bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell swelling induced by water and glycerol gradients in hepatocytes from warm‐and cold‐acclimated Hyla chrysoscelis, a freeze‐tolerant amphibian
Authors: Tsou PonTi , Koneru Geetha , Finatti Luiz , Bobka Kara , Frisbie James , Goldstein David L.
Published: June 2021

Gray tree frogs, Hyla chrysoscelis, survive freezing in part by accumulating cryoprotective glycerol, which is likely synthesized by hepatocytes. During cold acclimation, freezing, and thawing, glycerol and water must be transported into and out of those cells, likely via aqua/glyceroporins. We therefore hypothesized that hepatocytes of cold‐acclimated frogs have enhanced permeability to glycerol and water. We tested this hypothesis by assessing cell volume change in cells subjected to an osmotic or glycerol gradient. We perfused liver with media containing collagenase, dissociated hepatocytes in isosmotic media, and tracked cell swelling upon exposure to hypotonic challenge. In response to a glycerol gradient, cells from cold‐acclimated animals swelled and lysed more quickly than those from warm‐acclimated animals. This response was slower for cells tested at ~4°C and was inhibited by pre‐incubation with 0.3 mM HgCl2, which blocks aqua/glyceroporin‐mediated permeability. Both HgCl2 and incubation temperature had a lesser effect on the response to an osmotic gradient than to a glycerol gradient. These findings imply that cold‐acclimated hepatocytes have enhanced glycerol permeability conferred by aqua/glyceroporins, but that water permeability may occur via a separate pathway. Supported by NSF IOS‐1121457 to D. Goldstein and C. M. Krane.
Theory of Gastric Ventilation: Effects of Hyperoxia, Hypoxia, and Hyperbaric Exposure in the Rat
Authors: Held Heather E. , Landon Carol S. , Dean Jay B.
Published: June 2021

We previously described gastric ventilation, a phenomenon in which hypercapnia stimulates gastric acid secretion. In the present study, we tested the effects of the following on gastric acid secretion: normobaric normoxia (NN) hypobaric hypoxia (hh), normobaric hypoxia (Nh), hyperbaric hyperoxia (HH), hyperbaric hypercapnia (HC), and hyperbaric normoxia (HN). Two rats implanted with gastric catheters have completed hypobaric experiments; 1 completed hyperbaric experiments. In hypobaric experiments, minute ventilation (VE) was 74.4 ± 5.7 ml/min at NN (mean ± SE) and increased as expected to 81.6 ± 35.5 ml/min at hh and 76.1 ± 8.6 ml/min at Nh; pH appeared minimally affected: 3.7 ± 0.2 at NN, 3.8 ± 0.3 at hh, and 3.7 ± 0.4 at Nh. Hyperbaric exposures appeared to stimulate gastric acid secretion, though in the rat tested, the response appeared delayed until the following air period: pH was 4.52 at NN, 4.5 at HH, 3.94 at NN, 3.81 at HC, 3.64 at NN, 3.66 at HN, and 3.64 at the final NN. This suggests that gastric acid secretion responds to environmental stimuli, an important health consideration for those who routinely dive and who tend to retain CO2 while diving. Funded by ONR.
Analysis of MicroRNA Expression Profile in PBMCs of Hypertensive Patients
Authors: Pinho Maria Joao , Vaz‐da‐Silva Manuel , Soares‐da‐Silva Patricio
Published: June 2021

MicroRNAs (miRNAs) are endogenous repressors of gene expression and have been reported to play important roles in a number of biological processes. We assessed the miRNA system in Portuguese population of hypertensive subjects in order to investigate the potential of miRNA as biomarkers for essential hypertension (EH). Total RNA was isolated from PBMCs from a total of 66 hypertensive subjects (divided into: group 1, medicated HTA I and II; group 2, medicated HTA III and group 3, nonhypertensive with cardiac disease) and 13 non‐hypertensive subjects. Agilent Human miRNA Microarray 16.0 was used to detect the expression of miRNAs. MiRNA arrays showed that 117, 9 and 21 miRNAs were expressed at different levels in group1, group 2 and group 3, respectively. In addition to several miRNAs previously known to be associated with EH, 4 unidentified miRNA candidates were found to be dysregulated in group 1 samples. Among the novel candidates, the genes hsa‐miR‐4306, hsa‐miR‐146a, hsa‐miR‐146b‐5pn, has‐miR‐22 were upregulated. 14 MiRNAs differentially expressed in group 1 were distributed in 5 miRNA clusters in chromosomes 1, 7, 13 and X. We report four novel miRNA candidates in EH; these miRNA sequences require further validation and functional analysis to evaluate whether they play a role in the pathogenesis of EH.

