bluuebunny/crossref_metadata_2025 huggingface dataset:
Hemodynamic Regulation of Androgen Receptor‐dependent Inflammatory Signaling
Authors: Browning Elizabeth Anne , Helmke Brian Paul
Published: June 2021

The correlation between timing of vascular disease in women and decreased production of estrogen after menopause suggests that circulating androgens promote vascular inflammation in the absence of protective estrogens. We hypothesized that androgen (R1881) stimulates androgen receptor (AR) in human endothelial cells to induce inflammatory signaling in a manner that is modulated by an atheroprotective shear stress (SS) profile. R1881 treatment for 24 h caused 2‐fold upregulation of AR protein expression and increased AR nuclear localization, whereas steady unidirectional SS (15 dyn/cm2) for 24 h caused 1.2‐fold downregulation of expression and nearly 3‐fold decrease in nuclear localization. Atheroprotective SS prevented AR upregulation regardless of whether cells were pretreated with or sheared in the presence of R1881. R1881 treatment for 48 h induced a nearly 2‐fold increase in VCAM‐1 expression, suggesting that ligand‐activated AR promotes inflammatory signaling. SS abrogated the effects of R1881. These data are the first evidence that SS and ligand differentially regulate AR to modulate inflammatory signaling in human endothelial cells. Supported by NIH grant HL071958 and a UVa CVRC Partners Award.
Stereoselective hydrolysis of epoxy‐eicosanoids by mammalian soluble epoxide hydrolases
Authors: Schmidt Cosima , Fischer Robert , Markovic Marija , Morisseau Christophe , Hammock Bruce D. , Schunck Wolf‐Hagen
Published: June 2021

Various signalling pathways are stereoselectively modulated by epoxy‐eicosanoids which are themselves produced and degraded in a stereoselective manner by CYP enzymes and soluble epoxide hydrolases (sEH). We observed that the contractility of cardiomyocytes and their response to calcium‐overload is decreased by 11(S),12(R)‐epoxyeicosatrienoic acid (EET) and 17(R),18(S)‐epoxyeicosatetraenoic acid (EETeTr). 11(R),12(S)‐EET showed the opposite effect, the corresponding diols were inactive. Cardiac CYP enzymes equally produced both enantiomers of 11,12‐EET from arachidonic acid but converted eicosapentaenoic acid preferentially to 17(R),18(S)‐EETeTr. Purified recombinant sEH enzymes from human, rat and mouse unisonously showed a high stereoselectivity in terms of preferentially hydrolysing the S,R‐enatiomer of 11,12‐EET but the R,S‐enatiomer of 17,18‐EETeTr, e.g. the catalytic efficiency of mouse sEH was 7‐fold higher towards the R,S‐compared to the S,R‐enantiomer of 17,18‐EETeTr. The sEH enzymes also displayed moderate to strict stereoselectivities when hydrolyzing omega‐3 epoxy‐docosahexaenoic acid as well as other regioisomeric EETs. These data suggest that sEH enzymes do not simply reduce the total epoxy‐eicosanoid level but rather play an important role in adjusting the balance between active and inactive enantiomers and those with opposite biological activities.
Effects of EXRD 4605, a soluble epoxide hydrolase inhibitor, on angiotensin II dependent hypertension in rodents
Authors: Weldon Steven M , Matera Damian , Kerr Steven , Berger Valentina , Ingraham Richard , DeLeon Rodney , Madwed Jeffrey B , Kabcenell Alisa
Published: June 2021

EETs are vasoactive AA metabolites generated by the CYP450 enzymatic pathway. EETs exhibit vasodilation in vitro and can be further metabolized to their less active diols, DHET by soluble epoxide hydrolase, sEH. Angiotensin II induces sEH expression in rodents. We examined the effects of EXRD4605 (EX), a sEH inhibitor, on angiotensin II‐induced vascular and blood pressure (BP) responses in rodents. EX is a potent inhibitor of human (Kd=2 nM) and mouse (0.4 nM) sEH and exhibits a cellular ED50 of 2.4 nM. EX induces dose‐responsive (25%–175%) increases in gracilis muscle arteriolar diameters that is blocked in the presence of the CYP inhibitor miconazole. Administration of ang II (100–1000 ng/kg, IV) in anesthetized mice induces acute BP increases (25%–53%) that are blunted (29%*–32%*p<0.05) by pre‐treatment with EX (30 mg/kg). We hypothesized the attenuation of the BP increase is the result of increased EET by EX pretreatment. EX had no effect in sEH gene deleted mice suggesting the role of sEH in this response. In mice with established hypertension (angII 1 mg/kg/d), EX (10 mg/kg) reduced BP from 136 to 117* mmHg compared to 139 mmHg to 142 mmHg in vehicle control. We conclude that pharmacological inhibition of sEH with EX induces vasodilation in resistance vessels ex vivo and attenuates ang II‐dependent hypertension in vivo. These data support sEH as an intriguing target for blood pressure regulation and hypertension.
BLOOD PRESSURE AND RENAL FUNCTIONS IN MALE AND FEMALE sEH KNOCKOUT MICE
Authors: Yan Qingshang , Raksaseri Promporn , Weldon Steven M. , Kabcenell Alisa K , Hebert Steven , Wang Tong
Published: June 2021

