bluuebunny/crossref_metadata_2025 huggingface dataset:
Knockout of ATF1 leads to enhanced cardiac contractility and output
Authors: Matus Marek , Schulte Jan , Sur Huyen , Seidl Matthias , Schütz Günther , Schmitz Wilhelm , Müller Frank U.
Published: June 2021

The transcriptional regulation by the CREB/CREM/ATF1 family factors presents an important cAMP‐dependent signalling pathway and it might contribute to the progression of heart failure. Activating transcription factor 1 (ATF1) is expressed ubiquitously, however, its role in the heart remains unknown. To elucidate the cardiac consequences of ATF1 ablation, we studied the left ventricular (LV) function in adult animals (14–18 weeks old). LV catheterization and analysis of pressure‐volume loops showed increased maximal LV pressure (LVPmax), stroke work (SW) and cardiac output (CO) of ATF1−/− mice.

Experiments on isolated adult cardiomyocytes confirmed these findings. The cell shortening of ATF1−/− cells was increased (3.9±0.9★ vs. 1.5±0.2 in ATF1+/+, n=10–11), whereby the calcium transient (CaT) amplitude was not significantly altered (★p<0.05 vs. ATF1+/+).

Hence, the presence of ATF1 is essential for a regular cardiomyocyte function, however, the increased contractility of ATF1−/− hearts cannot be explained by increased Ca2+ release from the sarcoplasmic reticulum. Supported by DFG (MU1376/10‐3)
Insufficient Vasopressin Release From Adequate Pituitary Stores Contributes to Inappropriately Low Plasma Vasopressin Levels in the Acute Response to Septic Shock
Authors: Delaney Richard R , Holt Danielle B , Hashiro Glenn M , Sato Aileen K , Hernandez Claudia A , Uyehara Catherine F T
Published: June 2021

Inadequate endogenous vasopressin (eVP) levels have been well described in catecholamine resistant hypotension in the setting of septic shock. In a piglet model of endotoxin (ETX) induced septic shock, we examined possible mechanisms for why eVP may not achieve adequate levels to reverse hypotension. eVP response to a 37% decrease in mean arterial pressure achieved levels of 150 ± 10 pg/mL (n=45) which were not sufficient to raise blood pressure. Plasma VP (pVP) levels achieved with pharmacologically administered vasopressin (VP) at 1, 10, and 100 ng/kg/min revealed that pVP levels of at least 400 pg/mL were needed to reverse hypotension (p<0.05). For piglets given ETX, pituitary levels of VP were measured and found to still contain adequate stores (735 ± 51 ng/pit) although decreased from non‐ETX controls (1297 ± 290 ng/pit) . Next we examined whether eVP could be further released by other stimuli in the face of endotoxic hypotension. Dopamine was infused in piglets who had the same degree of hypotension as untreated controls and was found to further increase pVP to 203.5 pg/mL ± 34.5, p<0.02. Given these results it appears that the pituitary has the stores and the capacity to release more eVP in septic shock. The mechanism responsible for low levels of pVP therefore seems to be due at least in part to the inability of the pituitary to adequately release the hormone in response to endotoxin‐induced hypotension.
Calcium Uptake in Isolated Chick Intestinal Epithelial Cells: Role of β‐glucuronidase and TRPV6
Authors: Nemere Ilka , Khanal Ramesh C , Sterling Tremaine M , Smith Nathan M
Published: June 2021

We have postulated that calcium influx is mediated by PKA and efflux mediated by PKC (1). In the current work, enterocytes from adult birds lacking PKC(2) responded to 1,25D3 with enhanced 45Ca uptake, relative to controls. The steroid response was blocked by Ab 099 against the 1,25D3‐MARRS receptor, but not by 9A7 against the VDR. Likewise, in freshly isolated enterocytes preincubated with calphostin C, 1,25D3 treatment enhanced 45Ca uptake. Cells treated with 100 nM PMA exhibited rapid extrusion of 45Ca, whereas myo‐inositol was without effect. We have noted that in chick enterocytes cultured for 72 h, there is also a loss of steroid‐stimulated PKC activity(3). Under 72 h culture conditions, 1,25D3 also stimulated 45Ca uptake. In response to a report that ß‐glucuronidase (ßG) activates TRPV 5 in kidney, we determined that enterocytes treated with 1,25D3, PTH or forskolin had elevated release of ßG, and addition of ßG to enterocytes stimulated 45Ca uptake. siRNA against either ßG or TRPV6 inhibited steroid‐stimulated 45Ca uptake. Using confocal microscopy we observed steroid enhanced surface staining of anti‐TRPV6 or anti‐ßG due to 1,25D3. We conclude that PKC does regulate efflux while uptake involves PKA‐mediated release of ßG with subsequent activation of TRPV6.
LOW SALT DIET DURING PREGNANCY AND LACTATION DEVELOPS OBESITY IN FEMALE ADULT OFFSPRING.
Authors: Lopes Karen Lucasechi , Furukawa Luzia Naoko Shinohara , Heimann Joel Claudio
Published: June 2021

Objective: To evaluate the effect of low salt diet during the perinatal period on adult offspring.

Methods: Female Wistar rats were fed normal (NSD 1.3% NaCl) or low (LSD: 0.15% NaCl) salt diet from 8 to 18 weeks of age. At 12 weeks, they were matched. After birth, only 8 pups (4 males ‐ M and 4 females ‐ F) were kept and after weaning all offspring groups were fed standard chow. Body weight (BW) was measured at birth and every week thereafter. Adipose tissue mass and adiposity index (AI), adipocyte volume (AV), plasma leptin level (Lep) were measured in adult offspring (Off).

