypoxyprobe‐1 (pimonidazole hydrochloride, which demonstrates hypoxia by adducting with proteins in cells having oxygen concentrations <14 ìM) was applied topically to the isolated cremaster muscle. In the control group the hypoxyprobe‐1 was applied after 6 hours without any ischemia. Hydroxyprobe‐1 was localized by primary mouse monoclonal antibody.

Results: All cremaster muscles within the ischemic groups demonstrated hypoxia as indicted by the immunohistochemistry for hypoxyprobe‐1. No tissue hypoxia was seen in the control group.

Conclusions: We have proved our hypothesis that 2,4 and 6 hours of induced ischemia within the cremaster muscle results in local hypoxia which increases with increased ischemic time
Steroid ‐ induced fetal programming alters angiotensin II receptor subtype expression in the sheep kidney
Authors: Gwathmey TanYa M. , Shaltout Hossam A , Diz Debra I , Figueroa Jorge P , Rose James C , Chappell Mark C
Published: June 2021

Antenatal exposure to glucocorticoids, as administered for preterm delivery, results in elevated mean arterial pressure (MAP) in offspring associated with reductions in glomerular filtration, renal plasma flow and nephron number. To determine whether renal angiotensin receptor changes accompany the alterations in hemodynamics in adulthood, fetal sheep (N=11) were administered doses of 0.17 mg/kg betamethasone (BMX) or saline (control) at 80–81 days gestation (dG) with full term delivery (~145 dG). Conscious MAP obtained in adult (1.8 years of age) BMX sheep was higher than controls (97 ± 3, N = 6 vs. 82 ± 2 mm Hg, N = 4; P<0.01). Receptors in nuclear (NUC) and plasma membrane (PM) fractions were studied using 125I‐[Sar1, Thr8]‐Ang II and the antagonists Losartan (AT1) or PD123319 (AT2). BMX exposure increased the proportion of AT1 sites in cortex (NUC: 59 ± 4%; PM: 51 ± 5%) compared to controls (NUC: 28 ± 4%; PM: 25 ± 4%; P<0.001). Moreover, BMX reduced cortical AT2 sites (NUC: 25 ± 14%; PM: 41 ± 10%) vs. controls (NUC: 84 ± 2%; PM: 88 ± 2%; P<0.0001). In contrast to the cortex, medullary receptor subtypes were similar between groups. As we previously reported that AT1 receptor blockade reduces MAP in BMX‐treated adult sheep, an alteration in AT1: AT2 receptor ratio may be a key factor in steroid‐induced hypertension. Support: HL56973, HD047584, HD047584‐02S3 and HD017644.
DOCA unmasks salt‐sensitivity of blood pressure in mice lacking P2Y2 receptors
Authors: Rieg Timo , Schroth Jana , Insel Paul A , Vallon Volker
Published: June 2021

Extracellular nucleotides (e.g., ATP) regulate cells by activating nucleotide (P2) receptors. Mice that lack P2Y2 receptors (ko) have salt‐resistant hypertension, suppressed plasma aldosterone (aldo) and renin concentrations and lower renal expression of epithelial sodium channels (FASEB J 21:3717–26, 2007). Moreover, ko mice require smaller increases in plasma aldo to adapt renal Na+ excretion to restricted intake, suggesting facilitated renal Na+ retention. To test if down‐regulation of the mineralocorticoid system masks salt retention and salt‐sensitivity of blood pressure (BP) in ko mice, we administered deoxycorticosterone acetate (DOCA, 2.4 mg/day s.c. pellet/21 day release) or DOCA plus 1% NaCl in drinking water. WT or ko mice given DOCA and water to drink had no change in BP. However, ko and WT DOCA‐treated mice initially (on day 1) preferred 1% NaCl vs. tap water, consistent with DOCA‐induced salt appetite. WT mice responded similarly on subsequent days but ko mice preferred water. In spite of lower salt intake, ko mice had a greater BP increase than did WT (Δ BP vs. basal: 14±3 vs. 5±2 mmHg; p<0.05). If only able to drink 1% NaCl, WT and ko mice had similar salt intake but ko mice had a greater increase in BP than did WT (Δ BP vs. basal: 23±3 vs. 6±3 mmHg, p<0.05). These data indicate that suppression of the mineralocorticoid system masks salt retention and salt‐sensitivity of BP in mice that lack P2Y2 receptors.
A mathematical model of the blood pressure response to salt intake
Authors: Mangourova Violeta Ivanova , Ringwood John , Van Vliet Bruce
Published: June 2021