We investigated blood pressure (MBP), plasma electrolytes, acid‐base and renal functions in sEH+/+ and sEH−/− mice. In anesthetized male, MBP, GFR, and Na+ and K+ excretions were significantly reduced in sEH−/− than in sEH+/+. MBP was 87.9 vs. 107.0 mmHg, GFR was 0.57 vs. 0.81 ml/min/100g BW, EK was1.06 vs. 0.41 uEq/min/100g BW and FEK was 32.76 vs. 16.72%, respectively. The 24‐hour urine and ENa were also reduced in male sEH−/− compared to sEH+/+, indicating reduced MBP in male sEH−/− is not due to reduction of salt and water absorption in kidneys. In female mice, there were no significant differences in MBP, GFR, and ENa and EK. MBP was 96.6 vs. 101.4 mmHg, GFR was 0.98 vs. 0.88 ml/min/100g BW, EK was 0.69 vs. 0.74 uEq/min/100g BW and FEK was 15.5 vs. 22.3%, respectively. The 24‐hour urine and ENa were not significantly different between female sEH−/− and sEH+/+. All plasma electrolytes and acid‐base parameters were in the normal range, with no difference between all groups of mice. We conclude that male sEH−/− exhibit lower MBP and GFR and reduced ENa and EK with normal plasma electrolytes and acid‐base balance. Female sEH−/− mice show no significant changes in MBP, renal functions and acid‐base parameters, suggesting that EET activity may be more important in males in lowering blood pressure than in females and the reduction of blood pressure in anesthetized males is more likely via vascular effects of increased EETs in sEH−/− mice.
Metabolism of mammalian soluble epoxide hydrolase inhibitors: 1‐adamantyl‐1‐yl‐3‐dodecyl‐urea and its esters
Authors: Watanabe Takaho , Morisseau Christophe , Tsai Hsing‐Ju , Hammock Bruce
Published: June 2021

In mammals, soluble epoxide hydrolase (sEH) is involved in the metabolism of both exogenous and endogenous epoxides. The modulation of endogenous lipid epoxides using sEH inhibitors has therapeutic benefits in both hypertensive and inflammatory conditions. Understanding the route of metabolism should provide information leading to the design of more potent inhibitors. Metabolism of the lead compound, 1‐adamantyl‐1‐yl‐3‐dodecyl‐urea (ADU) and its esters, were investigated. Several metabolites of ADU, such as ω‐hydroxylated and ω‐carboxylate, were found in hepatic microsomes. The ratio of ω‐0/ω‐1 oxidation of ADU in human microsomes was different from rat and mouse. AUDA, which was metabolite from ADU, was potent and metabolically stable inhibitor. To improve solubility, we designed ester compounds. These ester compounds were immediately metabolized to AUDA in hepatic microsomes. AUDA was further metabolized to final metabolite (butyric acid metabolite (β‐oxidation)) in mouse urine.
Endothelium CYP1B1 Expression Mediates a Proangiogenic Phenotype
Authors: Sheibani Nader , Tang Yixin , Sorenson. Christine M
Published: June 2021

Cytochrome P450 (CYP) enzymes catalyze mono‐oxygenase reactions, which not only facilitate excretion of foreign and endogenous lipophilic compounds, but also affect synthesis of physiologically active chemicals. Metabolisms of arachidonic acid by CYP4A and 2C subfamilies generate intermediates with distinct roles in modulation of vascular tone and angiogenesis. CYP1B1 is expressed in endothelial cells (EC) including those in the retina. Furthermore, CYP1B1 deficiency has been associated with abnormal ocular development in the human and mouse. To gain insight into the role CYP1B1 plays in EC function, we have isolated retinal EC from CYP1B1+/+ and CYP1B1−/− mice. We show that CYP1B1−/− retinal EC are less adherent, less migratory, and fail to undergo capillary morphogenesis in Matrigel. CYP1B1−/− retinal EC expressed high levels of thrombospondin‐2 (TSP2) in their culture medium, which was almost absent in medium from CYP1B1+/+ retinal EC. TSP2 is a matricellular protein with potent antiangiogenic activity. Re‐expression of CYP1B1 in CYP1B1−/− retinal EC improved their ability to undergo capillary morphogenesis, and resulted in down‐regulation of TSP2 expression. Our data together indicate that CYP1B1 metabolism of endogenous substrates greatly impacts the EC adhesive and migratory phenotype, at least in part, through suppression of TSP2 production in retinal EC.
Arterial branching morphogenesis
Authors: Simons Michael
Published: June 2021

Branching is the key event in formation of vascular trees. Branching morphogenesis occurs not only during embryonic developments but also in adult tissues in response to a variety of signals such as organ growth, ischemia and tissue injury. Despite its obvious importance, little is known about signals inducing arterial branching, the intracellular signaling cascades responsible for it, or the cell types involved. While studying disruption of synectin gene expression, we noted, both in mice and in zebrafish models, significant abnormalities in arteriogenesis, including a reduction in the arterial tree size and a profound decrease in the number of arterial branches. Remarkably, neither venous nor lymphatic systems seem affected. Thus, it appears that synectin plays a key role in regulation of arterial differentiation as well branching.

Molecular defects in synectin−/− endothelial cells include decreased responses to VEGF, FGF2 and PDGF stimulation, abnormal activation and mis‐localization of activated Rac1 and decreased Erk‐1/2 activation. The latter appears to be particularly important in regulation of arterial morphogenesis.
Skeletal variations in the Fam53A mutant mouse.
Authors: Bergemann Andrew D. , Pagano Anthony S. , Reidenberg Joy S. , Simon Ruth , Balboni Armand L. , Laitman Jeffrey T.
Published: June 2021