Results (mean&SEM, (p<0.05) (n= 5): BW (g) was lower at the birth in LSD (M‐5.6±0.1 and F‐5.8±0.1) than in NSD Off (M‐6.5±0.1, F‐6.2±0.1). The Lep (ng/mL) was higher in LSD (5.3±0.1) than in NSD M Off (1.9±0.4), otherwise in LSD Lep was lower (1.7±0.6) than in NSD F Off (3.7±0.6) group. The AI was higher in LSD (8.3±0.6) compared to NSD F Off (6.3±0.6), but not in M Off. The retroperitoneal (RET) and brown adipose (BAT) tissue mass (g) were higher in LSD F Off compared to NSD F Off. AV (μm3) was higher in RET and lower in BAT in LSD compared to NSD F Off, but not in M Off.

Conclusions: sex‐specific effects of salt restriction during the perinatal period on adult fat content and distribution were observed. These results are important, once they unraveled a previously unknown mechanism of intrauterine programming of adult obesity.

Supported by FAPESP.
Different mechanisms of cGMP‐mediated relaxation in carbachol‐precontracted colon and jejunum smooth muscle from mice
Authors: Frei Eva , Huster Maria , Smital Petra , Schlossmann Jens , Hofmann Franz , Wegener Jörg W.
Published: June 2021

cGMP dependent protein kinase I (cGKI) in intestinal smooth muscle may induce relaxation via several mechanisms that include inhibition of intracellular Ca2+ release by phosphorylation of the inositol‐1,4,5‐phosphat receptor associated G‐kinase substrate (IRAG) and increasing myosin phosphatase activity by phosphorylation of the myosin phosphatase targeting subunit 1 (MYPT1). In the present study, we investigated whether one or both mechanisms are involved in cGMP‐mediated inhibition of carbachol‐stimulated contraction of colon and jejunum. In simultaneous recordings of tension and intracellular Ca2+ signals, 8‐Br‐cGMP reduced both carbachol‐induced tension and intracellular Ca2+ signals in colon muscle. In contrast, in jejunum muscle, 8‐Br‐cGMP reduced tension but did not change intracellular Ca2+ signals. In colon muscles from mice exhibiting a mutated IRAG (IRAG 12), 8‐Br‐cGMP did not reduce carbachol‐induced tension and Ca2+ signals. Inhibition of phosphatase activity by calyculin A abolished relaxation of carbachol‐induced contraction by 8‐Br‐cGMP in jejunum but not in colon muscle. Western blot analysis showed a larger signal for phosphorylated MYPT1 in carbachol‐stimulated jejunum than in colon muscle. These result suggest that cGMP/cGKI induces relaxation mostly by inhibition of intracellular Ca2+ release in colon and by activation of myosin phosphatase in jejunum.
Arginase inhibition increases the relaxation response to acetylcholine in murine mesenteric vessels
Authors: Wynne Brandi M. , Giachini Fernanda RC. , da Silva‐Santos Jose Eduardo , Tostes Rita C. , Webb R. Clinton
Published: June 2021

Endothelial dysfunction and smooth muscle hyper‐reactivity is characteristic of many vascular diseases. Arginase is a central enzyme in the urea cycle, hydrolyzing L‐arginine to urea and L‐ornithine. This process decreases arginine bioavailability for the production of nitric oxide (NO) by nitric oxide synthase (NOS). There is growing evidence that arginase plays a key role in vascular and endothelial dysfunction by decreasing NO production and uncoupling NOS leading to reactive oxygen species generation. Arginase expression and activity has also been shown to be increased in the large conductance vessels during hypertension. We tested the hypothesis that arginase inhibition in resistance arteries of the mesentery would enhance endothelium‐mediated relaxation. C57Bl6 mice were killed; 1st order mesenteric vessels were isolated and then subjected to standard muscle bath procedure. Concentration response curves (CRC) to phenylephrine and acetylcholine were performed with or without the arginase inhibitor, nor‐NOHA [3×10−4 M]. Vessels incubated with nor‐NOHA exhibited a leftward shift in the CRC to acetylcholine (p<0.005). These data suggest that arginase acts to limit the pool of arginine available for relaxation to acetylcholine in resistance arteries in mice. This work is funded by NIH HL074167.
Na+‐K+ ATPase and Ca2+ Clearance Proteins: A Coordinated Unit in Smooth Muscle?
Authors: Pritchard Tracy J.
Published: June 2021

Na+−K+ ATPase (NKA) has been hypothesized to be involved in hypertension in part by influencing Ca2+ handling via coupling to Na+−Ca2+ exchanger (NCX). The specific contribution of smooth muscle NKA to systolic blood pressure is not known with certainty. We previously developed mice that expressed a transgene for the NKA α2‐isoform using the smooth muscle SMP8 promoter (α2sm+ mice). These mice had a 2–7‐fold increase in α2‐isoform protein, and decreased systolic blood pressure and relaxation half‐times of aortic force. Surprisingly, α2sm+ mice also had a similar increase in the NKA α1‐isoform, not contained in the transgene. To elucidate mechanisms for the decreased systolic blood pressure, we measured NCX, sarcoplasmic reticulum Ca2+ ATPase (SERCA), and plasma membrane Ca2+ ATPase (PMCA) protein levels. There was a 2–5‐fold increase in SERCA, NCX, and PMCA in aortic and antral smooth muscle from α2sm+ mice compared to WT mice. In contrast, actin and myosin light chain were not changed in α2sm+ aorta, suggesting no vascular remodeling. Our data suggest that NKA may be involved in a larger, regulated Ca2+ clearing unit, consisting of at least NKA, NCX, SERCA, and PMCA, which may be important in coordinating control of [Ca2+]i. Supported by HL66044, HL61974.
Role of Exchange Protein Directly Activated by cAMP‐1 (Epac‐1) in Pulmonary Artery Smooth Muscle Cells: A New Target for Pulmonary Hypertension
Authors: Murray Fiona , Suda Ryan Y.S. , Do Thao , Yokoyama Utako , Thistlethwaite Patricia A. , Yuan Jason X‐Y , Insel Paul A.
Published: June 2021