Mathematical modeling is an invaluable tool for understanding the operation of complex physiological systems such as those regulating blood pressure (BP). While high salt intake has been implicated as a contributor to the development of essential hypertension, the dynamics and underlying mechanisms of salt‐induced hypertension remain unclear. Mathematical modeling of the BP response to changes in salt intake can provide better understanding of the mechanisms and time scales involved in the development of hypertension. Here we present our development of a mathematical model of the dynamics of salt‐induced hypertension. Model structure development was guided by general physiological principles combined with the need to adequately account for the dynamics of the original data. Model parameters were determined using numerical techniques and data from a number of experimental protocols involving Dahl salt‐sensitive and salt‐resistant rats. Our results suggest that salt‐induced hypertension may be modeled as a combination of several components, including (a) a rapid reversible effect of salt on BP, (b) a slow and irreversible component which may represent the effect of accumulating damage (renal lesions or vascular changes), (c) compensatory dynamics opposing BP increase. Our model may provide some insight into the etiology and epidemiology of salt related hypertension and its prevention. Funded by IRCSET.
Angiotensin II‐adenosine interaction via receptors and intracellular calcium in afferent arterioles
Authors: Lai Enyin , Patzak Andreas , Persson Erik
Published: June 2021

Adenosine and angiotensin II (Ang II) interact in the control of renal blood perfusion and glomerular filtration rate. Here we test the hypothesis that Ang II in low concentrations enhances adenosine vasoconstrictor effects. Further, we investigated cytosolic calcium transients and the role of adenosine and Ang II receptors in this context. Bolus application of Ang II (10−12 and 10−10mol/l) induced negligible vasoconstrictions in isolated perfused afferent arterioles of mice, while Ang II 10−8mol/l reduced diameters by about 35% within 2 min. The arteriolar response to cumulative adenosine applications (10−11 to 10−4 mol/l) was very small, but clearly enhanced by Ang II at 10−12,10−10, 10−8 mol/l, respectively. In Fura‐2 AM loaded arterioles, adenosine bolus application induced an increase of the calcium concentration in the arteriolar wall which was greater at 10−5 mol/l compared with 10−8 mol/l. Ang II (10−11 to 10−6 mol/l) induced a concentration dependent increase of cytosolic calcium concentration. Ang II receptor type 1 (AT1AR) antagonist ZD7155 (10−7 mol/l) nearly prevented this Ang II effect. Simultaneous application of Ang II enhanced the calcium concentration to 10−8 and 10−5 mol/l adenosine. The results show that Ang II enhances adenosine induced vasoconstriction even at low concentrations and that this effect goes along with increased calcium transients.
Gq‐coupled vasopressor receptors are essential mechanosensitive components for the myogenic vasoconstriction
Authors: Schnitzler Michael Mederos y , Storch Ursula , Meibers Simone , Essin Kirill , Breit Andreas , Gollasch Maik , Gudermann Thomas
Published: June 2021

The distal steps of the signalling cascade leading to myogenic constriction in vascular smooth muscle cells have been well investigated, while sensing and the proximal steps are enigmatic. Here we show that mechanical membrane stretch and osmotic cell swelling agonist‐independently activate Gq‐coupled receptors, and subsequently TRPC channels. Using BRET, β‐arrestin was shown to be recruited to the receptor by membrane stretch in a way similar to that seen on agonist stimulation. Simultaneous agonist and mechanical stimulation led to a leftward shift of the angiotensin II‐concentration response curve. Receptor activation and the subsequent signalling cascade could be blocked at different levels. We show that Gq‐coupled receptors like AT1 angiotensin II and V1A vasopressin receptors are activated by membrane stretch dependent on their receptor density. Elevated expression levels of AT1 receptors in A7r5 cells, as well as high endogenous expression levels in renal vascular smooth muscle cells were sufficient for mechanosensivity. In rat cerebral and mouse renal arteries, myogenic tone was profoundly diminished by incubation with the AT1 receptor inverse agonist losartan. Thus, Gq‐coupled receptors are important components of the mechanosensory complex in vascular smooth muscle cells.
Influence of sympathetic vasoconstriction on contraction‐ and mechanically‐induced rapid vasodilation in humans
Authors: Carlson Rick E , Pudwill Linnea , Kirby Brett S , Markwald Rachel R , Voyles Wyatt F , Dinenno Frank A
Published: June 2021