Supported by FCT (PIC/IC/83204/2007)
Preliminary Results on the Prevalence of Physiology Students’ Homeostatic Misconceptions
Authors: Wright Ann , McFarland Jenny , Cliff William , Michael Joel , Modell Harold , Wenderoth Mary Pat
Published: June 2021

We specifically asked forty‐seven physiology students in a survey about the core concept “homeostasis.” Physiology students (200 level) were asked to define homeostasis and describe the process by giving a physiological example of homeostasis. Finally, students chose whether heart rate, blood pressure, or both were homeostatically regulated and justified their answer. Definitions, examples of the homeostatic process, and justifications of whether heart rate, blood pressure, or both were homeostatically regulated were scored as Unclear Definition, Partially Complete Definition, or Complete Definition. For the most part, the results indicated that students’ understanding of homeostasis is incomplete and superficial. We are also concerned about the language students used to answer the questions. For example, we wonder what students mean by a “balanced internal environment?” At what level do they understand “balance?” The results also reveal the importance of incorporating many different examples of homeostasis as a general model during instruction to improve understanding. Supported by NSF grant DUE‐1043443.
Interactive computer tutorials illustrating a pedagogy promoting conceptual change
Authors: Modell Harold I , Michael Joel A
Published: June 2021

The traditional pedagogical paradigm for instruction provides the student with information and then tests to see if the student has acquired or can apply that information. Constructivism asserts that we build knowledge on an existing framework and that conceptual change occurs when the learner tests an existing mental model. Hence a more appropriate pedagogy may be one that encourages the student to make predictions based on existing mental models before presenting “new” information. We have developed a series of interactive computer‐based tutorials that illustrates a pedagogy based on this process of recognizing, testing and refining mental models. The exercises focus on core concepts in physiology. Students engage in a dialog in which they are presented with a situation and asked to make a prediction. The follow‐up response addresses that prediction and, in many cases, the implications of the prediction relative to the problem presented. The continued dialog iterates this process to help the student build a mental model that can be used to solve problems relating to the core concept. Topics covered include Determinants of Flow, Membrane Potentials, Elastic Structures, Homeostatic Control Mechanisms, Conservation of Mass, and Membrane Transport. Supported in part by NSF Grant #DUE‐1043422.
Skeletal measures dictating hip abductor force production during locomotion
Authors: Warrener Anna Gabriella
Published: June 2021

This study evaluated the relationship between skeletal dimensions of the hip and pelvis and biomechanical parameters dictating force production of the hip abductors during locomotion. Twenty‐eight subjects participated in this study (IRB, Washington University in St. Louis). Lower limb and trunk kinematics were determined during walking and running trials using high‐speed motion capture (Vicon, 200Hz) while ground reaction forces were simultaneously recorded (AMTI, model‐OR force‐plate, 1000Hz). Anatomical data for each subject was obtained from full lower body MRI (Avanto 1.5T, isotropic 1.7mm resolution). Results indicate that femoral neck length and biiliac breadth are both good predictors of the length of the hip abductor moment arm, but biacetabular width, generally considered a reliable proxy for the body weight moment arm, is not predictive of the true external moment arm about the hip during the course of stance phase. Sex differences were observed in the length of the hip abductor moment arm, with women having muscle moment arms an average of 1cm shorter than men. There were no sex differences in the length of the external moment arm at the hip in the coronal plane, although mass‐specific hip abductor force production was slightly higher in women because of shorter abductor moment arms. Research funded by: NSF #0850841, The Leakey Foundation, and The Wenner‐Gren Foundation.
Hydroxamates Inhibit EMMPRIN as well as ADAM17/TACE and MMPs
Authors: Gordon Marion K , DeSantis Rodrigues Andrea S. , Hahn Rita A. , Gerecke Donald R , Svoboda Kathy K H
Published: June 2021

