bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression, Purification, and Initial Characterization of the Diverse ADPGlucose Pyrophosphorylase from Thermodesulfovibrio yellowstonii
Authors: Berriman Michael , Kieu Tu , Cheever Tyber , Orry Andrew , Meyer Christopher R
Published: June 2021

ADP‐Glucose pyrophosphorylase (ADPG PPase, glgC gene product) catalyzes the rate‐limiting step of glucan biosynthesis in plants and bacteria. Structure‐function studies of ADPG PPases from diverse organisms will allow for the identification of the amino acids responsible for different characteristics and the subsequent engineering of a highly active form of the enzyme to increase the supply of renewable carbon. The glgC gene from Thermodesulfovibrio yellowstonii (Td.y.) was successfully cloned and expressed. This enzyme displays only ~30% identity to the well characterized E. coli and A. tumefaciens (Ag.t.) enzymes, and harbors unusual sequences compared to other ADPG PPases in regions known to be important for allosteric regulation. Interestingly, molecular modeling predicted that the structure was more similar to the potato tuber enzyme (PDB 1yp3) than the bacterial Ag.t. ADPG PPase structure (PDB 3brk). The enzyme was successfully purified by sonication, a heat step, anion exchange, and hydrophobic interaction chromatography. The Td. y. enzyme was found to be stable up to 75 °C, and exhibited optimal activity at pH 8.5 and 70 °C. The enzyme was also activated by 2 mM G6P (~5‐fold), FBP (~3‐fold), sulfate (~4‐fold) and strongly inhibited (80%) by 2 mM AMP, indicating a novel allosteric specificity pattern. Supported by NSF Award 0448676.
Evidence for Specific Roles of Arginines 26 and 38 in the Allosteric Regulation of ADP Glucose Pyrophosphorylase from Thermus thermophilus
Authors: Sanders Jamie , Doshi Dhaval , Botero Fabian , Truc Teddy , Orry Andrew , Meyer Christopher R
Published: June 2021

ADPGlucose Pyrophosphorylase (ADPG PPase) is an allosteric enzyme that catalyzes the rate‐limiting step in glucan synthesis, an attractive target for engineering to increase the production of renewable carbon. The thermophillic bacterial enzyme from Thermus thermophilus was found to be activated by G6P, F6P, and FBP and displayed optimal activity at 75°C. Based on alignment and molecular modeling studies, R26 and R38 were proposed to be invovled in allosteric regulation of this enzyme. Altered proteins (R26A and R38A) were generated, expressed, and purified. R26A and R38A enzymes displayed a significant decrease in Vmax and a decrease in the apparent affinity for substrates compared to the wild‐type enzyme. In contrast to the wild‐type, the presence of activators caused an increased apparent affinity for the substrate ATP for both enzymes. The R26A mutation resulted in an ~8‐fold decrease in apparent affinity for G6P at 37°C, and a ~30‐fold decrease at 75°C. The R38A enzyme showed ~6‐fold and ~8‐fold decreases in apparent affinity for G6P and F6P at 37°C, respectively. Preliminary data shows decreased apparent affinity for substrates at 75°C for both enzymes. The diminution of the Vmax is consistent with a role for these arginines in stabilizing an active conformation of the enzyme. In addition, R26 appears to play a role in the binding of G6P while R38 appears to facilitate binding of both G6P and F6P in the allosteric subsite(s). Complete kinetic characterization of the altered enzymes and double mutant R26A, R38A at 75°C is underway. Supported by NSF Award 0448676.
Allosteric reversion of Haemophilus influenzae β‐carbonic anhydrase by a proline shift variant
Authors: Rowlett Roger S , Hoffmann Katherine M , Million-Perez H. Rachael , Merkhofer Richard , Nicholson Hilary E
Published: June 2021

The type II β‐carbonic anhydrase from H. influenzae is an allosteric enzyme that is inhibited by its product, bicarbonate ion, and exhibits highly cooperative pH‐rate kinetics. A proline shift variant of this enzyme, P48S/A49P, has kcat and kcat/Km values that are 114% and 395% of the wild‐type enzyme, respectively, and shows a complete loss of pH‐rate cooperativity. The X‐ray structure of the P48S/A49P variant reveals that in each dimer of this homotetrameric protein, one chain has adopted the inactive, T‐state (characterized by a closed metal coordination site) while the other chain has adopted the active, R‐state (characterized by a zinc coordination site with an exchangeable water ligand). The R‐state chain is associated with N‐terminal helical elements of the neighboring chain. In the T‐state chain this helix interaction is not observed. Comparison of the structure of the R‐state chain of P48S/A49P variant and the associated helical elements from the neighboring chain to the wild‐type enzyme (which crystallizes in the T‐state) reveals that the N‐terminal helices in the P48S/A49P variant are displaced by as much as 6 Å compared to the wild‐type. This suggests that the N‐terminal helices may be important in intersubunit communication. This work is supported by NSF grants CHE‐0819686 and MCB‐0741396.
The effects of ‘Candidatus Liberibacter asiaticus’ infection on the protein expression profiles and nutrient status of citrus plants
Authors: Nwugo Chika Charles , Lin Hong , Duan Yong-Ping , Civerolo Edwin
Published: June 2021