Brief muscle contractions elicit a rapid vasodilation that appears due, in part, to mechanical effects on the vasculature. We tested the hypothesis that sympathetic tone modulates contraction‐ and mechanically‐induced rapid vasodilation in humans. We measured forearm blood flow (FBF; Doppler ultrasound) and calculated the vascular conductance (FVC) responses to a range of single, 1‐sec isometric contractions (10–75% maximum voluntary contraction; MVC) and mechanical compressions (50–300 mmHg) of the forearm. Contractions and compressions were performed during supine control conditions and during 10 degrees head‐down tilt (HDT; sympathetic inhibition; n = 10), and compressions were also performed during control and during −20 mmHg lower body negative pressure (LBNP; sympathetic activation; n = 10). HDT increased and LBNP decreased resting FVC by ∼20%, respectively. The immediate (1st cardiac cycle post contraction) vasodilator responses at 10 and 25% MVC were greater during HDT vs control, as were the total hyperemic responses to all contraction intensities (P<0.05). In contrast, alterations in sympathetic tone via HDT or LBNP did not influence the vasodilator responses to forearm compressions (all P>0.05). We conclude that sympathetic tone restrains contraction‐induced rapid vasodilation, but does not influence mechanically‐induced vasodilation in humans.

Supported by AG‐022337
GSK‐3β suppresses myogenic differentiation through negative regulation of NFATc3
Authors: Langen Ramon , van der Velden Jos , Willems Jodil , Kelders Marco , Schols Annemie
Published: June 2021

Skeletal muscle atrophy is a prominent and disabling feature in many chronic diseases. Prevention or reversal of muscle atrophy by stimulation of skeletal muscle growth is a potential therapeutic strategy. Glycogen Synthase Kinase 3beta (GSK‐3beta) has been implicated in negative regulation of skeletal muscle growth. The aim of this study was to investigate if inhibition of GSK‐3beta stimulates myogenic differentiation via Nuclear Factor of Activated T‐cells (NFAT).

GSK‐3beta knock‐down in C2C12 myoblasts or genetic deficiency in myogenically converted MEFs enhanced myotube formation and muscle specific gene expression during differentiation. GSK‐3beta deficient MEFs or myoblasts displayed enhanced nuclear translocation of NFATc3, and elevated NFAT‐sensitive promoter transactivation. Over‐expression of NFATc3 increased muscle gene promoter transactivation. Finally, stimulation of muscle gene expression observed following GSK‐3beta inhibition was strongly attenuated in NFATc3‐deficient myoblasts, indicating that this response requires NFATc3. In conclusion, negative regulation of myogenic differentiation by GSK‐3beta occurs through a transcriptional mechanism which depends on NFATc3. Inhibition of GSK‐3beta may be a potential strategy in prevention or treatment of muscle atrophy.

This work was supported by a research grant of the Netherlands Astma Foundation (NAF 3.2.02.63).
Changes in exercise tolerance and body composition in myostatin overexpressing transgenic mice
Authors: Porszasz‐Reisz Suzanne , Abraham Andrea , Hightower C. Makena , Porszasz Janos
Published: June 2021

