bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Conjugated Linoleic Acid (CLA) on Tumor Suppression in Canine Osteosarcoma Cells
Authors: Wong Janice Y , Park Yooheon , Park Yeonhwa
Published: June 2021

Cancer is one of the leading causes of death in the US both in men and women, second to the heart disease. Among types of cancer, osteosarcoma is the most common type of malignant bone cancer. Although there is substantial scientific research focusing on understanding mechanisms of osteosarcoma, the causes of osteosarcoma are largely unknown. One potential mechanism of osteosarcoma involves the non‐steroidal anti‐inflammatory drug‐activated gene 1 (NAG‐1), where NAG‐1 serves as the intermediate key regulator for tumor development and apoptosis for osteosarcoma. Conjugated linoleic acid (CLA) is a fatty acid with anticancer properties in vitro and in vivo studies that occurs naturally in many dairy and beef products. Thus the purpose of this study was to investigate the anticancer effects of CLA isomers on a canine osteosarcoma cell line, CLL 183. The trans‐10,cis‐12 CLA isomer effectively suppressed the tumor cell growth after 72 hour treatment, while the cis‐9,trans‐11 CLA isomer had no effect. Further experiments confirmed that the trans‐10,cis‐12 CLA isomer, but not the cis‐9,trans‐11 isomer, affects NAG‐1 expression. These results support our hypothesis that CLA's anticancer effects are in part mediated by controlling NAG‐1 expression.
Dried fruit consumption associated with reduced overweight or obesity in adults: NHANES, 1999‐2004
Authors: Keast Debra Rose , Jones Julie Miller
Published: June 2021

NHANES, 1999‐2004, data were used to examine the associations of dried fruit consumption with body weight and adiposity in adults 19+ years (n=13,292). Group definitions were non‐consumers vs. consumers of =1/8 equivalent cup fruit/day from dried fruit (eaten out of hand or contained in foods). Anthropometric assessments were overweight/obesity (BMI =25 kg/m2), abdominal obesity (waist circumference >102 cm, men; >88 cm, women), and skinfold measures. Sample‐weighted means, standard errors, and ANOVA (adjusted for covariates including physical activity and other lifestyle factors) were determined using SUDAAN. Between‐group difference in subscapular (‐1.0 mm, p<0.05), but not triceps skinfold was significant. Weight (‐2.7 kg, p<0.01), BMI (‐1.1 kg/m2, p<0.01), prevalence (%) of overweight/obesity (56.0 ± 2.4 vs. 65.8 ± 0.7, p<0.01); waist circumference (‐2.6 cm, p<0.01), and prevalence (%) of abdominal obesity (40.5 ± 2.3 vs. 49.4 ± 0.8, p<0.01) were all lower in dried fruit consumers than non‐consumers. Logistic regression also indicated dried fruit consumption was associated with reduced risks for overweight/obesity (OR=0.64, 95% CI = 0.51‐0.81), and abdominal obesity (OR=0.67, 95% CI = 0.54‐0.83). Dried fruit consumption was associated with lower body weight measures, and reduced abdominal obesity in adults. Supported by California Raisin Marketing Board
Shifts in Diet from High Fat to High Carbohydrate Improved Levels of Adipokines and Pro‐inflammatory Cytokines in Mice Fed a High‐fat Diet
Authors: Lee In Seok , Shin Gina , Kim Sung Joo , Choue Ryowon
Published: June 2021

High‐fat diets induce an expansion of the adipose tissue (AT) which is an important source of adipokines and pro‐inflammatory cytokines. The purpose of this study was to evaluate the effects of a shift from a high‐fat diet to high‐carbohydrate (CHO) diet on the blood levels of adipokines and pro‐inflammation cytokines in mice fed a high‐fat diet. Six‐week‐old male C57BL/6 mice were fed a high‐fat diet (40% of the total calories) for 9 weeks and then the diet was shifted to a high‐CHO diet (70% of the total calories) for 3 weeks. Body weight and epididymal adipose tissue weight as well as blood lipid levels were measured. The serum levels of adipokines and pro‐inflammatory cytokines were analyzed. Shifting the diet from high fat to high CHO significantly decreased body weight and epididymal fat. The blood levels of lipids (TG, total‐chol, and LDL‐chol) decreased. The blood levels of leptin and resistin significantly decreased after the diet was shifted to a high‐CHO diet however, the adiponectin concentrations did not changed. The serum levels of IL‐6 and TNF‐alpha. decreased by the high‐CHO diet. The serum levels of IL‐13 increased by the high‐CHO diet. Conclusively, reduction of body weight and epididymal fat through shifts from a high‐fat diet to a high‐carbohydrate diet effectively improved low‐grade inflammation states in mice fed a high‐fat diet.
An anatomically correct apron
Authors: Farias Anna C
Published: June 2021

A hand sewed, wearable, teaching tool which displays the contents of the abdomen with the peritoneal folds. This model demonstrates a 3D appreciation of abdominal organs with their peritoneal folds. Materials used are cloth of various textures, cushion filling material and bicycle tubing. Students are able to visualize, feel and appreciate the peritoneal fold and their relationships to supplement what they see in the anatomy laboratory. The few advantages of this model are as follows: A 20 minute substitute in the laboratory for a 2 hour lecture. One model can be used to show as little or great detail of the contents of the abdomen. The faculty and students found this model self explanatory and explicit.
Alternative electrode positioning for monitoring ECG with telemetry in freely moving rats
Authors: Huetteman Daniel Anthony
Published: June 2021