Dysfunctional cAMP signaling contributes to enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC) in pulmonary arterial hypertension (PAH). Exchange protein directly activated by cAMP (Epac, Epac‐1 and Epac‐2 are isoforms), a guanine nucleotide exchange factor for Rap, has been identified as a cAMP‐effector. We found that PASMC isolated from PAH‐patients have decreased mRNA and protein expression of Epac‐1, decreased Rap‐1 activation and blunted anti‐proliferative response to an Epac‐selective cAMP analog [8‐pCPT‐2Me‐cAMP (8Me), 50μM, 24 hr]. Hypothesizing that increasing Epac‐1 expression in PAH‐PASMC would enhance the anti‐proliferative effect of cAMP, we restored its expression using an Epac‐1 adenovirus. Adv‐Epac‐1 increased Epac‐1 protein expression in PAH‐PASMC 20‐fold compared to Adv‐Lac‐Z without altering Epac‐2 and decreased serum‐induced proliferation more in PAH‐ than control‐PASMC (64 ± 2.8% and 38.3 ± 1.7% respectively, compared to Adv‐Lac‐Z). Over‐expression of Epac‐1 enhanced the anti‐proliferative effect of 8Me in PAH‐PASMC by 22.3±2.9% and increased Rap‐1 activation to a level similar to control‐PASMC. We conclude that decreased Epac‐1 contributes to altered signaling and increased PASMC proliferation in PAH. Restoring Epac‐1 expression may be beneficial in PAH by enhancing the effectiveness of drugs that increase cAMP. Supported by NIH and ALA.
Periodic Dynamics of Rolling Leukocytes
Authors: Pickard John Edwin , Zarbock Alexander , Pospieszalska Maria K , Ley Klaus
Published: June 2021

At the time scale of individual bond interactions, leukocyte rolling consists of short periods of movement followed by relatively long pauses, a motion termed saltation. Prior to a bond breakage event the blood flow‐induced convective hydrodynamic forces are balanced by load‐bearing bonds and the cell is at rest. Dissociation of a load‐bearing bond results in rapid shear‐induced acceleration followed by rapid deceleration as the shear force, transmitted through the cell membrane, is redistributed among the adhesive bonds and the resting force balance is reestablished. This stop‐and‐go behavior is manifested as a periodic rolling motion and is governed by the timing of bond dissociation and the random distribution of leukocyte microvilli. By studying leukocyte rolling dynamics at high spatial and temporal resolution, we aim to elucidate the molecular dynamics of cellular adhesion. We employ fast imaging of leukocytes in a physiologically relevant whole blood‐perfused flow chamber in conjunction with edge‐based automated object tracking. Frequency analysis of position and velocity tracks is used to identify the lifetime and spacing of stressed molecular bonds. In neutrophils rolling on P‐selectin, distinct peaks were found which are consistent with the spacing of microvilli expressing small clusters of PSGL‐1, the main P‐selectin ligand. This work supported by NIH grants EB02185.
Control Entropy of mechanomyogram (MMG) and electromyogram (EMG) during fatiguing isometric muscle actions
Authors: Mcgregor Stephen James , Armstrong Jeffrey , Bollt Erik M
Published: June 2021

The purpose of this study was to determine if Control Entropy (CE) of MMG and EMG would decline during fatiguing isometric muscle actions. Procedures were approved by the Hope HSRB. Subjects performed one isometric leg extension (30° flexion) at the 1‐RM of the non‐dominant leg to volitional fatigue. MMG and EMG signals were recorded from the vastus medialis (VM) and rectus femoris muscles (RF) using commercial software (Biopac Systems, CA). The effort was divided into epochs (10% of time‐to‐fatigue) and mean and peak‐to‐peak (P‐P) amplitude MMG and EMG data were analyzed using repeated measures ANOVA (α=0.05). CE was calculated in Matlab (Mathworks, MA) using 2 and 8 bit partitions, and varying window lengths according to the following equation: CEKS[x(t)] =hKS[sign(dx/dt(t))]. P‐P amplitude of VM‐MMG and RF‐EMG declined (p<0.05), while RF‐MMG (p=0.58) trended lower over time. Mean amplitude of VM‐EMG declined (p<0.05). CE of the MMG declined over the course of the effort for males, but not for females. When accounting for gender, a significant effect was observed for P‐P of VM .MMG, RF‐EMG and mean VM‐EMG. In addition, CE of EMG exhibited an unexpected response whereby it increased at the onset of the muscle action. These results support previous reports of a differential MMG response in males vs females. The attenuated CE response of MMG in females vs. males may shed light on the nature of this difference between genders.
Leg exercise hyperemia: role of nitric oxide synthase and cyclooxygenase
Authors: Limberg Jacqueline K , Schrage William G , Wilkins Brad W , Johnson Christopher P , John Eisenach , Dietz Niki M , Curry Timothy B , Joyner Michael J
Published: June 2021

The key vascular signals regulating muscle blood flow during exercise are unclear. We tested the hypothesis that in young adults, leg blood flow (LBF) during exercise would be reduced independently by pharmacological inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX). In 13 young adults, we measured heart rate (ECG), blood pressure (femoral artery and vein), blood gases, and venous oxygen saturation (indwelling oximeter) during steady‐state dynamic (60/min) knee extension exercise. LBF was determined by Doppler ultrasound of the femoral artery. Exercise (20W) increased leg vascular conductance (LVC) from 4± 1 to 33± 3 ml/min/100mmHg. The NOS inhibitor L‐NAME reduced LVC by ∼10% to 30± 1. Infusion of the COX inhibitor, ketoralac (n=9), increased LVC from 30± 1 to 34± 2. L‐NAME infusion reduced venous oxygen saturation from 48 to 38%, suggesting greater extraction to maintain oxygen consumption. Control intra‐arterial infusions of acetylcholine (ACH) and nitroprusside (NTP) tested inhibitor efficacy. The change in LVC to two doses of ACh was blunted 53 and 15%, by L‐NAME+ketorolac infusion. In contrast, the LVC responses to NTP were unaltered. These data implicate a modest role for NO‐mediated leg exercise hyperemia in adults and a potentially modest LBF restraint by vasoconstrictor prostaglandins. Funded by the NIH HL‐46493 and the Minnesota Obesity Center.
Overexpression of Angiotensin II type 2 receptor (AT2R) in neonatal cardiomyocytes induces apoptosis
Authors: Qi Yanfei , Li Hongwei , Mecca Adam , Shenoy Vinayak , Sumners Colin , Katovich Michael
Published: June 2021

