bluuebunny/crossref_metadata_2025_split huggingface dataset:
Muscarinic M2 selective antagonism prolongs 5′‐AMP‐induced hypometabolism
Authors: Strahota Mary Caitlin , D’Alecy Louis George , Zwemer Charles Frederick 
Published: June 2021

Recent studies have shown that exogenous 5′‐adenosine monophosphate (5′‐AMP) induces a dose‐dependent hypometabolic state (HMS) in mice. Using indirect calorimetry, implantable telemetric probes, 5′‐AMP (913mg/Kg, i.p.), broad‐spectrum (atropine sulfate, 2mg/Kg i.p.), and subtype muscarinic antagonists (pirenzepine (M1), 0.2mg/Kg i.p. and methoctramine (M2), 0.2 mg/Kg i.p.), we tested the hypothesis that much of this effect in mice (C57BL6) was mediated by the parasympathetic nervous system. Atropine prolonged the HMS by 2.7 fold. Selective blockade of M2 similarly extended HMS while M1 blockade did not. Respiratory exchange ratio (RER) was elevated dramatically (≥ 1.5) during the HMS with atropine, but was lower (p=0.054) with M2 blockade suggesting a more adaptive steady state.These preliminary data suggest the 5′‐AMP‐induced HMS may be sustained by the withdrawal, inactivation, or blockade of tonic M2 effector mechanisms.
The Role of Reactive Oxygen Species in Renal Blood Flow Autoregulation
Authors: Just Armin , Arendshorst William J. 
Published: June 2021

Autoregulation of renal blood flow (RBF) is mediated by a myogenic response (MR), tubuloglomerular feedback (TGF) and by a third mechanism that is slower than MR and TGF. Reactive oxygen species (ROS) are known to contribute to acute agonist‐induced renal vasoconstriction and to enhance TGF. Little is known about the role of ROS in RBF autoregulation and underlying mechanisms.Autoregulatory mechanisms were assessed from the response of RBF to a rapid step‐increase in renal artery pressure in rats. MR was derived from resistance changes within the first 5 s, TGF from those between 5 and 25 s, and the third mechanism from 25–100 s. During control, overall autoregulation was 85±6% of perfect. MR provided autoregulatory efficiency of 63±6%, TGF 41±5% and the 3rd mechanism 5±6%. Inhibition of NAD(P)H oxidase by apocynin attenuated overall autoregulation to 62±7% (p<0.05) and MR to 39±4% (p<0.001) but barely affected TGF and 3rd mechanism (32±3 and 14±4%, p>0.06). Inhibition of nitric oxide synthase by LNAME markedly augmented MR (130±16%, p<0.001) leaving little room for TGF and 3rd mechanism. During LNAME, apocynin strongly blunted MR (to 56±11%, p<0.01), reversing the effect of LNAME. The superoxide dismutase (SOD) mimetic tempol tended to diminish MR similar to apocynin (38 vs. 52%, p>0.08).We conclude that ROS contribute to RBF autoregulation by strengthening MR in the normal kidney. This effect is mainly due to superoxide rather than H2O2 and does not require NO but instead is blunted by NO. Superoxide plays a major role in facilitating the modulation of MR by NO in the renal microcirculation.Supported by NIH (HL‐02334) and Arthur C Guyton Award.
Admitting Health Care Professionals Other Than Registered Nurses into a Physician Assistant Program: A Comparison Based on a Pathophysiology Course
Authors: McCleary Vikki L. , Hosford Charles C. , Aslagson Samantha M. 
Published: June 2021

In 2006, University of North Dakota Physician Assistant (PA) Program initiated a pilot project of enrolling, in addition to Registered Nurses (RN), other health care professionals (HCP). The purpose of this study is to begin assessing what effect this program change will have upon the PA pathophysiology course.Demographic data were obtained from those enrolled in the first class. Demographic and performance variables were analyzed using analysis of variance.Thirty‐one students (17 RN and 14 HCP) were admitted. Mean age upon matriculation was: RN ‐ 39.1 years; HCP ‐ 34.1 years. Mean years of clinical experience was 12.7 and 7.9 years respectively. Mean objective multiple‐choice exam scores were 77.7% (RN) and 81.6 % (HCP). Based upon exam scores only, 65% of RN and 43% HCP students would have performed below a grade of 79%.Sample data showed RNs have more years of clinical experience, are older, and have lower objective exam performance than HCP. Results from the analyses found moderate effect sizes, but due to the small group sizes power was low; therefore, results were not significant.The information obtained has stimulated an in‐depth discussion of course content, format and modes of delivery.
Reactive Oxygen Species (ROS) in Human Breast Cancer cell lines Differing in Malignancy: An Electron Paramagnetic Resonance (EPR) Study
Authors: Xing Guoqiang , Romanyukha Alex , Bunger Rolf 
Published: June 2021