This study reports our findings on cranial and postcranial skeletal anatomy of a new mouse line. Our laboratory discovered the Fam53A (DNTNP) gene, the founding member of the Fam53 family of transcription factors, as a marker of the developing dorsal neural tube. It has recently been determined that Fam53 genes participate in the maintenance of stem cell pluripotency. We have created mouse lines carrying targeted mutations of the Fam53A gene. Mice homozygous for the mutations either die in utero, or if they escape to be born, fail to thrive post‐natally. A proportion of the Fam53A mutant mice display skeletal problems, such as polydactyly and incisor overgrowth, reminiscent of the phenotype of Bmp4 hypomorphic mice. This observation is consistent with the known interaction of Fam53 members with SKIP, a modulator of BMP/TGFβ signaling, and suggests Fam53 proteins may be novel components of the BMP/TGFβ pathways. The human Fam53A gene maps to 4p16.3, and is one of a small number of genes deleted in all full‐blown cases of Wolf‐Hirschhorn Syndrome (WHS). As pleiotrophic skeletal defects are a classic feature of WHS patients, it is clinically important to define the anatomical changes that are present in the Fam53A mutant mouse.
In vitro migration of mesenchymal stem cells (MSC) promotes differentiation to chondrocytes
Authors: Khoury Joseph , Geuss Laura R. , Shortkroff Sonya , Berlowitz Tarrant Laurence J.
Published: June 2021

The capacity of MSC to differentiate into numerous cell types, including chondrocytes, has been well studied. However, the effects on differentiation of MSC during migration have yet to be elucidated. To assess migration, MSC or articular chondrocytes (hAC) were seeded in the upper portion of 8μm Boyden chambers with 500μl medium containing 1% BSA, chondrogenic medium with TGFβ (CCM), or basal medium with 10% FBS in the lower portion in the presence or absence of collagen scaffolds (HC) (n=6). Chambers were incubated for 16h prior to counting the migrated cells. To assess differentiation after migration, cells were plated in the upper chamber and allowed to attach for 48h. Then, HC discs were placed in the lower chamber with CCM or basal medium and chambers were cultured for 2 weeks; cell seeded HC discs served as controls (n=9). HC were assayed for sulfated glycosaminoglycan (S‐GAG) accumulation; processed for histology; total RNA was extracted and amplified by RT‐PCR. MSC and hAC preferentially migrated towards wells that contained HC (p<0.01). MSC that migrated produced 70% more S‐GAG than control and 2.43 fold more than hAC (p<0.05). By RT‐PCR, MSC that migrated into HC increased COL2A1 and AGC1 expression while hAC showed signs of dedifferentiation with increased COL1A1. This study reveals that migration of MSCs into a collagen scaffold can enhance differentiation into chondrocytes and ECM production.
Methamphetamine partitions dopamine transporter into a distinct membrane microdomain regulating its function
Authors: Khoshbouei Habibeh
Published: June 2021

Psychostimulants such as methamphetamine (METH) and amphetamine (AMPH) elicit their behavioral effects by acting on the dopamine (DA) transporter (DAT). To explore cellular mechanisms of the exaggerated behavioral responses induced by METH when compared to AMPH, we evaluated these two structurally similar agents on DAT‐mediated functions. Using whole‐cell voltage clamp coupled to microamperometry, we observed METH generates more currents, releases more DA than does AMPH, also accomplishes this at a more negative membrane potential, suggesting a fundamental difference between METH‐occupied and AMPH–occupied DAT. Furthermore, METH increases [Ca2+]i twice as effectively as AMPH, independent of changes in membrane potential. Fluorescence recovery after photobleaching experiments show that METH causes a decrease in the lateral mobility of DAT. These results support our hypothesis that METH causes DAT to partition into a spatially distinct membrane microdomain, promoting kinetically favorable interactions that are necessary to promote a high‐affinity state of the transporter stimulating the reverse transport process and increasing its function. Our findings demonstrate that METH occupancy of DAT evokes distinctive changes in electrical, chemical and lateral mobility of DAT that may, in future studies, be determined to underlie the exaggerated behavioral and addictive properties of METH.
Silencing the Noise: The development and application of small RNAs for genome scale research
Authors: Baskerville Scott
Published: June 2021

The rapid pace of developing siRNA‐mediated gene knockdown as a research tool has challenged biologists and chemists to develop strategies and techniques to simplify its use. Based on discoveries regarding the biogenesis and mechanism of action of both siRNAs and miRNAs, we have developed sophisticated siRNA designs that incorporate a combination of bioinformatics‐based approaches and chemical modifications to enhance their specificity, activity, and delivery. Advanced applications of these molecules include the use of both siRNAs and miRNAs in large scale functional screens. The strategies used in the design and the results of both miRNA and siRNA screens will be discussed.
How are chromosomes moved during mitosis? Biophysics at the kinetochore‐microtubule interface
Authors: Asbury Charles , Powers Andrew , Franck Andrew , Gestaut Daniel , Gonen Tamir , Davis Trisha
Published: June 2021

During cell division, chromosome movements are coupled to the growth and shortening of microtubule (MT) filaments, the tips of which transmit tension to specialized sites on each chromosome, called kinetochores. Recent work suggests that the Dam1 complex, a component of yeast kinetochores, participates in kinetochore‐MT attachment, force production, and regulation of attached MTs, perhaps by forming a ring encircling the MT. To test these hypotheses, we developed an in vitro motility assay where Dam1‐coated beads attach the tips of individual MTs. Like kinetochores, the beads remain tip‐bound and undergo movements driven by MT growth and shortening. By using a feedback‐controlled optical trap, we found that tension applied continuously through Dam1 decreases the likelihood that growing tips will begin to shorten, slows shortening, and increases the likelihood that shortening tips will resume growth. These findings demonstrate how Dam1 can contribute directly to MT‐driven force production and chromosome movement. They also show that tension can modulate filament dynamics directly, without need for additional MT‐modifying factors.
A Temporal Code to generate Specificity in Inflammatory Signaling
Authors: Hoffmann Alexander
Published: June 2021

The immune system responds specifically and appropriately to diverse pathogens. Strikingly, distinct gene expression programs all require the same transcription factor: NF‐kappaB.