In vitro experiments suggest that AT2R is involved in apoptosis of fibroblasts, neurons, and endothelial cells. These apoptotic effects are elicited by either Ang II stimulation or constitutive activation of the AT2 receptor. However, the apoptotic effects of AT2R in cardiomyocytes have not been elucidated. Therefore, we investigated the effects of AT2R overexpression in neonatal rat cardiomyocytes. Neonatal cardiomyocytes were isolated from 5‐day old SD rat pups. An AT2R recombinant adenovirus, Ad5‐CMV‐AT2R‐EGFP, was used to overexpress AT2R. At 48 hours post transduction, apoptosis was evaluated by a deoxynucleotidyltransferase‐mediated dUTP‐biotin nick end labeling (TUNEL) assay (for in situ detection of apoptotic cells). Apoptosis was quantified by percentage of positive TUNEL stained area using ImageJ software. Apoptosis was significantly higher in the AT2R overexpressed cardiomyocytes (2.24 %) compared to both PBS (0.46%) and GFP (0.32%) control groups (P<0.001). A decrease in cell density was evident only in the AT2R overexpressed group after 96 hours of viral transduction compared to the PBS and GFP control groups. We conclude that this decrease in cardiac myocyte density is likely due to constitutive pro‐apoptotic signaling via the AT2R.
Agmatine inhibits growth via a cell cycle arrest and confers resistance to apoptosis
Authors: Arndt Mary Ann , Parisi Eva , Isome Masato , Satriano Joseph
Published: June 2021

The arginine metabolite agmatine suppresses growth. Notably, this effect preferentially targets cells with high proliferative kinetics (Isome, et al, AJP, 2007). However, the mechanisms of this process have yet to be defined.

Here we demonstrate via FACS analysis a dose dependent G1 phase cell cycle arrest with agmatine administration. Western blot evaluations of G1 cell cycle proteins demonstrate decreases in cyclin D1 expression, phosphorylation state of the retinoblastoma protein, and cyclin A expression. We observe induction of cyclin kinase inhibitors p16 and p21, but not p53.

Agmatine administered to cultured cells results in neither necrosis nor apoptosis, as shown by DNA fragmentation and chromatin condensation assays. Unexpectedly, agmatine decreases the expression and activity of caspase‐3 in a dose and time dependent fashion. Furthermore, agmatine inhibits the induction of caspase‐3 activity in response to apoptotic mediators, camptothecin and 5‐fluorouricil.

CONCLUSIONS: Agmatine confers resistance to apoptosis and can suppress proliferation via a p53 independent cell cycle arrest. Aberrant apoptosis and/or proliferation are common pathologic complications. As previously shown in an in vivo model of glomerulonephritis (Ishizuka, JASN, 2000), agmatine may be a candidate for adjunctive therapy in renal disease.

Funding: NIH/NIDDK, VA
Thermal acclimation confers no tolerance to acute temperature change in cardiac myocytes from bluefin tuna
Authors: Galli Gina Lucia Jane , Shiels Holly Alice , Block Barbara
Published: June 2021

Pacific bluefin tuna, Thunnus orientalis, make extensive horizontal and vertical migrations where they inhabit a broad range of thermal niches. Although tuna are capable of conserving metabolic heat, the hearts of these fishes operate at near ambient water temperatures, and must therefore be able to function at a broad range of temperatures.

Study objective

to investigate the effects of thermal acclimation and acute temperature change on aspects of sarcolemmal and intracellular calcium cycling in isolated cardiac myocytes from the Pacific bluefin tuna.

Methods

Atrial and ventricular myocytes were isolated from bluefin tuna acclimated to either 15 or 23 °C, and the effect of experimental temperature (15, 19 and 23 °C) on the sarcoplasmic reticulum load (SRload) and L‐type calcium channel density (ICa) was assessed using whole‐cell voltage‐clamp.

Results

Thermal acclimation has little effect on ICa or the ability of the SR to accumulate calcium. Acute reductions in temperature significantly decreased both ICa and SRload in atrial and ventricular myocytes from both acclimation temperatures.

Conclusions

Acute reductions in temperature reduce calcium flux both across the sarcolemmal membrane and through the SR. This will place a significant burden on the heart of the diving bluefin tuna and may limit the amount of time these animals can spend in cold waters.
Dendritic Cell immuno‐activating receptor 1 – Characterization of a novel member of the C‐type Lectin family
Authors: Kaden Stefan A. , Vasters Katrin , Schmitz Juergen , Winkels Gregor
Published: June 2021

C‐type lectins (CLEC) comprise a superfamily of cell surface proteins, whose multifunctional capacity has been shown to address both, innate and adaptive immune response. Here, we describe mDCAR1 a novel CLEC belonging to the recently defined murine dendritic cell (DC) immuno‐receptor (mDCIR)/DC immuno‐activating receptor (mDCAR) family.

mDCAR1 expression was found to be restricted to mononuclear phagocytes in bone marrow. Extensive phenotypical characterization defined them as CCR2−CD62L−CX3CR1+Ly6C− cells, thereby belonging to the recently described ‘resident’ monocytes, which correspond to CD14+ CD16+ human monocytes. In contrast, the CCR2+CD62L+CX3CR1lowLy6C+ ‘inflammatory’ monocytes were mDCAR1−. Noteworthy, in thymus, mDCAR1 expression was found on CD11chighCD8alpha+CD205+CD11b−B220−BP‐1+ lymphoid‐related DC, which play an important role in central tolerance induction.

With respect to the classical function of CLEC as antigen uptake receptors for pathogens and self‐proteins, mimicking the ligation of mDCAR1 by labeling with specific mAb shows the internalization of the mDCAR1/ligand complex. Crosslinking of mDCAR1 further initiates signal transduction resulting in tyrosine‐phosphorylation of proteins.

