bluuebunny/crossref_metadata_2025 huggingface dataset:
Human a1A‐adrenergic receptor phosphorylation, internalization and desensitization.
Authors: Rocio Alcantara Hernandez
Published: June 2021

The a1A‐adrenergic receptor (a1A‐AR) is a G protein coupled‐receptor (GPCR) that signals through the Gq/ phospholipase C/ calcium release pathway. This adrenergic receptor subtype modulates important cellular responses and it is also involved in the pathogenesis of diseases. Phosphorylation of intracellular loops and the carboxyl terminal tail of a1A‐AR is part of their fine regulation, generally associated with desensitization/internalization processes, in which different protein kinases participate, Including second messenger‐activated protein kinases, such as protein kinase C isoforms (PKCs) and G protein‐coupled receptor kinases (GRKs).

In silico analysis of the human a1A‐AR amino acid sequenced, suggested a series of possible phosphorylation sites targeted by PKCs, GRKs and also for the glycogen synthase protein kinase‐3 (GSK3). GSK3 is an important element into signal transduction of diverse receptor systems, and the phosphorylation on specific substrates can modulates enzyme activity, subcellular localization, stability of the target protein and protein‐protein interactions. We observed using a1A‐AR immunopurification and mass spectrometry analysis that in cells treated with noradrenaline, oxymetazoline or phorbol esters four serines that are target of GSK3: S229, S258, S352 and S381, are phosphorylated. However, the specific GSK3 inhibitor, SB216763, did not block the a1A‐AR desensitization induced by noradrenaline or oxymetazoline. This suggested that other protein kinases play major roles in such agonist‐induced desensitization. Interestingly, phorbol ester‐induced a1A‐AR desensitization was increased by the GSK3 inhibitor. In cellulo colocalization assays of a1A‐AR‐GSK3, showed increased interaction of these proteins in the presence of agonists and phorbol esters. Interestingly, expression of a GSK3 dominant‐negative mutant interferes with a1A‐AR internalization in response to NA and OXY, but this effect does not occur when cells are pretreated with PMA. All these data suggest the possibility that GSK3 might play a role in the regulation of a1A‐AR function and localization.

Partially supported by Grants from Dirección General de Asuntos del Personal Académico (IN200718) and Consejo Nacional de Ciencia y Tecnología (Fronteras 882 and 253156).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Constitutive Activation of Stat Signaling in Fibroblast‐like Synoviocytes Derived from Patients with Juvenile Idiopathic Arthritis Is Inhibited by Methotrexate
Authors: Singh Rakesh , van Haandel Leon , Leeder J. Steven , Becker Mara , Funk Ryan
Published: June 2021

BACKGROUND

Methotrexate (MTX) is the cornerstone of treatment in the management of autoimmune arthritis, including juvenile idiopathic arthritis (JIA). Despite being in use for over 35 years the mechanism through which MTX inhibits inflammation remains unclear. In this study we investigate activation of JAK/STAT signaling in fibroblast‐like synoviocytes from patients with JIA and the effect of MTX.

METHODS

Synoviocytes were isolated from the synovial fluid of juvenile idiopathic arthritis patients following routine arthrocentesis and maintained in culture. Synoviocytes were treated with MTX and assessed for cellular levels of MTX and its polyglutamated metabolites, as well as intermediates of nucleotide biosynthesis, including dUMP and ZMP. Analytes were detected by UPLC‐MS/MS. JAK/STAT signaling was assessed in synoviocytes and A549 human lung carcinoma cells by Western blotting and immunofluorescence. Experiments were performed in triplicate and statistical testing was done by unpaired t test analysis.

RESULTS

MTX was actively taken up and metabolized in synoviocytes to form polyglutamated metabolites of MTX, however, MTX failed to cause measurable inhibition of de novo nucleotide synthesis by dUMP and ZMP analysis. Synoviocytes displayed a high level of STAT phosphorylation with 58.8±4.8% of total measureable STAT3 signal and 69.9±7.3% of total measureable STAT5 signal. In contrast, phosphorylation of STAT3 and STAT5 was undetectable in A549 cells and treatment with 100 ng/mL of recombinant IL‐6 resulted in a marked increase in STAT phosphorylation with 64.4±6.0% of measureable STAT3 signal and 76.6±8.5% of measureable STAT5 signal. Treatment with MTX resulted in inhibition of STAT3 and STAT5 phosphorylation by 38.1±3.4% (p<0.05) and 61.2±7.8% (p<0.05) in primary synoviocytes and 65.3±2.8 (p<0.05) and 92.8±8.1% (p<0.01) in IL‐6 stimulated A549 cells, respectively. In comparison, phosphorylation of STAT3 and STAT5 in response to IL‐6 stimulation in A549 cells was decreased by 91.6±11.3% (p<0.001) and 94.5±12.3% (p<0.001) by 50 μM of JAK Inhibitor I (CAS 457081‐03‐7). In A549 cells inhibition of STAT phosphorylation by MTX was completely reversed by co‐treatment with 100 μM folinic acid.

CONCLUSION

These findings support constitutive activation of JAK/STAT signaling in fibroblast‐like synoviocytes isolated from patients with JIA that is inhibited by treatment with MTX. Although the underlying biochemical mechanism through which MTX inhibits JAK/STAT signaling is not known, it appears to be dependent on the anti‐folate activity of MTX. Together, this data supports inhibition of JAK/STAT signaling as a possible mechanism of anti‐inflammatory activity of MTX.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhancing pre‐clinical in vitro cardiotoxicity assays with bioengineering strategies in a high‐throughput manner
Authors: Fisher Elliot , Ghazizadeh Hamed , Gray Kevin M , Geisse Nicholas A
Published: June 2021