Interestingly, different stimuli lead to different dynamic profiles of NF‐kappaB activity. In order to investigate the role of dynamic regulation further, we constructed a mathematical model that recapitulates the control of NF‐kappaB activation by the regulated synthesis and degradation of the three canonical IkappaB proteins.

Guided by computational simulations, we engineered a mutant cell line that is defective in NF‐kappaB temporally controlled amplitude modulation (AM); these cells are still responsive to inflammatory stimuli but, remarkably, lose stimulus‐specificity in gene expression. We therefore postulate the existence of a temporal code to generate stimulus‐specific inflammatory gene expression programs.

I will present computational tools to probe this stimulus‐specific temporal code. By perturbing the parameter space pharmacologically, we provide experimental support for computational predictions. Our studies suggest that the temporal code of inflammatory signaling can be harnessed to maximize the specific efficacy of pharmacological agents.
Structural studies of RGS9/Gβ5
Authors: Cheever Matthew , Snyder Jason T. , Gershburg Svetlana , Siderovski David P. , Harden T. Kendall , Sondek John
Published: June 2021

Regulator of G protein Signaling (RGS) proteins were originally identified as GTPase‐activating proteins (GAPs) for heterotrimeric G protein α subunits. However, many RGS proteins possess highly conserved domains in addition to the signature RGS box responsible for GTPase activation. For example, the R7 subfamily of RGS proteins (RGS6, −7, −9 and −11) contain a distinctive G‐γ‐like (GGL) domain that mediates specific and obligate heterodimer formation with the atypical G protein subunit, Gβ5. Recent work describing the functional and structural integration of Gβ5/R7 dimers within conventional GPCR machinery will be discussed.
Teaching Undergraduates Pharmacology using Flow Cytometry
Authors: Stoyan Tanja , Zhang Xin , Hoo Linda Soo , Williams Melanie , Thrower Doug , Vandenberg Carol
Published: June 2021

The flow cytometer is a mainstream instrument for analyzing cells and is widely used in research and clinical laboratories. However, its use in the teaching laboratory has been limited due to instrument cost and extensive training required. As a result, few biology and pharmacology students understand how this instrument works and how to interpret the data it generates. We have used a new generation flow cytometer to teach senior level undergraduate students basic principles of pharmacology. Based on protocols by Diane Fishwild et al. (2004, Guava Technologies, Inc.), we have developed two exercises for the teaching laboratory that study the effects of drugs on human cancer cells. In the first exercise students screen a library of 80 anti‐cancer and anti‐inflammatory drugs for their ability to induce apoptosis or cytotoxicity in human Jurkat cells. In a second computer‐based exercise, the students determine the EC50 values of different drugs with raw data generated by a flow cytometer. Besides learning the principles of flow cytometry, students learn to analyze large data files with intuitive flow cytometry analysis software, to organize scientific data in tables and graphs, and to draw scientific conclusions from their findings. We show that the flow cytometer is an excellent teaching tool, and that a new generation of user friendly instruments has made it easy to use the technique in the undergraduate laboratory.
How do inquiry‐ based physiology laboratories improve learning in majors versus non‐majors courses?
Authors: Knabb Maureen , Rieser‐Danner Loretta , Casotti Giovanni
Published: June 2021

We have incorporated inquiry‐based physiology labs in upper level majors and lower level non‐majors courses. Assessment consisted of oral presentations of experiments designed by students and pre‐ and post‐course surveys for both majors and non‐majors. Differences in assessment included inquiry‐based questions on written exams for non‐majors while majors had written lab reports, lab notebooks, and final projects. Assessment results of our pre/post surveys indicated that the change in overall confidence scores from pre‐ to post‐test were statistically significant each semester (p < .001 for 200 non‐ major students and 44 majors). The greatest increase in confidence ratings occurred for those items that were specifically targeted by our curriculum modifications. One important assessment difference between the majors versus non‐majors courses was in student's perceptions of the types of labs that assisted their understanding of physiological content. Majors identified the presentation labs while non‐majors preferred the non‐presentation labs for learning course material. We are currently investigating the use of alternate curricular strategies and assessment tools to effectively incorporate physiology content and inquiry process skills in both majors and non‐majors labs. NSF CCLI 0509161.
The epicardium has potential in cardiac healing and regeneration in mammals
Authors: Martin Jody Carl
Published: June 2021

The proepicardium is a small transient structure that develops at the base of the heart, adjacent to the septum transversum. It is made up of pluripotent cells that migrate to the developing heart to give rise to fibroblasts, vascular support cells, cardiomyocytes, and the epicardium. It is unclear if and when proepicardial cells lose their pluripotential.

After an ischemic event, such as a heart attack, the heart compensates for cardiomyocyte loss with fibrosis to maintain the integrity of the cardiac wall, preventing rupture. The result is a stiffer and less effective pump.

These experiments show the ability of embryonic epicardial cells to contribute to myocardial healing In Vitro after transmural laceration. Following injury, the whole embryonic heart is explanted and cultured. Further studies in adult mice show that epicardial cells are present at the site of injury in a surgical model of cardiac damage. Further studies will show the fate of cells arising from the epicardium that contribute to the healing response following cardiac injury.