Huanglongbing (HLB) is a lethal disease of citrus and the bacterium, ‘Ca. Liberibacter asiaticus’ (Las) is the most prevalent etiological agent of the disease worldwide. Here, proteomic analyses via 2‐DE and mass spectrometry were employed to elucidate the protein expression profiles in leaves of Las‐infected greenhouse‐grown citrus grapefruit and lemon plants that are pre‐symptomatic or symptomatic for HLB. We also applied ICP spectroscopy to identify the effect of the disease on the nutrient status of greenhouse‐grown citrus grapefruit, lemon, and trifoliate plants. 2‐DE analysis revealed over 200 and 70 protein spots in grapefruit and lemon leaves, respectively, that were differentially expressed due to Las infection compared to uninfected plants. Interestingly, LC‐MS analysis showed that chitinase and starch synthase are among a subset of proteins precociously induced in the leaves of Las‐infected grapefruit and lemon plants. Furthermore, nutrient status analysis of leaves suggests that active accumulations of Zn and Ca in lemon and trifoliate plants, respectively, are associated with citrus response to Las infection. In summary, our results provide novel insights into potential host‐specific response mechanisms associated with HLB, which is important for early detection and better nutritional management of the disease. This project was supported by the USDA, Agricultural Research Service.
The Linkage Between Citrate, pH Regulation and Protein Folding in Glyoxysomal Malate Dehydrogenase
Authors: Yanta Helen , Bell Ellis
Published: June 2021

Malate Dehydrogenase, a dimeric enzyme that catalyzes the NAD/NADH‐dependent reversible oxidation of malate into oxaloacetate, is involved in the Citric Acid and Glyoxylate Cycles as well as the malate‐aspartate shuttle. Citrate and pH appear to regulate the activity of glyoxysomal and mitochondrial MDH. The aim of this project is to study the linkage between pH and citrate regulation of Malate Dehydrogenase and protein stability and folding cooperativity. Over the pH range from 6.0 to 8.0 clear effects on secondary structure as shown by circular dichroism are seen. Thermal melts using CD at 222nm with data fitting via a four parameter Hill equation to give both Tm and a cooperativity parameter indicate effects on the unfolding cooperativity as well as the overall stability. Likewise, Citrate binding at various pH values clearly affects both, suggesting a linkage between secondary and tertiary structure stabilities, allosteric behavior and folding cooperativity.

This work is supported by NSF Grant MCB 0448905 to EB.
The Roles of Conserved Cysteine Residues in the Structure and Function of Glyoxysomal Malate Dehydrogenase
Authors: Jones Rachel , Bell Ellis
Published: June 2021

Bioinformatics analysis of malate dehydrogenases from both eukaryotic and prokaryotic sources indicate the existence of a number of cysteine residues that are highly conserved in mitochondrial and glyoxysomal forms of the enzyme (C282 and C295) as well as a cluster of cysteines (145, 151, 152) that are not conserved. In order to probe the roles of these conserved cysteines residues in both structure and folding of the enzyme as well as the catalytic activity, a number of site directed mutants (C145V‐C151S‐C152S triple mutant, C282S and L, and C295S) of watermelon glyoxysomal malate dehydrogenase have been constructed, expressed, purified and characterized. The resultant mutants are characterized for reactivity to DTNB (native protein reacts slowly indicating that one or more of the SH groups are somewhat accessible), secondary and tertiary structure using CD and CD/Thermal melts, and catalytic activity. Preliminary results indicate that all mutants have significant activity with differences in Km and Vmax. Similarly all mutants show evidence via CD for secondary structure but show some differences in stability and cooperativity of folding suggesting that none of the conserved cysteines are critical for activity but contribute to overall structure and function.

This work is supported by NSF Grant MCB 0448905 to EB
Structural investigations of ClC‐ec1, a large integral membrane protein, using solution‐state NMR and nanodisc technology
Authors: Chew Thomas , Abraham Sherwin , Elvington Shelley , Maduke Merritt
Published: June 2021

CLC antiporters mediate Cl−/H+ exchange across cell membranes in organisms ranging from E. coli to humans via coupling of protein conformational changes to ion binding, unbinding and translocation. X‐ray crystallographic structures have provided essential molecular pictures, but details of conformational changes remain elusive. We are using solution state NMR to probe conformational change in ClC‐ec1, a CLC homolog of known structure. 13C labeling of lysine residues is achieved by post translational reductive methylation. 1H‐13C HSQC spectra reveal reversible, substrate‐dependent spectral changes that may reflect conformational changes. To address the concern that the detergent‐solubilized protein is not in a native environment, we are also using nanodiscs, which consist of two amphipathic helices wrapped around a bilayer patch of lipids surrounding the membrane‐embedded protein. The monomeric, 13C‐methylated mutant form of ClC‐ec1 was incorporated into discs and tested for homogeneity and monodispersity. The 1H‐13C HSQC spectra show substrate‐dependent spectral changes in this system, although further experiments will be needed to determine if these represent functionally‐relevant conformational changes. These studies demonstrate the potential for using solution‐state NMR and nanodisc technology to study ClC‐ec1 in a native lipid environment.

Supported by: National Science Foundation
Continuing Studies on the Structural Stability of E. coli Alkaline Phosphatase by an Undergraduate Biochemistry Laboratory Course
Authors: Hazzard James T. , Hailey Alexander , Klein Mary E. , Korte Stepahnie R. , Mason Ashley M. , Narendran Nirushan H. , Schnoebelen Carly L.
Published: June 2021

On‐going investigations into the roles of the two disulfide bonds in E. coli alkaline phosphatase (AP) on structural stability and limited proteolytic degradation have been incorporated into a guided inquiry portion of an undergraduate biochemistry laboratory course. We studied the effects of reducing agents (DTT and TCEP) on the thermal and chemical (urea and guanidinium hydrochloride) denaturation of AP using a combination of tryptophan fluorescence and CD spectroscopy, also investigating the roles of Hofmeister series ions during thermal and chemical denaturation. We also present evidence for potential chaperonin‐like proteins found in the periplasmic lysates that contribute to the dramatic thermal stability of AP, relative to the highly purified commercial enzyme and used tandem mass spectrometry to identify likely stabilizing proteins. Being a portion of an undergraduate biochemistry laboratory course, these investigations represent data obtained from individual laboratory groups, that make oral presentations at the end of their research, providing a more real world approach to the educational experience. This work is supported, in part, by a grant from NSF (DUE 0837398).
Macronutrient composition of milk produced by Ngandu and Aka women in the Central African Republic
Authors: Williams Janet , Wilcox Jennifer , Meehan Courtney L , Brooker Sarah L , Hunt Katherine M , McGuire Mark A , McGuire Michelle K
Published: June 2021

