bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of C677T methylenetetrahydrofolate reductase (MTHFR) gene polymorphism on plasma homocysteine levels among Cuban Americans with and without type 2 diabetes
Authors: Huffman Fatma G , Zarini Gustavo G. , Shin Dong‐Ho
Published: June 2021

The study examined the influence of C677T MTHFR gene polymorphism on plasma homocysteine (Hcy) levels among Cuban Americans with (N=136) and without (N=136) type 2 diabetes (T2D). Subjects were randomly recruited from Miami‐Dade and Broward Counties, FL. Hcy was determined in fasting blood samples. MTHFR genotypes of the polymorphism C677T were analyzed from genomic DNA samples using a restriction enzyme (Hinf I). Statistical analyses used SPSS and included descriptive statistics and ANCOVA test controlling for age, gender, BMI, smoking, family history of CHD, intake of folate, vitamins B6 and B12. Prior to all statistical analyses plasma Hcy was log‐transformed to achieve normality. After adjusting for the control variables ANCOVA test indicated that non‐diabetics had higher log Hcy levels (2.20±.02) than T2D subjects (2.04±.03), (p=.001). Subjects with the TT genotype had higher log Hcy levels (2.19±.02) than those with CC genotype (2.11±.05), (p=.002). The interaction between diabetes status and MTHFR genotypes with log Hcy was not statistically significant. High Hcy levels among Cuban Americans with TT genotype were independent of their diabetes status. Our study agrees with previous reports with other ethnic groups indicating that high Hcy levels are found in persons with TT genotype.
Does dietary resistant starch mimic the effects of food restriction on the aging related co‐morbities?
Authors: Martin Roy J , Keenan Michael J , Tulley Ricard T , Raggio Anne M , Shen Li , McCutcheon Kathleen L , Ingram Donald , Zhou Jun
Published: June 2021

It is well established that caloric restriction attenuates many aging processes and increases lifespan in numerous species. Resistant starch (RS) is a dietary carbohydrate formed by non‐fiber glucose polymers that resist digestion in the small intestine and is fermented by bacteria in the large intestine to produce short chain fatty acids. Although the mechanism remains unclear, dietary resistant starches have been shown to decrease plasma cholesterol and triglycerides, increase insulin sensitivity, decrease fat storage, and produce anti‐cancer effects in humans. We propose that dietary RS will mimic the effects of food restriction on aging and life span. In this study, we report on the mining of a microarray data generated from cecal cells from rats fed control and RS diets using Applied Biosystems rat genomic chips and PANTHER software and validations by qPCR. The focus of this study was on pathways known to be involved in anti‐aging mechanisms. For example, glutathione S‐transferase, which is involved in detoxification pathways, is elevated 10‐fold by RS. The following pathways were found to be up‐regulated by RS: apoptosis, oxidative stress and p53. The following pathways were down regulated by RS: inflammation, and toll‐like receptors. These observations provide guidance for future studies on the impact of a resistant starch diet on anti‐aging mechanisms. Supported by National Starch and LSU AgCenter.
Testing A Dietary Supplement Mix For Safety and Efficacy
Authors: Hubbard Richard , Rea Brenda , Fukuda David
Published: June 2021

Nutritional supplements use in acutely ill patients, has been associated with statistically significant reduction in the number of non elective hospital readmissions (Gariballa et. al, AJM 2006:119:693). Also, nutritional importance of oral supplement use in lower‐limb ulcers was shown to be highly significant (Raffoul et al Nutr 2006;22:82) and multivitamin supplementation improved hematologic status in HIV‐infected women and children in Tanzania (Fawzi, et. al, Am J Clin Nutr 2007;85:1335). Twenty‐five healthy subjects were selected for this study. The 20 subjects who completing the study were 11 females and 9 males (ages 48‐ 84, mean of 67). Subjects were selected by random draw as Group A, and Group B, alternately by groups on and off supplements for 3 months. Supplements were taken with breakfast and supper with good tolerance. Supplements included: vitamins, minerals, and metabolic growth factors. C‐reactive blood levels were not significant (NS), early morning urine analysis for malondialdehyde (MDA) (NS), N‐terminal teleopeptides (NS), and the SF‐36 questionnaire for mental analysis (p<0.03) was significant for vegan females, and subjects stated they had more energy. Dietary questionnaires separated subjects as vegans or omnivores. Health questionnaires classified subjects for study participation.
Factors associated with use of dietary supplements in the US Army
Authors: Lieberman Harris R. , White Alan , Stavinoha Trisha , McGraw Susan , Hadden Louise , Marriott Bernadette
Published: June 2021

A survey of Dietary Supplement (DS) intake, demographic and lifestyle factors, exercise, and DS knowledge was conducted with 990 randomly selected Soldiers at 11 Army bases. Data were weighted to be representative of the active‐duty Army based on age, gender and rank. Fifty‐three percent of Soldiers consumed at least 1 DS/wk and 23% of users consumed 5 or more. The most popular DS were multivitamins (70%), protein and amino acids (35%) and individual vitamins and minerals (34%). Reasons stated for DS use were to: improve health (64%), give more energy (31%), improve muscle strength (25%), and enhance performance (17%). Regression modeling indicated that being older, higher educational attainment, having a higher BMI, and participation in strength training were predictors of DS use (p<0.05). Factors associated with use of 5 or more DS a week included longer duration of aerobic exercise and lower educational attainment (p < 0.01). Intake of protein and amino acid DS was associated with age 30‐39 yrs (p < 0.01), male sex (p < 0.05), longer duration of aerobic exercise (p < 0.01) and strength training (p < 0.01). Use of herbal supplements was associated with longer aerobic training (p < 0.01) and higher BMI (p < 0.01). Overall, only 41% of DS users reported they were extremely or very confident their DS worked as claimed, and 55% were extremely or very confident their DS were safe. Funded by US Army Medical Research & Materiel Command.
Relationships between body mass index, endogenous estrogen levels, and patterns of estrogen metabolism in Asian‐American women
Authors: Sue Laura , Fuhrman Barbara J. , Xu Xia , Gail Mitchell H. , Falk Roni T. , Wu Anna H. , Pike Malcolm C. , Keefer Larry K. , Veenstra Timothy D. , Hoover Robert N. , Ziegler Regina G.
Published: June 2021

