bluuebunny/crossref_metadata_2025_split huggingface dataset:
Immunohistochemical studies of Vascular endothelial growth factor and tumor suppressor protein P53 in mammary tumor of mice under the effect of green tea
Authors: Amin Afaf Mosad Amin 
Published: June 2021

This work aimed to study the effect of green tea extract on transplanted mammary tumor in Swiss mice through studying VEGF & P53. Male Swiss mice weighing 25–30 g were used in this work. They were divided into 3 equal groups 12 animals in each group. The first group was injected i.m with 1/106 MT cells/0.1 ml saline solution/mouse for 2 weeks. The second group was injected with tumor cells as in the 1st group and kept on 5mg green tea extract in 0.2ml dist .water/mouse via gavage for 2 weeks. The third group received 5mg GT extract in 0.2 ml distilled water/mouse daily for two weeks before tumor injection. Six animals from each group were sacrificed after 1 and 2 weeks from tumor injection. The tumor was measured by a caliper and its size was calculated using the formula: 3/4Π r3. The tumor specimens were processed for immunohistochemical studies of VEGF and P53.The results of the present study showed that the angiogenic growth factor VEGF, the key signal used by oxygen deprived cells promote the growth of blood vessels which increase the ability of the mammary tumor to grow. Our observations have raised the possibility that animals received GT extract before tumor injection showed remarkable reduction in the size of the tumor. As regards P53, a tumor suppressor protein. Immunohistochemical stained sections of MT using monoclonal P53 antibody revealed its low expression in tumor cells and its over expression after application of GT extract suggesting its important role to arrest the cell cycle. This ceasing of the cell cycle forced the cell to self destruction by inducing apoptosis through p53 dependent pathway.
The SWIM domain of MEKK1 is required for complex formation with Jun that controls Fra‐2 ubiquitination
Authors: Cuevas Bruce Daniel , Duellman Tyler 
Published: June 2021

The MAPK kinase kinase (MKKK) MEKK1 is unique among kinases in that it contains E3 ubiquitin ligase activity. MEKK1 has been reported to mediate ubiquitination of c‐Jun, thus requiring MEKK1/Jun complex formation, but the nature of that complex has not been described. The objective of this study was to determine if Jun forms a complex with MEKK1, and if so, characterize that interaction. Here we report that MEKK1 directly associates with the DNA‐binding region (DBD) of Jun, and this complex requires the MEKK1 SWIM domain, the function of which was previously unknown. We also demonstrate that direct interaction between the SWIM domain and AP‐1 proteins is limited to Jun proteins, and this requires amino acids conserved in the DBD of Jun, but not Fos proteins. Finally, we demonstrate that the Fos family protein Fra‐2 associates with the MEKK1 complex via Jun, and that Jun is required for MEKK1‐dependent Fra‐2 ubiquitination. Altogether, our data show that, in addition to regulating AP‐1 phosphorylation through control of MAPK activity, MEKK1, Jun, and Fra‐2 form a multi‐protein complex that regulates Fra‐2 ubiquitination.This work was supported by a grant from the National Institutes of Health.
Butyrate uptake and effect on proliferation, differentiation, viability and apoptosis of Caco‐2 cells: influence of polyphenols
Authors: Gonçalves Pedro , Araújo João JR , Martel Fátima 
Published: June 2021

The short‐chain fatty acid butyrate (BT) is one of the main end‐products of anaerobic bacterial fermentation of dietary fibre within the human colon. Among its recognized effects, BT is known to inhibit colon carcinogenesis. Polyphenols have several properties, including anti‐oxidant, anti‐carcinogenic and anti‐inflammatory activity. The aim of this study was to investigate the influence of some polyphenols upon the apical uptake of BT into human colonic adenocarcinoma Caco‐2 cells, and to evaluate their influence upon the effect of BT on proliferation, differentiation, viability and apoptosis.In the first part of the study, we demonstrated that MCT1‐mediated apical uptake of 14C‐BT in Caco‐2 cells is modulated by either acute or chronic exposure to some polyphenols, and that resveratrol acts as a competitive inhibitor of 14C‐BT uptake. In the second part of the study, we evaluated the effect of those polyphenols that chronically increased the apical uptake of 14C‐BT, alone or together with BT, on cell viability, cell proliferation, differentiation and apoptosis. In general, combination of polyphenols with BT did not significantly modify the changes in proliferation, differentiation, viability and apoptosis induced by BT alone. In conclusion, changes in uptake of BT induced by polyphenols do not correlate with changes on cell viability, cell proliferation, differentiation and apoptosis.
Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4‐(4‐Chlorophenyl)imidazole at 2.0 Å Resolution
Authors: Gay Sean C. , Shah Manish B. , Talakad Jyothi C. , Maekawa Keiko , Roberts Arthur G. , Wilderman P. Ross , Sun Ling , Yang Jane Y. , Huelga Stephanie C. , Zhang Qinghai , Stout C. David , Halpert James R. 
Published: June 2021