This study was designed to (1) test the feasibility of collecting human milk in extremely remote locations for analysis of macronutrient and microbiological composition and (2) conduct a preliminary trial of the collection procedures in two populations living in the Central African Republic (CAR). Milk samples were preserved with Microtab II tablets (2‐bromo‐w‐nitropropase‐1,3‐diol and natamycin), stored at 99°F, and analyzed for lipid, lactose, and protein at various time points using standard methodologies. Milk lipid, protein, and lactose contents remained stable for over 10 wk (2.4% ± 0.4, 17.4 ± 2.6 mg/mL, 78.6 ± 2.0 g/L, respectively). Milk was then collected from 13 women (25 ± 5 y; 5.2 ± 4.3 mo postpartum) in the Ngandu farming community and 17 women (27 ± 8 y; 15.2 ± 9.3 mo postpartum) from the Aka tropical forest foragers in the CAR. Milk was hand expressed, aseptically transferred into sterile tubes containing bronopol, stored at ambient temperature (~ 95°F) for ~ 3 wk and then stored at −20ºC prior to analyses. Milk components measured included total lipid (3.3 ± 1.5%), protein (15.5 ± 3.3 mg/mL), and lactose (85.3 ± 10.7 g/L). These results indicate that preservation of milk with bronopol tablets allows its collection and storage in remote field locations without refrigeration for the study of various aspects of milk composition and their relationships to maternal and infant health. This work was supported by the Bill and Melinda Gates Foundation, the National Science Foundation (BCS – 0955213).
A food based intervention for drug‐naïve HIV+ women living in Turbo, Kenya
Authors: Ettyang Grace , Neumann Charlotte , Siika Abraham , Nyandiko Winstone , Ernst Judith
Published: June 2021

Objective

To determine the contribution of a food intervention to nutrient intakes.

Methods

During 2008–2010, 225 drug naïve HIV+ (WHO Stage 1 or 2; CD4 >250), 19–45 yrs, clients at the USAID‐Academic Model Providing Access to Healthcare (AMPATH) clinic at Turbo enrolled in a randomized trial of beef vs soy vs wheat biscuit supplements. Height and weight were obtained with standardized procedures. Dietary patterns were summarized from food questionnaires. Nutrient intakes were determined with customized computer soft ware. The adequacies of nutrient intakes were determined with the probability approach and estimated average requirement (EAR).

Results

At baseline, the women mean (±SD) age, body mass index and CD4 were 36.4(7.5) yrs, 22.3(3.6) and 492(191) cells/μL, respectively. Inadequate intakes of 100% occurred for iron, zinc and vit. A. Biscuits were well tolerated and those with beef provided vit. A, selenium, zinc, and vit. B12 at 22%, 24%, 37% and 50% of EAR respectively; soy biscuit provided selenium > EAR.

Conclusions

The beef biscuit provided drug naïve HIV+ Kenyan women micronutrients, whose deficiencies are linked to HIV disease progression. Improved nutrition status and slowed disease progression are expected outcomes from this feeding trial which is near completion. Supported in part by USAID Grant No. PCE‐G‐ 00‐98‐00036‐00, 1R01HD57646‐01A1 (CFDA #93.865) and Heifer Project International.
How Does Iron Deficiency Affect Infant Memory? A model of brain networks and the physiological effects of iron deficiency
Authors: Hammons Julie , Wenger Michael , Robertson Steven , Haas Jere
Published: June 2021

Infants of weaning age (4–7 mo.) have a high risk for iron deficiency (ID). Both human and animal studies suggest that ID causes cognitive—specifically memory—deficits that may persist after iron repletion and into adulthood. Previous reserach in humans is limited by non‐specific measures of cognition, which cannot link ID to decreased memory performance via specific brain abnormalities. To clarify the mechanisms by which ID causes memory deficits, we developed a theoretical model of the neural circuits involved in attention and memory most likely to be affected by ID. Using the model, we predicted the hypothesized electrophysiological output produced by an ID infant brain during a simple memory task. The model predicts the effects of ID on myelination, and neurotransmitter production and availability. We validated the model with data from a study of memory performance and simultaneous brain functioning in ID infants. Data were fit to the model to 1) determine goodness‐of‐fit, with implications for which mechanisms contribute most to memory decrements and 2) improve the model and our ability to predict the effects of ID on infant memory performance and development. Supported by NSF.
α‐Tocopheryl phosphate – an activated form of vitamin E important for angiogenesis?
Authors: Zingg Jean-Marc , Meydani Mohsen , Azzi Angelo
Published: June 2021