In our population‐based case‐control study of breast cancer in Asian‐American women, body mass index (BMI) was positively and significantly associated with risk of both premenopausal and postmenopausal disease. Conversion of androgens to estrogens by aromatase in adipose tissue is believed to explain the association in postmenopausal women. Using the Asian‐American controls in our study, including 262 premenopausal and 164 postmenopausal women of Chinese, Japanese, and Filipino ancestry, we explored the associations between BMI and absolute and relative levels of 15 estrogens and estrogen metabolites (EM) in 12‐hour urines. The EM were assayed by a recently developed and validated liquid chromatography‐tandem mass spectrometry method with high sensitivity, accuracy, and reproducibility. Estrone and estradiol, as a proportion of total EM, were each positively associated with BMI in both premenopausal (r = 0.14 and 0.16, respectively) and postmenopausal (r = 0.16 and 0.09) women. These associations were strongest among premenopausal women with BMIs =20.0 kg/m2 and postmenopausal women with BMIs =21.0 kg/m2. The proportion of EM in the 2‐hydroxylation pathway, particularly the potentially carcinogenic catechol estrogens, was inversely associated with BMI in pre‐ and postmenopausal women (both r = ‐0.18). We are continuing to explore the relationships between BMI and patterns of estrogen metabolism.
The Impact of Egg Consumption on Heart Health using the NHANES III Follow‐up Survey
Authors: Scrafford Carolyn , Tran Nga , Barraj Leila
Published: June 2021

The association between egg consumption and heart health was evaluated using the NHANES III (1988‐94) Follow‐up Survey and FFQ. Egg consumption was obtained from the 30‐day FFQ administered to participants within the NHANES III survey. Adults 17 years and older were followed for mortality from all causes. There were 366 deaths from CHD and 137 deaths due to stroke among 14,946 subjects included in the analysis. Estimated rate ratios (RR) of death from CHD or stroke associated with categories of egg consumption were estimated using Cox proportional hazards regression. Multivariate models show no increased risk of death from CHD with increased egg consumption (Males: RR = 1.40, 95% CI 0.83‐2.85; Females: RR = 0.93, 95% CI 0.30‐2.84). There was a slight reduction in risk of mortality from stroke in males among consumers of 1 to 6 eggs/week compared to <1 egg/week consumers (RR = 0.93, 95% CI 0.87 ‐ 0.996). Females showed a significant reduction in risk of stroke among consumers of 1 to 6 eggs/week (RR = 0.89, 95% CI 0.83 ‐ 0.95) and 1 or more egg/day (RR = 0.84, 95% CI 0.71 ‐ 0.99). These results showing no association between egg consumption and increased risk of death from CHD or stroke agree with the conclusions in previous studies using the NHANES I Follow‐up Survey, the Health Professionals and Nurses' Health Study.

Funding sources: Egg Nutrition Center and NIH Training Grant T32HD046405.

Grant Funding Source

T32HD046405
FSH enhancing the RANKL‐induced Osteoclastogenesis
Authors: Wu Yu‐Wei , Chiu Li‐Shuan , Chen Shih‐Ching , Tsai Yu‐Hui
Published: June 2021

Recent studies argue whether the declined estrogen or the increased follicle stimulating hormone (FSH) levels is responsible for the postmenopausal osteoporosis. Our recent efforts have been devoted to examining the role of FSH in bone resorption. To test the possible effect of FSH, RAW 264.7 cells was treated with 0, 25, 50, 100 ng/ml RANKL, with/without 0, 5, 30, 100 ng/ml FSH. The RANKL‐induced osteoclastogenesis is in a dose‐dependent manner. FSH alone did not induce the formation of osteoclast. However, FSH augments the RANKL‐induced osteoclastogenesis. The differentiation markers of osteoclast are also examined in this study. The TRAP activity could be stained on the multinucleated osteoclast cells. In addition, the mRNA expressions of RANK, TRAP, cathepsin K, osteopontin and MMP 9 in RANKL‐treated RAW 264.7 cells were also examined by PCR analyses. The combination of RANKL and FSH treatment increases these marker mRNA expressions, except MMP 9 mRNA, compared to those of the RANKL alone group. The protein levels of these osteoclast markers related to the dose of FSH are currently undertaken. The results appear to support the observation reported by Sun et al. [Cell 125:247‐60, 2006]. Our data up to now indicate that FSH appears to modulate RANKL‐induced osteoclastogenesis and thus affect bone mass. Supported by NSC 97‐3112‐B‐038‐003 (YHT).
The role of 1α, 25 dihydroxyvitamin D on muscle hypertrophy and insulin signaling
Authors: Pinkston Catherine Marie , Teegarden Dorothy
Published: June 2021

Clinical studies suggest that vitamin D may improve muscle function and insulin sensitivity. The aim of this study was to investigate the effect of the active metabolite of vitamin D, 1α, 25 dihydroxyvitamin D (1,25D), on muscle hypertrophy and insulin signaling in differentiated C2C12 myocytes. Co‐treatment of cells for 72 hr with 1,25D (10 nM) + insulin (100 nM) resulted in an increase in hypertrophy (21.46±0.27 protein/DNA) compared to insulin (15.76±1.09, p=0.005) or 1,25D treated cells (14.27±1.68). 1,25D + insulin treatment also increased basal insulin signaling (phosphorylated AKT/total AKT protein levels, 2.1±0.18 fold) compared to insulin (1.06±0.23, p=0.01) or 1,25D (1.03±0.08, p=0.003). In addition, insulin receptor mRNA expression levels increased following 72 hr treatment with 1,25D (4.73±1.70 fold, p=0.02) compared to vehicle (1.00±0.29). Consistent with this, 1,25D treatment for 72 hrs followed by 20 minute insulin stimulation increased phosphorylated AKT/total AKT (3.41±0.46 fold) compared to vehicle treated cells (1.00±0.91, p=0.03). This study suggests 1,25D enhances muscle hypertrophy as well as basal and insulin‐stimulated insulin signaling, potentially by increasing the expression of the insulin receptor in skeletal muscle cells. These results may, in part, explain the beneficial effects of vitamin D on muscle and insulin resistance. Supported by NIH DK069965.