Stem‐cell based in vitro cardiotoxicity assays hold great promise for mitigating the cost of drug development. These assays have the potential to be more predictive of drug arrhythmogenicity than contemporary screening methods which rely on singe ion channel recordings. However, stem cell assays have several important shortcomings when detecting and classifying the risk profile of several known compounds. One likely source of this error is the maturity of the cells; most phenotypes are indicative of a fetal developmental stage. To enhance the predictive power of stem‐cell based assays, we have implemented bioengineering techniques to control the cell's culture surface. We utilized standard photolithography techniques to create nanoscale biomimetic grooves that mimic the shape and structure of collagen on a polymer‐coated glass layer. The fabrication technique allows for generation of this pattern on standard formats and permits use of high‐NA fluorescence microscopy. Using this approach we demonstrate that biomimetic engineering enhances several structural and biochemical phenotypes including sarcomere spacing, myofibril alignment, and sarcomere width. We extended this technique to pattern the surface of micro‐electrode arrays, and demonstrate that these ECM‐based cues enhance the electrophysiological response of cardiomyocytes to various drugs of known action that fail to elicit in vivo responses to drugs in vitro and recapitulated physiological IC50s when compared to traditional assays.

Support or Funding Information

NIH SBIR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vitamin A content of beef and chicken liver revisited: variability and its implications for complementary feeding programs for infants.
Authors: Mazariegos Manolo , Romero‐Abal Maria Eugenia , Solomons Noel W. , Craft Neal E.
Published: June 2021

Background:

Because beef (BL) and chicken liver (CL) are rich sources of Vitamin A (VA) and of key minerals, their consumption has been proposed in food‐based complementary feeding programs. Literature values for VA content and its variability in liver are inconsistent.

Objectives:

To determine the VA content and its variability in BL and CL.

Methods:

13 samples each of BL and CL were purchased in Guatemalan markets. Each sample was split and analyzed by HPLC (Craft Technologies), both as raw (R) and steamed (S).

Results:

Mean±SD of BL content of VA (ug/100g) at 21776±9433 (CI, 95%, 17882 – 25670; SE, 1887) tended to be greater than CL at 17734±6232 (CI, 95%, 15102 – 20366; SE, 1272) (p>0.05). The respective CVs for BL and CL were 43.3%, and 35.1%. R and S contents were comparable in both BL and CL. VA content in BL and CL was much higher than comparative USDA values (2005).

Conclusions:

To meet 500 ug RDAs for VA in infants, it would require 2.3 – 3.3 g/d or 16 – 23 g/wk of BL. DRIs for Fe and Zn would be unattainable from liver without surpassing the 600 ug ULs for retinol. The high content of VA in liver and the variability attests its potential, but raises a note of caution for its use in programs combating infant micronutrient malnutrition.

Sponsored by the Sight and Life
Volatile organosulfur and nutrient compounds from garlic by cultivating areas and processing methods
Authors: Chang Kyung Ja , Cheong Sun Hee
Published: June 2021

The objective of this study was to extract of organosulfur compounds of garlic according to cultivating areas and processing methods by solvent extraction and analysis by GC‐MS. The crude fat and crude carbohydrate contents in Danyang garlic were significantly lower compared to Seosan and Uisung garlic. However, the calcium content in Danyang garlic was the highest among groups. The crude protein and the crude carbohydrate contents of freeze‐dried garlic powder were significantly higher than those of garlic cloves and garlic vines. The crude fat and the crude fiber contents of garlic cloves were significantly lower than those of garlic vines. Solvent extract of homogenates of fresh garlic cloves by cultivating area was subjected to GC‐MS analysis, which showed major volatile sulfur compounds such as diallyl disulfide (DADS), diallyl sulfide (DAS), diallyl trisulfide (DATS), methyl allyl disulfide (MADS) and methyl allyl trisulfide (MATS). Major sulfur compounds from freeze‐dried garlic powder were identified to be allyl monosulfide (AMS), MADS, dimethyl trisulfide (DMTS) and DATS. The DADS and DMDS contents of Uisung garlic were significantly lower than the others. As for DAS content, Danyang garlic was significantly higher than the others. As for processing methods, the DADS, DAS and DMDS contents of garlic juice and garlic vines were higher compared to garlic sediment, freeze‐dried garlic and thermal‐dried garlic. Our results indicated that garlic vines and garlic juice as well as garlic cloves have considerable organosulfur compounds with bioactivities. Therefore, these data may be useful for recycling the garlic vines and controlling the quality and biological activities of garlic in various food industries.
Identification of novel antagonists of CB1 cannabinoid receptors
Authors: Burstein Ethan S , Ott Thomas R , Bulow Anne , Ek Fredrick , Ma Jian‐Nong , Pettersson Hanna , Jensen Jacob , Ottesen Lars , Owens Michelle L , Clemons Bryan , Bertozzi Fabio , Johnson Robert W , Sweetnam Timothy , Currier Erika , Del Tredici Andria L , Schiffer Hans H , Piu Fabrice , McFarland Krista , Tabatabaei Ali , Gardell Luis R , Olsson Roger , Bonhaus Doug
Published: June 2021

CB1 cannabinoid receptor antagonists have therapeutic utility in CNS as well as metabolic disorders. We identified multiple novel structural classes of CB1 antagonists and characterized a compound called AC‐462 in detail. AC‐462 is structurally distinct from rimonabant and other biarylpyrazoles, utilizing a dibenzothiazepine structure as its scaffold. The binding Ki's of AC‐462 to human and rat CB1 receptors are 0.2 and 2 nM, respectively. AC‐462 is highly selective for CB1 receptors over CB2, and over 60 other GPCRs, transporters and ion channels. AC‐462 has pro‐cognitive actions in radial arm maze and novel object recognition paradigms and augments apomorphine‐induced rotations in the 6‐hydroxy dopamine lesion model of Parkinson's disease. AC‐462 reverses CP 55,940 induced analgesia and hypothermia in mice, and is orally active as an appetite suppressant, reducing food intake in fasted Sprague‐Dawley rats. Once daily dosing of AC‐462 to Zucker rats over 14‐days significantly decreased body weight compared to vehicle‐treated animals. A large number of analogs of AC‐462 with similar in vitro and in vivo activities were produced. One of these analogs called AC‐999 produced robust decreases in body weight of diet‐induced obese (DIO) mice dosed orally qd over 14‐days. These results demonstrate that AC‐462 and its analogs have therapeutic potential for a wide array of indications.
Initial synthesis and characterization of an alpha7 nicotinic receptor cellular membrane affinity chromatography column: Effect of receptor subtype and cell type
Authors: Moaddel Ruin , Oliveira Regina , Kimura Tomoko , Hyppolite Patrick , Juhaszova Magdalena , Bernier Michel , Wainer Irving
Published: June 2021