Vitamin E (α‐tocopherol, αT) was originally discovered as a dietary factor essential for reproduction in rats. The molecular basis for reproductive failure during vitamin E deficiency was later described to be associated with impaired formation of an extensive placental vascular network. Recently, small amounts of a novel αT derivative, alpha‐tocopheryl phosphate (αTP), have been detected in plasma and animal tissues as well as in foods. We have now assessed the effects of αT and αTP on gene expression in THP‐1 monocytes using gene arrays. The expression of the vascular endothelial growth factor (VEGF) was induced by αTP at mRNA and protein level leading to enhanced angiogenesis in HUVEC cells. Induction of VEGF by αTP was the consequence of stimulation of the PI3K/Akt signalling pathway. The treatment with αTP, but not with αT, significantly induced a VEGF‐promoter luciferase construct transfected into HEK293 cells. We propose that αT is activated by phosphorylation to αTP which may act as an active cofactor or lipid mediator influencing cellular signaling and gene expression leading, in selected tissues, to increased angiogenesis and most likely vasculogenesis. Increased VEGF expression induced by αTP may explain not only the essential effects of vitamin E on reproduction, but also increased cell survival during ischemia/reperfusion injury in brain and muscle cells. Supported by USDA Contract #58‐1950‐7‐707.
Proanthocyanidins stimulate an inflammatory response in a pig intestinal epithelial cell line (IPEC‐1 cells)
Authors: Monagas Maria , Schoene Norberta , Urban Joseph , Solano-Aguilar Gloria
Published: June 2021

Proanthocyanidins are oligomers and polymers of flavan‐3‐ols that are amongst the most abundant and bioactive polyphenols in the human diet. IPEC‐1 cells are a non‐transformed, non‐tumorigenic cell line derived from pig ileum commonly used as a model to evaluate intestinal epithelial cell activity in vitro. In the present work, IPEC‐1 cells were used to determine the effect of proanthocyanidins on immune and inflammatory gene expression. Cells were treated with highly purified proanthocyandin extracts from cranberry and grape seed under non‐stimulated and LPS‐stimulated conditions during a 48 h total incubation period at 37°C. Inflammatory gene expression levels for ICAM‐1, CCL28, IL‐8, TLR4, TNF‐α, IL‐25, IL‐6, CCL20, MUC‐2, IL12p40, CCL2 (MCP‐1), and PTGS2 (COX‐2) were analyzed by real‐time RTPCR, and IL‐8 protein production in cell supernatants was determined by ELISA. Our results showed no significant change in cell viability or apoptosis, but ICAM‐1, CCL20, and IL‐8 gene expression was up‐regulated in response to proanthocyanidin extracts. Increased protein levels of IL‐8 were consistent with increased gene expression. Our results suggested that proanthocyanidins stimulate components of the inflammatory response of IPEC‐1 cells. This effect might be beneficial for certain immune‐related diseases. Funded by the Spanish MICINN (AGL2010‐17499) and ARS #1235‐51530‐053.
Quantitative assay for evaluating anti‐clumping reagents
Authors: Zem Gregory , Khurrum M. , Saab L. , Nazari S , Tonyan O , Shahbazian Az , Alvarez G. , Leyva Y , Azatyan A , Majd A , Moshtael M. , Shafagh S , Farivarmoheb N , Azalbar S , Harutyunyan K. , Sabbaugh A , Kirungi C , Dzhambazian A , Khachatoorian C , Miranda S , Sherazi Z , Altunyan M. , Iriana N , Aghdasi O , Dorian J. , Ter-Papyan H. , Zadori N , Hernandez F. , Oppenheimer SB
Published: June 2021

A model system was developed to quantitatively identify reagents that unclump cells (yeast, Saccharomyces cerevisiae). Biofilms, infections and cancer cells in the circulation often involve a clumped or adhering state. Reagents that unclump cells might help lead to the development of anti‐biofilm, anti‐infection and anti‐cancer cell survival drugs. Various sugars and an amino acid (0 mg/ml – 20 mg/ml) were tested for their ability to unclump low pH (2.4), flocculated, formaldehyde (1%) fixed yeast in distilled water. The yeast with and without the reagents were stirred in 0.5 ml droplets on glass microscope slides in distilled water and percentages of single yeast cells (vs clumped yeast) were recorded over a 60 min time course by focusing on the edge of the droplets in 923 total trials. Alpha methyl glucose, alpha methyl mannose and D‐glucose increased the percentage of single cells over controls in the absence of reagents (p less than 0.05), while L‐arginine did not and D‐fucose, alpha lactose and sucrose had inconsistent effects. Most important in this study was the development of an essentially no‐cost, remarkably simple assay, to quantitatively assess reagents that could clump or unclump cells (supported by NSF Presidential Award 0731633, NIH NIGMS SCORE S0648680, RISE, MARC, Joseph Drown Foundation, Sidney Stern Memorial Trust).
Resistance to multi organ damage after hemorrhagic shock induced ischemia/reperfusion in arctic ground squirrels
Authors: Bogren Lori , Olson Jasmine , Carpluk JoAnna , Moore Jeanette , Drew Kelly L
Published: June 2021

During hemorrhagic shock (HS), the body undergoes global ischemia as blood pressure drops below the threshold at which tissues can be adequately perfused. Resistance to ischemia/reperfusion (I/R) injury is a characteristic of hibernating mammals. The present study sought to determine if arctic ground squirrels (AGS) are protected from HS induced I/R and if any protection is dependent upon their hibernation season. Rats, euthermic AGS, and interbout arousal AGS were subject to HS by withdrawing blood to a mean arterial blood pressure (MAP) of 35 mmHg. Low MAP was maintained for 20 min and then the animals were reperfused with Ringers. The animals’ temperature was maintained at 36.5–37.5 °C. After reperfusion, animals were monitored for 3 hrs then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis. In addition, a group rats and AGS were monitored for three days after HS to access survival and physiological impairment. AGS were able to survive three days beyond HS with no impairment noticeable after 12 hrs whereas rats did not survive to 12 hrs. Regardless of season, blood chemistries indicate no organ damage in the ground squirrel while the rat showed signs of liver damage. In addition, both winter and summer AGS had a lower cytokine response after HS than did rats. Funded by USAMRMC W81XWH009‐2‐0134.
Tubular Fluid Oscillations Mediated by Tubuloglomerular Feedback in a Short Loop of Henle
Authors: Ryu Hwayeon , Layton Anita T.
Published: June 2021