Grant Funding Source

NIH DK069965
Quantitative proteomic analysis of progressive steatosis in human hepatocytes using an iTRAQ‐coupled 2D LC‐MS/MS approach
Authors: Moore J. Bernadette , Spanos Christos
Published: June 2021

First described less than 30 years ago, non‐alcoholic fatty liver disease (NAFLD) is now the most common reason for referral to hepatology clinics. A disease spectrum, NAFLD progresses from the relatively benign steatosis to non‐alcoholic steatohepatitis (NASH) which can lead to fibrosis, cirrhosis and end stage liver disease. While a "two‐hit" model of NAFLD progression has been proposed, mechanisms mediating progression remain unknown. The objective of this study was to identify protein changes during NAFLD progression in an in vitro model of progressive steatosis. Treatment of HuH‐7 human hepatocytes for 24 hrs with increasing doses (100, 150 and 200 μM) of palmitate resulted in progressive intracellular steatosis (1.6, 2.3 and 4‐fold over vehicle; p<0.05) and progressive cytotoxicity (10, 20 and 34% versus 3% in vehicle; p<0.001). Use of iTRAQ isobaric tags in a multiplexing approach along with 2D LC‐MS/MS identified proteins altered during progressive steatosis. Confirmation of proteins altered during palmitate‐induced steatosis may identify candidate biomarkers and mechanisms of NAFLD progression. This research was supported by University of Surrey pump‐priming funds.
Effects of long‐term storage and delayed hemolysis of whole blood on folate species by LC‐MS/MS
Authors: Fazili‐Qari Zia , Pfeiffer Christine M , Zhang Mindy
Published: June 2021

Whole blood (WB) folate stability during sample collection, hemolysis and analysis is critical. To facilitate logistics in the field, we investigated whether storage of intact WB at ‐70°C for up to 2 years is feasible. We used 13 WB samples each for subjects with C/C and T/T genotype of the MTHFR C677T polymorphism. Hemolysates were prepared in 1% ascorbic acid (1/11 dilution) at two pH values and incubated for 4‐h/37°C (pH 4.0) or 3‐h/37°C (pH 4.7), then extracted and analyzed for folate species by LC‐MS/MS at 0, 6, 20, and 24 months. Compared to baseline, total folate (TFOL) levels varied as follows: at pH 4.0, ‐4% to ‐13% for C/C samples and ‐7% to +10% for T/T samples; at pH 4.7, ‐11% to ‐12% for C/C samples and ‐6% to +1% for T/T samples. Interestingly, the variation from baseline appeared slightly larger for C/C compared to T/T samples and the drop in TFOL for C/C samples was mainly attributable to a decrease in 5‐methyltetrahydrofolate. At pH 4.7 we noted an interconversion of tetrahydrofolate to 5,10‐methenyltetrahydrofolate for T/T samples over the course of storage with no effect on TFOL levels. Maintaining a short thaw time (=1 h) to avoid folate degradation is very critical (1‐h thaw time: 1% loss of TFOL for C/C and 2% for T/T; 2‐h: 7% for C/C and 21% for T/T, respectively; 3‐h: 24% for C/C and 51% for T/T, respectively). These data show that intact WB can be stored frozen for up to 2 years with only small losses in TFOL.
A comparison of the bioavailability of sulforaphane from broccoli sprouts and a semi‐purified broccoli powder rich in glucoraphanin in healthy human males
Authors: Cramer Jenna M. , Jeffery Elizabeth H.
Published: June 2021

Glucoraphanin (GP) from broccoli exhibits properties of cancer prevention only after hydrolysis to sulforaphane (SF) by the plant enzyme myrosinase. Four healthy males were each fed four meals containing different broccoli preparations: broccoli sprouts, a novel semi‐purified broccoli powder lacking myrosinase (GP‐powder), broccoli sprouts combined with GP‐powder (mix), and a control meal. During the following 24 h, SF recovery in urine was 80%, 50%, and 22% of the GP ingested from sprout, mix and GP‐powder meals, respectively. Recovery of SF was delayed significantly from GP‐powder (only 1/5 of total during the first 6 h), compared to the sprouts and mix meals (2/5 of total during the first 6 h). Preliminary plasma analysis of SF metabolites revealed a similar delayed appearance from the novel GP‐powder. Liver analysis showed no indication of toxicity of any treatment at 24 hours. Whereas these data indicate a higher effectiveness of broccoli sprouts, they also suggest a delayed or prolonged effect from consumption of the novel GP‐powder from broccoli. Supported by Caudill Seed Inc., Louisville, KY.
Blueberry‐Enriched Diets Reduce Metabolic Syndrome and Insulin Resistance in Rats
Authors: Seymour Mitchell , Tanone Ignasia , Lewis Sarah , Urcuyo‐Llanes Daniel , Bolling Steven F , Bennink Maurice R
Published: June 2021