To study corneal wound healing, organ cultures were injured with nitrogen mustard (NM). This blistering agent causes the epithelium to separate from the stromal layer. Separation is in part due to the activation of ADAM17/TACE. We found that ADAM17 is activated in rabbit corneal organ cultures immediately after NM exposure, and remains activated even at 24 hr post exposure. To inhibit ADAM17 and promote epithelial‐stromal integrity after NM exposure, the hydroxamates olvanil OH (NDH4409) and retro olvanil 8 (NDH4417) were tested for their ability to inhibit ADAM17 and reduce collagen XVII cleavage. The hydroxamates were applied 4 times to organ cultures over the course of 22 hrs, beginning 2 hours post NM exposure. Epithelial‐stromal separation at 24 hr was significantly reduced by the hydroxamate treatments. Surprisingly, both drugs also reduced expression of the matrix metalloproteinase (MMP) inducer, EMMPRIN. Thus, the inhibition of MMP‐9 observed after hydroxamate treatment was likely caused by both direct inhibition of the catalytic zinc as well as the reduction of EMMPRIN. In conclusion, hydroxamates inactivate ADAM17, MMPs, and also preserve corneal epithelialstromal integrity by attenuating the vesicant‐induced upregulation of EMMPRIN. Supported by UMDNJ‐Rutgers Counteract Center of Excellence NIAMSD U54AR055073; NIH NEI EY009056; and NIEHS P30ES005022
Regional material heterogeneity in craniofacial cortical bone of the genus Pan
Authors: Gharpure Poorva Hemant , Dechow Paul C
Published: June 2021

Examination of craniofacial biomechanics necessitates regional material properties data as a foundation. We studied the pattern of material properties in the craniofacial complex of the genus Pan. Our aim was to characterize the elastic properties of the chimpanzee craniofacial cortical bone regionally. Former studies substantiate similarities in patterns of these properties within closely related primate species. Specimens were harvested from the craniofacial cortical bone of five adult chimpanzees. A continuous wave ultrasonic technique was used to measure velocities. Elastic coefficients and technical constants derived from mathematical equations quantified elastic properties. Regional comparisons of data using one‐way ANOVA revealed variations in elastic properties. Significant differences were observed in regional density (P< 0.001). The alveolar region displayed the least thick (mean=1.35 mm) and dense (mean=1630mg/cm3) cortical bone. Results for elastic moduli are significant E1 (P<0.007), E2 (P<0.009) and E3 (P<0.001). Shear moduli were prominent in all of the planes; larger values of G23 indicate stiffer bone. Poisson's ratio was also observed to be significant in v23 (P< 0.019). Greatest anisotropy was seen in the zygomatic arch (E2/E3=0.61). Findings suggest that mechanical properties of cortical bone in the chimpanzee craniofacial skeleton are influenced by its density, architecture and loading patterns in response to functional demands of the cranium.

Grant Funding Source: NSF Hominid grant 0725141
C8‐Arylguanine modified oligonucleotides: Tools for investigating Z‐DNA formation
Authors: Train Brian Christopher , Vongsutilers Vorasit , Thomsen Nissa M. , Gannett Peter M.
Published: June 2021

DNA has many different conformations however only Z‐DNA adopts a left‐handed helix. The zigzag phosphate backbone of Z‐DNA brings the negatively charged phosphate residues closer together rendering Z‐DNA a higher energy form of DNA. Z‐DNA has been implicated in carcinogenesis from its role in gene expression and mutagenesis because Z‐DNA formation can stimulate large scale gene deletions, translocations, and rearrangements in vivo. The inability to generate a stable Z‐DNA structure under physiological conditions has made investigation of B‐ to Z‐DNA interconversion difficult since Z‐DNA requires external stimuli to stabilize its formation like chemical modifications or specific proteins known to stabilize Z‐DNA, Z‐DNA binding proteins (ZBP). We have demonstrated that C8‐arylguanine DNA adducts, derived from carcinogenic arylhydrazines, drive Z‐DNA formation and the addition of MgCl2 stabilizes the Z form under physiological conditions. The conformational effects of the C8‐arylguanine mutation were determined by nuclear magnetic resonance (NMR) and circular dichroism (CD). In addition, preliminary results using in silico molecular dynamics and CD have demonstrated that ZBPs prefer to bind to the C8‐arylguanine modified DNA. C8‐Arylguanine can be used as a tool to clarify the mechanism of ZBP mediated B‐ to Z‐DNA transition. The current view fails to explain the ZBP selectivity for the Z form and leads to a contradictory conclusion that ZBPs cannot bind Z‐DNA. C8‐Arylguanine modified oligonucleotides can be used to fully elucidate the binding of ZBPs to DNA, as both the B and Z forms can be generated under physiological conditions, using surface plasmon resonance (SPR). This may reveal the underlying factors that control ZBP‐DNA interactions which could become a potential therapeutic target. (Supported by a fellowship to BCT (HEPC.dsr.09013) and (NSF EPS‐1003907)
Tumor suppressor WWOX participates in cell/cell recognition and migration
Authors: Chen Yu‐An , Chou Pei‐Yi , Hsu Li‐Jin , Chang Nan‐Shan
Published: June 2021