The tubuloglomerular feedback (TGF) system in the rat kidney, which is a key regulator of filtration rate, has been shown in physiologic experiments to mediate oscillations in tubular fluid pressure and flow, and in NaCl concentration in tubular fluid of the loop of Henle. We developed a mathematical model of the TGF system that represents NaCl transport along a short loop of Henle with compliant walls. A bifurcation analysis of the TGF model equations was performed by deriving and finding roots of the characteristic equation arising from a linearization of the model equations. The analysis revealed a complex parameter region that allows a variety of qualitatively different model equations, including stable time‐independent steady‐state solution and oscillatory solution. Model results suggest that a higher thick ascending limb (TAL) NaCl active transport rate or a smaller TAL radius near the loop‐bend gives rise to stable oscillatory solutions at sufficiently high TGF gain values, even with zero TGF delay. Also, tubular wall compliance increases the tendency of the TGF system to oscillate. This research was supported in part by NIH grant DK‐89066 and by NSF grant DMS‐0715021.
Interactions between Tubuloglomerular Feedback and the Myogenic Mechanism of the Afferent Arteriole
Authors: Sgouralis Ioannis , Layton Anita T.
Published: June 2021

We have formulated a mathematical model for the rat afferent arteriole (AA), glomerulus, and short loop of Henle, and used that model to study the interactions between the tubuloglomerular feedback (TGF) and myogenic mechanism, the two key mechanisms that mediate renal autoregulation. Blood flow is described by Poiseuille flow. The AA model consists of a series of arteriolar smooth muscle cells, each of which represents ion transport, cell membrane potential, cellular contraction, gap junction coupling, and wall mechanics. The myogenic response representation is based on the hypothesis that the voltage dependence of calcium channel openings responds to transmural pressure so that the vessel constricts when pressure increases. The glomerular filtration model is based on the model by Deen et al. (AJP 1972). The TGF model represents the pars recta, descending limb, and thick ascending limb, and predicts tubular fluid flow rate and [Cl−] along the loop. The model can be used as a fundamental component in a multi‐scale renal microvasculature model for investigations of pathogenesis of hypertensive renal diseases. This research was supported in part by NIH grant DK‐89066 and NSF grant DMS‐0715021
Functional characterization of the interaction between oxidative and xenobiotic stress response proteins SKN‐1/Nrf and WDR‐23 in C. elegans
Authors: Leung Chi Kwan , Choe Keith Patrick
Published: June 2021

The conserved cap ‘n’ collar transcription factors SKN‐1 and Nrf regulate development, cytoprotection, and drug metabolism in animals ranging from nematodes to mammals. In C. elegans, SKN‐1 activity and abundance are increased during cellular stress by unknown mechanisms. We recently defined WDR‐23 as a conserved WD40‐repeat protein that interacts with SKN‐1 to target the factor for proteasomal degradation in C. elegans. Interestingly, WDR‐23 contains multiple cysteine residues that could function as redox sensors. SKN‐1 activity is also regulated by multiple protein kinase pathways, including the conserved GSK‐3, p38 MAPK, and SGK‐1/AKT‐1/2 insulin‐like signaling pathways. To provide mechanistic insight into how environmental stress and phosphorylation regulate SKN‐1, we are 1) identifying minimal binding motifs, 2) defining the biochemical relevance of mutations in SKN‐1 or WDR‐23 that alter pathway function in vivo, and 3) determining if oxidants directly modify WDR‐23. This work is funded by NSF grant IOS‐1120130 to KPC.
Intermittent activation of peripheral renin‐angiotensin system (RAS) elicits sympathetic long term facilitation (LTF)
Authors: FONG ANGELINA Y , Xing Tao , Bautista Tara G , Zogovic Branimir , Pilowsky Paul M
Published: June 2021

We recently reported that acute intermittent hypoxia (AIH, 10×45s of 10% O2, 5 min intervals) produces LTF of splanchnic sympathetic nerve activity (sSNA), independent of respiratory LTF (Xing & Pilowsky, 2010, J Physiol, 588:3075). We hypothesised that sympathetic LTF (sLTF) is the result of intermittent activation of RAS, by renal hypoxia caused by AIH‐evoked hypotension. We recorded phrenic nerve activity (PNA) and sSNA, in anesthetised (pentobarbitone, 60mg/kg, ip), vagotomised, and ventilated Sprague‐Dawley rats. Intermittent bolus injections of angiotensin II (Ang, 10×35pmol in 0.1ml, iv), at the same 5 min intervals as AIH, elicited sLTF (+42.4 ±11.5%, n=5). Intermittent phenylephrine (PE, 10× 25μg in 0.1mL, iv) also produced sLTF (+72.4±21.6%, n=5), possibly via renal vasoconstriction induced renin release. Intermittent sodium nitroprusside induced hypotension (10×50μg in 0.1mL, iv) did not elicit sLTF (+2.4±5.6%, n=5). Infusion of Ang (350pmol in 1mL) or PE (250μg in 1mL) over 10 mins, also did not cause sLTF, indicating that intermittent stimulation of RAS is essential for sLTF to develop. PNA was unchanged in all groups. This data supports the idea that sLTF and respiratory LTF are mediated by separate mechanisms and that intermittent stimulation of peripheral RAS may be sufficient to elicit sLTF. This data may be relevant to sympathetic overactivity in sleep apnea patients. Funded by NH&MRC, ARC.
Alpha1A Adrenergic Receptor Stimulation Improves Mood in Mice
Authors: Davis Bethany A. , Luger Elizabeth J. , Fossen Anna H. , Collette Katie M. , Wilkie Lee N. , Poitra Shelby L. , Haselton Jim R. , Perez Dianne M. , Doze Van A.
Published: June 2021