Objective:

In this study, cellular membrane fragments from SH‐EP1‐pCEP4‐hα7 and α7 HEK‐293 cell lines were used to synthesize cellular membrane affinity chromatography (CMAC) columns containing functional α7 nicotinic acetylcholine receptors, CMAC(α7 nAChR) columns.

Methods:

The synthesis of stable columns required the addition of cholesterol to the 2% cholate solubilization/immobilization (s/i) buffer and to the mobile phase. In addition, when membranes from the SH‐EP1 cell line were used L‐α‐phosphatidyl serine and L‐α‐phosphatidyl ethanolamine also had to be added to the s/i buffer.

Results:

A CMAC(α4β2 nAChR) column was prepared using membrane fragments from a SH‐EP1‐pCEP4‐hα4β2 cell line and this process required the addition of L‐α‐phosphatidyl serine and L‐α‐phosphatidyl ethanolamine to the s/i buffer, but not cholesterol. The s/i buffers from the 3 columns were compared with the s/i buffer utilized in the preparation of a CMAC (α4β2 nAChR) column prepared using an α4β2 HEK‐293 cell line, which required no additions to the 2% cholate s/i buffer.

Conclusion:

The data demonstrates that both cell type and receptor type affect the protocol required to produce a stable CMAC column and that, at the current time, the development of an optimum immobilization protocol is an empirical process.
A Genetic Deficiency in Presynaptic Choline Uptake Leads to Resting Tachycardia and Diminished Vagal Reserve
Authors: English Brett A , Keller Nancy R , Appalsamy Martin , Ruggerio Alicia , Bazalakova Mihaela , Lund David , Robertson David , Blakely Randy D
Published: June 2021

The parasympathetic branch of the autonomic nervous system projects to the heart via the vagus nerve. Vagal efferents into the heart produce bradycardia through release of acetylcholine (ACh). The presynaptic synthesis of ACh is believed to be dependent on the uptake of extracellular choline via the hemicolinium‐3‐sensitive choline transporter (CHT). Attenuated parasympathetic tone has been shown to be a high risk indicator for increased mortality in patients with cardiac disease. Our studies have examined the impact of a genetic loss of CHT on cardiac function in mice. Cardiac synaptosome preparations made from CHT heterozygous (Het) mice have significantly reduced cardiac tissue levels of ACh as well as elevated choline levels, along with elevated urinary NE levels compared to wildtype mice. Conscious CHT Het mice monitored via telemetry exhibit significantly higher resting heart rates, but display similar mean blood pressure compared to wildtype mice. Functional cardiac studies using echocardiography show that CHT Het mice also exhibit reduced fractional shortening and increased left ventricular internal dimensions, but normal cardiac output. Consistent with reductions in parasympathetic tone, CHT Het mice exhibit an attenuated baroreceptor‐mediated reflex. During recovery from treadmill exercise, CHT Het mice show reduced heart rate recovery, indicative of reduced vagal feedback for HR control.
Roles of nitric oxide and K+ channels in the vasodilatory action of nicotine in the rat renal vasculature
Authors: El‐Mas Mahmoud M , El‐gowilly Sahar M , Goher Eman Y , Ghazal Abdel‐Rheem M
Published: June 2021

We investigated the mechanism(s) of the renal vasodilatory action of nicotine in rats. Nicotine vasodilation in phenylephrine‐preconstricted perfused kidneys was evaluated in the absence and presence of inhibitors of nitric oxide synthase (NOS), K+ channels, cholinergic or adrenergic activity. Nicotine infusion (5×10−5, 1×10−4−5×10−4 M) produced concentration‐dependent decreases in the renal perfusion pressure. Endothelium denudation with 3‐[(3‐cholamidopropyl)‐dimethyl‐ammonio]‐1‐propane‐sulfonate (CHAPS, detergent), or inhibition of NOS (NG‐nitro‐L‐arginine), guanylate cyclase (methylene blue) or muscarinic receptors (atropine) almost abolished nicotine vasodilation. The latter was also attenuated after blockade of ATP‐sensitive (KATP, glibenclamide) or inward rectifier (Kir, BaCl2) K+ channels but remained unaltered after blockade of calcium (tetraethylammonium) or voltage (4‐aminopyridine) dependent K+ channels. Hexamethonium, propranolol, guanethidine, or the use of KCl‐preconstricted kidneys reduced nicotine vasodilation in contrast to no effect for diclophenac or neostigmine. These findings implicate cholinergically (muscarinic)‐mediated activation of endothelial NOS and KATP and Kir channels in the renal vasodepressor effect of nicotine. Neurogenic NO also contributes to nicotine vasodilation.