Metastatic cancer cells are essentially devoid of tumor suppressor WWOX expression, and this appears to be associated with their enhanced migration. A portion of WWOX is localized at the cell membrane/cytoskeleton area. By time‐lapse microscopy, we showed that wild type Wwox+/+ mouse embryonic fibroblast (MEF) cells migrated collectively, whereas the knockout Wwox−/− cells migrated individually. When wild type MEF cells encountered knockout cells, the knockout cells failed to recognize the wild type, although they are from the same mouse strain. The knockout Wwox−/− MEF cells reached out with their pseudopodia to touch the wild type from a distance (5–20 μm), and then moved backward significantly faster and divided simultaneously. The event was also shown in many WWOX‐negative malignant cancer cells upon encountering with WWOX‐expressing cells. Restoration of WWOX with ectopic gene expression or treatment with an inhibitor of DNA methylation restored collective migration. Molecular analysis revealed that when cells overexpressing a short N‐terminal segment of WWOX, they attracted upcoming visiting cells. But, they repelled the visitors if a short segment in the C‐terminal SDR domain of WWOX was overexpressed. Thus, WWOX participates in cell/cell recognition, and conformational alteration of membrane‐associated WWOX affects cell behavior. (Supported in part by NHRI, NSC and DOH, Taiwan, and DoD, USA)
Thermodynamic Examination of the 4×2 Bulge of Leadzyme
Authors: Frost Arian Cai , Grover Neena
Published: June 2021

The leadzyme is an in vitro selected ribozyme that contains an asymmetrical 4×2 bulge. It is cleaved by divalent lead ions between nucleotides C6 and G7. Three bases in the bulge, C6, G9, and G24, are conserved. A 1.8 Å crystal structure solved in presence of Mg2+ and Sr2+ shows metal ion binding sites in the bulge and helical regions. Lead cleavage is out‐competed and inhibited by Mg2+ ions. The bulge region of the leadzyme is expected to form different structures under different ionic and pH conditions. A25+ • C6 non‐canonical base pair is expected to form in the bulge and shows two hydrogen bonds in NMR structure and one in the crystal structure. Thermodynamic stability and ion binding of wild type, a control helix, and modifications in the conserved and non‐conserved regions are examined by thermal denaturation experiments. Constructs were denatured in various concentrations of Mg2+ ions and 1 M salt in two different pH conditions. The helix is more stable than the wild type; penalty for breaking the helical structure by insertion of a 4×2 bulge is ~3 kcal/mol. The stability of wild type is not pH dependent. The stability of the modified constructs in high salt conditions varies depending on pH. Several bulge modifications indicate pH dependent Mg2+ stabilization. A detailed analysis of thermodynamic results will be presented. This work was supported by NSF grant, MCB‐0950582 to NG.
Characterization of an archaeal ubiquitin‐like protein SAMP1
Authors: Hepowit Nathaniel Lantaca , Miranda Hugo Venicio , Maupin‐Furlow Julie
Published: June 2021