The expression pattern of mDCAR1 and the evidence of a classical antigen‐uptake mechanism are first steps to uncover the role of this representative of the CLEC receptor family.
Modelling glioma growth and invasion in Drosophila
Authors: Paulus Werner , Witte Hanna , Jeibmann Astrid , Klaembt Christian
Published: June 2021

Drosophila melanogaster model systems are characterized by easy handling, rapid generation time, and a wide armamentarium of genetic techniques. Furthermore, many molecular pathways are highly conserved between invertebrates and humans. While fly models have been established for type 1 neurofibromatosis, tuberous sclerosis and neurodegenerative disorders, no fly model of glioma is available so far. Using the Gal4‐UAS system we have produced several lines of transgenic flies by overexpression or RNAi‐mediated inactivation of homologs of human “glioma genes” (EGFR, PI3K, TP53, PTEN, PDGFR and RAS genes) under control of the glia‐specific promoter reversed polarity (repo). Glial expression of activated Egfr, activated Pvr (PDGFR and VEGFR homolog), wild type Pi3k and wild type Ras2 resulted in markedly increased numbers of glial cells as determined by morphometry of eye imaginal disk and optic stalk glia of 3rd instar larvae. Remarkably, glial cells ectopically migrated along nerve tracts. Larval lines overexpressing activated EGFR and Pvr showed fused brain lobes and thickened peripheral nerves. Administration of the EGFR tyrosine kinase inbibitor gefitinib and the PI3 kinase inhibitor wortmannin reverted larval neuropathology. These Drosophila models should be useful in genetic screens for revealing interacting genes involved in gliomagenesis, as well as in experimental therapy approaches.
Effect of Soy Isoflavone Supplementation on Menopausal Quality of Life
Authors: Amato Paula , Young Ronald , Lewis Richard , Murray Michael , Cramer Margaret , Steinberg Francene , Barnes Stephen , Ellis Kenneth , Fraley Kennard , Konzelmann Karen , Smith E O'Brian , Wong William , Fischer Joan
Published: June 2021

Recent clinical trials have found an increased risk of health problems in women using menopausal hormone therapy. As a result, women are in search of alternative strategies to improve their quality of life. The purpose of this study was to assess the effect of soy isoflavone supplementation on quality of life in postmenopausal women. 403 healthy postmenopausal women (54.0+4.0 yrs) were enrolled in a 2 year multicenter, randomized, controlled trial designed to evaluate the effect of soy isoflavone supplementiation on the prevention of osteoporosis. Quality of life was assessed as a secondary outcome and was measured using the validated Menopause‐Specific Quality of Life Questionnaire (MENQOL) administered at baseline and at annual intervals. Participants were randomly assigned to receive soy isoflavones 80 mg/d, soy isoflavones 120 mg/d, or placebo. Data were analyzed using an analysis of variance (ANOVA). Baseline characteristics were similar among the three groups. There was a non‐significant decrease i.e. improvement, in the vasomotor domain score at one and two years compared to baseline in all three groups. However, there were no differences among groups. In conclusion, soy isoflavones provide no added benefit over placebo in terms of qualifty of life as measured by the MENQOL. Supported by IFAFS 2001‐52102‐11255 USDA CSREES.
Energy Intake and HIV Disease Progression in Asymptomatic ARV‐Naive HIV+ Adults in Botswana
Authors: Baum Marianna K , Etengoff Kirstin , Marlink Richard , Rafie Carlin , Makhema Joseph , Burns Patricia , Thomas Ann , Tsalaile Lesedi , Sales Sabrina , Campa Adriana ,   Botswana Dikotlana Study Team
Published: June 2021

Objective

Adequate nutrition is needed for optimal immune response. We examined the relationship of energy intake to disease progression in asymptomatic HIV+ adults in Botswana.

Methods

After obtaining informed consent from 806 HIV+ adults, data on demographic and nutritional status were collected. Blood was drawn for CD4 cell count and viral load. 24‐hour dietary intakes were analyzed with the NutraBase Nutrition 7 program.

Results

Of 806 participants, 26.2% (N=211) were male. Their mean age was 34±8.1 years, mean BMI was 24.45±5.0 kg/m2, CD4 cell count 462.5±181 cells/μl, and viral load 4.1±0.89 log10 copies/mL. Increasing energy intake by 1000 kcal significantly predicts an increase of 20 CD4 cells (p=0.044) and a decrease of viral load by 0.14 log10 copies/mL (p=0.0089) after adjusting for age and gender using the Generalized Linear Model. Participants with adequate protein intake (≥46g for women and ≥56g for men), had significantly higher mean BMI (24.7±4.9) compared to those with inadequate intake (23.5±5.1, p=0.038).

Conclusions

Overall calorie intake significantly predicted the indices of disease progression in an adult asymptomatic, ARV‐naïve HIV+ cohort in Botswana. Further research investigating the effect of dietary support on attenuating HIV disease progression is needed.

Support

Funded by NIDA
Spliceform specific Coxsackie‐adenovirus receptor interactions
Authors: Excoffon Katherine Ashbourne , Gansemer Nicholas , Zabner Joseph
Published: June 2021