These preliminary results suggest that epicardial cells are able to migrate and differentiate in response to injury. The epicardium may be a source of pluripotent cells that, with the right encouragement, may have the ability to contribute new vasculature as well as new myocytes after an ischemic event.
Self orientation of cell and cell‐secreted collagen in a tissue‐engineered cornea grown on microstructured polystyrene
Authors: Auger François A. , Veres Teodor , Cui Bo , Deschambeault Alexandre , Carrier Patrick , Germain Lucie , Guillemette Maxime D.
Published: June 2021

The corneal stroma is composed of many layers of fibroblasts and collagen that are oriented longitudinally within a layer and the layers display a shift of approximately 60‐degree angle between each other. This type of structure is known to be responsible for the transparency of the cornea. Using the self‐assembly approach to cultured cell sheets, we evaluated how cell‐secreted collagen reaction could be influenced by external physical cues. Microstructured polystyrene samples used for cell culture were prepared by hot embossing, replicated and transferred from a Si master of different grating periods and linewidths to a polystyrene substrate. We observed that cell division occurred only in the longitudinal axis of the gratings instead of the random cell orientation seen in the control. Cells forming the first layer on the substrate aligned themselves in the same longitudinal direction as the gratings. We also observed that after 7 days in culture the cells of the second layer, forming on top of the first one, aligned themselves at an angle of approximately 60 degree from the first one, and so did the cell‐secreted collagen fibres. These results are very promising for the reconstruction of autologous tissue‐engineered cornea since optimization of transparency is of paramount importance for the patients who will benefit from this type of surgery.

Financial support is provided by the National Research Council‐Canadian Institutes of Health Research (grant 66473).
High specificity and sensitivity of olfactory bulb synucleinopathy for Lewy body disorders
Authors: Beach Thomas G , White Charles L , Hladik Christa , Sabbagh Marwan N , Connor Donald J , Caviness John N , Sue Lucia I , Sasse Jeanne , Bachalakuri Jyothi , Akiyama Haru , Adler Charles H
Published: June 2021

Recent reports have suggested a high prevalence of olfactory bulb synucleinopathy in Lewy body disorders but there have been no estimates of the sensitivity or specificity of this as a diagnostic test. We performed α‐synuclein immunostaining on paraffin‐embedded sections of olfactory bulb from a series of neuropathologically‐diagnosed subjects from the Sun Health Research Institute Brain Donation Program/Arizona Parkinson's Disease Consortium. Included in the series were 76 normal elderly individuals, 8 subjects with incidental Lewy body disease (ILBD), 63 with Parkinson's disease (PD) and 79 with dementia with Lewy bodies (DLB). Sections were stained with a sensitive method employing pretreatment with proteinase K. The ratios of positive to negative olfactory bulb staining for each diagnostic category were: normal 5/71; ILBD 8/0; PD 57/6; DLB 74/5. An α‐synuclein‐positive olfactory bulb has 92% sensitivity and 93% specificity for the diagnosis of PD versus normal while for DLB it is 94% and 93%, respectively. As all 8 cases of ILBD had a positive olfactory bulb it appears that olfactory bulb synucleinopathy may also be a reliable marker of presymptomatic Lewy body disorders. Supported by the AZ Dept. of Health Services, NIA P30 AG19610 and the Prescott Family Initiative of the Michael J. Fox Foundation.
Expression of alpha‐synuclein (AS) in autonomic neurons is increased in Parkinson's disease (PD)
Authors: Munoz David G , Gold Andrea
Published: June 2021

Background and objective

We have previously shown that expression of the main protein constituent of Lewy bodies, AS, is detectable in autonomic neurons in a subset of the adult population. The objective was to determine wheteher expression has greater prevalence or intensity in PD than in the general population or patients with Alzheimer's disease (AD)

Methods

Archival autopsy samples of colon obtained from adult patients with pathologically confirmed PD, AD, or no neurological disease were immunostained with anti AS antibody KM51 diluted 1:70 and visualized with a MACH 3 polymer kit. The expression was scored as absent (AS‐), or present (AS+) in perikarya or terminals, semiquantitevely rated 1 to 3.

Results

Just over ½ of 70 subjects with no neurological disease were AS+: neither prevalence nor intensity varied by age over 40 years. Differences between sexes were not significative (NS). All 10 PD patients were AS+ as compared to 25 of 54 age‐matched subjects (p< 0.002). The average intensity was also significantly greater (p< 0.002). In contrast 3 of 8 AD cases were AS+ (NS as compared with normal population)

Conclusion

AS+ in autonomic neurons is common in the adult population. This trait is significantly more prevalent and intense in PD, but not in AD. Either the development of PD increases AS expression, or PD preferentially develops in AS+ subjects
Tissue Inhibitor of Metalloproteinase‐1 Levels Predict Left Ventricular Dilation following Myocardial Infarction in Mice
Authors: Escobar Gladys Patricia , Dai Qiuxia , Lindsey Merry
Published: June 2021

