bluuebunny/crossref_metadata_2025_split huggingface dataset:
Corticosteroid Biosynthesis Revisited: Substrate Specificity of Steroid 21‐Hydroxylase
Authors: Loke Steffen , Stoll Anna , Joseph Jan Felix , Machalz David , de la Torre Xavier , Botrè Francesco , Wolber Gerhard , Bureik Matthias , Parr Maria 
Published: April 2020

ObjectiveCytochrome P450 enzymes (CYPs) are capable of catalyzing regio‐ and stereo‐specific oxy functionalization reactions which otherwise are major challenges in organic chemistry. In order to make the best possible use of these biocatalysts it is imperative to understand their specificities.Human CYP21A2 (steroid 21‐hydroxylase) is known as one important enzyme in the biotransformation of progestins into corticosteroids. Humans with a reduced activity of steroid 21‐hydroxylase suffer from congenital adrenal hyperplasia (CAH), that is characterized by reduced production of cortisol. Due to feedback mechanisms of the hypothalamic pituitary adrenal axis, increased concentrations of progestins, but also androgens resulting from alternative metabolic pathways are observed in these patients.As enzymes in steroid biosynthesis are generally considered to show high substrate selectivity a closer investigation on structure requirements of steroid 21‐hydroxylase was conducted.MethodsBiotransformation experiments were performed using a fission yeast expressing human CYP21A2 (CAD75) in a whole‐cell biotransformation assay
[1]. Progesterone, pregnenolone, and their corresponding 17‐hydroxy analogues were used as substrates. Product formation was monitored by GC‐MS. Molecular docking experiments were performed using GOLD software using the X‐ray structure of CYP21A2 co‐crystallized with progesterone (17, PDB entry 4Y8W
[2]) as protein conformation.ResultsAn investigation on A‐ring requirements yielded that the presence of a 3‐oxo group is a strict prerequisite for a CYP21A2 substrate
[1]. In line with these findings the incubation of the endogenous progestins pregnenolone (3β‐hydroxy‐pregn‐5‐en‐20‐one) and 17α‐hydroxypregnenolone (3β,17α‐dihydroxy‐pregn‐5‐en‐20‐one) did not result in successful 21‐hydroxylation, while progesterone (pregn‐4‐ene‐3,20‐dione) and 17‐hydroxyprogesterone (17α‐hydroxy‐pregn‐4‐ene‐3,20‐dione) were hydroxylated at C‐21.Molecular docking experiments supported the structure requirements. Apparently, the interaction of the carbonyl group at C‐3 of the substrate to the side‐chain Arg234 of the enzyme is indispensable.ConclusionsThe results are opposed to actual scientific view of steroid biosynthesis which for example supposes formation of 21‐hydroxypregnenolone is also catalyzed by CYP21A2
[3]. They also help to understand metabolic pathways of steroids in the human body and may be useful in further synthesis experiments due to the use of enzymes as biocatalysts.
Carboxyspermidine Dehydrogenase of Spermidine Biosynthesis from Bacteroides fragilis
Authors: Todd Kaleb Jonathan , McFarlane Jeffrey 
Published: April 2020

The human gut microbiome performs a host of metabolic processes which remain largely unstudied, many of which have implications for human health. For example, human cells uptake the microbial polyamine spermidine (
1), and increased spermidine levels are associated with the progression of pancreatic and colon cancers (
2,
3). Thus gut microbe spermidine biosynthesis is a potential therapeutic target. Spermidine is produced by the dominant gut microbes via a unique enzymatic pathway (
4). The first enzyme in this pathway is carboxyspermidine dehydrogenase (CASDH), an NAD(P)H‐dependent saccharopine dehydrogenase expected to catalyze the reductive condensation of either 1,3‐diaminopropane or putrescine with L‐aspartate semialdehyde to form carboxynorspermidine or carboxyspermidine, respectively (
5). CASDH enzymes involved in spermidine biosynthesis have not been structurally or kinetically characterized from any species. We propose the characterization of CASDH from a dominant gut microbial species using X‐ray crystallography and steady‐state kinetic analysis. The CASDH gene from Bacteroides fragilis was chosen as this species is known to adhere to colon cancer epithelial cells and has been associated with tumor progression. We have successfully expressed BfCASDH in an E. coli BL21 expression cell line and purified it via affinity chromatography. Crystallization trials and the development of kinetic assays are currently underway. X‐ray crystal structures of BfCASDH will reveal the active site structure and the amino acid residues involved in binding and catalysis. In addition, our kinetic assays will confirm the preferred substrate identities and enzymatic activities of the BfCASDH. These data will contribute basic science knowledge necessary for the future explorations of potential inhibitors.Support or Funding InformationKT was supported by NIH BLaST Undergraduate Research Award RL5GM118990. BLaST is supported by the NIH Common Fund, through the Office of Strategic Coordination, Office of the NIH Director with the linked awards: TL4GM118992, RL5GM118990, & UL1GM118991.
SPARC: Topographical Organization and Morphology of Substance P‐IR Axons and Terminals in the Whole‐Mount Stomach of Rodents
Authors: Ma Jichao Mark , Chen Jin , Powley Terry L. , Cheng Zixi Jack 
Published: April 2020