The Coxsackievirus and Adenovirus Receptor (CAR) is both a homotypic cell adhesion molecule and a viral receptor. The murine gene for CAR, Cxadr, consists of 8 alternatively spliced exons terminating in either exon 7 (CAREx7) or exon 8 (CAREx8). Both splice forms contain a C‐terminal PDZ binding domain and differ only in the last 26 (Exon 7, ‐GSIV) or 13 (Exon 8, ‐ITVV) amino acids. The gene encoding human CAR, Cxadr, was previously described as 7 alternatively spliced exons. We hypothesized that a human CAREx8 isoform exists, has similar virus receptor characteristics to CAREx7, but differs in other biological aspects. Human CAREx8 was cloned from primary human airway epithelia, inserted into an expression vector and evaluated for localization, adenovirus gene transfer and PDZ‐interaction. CAREx7 and CAREx8 showed similar localization and adenovirus infection in non‐polarized cells. In polarized cells, CAREx7 localized exclusively to the basolateral membrane. In contrast, CAREx8 showed apical localization and mediated apical adenovirus infection. Moreover, whereas co‐expression of CAREx7 with the PDZ‐domain containing protein MAGI‐1b altered the localization of MAGI‐1b from diffusely cytoplasmic to cell junctions, co‐expression of CAREx8 with MAGI‐1b resulted in the loss of CAREx8. This loss required both the PDZ interaction and the upstream 9 amino acids. In conclusion, human CAR is an 8 exon alternatively spliced protein yielding isoforms with distinct localization and interactions. Furthermore, CAREx8 may be responsible for respiratory viral infection. (NIH)
t10c12 CLA isomer prevents age associated bone loss by modulating osteoclastogenesis
Authors: Rahman Md Mizanur , Bhattacharya Arunabh , Halade Ganesh , Banu Jameela , Fernandes Gabriel
Published: June 2021

Osteoporosis is one of the major causes of morbidity in the elderly. Inflammation exerts a significant influence on bone turnover, inducing the chronic form of osteoporosis. Dietary nutrition has the capacity to modulate inflammatory response. Therefore, nutritional strategies and lifestyle changes may prevent age‐related osteoporosis, thereby improving the quality of life of the elderly population. Conjugated linoleic acid (CLA) has been shown to positively influence calcium and bone metabolism. Earlier we have shown that CLA (equal ratio of c9t11 and t10c12 isomers) can protect age associated bone loss by modulating inflammatory markers and osteoclastogenesis. However, c9t11 and t10c12 CLA individual isomers differentially regulate functional parameters and gene expression in different cell types. Therefore, the present study was undertaken to examine the differential effect of c9t11 and t10c12 CLA isomers on bone mineral density (BMD) in middle‐aged (12 mo) C57BL/6 female mice. After 5 months on diet, t10c12 CLA‐isomer fed mice maintained a significantly higher BMD in femoral, tibial and lumbar regions than c9t11 and corn oil (CO)‐fed mice. The increased BMD was accompanied by a decreased activity of osteoclastogenic factor, RANKL and decreased osteoclast activity as measured by TRAP5b in serum. A significant reduction of osteoclasts differentiation was also observed in RANKL stimulated RAW 264.7 cells in t10c12 CLA isomer treated culture as compared to c9t11 CLA isomer or linoleic acid treated culture. In conclusion, these findings suggest that t10c12 isomer is the most potent CLA isomer to prevent the bone loss by modulating osteoclastogenesis. Research support: NIH R21 AG027562.
Expression of lipid metabolism genes in skeletal muscle after weight loss in post‐obese insulin resistant dogs
Authors: Leray Veronique , Serisier Samuel , Nguyen Patrick
Published: June 2021

Overweight and obesity are associated with insulin resistance, and impaired lipid and glucose metabolism in insulin target tissues. We have previously showed in dogs that a weight loss was accompanied by changes in the expression of lipogenesis and lipolysis in fat and liver. The aim of this study was to evaluate the effect of weight loss on insulin sensitivity and expression of these genes in skeletal muscle.

Obese dogs were given a hypoenergetic diet in order to induce weight loss. When dogs were obese and then when they had reached their healthy body weight, insulin sensitivity was assessed using the euglycemic hyperinsulinemic clamp. mRNA expression of IRS‐2, SREBP, ACC, FABP, ADRP, PEPCK, perilipin and HSL was semi‐quantified using real time RT‐PCR in skeletal muscle.

Dogs' body weight was 25 % lower at the end of the slimming period compared to obese state. Insulin sensitivity was higher (+ 40 %). In skeletal muscle, the mRNA expression of perilipin and ADRP was significantly lower (− 50 %). The expression of genes involved in lipid metabolism did not change.

These results showed a decrease in perilipin and ADRP expression, which could reflect the lower lipid content in skeletal muscle after weight loss. They also show that weight loss did not affect the expression of lipid metabolism genes in skeletal muscle, although insulin sensitivity recovery, contrary to our previous results in fat and liver.
Characterization of SV40 T‐antigen G‐quadruplex Helicase Activity Using SPR
Authors: Brennan Jarryd , Karr Jessica R. , Jasheway Karl , David Wendi M.
Published: June 2021

Simian Virus 40 (SV40) is a double stranded DNA virus helpful as a model system for eukaryotic cellular DNA replication. It encodes a Large T‐antigen (T‐ag) protein, which is essential for viral genome replication and regulation of the host cell cycle. Acting as a helicase enzyme, T‐ag is able to unwind various forms of DNA. Guanine rich sequences of DNA capable of forming G‐quadruplex knot‐like structures have been identified in several eukaryotic oncogene promoter regions, as well as in the SV40 virus genome, and may possibly influence transcription activity. T‐ag forms an active hexameric unit in the presence of ATP, and was demonstrated to bind single‐strand, duplex, and G‐quadruplex DNA using real time surface plasmon resonance (SPR). Probing with single strand binding protein (SSB) confirmed that T‐ag activity resulted in the unwinding of an intermolecular quadruplex.
ChoD Is Important for Lipid Modification in M. smegmatis
Authors: Gao Jin , Sampson Nicole S.
Published: June 2021