Small archaeal modifier protein (SAMP1) structurally resembles the eukaryotic ubiquitin (Ub) and bacterial MoaD, sharing with them the β‐grasp fold and C‐terminal GlyGly motif. Our study was aimed to understand the function of SAMP1 using the haloarchaeon Haloferax volcanii as a model system. We demonstrated in vitro that an archaeal homolog of the ubiquitin‐activating enzyme E1 (named ubiquitin‐like activating enzyme of archaea or UbaA) activates SAMP1 in an ATP‐ or GTP‐dependent manner. LC‐MS/MS analysis shows that SAMP1, like Ub, is conjugated to the Lys of its protein substrates by a mechanism requiring UbaA. Cleavage of SAMP1‐conjugates by a JAB1/MPN/MOV34 protease (HvJAMM1) provides evidence that sampylation is reversible. SAMP1 is also found essential for MoCo‐dependent dimethyl sulfoxide reductase activity, suggesting that SAMP1 functions similarly with MoaD in MoCo biosynthesis. Overall, the shared functional similarities of SAMP1 with Ub and MoaD provide a physiological framework in understanding the evolutionary relationship between two divergent biochemical pathways: protein conjugation and sulfur transfer. This work is funded by NIH (ROI GM057498) and DOE (DE‐FG02–05ER1560).
The Ubiquitin Conjugating Enzyme UbcH7, controls cell migration
Authors: Whitcomb Elizabeth A. , Le Feuvre Aurelie , Taylor Allen
Published: June 2021

Post translational modification by ubiquitination can target proteins for degradation, allow the interaction of proteins to form complexes or direct relocalization of proteins to different subcellular compartments. As such, ubiquitin controls a variety of essential cellular processes. Previously we demonstrated that the ubiquitin conjugating enzyme, UbcH7 controls both the entry into and progression through S phase of the cell cycle (Whitcomb et. al 2009 Mol. Biol. Cell v20:1–9). In our search for cell cycle regulatory proteins which might be involved in the UbcH7‐mediated control of the cell cycle, we discovered that UbcH7 affects the level of the cyclin dependent kinase inhibitor, p27, but does not affect the similarly regulated cyclin dependent kinase, p21. Interestingly, depletion of UbcH7 affects p27 levels primarily in the cytosol. While nuclear p27 controls the G1 to S transition of the cell cycle through inhibition of the cyclin dependent kinases, cytosolic p27 plays a role in cell migration. Thus, we asked whether manipulation of UbcH7 also affected cell migration and find that depletion of UbcH7 inhibits cell migration. These results suggest that UbcH7 might be an attractive drug target as it plays roles in controlling both the cell cycle and cell migration.

This work was supported by grants NIH EY13250 and USDA 581950–5100‐060–01A to AT.
Developing a biological reducing system for use at elevated temperatures
Authors: Cramer Julia , Fugate Corey , Jarrett Joseph
Published: June 2021

S‐Adenosylmethionine (AdoMet)‐dependent radical enzymes constitute a rapidly expanding family of enzymes, the Radical SAM superfamily, that are involved in a variety of biological pathways. A defining characteristic of AdoMet‐dependent radical enzymes is the ability to use a reduced [4Fe‐4S] cluster to reductively cleave AdoMet, producing methionine and a 5′‐ deoxyadenosyl radical. This highly oxidizing radical intermediate initiates chemistry by abstracting a hydrogen atom from the substrate. Currently, the most common experimental means of reducing the [4Fe‐4S] cluster are the use of chemical reductants such as sodium dithionite, or the biochemical reductant flavodoxin. In the E. coli reducing system, NADPH reduces ferredoxin(flavodoxin) oxidoreductase (FNR), which then reduces flavodoxin (Fld) or ferredoxin (Fd). The E. coli system has drawbacks including thermal instability, and poor reaction kinetics. The development of a reducing system involving enzymes from thermophilic photosynthetic cyanobacteria will offer solutions to these problems, as such enzymes are stable and functional at elevated temperatures and exhibit rapid turnover due to their involvement in photosynthetic electron transport. We report that FNR from the thermophilic cyanobacterium, Thermosynechococcus elongatus, can be reduced by NADPH and rapidly transfers electrons to E. coli flavodoxin as well as T. elongatus ferredoxin. The component proteins are stable and active up to at least 65 °C. A heterologous reducing system comprised of NADPH, T. elongatus FNR, and E. coli flavodoxin is able to support the catalytic activity of biotin synthase from E. coli, as well as from thermophilic bacteria, including Methylococcus capsulatus. (Supported by NSF Grant MCB 09–23829 to J.T.J.)
Crosslinking Studies to Probe Substrate Binding by Soybean Lipoxygenase‐1
Authors: Howell Rebecca Anne , Clapp Charles H.
Published: June 2021