The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4‐(4‐chlorophenyl)imidazole has been determined using x‐ray crystallography to 2.0 Å resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6‐4‐CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. Based on the new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen‐bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine and clopidogrel to bind to the 2B6 active site in the expected orientation. However, the inability to dock the larger ligands raloxifene and itraconazole in favorable conformations suggests that 2B6 undergoes some degree of structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.NIH grants ES003619, ES006676
Chocolate Milk Consumption During Recovery from Endurance Exercise Affects Intracellular Events of Skeletal Muscle Protein Synthesis and Proteolysis
Authors: Lunn William R , Colletto Megan R , Karfonta Kirstin E , Anderson Jeffrey M , Carbone John W , Rodriguez Nancy R 
Published: June 2021

PURPOSEThe aim was to determine effects of fat‐free chocolate milk consumption during recovery from endurance exercise on intracellular proteins (ICP) influencing skeletal muscle synthesis and proteolysis.METHODS8 active males (23.7 ± 1.6 y, 76.0 ± 3.8 kg, BMI = 24±1, 14.4 ± 2.0% body fat, VO2peak = 53.1 ± 1.6) participated in a randomized, crossover‐design study during which they consumed a eucaloric diet for 2wks. Protein intake was 1.5 g·kg−1. After each week, participants consumed 16 oz. of either fat‐free chocolate milk (MILK) or a non‐nitrogenous, isocaloric control beverage (CON) after a 45‐min run at 65% of VO2peak. Protein expression of PI3K and relative phosphorylation of eEF2, mTOR, eIF4E‐BP1, and rpS6and relative phosphorylation of Akt and FOXO3a, total protein expression of ubiquitin and PSMA2, and activity of caspase‐3 and the 26S proteasome were quantified for protein synthesis and proteolysis, respectively, from muscle biopsied during 3 h of recovery.RESULTSSignificant results for 0 to 3 h recovery (p<0.05): FOXO3a (Ser253) decreased (1.39 ± 0.14 to 0.98 ± 0.10 AU) and proteasome core activity increased (1.79 ± 0.22 to 2.30 ± 0.19 RFU) for CON vs MILK. Caspase‐3 activity decreased for MILK (3.71 ± 0.61 to 1.68 ± 0.37 AU). Akt (Ser473) was reduced for MILK and CON (0.76 ± 0.13 to 0.27 ± 0.08 AU and 0.72 ± 0.21 to 0.29 ± 0.12 AU, respectively). eIF4E‐BP1 (Thr37/46) increased for MILK (0.63 ± 0.03 to 0.88 ± 0.08 AU).CONCLUSIONFindings suggest that drinking fat‐free chocolate milk after endurance exercise attenuates ICP activity specific to proteolysis and enhances that of protein synthesis during recovery.
Amino Acid Transporter Expression is Increased in Human Skeletal Muscle Following Essential Amino Acid Ingestion
Authors: Drummond Micah J , Glynn Erin L , Fry Christopher S , Volpi Elena , Rasmussen Blake B 
Published: June 2021

Essential amino acids (EAA) stimulate skeletal muscle mTORC1 signaling and protein synthesis in humans. It has recently been reported that amino acid transporters may play a role in the regulation of mTORC1 signaling and translation initiation. The purpose of this study was to profile the expression of several amino acid transporters in human skeletal muscle before and after the ingestion of EAA. Muscle biopsies were obtained from the vastus lateralis of 7 young adult subjects (3 male; 4 female) before and 1–3h after ingesting 10 grams of EAA. Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques and qRT‐PCR was used to determine mRNA expression for L‐type (LAT1/SLC7A5, CD98/SLC34A2, LAT3/SLC7A7), sodium‐coupled neutral (SNAT2/SLC38A2 and SNAT4/SLC38A4), proton‐assisted (PAT1/SLC36A1 and PAT2/SLC36A2), cationic (CAT2/SLC7A2), and excitatory (EAAT1/SLC1A3) amino acid transporters. EAA ingestion increased blood leucine concentration, delivery to muscle, transport into muscle, muscle concentration and muscle protein synthesis in association with an increase in LAT1, CD98, SNAT2, and PAT1 expression during the first hour post‐ingestion (P<0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle amino acid transporter expression which likely plays a key role in stimulating anabolic signaling and muscle protein synthesis.Supported by NIA P30 AG024832 (MJD) & NIAMS R01 AR049877 (BBR)
Systemic Clearance of Fluorescent Dextrans after Hemorrhage in Rats
Authors: Carvalho Helena , Woodruff Aleesha R , Simmons Kristin L , Pittman Roland N 
Published: June 2021