The development of breast cancer is a multifactorial process whose mechanism is still largely unknown. We hypothesized that ROS and oxidative stress may play a critical role in breast cancer etiology and progression and be associated with the proliferation potencies of breast cancer cells. By using the electron paramagnetic resonance (EPR) spin trapping technique, we did real‐time quantitative estimation of ROS production and thus the oxidative stress in three human breast cancer cell lines with known different malignancies (MD‐MBA 468>MCF‐7>T47D). Our data showed the lowest basal superoxide radical (O2−) level in the more malignant MD‐MBA 468 breast cancer cells compared to the other two cell lines. The T47D cells, with the least malignant potency, showed the highest basal level of ROS. Treatment of either 14h hypoxia (1% O2) or 8 Gy gamma‐ray irradiation increased superoxide levels in T47D and MCF‐7 cells but not in the more advanced MD‐MBA 468 cells.Conclusion: more malignant human breast cancer cells are characterized with a reduced ROS production. Further study in this line will advance our understanding about the role of ROS in the carcinogenesis and cancer cell apoptosis.
AMP Kinase (AMPK) activator 5‐aminoimidazole‐4‐carboxamide‐1‐β‐D‐ribofuranoside (AICAR) alters vascular smooth muscle vasomotor responses in thoracic aorta rings from spontaneously hypertensive (SHR) and Wistar‐Kyoto (WKY) rats
Authors: Ford Rebecca J , Rush James W.E. 
Published: June 2021

The metabolic regulatory role of AMPK is well established but potential mechanisms of AMPK in vascular control are not well defined. Aortic rings were isolated from 16–20wk old male SHR and WKY rats and assessed using myography to determine potential vasomotor effects of AICAR. There was a significant drop in resting tension after endothelium‐denuded rings were incubated with AICAR (A; 2mM, 30 min) in SHR (control (C) = −0.14±0.06g, A = −0.97±0.09g, p<0.01) but not in WKY vessels (C = −0.26±0.06g, A = −0.24±0.08g, p=NS, strain*drug p<0.01). Both SHR and WKY denuded vessels relaxed upon exposure to AICAR following phenylephrine pre‐constriction (PE 10−7M; SHR: C = 2.1±2.2%, A = 41.4±11.9%, p<0.01; WKY: C = 3.0±2.2%, A = 23.7±9.7%, p<0.01). In denuded vessels pre‐exposed to AICAR, potassium chloride dose‐response curves were right‐shifted in both SHR and WKY (SHR C vs A p<0.01, WKY C vs A p<0.01) and developed tension to increasing PE concentrations was blunted in both strains at all concentrations (max tension SHR: C = 2.07±0.19g, A = 1.14±0.21g, p<0.01; WKY: C = 2.57±0.19g, A = 1.98±0.21g, p<0.01, strain*drug p<0.01). These results suggest that AMPK can influence vasomotor function in SHR and WKY aortic rings.Funded by the Heart and Stroke Foundation of Ontario
Incorporating radiology into medical gross anatomy: A study of its efficacy in learning spatial relationships
Authors: Lufler Rebecca Sage , Zumwalt Ann , Hoagland Todd M 
Published: June 2021

Radiological imaging is the depiction of anatomical structures and the spatial relationships among them. Visual‐spatial ability (VSA), the ability to mentally manipulate objects in 3D, is an important skill for medical students to apply their anatomical knowledge in the clinical setting. To date, no studies have investigated the effects of cadaver specific CT scans in the gross anatomy lab on students' learning and VSA. We hypothesize that there will be a positive relationship between VSA and practical exam scores, and that use of CT scans will be associated with practical exam scores and increases in students' VSA. 183 first year medical students were provided with cadaver CT scans, and students self selected into groups based on their time spent viewing the scans in lab. VSA was assessed by the Mental Rotations Test, given during orientation and at the end of the course. During the Back and Extremities section, 35% of the students used the CT scans in lab. Preliminary data suggest students who used the CT scans scored significantly higher on the practical exam (p=0.0086). Average VSA of those who used the CT scans was slightly lower than those that did not, but there was a positive correlation between VSA and practical exam scores (p=0.019, r=0.31). This research is supported by the LWW/AAA Education Research Scholarship.Grant Funding SourceLWW/AAA Education Research Scholarship
Two Distinct Sites on Gβγ are Required for Binding to the N‐Terminus Versus the Activation Site on Adenylyl Cyclase
Authors: Brand Cameron S. , Sadana Rachna , Malik Sundeep , Smrcka Alan V. , Dessauer Carmen W. 
Published: June 2021