Implantable radio‐telemetry allows researchers to collect chronic electrocardiographic (ECG) data from freely moving animals in their home cage. ECG data is typically collected using wire electrodes placed subcutaneously in a lead II configuration. Unfortunately, subcutaneous leads in an ambulatory animal can sometimes be characterized by low signal amplitude, inconsistent waveform characteristics, and high background noise during voluntary muscle movement. These properties can contribute to the inability of researchers to consistently rely on automated software analysis packages to analyze ECG waveforms. This study compares five methods of ECG lead attachment: three using lead II electrode configuration, one previously published base/apex arrangement (Sgoifo, 1996) and a novel trans‐esophageal coaxial ECG lead. The ECG data were analyzed using automated feature detection software (P3P 4.9) and recommendations are presented for optimizing electrode placement for chronic telemetric monitoring of ECG in rats and mice.
Cyclohexanone contamination in extracorporeal circuits mimics cardiovascular elements of SIRS
Authors: Thompson‐Torgerson Caitlin S. , Champion Hunter C. , Santhanam Lakshmi , Harris Z. Leah , Shoukas Artin A.
Published: June 2021

Extracorporeal circulation provides critical life support but also introduces a host of unique morbidities collectively known as the Systemic Inflammatory Response Syndrome (SIRS), characterized by edema and cardiovascular/pulmonary/renal/neurological dysfunction. We tested the hypothesis that cyclohexanone (CHX), a solvent used in production of extracorporeal circuits, leaches into circuit fluids and can replicate SIRS‐like cardiovascular morbidities. Circuit fluid samples were analyzed with gas chromatography‐mass spectrometry, showing CHX contamination (210‐3694 μg/L). In vivo rat studies were conducted (n=49) to determine the effects of a bolus intravenous CHX infusion (210 μg/l, a conservative clinical exposure) on cardiovascular function, baroreflex responsiveness (arterial pressor response to carotid occlusion), and edema formation (wet:dry ratios of organ weights). CHX infusion led to systemic hypotension; pulmonary hypertension; depressed contractility, heart rate, stroke volume, and cardiac output; and elevated vascular resistance (all p<0.05). The arterial pressor response to carotid occlusion was dampened after CHX infusion (p<0.05). CHX infusion led to higher wet:dry weight ratios (p<0.05). CHX can reproduce SIRS‐like cardiovascular, neurological, and edema sequelae and may operate as a putative trigger of SIRS. (The Joyce Koons Family Cardiac Endowment Fund)
Low dose but not high dose of 17â‐estradiol prevents cardiac fibrosis in DOCA‐salt model of hypertension in rats
Authors: Bruce Erin , Shenoy Vinayak , Grobe Justin L. , Qi Yanfei , Ferreira Anderson J. , Collamat Golda , Katovich Michael
Published: June 2021

The current study was designed to investigate the effects of 17ß‐estradiol (E2) on cardiac remodeling. To address this issue, we evaluated cardiac fibrosis and hypertrophy in deoxycorticosterone acetate (DOCA)‐salt treated rats with chronic, six‐week administration of two different doses of E2. Female SD rats were ovariectomized bilaterally (Ovex). Hypertension was induced by subcutaneous implantation of DOCA‐pellet in uninephrectomized animals, which were later maintained on 1% saline. The rats were randomly assigned to one of the following groups: Ovex control; Ovex + DOCA; Ovex + DOCA + E2 (low dose); or Ovex + DOCA + E2 (high dose). Blood pressure measurement showed a significant increase in DOCA treated rats over a six week period, which was not altered by estrogen replacement. No difference in cardiac function was observed among the different groups when assessed using isolated rat heart Langendorff apparatus. Both doses of estrogen attenuated cardiac hypertrophy observed in the DOCA treated animals. However, when measuring cardiac fibrosis induced by DOCA treatment, only the low dose E2 therapy showed a protective effect. The high dose E2 therapy failed to exhibit anti‐fibrotic effects. These findings suggest that estrogen replacement therapy may have blood‐pressure independent cardio‐protective effects that are dose‐dependent.
Investigation of the Naviculocuboid Joint Reveals a Functional Coalition
Authors: Greiner Thomas M , Ball Kevin A
Published: June 2021

Fourteen cadaveric specimens were prepared by removing soft tissue, so that only ligaments remained to insure joint integrity. Rigid marker clusters were inserted into the tarsal bones. An OptoTrak system was used monitor each specimen through three standardized motions: plantarflexion/dorsiflexion, inversion/eversion and medial/lateral rotation. Results show identifiable rotations occur at the talonavicular and calcaneocuboid joints, especially during the inversion/eversion driving action. However, the rotational motions at these two joints do not synchronize. The implication is that a compensatory motion occurs between the navicular and cuboid. Our investigation of that motion, however, shows that it does not exist. For almost all individuals, no measureable motion could be detected at the naviculocuboid articulation. This suggests that the two bones act as a single functional unit within the foot, essentially acting as if there were an osseous coalition between them. Further research is necessary to identify joint compensations that allow for the independent movement of the medial and lateral midtarsal joints.
Aging and vascular endothelial cell senescence: potential role of SIRT1 in development of inflammation and dysfunction
Authors: Blimline Mark , Seals Douglas , Nelson Keri , Rippe Catarina
Published: June 2021

Aging leads to vascular endothelial dysfunction, but the mechanisms involved are incompletely understood. We assessed the effects of aging on endothelial cell (EC) senescence in vivo and the possible role of the anti‐senescence and anti‐inflammatory deacetylase, SIRT1, in vitro. Compared with young adult (5 mo) mice, old (30 mo) mice had reduced endothelium‐dependent dilation and a higher % of aortic EC that stained for the senescence marker p16INK4a (31 vs. 6). "Aged" cultures of human aortic EC (HAEC) (35±2 population doublings, PDL) had a higher % of cells stained for p16INK4a (46 vs. 7) and (beta)‐galactosidase, a 2nd marker of senescence (57 vs. 6), than "young" cultures (20±1 PDL). SIRT1 expression was 55% lower in senescent HAEC (0.45±0.08 vs. 1.00 ± 0.10 AUs, P<0.002). Senescent HAEC demonstrated reduced expression of the anti‐inflammatory proteins interleukin‐10 (0.6±0.05 vs. 1.0±0.2 AUs, P<0.05) and interleukin‐1 receptor‐α (0.7±0.05 vs. 1.0±0.2 AUs, P<0.05), and increased expression of the inflammatory protein monocyte chemoattractant protein‐1 (1.6±0.1 vs. 1.0±0.1 AUs, P<0.005). In conclusion, the % of senescent arterial EC in vivo is greater in old age. A decrease in SIRT1 may play an important role in EC senescence and contribute to development of vascular endothelial inflammation and dysfunction.