Several studies suggest that anthocyanin intake could modify risk factors for metabolic syndrome. Blueberries are a rich, whole‐food source of anthocyanins. We tested the effect of a semi‐purified diet with added whole blueberry powder (2% w:w) or a carbohydrate‐matched control diet on factors of metabolic syndrome in the obesity‐prone Zucker Fatty rat, fed either a low fat (10% fat) diet or a high fat (45% fat) diet. After 90 days, both the low fat blueberry (LFB) and high fat blueberry (HFB) groups had reduced abdominal fat compared to controls, and the LFB group had reduced body weight and % fat mass. Both LFB and HFB groups had significantly reduced triglycerides, total cholesterol, fasting insulin, and fasting glucose. LFB and HFB also had improved glucose clearance, as measured by a glucose tolerance test. Given the improved glucose control, we then measured mRNA transcripts in skeletal muscle and retroperitoneal fat related to glucose and lipid uptake and metabolism by PCR Array. The results suggest that regular intake of blueberry reduced several key risk factors for cardiovascular disease and metabolic syndrome. Furthermore, these benefits were augmented when fed a low fat diet. Finally, blueberry diet conferred tissue changes in several genes related to lipid and glucose metabolism, which could support the in vivo results of greater insulin sensitivity and reduced fat accumulation.
Raphasatin Degradation Product Induces Detoxification Enzyme Activity
Authors: Scholl Chris , Hanlon Paul , Barnes David
Published: June 2021

Epidemiologic studies suggest that cruciferous vegetable consumption lowers cancer risk, likely through the induction of detoxification enzymes. Glucosinolates are defensive secondary metabolites found in cruciferous vegetables which yield several myrosinase metabolites upon tissue disruption including isothiocyanates (ITC). ITCs have been shown to induce phase I and phase II detoxification enzymes both in vivo and in vitro. Spanish black radish (SBR) (Raphanus sativus L. var. niger) is a member of the Cruciferae family that has a high concentration of a glucosinolate unique to the radish family, glucoraphasatin (GRH). The predominant myrosinase‐metabolite of GRH hydrolysis is 4‐methylthio‐3‐butenyl isothiocyanate (raphasatin). Raphasatin was undetectable in 24‐hour hydrolysis of whole radish preparations. Raphasatin levels peaked 30 minutes after the initiation of myrosinase‐catalyzed hydrolysis of purified GRH, but then rapidly decreased and were undetectable after 24 hours using GC‐MS. This 24‐hour myrosinase hydrolysis of GRH induced quinone reductase activity dose‐dependently in the human hepatoma HepG2 cell line. These data suggest that a raphasatin degradation product is able to induce phase II detoxification enzymes in a manner similar to raphasatin.
New Data on Human Cytokine Changes with Age
Authors: Henkin Robert I. , Schmidt Loren , Velicu Irina
Published: June 2021

Changes in cytokine activity with age are well known. Some cytokines have been reported to increase with age, others have been reported to decrease while still others may not change at all. T lymphocyte activity has been reported to decrease with age consistent with findings that the elderly are more susceptible to infection and neoplasias than are younger subjects. Most of these cytokine changes were observed in stimulated activity in various hematological system function over limited age ranges. We measured the cytokines IL‐1á, IL‐1 â, L‐1ra, IL‐1 RII, IL‐2, IL‐2R, IL‐6, L‐10, IL‐18, TNF‐á, IFN‐â and IFN‐ã at progressive age intervals beginning at age <30y and extending at 10 year intervals to age >70y by use of a sensitive 96 plate spectrophotometric ELISA technique in four stable biological fluids ‐ plasma, urine, saliva and nasal mucus. Measurements were made in 79 subjects with hyposmia (loss of smell) but who were otherwise in good health. Results indicated that levels of their cytokines varied differentially with age in complex patterns both within and between each biological fluid studied. These results are the first to compare cytokine levels with age in these four biological fluids over an extended age range and indicate significant differences from results previously published in levels of cytokines measured over limited age ranges in various hematological systems.
COMBINATION OF HIGH SALT INTAKE WITH DIABETES IN AUTONOMIC MODULATION OF THE CARDIOVASCULAR SYSTEM IN RATS
Authors: Farah Vera , Fiorino Patricia , Yokota Rodrigo , Araujo Iara Cristina , Fonteles Manassés Claudino
Published: June 2021

The purpose of this study was to evaluate the cardiovascular function induced by high salt intake in diabetic rats. Male Wistar rats (n=6, each group) were divided in: Control (CG), without treatment; Diabetic (DG), induced by streptozotocin (STZ 50mg/kg, 21 days) and Diabetic and salt (DSG), diabetes induced by STZ and salt added to the drinking water (1%) during the last 10 days. Arterial pressure (AP) was recorded and processed with a data acquisition system. Systolic arterial pressure (SAP) and pulse interval (PI) time series were submitted to autoregressive spectral analysis to determine SAP and PI variability in time (variance) and frequency domains. AP was reduced in DG (95±2 mmHg) and increased in DSG (119±4 mmHg vs ), when compared with CG (106±1 mmHg). Heart rate was reduced in DG (286±10 bpm vs 352±5 bpm, CG). In DG, there was a decrease in SAP variance (3±0.6 mmHg2 ) and its low frequency domain (LF) (1±0.2 mmHg2 ) when compared with CG (9±0.6 and 6±1 mmHg2 , variance and LF, respectively). High salt intake restored the SAP variance and its LF domain in DSG. Salt intake associated with diabetes increased PI variance (41±8 vs 12 ± 1 ms2, DSG vs CG) and both frequency domains, LF (3.8±2 vs 1.7 ±1 ms2, DSG vs CG) and high frequency (28±7 vs 8 ±1 ms2, DSG vs CG). The key findings of the present study were that high salt intake associated with diabetes modulate the autonomic input of the cardiovascular system induced by diabetes.
SKCa and IKCa channels are involved in epithelium‐dependent relaxation of rat bronchioles
Authors: Kroigaard Christel , Dalsgaard Thomas , Simonsen Ulf
Published: June 2021