Myocardial infarction (MI) is a leading cause of congestive heart failure. Currently, biomarkers to predict adverse left ventricle (LV) remodeling post‐MI are lacking. The objective was to evaluate extracellular matrix (ECM) and adhesion molecule (AM) gene levels post‐MI to determine biomarkers of LV remodeling. We evaluated unoperated (n=6) and 7 days post‐MI mice (n=7) by echocardiography. Remote non‐infarcted (LVR) and infarct region (LVI) were examined and compared to unoperated LV control (LVC). RT2 profiler PCR array for ECM and AM was used to determine differentially expressed gene levels in post‐MI LV. The most upregulated gene was α1 collagen I, which increased from 6.0±0.2 normalized threshold cycles (ΔCt) in LVC to 2.6±0.8 normalized ΔCt in LVR to 0.5±0.9 normalized ΔCt in LVI (p<0.001 between all groups). The square of the Pearson product moment correlation coefficients were calculated for end diastolic dimensions (EDD) (y) and gene levels (x). Positive correlations were found for α1 collagen I (R=0.70), α1 collagen II (R=0.72), periostin (R=0.70), and tissue inhibitor of metalloproteinase‐1 (TIMP‐1; R=0.70; all p<0.001). Plasma TIMP‐1 levels increased from 0.83±0.05 ng/mL in LVC mice to 3.70±0.63 ng/mL and MI mice (p<0.01). Plasma TIMP‐1 levels correlated significantly with EDD (R=0.697; p<0.001). Together, our data suggest TIMP‐1 is a useful plasma biomarker for LV diastolic dimension changes.
Chondrichthyans have a bulbus arteriosus at the arterial pole of the heart
Authors: Durán Ana Carmen , Rodríguez Cristina , Fernández Borja , de Sola Luis Gil , Macías David , Arqué Josep Maria , Sans‐Coma Valentín
Published: June 2021

The aim was to test our hypothesis that the bulbus arteriosus is a genuine arterial pole component of the primitive heart of gnathostomates. We examined adult and embryonic hearts of a number of chondrichthyan species by means of histochemical and immunohistochemical techniques. In all cases, a conspicuous intrapericardial, nonmyocardial, tubular segment connected the myocardial conus arteriosus with the ventral aorta. This segment could be well distinguished from the aorta because it 1) showed a different arrangement of the tissue layers; 2) was covered by epicardium; and 3) was crossed by the coronary arteries. Experimental in vivo labelling with the fluorescent nitric oxide marker 4,5‐diaminofluorescein diacetate indicated that this segment is homologous to the bulbus arteriosus of teleosts. Therefore, we conclude that the bulbus arteriosus is not an apomorphy of the actinopterygians. It appeared in an earlier period of the vertebrate story. In addition, our findings, together with data from the literature, suggest that, from the evolutionary view, the bulbus arteriosus of the chondrichthyans is the precursor of the basal portions of the aorta and pulmonary artery of tetrapods, a fact which raises the question whether there is a secondary heart field in phylogenetically basal vertebrates. This study was supported by grant CGL06‐00693 (Ministerio de Educación y Ciencia, Spain).
Vascularization fo the cardiac conduction system in humans by the right coronary artery
Authors: Herrera Patricia M , Rojas Adelina , Cichero Carlos F. , Barceló Adrián J. , Arteaga Manuel , Abuin Gustavo
Published: June 2021

The vascularization of the cardiac conduction system is a topic frequently mentioned and scarcely photographed, the purpose is to demonstrate in pictures the irrigation of the right and left branch of the His bundle ( atrioventricular junctional tissue ) by the superior septal branch of the right coronary artery.

Material and Method

Twenty fresh human hearts were perfunded with Microfil ® with the method and dissection previously published by our group.

Results

We demonstrate with photographs the irrigation of the cardiac conduction system by the right coronary artery in our cases.

Conclusions

This would explain the variation in presentation of the AV block associated with the acute myocardial infarction with the oclusion of the right coronary artery in its origin or near. The importance of this vessel is relevant in Ross technique and other cardiac surgery procedures.
Angioarchitecture of the root of the equine penis
Authors: Freeman Larry Eugene
Published: June 2021

Equine penile vascular casts were prepared by perfusion with Batson's compound via the abdominal aorta in post mortem specimens of adult male ponies to clarify vascular features of the penile root (bulb and crura). Casts revealed vascular details of (1) the bulb and corpus spongiosum penis (CSP), the bulb having slightly larger diameter than the continuing CSP; (2) vascular spaces of the two crura, each with the proximal‐most part of the corpus cavernosum penis (CCP), and (3) proximal portion of the penile body, indicated by fusion of the two CCP. Each internal pudendal artery gave rise to only the artery of the bulb; there was no branch that supplied the CCP at the crus or that continued distally as the dorsal artery of the penis as found in other domestic mammals. The deep artery of the penis into the CCP for effecting penile erection was found to come solely from a branch of the obturator artery. In contrast to illustrations/descriptions in many veterinary anatomy atlases/texts, we were unable to demonstrate any anastomostic connections between the internal pudendal arterial and obturator arterial supply to the penile root. Multiple small, short veins passed from each crus to join the ipsilateral side of the plexiform dorsal vein at ischial arch level. Multiple small short veins rather than one large vein may enhance restriction of venous outflow from the crus during ischiocavernosus muscular contraction for erection.
3D‐Micro‐Ultrasound Documents Diltiazem Mediated Changes in Central Aortic Hemodynamics
Authors: Bunzel Michelle M , Campbell Barry , Azer Karim , Tong Christopher , Ramcharan Sharmilee , Saini Kavita , Fancourt Craig , Wang Shubing , Walker Matthew
Published: June 2021

3D‐micro‐ultrasound imaging has emerged as a valuable research tool to co‐register vascular physiology. This co‐registration allows serial interrogation of arterial wall integrity, blood flow and stiffness. We quantified hemodynamic changes associated with the acute administration of Diltiazem, to benchmark high throughput imaging preclinically.

Six male SD rats implanted with DSI pressure probes in the femoral artery were imaged using the VisualSonics Vevo770 (25MHz probe) system. Baseline Doppler measurements in the abdominal aorta were acquired followed by administration of 3mpk Diltiazem i.v. Measurements were then acquired during the acute phase of drug administration and every 5’ over a 60’ time course. Administration resulted in an increase from baseline in cardiac period, diastolic time and Doppler peak‐to‐peak/valley‐to‐valley interval over 60’. Heart rate decreased relative to baseline as did the mean Doppler velocity and max dp/dt (positive/negative). Decreases in systolic BP, diastolic BP, MAP and pulse pressure were also seen.