Supported by Faculty of Pharmacy, Univ. of Alexandria, Egypt.
Melatonin as an Adjuvant Therapy Against Lung Paraquat Toxicity in Rats
Authors: Korkmaz Ahmet , Gocgeldi Ercan , Uysal Bulent , Ogur Recai , Reiter Russel J , Oter Sukru , Yesilyurt Ozgur , Hasde Metin
Published: June 2021

The intake of paraquat (PQ) causes severe tissue injury leading to numerous fatalities. Considering that PQ toxicity involves the production of reactive oxygen and nitrogen species, transcription factors and inflammatory cytokines, it may be hypothesized that combination a potent antiinflammatory and antioxidant agent may counteract more of PQ's effects than an antiinflammatory agent alone. For this purpose, combination of dexamethasone (Dex) and melatonin (Mel) was compared with Dex alone. Forty male Wistar rats were divided into control, PQ, Dex only and Dex plus Mel groups. Animals were given a toxic dose of i.p. 19 mg/kg PQ dissolved in saline. Control animals were injected the same amount of saline. 1 mg/kg Dex was administered two h after PQ administration. In the combination treatment group 20 mg/kg Mel was given with Dex. Drugs were given every 12 h for a total of 6 doses. Five animals in PQ group and three animals in Dex only group died by the end of the study. No death occurred in the Dex+Mel group. Dex exerted improvements several oxidative and antioxidative parameters. However, combination treatment provides beneficial effects against PQ toxicity far greater than Dex alone. This difference was apparent when tissues were histologically compared. These results suggest that the reduced dose of corticosteroids with Mel is a safe treatment against PQ toxicity.
A comparison of the protective effects of neostigmine and physostigmine on Delphinium barbeyi poisoning in cattle
Authors: Green Benedict Thomas , Cook Daniel , Pfister James A. , Welch Kevin D. , Lee Stephen T. , Stegelmeier Bryan L. , Gardner Dale R. , Panter Kip E.
Published: June 2021

The N‐(methylsuccinimido) anthranoyllycoctonine type (MSAL) alkaloids in toxic larkspur spp. are potent nicotinic cholinergic receptor antagonists with curare‐like effects at high doses. We addressed the hypothesis that neostigmine would be as effective as physostigmine, at reversing the effects of MSAL alkaloids from D. barbeyi in beef cattle. Cattle were dosed p.o. with dried‐ground larkspur containing 5.8 mg/g MSAL alkaloids and changes in heart rate and the evoked‐electromyographic response of the anal sphincter was monitored at 24 h. Treatment with MSAL alkaloids over a dose‐range of 0.45 – 11.77 mg/kg resulted in a dose‐dependent increase in heart rate, and a decrease muscle tone. Neostigmine (0.04 mg/kg) given i.v., 24 h after dosing with 10.38 mg/kg MSAL alkaloids decreased heart rate but had no effect on muscle tone. Neostigmine (0.02 mg/kg) given i.m. reversed the curare‐like effects of a high dose (14.54 mg/kg) of MSAL alkaloids within 15 minutes. Conversely, physostigmine (0.04 mg/kg) given i.v., 24 h after dosing with 10.38 mg/kg MSAL alkaloids had no effect on heart rate but increased muscle tone by 2‐fold. These results suggest that in an experimental setting, neostigmine is more effective than physostigmine at reversing the peripheral effects of MSAL alkaloids on the heart and that it can reverse the curare‐like effects of high doses of MSAL alkaloids in cattle. Research supported by USDA‐ARS.
Impaired contribution of voltage‐dependent K+ channels to ischemic coronary vasodilation in Ossabaw swine with metabolic syndrome
Authors: Borbouse Lena , Payne Gregory A. , Dick Gregory M. , Sturek Michael , Tune Johnathan D.
Published: June 2021

This study assessed the contribution of large conductance Ca2+‐activated (BKCa) and voltage‐dependent (KV) K+ channels to coronary ischemic vasodilation in lean and metabolic syndrome (MetS) Ossabaw swine. Experiments were conducted in anesthetized, open‐chest swine fed a normal maintenance diet or an atherogenic diet over 9 weeks that induces MetS. Ischemic vasodilation was assessed by measuring the reactive hyperemic response to a 15‐second occlusion of the left anterior descending coronary artery. Mean body weight was 36 ± 2 kg in lean (n = 8) and 54 ± 3 kg in MetS (n = 7) swine. BKCa inhibition with penitrem A (10 μg/kg iv) had no effect on peak coronary blood flow in either lean (2.9 ± 0.3 ml/min/g) or MetS (2.6 ± 0.3 ml/min/g) swine. The duration of the reactive hyperemic response was also unaffected by penitrem A in lean (94 ± 10 sec) and MetS (88 ± 8 sec) swine. Subsequent inhibition of KV channels with 4‐aminopyridine (0.3 mg/kg, iv) significantly reduced the duration of reactive hyperemia in lean (66 ± 5 sec) but not in MetS swine (77 ± 12 sec). These data indicate that BKCa channels do not contribute to ischemic vasodilation in lean or MetS Ossabaw swine. In addition, our findings reveal that KV channels play a prominent role in coronary ischemic vasodilation in lean, but not MetS, swine suggesting putative mechanisms of ischemic dilation are switched in early obesity and MetS. (Support: HL67804, RR013223, HL062552)
Human saphenous vein relaxation in response to sodium nitroprusside correlates with systolic blood pressure and phosphorylated heat shock protein 27
Authors: Morley Erica L , Thresher Jeffrey , Grow Leslie , Wheatley Grayson , Komalavilas Padmini , Brophy Colleen M
Published: June 2021

Stress has been shown to cause hypertension and increase heat shock protein 27 (HSP27) expression and phosphorylation in animal models. Impaired relaxation of vascular smooth muscle occurs during hypertension and may be associated with HSP27 expression and/or phosphorylation. Human saphenous vein (HSV) was used as a model system to examine impaired relaxation and HSP27 expression and phosphorylation in a hypertensive patient population. Relaxation of HSV samples was determined by measurements of tension produced in a muscle bath in response to submaximal norepinephrine dose followed by a sodium nitroprusside dose curve. Relaxation was found to be significantly inversely correlated with systolic blood pressure at patient admission (R2=0.6175, n=10, p<0.05). Western blotting for HSP27 and phosphorylated HSP27 revealed a significant inverse correlation between relaxation and the amount of phosphoryated HSP27 present in the HSV samples (R2=0.8699, n=5, p<0.05). Hence this study provides a link between hypertension, HSP27 phosphorylation, and impaired relaxation in human vascular tissue. The mechanism for this impaired relaxation may involve the expression and/or phosphorylation of HSP27 since phosphoHSP27 also appears to be associated with impaired relaxation. Support: NIH RO1 70715
Absence of an anorectic effect in lesioning of the hypothalamic paraventricular nucleus (PVN) with neuropeptide Y ‐saporin (NPY‐SAP)
Authors: Xu Guang , Wu Mingfei , Mack Serdia O
Published: June 2021