An intact endothelial glycocalyx is associated with vascular homeostasis, and its disruption leads to increased vascular permeability. We evaluated the systemic clearance of macromolecules from the circulation after hemorrhage. We injected a dextran mixture (0.5ml of 1.2 mg/ml FITC‐labelled 500 kDa dextran (Dex‐500) and 0.5 ml of 2.5 mg/ml Texas Red‐labelled 40 kDa dextran (Dex‐40)) i.v. in anesthetized Sprague‐Dawley rats (250–350 g). Hemorrhage was induced by withdrawing 40% of blood volume over a 30 min period. After 30 min of stabilization at the end of hemorrhage, rats received the dextran mixture (1 ml/300 g rat). Blood samples were collected at 1, 5, 10, 15, 20 and 30 min and then centrifuged. Plasma fluorescence was analyzed in a luminescence spectrometer at 490/535 nm (ex/em) for Dex‐500 and 595/615 nm (ex/em) for Dex‐40. Hemorrhage led to a drop in arterial pressure from 110 ± 10 mmHg to 50 ± 8 mmHg. The clearance rate of Dex‐40 increased by 33% and Dex‐500 by 9% compared with the control group. Under control conditions Dex‐40/Dex‐500 clearance ratio was 1.36 and increased to 1.66 after hemorrhage; thus, the permeability to Dex‐40 increased more than the permeability to Dex‐500 following hemorrhage. The increased clearance rates of these macromolecules are consistent with degradation of the glycocalyx following hemorrhage, compared to control, based on its role as a permeability barrier to macromolecules.Support: HL079087 from NHLBI
Role of endothelial surface layer in mediation of flow‐induced dilation of isolated arterioles
Authors: Marki Alex , Gara Edit , Veresh Zoltan , Seress Laszlo , Toth Peter , Koller Akos 
Published: June 2021

Vascular endothelial cells respond to increases in wall shear stress (WSS) by releasing vasoactive factors. However, it is less known how WSS signal is sensed by the endothelial cells. Recent studies implied that the endothelial surface layer (ESL) senses changes in WSS and transmits the signal towards intracellular enzymes responsible for producing these factors. We hypothesized that degradation of ESL inhibits flow‐induced dilation of arterioles. Thus the effect of step‐wise increases in intraluminal flow were investigated on the diameter of isolated gracilis muscle arterioles of rats (passive diameter ~200μm, active diameter ~150μm) in the absence and presence of intraluminal hyaluronidase (1.5 and 15 U/ml, for 30 minutes) known to degrade hyaluronic acid of the ESL. In control conditions acetylcholine (ACh) and flow elicited dilations (20±6% and 20±2%, respectively). Presence of 1.5 U/ml hyaluronidase reduced ACh‐induced dilations, but did not affect flow‐induced responses. In contrast, 15 U/ml hyaluronidase elicited significant reduction of both Ach‐ and flow‐induced dilations (to 7±2% and to 2±1%), whereas vasomotor responses of smooth muscle remain intact. These results provide further support for the potential role of the ESL in sensing and transmitting changes in WSS.(Grants: Hungarian Nat. Sci. Res. Fund.‐OTKA K71591, K67984, AHA Founders Aff., 0855910D)
ROLE OF ENDOTHELIAL CANONICAL TRANSIENT RECEPTOR POTENTIAL (TRPC) CHANNELS IN ATP‐MEDIATED VASORELAXATION
Authors: Marrelli Sean P , Kochukov Mikhail Y , Noel Rebecca C , Mbaka Maryann M , Chen Jie 
Published: June 2021