Our novel assessment of the anti‐hypertensive vascular and cardiac effects of Diltiazem is attributable to its primary indication and consistent with the literature. Evaluation of vascular structure and function in real, fiducially aligned, spatial and temporal resolution, unveils a powerful imaging tool for the in vivo characterization of compound efficacy.
Sympathetic dysautonomia despite the absence of sympathetic neuron loss in Egr3‐deficient mice
Authors: Tourtellotte Warren Gerard , Honasoge Avinash , Eldredge‐Tudor Laurie
Published: June 2021

The sympathetic nervous system (SNS) is essential for tissue and organ homeostasis. Despite the fact that it is the target of a wide variety of congenital and degenerative diseases, the mechanisms involved in sympathetic neuron differentiation and target tissue innervation are still very poorly characterized. Sympathetic neurons depend upon target tissue‐derived trophic factors such as nerve growth factor (NGF) for survival and differentiation, but the downstream signaling molecules that regulate new gene expression have not been fully defined. We recently identified the NGF‐regulated transcription factor Egr3 as an essential mediator of normal SNS development. Egr3‐deficient mice can survive to adulthood but they have profound physiologic dysautonomia reminiscent of human disease. The dysautonomia is associated with sympathetic neuron loss and abnormal target tissue innervation. Thus, it has not been clear whether Egr3 is primarily involved in gene regulation related to sympathetic neuron survival, differentiation or both. To examine whether Egr3 has a direct role in sympathetic neuron survival, Egr3 knockout mice were mated to mice lacking the pro‐apoptotic molecule Bax. We found that both physiologic and Egr3‐dependent sympathetic neuron death was completely inhibited in Egr3−/−:Bax−/− double knockout mice, yet the mice still had dysautonomia and target tissue innervation defects. These results demonstrate that Egr3 has an essential role in sympathetic axon outgrowth and target tissue innervation that is independent of any role it may have in sympathetic neuron survival. Moreover, the results reaffirm the concept that human dysautonomias, which are virtually always associated with neuron loss, are not necessarily caused by primary defects in neuron survival. Support: NIH and HHMI.
CD133 RNA expression marks a rare and widely distributed cell population in the adult mouse brain
Authors: Olausson Karl Holmberg , Learner Emily , Ligon Keith Lloyd
Published: June 2021

The transmembrane glycoprotein, CD133, has been used as a marker for isolation of stem/progenitor cells from normal tissues and human tumors, including the central nervous system. However, in vivo studies of the normal CNS cell types that express CD133 protein have yielded controversial results, potentially due to limitations inherent to protein analysis by immunohistochemistry. As an alternative, we have used RNA in situ hybridization to examine CD133 expression during embryonic and postnatal mouse development. During early development, CD133+ cells are abundant within the dorsal neocortical ventricular zone. In contrast, adult CD133+ cells are a rare, but widely distributed, population of cells found throughout all regions of the adult CNS. Adult CD133+ cells are more frequently seen within compact white matter tracts but are rarely found in stem/progenitor cell niches (SVZ, SGZ) of the adult brain. Co‐localization of CD133 RNA with well‐characterized CNS progenitor and lineage markers shows that normal CD133+ cells express the progenitor cell marker Olig2, an association previously reported in human glioma stem cells. In summary, these findings suggest that CD133 may mark a unique population of distributed CNS progenitor cells whose normal in vivo potential remains to be elucidated.
Identification of Residues in SV40 Large Tumor Antigen Important for DNA Replication and Topoisomerase I Binding
Authors: Neal Krista , Simmons Daniel
Published: June 2021

Eukaryotic DNA replication is a vital molecular process which is not fully understood. Simian Virus 40 provides an ideal model system. Its genome encodes the protein large T‐Antigen (T‐Ag), which orchestrates the initiation of replication. T‐Ag interacts with host cellular protein Topoisomerase I (Topo I). Topo I binds a T‐Ag monomer at two sites, one near the N‐terminal domain (residues 83–160) and one at the C‐terminal end (resides 602–708). Preliminary data suggests that T‐Ag's J‐domain and hinge region residues 101–109, may be involved in Topo I binding. We generated single point mutations in full‐length T‐Antigen and in a N‐terminal construct that contains residues 62–262. In vitro replication assays identified three mutants, N99S, L103Q, and C105F to be replication deficient. Furthermore, DNA replication was not stimulated by the addition of Topo I, as is WT T‐Ag. ELISAs showed mutant T‐Ags N99S, E100A, L103Q, F104V, and E107G have a decrease in Topo I binding as compared to the WT fragment. These data indicate that this region is important for Topo I binding and residues N99 and L103 may be necessary for DNA replication. Other assays will be performed to verify that the mutants’ deficiency in DNA replication is due to a defect in Topo I binding and not to other causes. This work is funded by a PHS Grant from the National Cancer Institute to Dan Simmons and a Howard Hughes Medical Institute Scholarship to Krista Neal.
A real‐time FRET assay to measure the temporal order of events in the clamp loading reaction catalyzed by the Escherichia coli gamma complex
Authors: Bloom Linda B. , Anderson Stephen G. , Paschall Christopher O. , Thompson Jennifer A.
Published: June 2021