We investigated whether Ca2+‐activated K+ channels of small (SKCa) and intermediate (IKCa) conductance are present, and in bronchioles can be modulated by an opener, NS309 (6,7‐dichloro‐1H‐indole‐2,3‐dione 3‐oxime). Immunohistochemical studies were conducted, and segments of rat bronchioles (847 ± 25 μm, n=51) were mounted in microvascular myographs for isometric tension recordings. The effect of NS309 was compared to that in pulmonary arteries. Immunoreaction for IKCa and SKCa3 was found in rat bronchiole epithelium. In 5‐HT (1 µM)‐contracted rat bronchioles, NS309 (0.01‐10 µM) and a β2‐adrenoceptor agonist, salbutamol, induced concentration‐ and epithelium‐dependent relaxations. An inhibitor of cyclooxygenase, indomethacin (3 µM), a NO‐synthase inhibitor, L‐NNA (100 µM) and a CYP2C9 inhibitor, sulphaphenazole (100 µM), did not reduce NS309 relaxation. In contrast to a blocker of large‐conductance KCa (BKCa) channels, iberiotoxin (0.1 µM), a blocker of IKCa and BKCa channels, charybdotoxin (0.07 µM) and a blocker of SKCa channels, apamin (0.5 μM), reduced salbutamol and NS309‐induced relaxation. The study suggests that IKCa and SKCa3 channels are located in rat bronchiole epithelium, and that activation of SKCa and IKCa channels by NS309 leads to epithelium‐derived hyperpolarizing factor (EpDHF)‐type relaxation of bronchioles analogue to the EDHF‐type relaxation in rat pulmonary arteries.
Subcellular localization of PDE5 allows for select cGMP signaling in discrete microdomains in human aortic smooth muscle cells
Authors: Wilson Lindsay S , Jimmo Sandra L , Maurice Donald H
Published: June 2021

Cyclic GMP (cGMP) is a ubiquitous intracellular messenger that acts to integrate and translate the information encoded by extracellular messenger molecules including nitric oxide. Recently we demonstrated that specific localization of phosphodiesterase 5 (PDE5) allowed spatial and temporal regulation of cGMP signaling in platelets. Specifically, PDE5 was identified as an integral component of a Protein Kinase G (PKG) and an inositol‐1,4,5‐trisphosphate receptor (IP3R) signaling complex where it acts to selectively regulate IP3 mediated Ca2+ release. Subsequently, we have investigated the role of PDE5 in human aortic smooth muscle cells (HASMC). The presented findings demonstrate that HASMC contains two pools of PDE5. One pool of PDE5 is tethered in the same cGMP‐signaling complex consisting of PDE5, PKG and IP3R and is responsible for regulating IP3 mediated Ca2+ release. Interestingly, we provide evidence that the untethered PDE5 plays an essential role in regulating proliferation in these cells. Inhibition of PDE5 in HASMC increased DNA synthesis measured by (3H) thymidine incorporation. Our findings provide insight into the role of specific pools of PDE5 in these cells and suggest that discrete domains of cGMP exist in HASMC. We suggest that by further elucidating the role of PDE5 we may define a molecular mechanism by which PDE5 inhibition can differentially impact selected cellular functions in these cells.
Regulation of the G protein βγ subunits through the covalent modification of Gβ
Authors: Dessal Axel Lionel , Smrcka Alan
Published: June 2021

The βγ‐subunits of G protein (Gβγ) dissociate from the a‐subunit (Ga) upon activation of G protein‐coupled receptors. Once free, Gβγ is able to regulate many target proteins, including phospholipase Cβ2 (PLCβ2), phosphoinositide 3‐kinase γ (PI3Kγ) and G protein‐coupled receptor kinase 2 (GRK2). Because overactivation of Gβγ signaling has been implicated in the pathophysiology of several diseases, inhibitors of Gβγ might be very valuable from a pharmacological point of view. Our group has used a combination of virtual and manual screening to find small molecules that bind to a protein‐protein interaction "hot spot" on the β subunit. These compounds inhibit the binding of a phage displaying the SIGKAFKILGYPDYD peptide (SIGK) to Gβγ in an enzyme‐linked immunosorbent assay (ELISA) at concentrations in the nM to µM range. Recently, we discovered that some of the small molecules, like cystamine and selenocystamine, disrupt Gβγ function by a redox mechanism. Their effect can be reversed by the addition of dithiothreitol (DTT). Mass spectrometry indicates that selenocystamine forms a selenylsulfide bridge at preferentially one position, and mutagenesis studies suggest that both cystamine and selenocystamine act on cysteine 204 of the βsubunit. This is the first evidence that protein‐protein interactions with Gβγ can be regulated in a reversible redox manner.

This work was supported by NIH grant GM60286‐08.
Expression of the Zebrafish Prolactin Receptor‐α Gene: Regulation Via MicroRNA Targeting
Authors: Lyon Raymond Stewart
Published: June 2021

Prolactin is best understood for its roles in reproduction including pregnancy and lactation. Recent studies, however, have shown that prolactin is also necessary for proper embryonic development including brain morphogenesis, eye development, and overall growth. Prolactin has also been shown to stimulate olfactory neurogenesis in mice and forebrain development in rats. Prolactin receptor is enriched in zebrafish olfactory tissue, but the role of prolactin in this tissue has not been elucidated. Members of the microRNA‐200 family have been shown to have an important role in olfactory neurogenesis. These microRNAs have been identified as being responsible for terminal differentiation of olfactory neural precursors in both mouse and zebrafish. The precise transcriptional products targeted by these microRNAs have not been completely elucidated. This study aims to investigate the potential for microRNA regulation of the prolactin receptor, particularly within the olfactory tissue of zebrafish. Using a dicer mutant line of zebrafish, lacking all mature microRNA, PRLR‐α expression will be measured and compared to the expression in wild type zebrafish. RT‐PCR, qRT‐PCR, and western blotting have been utilized thus far. It has been determined that PRLR‐α expression is significantly up‐regulated in the dicer mutant zebrafish, indicating some direct or indirect control of the PRLR‐α gene via microRNA targeting.
Effects of pregnanolone on the retention of tandem response sequences in male rats
Authors: Amato Russell J , Winsauer Peter J
Published: June 2021