Soybean lipoxygenase‐1 (SBLO‐1) catalyzes the oxygenation of polyunsaturated fatty acids into conjugated diene hydroperoxides. The three dimensional structure of SBLO‐1 is known, but it is not certain how substrates bind. One hypothesis involves the transient separation of helix‐2 and helix‐11 located on the exterior of the molecule in front of the active site iron. To test this hypothesis, site‐directed mutagenesis was used to create a cysteine mutation on each helix, which could allow for the formation of a disulfide linkage. Disulfide formation between the two cysteines in the T259C, S545C mutant was found to be unfavorable. Treatment of this mutant with the crosslinker 2,3‐dibromomaleimide (DBM) resulted in a 50% reduction in catalytic activity. No loss of activity was observed when the single mutant S545C or the wild type was treated with DBM. Additionally, no loss in activity occurred when the double mutant was treated with N‐ethylmaleimide. The results suggest that restricting the mobilities of helices 2 and 11 reduces but does not eliminate catalytic activity. Funded by NSF grant CHE‐1213262.
Inhibition of Soybean Lipoxygenase‐1 by N‐oleoyl‐Dtryptophan
Authors: Johnson Cody L. , Clapp Charles H.
Published: June 2021

Soybean lipoxygenase‐1 (SBLO‐1) is a nonheme iron protein that catalyzes the oxygenation of polyunsaturated fatty acids using molecular oxygen, resulting in conjugated diene hydroperoxides. Although the three‐dimensional structure of SBLO‐1 is known, it is not certain how substrates bind. Following the discovery that N‐linoleoyl‐ D‐tryptophan is an excellent substrate for SBLO‐1, N‐oleoyl‐ D‐tryptophan (ODT) was synthesized as a potential competitive inhibitor. Potent inhibition was observed at micromolar concentrations of ODT. Kinetic analysis demonstrated that ODT had only small effects on Vmax, indicating that the inhibition is nearly competitive; however, the inhibition is strikingly non‐linear, which implies that OTD and substrate can bind simultaneously. Similar behavior was observed with N‐oleoyl‐ L‐tryptophan and N‐oleoyl‐D‐phenylalanine. Slow oxygenation of ODT was detected by HPLC, which indicates that ODT can access the active site. However, the inhibition kinetics imply that ODT binds more effectively somewhere else. Funded by NSF grant CHE‐1213262.
Conditional protein splicing via disulfide bond formation
Authors: Nicastri Michael C. , Xega Kristina , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Inteins are intervening polypeptides that self‐catalyze their own excision from the flanking polypeptides, or exteins, in a process called protein splicing. An intein interrupting the DNA PolII in Methanoculleus marisnigri (Mma) can facilitate protein splicing when over‐expressed in E. coli. However, when overexpressed in gor– E. coli, we isolate mostly unspliced precursor. This precursor can be induced to undergo N‐terminal cleavage upon in vitro incubation with 1,4‐dithiothreitol. Unusual migration on SDS‐PAGE, resolvable by reduction, suggests the presence of a disulfide bond in the unspliced precursor. Mutagenesis analysis indicates that the Cys residues involved in the bond are within the intein, suggesting that this inducible intein may be able to regulate the expression of foreign flanking exteins.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and by the Camille and Henry Dreyfus Foundation.
Expression and auto‐processing of hedgehog‐like proteins from Brugia malayi and Cryptosporidium
Authors: Cahn Sarah A. , 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 sterolrecognition 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 and from two species of Cryptosporidium. We have created E. coli expression vectors that encode fusion proteins of either an N‐terminal His tag or GB1 tag to the full signaling and HINT domains. We are 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 and the Camille and Henry Dreyfus Foundation.
Splicing of a non‐canonical class three intein from Clostridium thermocellum
Authors: Pusztay Jennifer M. , Drago Matthew J. , Powers Taryn L. , Schufreider Ann K. , Connor Katherine R. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

Protein splicing is a post‐translational event by which an intervening polypeptide, called an intein, facilitates its own excision from the flanking polypeptides, called the exteins, and the ligation of the exteins. The first step of protein splicing is an amide to ester or thioester rearrangement of the peptide bond linking the N‐extein and intein, facilitated by the N‐terminal Ser or Cys of the intein. The Clostridium thermocellum TerA protein is interrupted by an intein that lacks the N‐terminal nucleophile. The TerA intein splices as a class three intein by bypassing the first step in the protein splicing mechanism and using an internal Cys to facilitate attack at the N‐terminal splice junction. We have examined the influence of conserved residues on the catalysis of this alternative mechanism.