A fluorescence resonance energy transfer (FRET) assay was developed to monitor protein‐protein interactions between the E. coli γ complex clamp loader and the β clamp directly in solution and in real time. A fluorescent donor is placed on the δ’ subunit, a unique subunit in 7‐subunit clamp loader. The clamp is a homodimer, and each monomer is symmetrically labeled with a nonfluorescent quencher. This FRET assay, in combination with real‐time DNA binding and ATP hydrolysis assays, was used to dissect the temporal order of events in the clamp loading reaction that occur when a pre‐formed clamp loader•clamp complex binds DNA. DNA binding initiates hydrolysis of three molecules of ATP, which in turn, triggers the clamp loader to release DNA and then the clamp. These results will be discussed in terms of a model in which interactions that the clamp loader makes with individual molecules of ATP (or ADP), the clamp, and DNA promote a series of conformational changes in the clamp loader. These changes modulate interactions with the next binding partner to favor a defined temporal order of events that leads to a productive clamp loading reaction.
PREFERENTIAL DIMETHYLATION OF HISTONE H4‐LYSINE 20 BY SUV4‐20
Authors: Yang Hongbo , Pesavento James J. , Wallrath Lori L. , Kelleher Neil L. , Mizzen Craig A.
Published: June 2021

Mounting evidence suggests mono‐ (1m), di‐ (2m) and trimethylation (3m) of histone H4 at lysine 20 (K20) in higher eukaryotes are functionally distinct, involving in gene activation, DNA damage responses, and heterochromatin function, respectively. The SET domain proteins PR‐Set7 and Suv4‐20 have been implicated in forming 1mK20‐H4 and 3mK20‐H4, respectively, but enzymes for 2mK20‐H4 have not been unequivocally identified. Using RNAi targeting SET domain proteins repertoire in Drosophila in conjunction with Top Down mass spectrometry in a systematic screen for K20 methyltransferases, we discovered Suv4‐20 preferentially forms 2mK20‐H4 accounting for more than 80 % of all H4 in addition to forming 3mK20‐H4 comprising less than 5 % of all H4 in human and Drosophila cells. HP1 is required for normal levels of 3mK20‐H4, but not 2mK20‐H4, suggesting HP1 regulates Suv4‐20 product specificity. Marked alterations in K20 methylation levels due to Suv4‐20 depletion did not affect H4‐K16 acetylation, revealing these modifications are not competitive in vivo. Our data implies Suv4‐20 proteins have at least two distinct functions: directing dimethylation throughout the genome to facilitate DNA damage responses or other processes and directing rarer trimethylation to contribute to telomere function or other processes localized at heterochromatin.

Work is supported by Roy J. Carver Charitable Trust (grant 04‐76).
Synergistic regulation of erythropoietin receptor (EPO‐R) expression by sense and antisense EPO‐R transcripts in the canine lung
Authors: Zhang Quiyang , Zhang Jianning , Moe Orson W. , Hsia Connie C.W.
Published: June 2021

We had found that EPO‐R protein is upregulated in growing canine lungs following pneumonectomy (PNX) (Am J Physiol 287:L1107, 2004). We also found both sense and anti‐sense EPO‐R transcripts (sEPO‐R and asEPO‐R) to be upregulated in post‐PNX remaining lungs. We hypothesized that sEPO‐R and asEPO‐R interactions modulate EPO signaling in lung growth. We cloned a canine asEPO‐R cDNA, fully complementary to the sense strand of EPO‐R gene starting from 2.5 kb 3′‐ to the sense stop codon, and extending into the 5′UTR of sEPO‐R transcript. The asEPO‐R transcript harbors two putative open reading frames but it is not clear if they translated. The asEPO‐R and sEPO‐R transcripts co‐localize to bronchiolar epithelium and alveolar capillary endothelium. In HEK 293 cells transfection with sEPO‐R (+FLAG tag) cDNA alone increased EPO‐R protein expression, detected by anti‐EPO‐R and anti‐FLAG. At a constant sEPO‐R cDNA level, co‐transfection with escalating levels of asEPO‐R cDNA further increased recombinant EPO‐R protein expression. Thus, both sEPO‐R and asEPO‐R transcripts contribute to in vivo EPO‐R upregulation in the remaining lung post‐PNX. These data demonstrate novel synergism between complementary sense‐antisense transcripts in response to physiological stimulation in a robust model of induced lung growth. Supported by NHLBI RO1 HL40070, 62873, 45716, 54060, DK48482, DK‐20543 and Simmons Family Foundation.
Regulated Alternative Splicing During Myogenesis
Authors: Hall Megan P. , Nagel Roland J. , Ares Manuel
Published: June 2021

Transcriptional analysis of the human genome reveals that alternative pre‐mRNA splicing (AS) occurs in approximately 60% of genes. AS contributes to human proteome complexity and there are examples of AS regulation contributing to proper development in multicellular organisms. While many studies described how developmental gene expression changes affect stem cell identity, pluripotency and differentiation, few addressed the role of AS regulation in cell fate decisions. Recent genome‐wide methods for analyzing AS, such as alternative splicing microarrays, permit identification of numerous regulated AS events in response to cellular stimuli. We are examining AS regulation during development using mouse myoblast (C2C12) cells. We identified hundreds of AS events that are regulated during myogenesis or osteogenesis. Motif searching revealed an ACUAA motif present downstream of exons whose inclusion increases during myogenesis. We previously identified this motif near exons activated in adult muscle compared to other mouse tissues using microarrays (1). Recently other groups (2–4) found a similar motif enriched in conserved regions near alternative exons. We selected the Capzb transcript, containing three ACUAA‐like elements downstream of its regulated exon, for further study. Using transiently transfected wild type and mutant reporter constructs, we found that the highly conserved ACUAA‐containing region, but not two other conserved regions, is required for proper exon inclusion. These motifs are also required for inclusion in non‐muscle cells, indicating that this motif might function generally downstream of regulated exons. We are currently attempting to identify ACUAA‐interacting factors.