Afferent fibers innervate the stomach. However, very little is known about the topographical distribution and morphological terminal structures of gastric nociceptive axons and terminals in rodents. In this study, we used Substance P (SP) as a marker to label afferent fibers in the whole‐mount dorsal and ventral stomach including longitudinal and circular muscles and the myenteric nervous system in C57BL/6J mice and SD rats. The whole‐mount dorsal and ventral stomachs were processed with the substance P (SP) primary antibody followed by the secondary antibody, and then examined using confocal microscopy. We found that: 1) SP‐IR axons and terminals extensively covered the fundus, corpus and antrum of the dorsal and ventral stomach. 2) SP‐IR varicose axons ran in parallel with the longitudinal as well as circular muscles. 3) SP‐IR varicose axons formed dense rings of terminal varicosities around individual neurons in the myenteric plexus. 4) The density of SP‐IR axons and terminals varicosities was different in the fundus, corpus and antrum. Collectively, our data showed for the first time the comprehensive topographical distribution and morphology of SP‐IR axons and terminals in different regions of the stomach. This knowledge will provide an anatomical basis for differentiated nociceptive gastric afferent functions in rodents.Support or Funding InformationNIH HEAL/SPARC Program NIH 1 U01 NS113867‐01 and NIH R15 HL137143‐01A1.
Laryngeal Adductor Reflex Motor Bursts Rapidly Oscillate in the Cat
Authors: Shen Tabitha Y. , Rose Melanie J. , Olsen Wendy L. , Wang Wei , McLeod Justin C. , Segers Lauren S. , Nuding Sarah C. , Pitts Teresa G. , Poliacek Ivan , Gestreau Christian , Morris Kendall F. , Musselwhite M Nicholas , Kermit Kofi K. , Bolser Donald C. 
Published: April 2020

The laryngeal adductor reflex (LAR) is a protective behavior induced by stimulation of upper airway afferents. Laryngeal afferent information reaches interneurons in the medulla via the superior laryngeal nerve (SLN). Expiratory laryngeal motoneurons (ELMs) are subsequently excited causing contraction of the thyroarytenoid (TA) and posterior cricoarytenoid (PCA) muscles. The LAR consists of a short latency (~10 ms), short duration (~10 ms) burst (R1) and a longer latency (~40ms) burst (R2) in laryngeal electromyogram (EMG) activity. We have shown that R1 LAR magnitude undergoes frequency‐dependent depression in response to electrical stimulation (eStim) of the SLN. Based on preliminary observations, we hypothesized that the R1 component of the LAR induced by SLN eStim oscillates. In anesthetized, spontaneously breathing cats (n=12: 7 males, 5 females), we recorded EMG activity from the TA bilaterally and the PCA unilaterally. The SLN was electrically stimulated at 2 or 20 Hz to evoke TA LAR ipsilateral to stimulation (iTA). No sex differences in TA or PCA LAR EMG magnitude were observed. Consistent with previous findings, TA LAR magnitude differed with breathing phase; normalized iTA LAR magnitude was 10% larger during expiration compared to inspiration. TA LAR contralateral to stimulation (cTA) and ipsilateral PCA LAR magnitudes were not breathing phase dependent. TA and PCA LAR EMG oscillations were highly variable across animals (range: 244 to 1140 Hz). Mean iTA EMG frequency was decreased at 20 Hz (2 to 20 Hz: 616 to 518 Hz, p=0.05). Mean cTA periodicity trended down at 20 Hz (2 to 20 Hz cTA: 647 to 531 Hz, p=0.07). In separate decerebrate, paralyzed cats (n=13), extracellular recordings of neurons in the ventral respiratory column (VRC) with multiple electrode arrays were conducted. Peristimulus time histograms provided evidence that ELMs oscillated at frequencies following SLN eStim similar to the lower range of periodicities in TA EMGs in unparalyzed animals. A subset of neurons recorded in the VRC were excited or inhibited by SLN eStim at short latencies. Spike train analysis suggested that ELM oscillations could be accounted for by synaptic inhibition and excitation from laryngeal‐responsive VRC inspiratory, expiratory, and non‐breathing modulated neurons. We conclude that the LAR is temporally regulated by a complex, anatomically distributed brainstem network that is capable of strong synchrony in response to stimulation of laryngeal afferents. We speculate that the function of this laryngeal‐responsive network is to exert tight regulatory control over LAR patterning.Support or Funding InformationSupported by NIH HL131716 and 3OT2OD023854
Molecular Transducers of Physical Activity Consortium (MoTrPAC) Progress
Authors: Joseph Lyndon , Williams John P. ,   The MoTrPAC Consortium
Published: April 2020