The PVN is an important integrative site for feeding behavior, energy homeostasis and autonomic control. We investigated selective lesioning of cells in the PVN with NPY‐SAP to determine the neurotoxicity of the conjugate and its effect on body weight and food intake. Based on our dose response studies, we determined that PVN tissue remained intact with microinjection of 50 ng (100 μl injection volume) of NPY‐SAP. Young male rats injected bilaterally with NPY‐SAP or saporin (control)were allowed to eat and drink ad libitum. Food and water intake, body weight and body temperature were measured for 8 weeks after treatment, and there were no differences (P>0.05) between the groups. Abnormal morphology of neurons in the medial parvocellular region of the PVN, which contains pre‐autonomic cells, was apparent 8 weeks after injection. The effect of chemical lesioning on autonomic function was assessed by challenging unrestrained conscious rats with hypercapnic or hypoxic stress. Ventilatory sensitivity to these stimuli was altered as early as 2 weeks after treatment. In conclusion, we did not find an anorectic effect with selective lesioning of PVN cells that are responsive to NPY. However, our data indicate that NPY acting on PVN cells may play a role in modulating respiratory drive. Supported by NIH NS45859, and NINDS/NCRR U54NS39407
Contributions of CO2, pHi, and pHo to the signaling pathway of chemosensitive neurons in the locus coeruleus
Authors: Hartzler Lynn K , Dean Jay B , Putnam Robert W
Published: June 2021

Central chemosensitive neurons increase their firing rate (FR) upon exposure to hypercapnic acidosis (HA: 15% CO2, pHo 7.0). Multiple signaling pathways have been proposed to account for this increase including changes in Ca2+, CO2, pHi, and pHo. To determine the relative contributions of ΔpHi, ΔpHo, and CO2 to an increased FR, we used the rapid diffusion of a weak acid technique to clamp pHi. A change in pHi is believed to be the major signal in the chemosensitive (CS) response due to strong correlations between the degree of intracellular acidification and the increase of FR in HA (P<0.05). In fact, an intracellular acidification (0.24 ± 0.04 U) with no change in pHo or CO2 elicits an increase in FR of 1.4 ± 0.5 Hz. However, this FR response can still be elicited without any change in pHi (1.4 ± 0.1 Hz). Only when both pHi and pHo are clamped, does hypercapnia not result in an increase in FR (0.4 ± 0.3 Hz in 5% CO2 to 0.16 ± 0.1 Hz in 15% CO2). Thus, the increase in FR seen upon HA with pHi clamped is likely due to the change in pHo (0.45 U) during the HA exposure. Our data suggest that ΔpHi and ΔpHo can both lead to an increase in FR in response to HA, while CO2 alone does not appear to have a significant contribution. It appears that ΔpHi or ΔpHo saturates the FR response because ΔFR with HA is not different than ΔFR with ΔpHi only or ΔpHo only. These data strongly support the multiple factors model of chemosensitivity. [NIH grants RO1 HL56683 and F32 HL80877].
Inhibition of PDE7 activity blunts calcium‐mediated permeability in pulmonary arterial endothelial cells
Authors: Zhu Bing , Rich Thomas
Published: June 2021

Intracellular concentrations of cAMP and Ca2+ regulate the permeability of pulmonary endothelium. For example, in pulmonary arterial endothelial cells (PAECs), activation of store‐operated Ca2+ channels leads to increased endothelial permeability. Pretreatment with rolipram, a selective PDE4 inhibitor, prevents increases in permeability. Recently, we demonstrated that PAECs also express the rolipram‐insensitive, cAMP‐specific PDE7. PDE7A and B proteins were detected in soluble and particulate fractions, respectively, using Western‐blot analysis. The subcellular distributions of PDE7A and 7B were determined using immunocytochemistry. We found that PDE7B is primarily at the plasma membrane, whereas PDE7A has a cytosolic distribution. PDE7 comprises ~50% of cAMP‐hydrolytic activity in soluble fractions and ~70% in particulate fractions. To elucidate the functional roles of PDE7 in PAECs, PDE7A levels were lowered by stable expression of shRNA. PDE7A shRNA cells had lower PDE7A expression levels, and lower, rolipram‐insensitive cAMP‐PDE activity in soluble fractions. No changes in PDE7B expression were observed. Ca2+‐induced increases in endothelial permeability were significantly reduced in PDE7A shRNA cells. These results suggest that PDE7A activity plays a key role in regulation of Ca2+‐mediated permeability of the pulmonary endothelium. HL66299 and HL074278.
Phenotyping a mouse model of pre‐eclampsia using sonography
Authors: Woods Ashley K , Franzek Elizabeth M , Sharma Ram V , Davisson Robin L
Published: June 2021

Our lab discovered a mouse strain (BPH/5) that spontaneously develops symptoms of pre‐eclampsia during pregnancy. We hypothesized that significant fetal phenotypes in BPH/5 pups would be apparent in early gestation. We have previously seen placental abnormalities in tissue sections as early as d9.5. To visualize the embryonic health status throughout gestation, ultra‐high frequency ultrasound was used to characterize BPH/5 pregnancies. Implantation sites were analyzed by exteriorizing the uterus, and gathering sonographic data for each embryo at gestation day 10.5. Three distinct fetoplacental phenotypes emerged in BPH/5 embryos: Class 1 embryos show normal development, Class 2 show challenged development, and Class 3 fail to develop. Class 1 BPH/5 and C57 embryos showed no differences in heart rate (measured as a fraction of maternal HR), however, Class 2 BPH/5 embryos showed a decrease in HR compared to C57 controls (Class 2 BPH/5: 8.8±0.7 %mat HR, n=6, C57: 15.2±0.1 %mat HR, n=21, p<0.05). We observed a decrease in crown rump size between both Class 1 and Class 2 BPH/5 embryos compared with C57 controls (Class 1 BPH/5 3.57±0.12mm;n=20, Class 2 BPH/5 2.38±0.12mm; n=4, and C57 4.12±0.07mm; n=13, p<0.001). These data show that ultrasound imaging is a useful means of phenotyping BPH/5 implantation sites throughout gestation, and gaining insight into molecular mechanisms of pre‐eclampsia.
Involvement of ezrin in cold storage preservation injury in renal proximal tubule epithelial cells: Possible structural and signaling roles
Authors: Mangino Martin J , Tian Tao
Published: June 2021