Swedish Research Council; NIH AG006537, AG013038, AG022241, RR00051
A congenic segment (<61kb) containing a single gene from a normotensive rat increases hypertension of the Dahl Salt‐sensitive (S) rat
Authors: Gopalakrishnan Kathirvel , Yerga‐Woolwine Shane , Farms Phyllis , Joe Bina
Published: June 2021

Genetic analysis of rat chromosomes and construction of congenic strains to identify the alleles that are responsible for abnormalities in blood pressure (BP) has lead to mapping of several BP quantitative trait loci (QTLs). Our laboratory has continued to contribute to the identification of several BP QTLs through the analysis of congenic strains of the hypertensive S rat containing segments of the genome from different relatively normotensive rats. Contrary to expectations of lowering BP of the S rat by introgression of congenic segments from normotensive rats, previously a BP QTL was mapped wherein alleles of the LEW rat within a 104kb (70,996,834‐71,100,513bp) region increased the BP of the S rat. Since salt‐sensitivity was an important aspect of the original selection process of the S rat, in this report, we evaluated the salt‐sensitivity of this QTL and determined that the BP increasing effect of this QTL region is independent of dietary salt. The 104kb congenic region contained four predicted genes‐ LOC688779, Lig3, Rffl and Rad51l3, three of which (LOC688779, Lig3 and Rffl) were examined and found not to harbor any exonic single nucleotide polymorphisms (SNPs). Intronic and intergenic SNPs were utilized to further resolve the region to 60,906bp containing a single predicted gene‐ Rffl. To date, 15 SNPs have been detected within the introns of Rffl. At least two alternatively spliced forms of Rffl were identified in both S and S.LEW rats. A detailed analysis of intronic variants of Rffl and other SNPs located within the <61kb QTL region will be presented. This work was supported by NIH Grant HL020176.
Transforming Growth Factor‐β Regulates Valve Interstitial Cell Growth in Vitro
Authors: Li Chen Xi , Gotlieb Avrum I
Published: June 2021

Background

The effect of transforming growth factor beta (TGF‐β) on VIC growth independent of injury is poorly understood. We showed that in the early stages of VIC wound repair TGF‐β promotes VIC proliferation and repair (A. Liu et al. Am J Pathol 2008; 173:1275–1285). In contrast, we found that under normal non‐wound conditions TGF‐β decreases cell number, alters cell morphology, and increases cell adhesion. In this study we set out to investigate the molecular mechanisms responsible for the latter observations.

Materials and Methods

SB431542 is a potent inhibitor of the TGF‐β receptor I. Low density cultures were treated with TGF‐β or SB431542 and their respective controls. The extent of retinoblastoma protein (pRb) phosphorylation and expression of cyclin D1, CDK4, and p27 were compared using Western Blotting.

Results

TGF‐β treatment inhibits VIC proliferation by under‐phosphorylation of p‐Rb at Ser807/811, which could be reversed by receptor signaling inhibition. There is also a concomitant downregulation of cyclin D1/CDK4 and upregulation of their inhibitor, p27. The decrease in cell number caused by exogenous TGF‐β can be abolished by the inhibitor.

Conclusion

TGF‐β regulates VIC growth in vitro by inhibiting proliferation in low‐density monolayer while stimulating proliferation at the wound edge following injury.
Aquaporin‐9 expression in malignant glioma
Authors: Jelen Sabina Katarzyna , Rützler Michael
Published: June 2021

Recent studies described enhanced expression of the water channel aquaporin 9 (AQP9) in biopsies of glioblastoma patients. This may indicate a function for AQP9 in glioma progression as water channels are known to contribute to cell migration. Furthermore, AQP9 is a glycerol and lactate channel and could play a role in the energy metabolism of malignant glioma cells.

Glial tumors usually consist of several cell types, including malignant glial cells, endothelial cells, microglia and other cells of the immune system. Currently, the specific cellular localization of AQP9 has not been determined. The aim of our studies was to determine the cellular expression of AQP9 in glioblastoma biopsies. Furthermore, we are currently investigating the cellular expression of AQP9 in various murine models of glioblastoma.

Human tissue revealed positive AQP9 labeling in a subpopulation of tumor cells. Moreover, we discovered strong immunolabeling of AQP9 co‐localized with the neutrophil marker CD15. Similar to the situation in human tumors, AQP9 was expressed in neutrophils invading murine Gl261 derived tumors. However, Gl261 derived cells did not express AQP9.

We conclude, that AQP9 is expressed in human glioma cells and tumor associated neutrophils. Furthermore, the Gl261 murine model of glioblastoma may be suitable to study a function of AQP9 in tumor associated neutrophils.

Study supported by Lundbeck Foundation
Sodium channel activator‐induced neurite outgrowth is NMDA receptor dependent and involves a TrkB‐PLCγ‐PI3K‐Akt pathway
Authors: Jabba Sairam V , Murray Thomas F
Published: June 2021