Physical activity is beneficial to human health and well being across the lifespan. The numerous benefits of regular physical activity have long been recognized. Despite this, most exercise studies are associational and the molecular mechanisms that are the bases for the beneficial effects remain obscure as are the mechanisms of multi organ communications and benefits.The NIH Common Fund initiated the Molecular Transducers of Physical Activity Consortium (MoTrPAC) in December 2016 by issuing 19 grants to 37 Principal Investigators from 23 institutions. Fundamentally, MoTrPAC is a large discovery project the goals of which are to:

Aim 1: Assemble a comprehensive map of the molecular changes that occur in response to exercise and provide insights into how they are altered by age, sex, body composition and fitness level.
Aim 2: Develop a user‐friendly database to facilitate investigator‐initiated studies and catalyze the field of physical activity research whereby researchers can develop hypotheses exploring novel mechanisms by which physical activity improves or preserves health.This project will explore and document changes in molecules mobilized in blood, muscle and fat in humans as well as 15 additional tissues for rats in response to different exercise regimes. The human studies are a multi‐center clinical trial cohort of people of both sexes from 10–80 years of age and recruitment has begun. Preclinical Animal Study Sites (PASS) have conducted treadmill running experiments in male and female 6 and 18‐month old F344 rats and harvested tissues from control inactive rats and rats at seven time‐points following a single 30 minute acute bout of treadmill running (70% VO2max) in their active phase (dark). A separate group of rats were subjected to an intensive (also 70% VO2max) and progressive run training program of 5 day/week for 1,2, 4, or 8 weeks, also in their active phase, with 18 tissues collected per rat. Multiple state of art and omics platforms including genomic, transcriptomic, epigenomic, proteomic and metabolomics are being employed to define and discover the molecules mobilized in response to exercise that will serve as a valuable resource for the research community and support future investigator‐initiated studies to understand the molecular mechanisms of exercises effects. PASS tissues not analyzed are stored in the MoTrPAC BioRepository and will be available for ancillary studies. This presentation will summarize data from the initial public data release (November 15th, 2019; five most abundant tissues). Data will be available to the extramural community to collaborate with the MoTrPAC investigators to explore innovative mechanisms to expand the impact of the MoTrPAC studies.Support or Funding InformationNIH Common Fund
Inhalation of Air Particulate Matter Accelerates Neuroinflammation and Amyloid‐beta Accumulation in APP/PS1 Mice
Authors: Mackos Amy , Sahu Bijayani , O’Piela Devin , Combs Colin , Wold Loren 
Published: April 2020

Alzheimer disease (AD) which is a progressive, neurodegenerative disease characterized by neuroinflammation and accumulation of amyloid‐β (Aβ) fibers leading to neuronal death. It is theorized that a combination of host genetics and environmental factors contribute to AD development and progression, however the trigger for neuroinflammation and Aβ development has remained elusive. One such environmental exposure linked to AD development and progression is air pollution. Thus, we designed a study to test our hypothesis that air particulate matter exposure accelerates AD development and progression. Male, wild type (WT) C57Bl/6 and AD transgenic mice, APPswe/PSEN1dE9 (APP/PS1), were exposed to either filtered air (FA) or air particulate matter sized under 2.5 μm (PM2.5) for 6 hrs/day, 5 days/week for 3 months. Brain tissue was collected at the end of the 3 month exposure and was either flash frozen or formalin‐fixed. Results demonstrated a significant increase in hippocampal Aβ accumulation in PM2.5‐exposed APP/PS1 mice over that of FA‐exposed APP/PS1 animals. PM2.5‐exposed WT mice did not develop Aβ plaques. Immune markers CD68 (tissue macrophage) and IBA1 (activated microglia) and the glial marker GFAP (activated astrocytes), as assessed by immunohistochemistry, were also significantly increased in PM2.5‐exposed APP/PS1 mice over their FA‐exposed counterparts. PM2.5‐exposure did not lead to enhanced levels of CD68, IBA1, or GFAP in WT mice. Taken together, these data suggest that airborne particulate matter has the propensity to alter the brain environment in mice genetically predisposed to develop Aβ in a way that mimics early AD‐like pathology.Support or Funding InformationFunding support from AG057046 to LEW and CKC.
Allosteric uncoupling of microtubule depolymerase activity from motility in human Kinesin‐5
Authors: Downs Micquel , Kim Catherine , Kim Elizabeth , Liu Liqiong , Parameswaran Sreeja , Kim Sunyoung , Wojcik Edward 
Published: April 2020