New targets are needed to develop antimicrobial therapies to combat M. tuberculosis (Mtb), especially the multi and extremely drug resistant organisms (MDR‐TB and XDR‐TB). Mycobacteria have been reported to oxidize 3β ‐hydroxysterols to 3‐ketosteroids, but the enzyme(s) responsible for this activity have not been identified in mycobacterial species. Rv3409c, from Mycobacteria tuberculosis H37Rv genome is annotated as choD, a putative cholesterol oxidase. The choD gene was heterologously expressed behind an acetamidase promoter with a C‐terminal hexa‐histidine tag in Mycobacterium smegmatis mc2 155. Using established cholesterol oxidase assays that follow the formation of the by‐product H2O2 or final product cholest‐4‐en‐3‐one, there was no cholesterol oxidizing activity detected. The choD gene is also conserved in M. smegmatis, M. leprae and Rhodococcus equi. The M. smegmatis orthologue has 83% amino acid identity with choD from M. tuberculosis. The M. smegmatis choD transposon mutant Myc11 can still catabolize cholesterol. Moreover, Myc11 has significantly different colony morphology than wild‐type as observed by light microscopy. The total lipids from both M. smegmatis WT and Myc11 were extracted and analyzed by MALDI‐TOF MS and TLC. There are distinct differences between the Myc11 lipid profile and the wild‐type lipid profile. Based on our results, we propose that choD is not a cholesterol oxidase, but rather plays a role in lipid cell wall modification or synthesis.
Human mevalonate diphosphate decarboxylase: Functional tests, structural progress, and assignments of active site residues
Authors: Miziorko Henry M. , Voynova Natalia E. , Fu Zhuji , Battaile Kevin , Kim Jung‐Ja P.
Published: June 2021

Expression in E. coli of his‐tagged human mevalonate diphosphate decarboxylase (hMDD) has expedited enzyme isolation, characterization, functional investigation, and crystallization. hMDD exhibits Vm = 6 U/mg; Km for ATP is 0.7 mM; Km for (R,S) mevalonate diphosphate is 29 uM. Diphosphoglycolyl proline is a competitive inhibitor (Ki = 2 uM) against MPP. Several conserved polar residues that, based on sequence alignment and structural overlay of yeast MDD and a mevalonate kinase‐ATP complex, are near the active site were mutated to test functional importance. R161Q exhibits ∼1000‐fold diminution in specific activity, although it binds fluorescent substrate analog, TNP‐ATP, like wild‐type enzyme. N17A exhibits a 50‐fold inflation in Km for mevalonate diphosphate. Crystallized hMDD (P2(1); a=87.0, b=53.2, c=97.8 Å, and beta=107.3°) diffracts to 2.4Å. Molecular replacement methods produced an initial structural model; refinement is in progress. Based on structural information, R161 and N17 map in the interior of the active site cleft. Predicted proximity (∼3 Å) of R161 to N17 and functional characterization of R161Q and N17A are compatible with their roles in binding/orientation of mevalonate diphosphate.
Carbon‐13 NMR studies on the glutamate metabolism of glutamate dehydrogenase mutants of yeast
Authors: Rajput M. Zak , Trotter Pamela J.
Published: June 2021

Saccharomyces cerevisiae has three glutamate dehydrogenase enzymes encoded by the GDH1 and GDH3 (NADP+‐dependent) and GDH2 (NAD+‐dependent) genes, all of which are essential in the interconversion of alpha‐ketoglutarate, a citric acid cycle intermediate, and glutamate. In this study, strains carrying single disruptions in the GDH genes were examined. Previous studies have determined GDH activity in glucose‐grown gdh1Δ cells is <5% as compared to wild type, while activity in gdh2Δ and gdh3Δ cells is comparable to wild type. The effects of these mutations in cells pre‐grown in non‐fermentable carbon sources (acetate, raffinose, ethanol) were studied by the utilization of [1, 2‐13C] acetate, which is metabolized by the citric acid cycle and converted to glutamate. Time trials were also conducted utilizing the labeled acetate precursor. Significant enrichment of the multiplet carbon peaks in glutamate of gdh1Δ strains indicated prolonged recycling of the glutamate backbone through the citric acid cycle before conversion to glutamate. Strains carrying gdh2Δ or gdh3Δ disruptions exhibited enrichment patterns comparable to wild type cells. These data support the primacy of Gdh1p in glutamate synthesis in a non‐fermentable carbon source. (Supported by NIH‐GM069372 to P.J.T.).
Hyperleptinemia induced adiponectin resistance in rat skeletal muscle cells
Authors: Fang XiangPing , Fetros Joyce , Sweeney Gary
Published: June 2021

Adiponectin and leptin have been shown to regulate energy metabolism in skeletal muscle. However, little is known regarding their signaling crosstalk. Hyperleptinemia is a common feature in human obesity and here we investigated effects of leptin (60 nM, 24 hours) on functions of recombinant globular (gAd) and full‐length multimers (fAd) of adiponectin in L6 skeletal muscle cells.

Real‐time PCR showed that mRNA expression levels of adiponectin receptor 1 (AdipoR1) and AdipoR2 were reduced after leptin treatment. Correspondingly, effects of both gAd and fAd on glucose and fatty acid uptake and oxidation were attenuated. AMPK is thought to be an important mediator of metabolic effects of adiponectin and here we showed that Compound C (AMPK inhibitor) pretreatment prevented stimulation of fatty acid uptake by fAd. Importantly, the activation of AMPK and ACC by gAd and fAd was attenuated by leptin. APPL1 was recently identified as an adaptor protein interacting with AdipoR and downstream targets such as Akt. Here we observed that leptin also regulated Ad‐stimulated binding of APPL1 to these targets.

These results suggest that hyperleptinemia can induce resistance to gAd and fAd in skeletal muscle cells, possibly due to the reduction of AdipoR expression.
Efficient Incorporation of Unnatural Amino Acids in Yeast, Mammalian Cells, and Stem Cells
Authors: Wang Lei , Wang Wenyuan , Takimoto Jeffrey K , H Qiang Wang. Jack
Published: June 2021

To study biological processes in various cell types using unnatural amino acids, we developed new methods for efficient incorporation of unnatural amino acids into proteins in yeast, mammalian cells, and stem cells. A general method was developed to express orthogonal prokaryotic tRNAs in eukaryotic cells using special polymerase III promoters. Mutant synthetases specific for unnatural amino acids were evolved in yeast and transferred for use in mammalian cells and stem cells. Other factors that decrease the incorporation efficiency were also addressed. Supported by the Searle Scholar Program and the Beckman Young Investigator Program.
Thymoquinone inhibits angiogenesis and prostate tumor growth by suppressing MAPK signaling pathways
Authors: Yi Tingfang , Cho SungGook , Yi Zhengfang , Luo Weijia , Wang Ying , Sethi Gautam , Aggarwal Bharat B. , Liu Mingyao
Published: June 2021