Calcium influx through N‐methyl D‐aspartate receptors (NMDAR) regulates signaling cascades that underlie activity‐dependent neuronal development, including the expression and release of BDNF with activation of TrkB pathway. BDNF‐TrkB mediated neuronal development primarily involves three pathways: the PI3K, MAPK and PLCγ systems. Voltage‐gated sodium channel (VGSC) activators promote neuronal development by increasing [Na+]i and upregulating NMDAR function. We examined the effect of the sodium channel activator veratridine (VRT) on neurite outgrowth (NOG) and BDNF‐TrkB signaling in DIV1 cerebrocortical neurons. VRT enhanced NOG and this response was dependent on NMDARs and TrkB signaling. Inhibitors of NMDAR (MK‐801), TrkB (K‐252a), PI3K (LY294002), and PLCγ (U73122) inhibited VRT‐enhanced NOG. VRT increased [Na+]I and [Ca2+]i. VRT‐induced elevation of Ca2+ involved VGSCs, NMDARs and PLCγ sensitive intracellular Ca2+ stores. VRT stimulated phosphorylation of TrkB (Y816: activates PLCγ) and Akt, a downstream effector of the BDNF‐TrkB‐PI3K pathway. VRT‐induced Akt phosphorylation involved VGSC‐NMDAR‐CaMKK‐PI3K signaling. VRT also activated ERK1/2 and CREB: downstream effectors of the MAPK pathway. These data suggest that VRT‐enhanced NOG involves elevation of [Na+]i, and activation of NMDAR‐BDNF‐TrkB signaling with subsequent engagement of downstream PLCγ, PI3K and MAPK pathways.

NIH RO1 NS053398‐06
Biphasic influence of sodium channel activation on neuronal structural plasticity
Authors: George Joju , Murray Thomas F
Published: June 2021

Neuronal activity regulates neuronal development and synaptic plasticity through calcium‐dependent signaling pathways. We used brevetoxin‐2 (PbTx‐2), an activator of voltage‐gated sodium channels (VGSCs), to mimic the influence of neuronal activity. PbTx‐2‐ enhanced neurite outgrowth exhibited a biphasic concentration‐response profile (George et al., 2009) in murine cerebrocortical neurons. N‐methyl D‐aspartate (NMDA) also exhibited a biphasic concentration‐response profile on neurite outgrowth. We therefore hypothesized that the PbTx‐2 enhanced neuronal structural plasticity may be NMDA receptor (NMDAR) dependent. The influence of PbTx‐2 on dendritic arbor complexity and synaptogenesis also followed a biphasic pattern. These data are consistent with the hypothesis that PbTx‐2 enhanced neuronal plasticity involves NMDAR dependent signaling. PbTx‐2 exposure produced concentration‐dependent increments in intracellular [Ca2+] and [Na+]i. Pharmacological studies showed that PbTx‐2‐induced Ca2+ influx primarily involves VGSCs and NMDARs. Moreover, a subthreshold concentration of PbTx‐2 increased the maximum response for NMDA‐induced Ca2+influx. These results suggest that PbTx‐2 enhances NMDAR function. The ability of PbTx‐2 to activate VGSCs with attendant increments in [Na+]i represents a novel strategy to upregulate NMDAR signaling.
Examination of the Neuroprotective Effects of Various Formulations of Curcumin Against Methylmercury induced Toxicity in Human Neuroblastoma (SH‐SY5Y) Cells
Authors: Petroni Daniel H , Mondal Debasis , George William J.
Published: June 2021

Methylmercury (MeHg) has been shown to cause numerous effects including oxidative stress and tau phosphorylation. Curcumin, the active ingredient of tumeric, has been reported to have antioxidant activity. A major limitation to therapeutic use of curcumin is its relatively low bioavailability. Strategies to increase curcumin's bioavailability include encapsulation within a liposome and/or binding to cyclodextrin. This study was conducted to assess the efficacy and potency of 4 forms of curcumin including: liposomal curcumin (LC), cyclodextrinated curcumin (CDC), liposomal cyclodextrinated curcumin (LC‐CDC) and DMSO‐dissolved curcumin (DC) against the toxic effects of MeHg in human neuroblastoma cells (SH‐SY5Y). The minimal concentrations found to produce complete protection against the effects of MeHg for DC, LC and LC‐CDC were 0.92μg/ml, 0.23μg/ml, 0.11μg/ml, respectively (n=9 p<0.001). The above doses of DC, LC and LC‐CDC completely attenuated MeHg induced tau phosphorylation (n=9, p<0.01). CDC did not protect the cells from the effects of MeHg at any tested dose (n=9). Results of this study indicate that curcumin was protective against the effects of MeHg and that LC‐CDC was significantly more potent than other formulations of curcumin. These results support the hypothesis that curcumin may be a useful tool to treat methylmercury induced neurological diseases.
Pentoxifylline exerts a neuroprotective effect on LPS‐injured PC 12 cells
Authors: Benzeroual Kenza E , Muchhala Sameer
Published: June 2021

Inflammatory immune mechanisms play a central role in the pathogenesis of Alzheimer's disease (AD). Recent reports have established the role of TNF‐alpha in neuronal dysfunction. This study evaluated the neuroprotective effects of pentoxifylline, phosphodiesterase E4 inhibitor, in LPS‐treated PC12 cells. Cells were pretreated with varying doses of Pentoxifylline (0.1–1mM) for 2 hours before stimulation with LPS for 48 hours. Cell viability increased with 1 mM pentoxifylline, as measured with MTT assay. Furthermore, western blot analysis showed a dose‐dependent reduction in the expression of TNF‐alpha. In parallel, agarose gel electrophoresis demonstrated that neurotoxicity induced with LPS triggered apoptosis (characterized by DNA fragmentation) and pentoxifylline exerted its protective effect through inhibition of the apoptotic pathway (reduction in DNA fragmentation). This anti‐apoptotic effect of Pentoxifylline was confirmed by the decrease in the expression of pro‐apoptotic protein Bad, the increase in the expression of antiapoptotic protein BcL2 on western blot analysis, and the reduction of caspase 3 levels as measured by caspase‐3 assay. The present study indicates that Pentoxifylline may be a promising approach for the treatment of AD, through inhibition of apoptosis. Study supported by LIU‐College of Pharmacy.
Proteomic Analysis of Changes Mediating Tolerance to Dopamine D1 Agonists: Implications for Parkinson's Disease (PD)
Authors: Boyd Kevin N , VanGuilder Heather D , Freeman Willard M , Mailman Richard B
Published: June 2021