Adenosine triphosphate (ATP) is a physiological agonist that is released from multiple sources in the vascular system including red blood cells, endothelial cells, and vascular smooth muscle cells. ATP can elicit an endothelium‐dependent vasorelaxation, however, the mechanism is still unresolved. The present study examines the role of TRPC channels in the mechanism of ATP‐mediated vasorelaxation.Experiments were performed with mouse aorta and aorta endothelial cells. Aorta contractions and relaxations were measured by artery myography. Freshly isolated endothelial cells were used for the measurement of endothelial calcium by fluorescent dye (Fura 2) and non‐selective cation currents by whole cell patch clamp.ATP produced a concentration‐dependent (EC50 = 2 μM) relaxation in phenylephrine (3 μM) pre‐contracted arteries. In isolated endothelial cells, ATP (10 to 100 μM) stimulated a non‐selective cation current (IATP) that was inhibited by La3+ or Gd3+ (each 100 μM). ATP also produced an increase in endothelial calcium that was inhibited by La3+ (100 μM) or 2‐APB (100 μM). In summary, we present evidence that ATP stimulates vasorelaxation subsequent to endothelial cell calcium entry via TRPC‐like channels. Further studies with genetic mouse models will test the roles of specific TRPC channels in the mechanism.Supported by NIH R01 HL088435 to SPM.
O‐GlcNAc signaling regulates cardiac hypertrophy via NFAT activation
Authors: di Tarso Fernandes Facundo Heberty , Eli Brainard Robert , Jones Steven Paul 
Published: June 2021

Calcineurin regulates the dephosphorylation and nuclear translocation of the cardiac hypertophy transcription factor, Nuclear Factor of Activated T‐cells (NFAT). O‐linked beta‐N‐acetyl glucosamine (O‐GlcNAc) has emerged as a highly dynamic post‐translational modification of serine and threonine residues regulating physiological and stress processes. Here, we show that hypertrophy (via phenylephrine, 100 μM – 6 hours) augmented O‐GlcNAc signaling and, through the use of an NFAT‐luciferase reporter, was associated with NFAT activation in Neonatal Rat Cardiac Myocytes. Adenoviral O‐GlcNAcase overexpression or hexosamine pathway inhibition (i.e. reduction in O‐GlcNAc signaling) reversed hypertrophy‐induced NFAT activation similar to the prototypic calcineurin inhibitor, cyclosporin A. Interestingly, the O‐glycosylating enzyme O‐GlcNAc Transferase augmented O‐GlcNAc levels and activated NFAT, even in the absence of hypertrophic stimulation. Hearts from mice subjected to pressure overload induced hypertrophy (i.e. Transverse Aortic Constriction) exhibited augmented O‐GlcNAc levels and elevated ANP and BNP expression. Taken together, these results demonstrate a critical role of O‐GlcNAc signaling in NFAT activation during hypertrophy and provide evidence that O‐GlcNAc is coordinated with the onset and progression of cardiac hypertrophy. This represents a potentially significant and novel mechanism of cardiac hypertrophy, which may be of particular interest in studies of diabetic cardiomyopathy.Financial support: NIH, American Heart Association
Diosgenin suppress cardiac apoptosis and enhance survival pathway in ovariectomized rats
Authors: Lee Shin‐Da , Tasi Ching‐Yi , Ou Hsiu‐Chung , Huang Chih‐Yang 
Published: June 2021

ObjectiveThe study was to evaluate the effects of diosgenin on cardiac apoptotic and survival pathways in bilateral ovariectomied animal model.MethodsSixty female Wistar rats at 6–7 months of age were divided into sham‐operated group (Sham), bilateral ovariectomized group (OVX) for 2 months, and OVX groups with 10 mg/kg and 50 mg/kg diosgenin daily (Ovx 10, Ovx 50) in the 2nd month. The excised hearts were measured by heart weight index, hematoxylin‐eosin staining, positive TUNEL assays, and Western blotting.ResultsDiosgenin decreased OVX‐induced cardiac TUNEL‐positive apoptotic cells. Diosgenin decreased OVX‐induced Fas ligand, Fas death receptors, Fas‐associated death domain (FADD), activated caspase 8, and activated caspase 3 (Fas pathways). Diosgenin decreased OVX‐induced Bax, Bad, Bax‐to‐Bcl2 ratio, activated caspase 9, and activated caspase 3 (mitochondria pathway). Diosgenin increased ER‐beta, IGF1,IGF1R, PI3K, and Akt(survival pathway).ConclusionsDiosgenin suppressed ovariectomy‐induced cardiac Fas‐dependent and mitochondria‐dependent apoptotic pathways and enhance IGF‐1 related survival pathway. The findings may provide one of possible therapeutic approaches of diosgenin for potentially preventing cardiac apoptosis after bilateral ovariectomy or post‐menopause.
Postnatal maturation and hypoxic acclimatization modulate PKG‐mediated activation of BK channels in ovine cerebral arteries
Authors: Williams James M , Stockman Sara L. , Lincoln Thomas M. , Pearce William J. 
Published: June 2021