The role of alpha1‐adrenergic receptors (alpha1‐ARs) in cognition and mood is controversial, likely due to past use of non‐selective agents. Activation of the alpha1A‐AR subtype was recently shown to increase neurogenesis, which is linked to cognition and mood. We studied the effects of chronic alpha1A‐AR stimulation using transgenic mice engineered to express a constitutively active mutant (CAM). Previously, we found that CAM‐alpha1A‐AR mice demonstrated enhancements in several behavioral models of learning and memory. We now report that CAM‐alpha1A‐AR mice exhibited antidepressant and less anxious phenotypes in several behavioral tests when compared to normal wild‐type littermate control mice. Furthermore, the reduction in anxiety‐ and depressive‐like behaviors was present in both sexes and across all ages (young to old). Our results suggest that chronic alpha1A‐AR stimulation improves cognitive function and mood. These results suggest that long‐term alpha1A‐AR stimulation may afford a potential therapeutic target for treating anxiety and depression. This strategy may also be effective in counteracting the decline in cognitive function and mood associated with aging and neurological disorders. This study was supported in part by NSF CAREER Award 0347259, NSF REU Site Grant 0851869, UNDSMHS REFUNDU program, NIH NCRR INBRE Grant P20RR016471, and NIH Grant RO1HL61438.
Web delivered program to demonstrate a pedagogy promoting meaningful learning
Authors: Modell Harold I. , Michael Joel A. , Knoche Craig
Published: June 2021

Meaningful learning results when acquired information is used to accomplish a goal or solve a problem. Meaningful learning is an iterative process in which the learner (1) recognizes his/her existing mental model, (2) tests the mental model, and (3) refines the mental model. Helping the learner to engage in this process requires a pedagogy in which the instructor assesses the current knowledge of the learner, presents him/her with opportunities to apply this knowledge (e.g. make a prediction), and helps the learner refine his/her mental model before making another prediction. We have developed a web delivered computer program that helps students study mechanisms underlying pressure and volume changes occurring during a cardiac cycle. The program engages the student in a dialog that explores common misconceptions related to the cardiac cycle and helps her/him generate a Wiggers diagram. In an assessment component of the program, the student predicts the nature and site of valve pathophysiology given a Wiggers diagram or predicts the Wiggers diagram resulting from a specific valve pathophysiology. The student is able to test these hypotheses using a simulation of the cardiovascular system.

Supported in part by NSF Grant #DUE‐1043422.
A new pedagogy for ion channels: they're permeases, not pores!
Authors: Nelson Peter Hugo
Published: June 2021

A recently proposed kinetic model provides a simple conceptual framework for teaching students the fundamentals of ion channel permeation. The association/dissociation (A/D) model is based on the known structure of ion channels and is supported by single‐channel permeation experiments and a large group of molecular simulations of potassium channels. Ion channel permeation is treated as an enzymatic process. The active site of the ion channel permease is the selectivity filter (SF) that normally contains helper ion(s) of the same species as the permeant ion. During association all the SF ions move together in a concerted manner as the substrate ion shunts on the helper ion(s) to form a conductive SF state. Dissociation is also a concerted process in which all the SF ions move together to shunt off an ion from either end of the SF. Dissociation of a helper ion from the far end of the SF completes the permeation cycle. The A/D model exhibits universal Michaelis‐Menten kinetics with a voltage‐dependent maximum permeation velocity and a voltage‐dependent Michaelis concentration. The ion channel is “autoselective”, as only ions of the same species as the helper ion(s) can form a stable conductive state. The model also provides a simple kinetic explanation for the Nernst potential – without relying on any thermodynamic arguments. Support from the NSF (Grant DUE‐0836833) is gratefully acknowledged – see http://circle4.com/biophysics.
Conceptual frameworks and misconceptions associated with core principles of physiology, including homeostasis
Authors: McFarland Jenny , Michael Joel , Wenderoth Mary Pat , Modell Harold , Wright Ann , Cliff William
Published: June 2021

The core principles of physiology are those that physiology students should understand and be able to use. We have ‘unpacked’ three of the most important core principles (Flow Down Gradients, Homeostasis and Cell‐Cell Communications) into their component ideas and thereby articulated a conceptual framework for each. Physiology faculty at 2‐and 4‐year colleges, universities and medical schools were surveyed and asked to identify the importance of each of the component ideas; the result of our first survey on ‘Flow’ was reported last year. Amongst the >40 responses to our survey on unpacking Homeostasis, there was strong agreement on the importance of the first level of component ideas within the conceptual framework. For example, ~80% agreed that the component idea that “Homeostatic processes require a sensor” was essential to understanding this core concept, however, less that a third responded that sub‐component idea ‘Sensory receptors may be in different, distant locations in the body’ was essential. We have also identified misconceptions associated with some core principles and linked these to our unpacked conceptual frameworks. We are using the conceptual frameworks and the misconceptions to develop a conceptual assessment of physiology (CAP) instrument (i.e., a concept inventory). Supported by NSF grant DUE‐1043443.
Comparative analysis of masticatory apparatus features in neonatal common marmosets (Callithrix jacchus) and cotton‐top tamarins (Saguinus oedipus)
Authors: Mork Amy Lovejoy , Taylor Andrea B , Vinyard Christopher J
Published: June 2021

Marmosets are unique among callitrichids in habitually gouging trees with their anterior teeth to stimulate exudate flow. Previous analyses suggest that the masticatory apparatus of adult marmosets displays features facilitating relatively large jaw gapes, but decreased load‐resistance ability relative to closely‐related non‐gouging tamarins. We analyzed masticatory apparatus features of neonatal marmosets and tamarins to determine if the morphological differences observed between adults are present at birth. If marmoset infants show similar morphological divergence as adults, then the early appearance of these features suggest they are potential adaptations for tree gouging at wide gapes.