Myostatin (Mst) regulates skeletal muscle mass. Mst‐null (KO) mice have double muscle size, while Mst overexpression leads to skeletal muscle wasting. We aimed at identifying the role of Mst in adult‐onset atrophy, hypertrophy, physical performance and the involved molecular cascades. Fiber type composition of Mst overexpressing transgenic (Tg), Mst KO and WT male 1 year old mice (n=6 in each group) was assessed by immunohistochemistry, MHC isoforms by gel electrophoresis and gene expression levels by real time RT‐PCR from muscle tissues. Exercise tolerance was measured by incremental treadmill tests performed to exhaustion 3 times a week for 8 weeks. The initial speed was set to 8 m/min at no inclination. The speed and inclination were subsequently incremented by 4 m/min and 5% every 5 min. Exercise tolerance of KO animals was significantly lower than Tg animals (1.5±1.6 mJ/g vs. 7.3±4.1 mJ/g, P<0.05). In Tg animals the abdominal fat increased by 150% while the muscle mass decreased by 25%. Compared to wild type animals, the fast type fibers decreased by 22% in Tg animals while it increased by 25% in KO. The amount of MHC IIb protein was 2.2 fold higher in KO mice and MHC I was 1.8 fold higher in Tg animals. The Tg mice had a 37% increase in Mst mRNA expression and Akt2 expression was down‐regulated. We conclude that Mst plays a crucial role in regulating body composition, muscle structure and physical performance.
New metabolic 13C isotopomer modeling approach for elucidating brain neuron‐glia metabolism
Authors: Shestov Alexander A , Ugurbil Kamil , Henry Pierre‐Gilles
Published: June 2021

Current brain two‐compartment neuronal‐glial metabolic models [1] use total 13C turnover curves at each carbon position (“positional models”) but do not take advantage of the additional information from 13C isotopomers, which appear as multiplets in 13C spectra and can now be measured and quantified in vivo [2], to determine metabolic fluxes, such as the glutamine‐glutamate neurotransmission flux VNT. We developed a new neuronal‐glial metabolic model (“isotopomer model”) capable of taking into account the additional information from 13C‐13C multiplets. The new metabolic model allows fitting of each dynamic NMR multiplet curve e.g. for glutamate obtained during i.v. C1,6‐glucose infusion (for example GluC4S, Glu‐C4D43, etc, Fig. 1). Monte‐Carlo simulations show that the new model leads to a significant improvement in the precision of metabolic fluxes (Fig. 2). For example, the standard deviation on VNT was 670% with the positional model and just 19% with the isotopomer model.

Conclusion

: we developed a new metabolic model to take into account the additional information from 13C isotopomers, which appear as multiplets in 13C spectra. The additional information provides significantly increased precision in fitted metabolic fluxes.

This work was supported by NIH R01NS38672 and P41RR08079.
Histamine regulates heart rate during digestion in pythons
Authors: Skovgaard Nini , Wang Tobias
Published: June 2021

The intrinsic heart rate of most vertebrates, including humans, is elevated during digestion, suggesting that a non‐adrenergic‐non‐cholinergic (NANC) factor contributes to the postprandial tachycardia. The NANC factor(s) may be released from the digestive organs, but the regulating factors remain elusive and difficult to identify. Pythons can ingest very large whole meals and digestion is associated with a marked rise in metabolism that is sustained for several days. The metabolic rise causes more than a doubling of heart rate and a four‐fold rise in cardiac output. This makes the python an interesting animal to investigate the postprandial tachycardia. We measured blood pressure and heart rate in fasting Python regius and at 24 and 48h after ingestion of a meal amounting to 25% of body weight. Digestion caused heart rate to increase from 25 to 56 min−1, while blood pressure was unchanged. The postprandial rise in heart rate was partially due to a doubling of the intrinsic heart rate (from 28 to 55 min−1), revealed after double autonomic block. The H2‐antagonist did not affect heart rate of fasting snakes, but decreased heart rate by 15–20 min−1 at 24h into digestion, while it had no effects at 48h. Thus, the histaminergic tone on the heart rose from none to 30% at 24h, but vanished after 48h. Bolus injection of histamine exerted a positive chronotropic effect in fasting snakes, but the effect was abolished during digestion. Our study strongly indicates that histamine regulates heart rate during the initial phase of digestion in pythons.