NO‐dependent relaxation of ovine cerebral arteries is enhanced during postnatal maturation but is attenuated by acclimatization to high altitude. Because NO‐mediated vasorelaxation is mediated by activation of PKG, which in turn activates large conductance BK channels, we hypothesized that the effects of both maturation and chronic hypoxia on NO‐dependent relaxation are mediated by modulation of PKG effects on BK channels. In middle cerebral arteries from normoxic and chronically hypoxic fetal and adult sheep, iberiotoxin (IBTX, an inhibitor of BK channels) enhanced the maximum contractile response to 5HT ≈10% in both age groups, but 8pCPT‐cGMP (8pCPT, a PKG activator) decreased this response ≈15% in fetal and ≈20% in adult arteries. Addition of 8pCPT followed by IBTX produced responses similar to controls in both age groups. In arteries treated with cumulative log dose increases of IBTX before or after incubation with 8pCPT, IBTX significantly enhanced basal tone ≈40% in both age groups. Preincubation with 8pCPT attenuated the contractile responses to IBTX by ≈70% in fetal and ≈90% in adult arteries. Addition of 8pCPT following maximum responses to IBTX decreased tone ≈30% in fetal and ≈40% in adult arteries. These results indicate that PKG enhances BK channel activity more in adult than fetal arteries, and markedly less in hypoxic than normoxic arteries.Supported by USPHS Grants HL54120, HD31266, & HL64867.
Cardiac sympathetic activity assessed by heart rate variability indicates myocardial ischemia on cold exposure in diabetes
Authors: Sasaki Keita , Matsushita Shonosuke , Sakakibara Yuzuru 
Published: June 2021

PurposeCold exposure is one of the major risk factors for cardiac events in diabetes. The aim of this study is how cardiac sympathetic nerve activity (SNA) is affected by temporal cold exposure in diabetes using heart rate variability (HRV).MethodsMale Wistar Rat (n=12) were divided into two groups: Control and Diabetes. Diabetes was induced by streptozocin. Lower extremity was sprayed for cold vapor for 5 seconds. Rectal and subcutaneous tissue temperature, flow velocity of lower extremity using laser Doppler flow meter, frequency domain analysis of HRV and ST change were measured up to 15 min from initial cold exposure.ResultsSNA was able to define as a power between 0 and 0.2 Hz in HRV by use of propranolol (60mg/kg). SNA was enhanced in diabetes than control (p<0.05) associated with reduction of blood flow (p<0.05). SNA was decreased by cold exposure in control (p<0.005), however, diabetes did not show significant reduction. SNA was enhanced significantly in diabetes at 1, 5, 10 and 15 min after cold exposure compared with control (p<0.01, p<0.005, p<0.05, p<0.0005, p<0.01, respectively). ST change was significantly larger in diabetes than control in 1 and 5 min after cold exposure (p<0.001, p<0.005).ConclusionCardiac SNA was decreased after cold exposure to lower extremity in control. In contrast, cardiac SNA was enhanced in diabetes than control, which may lead to cardiac ischemia in cold exposure in diabetes.
THE CORONARY ENDOTHELIUM AS A FUNCTIONAL AND PHYSICAL BARRIER FOR ENDOCRINE ANGIOTENSIN II
Authors: Tirado David Torres , Hernandez Jesus Castillo , Ahumada Erika Chi , Diaz Juan Ramiro , Rubio Rafael 
Published: June 2021

Circulating/endocrine Angiotensin II (AII) may remain intravascularily, does not diffuse across the vessel wall and acts only in the luminal endothelial membrane (LEM). We synthesized 15,000 kDa polymers of AII (AII‐Pol) and Saralasin (Sar‐Pol) that due to their size, remain intravascularily and act solely in the LEM's AII receptors (L‐AT1).In isolated perfused rat hearts AII and AII‐Pol both induces equally a positive inotropism and vasoconstriction. However, their ways of action differ; AII causes desensitization but AII‐Pol does not. Simultaneous infusion of both agonists show that as AII‐Pol/Ang II ratio raises, AII's desenzitization dwindles and is blocked. Desensitization is attributed to L‐AT1 internalization and is expected AII to induce L‐AT1 internalization but not so AII‐Pol. In LEM sampled during sustained stimulation with AII or AII‐Pol we show AII decreases L‐AT1 expression but no so AII‐Pol. The effects of both agonists were blocked by the non‐permeant Sar‐Pol. These results indicate that Ang II and Ang II‐Pol act and compete for the same L‐AT1.To demonstrate the action of AII is restricted to L‐AT1, we intracoronary infused biotin labeled AII (AII‐B). Micrographs reveal AII‐B retained luminally and co localized with endothelial markers.We conclude that the coronary endothelium is a functional and physical barrier for circulating AII. Funded by CONACYT SEP‐42567, 2004 C01 156.