With increasing interest in using neuroactive steroids therapeutically, greater understanding of the effects of these substances on learning and memory is needed. Neuroactive steroids have effects at both the NMDA and GABAA receptor complexes, and therefore, may have the capacity to alter memory processes. To specifically assess the effects of a neurosteroid on memory, male Long‐Evans rats responding under a retention procedure were administered pregnanolone (1 ‐ 18 mg/kg). During the procedure, a tandem response sequence was acquired and then retested after delays of 30 (n=12) or 180 (n=12) minutes. Responding during both sequence acquisition and retest was maintained under a second‐order fixed‐ratio (FR) 2 schedule of food reinforcement and incorrect responding (errors) produced a 5‐sec timeout. Retention of the sequence was quantified as percent savings in errors to criterion and the 180‐min delay interval alone produced a time‐dependent decrease in retention. Pregnanolone produced a dose‐dependent decrease in the response rate, accuracy, and percent savings. However, decreases in retention occured at doses lower than those that disrupted response rate and accuracy. These data indicate that the neurosteroid pregnanolone potently disrupts retention and that this effect may be an important consideration in the evaluation of neuroactive steroids as therapeutics. Supported by DA 019625
Prenatal methanol exposure alters expression of dopaminergic markers in the striatum of a parkinsonian mouse model
Authors: Mackey Veronica R. , Charlton Clivel G.
Published: June 2021

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a loss of substantia nigra (SN) dopaminergic neurons. The etiology of PD remains unknown. This study proposes that prenatal exposure to methanol (MeOH) will sensitize the developing dopamine system to post‐natal challenges. Pregnant mice were treated with MeOH (4 mg/kg) during gestational days 8‐12. At 12 weeks of age, the control and MeOH exposed offspring were treated with 1‐methyl‐4‐phenyl‐1,2,3,6,‐tetrahydropyridine (MPTP) for 7 days. Markers of the dopaminergic system such as tyrosine hydroxylase (TH), aromatic amino acid decarboxylase (AADC) as well as dopamine (DA) and its metabolites were determined in the striatum of control and MeOH offspring. The results show that striatal TH and dopamine were decreased by 25% and 44.2% respectively, in MeOH offspring. DA, TH and LAAD expression were decreased in a dose dependent manner by MPTP in both the control and MeOH offspring. The MeOH groups with an already 25% reduction, showed a reduced rate of decline of TH or resistance to MPTP. In summary, evidence from preliminary histological study indicates that MeOH causes a failure of TH cell migration into SN compacta. This may reduce the TH projection fibers to the striatum and code for a more resilient population of TH cells. The outcome is a positive test of concept that a sensitization stage underlies PD.
The cannabinoid CB1 receptor system modulates behavioral phenomena predictive of relapse during extinction of self‐administration in mice
Authors: Ward Sara Jane , Rosenberg Marisa , Dykstra Linda , Walker Ellen
Published: June 2021

The endogenous cannabinoid system plays a defined role in the extinction of fear conditioning and spatial memory. It is also well‐established that manipulation of CB1 receptors modulates positively‐reinforced behaviors such as drug self‐administration in laboratory animals, but whether and how the CB1 system regulates extinction of such behaviors is poorly understood. We investigated whether the CB1 receptor antagonist SR141716 affects several extinction behaviors and/or the rate of extinction learning in mice trained to self‐administer palatable food (32% corn oil or 50% Ensure p.o.) or cocaine (0.3 mg/kg/inf i.v.). Following stable self‐administration behavior, mice were pretreated prior to daily extinction sessions with 3.0 mg/kg SR141716 or vehicle, and effects on initial extinction burst responding, spontaneous recovery, CS‐ or prime‐induced reinstatement, and daily extinction rates were assessed. SR141716 pretreatment attenuated initial burst responding, spontaneous recovery, and CS‐ and prime‐ induced reinstatement, and SR141716‐treated mice also required fewer trials to reach daily extinction criterion than vehicle‐treated mice. Taken together, these data suggest that in addition to attenuating the primary reinforcing effects of both palatable foods and drugs of abuse, CB1 receptor antagonism can attenuate relapse behaviors during extinction and enhance extinction learning.
Role of sigma receptors in attenuation of methamphetamine‐induced neurotoxicity in vitro
Authors: Vedam Shreedeepalakshmi , Mastumoto Rae R. , Coop Andrew
Published: June 2021

Methamphetamine (Meth) abuse is a growing health concern because of its capability to cause neurotoxicity and death. Currently, no pharmacotherapy exists to treat Meth‐ induced neurotoxicity. Initial drug targets focused on dopamine, which were limited in effectiveness; alternative targets for medication development are sigma receptors due to Meth's interactions with them. The goal of this study was to evaluate the involvement of sigma receptors in Meth‐induced neurotoxicity in vitro. NG108‐15 cells were incubated with various concentrations of Meth up to 48 hrs. Cytotoxicity was quantified using cell morphology scores. Cultured cells treated with Meth exhibited significant worsening of cells morphology scores in a time‐ and concentration‐dependent manner. AC927, a sigma receptor antagonist attenuated the cytotoxicity of Meth at concentrations that did not significantly alter cell morphology. Together, the results suggest that NG108‐15 cells can serve as an in vitro model for studying Meth‐induced neurotoxicity and AC927 can serve as a good lead molecule for developing pharmacotherapies for Meth.

Funding: NIH DA011979, DA013978.
Modeling Skeletal Muscle Angiogenesis from the Molecular to the Tissue Level
Authors: Qutub Amina Ann , Liu Gang , Vempati Prakash , Popel Aleksander S.
Published: June 2021

We present a multiscale model of angiogenesis that integrates individual modules representing blood flow, oxygen transport, growth factor distribution and signaling, cell sensing, cell movement and cell proliferation. This integration will be used to better understand underlying biological mechanisms from the molecular level up through the organ systems level; examine the effects of pathological conditions on skeletal muscle capillary growth; and test new therapeutic strategies. Model methodology includes partial differential equations, stochastic models, complex logical rules, and agent‐based architectures. Spatial and time scales range from ligand‐receptor interactions and intracellular signaling occurring on the order of seconds and minutes, to cell‐level movement and cell‐matrix interactions over minutes to hours, to vessel formation and tissue level responses over hours to days.