The heterotrimeric G protein α and βγ subunits are important regulators of adenylyl cyclase (AC), which controls synthesis of the second messenger cyclic AMP. In the inactive state, critical effector sites on Gα and Gβγ are not available. When activated, the subunits either dissociate or rearrange to expose these sites to effectors. However, we have previously shown that AC5 binds to inactive Gs heterotrimer at its N‐terminus (NT), while Gβγ conditional stimulation of AC5 is independent of this binding site. Both FRET and BiFC in live cells support interactions between Gβγ and AC5 that are at least partially dependent on the NT. We hypothesize that Gβγ utilizes two different surfaces to interact with the NT versus the activation sites on AC5 and AC6. In support of this hypothesis, mutations of the Gβγ effector surface were tested for Gβγ conditional stimulation of AC5 and for binding to AC5‐NT. Several Gβγ mutants, including W99A, are unable to support Gβγ activation, but have no effect on Gβγ binding to AC5‐NT. We have also utilized the SIGK peptide as a competition inhibitor of Gβγ “hotspot” interactions. SIGK is a strong inhibitor of Gβγ stimulation of AC6, but has little effect on Gβγ binding to AC6‐NT. Furthermore, Gβγ binds to the NT of many AC isoforms, suggesting that AC anchoring of heterotrimer may be a common theme for ACs to facilitate regulation by both α and βγ subunits.Supported by NIH grant GM60419 and AHA.
Alternative CYP3A4 mRNA Isoforms Are Related to Hepatocyte Differentiation, Liver Development, and Response to Drugs
Authors: Li Dan , Gaedigk Roger , Hart Steven N. , Tregear Hans , Leeder J Steven , Zhong Xiao‐bo 
Published: June 2021

Cytochrome P450 3A4 (CYP3A4) metabolizes over 50% of prescribed drugs. CYP3A4 activity changes during liver development and the administration of drugs. Alternative mRNA isoforms occur in over 90% of human genes and are frequently observed in cells responding to developmental and environmental signals. The purpose of this study was to identify CYP3A4 mRNA isoforms in liver and characterize their differential expression during liver development, hepatocyte differentiation, and in response to drugs. cDNA cloning and RNA sequencing (RNA‐Seq) were used to identify CYP3A4 mRNA isoforms. Three isoforms were found in human HepaRG cells and liver tissues, one represented a canonical full length mRNA, one had a shorter 3′‐UTR, and one contained a partial intron 6 retention. The alternative mRNA isoforms were validated by either rapid amplification of cDNA 3′‐end (3′RACE) PCR or end‐point PCR. Quantification of the isoforms by real‐time PCR revealed that expression levels of the shorter 3′‐UTR isoform increased significantly more than the isoform with the canonical 3′‐UTR during liver development, hepatocyte differentiation, and in response to rifampicin, phenobarbital, and dexamethasone.ConclusionThis study identified two novel CYP3A4 mRNA isoforms. The isoform with the shorter 3′‐UTR might be involved in the regulation of CYP3A4 gene expression during liver development and the administration of some drugs.This study was supported by 1R01GM087376‐01A1, 1R01ES019487‐01A1, and 5P20‐RR021940 (to X.B.Z), 1R01HD058556‐01A1 (J.S.L), and Lila Self Graduate Fellowship from the University of Kansas (to S.N.H.)
The ABC transporter Mrp4 (Abcc4) plays a crucial role in normal testosterone production
Authors: Morgan Jessica A , Adachi Masashi , Boyd Kelli , Neale Geoff , Wang Yao , Cheepala Satish , Scheffer George L , Schuetz John D 
Published: June 2021