Full dopamine D1 agonists have antiparkinson effects that equal or exceed those of L‐DOPA in PD. No full D1 agonists are currently approved for PD, in part because some prior drugs caused rapid tolerance. Previously, we have shown that tolerance requires constant D1 receptor occupation for >22 h regardless of which D1 agonist was administered. For experimental reasons, the present study focused on the long‐acting drug A77636. We used 2‐DIGE and MS/MS to study the striatal synaptoproteome of three groups of male Sprague‐Dawley rats: 1) control: saline (0 h) + saline (24 h); 2) naïve: saline (0 h) + A77636 (1 mg/kg, 24 h); and 3) tolerant: A77636 (0 hr) + A77636 (24 h). Rats were euthanized 2 h after the last injection. Among other changes, we found a switch from Gαolf‐coupling (predominant in naïve rats) to GαS‐coupling after tolerance. These proteomic results are critical to elucidating the functional changes that mediate tolerance to full D1 agonists and could assist in the development of functionally selective D1 agonists that do not cause tolerance. In addition, these data may be critical in predicting the consequences of using D1 agonists constantly to improve function mediated by the prefrontal cortex and/or hippocampus.
Identification of a novel small molecule that targets the extracellular regulated kinase‐2 (ERK2) FXFP motif docking site and inhibits substrate phosphorylation and cell proliferation
Authors: Zhang Jun , Burkhard Kim , Nevels Kerrick , St John Franz , MacKerell Alexander , Shapiro Paul , Pozharski Edwin
Published: June 2021

Activation of the ERK/ MAPK signaling pathway is involved in a broad range of cellular processes such as growth, differentiation, death, metabolism, and stress response et al. ERK1/2 proteins play the central role in this signaling pathway by integrating a variety of extracellular signals and distributing the integrated signals to different downstream tiers. ERK1/2 proteins exert their biological activities by phosphorylating and regulating dozens of substrates. The efficiency and specificity of substrate phosphorylation by ERK1/2 are largely dependent on docking domains that facilitate interactions with substrates containing unique binding motifs. To screen for small molecule compounds that selectively manipulate ERK interactions with substrates containing distinct binding motifs, computer aided drug design (CADD) was used to predict small molecules that bind to ERK2 on regions involved in substrate interactions. One compound (2.3.2) was identified and showed high nanomolar binding affinity to ERK2. Compound 2.3.2 selectively inhibited phosphorylation of the DEF motif ERK substrate ELK‐1 as compared to the DEJL motif substrate RSK‐1 and reduced HeLa cell proliferation (IC50~ 1μM). We further co‐crystalized 2.3.2 with ERK2 and solved the structure at 2.2 Å by x‐ray crystallography. The crystal structure confirmed that 2.3.2 binds to ERK2 in the region involved in DEF motif substrate interactions. These findings demonstrate the identification and use of novel small molecules to selectively disrupt ERK activation of substrates involved in cell proliferation.
Molecular Mechanisms Regulating Drug Metabolizing Enzyme Ontogeny
Authors: Hines Ronald N , Shadley Jeffery D , Giebel Nicholas , Le Min , Dorko Kenneth , Strom Stephen C
Published: June 2021

The study objective was to evaluate changes in histone marks and the associated changes in chromatin structure as a mechanism controlling drug metabolizing enzyme ontogeny. The relative enrichment of histone marks was determined using chromatin immunoprecipitation with pooled chromatin isolated from 1st trimester primary human fetal or adult hepatocytes with quantitation by RT‐PCR. CYP3A4 expression increases at least 20‐fold between the third trimester and the adult (mean ± SD = 4.5 ± 2.1 versus 89.6 ± 64.0 pmol/mg microsomal protein, respectively). Evaluating the CYP3A4 CLEM4 domain located from position −11,420 to −10,883 and containing 3 USF1, 2 HNF4α, 1 HNF1α, and 1 AP1 elements revealed a 6‐ to 14‐fold enrichment of the H3K27me3 histone mark in fetal versus adult hepatocytes. In contrast, a 1.5‐ to 3‐fold enrichment of the H3K4me1 histone mark was observed in adult versus fetal hepatocytes. A 100‐fold difference in USF1 enrichment was observed in adult versus fetal hepatocytes. Similar results were observed for the presence of these same histone marks and the binding of C/EBPβ in the regulatory domain located between CYP3A4 position −5900 and −5700. Given the association of H3K27 and HeK4me1 with condensed and open chromatin, respectively, tThese data are consistent with changes in chromatin structure having a dominant role in regulating CYP3A4 ontogeny. Supported in part by PHS grant GM081344.
Cobalt chloride induces the reduction of fibroblast growth factor 21 in Caco‐2 cells through HIF and PPAR‐α and –γ pathway
Authors: Liu Yanlong , Wang Yuhua , McClain Craig , Feng Wenke
Published: June 2021

Fibroblast growth factor 21 (FGF21) has emerged as an important metabolic regulator of glucose and lipid metabolism. Previously, FGF21 present in the adipose tissue regulates lipolysis, and in the liver increases fatty acid oxidation. FGF21 also is present in the intestine. Little is known about FGF21 in intestine. Intestinal epithelial cells are positioned between an anaerobic lumen and a highly metabolic lamina propria, which has a steep physiologic oxygen gradient. Physiological and pathological hypoxia induces the hypoxia‐inducible factors (HIF). We evaluated how the hypoxic state regulates FGF21 in intestine‐like Caco‐2 cells. Caco‐2 cells were treated with cobalt chloride, a chemical hypoxia mimetic. Cobalt chloride treatment decreased mRNA levels of FGF21, but increased mRNA levels of Glut‐1, a glucose transporter transcriptionally regulated by HIF, in a time dependent manner. Inhibition of HIF and transfection of a dominant negative HIF‐1α plasmid eliminated this effect. Further studies showed that cobalt chloride treatment reduced the mRNA levels of PPAR‐α and –γ, of which HIF is a negative regulator. In addition, cobalt chloride treatment decreases the mRNA levels of FGF21 in mouse colon. Hypoxia reduced FGF21 is, at least in part, mediated via HIF and PPAR–α and –γ pathway. Further study is needed to determine the intestinal metabolic change regulated by FGF‐21 under hypoxic conditions.
Acrolein‐induced hepatotoxicity: role of mitochondrial dysfunction and endoplasmic reticulum stress
Authors: Joshi‐Barve Swati , Avila Diana , Zhang Jingwen , Barve Shirish , McClain Craig
Published: June 2021

Background

Acrolein is a common environmental, food and water pollutant and a major component of cigarette smoke. It is produced endogenously by the metabolism of several drugs and amino acids and has also been identified as both a product and initiator of lipid peroxidation. Acrolein is known to be cytotoxic to many cell types including hepatocytes; however the underlying molecular mechanisms are not fully understood.