Renal transplantation is the treatment of choice for patients with end‐stage renal disease. Preservation injury is caused by ischemia/reperfusion and hypothermia during cold storage, which leads to early graft loss and delayed graft function. Advancements in organ preservation are limited by our understanding of basic root mechanisms that cause this injury. We hypothesize that sub‐lamellar cytoskeletal protein failure is involved and studied this in isolated rabbit renal proximal tubules and epithelial cells. Tubules cold stored in UW solution released ezrin from cytoskeletal actin attachments. Ezrin phosphorylation at T567 and Y353 decreased with progressive cold storage ischemia, which controls both ezrin binding to the cytoskeleton and survival signaling through Akt, respectively. Tubule epithelial cell death at reperfusion after cold storage ischemia was almost exclusively by apoptosis (annexin‐V FACS analysis). Transfection of primary renal epithelial cells with wild‐type mammalian ezrin cDNA increased survival and viability after cold storage ischemia and reperfusion and increased Akt phosphorylation (IHC). Conversely, inhibition of ezrin expression with specific siRNA duplexes decreased cell survival and viability. In conclusion, inactivation of ezrin during preservation may cause renal preservation injury by altering membrane structure and by reducing survival signaling.
Effects of prenatal stress on cardiovascular regulation in offspring
Authors: Hines Tina
Published: June 2021

Certain prenatal disorders increase risk for cardiovascular disease in adult offspring. We tested the effect of reduced uterine perfusion (RUP) during pregnancy on arterial pressure (AP), baroreflex and stress regulation in adult rat offspring.

Uterine vessels were clipped (RUP) on gestational day 14. Resting AP, baroreflex control of heart rate (HR), and AP, HR and glucose and corticosterone (cort) responses to 60‐min. restraint stress were measured in conscious chronically instrumented offspring. Baroreflex control of renal sympathetic nerve activity (RSNA) was assessed in anesthetized offspring.

Resting AP was ~10mmHg higher in RUP offspring from 8–12wks in males and 8–20 wks in females. Baroreflex control of HR and RSNA was similar in all groups of 8wk offspring. AP and HR at 10 and 60 min. of restraint were also similar among offspring groups. Glucose levels after 10 min. of restraint were lower in RUP male offspring, and plasma cort was lower after 60 min in this group compared with control male offspring. The cort response to stress peaked at a higher level in RUP female offspring at 10 min. and remained higher than control at 60 min (~140ug/dl).

Resting AP is modestly increased in adult male and female RUP offspring. Baroreflex regulation of HR and RSNA does not appear to be adversely affected in these rats. Results suggest sex differences in metabolic and hormonal responses to stress in RUP offspring. NIH NR04184.
Changes in gain of baroreflex control of heart rate during delivery and the early post partum period of the rat
Authors: Goldman Robert K , Azar Afaf S , Mulaney Julia M , Brooks Virginia L
Published: June 2021

Pregnancy decreases baroreflex sensitivity (BRS) at end gestation (EG) in several species. However, the effects of labor and delivery on BRS, and the time at which BRS normalizes during the postpartum (PP) period, are unknown. Because oxytocin, which influences BRS, is released during labor and delivery, we hypothesized that this release may alter BRS. In addition, the maternal plasma levels of placental hormones rapidly normalize PP, suggesting that BRS will improve quickly after delivery. To test these hypotheses, blood pressure (BP) was measured continuously by telemetry (1000 Hz) in female rats (n=5) during the non‐pregnant state (NP), EG, delivery and days 2–10 PP. Pups were removed soon after delivery. Cardiovagal BRS was estimated using the sequence method, which selects spontaneous relationships between ramps in systolic BP and associated changes in pulse interval. Compared to NP, BP and BRS were decreased, and HR was increased, at EG (all P<0.05; Table:*). However, during delivery BRS increased from 1.23±0.09 to 1.65±0.08 msec/mmHg (first 2 hr; P<0.01), but rapidly decreased again (to 1.26±0.7 msec/mmHg) within 2 hr after delivery. By PP3, MAP and HR were recovered (P<0.05 compared to EG; Table,†); however, BRS did not increase significantly until PP6 (P<0.05; Table, †).

In conclusion, cardiovagal BRS increases during delivery, and the mechanism may involve increased oxytocin. In addition, the prolonged time for BRS to recover during PP, in contrast to BP and HR, suggests that different mechanisms are responsible. Supported by HL70962, AHA and Collins Fdn.
Glycemic Index Reduction by a Viscous Polysaccharide Blend Independent of Food Form: Determination of the Glycemic Reduction Index Potential (GRIP)
Authors: Jenkins Alexandra , Vuksan Vladimir , Kacinik Veronica , Lyon Michael
Published: June 2021

Objectives: To determine the extent to which a novel viscous polysaccharide blend (VPB), reduces post prandial glycemia and the glycemic index (GI) when added to either a liquid or a solid meal. VPB is commercially known as PGXTM.