This material is based upon work supported by the National Science Foundation under grant MCB‐0950245 and the Camille and Henry Dreyfus Foundation.
Evolution of the respiratory system in Testudines: anatomy and phylogeny
Authors: Schachner Emm R , Lyson Tyler R , Farmer CG
Published: June 2021

The lung is among the most morphologically diverse vertebrate organs, but the underpinnings (e.g., environmental, phylogenetic etc.) of this diversity are not well understood. We analyzed the pulmonary anatomy of a phylogenetically broad range of morphologically diverse turtles that occupy a wide range of habitats to determine the factor(s) that influence pulmonary anatomy. The anatomy of the primary secondary, and tertiary pulmonary bronchi were visualized as 3D surface models using medical grade computed tomography (CT). Preliminary results indicate that certain aspects of lung anatomy are correlated with carapace morphology and thus ecomorph including: 1) the number of lateral secondary bronchi, 2) presence/absence of ventrally projecting sac‐like secondary bronchi, and 3) the morphology of the tertiary bronchi. This suggests that lung evolution in turtles was influenced by environmental as well as genetic factors. These data also demonstrate that certain aspects of lung anatomy are not reliable phylogenetic characters until the relationship between the respiratory system and the environment in sauropsids is resolved.

Grant Funding Source: National Science Foundation, American Association of Anatomists
Role of C‐terminus tail into the ligand binding and releasing mechanisms of Antheraea polyphemus Pheromone binding protein1 (ApolPBP1)
Authors: Mazumder Suman , Katre Uma , Mohanty Smita
Published: June 2021

In moths, pheromone binding proteins (PBPs) transport the hydrophobic pheromone molecules to the membrane‐bound receptors across the aqueous sensillar lymph. We already showed that recombinant Antheraea polyphemus PBP1 (ApolPBP1) expressed with hydrophobic molecule(s) endogenous to the Escherichia coli which keeps the protein in the ligand‐bound conformation at high pH but switches to the ligand‐free conformation at low pH. For the first time, we showed that the delipidated (without the ligand) ApolPBP1 exists primarily in the free form at all pH levels. We also showed the role of Histidine (His70 and His95) residues presented at one end of the binding pocket. Here we are discussing the role of C‐terminus tail located at the other end of the pocket pocket by solution NMR and binding of 1‐aminoanthracene (AMA) by fluorescence spectroscopy. We found out that C‐terminus truncated proteins showed reduced binding affinity towards the ligand at all pH levels, which indicates that apart from helping in the ligand releasing mechanism, C‐terminus may play a role in ligand binding as well. We also examined the role of three charged residues (Asp132, Glu137 and Glu141) present inside the C‐terminus tail. We discovered that, single mutations (D132N, E137Q and E141Q) and double mutations (D132NE137Q and E137QE141Q) have no effect on the conformation of the protein. They behaved as the wild type protein.

This research was financially supported by United States Department of Agriculture PECASE award 2003–35302‐12930, and grant 2011–65503‐20030, National Science Foundation grant IOS‐0628064, and National Institutes of Health grant DK082397 to Smita Mohanty.
TLC‐based assay of ganglioside synthesis
Authors: Czarnowski Megan N , Ettinger Anna C , Moore Kathleen H
Published: June 2021