Methods

Human hepatic cells (HepG2) were used to measure acrolein‐induced hepatotoxicity, and to study endoplasmic reticulum stress (ERS) and mitochondrial death pathways and the induction of apoptosis.

Results and Conclusions

Acrolein exposure caused a dose‐dependent decrease in intracellular antioxidant glutathione and a loss of survival of hepatic cells. Cell death was apoptotic at moderate and necrotic at higher cytotoxic concentrations of acrolein. Additionally, acrolein disrupted mitochondrial integrity/function as seen by fluorescence microscopy and ATP depletion. Acrolein activated ERS proteins PERK, eIF2α and ATF3, 4, 6, and also activated the kinases JNK and p38 MAPK. We postulate that acrolein induced disruption of both mitochondria and ER contributes to hepatotoxicity. Our study defines basic mechanisms involved in liver injury due to oxidative stress molecules and reactive aldehyde pollutants such as acrolein. This study was funded by NIH and VA.
Proanthocyanidin protects intestine and remote organs against mesenteric ischemia/reperfusion injury
Authors: Sizlan Ali , Guven Ahmet , Uysal Bulent , Yanarates Omer , Atim Abdulkadir , Oztas Emin , Cosar Ahmet , Korkmaz Ahmet
Published: June 2021

BACKGROUND

Intestinal ischemia/reperfusion (IR) induces a systemic inflammatory response and releases harmful substances that may affect the function and integrity of distant organs such as lung, liver, and kidney. We conducted this study to find out if proanthocyanidins (PA) has protective effects against mesenteric IR injury and mesenteric IR‐induced intestinal and distant organ injury.

MATERIALS AND METHODS

Thirty‐two Sprague‐Dawley rats were divided into four groups: control, control + PA, IR, IR + PA. The IR and IR + PA groups were subjected to mesenteric arterial ischemia for 60 min and reperfusion for 6 h. The Control + PA and IR + PA groups were administered PA (100 mg/kg/day via oral gavage) for 7 days prior to injury insult. We collected ileal and distant organ tissues, such as pulmonary, hepatic, and kidney specimens to measure tissue levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GPx), and nitrite plus nitrate (NO(x)), and we then evaluated histological changes.

RESULTS

In the IR group, significant increases in MDA and NO(x) levels and significant increases in SOD and GPx activities of intestine, liver, kidney, and lung were observed. The MDA and NO(x) levels were significantly lower, as were the SOD and GPx activities in the IR + PA group than that in the IR group. Although the intestine and distant organs damage scores were significantly higher in the IR group, these injuries were prevented by PA in the IR + PA group.

CONCLUSIONS

This study demonstrates that PA has a significant effect in the protection of the intestine and the remote organs against mesenteric IR injury.
Potential involvement of actin cytoskeleton reorganization in generation of the arterial myogenic response
Authors: El‐Yazbi Ahmed Fawzy , Walsh Emma , Walsh Michael , Cole William
Published: June 2021

The myogenic response of resistance arteries to intravascular pressure is due to cellular processes intrinsic to vascular smooth muscle cells. Myogenic constriction of cerebral arteries involves the activation and inhibition of myosin light chain kinase and phosphatase, respectively, to increase myosin light chain phosphorylation (pLC20) and permit cross‐bridge cycling. However, equivalent levels of pLC20 are detected at 80 mmHg when pressurized vessels flash‐frozen prior to or after spontaneous development of myogenic tone during the equilibration period are compared. Also, myogenic constriction evoked by a pressure step to 80 mmHg is observed following a maximal increase in pLC20 (~50%) due to 5‐HT stimulation, but there is no further increase in pLC20, or change in p‐caldesmon or p‐calponin. Latrunculin B (10 μM), that binds free G‐actin, had no effect on constriction due to 5‐HT or 80 mmHg alone, but it reversed contractions due to the combination of 5‐HT and 80 mmHg, or pressure alone at 120 mmHg, that are associated with maximal levels of pLC20. Our findings indicate that re‐establishment of myofilament‐actin cytoskeleton contacts during equilibration is necessary for myogenic constriction, and dynamic reorganization of the cytoskeleton may be required to faciltiate increased force generation in the myogenic response when peak levels of pLC20 are attained. (Supported by CIHR MOP‐97988 and AIHS).
Dantrolene and nimodipine inhibits vasoconstriction of basilar artery in a rat subarachnoid hemorrhage model
Authors: Soydan Guray , Xu Jian , Sims John Randall
Published: June 2021