Human Kinesin‐5 (Eg5) has a large number of known allosteric inhibitors that disrupt its mitotic function. Small‐molecule inhibitors of Eg5 are candidate anti‐cancer agents and important probes for understanding the cellular function. Here we show that Eg5 is capable of more than one type of microtubule interaction, and these activities can be controlled by allosteric agents. While both monastrol and S‐trityl‐L‐cysteine (STC) inhibit Eg5 motility, our data reveal an unexpected ability of these loop5 targeting inhibitors to differentially control a novel Eg5 microtubule depolymerizing activity.Remarkably, small molecule loop5 effectors are able to independently modulate discrete functional interactions between the motor and microtubule track. We establish that motility can be uncoupled from the microtubule depolymerase activity and argue that loop5‐targeting inhibitors of Kinesin‐5 should not all be considered functionally synonymous. Also, the depolymerizing activity of the motor does not contribute to the genesis of monopolar spindles during allosteric inhibition of motility, but instead reveals a new function. We propose that, in addition to its canonical role in participating in the construction of the three‐dimensional mitotic spindle structure, Eg5 also plays a distinct role in regulating the dynamics of individual microtubules, and thereby impacts the density of the mitotic spindle.
Nutraceuticals Derived From Pomegranate Selectively Enhance Vitamin D Receptor Signaling to Amplify Key Vitamin D Target Genes
Authors: Livingston Sarah , Lucas Daniel , Sabir Marya S. , Mallick Sanchita , Purdin Hespera , Nidamanuri Sree , Whitfield G. Kerr , Haussler Carol A. , Haussler Mark R. , Jurutka Peter W. 
Published: April 2020

The active vitamin D hormone, 1,25‐dihydroxyvitamin D (1,25D), mediates its biological effects by binding to the nuclear vitamin D receptor (VDR) and promoting heterodimerization with retinoid X receptors (RXRs). Various nutraceuticals, including resveratrol and curcumin, have been postulated to interact with the vitamin D signaling pathway. More recently, health benefits attributed to pomegranate have been associated with its high content of polyphenols, specifically ellagitannins, which are metabolized by the gut microbiota to produce Urolithin A (UA). In this study, we investigated the ability of UA to modulate 1,25D signaling via transcription and qPCR assays. We hypothesized that 1,25D in combination with urolithin will stimulate VDR activity more than 1,25D alone, increasing transcription of 1,25D target genes. The increased activation of anti‐oxidation genes by 1,25D and UA could help combat reactive oxygen species and attenuate cellular “aging”. Our results indicate that there is a significant increase in VDR transcriptional activity when HEK‐293 kidney cells are treated with UA in conjunction with 1,25D at several 1,25D andUA concentrations. A similar effect was also observed using three structurally‐distinct 1,25D response elements. The potentiation of VDR activity via UA retains receptor‐selectivity since UA did not promote further activation of other receptor‐ligand complexes including estrogen‐ER, rexinoid‐RXR and TO‐LXR. Finally, amplification of serotonin production in brain cells was greatest with a combined cocktail of 1,25D/UA/resveratrol via qPCR (TPH2 gene) and ELISA (5‐HT). In order to probe the molecular mechanism of UA amplification in VDR activity, we tested VDR‐RXR heterodimerization and observed no effect of UA. However, UA activates AMP‐kinase, and we found potential AMP kinase phosphorylation consensus sites in the VDR protein sequence. Collectively, our novel results position gut microbiota‐derived UA as a putative VDR modulator, suggesting that the influences of 1,25D and UA converging on VDR may potentially mediate anti‐aging and promote longevity.Support or Funding InformationFinancial support/grant sponsor: National Institutes of Health; grant numbers: DK033351 and CA140285
Characterizing the Interaction Between CowN and Nitrogenase
Authors: Lee Terrence , Chong Kiersten , Garcia Chloe , Strul Max , Wong Emily , Kharwa Ruchita , Bretzing Kevin , Owens Cedric 
Published: April 2020