We introduce the individual modules and describe our methodology for integration, which involves wrapping models (written in different programming languages) in a Java‐based dynamic link library format that allows for a central controller to readily pass parameters between the models. We present results from the coordination of modules: the effects of hypoxia in skeletal muscle from the molecular‐detailed changes in HIF1‐initiated VEGF secretion and VEGF‐VEGFR signaling; to endothelial cell activation, cell movement in response to VEGF gradients, and capillary branching as a function of the Notch ligand DLL4; to the resulting new capillary network and altered pattern of blood flow. This angiogenesis multiscale modeling and integration will be a tool in rational drug development and therapeutic regime design, and encourage related angiogenesis experiments.
Pro‐angiogenic and anti‐angiogenic therapies targeting the VEGF‐VEGFR system
Authors: Mac Gabhann Feilim , Popel Aleksander S
Published: June 2021

The vascular endothelial growth factors (VEGFs) have become important therapeutic targets in multiple diseases. VEGF antagonists have been approved for use in several cancers and age‐related macular degeneration, to reduce ectopic microvasculature. Gene and protein delivery of VEGF and VEGF agonists have been proposed as therapeutic strategies to increase microcirculatory flow and oxygen delivery for diseases of hypovascularization (notably coronary and peripheral arterial diseases), with mixed results so far. We have developed computational models of VEGF and its receptors, and used them to predict optimal therapeutic targets. Surprisingly, for both pro‐ and anti‐angiogenic therapies, targeting of VEGF receptors ‐ rather than, or in addition to, the ligands ‐ produced better outcomes. In addition, both microenvironment and interpersonal variability significantly impact the efficacy of the therapies in a predictable way, indicating benefits of a combined experimental‐computational approach (personalized medicine). Using these models, we identify therapy‐responsive subgroups and lay out basic principles for therapeutic targeting of a cytokine system. Support provided by: NIH‐R33‐HL087351, NIH‐R01‐HL079653 and NIH‐T32‐HL007284.
Hypertension and Vascular Remodeling Depend on the Immunological Background in Mice
Authors: Bakker Erik , Taherzadeh Zhila , de Vos Judith , Brewster Lizzy , van Montfrans Gert , VanBavel Ed
Published: June 2021

Hypertension is associated with inward remodeling of small arteries. We propose that vascular remodeling relates to inflammation of the vessel wall and depends on the immunological background. We determined cardiovascular parameters and inflammatory responses in mice, which received NG‐nitro‐L‐arginine methyl ester (L‐NAME) to induce hypertension. Two strains of mice with a genetically different immune background were used: C57BL/6 mice, a T‐helper 1 dominant phenotype (n=14); and BALB/c mice, a T‐helper 2 dominant phenotype (n=40), additionally treated or not with TCR peptides to promote a T‐helper 1 response. After 4 weeks, L‐NAME treatment induced a significant increase in mean arterial blood pressure in C57BL/6 mice (+53%), but not in BALB/c mice (+8%; NS), unless treated with TCR peptides (+13%). Arteries from C57BL/6 mice showed an increase in the wall‐to‐lumen ratio, which was absent in BALB/c mice, unless treated with TCR peptides. In both strains, a complex inflammatory response was found after 3 days of L‐NAME treatment, which had returned to baseline values after 4 weeks. The inflammatory response was similar in the two strains, except for the leukocyte marker CD11b, which showed an increased expression in C57BL/6 only. Confocal microscopy confirmed the presence of CD11b+/CD68− leukocytes in the vessel wall. These data show that vascular remodeling and hypertension are strain dependent. Mice with a T‐helper 1 phenotype are highly susceptible to the development of vascular remodeling and hypertension.
Changes in muscle carnosine of subjects with 4 weeks supplementation with a controlled release formulation of beta‐alanine (Carnosyn™), and for 6 weeks post
Authors: Harris Roger C , Jones Glenys A , Kim Hyo J , Kim Chang K , Price Karen A , Wise John A
Published: June 2021

Muscle Carnosine (M‐Carn) synthesis is limited by the availability of ?‐Alanine ( ?‐Ala) but can be increased by supplementation with ?‐Ala. This is of interest to athletes since increased levels of muscle M‐Carn have previously been shown to improve exercise performance. Above 10mg/kg bwt, however, ?‐Ala in solution or rapidly‐dissolving capsules causes symptoms of paraesthesia, despite the amount being less than that available from normal meat ingestion.

PURPOSE

To investigate the change in M‐Carn in subjects supplemented with a controlled‐release ?‐Ala formulation.

METHOD

Seven males received 2x800mg ?‐Ala controlled release tablets, 4/d for 4w. Controlled release tablets were Carnosyn(tm) supplied by Collegiate Sports Nutrition. A muscle biopsy from the vastus lateralis was taken at 0 and 4w, and 3 and 6w post supplementation. No control group was included as several previous studies have demonstrated no change in M‐Carn without ?‐Ala supplementation.

RESULTS

Subjects did not experience paraesthesia. Mean (±SD) M‐Carn at 0 and 4w, and, 3 and 6w post supplementation were 25.9±4.3, 41.3±5.5, 38.3±6.7 and 35.4±5.5 mmol.kg−1 dry muscle. Post supplementation M‐Carn declined via first‐order kinetics with a half‐life (t½) determined to be 8.6w.