Methods: Two groups of 10 healthy subjects (5M, 5F; age, 35.6 ± 13.2 y; BMI 24.6 ± 2.1 kg/m2; and 3M, 7F; age, 33.5 ± 11.1y; BMI 26.3 ± 5.2 kg/m2), participated in an acute randomized controlled study. Zero, 2.5, 5 and 7.5 g of VPB was added to either a glucose drink or white bread (WB) and margarine (Marg). All meals contained 50g of available carbohydrate. Capillary blood samples were taken fasting and at 15, 30, 45, 60, 90 and 120 min after the start of the meal. Control meals (glucose or WB) were repeated 3 times.

Results: Addition of VPB to glucose and white bread significantly reduced blood glucose iAUC irrespective of dose (p<0.01). The GI of glucose alone, and glucose and 2.5g, 5g or 7.5g were 100, 84, 78 and 73 respectively, the GI of the white bread, WB+Marg, WB and 2.5, 5g or 7.5g of VPB were 71, 67, 48, 37, and 34 respectively. The Glycemic Reduction Index Potential (GRIP) per g of VPB was 4.9±0.9 with glucose and 7.0±1.3 GI units with WB.

Conclusion: The novel viscous fiber blend, is highly effective in lowering the glycemic index of a food irrespective of food form. Estimated GRIP for the VPB was 5 units when added to liquid and 7 units when added to a solid food.

Sponsor: InovoBiologic, Inc, Calgary
The depletion of maternal stores of lutein and zeaxanthin during pregnancy and lactation
Authors: Connor William E. , Bezzerides Eugenia , Wang Yingming , Connor Sonja L.
Published: June 2021

Age‐related macular degeneration has a greater incidence in women who have had multiple pregnancies and who breast‐fed. Pregnancy and lactation might deplete lutein and zeaxanthin stores since they are transferred via the placenta to the fetus and through lactation. Plasma was obtained from 11 women during the last trimester of pregnancy and after four and 12 weeks lactation. Never pregnant women served as controls. Carotenoids were analyzed by high‐pressure liquid chromatography. Pregnant and never‐pregnant women had similar plasma values for lutein (18.4 ± 8.3 mcg/dL pregnant vs. 18.4 ± 12.6 never‐pregnant) and zeaxanthin (4.0 ± 2.1 mcg/dL. pregnant vs. 3.0 ± 1.5 mcg/dL never‐pregnant). Plasma lutein and zeaxanthin values were lower in women who had given birth ≥3 times compared to women who had given birth to ≤2 times (plasma lutein, 10.5 vs. 15.8 mcg/dL respectively, p< 0.01; plasma zeaxanthin 2.4 vs. 3.2 mcg/dL, p<0.03). After 4 weeks lactation, plasma values were lower for lutein (11.0 ± 6.6 μg/dL, p= 0.000) and zeaxanthin (2.3 ± 1.3 mcg/dL, p< 0.001). β‐cryptozanthin declined similarly, but α and β‐carotene increased significantly (33 and 68 percent respectively). Lycopene did not change. In conclusion, plasma lutein and zeaxanthin were 40 percent lower during lactation suggesting that depletion of these xanthophylls might have a role in macular degeneration later in life. USDA grant
Food Choices of Food Stamp Participants: Opportunities for Policy and Educational Interventions
Authors: Fox Mary Kay , Cole Nancy
Published: June 2021

The overarching aim of the Food Stamp Program (FSP) is to ensure that low‐income households have enough food to eat. Over time, as evidence has accumulated about the role diet plays in the development of chronic disease, the program's goals have shifted to include an interest in the quality of foods eaten by FSP participants. Interest in diet quality has also been fueled by the ongoing obesity epidemic in the U.S., the high prevalence of obesity among some segments of the FSP population, and speculation that participation in the FSP and other Federal food assistance programs may be contributing to this problem Recently, some health advocates have recommended that the FSP implement more specific strategies to promote healthful food choices among FSP participants.

This study analyzed data from the National Health and Nutrition Examination Study (NHANES 1999–2006) to examine dietary patterns and food choices of FSP participants. Analyses revealed that the subgroup of FSP participants with the most problematic dietary patterns is adult females. Additional analyses compared dietary patterns of overweight and obese females to identify specific food choices that might be targeted in educational or policy interventions to improve overall diet quality of this high‐risk subgroup of FSP participants.
Almonds and Biomarkers of Lipid Peroxidation: A Randomized Controlled Cross‐over Trial
Authors: Josse Andrea R. , Kendall Cyril W. C. , Marchie Augustine , Nguyen Tri H. , Blumberg Jeffrey B. , Lapsley Karen G. , Jenkins David J. A.
Published: June 2021

Background: Nut consumption is associated with a reduced risk of coronary heart disease (CHD). Almonds, in addition to their cholesterol‐lowering properties, have been shown to reduce oxidized LDL concentrations. However, their effect on other markers of lipid peroxidation is unknown.

Methods: Twenty seven men and women with hyperlipidemia consumed 3 iso‐energetic (mean 423 kcal/d) supplements as part of their therapeutic diets for 1 month each. Supplements consisted of full‐dose almonds (73±3g/d), half‐dose almonds plus half‐dose muffins and full‐dose muffins. Subjects were assessed at wks 0, 2 and 4.

Results: At 4 wks, the full‐dose almonds significantly reduced serum concentrations of malondialdehyde (MDA) (P=0.040) and creatinine‐adjusted urinary isoprostane output (P=0.026) compared to the full‐dose muffins. No effect was seen in serum levels of α‐ or γ‐ tocopherol, adjusted or unadjusted for total cholesterol.

Conclusion: Almond consumption significantly decreased oxidative damage to 2 additional biomarkers of lipid peroxidation, serum MDA and urinary isoprostanes, further supporting our previous finding that almonds reduced the oxidation of LDL. This provides a further mechanism, in addition to cholesterol‐lowering, that may account for the reduction in CHD risk associated with nut consumption.