We examined six stillborn common marmosets (Callithrix jacchus) and six cotton‐top tamarins (Saguinus oedipus). We microCT scanned the jaws, quantified fiber architecture of masticatory muscles and estimated symphyseal strength in simulated in vitro loading.

We found few differences in mandibular cross‐sectional geometry, symphyseal strength or jaw‐muscle architecture between marmoset and tamarin neonates. Collectively, these findings suggest that at least some of the postnatal changes in the marmoset mandible and jaw‐muscle architecture that facilitate wide gapes, but reduced load‐resisting abilities compared to tamarins, arise from phenotypic plasticity related to gouging at wide gapes.

Grant Funding Source: This work was funded by grants from the National Science Foundation (BCS‐0959438; BCS‐0412153).
The Role of Vascular Endothelial Growth Factor in Mechanically Stimulated Muscle Derived Stem Cell Repair
Authors: Beckman Sarah , Mlakar Logan , Huard Johnny
Published: June 2021

We have previously shown that muscle‐derived stem cells (MDSCs) are significantly better than myoblasts at maintaining cardiac function after infarction, which is likely due to higher survival and vascular endothelial growth factor (VEGF) secretion. Mechanical stimulation increases VEGF secretion, and after mechanically preconditioning MDSCs and injecting them into an acute model of cardiac infarction function is improved. Therefore, we hypothesized that the benefits seen from mechanical stimulation is due to an increase in VEGF, and if we block VEGF we will not see an improvement in repair. To test this we have mechanically stimulated MDSCs transduced with the soluble VEGF receptor antagonist sFlt1 and tested their regenerative potential in the skeletal muscle of a mouse model of Duchene Muscular Dystrophy. We have found that mechanically stimulating MDSCs resulted in an increase in angiogenesis in the engraftment area. The MDSCs transduced with sFlt1 resulted in less angiogenesis, and the mechanically stimulated MDSCs transduced with sFlt1 showed no difference in angiogenesis from the un‐stimulated sFlt1 group. There also appears to be little difference between fibrosis in the sFlt1 stimulated and un‐stimulated groups. Therefore, VEGF appears to be a critical factor in angiogenesis following cell transplantation in mechanically stimulated MDSCs.

Grant Funding Source : DOD
Architecture of the Mediator Head Module
Authors: Takagi Yuichiro , Imasaki Tsuyoshi , Cai Gang , Yamada Kentaro , Berger Imre , Asturias Francisco
Published: June 2021

Mediator is a key regulator of eukaryotic transcription, connecting activators and repressors bound to regulatory DNA elements with RNA polymerase II (Pol II). In the yeast S. cerevisiae, Mediator is organized into three modules (Head, Middle/Arm and Tail) that together comprise 21 subunits with a total mass over 1 MDa. Our understanding of Mediator assembly, and of its role in regulating transcription, has so far been hampered by limited structural information. We solved the crystal structure of the Mediator Head module (7 subunits, 223 kDa) at 4.3 angstrom resolution, revealing three distinct domains (Fixed jaw, Movable jaw, and Neck) that come together to form a flexible central joint region. Strikingly, we discovered a novel structure in the Neck domain, in which a total of ten helices from five different subunits form a large multi‐helical bundle as a single structural unit. This unique helical bundle structure ensures stable assembly of the Head subunits and provides binding sites for general transcription factors and Pol II. Mapping previously identified genetic mutations onto our Head structure suggests that the Head module may juxtapose TFIIH and the carboxyl‐terminal domain (CTD) of the largest subunit of Pol II (Rpb1) to facilitate CTD phosphorylation. These results reveal architectural principles underlying the role of Mediator in the regulation of gene expression. Supported by NSF, AHA and NIH
The role of protein sequence and global conformation in DNA binding specificity of members of the Cro family
Authors: Nelson Michael R , Olson Keith , Cordes Matthew H.J.
Published: June 2021

The Cro family of bacteriophage DNA‐binding proteins demonstrates the substantial conformational changes that can occur in protein evolution while the primary sequence is significantly conserved. Xfaso 1 and Pfl 6 of the Cro family are α‐helical and mixed α‐helical/β‐sheet, respectively, despite sharing 40% sequence identity. Both proteins are transcription factors that use a helix‐turn‐helix (HTH) motif as their primary DNA recognition moiety. However, the natural consensus DNA sequences of the proteins are different at three positions in each seven base‐pair half site. Fluorescence anisotropy measurements showed that wild‐type (WT) Xfaso 1 and Pfl 6 bound their cognate sites with dissociation constants (KD) of 230 nM and 56 nM, respectively. WT Pfl 6 bound its noncognate site with KD = 1.99 μM and WT Xfaso 1 did not bind its noncognate site at all. To explore whether this change in binding specificity is due to differences in sequence in the HTH region or to global differences in their tertiary structures, mutations were introduced into the HTH region of both proteins in order to equalize the binding region sequence while retaining global structure. The binding affinities of these variant proteins against the consensus binding sites were tested and preliminary results suggest that mutation of the Xfaso 1 sequence toward Pfl 6 allows it to bind the Pfl 6 consensus site. Funded by NSF (MCB‐0647390) and HHMI (52005889).
Expression and auto‐processing of hedgehog‐like proteins from Brugia malayi
Authors: Savidis George , Drago Matthew J. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Hedgehog proteins have an N‐terminal signaling domain and a C‐terminal auto‐processing domain, called the HINT domain (for Hedgehog‐INTein, as it shares sequence and structural similarities to self‐splicing inteins). The HINT domain has a C‐terminal sterol‐recognition region (SRR) not shared with inteins. We will analyze the post‐translational cleavage between the signaling and HINT domains from a hedgehog‐like protein from the parasitic nematode Brugia malayi. We have created E. coli expression vectors that encode fusion proteins of 1) an N‐terminal maltose‐binding protein to the HINT domain, or 2) an N‐terminal His‐tag to the signaling and HINT domains. We have created both fusion proteins with and without the SRR. We are currently optimizing expression and purification conditions for these proteins, with the aim to study the role of conserved intein residues in the hedgehog‐processing mechanism.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KVM), and a Sherman Fairchild Foundation Summer Research Scholarship (GS).
Expression of a Tetradomain Fragment from a Polyunsaturated Fatty Acid Synthase with Dehydratase Activity
Authors: Oyola-Robles Delise J , Rodriguez-Guilbe Maria M , Bermudez Mei-Ling , Rivera-Diaz Monica , Carballeira Nestor M , Baerga-Ortiz Abel
Published: June 2021