Cerebral vasoconstriction is associated with increased cytosolic Ca+2 concentration in vascular smooth muscle, presumably due to Ca+2influx and Ca+2release from intracellular stores. We have previously shown that dantrolene has synergistic effects with nimodipine against serotonin (5‐HT)‐induced vasoconstriction in rat isolated cerebral arteries. We aimed to investigate the effects of dantrolene and nimodipine in a rat subarachnoid hemorrhage (SAH) model. Using an infusion pump 400μl blood or saline was given with intracysternal injection to Sprague‐Dawley rats. After 48 hours brain was removed and basilar artery was isolated and mounted into a wire myograph. Vasoconstriction of basilar artery was measured while exposed to KCl and 5‐HT in the presence or absence of dantrolene (10, 100μM) and/or nimodipine (30μM). Compared to saline group, 5‐HT‐induced vasoconstriction increased in SAH. Dantrolene significantly inhibited the constriction to 5‐HT whereas it was not affected the constriction to KCl. Nimodipine inhibited both KCl and 5‐HT constrictions significantly. When given together with dantrolene, nimodipine did not show an additional inhibition in KCl and 5‐HT‐induced vasoconstrictor responses. Our results suggest that dantrolene and nimodipine inhibit 5‐HT‐induced vasoconstriction but they do not have a synergistic effect in basilar artery of a SAH model in rat.
Hyperbaric oxygenation reduces exercising forearm blood flow in humans
Authors: Casey Darren P. , Joyner Michael J. , Claus Paul L. , Bigelow Maureen L. , Fuqua William R. , Balgeman Jon R. , Curry Timothy B.
Published: June 2021

Hypoxia during exercise augments vasodilation and blood flow to ensure adequate delivery of O2 to active muscles. However, the impact of hyperoxia on skeletal muscle blood flow during exercise is poorly understood. Therefore, we tested the hypothesis that the hyperemic response to exercise with hyperbaric hyperoxia would be blunted compared to exercise during normoxia. Seven subjects (6M/1F; 26 ± 2 years) performed forearm exercise (20% of maximum) under normoxic and hyperoxic conditions. Forearm blood flow (FBF; ml/min) was measured using Doppler ultrasound. Forearm vascular conductance (FVC; ml/min/100mmHg) was calculated from FBF and blood pressure (mmHg; brachial arterial catheter). Studies were performed in a hyperbaric chamber with the subjects supine at 1 ATA (sea level) and 2.8 ATA while breathing normoxic (21% O2, 1 ATA; FIO2 ≈ 150 mmHg), hyperbaric normoxic (7% O2, 2.8 ATA, FIO2 ≈ 150 mmHg) and hyperoxic (7% O2, 2.8 ATA, FIO2 ≈ 2100 mmHg) gas. The change in FBF and FVC (Δ from rest) during exercise under normoxia and hyperbaric normoxia did not differ (P > 0.05). However, the ΔFBF and ΔFVC during exercise under hyperoxia were attenuated by ~18–29% compared to both normoxic conditions (P ≤ 0.01). Our data suggest that exercise hyperemia in skeletal muscle is highly dependent on oxygen availability during hyperoxia as well as hypoxia.

NIH HL46493 (MJJ), AR55819 (DPC) and RR‐024150.
Dissociation between Changes in Metabolism and Blood Flow During Coronary Artery Stenosis
Authors: Kudej Raymond K. , Zhao Xin , Vatner Dorothy E. , Vatner Stephen F. , Lewandowski E. Douglas
Published: June 2021

We previously reported that the oxidation of short chain fatty acids displaced that of long chain fatty acids (LCFA) similarly in subendocardium (ENDO) and subepicardium (EPI) layers during coronary artery stenosis, where blood flow fell significantly in ENDO but was preserved in EPI. The goal of this study was to determine if this transmural dissociation between metabolism and flow was a more generalized paradigm. Accordingly, we studied LCFA oxidation by infusing 13C palmitate intracoronary to 5 control conscious, chronically instrumented pigs and to 5 pigs with sublethal left anterior descending coronary artery stenosis (STEN). STEN reduced coronary blood flow by 40%, but reduced tissue blood flow, measured with microspheres, by 53% (p<0.05) in ENDO and not significantly, by 16% in EPI. 13C NMR of tissue extracts demonstrated similarly decreased contributions of palmitate to mitochondrial oxidation, via acetyl CoA production, in ENDO vs EPI during STEN (13% vs. 17%). STEN also induced similar increases in glycolytic end products between ENDO and EPI: Lactate increased from 5.7 to 17.1 μmol/g in ENDO and from 5.0 to 12.5 in EPI; Alanine increased from 4.1 to 9.9 μmol/g in ENDO and from 4.4 to 8.4 in EPI. Thus, the findings in ENDO are consistent with the ischemia induced by STEN, but were unexpected in EPI, where tissue blood flow was preserved, suggesting a novel mechanism for transmural metabolic cross talk.
Hawthorn extract increases coronary flow in the isolated perfused rat heart
Authors: Frey Aaron D , Geisbuhler Timothy P.
Published: June 2021

Hawthorn extract (Crataegus monogyna leaves/flowers) is used in Europe as an alternative therapy to treat cardiovascular diseases Previous studies performed in this laboratory have shown that hawthorn extract suspended in dimethyl sulfoxide (DMSO) causes coronary vasodilation, followed by a reduction in coronary flow to nearly zero. In the present study, we sought to examine whether hawthorn extract suspended in water (aqueous hawthorn) would still cause coronary vasodilation without the late phase decrease in flow observed using the DMSO‐suspended product. We used isolated perfused rat hearts (Langendorff) and an in line flow sensor to measure changes in coronary flow. Hawthorn extract was suspended in water, centrifuged at 13,000×g, and the supernatant added to the heart preparation. We found that hawthorn extract increased coronary flow in a dose‐dependent manner. Maximum vasodilation was achieved at a lower dose of aqueous hawthorn than that required for the DMSO‐suspended product. Further, the late‐phase decrease in flow observed with DMSO‐suspended hawthorn extract was not observed with the aqueous suspension. We conclude that hawthorn extract suspended in water is an effective coronary vasodilator, and is a more realistic preparation for vasodilation than the DMSO‐suspended extract. [Supported by the ATSU Biomedical Research Fund; extract supplied by Willmar Schwabe GMbH, Karlsruhe, GER].
Differential regulation of vascular growth through cGMP/PK‐G/PK‐A signaling
Authors: Joshi Chintamani N , Martin Danielle N , Fox Jonathan C , Adderley Shaquria P , Tulis David A
Published: June 2021