Supported by the Danish Research Council.
Oxidized extracellular redox state increases Nox1 expression in vascular smooth muscle cells
Authors: Stanic Bojana , Miller Francis
Published: June 2021

Redox‐sensitive cell signaling pathways are susceptible to the redox state of thiol/disulfide couples. A more oxidized extracellular redox state (Eh) has been associated with cardiovascular disease. We examined the effects of Eh on expression of the NADPH oxidase catalytic subunit Nox1 in smooth muscle cells (SMC). Murine aortic SMC were exposed to Eh between −150 mV (reduced) and 0 mV (oxidized) by altering the concentration of cysteine and its disulfide, cystine, the predominant redox couple in plasma. Oxidized Eh rapidly induced phosphorylation of EGFR, which was attenuated by the metalloproteinase inhibitor GM6001, and caused phosphorylation of ERK1/2 and PI3K/Akt, which were attenuated by AG1478, an inhibitor of EGFR phosphorylation. In addition, oxidized Eh caused phosphorylation of PKA and the transcription factors activating transcription factor‐1 (ATF‐1) and cAMP‐response‐element‐binding protein (CREB). Inhibition of EGFR phosphorylation blocked ATF‐1 but not CREB phosphorylation, whereas the PKA inhibitor H89 suppressed both CREB and ATF‐1 phosphorylation. Compared with reduced Eh, oxidized Eh increased SMC Nox1 mRNA expression after 24 hours by 2.4‐fold, which was prevented by pretreatment with GM6001 or AG1478. These data identify a novel mechanism whereby “oxidative stress” can induce Nox1 expression through signal transduction pathways involving EGFR.
Failure of osteoclastic bone resorption in the op rat is not due to a mutation within the coding region of the Abca3 gene.
Authors: Dobbins David E.
Published: June 2021

The spontaneous mutation in op rats produces an animal with severe osteopetrosis. This mutation has been localized to a 0.51 cM portion of RNO10. The Abca3 gene resides within this region and would be considered a functional candidate for op. In this study, we tested if this gene could be responsible for the osteopetrosis seen in the op rat. M‐13 tailed primer sets were designed from rat genomic sequence to cover the 32 exons of the rat Abca3 gene. These primer sets were amplified by PCR against genomic DNA from affected mutant animals and the two parental strains, BN and LEW. Mutational analysis of the Abca3 sequences from the three strains failed to reveal significant differences between the affected mutant animals and the two parental strains although multiple single nucleotide polymorphisms between BN and the other two strains were identified. These data indicate that the osteopetrosis seen in the op rat is not the result of a mutation within the coding region of the Abca3 gene. Analysis of additional candidate genes will be required to identify the causative mutation in this animal model of bone disease. Elucidation of the op mutation may indicate novel therapeutic paradigms in osteopetrosis, osteoporosis and rheumatoid arthritis where failed bone homeostasis is an instigating or exacerbating component of the disease process.
Novel role of cholesterol sulfate in gene regulation during skin development
Authors: Nakae Hanako , Hanyu Osamu , Fuda Hirotoshi , Strott Charles A
Published: June 2021

Cholesterol sulfate (CS) is prominent in the epidermis where it accumulates during skin development. Cholesterol sulfotransferase (SULT2B1b), which produces CS localizes to the granular layer of the epidermis coinciding with the highest CS content. Localization of SULT2B1b is similar to that of the barrier protein filaggrin, a late marker of keratinocyte differentiation. CS, based on crystallographic data, binds with high affinity to the retinoic acid receptor‐related orphan receptor alpha (RORα) moreover, binding translates into increased transcriptional activity. Using normal human epidermal keratinocytes (NHEK) in primary culture, CS stimulates profilaggrin (filaggrin precursor protein) mRNA expression in a dose‐dependent manner, and hypothesized that CS by binding to the nuclear receptor RORα stimulates profilaggrin gene transcription. This hypothesis was tested by knockdown experiments directed against mRNAs for RORα and SULT2B1b using NHEK during calcium‐induced differentiation. Concomitant with siRNA‐induced reduction in either RORα or SULT2B1b expression, a decrease in profilaggrin mRNA expression of up to 80% was observed. Furthermore, during RORα knockdown CS was unable to stimulate profilaggrin mRNA expression. These studies suggest that CS regulates the gene for profilaggrin via its ability to interact with the nuclear receptor RORα, an action reminiscent of a typical hormone.