The primary site of testosterone biosynthesis in males occurs in the Leydig cells of the testes. Accordingly, circulating levels of testosterone exhibit a developmental pattern that correlate to the amount of testosterone produced. We show that, in the testes of both mouse and man, Mrp4 is almost exclusively expressed in the Leydig cells. Prior to puberty, despite normal Leydig cell numbers, Mrp4−/− mice have dramatically reduced serum testosterone concentrations when compared to their Mrp4+/+ counterparts. Consistent with this finding, Mrp4−/− mice exhibited both impaired spermatogenesis and prostate development. Nonetheless, adult Mrp4−/− animals are fertile with no obvious reproductive defects. After puberty, circulating testosterone concentrations of the Mrp4−/− mouse rise to levels comparable to those of the Mrp4+/+ mouse. In the Mrp4−/− mouse we have identified a developmental mechanism that accounts for the low levels of testosterone prior to puberty and the unexpected subsequent rise post‐puberty. These findings indicate that Mrp4 function has a crucial role in the appropriate developmental production of systemic testosterone.Funding provided by the NIH and ALSAC.
Association between Central Artery Stiffness and Cerebral Perfusion in Deep Subcortical White Matter
Authors: Tarumi Takashi , Shah Furqan , Tanaka Hirofumi , Haley Andreana 
Published: June 2021

BackgroundCentral arterial stiffness is a strong independent risk factor for cerebrovascular disease. Animal study demonstrated that higher pulse pressure in cerebral artery is associated with hypertrophic remodeling of the microvasculature, leading to increased vascular resistance to the blood flow. Cerebral hypoperfusion is considered the primary cause of cerebrovascular disease including stroke, vascular dementia, and Alzheimer's disease. The aim of this study was to determine whether central arterial stiffness is associated with cerebral perfusion in human.MethodsA total of 35 middle‐aged adults (15 men and 20 women; 49±7 yrs) were studied. Central aortic stiffness was determined by carotid‐femoral pulse wave velocity. With quantitative perfusion image technique using functional MRI, regional cerebral perfusion in deep subcortical white and gray matter was measured.ResultsCarotid pulse pressure and carotid‐femoral pulse wave velocity were significantly associated with the perfusion in the frontal white matter (r = −0.36 and −0.61 respectively, P<0.05). Hierarchical linear regression analysis revealed that carotid‐femoral pulse wave velocity significantly explains 22 % of variability in the frontal white matter perfusion (β = −0.67, P<0.05), independently from age and blood pressure.ConclusionsCentral aortic stiffness is strongly associated with the cerebral perfusion in frontal white matter even after the adjustment for age and blood pressure.
Tetrahydrobiopterin‐dependent Alkylglycerol Monooxygenase: From Gene Identification to Novel Insights into Etherlipid Degradation
Authors: Watschinger Katrin , Keller Markus A. , Golderer Georg , Hermann Martin , Hermetter Albin , Werner‐Felmayer Gabriele , Werner Ernst R. 
Published: June 2021

Alkylglycerol monooxygenase is the only enzyme able to degrade alkylglycerols by cleaving the ether bond in a tetrahydrobiopterin‐dependent manner yielding a toxic aldehyde which is further oxidized by fatty aldehyde dehydrogenase.Using candidate gene expression we have recently identified the gene coding for alkylglycerol monooxygenase (Watschinger K. et al, Proc Natl Acad Sci U S A, 2010, 107:13672‐7). Here, by silencing of TMEM195 in murine macrophage RAW 264.7 cells using lentiviral constructs we confirm that this gene is responsible for tetrahydrobiopterin‐dependent alkylglycerol cleavage.To investigate the metabolic interplay with fatty aldehyde dehydrogenase we transiently expressed fluorescently‐tagged constructs of alkylglycerol monooxygenase with one of the two splice variants of fatty aldehyde dehydrogenase and analyzed the localization by live cell confocal microscopy. While the major splice variant of fatty aldehyde dehydrogenase (V2) is expressed together with alkylglycerol monooxygenase in the endoplasmic reticulum, the second splice variant (V1) was located exclusively to peroxisomes. This allows the conclusion that the V2 variant is responsible for detoxification of the aldehyde produced by alkylglycerol monooxygenase whereas the V1 variant could be responsible for aldehyde elimination originating from other sources.Supported by the Austrian Science Fund (FWF) P22406.
Zfp206: A novel regulator of adipocyte differentiation potential and cell size
Authors: Pekala Phillip H. , Karschner Vesna A. , Metcalf Mark , Murrell Rachel , Houston Melanie , Sauer Katherine , Stanton Larry 
Published: June 2021