Nitrogenase catalyzes the conversion of atmospheric dinitrogen into ammonia. The enzyme produces ammonia under ambient conditions using the free energy of ATP to drive dinitrogen’s reduction. The most common form of nitrogenase is molybdenum nitrogenase (Mo‐nitrogenase). Mo‐nitrogenase is inhibited by the ubiquitous pollutant carbon monoxide (CO). To prevent inhibition of Mo‐nitrogenase by CO, nitrogen‐fixing bacteria produce the protein CowN and in its presence, Mo‐nitrogenase avoids inhibition by CO and remains active. However, the mechanism by which CowN protects Mo‐nitrogenase is unknown. Enzymatic assays suggest that CowN and Mo‐nitrogenase interact with a K‐d of approximately 5–10μM. Here, we present data from crosslinking and pulldown assays that were used to determine how CowN interacts with Mo‐nitrogenase. Crosslinking assays using the heterobifunctional crosslinker EDC showed no binding between CowN and Mo‐nitrogenase in the presence and absence of CO gas. Pulldown assays using Ni‐NTA resin and a His‐tagged CowN bait also did not show significant binding between CowN and Mo‐nitrogenase. These assays indicate that CowN may not have a strong binding affinity to Mo‐nitrogenase to be detected by crosslinking or pulldown assays. Future work will investigate potential nitrogenase‐CowN interaction using fluorescence anisotropy.Support or Funding InformationThis research was supported by the Chapman Center for Undergraduate Excellence, USDA‐NIFA (Grant no. 2015‐67012‐22895) and the National Science Foundation (Grant no. 1905399).
Deciphering the isoform code contained in Vo a‐subunit isoforms of the V‐ATPase
Authors: Kane Patricia M , Jaskolka Michael C. , Tuli Farzana , Mitra Connie , Banerjee Subhrajit , Tarsio Maureen 
Published: April 2020

V‐ATPases are highly conserved proton pumps that acidify multiple organelles, including lysosomes, endosomes, the late Golgi apparatus, synaptic vesicles, and other regulated secretory granules. Precise tuning of the luminal pH of these organelles is critical for function, but the factors governing organelle pH control are not completely understood. V‐ATPases are multisubunit complexes consisting of a peripheral subcomplex (V1) that contains sites for ATP hydrolysis and an integral membrane subcomplex (Vo) that contains the proton pore. The Vo a‐subunit is the largest subunit. It is comprised of an N‐terminal cytosolic domain and a C‐terminal domain that forms part of the proton pore. Most organisms encode multiple isoforms of the Vo a‐subunit that exhibit organelle‐specific localization. We hypothesize that the N‐terminal (NT) domains of a‐subunit isoforms participate in distinct cellular interactions that are critical for isoform‐specific V‐ATPase localization, activity, and regulation. In yeast cells, there are two organelle‐specific isoforms of the Vo a‐subunit, Vph1 and Stv1. Vph1‐containing V‐ATPases transit through the secretory pathway en route to the lysosome‐like vacuole. Their activity is regulated by reversible disassembly in response to glucose levels, and they require interaction with the yeast RAVE (regulator of acidification of vacuoles and endosomes) for both their initial biosynthetic assembly and for glucose‐dependent reassembly. The Vph1NT domain binds directly to RAVE and is responsible for glucose‐sensitive interactions with the RAVE complex. In addition, the vacuolar lipid PI(3,5)P2 promotes assembly and activity of Vph1‐containing V‐ATPases. Mutations in Vph1NT compromise PI(3,5)P2‐induced activation, suggesting direct binding to lipid. In contrast, Stv1‐containing V‐ATPases assemble and function in the absence of the RAVE complex, and Stv1NT does not bind to RAVE. However, Stv1NT binds to the Golgi‐enriched lipid PI(4)P, and mutations that abolish PI(4)P binding compromise Golgi retention of Stv1‐containing V‐ATPases. The aNT domains of the four human Vo a‐subunit isoforms exhibit differential recognition of phosphoinositide lipids. Some human aNT domains interact with the human homologue of the RAVE complex. These data indicate that NT domains of Vo a‐subunit isoforms encode information for localization and regulation of V‐ATPase activity that help determine organelle pH and protect cells from stress.Support or Funding InformationNIH R01 GM127364 and NIH R01 GM126020