Thymoquinone (TQ), the main component of Nigella sativa (a herb medicine used for anti‐inflammatory for thousands of years), has been reported to inhibit proliferation of many tumor cells by suppressing TNF‐induced NF‐kB signaling pathway and arresting androgen receptor and E2F‐1 expression. In this study, we examined the effects of thymoqinone on human umbilical vein endothelial cell (HUVEC) proliferation, migration, invasion, and angiogenesis using in vitro and in vivo approaches. We found that TQ dramatically inhibited HUVEC proliferation, migration, invasion, and tube formation at significantly lower concentration (∼10 fold lower) compared to human prostate cancer cells. 100 nM TQ activated apoptosis in 45 ± 2.6% HUVEC cells, but in only 4.8 ± 1.8% PC3 cells. Furthermore, we demonstrated that TQ dramatically inhibited HUVEC invasion, tube formation, and angiogenesis in vitro by aortic ring assay and in vivo by matrigel plug assay. In a xenograft prostate tumor model, we found that TQ significantly inhibited tumor angiogenesis and prevented tumor growth by inhibiting the activation of FAK, c‐Src, ERK, and MAPK signaling pathway.
TIPE2, a novel negative regulator of innate and adaptive immunity that maintains immune homeostasis
Authors: Sun Honghong , Gong Shunyou , Carmody Ruaidhri , Li Li , Chen Youhai H.
Published: June 2021

Immune homeostasis is essential for the normal functioning of the immune system and its breakdown leads to fatal inflammatory diseases. We report here the identification of a novel member of the tumor necrosis factor‐induced protein‐8 family, designated TIPE2 that is required for maintaining immune homeostasis. TIPE2 is preferentially expressed in lymphoid and inflamed tissues and its deletion in mice leads to multi‐organ inflammation, splenomegaly and premature death. TIPE2‐deficient animals are hypersensitive to septic shock, and TIPE2‐deficient cells are hyper‐responsive to Toll‐like receptor (TLR) and T cell receptor (TCR) signaling. Importantly, TIPE2 binds to caspase‐8, and inhibits activating protein‐1 and nuclear factor‐kB activation while promoting Fas‐induced apoptosis. Inhibiting caspase‐8 significantly blocks the hyper‐responsiveness of TIPE2‐deficient cells. These results establish that TIPE2 is an essential negative regulator of TLR and TCR signaling, and its selective expression in the immune system prevents hyper‐responsiveness and maintains immune homeostasis.
Molecular interactions regulating RIG‐I signaling to the innate antiviral response
Authors: Hiscott John , Paz Suzanne , Vilasco Myriam , Arguello Meztli , Lacoste Judith , Zhao Tiejun , Meurs Eliane , Lin Rongtuan
Published: June 2021

Cytosolic sensing of incoming RNA virus infection is recognized by the DExD/H box RNA helicases RIG‐I and Mda5. The mitochondrial adaptor MAVS links sensing of viral RNA by RIG‐I/Mda5 to downstream signaling, resulting in the induction of pro‐inflammatory and antiviral genes by NF‐ κB and IRF respectively. We have demonstrated that NEMO, the adaptor of the IKK complex, induces activation of IRF3/7, acting downstream of RIG‐I and MAVS but upstream of TBK1/IKKε. NEMO requires TANK adaptor to mediate recruitment of the kinases and phosphorylation of IRF3/7. Thus, NEMO bridges the NF‐κB and IRF components of RIG‐I with the formation of a multimeric kinase complex.

Ubiquitination plays a central role in regulating the activity of the RIG‐I pathway. MAVS undergoes K63 ubiquitination at a single Ser500 residue, adjacent to the MAVS transmembrane domain and a TRAF interacting motif (TIM). K63‐linked ubiquitination of MAVS mediates recruitment of IKKε, but not TBK1, to the mitochondrial compartment. Recruitment of TRAF3 to the C‐terminal TIM motif of MAVS is sterically hindered by IKKε, but is relieved by mutation of K500. Interestingly, the MAVS K500R mutant did not affect IFN gene activation, but rather appears to alter processing of NF‐κB2p100 in the non‐canonical NF‐κB pathway. Thus, MAVS ubiquitination provides another level at which to regulate the RIG‐I response to virus infection.

This research was supported by grants from CIHR and NCIC.
Effect of a Spanish Diabetes Education on Dietary Changes and Diabetes Control
Authors: Salto Lorena , Beeson Lawrence , DeLeon Marino , Cordero‐MacIntyre Zaida
Published: June 2021

O: To assess the impact of culturally appropriate and language‐sensitive diabetes education on diabetes control in a Hispanic diabetic cohort.

M: Twenty six subjects (13 M & 13 F), ranging from 29–75 years, participated in a 3‐month study. Subjects were enrolled from San Bernardino and Riverside counties. Waist and hip circumferences were measured. The Kolmogorov‐Smirnov Test was used for normality and the Paired Samples T‐test to compare baseline and three‐month means. Spearman's correlation coefficient was used to determine the linear relationship between laboratory results and dietary changes (Southwestern FFQ).

R: The group had a baseline fasting blood glucose mean of 158 mg/dL, a baseline HbA1c mean of 8.5%, and insulin mean of 14.7 uU/mL. There was a significant decrease in HbA1c % (8.5% ± 2.3 vs. 7.6%±1.6 p=.007) in both genders. There was a significant decrease in hip circumference (cm) (107.2±14 vs. 105.8±13 p=.044). Though not significant, there was also a decrease in FBG (158±83 vs. 152±77, p=0.558). There were significant correlations, at 3 months, between scale weight (kg) and dietary cholesterol (mg) (r=.456, p=.019), BMI (kg/m2) and dietary cholesterol (r=.404, p=.041), BMI and % calories from saturated fat (r=.424, p=.031), insulin (uU/mL) and supplement chromium use (r= −.451, p=.021).

C: The program had a beneficial effect on dietary changes and blood sugar control.