Funding support: Almond Board of California
Homocysteine lowering with folate, vitamin B12 and vitamin B6 does not alter the proportion of n‐3 long chain polyunsaturated fatty acids (LCPUFA) in plasma phosphatidylcholine.
Authors: Crowe Francesca , Skeaff C Murray , Williams Sheila , Green Tim
Published: June 2021

There is evidence to suggest that folate, homocysteine or both affect tissue n–3 LCPUFA composition. We aimed to determine whether homocysteine‐lowering with folate, vitamin B12, and B6 affect the tissue composition of n‐3 LCPUFA in plasma phosphatidylcholine. We conducted a randomized placebo controlled clinical trial involving 253 participants, 65 y or older, with plasma homocysteine concentrations of at least 13 μmol/L. Participants in the vitamin group (n=127) took a supplement containing 1000 μg folate, 500 μg vitamin B12, and 10 mg vitamin B6 for 2y. The fatty acid composition of plasma phosphatidylcholine was measured at baseline and at 2 y. Plasma homocysteine concentrations during the course of the study were, on average, 4.4 μ mol/L lower in the vitamin group than in the placebo group. There were no significant differences in the proportion of eicosapentaenoic, docosapentaenoic or docosahexaenoic acids in plasma phosphatidylcholine between the vitamins and the placebo group at 2 y; the differences after adjusting for baseline values and sex were −0.03 (99% CI: −0.22, 0.16), 0.03 (−0.03, 0.09) and −0.02 (−0.27, 0.24) mol%, respectively. Lowering plasma homocysteine concentrations with B‐vitamins had no effect on the proportion of n‐3 LCPUFA in plasma phosphatidylcholine.
Deficiency of NRF2 predisposes mice to Pseudomonas aeruginosa induced pneumonia
Authors: Thimmulappa Rajesh , Sussan Thomas , Biswal Shyam
Published: June 2021

Rationale: Pseudomonas aeruginosa is the most common gram‐negative organism associated with hospital acquired pneumonia. NRF2 maintains cellular redox homeostasis by regulating antioxdative genes, and it protects from oxidative stress and dysregulation of the innate immune response. We have previously demonstrated increased mortality of NRF2−/− mice to endotoxin mediated septic shock and cecal ligation and puncture‐mediated polymicrobial sepsis. The present study was designed to investigate the role of NRF2 in P. aeruginosa induced pneumonia and mortality. Methods: NRF2−/− and wild type mice were administered with P.aeruginosa intranasally. Mortality, pulmonary inflammation, and bacterial counts were measured. Results: P.aeruginosa caused greater and earlier mortality in NRF2−/− mice, compared to wild‐type mice. Lungs of NRF2−/− mice were associated with enhanced neutrophil recruitment, compared to wild‐type mice. Lungs from NRF2−/− mice contained increased bacterial counts, and were associated with significantly lower antioxidative gene expression and increased oxidative stress. Conclusion: NRF2 plays a central role in protecting from P. aeruginosa‐induced pneumonia and mortality.
Selective and important contribution of ready‐to‐eat cereals to the nutrient intake of Guatemalan schoolchildren
Authors: Montenegro‐Bethancourt Gabriela , Vossenaar M , Doak Colleen M. , Solomons Noel W.
Published: June 2021

Background:

Ready‐to‐eat cereals (RTEC) are recognized as an important source of micronutrients.

Objectives:

To determine dietary and micronutrient contributions of RTEC from a 1‐day intake survey of schoolchildren in an urban Guatemalan area.

Methods:

Data were obtained in a cross‐sectional study of 449 students, boys and girls, stratified by SES in private (n=230) or public (n=219) schools. Intake estimation comprised a single 24‐h pictorial self‐recording, supplemented by a dietitian interview to quantify amounts and check for completeness. Nutrient content was obtained from product labels. The nutrient contribution presented did not include the milk usually consumed with RTEC. The 2004 WHO recommended nutrient intakes (RNI) were used to assess nutrient adequacy.

Results:

RTEC were mentioned at least once on 183 child‐days (41%): 93% at breakfast. From the 7 RTEC varieties reported, cornflakes was most common (70%); it was first source of Fe, folate and thiamin. RTEC made important contributions (17‐21%) to the RNI for Fe, folate and vit D. Cereal consumption was significantly higher in the private school sample.

Conclusions:

The main benefit from added micronutrients in RTEC goes to higher rather than lower income children.

Funded in part by Kellogg Co, Mexico
Place of birth Matters: Adverse dietary profiles observed for US born compared to non‐US born Latino populations
Authors: Duffey Kiyah J. , Gordon‐Larsen Penny , Ayala Guadalupe X. , Popkin Barry M.
Published: June 2021

Place of birth Matters: Adverse dietary profiles observed for US born compared to non‐US born Latino populations Background: While several studies have examined acculturation and obesity, minimal research on the diet composition of US‐ and non‐US born Latino subpopulations has been undertaken.

Objective: To examine differences in composition of US and non‐US born Mexican‐American (MA) and other Hispanic‐American (OHA) adults. Methods: We used NAHANES 1999‐2004 data from MA (n=3375) and OHA (n=622) adults aged 18‐74. Dietary data were collected with a single 24‐hour recall, and grouped using the UNC‐food grouping system. All results weighted to be nationally representative with significance p<0.05.

Results: Compared to their non‐US born counterparts, US born MA and OHA adults consumed more total energy (+63 and +212 kcals respectively) and added sugar (+12 and +40 grams, respectively), with differences in percent of energy by food group. Among MA, those who were US versus non‐US born consumed a greater percent of energy from food obtained away‐from‐home (+2.95%), high‐fat salty snacks (+2.1%), mixed dishes (+1.15%) and desserts (+0.78%). Conversely, US born MA consumed less energy from fruits & vegetables (‐3.04%), soy & legumes (‐2.51%), soups (‐1.76%), and whole‐fat milk (‐1.36%). Similar trends were observed for OHA. US vs. non‐US born OHA consumed more soda & fruit drinks (+3.39%).Conclusion: Adverse diet profiles of US‐born Latinos, particularly MA, may underlie the higher obesity rates seen in this population.

Grant Funding Source: NIH CA109831‐01