Polyunsaturated fatty acids (PUFAs) are important components of human health and important ingredients in biodiesel preparations. PUFAs from deep‐sea bacteria are synthesized by a modular polyketide synthase, which contains several domains including two dehydratase (DH) domains responsible for the introduction of double bonds. In order to study double bond formation in PUFAs, we have expressed and purified the individual protein fragments from a deep‐sea PUFA synthase. Protein constructs were designed using a bioinformatic tool for the prediction of protein linkers which revealed the presence of two previously uncharacterized pseudo‐domains. The resulting design comprising two putative DH domains in tandem was expressed in E coli, purified by chromatography and assayed against surrogate substrates by UV spectroscopy. Results showed that the designed construct is more active against CoA‐linked substrates than for N‐acetylcysteamine thioester, as revealed by reaction kinetics. Additionally, E coli strains engineered to over‐express DH1‐DH2 can produce 3–5 times more FAs than the wild‐type strain, suggesting that dehydration may be rate‐limiting. We anticipate this result can be implemented to drive the production of FAs in bacteria and be a viable option for the production of biodiesel precursors. This work was funded by Grant CHE0953254 from the NSF and MBRS‐RISE Program (R25GM061838) of the UPR‐MSC.
Side‐chain cyclization of Gln or Asn residues coupled to peptide bond cleavage in an intein and a model peptide
Authors: Urbanski Laura M. , Chin Stacy L. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational event by which an intervening polypeptide, or intein, facilitates its own excision from the flanking polypeptides, or exteins, and the ligation of the exteins. An epimerase found in Trichodesmium erythraeum contains two inteins, Ndse1 and Ndse2. The first intein, Ndse1, can facilitate protein splicing efficiently in E. coli, even with a native C‐terminal Gln replacing the highly conserved Asn. In addition, activity of the intein may be influenced two downstream Cys residues and the presence of disulfide bonds, as we observed differential reactivity in inteins expressed under reducing and non‐reducing conditions. Substitution of Gln for Asn results in an increase in the rate of C‐terminal bond cleavage for the PolII intein from Pyrococcus abyssi. To study side chain cyclization in a model system, we obtained three pentapeptides of Abz‐His‐Xxx‐ Src‐Lys, where Xxx is Gln, Asn or Ala. Dnp, dinitrophenyl, is attached to the C‐terminal Lys and ortho‐aminobenzoic acid (Abz) serves as the N‐terminal residue. Peptide bond cleavage separates the Abz fluorophore from the Dnp quencher.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245, the Camille and Henry Dreyfus Foundation (KM), and a Holy Cross Alumni/Parents Summer Research Scholarship (LU).
Calcium‐Dependent Interactions and Electron Transfer in NADPH Oxidase 5
Authors: Wei Chin-Chuan , Reynolds Nicole , Palka Christina
Published: June 2021

Superoxide generated by NADPH oxidase 5 (NOX 5) is of growing importance for physiology and pathobiology. The NOX5 activity appears to be regulated by its self‐contained calcium binding domain (CaBD), through an interaction with its flavoprotein domain. Here, using fluorescence we showed that such domain‐domain interaction is Ca2+‐dependent. The resulting interaction enhances the electron transfer from NADPH to FAD in cytochrome C assay and gates heme reduction using full‐length NOX5. Our data support the published results showing that CaBD acts as an activator by binding the inhibitory loops in the flavoprotein, and thus lifting the blockage of electron transfer for superoxide production. CaBD contains two half domains, in which the N‐terminal half contains the Ca2+‐specific EF‐hands and The C‐terminal half contains the Ca2+/Mg2+ mixed EF‐hands. The half domains alone showed no interaction with the flavoprotein. However, the calcium knock‐out mutant, CaBD(E99Q/E143Q) in which the E residues at the –z position in 3rd and 4th EF‐hands were replaced with Q, retains its interaction with the flavoprotein, suggesting that its N‐terminal half is critical for regulating electron transfer. Currently we are investigating other constructs, such as CaBD(E31Q/E64Q) and transmembrane‐deleted NOX5, for the studies of domain‐domain interaction and electron transfer. Furthermore, we include calmodulin (CaM) in our existing system to investigate a possible ternary complex formation (CaBD/flavoprotein/CaM) using FRET and for determining its effects on electron transfer as it has been shown that CaM interacts with the flavoprotein. This work is supported in part by National Science Foundation CCLI grant.