CONCLUSION

The controlled release ?‐Ala formulation was effective in raising M‐Carn. Following supplementation, levels of M‐Carn declined slowly with a t½ ~9w.
Caveolin‐3 KO Mice Develop Dyslipidemia with Impaired Substrate Utilization in Skeletal Muscle.
Authors: Capozza Franco , Trimmer Casey , Lisanti Michael P
Published: June 2021

Caveolin‐3 (Cav‐3) is expressed predominantly in skeletal muscle fibers where it drives Caveolae formation at the muscle cell's plasma membrane (sarcolemma). We and others have previously shown that mice with a genetic ablation of Cav‐3 develop glucose intolerance, insulin resistance, and obesity. The main objective of this study is to determine the effect of Cav‐3 ablation on whole‐body/tissue lipid metabolism. To address this issue, several metabolic parameters were analyzed in the plasma of young and older Cav‐3 KO mice. In addition, by employing radiolabeled metabolic substrates, we directly determined the contribution of skeletal muscle to the plasma dyslipidemia of Cav‐3 KO mice. In summary, the current study shows that an absence of Cav‐3 leads to dyslipidemia in mice. The primary defect can be localized to the skeletal muscle and is secondary to altered muscle substrate utilization caused by Cav‐3 ablation.

Support: American Heart Association; BGIA
Hypergravity resistance training on a human powered centrifuge
Authors: Baker Michael John , Blank Jason , Kreitenberg Arthur , Adams Gregory , Haddad Fadia , Qin Anqi , Baldwin Kenneth , Caiozzo Vincent
Published: June 2021

Microgravity exposure results in muscular, cardiovascular, and skeletal deconditioning and loss of exercise capacity. The Space Cycle is a human powered centrifuge that can be configured for aerobic or resistance training to offer an integrative countermeasure to microgravity. Healthy male subjects performed 2 weeks of squat resistance training on the Space Cycle (SC, n=9) or using free weights (FW, n=9). Subjects completed 9 workouts, each consisting of 3 sets of squats at 70‐90% of individual 10 repetition maximum (10RM). SC subjects performed squats at 2.3 to 3.9 +Gz (measured at the feet) without additional weights. Biopsies were taken from the right vastus lateralis prior to, 4 h after, and 24 h after the first and last workout for evaluation of acute and chronic cellular responses to SC training. RNA was isolated from biopsy samples, cDNA prepared using RT‐PCR, and gene expression analyzed. Collagen IIIα1 expression was elevated two‐ to three‐fold after two weeks in both training groups. Importantly, no significant differences in collagen IIIα1 were seen when comparing FW and SC groups. Gene expression changes support the efficacy of the Space Cycle as a gravity‐independent resistance training device. Supported by NASA MA00403 and NIH M01 RR00827.
Interleukin‐6 Signaling Mediates Human Muscle Satellite Cell Proliferation Following Acute Muscle Damage
Authors: McKay Bryon Robert , De Lisio Michael , Johnston Adam P. W. , Phillips Stuart M. , Tarnopolsky Mark A. , Parise Gianni
Published: June 2021

Interleukin‐6 (IL‐6) has been implicated in satellite cell (SC)‐mediated muscle hypertrophy in animals; however, the role of IL‐6 in the SC response to acute injury in humans remains to be elucidated. Eight subjects (22 ± 1 y; 79 ± 8 kg) performed 300 maximal leg lengthening contractions (3.14 rad.s−1). Blood and muscle samples were collected before and at 4 (T4), 24 (T24), 72 (T72), and 120 hr (T120) post intervention. IL‐6, IL‐6 receptor (IL‐6R), Cyclin D1, SOCS3 mRNA and serum IL‐6 protein were measured. JAK2 and STAT3 phosphorylated and total protein and immunohistochemistry for phosphorylated STAT3 (p‐STAT3), IL‐6 and Pax7 were analyzed across all time‐points. Serum IL‐6 increased 200% by T4, while IL‐6 mRNA increased 4.5‐fold at T4, and IL‐6R mRNA increased 5‐fold at T4. Serum IL‐6 strongly correlated to muscle IL‐6 mRNA (r2 = 0.89, p = 0.02). The number of Pax7+ cells (% myonuclei) increased 74% at T24, peaking at T72 (157% increase from PRE). SC that were IL‐6+ (% of Pax7+ cells) increased from 2% at PRE, to 60% at T4 and peaked at T24 (80% of SC). By T72, IL‐6+ SC dropped to PRE values. There was no change in JAK2, p‐JAK2, STAT3 or p‐STAT3 whole muscle protein; however, p‐STAT3 localized to the SC at T4. Cyclin D1 increased T4 and T24 (6‐fold), and SOCS3 increased 6‐fold at T24. These data suggest that IL‐6, signaling via the JAK/STAT3 pathway, mediates SC proliferation following acute myotrauma in humans.

Supported by NSERC
Regulation and function of NFAT5 in medullary thick ascending limb (mTAL) cells
Authors: Hao Shoujin , Battula Sailaja , Ferreri Nicholas R.
Published: June 2021

The molecular mechanism and function of NFAT5 in mTAL cells has not been fully explored. In this study,RT‐PCR analysis was performed on primary cultures of mouse mTAL cells and freshly isolated mTAL tubules to determine which Na+K+2Cl‐ cotransporter type 2 (NKCC2) isoforms are present. The results show that primary cultures of mTAL cells and freshly isolated mTAL tubules express NKCC2 isoforms A and F, but not B, the expression of which is restricted to the macula densa. The data also indicate that the abundance of NKCC2A and NFAT5 mRNA significantly increased when mTAL cells were exposed to a change in osmolality from 300 to 500 mosmol/kg H2O, produced with NaCl for 2 or 4 hours, respectively. Moreover, transient transfection of cells with shRNA NKCC2A prevented the increase in NFAT5 in response to hypertonic stress, suggesting a role for NKCC2A in the regulation of NFAT5 in these cells. DNA degradation was attenuated and cell viability improved with an NFAT5 overexpression vector, but not empty vector, when osmolality was changed from 300 to 500 mosmol/kg H2O for 48 hr. Overexpression of DN‐NFAT5 attenuated calcium sensing receptor‐mediated Cl‐ influx by 42%, suggesting that a NFAT5‐dependent mechanism contributes to the regulation of Cl‐ transport in mTAL cells. Elucidating NFAT5‐dependent pathways in mTAL cells will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.