The 3T3‐L1 differentiation program leading to the adipocyte phenotype is controlled by a cascade of transcription factors, particularly PPARγ and the family of C/EBP isoforms. Superimposed on this framework we have recently identified the transient expression of the embryonic stem (ES) cell transcription factor, Zfp206, proposed to maintain the differentiation potential of ES cells. In the 3T3‐L1 cells, Zfp206 is expressed at confluency and is attenuated by day 3 as expression switches to Zscan10.201, a splice variant lacking the entire zinc‐finger DNA binding domain which we propose functions as a dominant negative. Our hypothesis is that expression of Zfp206 is essential for maintenance of the differentiation potential and that as terminal differentiation is established with the expression of PPARγ, Zfp206 expression is diminished. The transient expression of the splice variant ZScan10.201 insures the effects of Zfp206 are negated. In murine C/EBPβ−/− ES cells that will not differentiate, Zfp206 was observed to be expressed but did not down regulate, consistent with C/EBPβ involvement in control of expression.Human preadipocytes also expressed Zfp206 and down regulated expression as the cells differentiated to adipocytes similar to the 3T3‐L1 cells. Suppression of Zfp206 expression using shRNA in the 3T3‐L1 cells, resulted in a marked decrease in cell size as well as an early onset of adipogenesis. Thus, Zfp206 appears to determine both cell fate and cell size. Supported in part by NIH DK088666.
Effective preparation method of human catalytic antibody light chains
Authors: Uda Taizo , Honjo Eijiro , Hifumi Emi 
Published: June 2021

We have successfully produced mouse type catalytic antibody light chains (antigenases) by immunizing ground‐state peptides or proteins. The antigenase possesses the ability to enzymatically decompose a targeting molecule using the catalytic triad formed in the structure. In the case of human antibody light chains, the catalytic triad was mostly encoded in Subgroup II, suggesting that the light chains belonging to the subgroup should exhibit enzymatic functions.In order to verify the above concept, we amplified an antibody germline gene as the cDNA belonging to Subgroup II, which was taken from human leucocyte. The cDNA was inserted in pET system, followed by a transformation to express the protein (human antibody light chain) in E.coli. We could recover the protein with very high purity by passing two affinity columns. Its catalytic activity was measured using synthetic substrate (peptide‐MCA) to monitor the fluorescence generated by the cleavage of the peptide‐MCA bond, suggesting the human antibody light chain exhibited the amidase activity. Furthermore, the light chains showed other interesting enzymatic reaction in addition to the amidase activity.
C2 Domains: Versatile Lipid Binding Modules
Authors: Stahelin Robert V , Gunasekaran Christopher , Yu Hui 
Published: June 2021

The C2 domain (~130 residues) was originally identified as one of two conserved regulatory domains of Ca2+‐dependent protein kinases C. A large number of proteins containing the C2 domain have been identified since, and most of them are involved in signal transduction or membrane trafficking. Early experiments demonstrated C2 domains could bind bulk phospholipids such as phosphatidylserine or phosphatidylcholine with high affinity and specificity, while more recently; C2 domains have been shown to interact with different phosphoinositides and/or sphingolipids in both Ca2+‐dependent and ‐independent manners. C2 domains have also been shown to bind specific proteins, interact with phosphotyrosine, induce membrane curvature changes, participate in intramolecular interactions, and interact with viral proteins.How the multifaceted C2 domains achieve diverse signaling and interaction properties is only beginning to come to fruition. We have begun a comprehensive and rigorous experimental, computational, single molecule, and structural analysis of the mammalian C2 domain family, which will lead to a better understanding of how these properties manipulate the full‐length proteins' functions.
The Molecular Basis of Ceramide‐1‐Phosphate Recognition by Peripheral Proteins
Authors: Ward Katherine Elizabeth , Sudhahar Christopher G. , Olmstead Deborah N. , Stahelin Robert V. 
Published: June 2021

The sphingolipid ceramide‐1‐phosphate (C1P) plays a critical role in the cellular signaling that mediates inflammation, cell proliferation and phagocytosis. C1P has been shown to increase the activity of cytosolic phospholipase A2 α (cPLA2α) as well regulate its translocation to cellular membranes, a process that promotes inflammation through the production of arachidonic acid. In this study we have resolved the first C1P binding site of a peripheral protein. Using NMR we demonstrate the cPLA2α C2 domain interaction site for C1P by mapping chemical shifts. Subsequently, this novel‐binding site is confirmed with in vitro biophysical and biochemical analysis as well as molecular dynamics simulations. To search the human genome for other C1P binding proteins we have performed proteomic studies to began high throughput characterization of the conserved nature of C1P binding.Funding source: American Heart Association SDG0735350N (R.V.S)