Vascular smooth muscle (VSM) proliferation and migration are vital components in vessel remodeling. Cyclic nucleotide signaling in cardiovascular tissues is protective and serves as a therapeutic target. The present work analyzed the effects of the recently‐described soluble guanylate cyclase stimulator BAY 41‐2272 (BAY) on vascular growth using a balloon injury model and primary VSM cells. In rat carotid arteries, perivascular application of BAY post‐injury reduced neointimal growth by ~ 40% compared to controls after 2 weeks. In rat primary VSM cells, cyclic GMP content peaked (248x) 20 min after BAY treatment and remained elevated (140x) through 60 min. BAY did not, however, affect cyclic AMP levels or cell viability. In‐Cell Western analyses showed increases in VASP phosphorylation (p‐VASP) at Ser 239 (3x) and Ser 157 (2x), respective markers of cyclic GMP‐ and cyclic AMP‐directed protein kinases. The selective PK‐G inhibitor DT‐2 reversed the BAY‐mediated increase in p‐VASPSer 239. BAY (10μM) reduced proliferation by 40% (p<0.05) after 72 h, which was reversed by PKI only. BAY also inhibited PDGF‐induced VSM cell migration by 40% (p<0.001) after 6 h which was completely inhibited by DT‐2. These findings provide direct evidence for PK‐G‐specific anti‐migratory and PK‐A specific anti‐proliferative actions of BAY in VSM, supporting its therapeutic potential in vasoproliferative disorders.
Novel insights into the role of glucose metabolism in regulating vascular smooth muscle cell phenotype and proliferative capacity
Authors: Salabei Joshua Kuimeta , Bhatnagar Aruni , Hill Bradford G
Published: June 2021

Abnormal vascular smooth muscle cell (VSMC) proliferation is a hallmark in the pathogenesis of vascular disease. Atherosclerosis or arterial damage due to angioplasty results in exposure of medial smooth muscle cells to growth factors such as platelet‐derived growth factor‐BB (PDGF‐BB); these growth factors promote a cellular phenotype switch from a contractile phenotype to a proliferative, synthetic phenotype. However, the mechanisms by which growth factors cause this phenotype switch remain unclear. We hypothesized that glucose metabolism is a primary regulator of PDGF‐BB‐induced VSMC phenotype and proliferative capacity. Treatment of VSMCs with PDGF‐BB (10 ng/ml) significantly increased glycolytic flux, cell proliferation, and the expression of osteopontin and vimentin—both of which are markers of the synthetic VSMC phenotype. Inhibition of glycolysis with koningic acid, an irreversible inhibitor of GAPDH, did not cause cell death, but decreased proliferation and prevented the upregulation of osteopontin and vimentin. Koningic acid treatment significantly increased the levels of proteins modified by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) but did not affect PDGF‐BB‐induced phosphorylation of Erk1/2 or Akt. Treatment of VSMCs with inhibitors of the O‐GlcNAcase enzyme, PUGNAc or Thiamet‐G, increased protein O‐GlcNAcylation and maintained cells in a quiescent, contractile phenotype despite their stimulation with PDGF‐BB. These data suggest that glucose metabolism regulates cell growth and that the hexosamine biosynthetic pathway is fundamental for maintaining a quiescent phenotype. Understanding how cellular energetics integrate with glucose‐derived metabolic signaling should aid in developing therapeutic strategies to prevent VSMC hyperproliferation, especially in the context of metabolic diseases such as diabetes.
Rab38 regulates lamellar body size and number in lung alveolar type II cells
Authors: Zhang Linghui , Yu Kevin , Roberts Kyle , Hong Nankang , DeBolt Kristine , Tao Jianqin , Huang Shaohui
Published: June 2021

The lung‐restricted small GTPase Rab38 has recently been shown to play key roles in surfactant trafficking and homeostasis in alveolar type II (ATII) cells. In this study, we found significantly enlarged but reduced number of lamellar bodies (LBs) in ATII cells isolated from Fawn‐hooded hypertension (FHH) rats containing a Rab38‐null mutation. This is accompanied by simplification of alveolar structures and thickening of lung small artery walls even in 10‐week‐old FHH rats. Interestingly, Rab38 expression is restricted to epithelium but not endothelium cells, and declines rapidly upon ATII cell culture. Transiently expressed EGFP‐Rab38, even at minimum expression level indicated by EGFP fluorescence close to background signals, consistently labels the limiting membranes of ~30% LBs in ATII cells isolated from WT and FHH rats. Such localization is abolished by point mutations (G19V & T23N) to the GTPase, suggesting the importance of an intact prenylation mechanism and GTPase activity. Consistently, we found endogenous Rab38 in the LB fraction isolated from the WT but not FHH rats. Importantly, reintroducing EGFP‐Rab38 back to the FHH ATII cells rescues the enlarged LB phenotype. Supported by NIH 5T32HL007748 and P01HL19737.
Morphologycal and morphometric analysis of Sural nerve in newly weaned spontaneously hypertensive rat (SHR) compared with normotensive Wistar Kyoto rat (WKY)
Authors: Sanada Luciana Sayuri , Salgado Hélio César , Fazan Valéria Sassoli
Published: June 2021

Previous data from our laboratory showed that the sural nerve from 20 weeks old spontaneously hypertensive rat (SHR) present alterations of myelinated fibers and endoneural vessels. To investigate whether these differences were caused by hypertension or were due to strains differences, the aim of the present study was to compare the aspects of the sural nerve from newly weaned SHR, which had not developed hypertension, with WKY. Rats aged 5 weeks (N=6 in each group) were used. After arterial pressure and heart rate recordings, right and left sural nerves were removed and prepared for light microscopy. Morphometric analysis was performed with the aid of computer software. Arterial pressure levels were similar between SHR and WKY. There were no differences in morphological and morphometric parameters of fascicular area, myelinated fiber number and density in both strains. Nevertheless, the morphometric parameters of myelinated fibers were different between SHR and WKY, despite no alterations on the endoneural vascularization in both strains. These results are suggestive of intrinsic differences on the myelinated fibers between SHR and WKY even before the development hypertension.