Ganglioside content in cultured dairy products is significant and may contribute to antimicrobial effects associated with such foods (J Food Comp Anal13: 783, 2000). The source of gangliosides is unclear as dairy products are generated from ganglioside‐free components (skim milk and culture bacteria). By a terminal synthetic reaction, sialyltransferase (ST) transfers N‐acetylneuraminic acid (NANA) to lactosylceramide (LC): CMP‐NANA + HO‐ lactosylceramide → NANA‐lactosylceramide + CMP. Formation of gangliosides (eg NANA‐LC) is visualized by thin‐layer chromatography (TLC) via resorcinol staining. Incubation conditions have been optimized. Analysis with multiple NANA acceptors (LC solution, whey, dialyzed yogurt, and milk) indicates that milk contains an appropriate acceptor, yielding a NANA containing product (putative ganglioside) with an Rf (0.71) that is distinct from the Rf (0.63) of free NANA. Now that it has been determined that appropriate NANA acceptors are inherent in milk, the presence of ST in dairy components can be assessed. Rich ganglioside content may be a synergistic by‐product among dairy components. (Supported by OU Provost's Undergraduate Research Award)
Multiple receptors for sulfated Lewis X trisaccharide and sialylated N‐acetyllactosamine are present on the boar sperm plasma membrane contributing to oviduct reservoir formation
Authors: Silva Elena , Miller David , Bovin Nicolai
Published: June 2021

After insemination, a sperm reservoir is established in the oviduct by sperm binding to oviductal glycans containing sulfated Lewis X (sLeX) and biantennary sialylated N‐acetyllactosamine (SiLN) motifs. Based on a binding assay to fluoresceinated sLeX, SiLN or N‐acetyllactosamine (LN) glycans, we localized glycan receptors on the sperm plasma membrane. Fluorescent staining revealed two binding regions: the anterior head region (AR) or both the anterior and posterior region (APR). sLeX bound preferentially to the AR (71.0% of sperm) and LN to the APR (98.9% of sperm) whereas SiLN bound to both regions, suggesting the involvement of two distinct receptors. Sperm preferentially bound to sLeX and SiLN compared to LN (61.5, 61.7 and 25.2% of sperm bound respectively, P=0.001), indicating the necessity of sialic acid and multivalency for optimal glycan recognition. Glycan blot analysis identified several proteins that bind specifically to sLeX with apparent molecular weights of 250, 130, 80, 55 and 47 kD. Bands of 70 and 37 kD recognized sLeX and SiLN, whereas a 15kD band recognized all three motifs. Based on these results, we determined that sperm recognition of the sLeX motif is mediated by multiple receptors that may also recognize the SiLN motif. We propose a binding model in which Lewis X and SiLN receptors are components of sperm adhesion to the oviduct. Supported by USDA Grant no. 2011–67015‐20099.
The Isothiocyanate, Sulforaphane, Alters the Acidification of the Vacuole to Trigger Yeast Cell Death
Authors: Murphy Michael Dennis , Tucker Douglass W. , Wilcox Alexander , Thomas Stacy K , Laprade David , Austriaco Nicanor
Published: June 2021

The isothiocyanate, sulforaphane (SFN), isolated from cruciferous vegetables is a potential chemotherapeutic agent. Recent studies in mammalian cells have suggested that SFN works by causing cell cycle arrest and/or initiating programmed cell death (PCD). In order to identify genes that may be involved in the eukaryotic cell's response to SFN, we initiated a genetic screen using the yeast knockout (YKO) library of the budding yeast, Saccharomyces cerevisiae, to identify loss of function mutants that are hypersensitive to SFN. We have identified 311 SFN‐sensitive and 11 SFN‐resistant mutants in yeast ORFs that had been previously linked to vacuolar acidification suggesting that SFN kills cells by altering the acidification of the yeast vacuole. Staining with BCECF‐AM confirmed that SFN makes yeast vacuoles more basic. Strikingly, cells cultured in lethal concentrations of benzyl isothiocyanate and phenethyl isothiocyanate did not exhibit changes in vacuole acidification, suggesting that this trigger of programmed cell death is unique to sulforaphane. [Our laboratory is supported by the following grants: NIGMS R15 GM094712, NSF MRI‐R2 0959354 and NIH Grant 2 P20 RR016457 to the Rhode Island INBRE Program]
Essential Concepts & Underlying Theories from Physics, Chemistry, and Mathematics for the Molecular Life Sciences
Authors: Wright Ann , Bell Ellis , Provost Joseph , Roecklein‐Canfield Jennifer A.
Published: June 2021

Over the past two years, through a NSF RCN‐UBE grant, the ASBMB has held regional workshops for faculty members from around the country. The workshops focused on developing a list of Core Principles in Biochemistry and Molecular Biology, Essential Concepts & Underlying Theories from Physics, Chemistry, and Mathematics, and a list of Foundational Skills that all Biochemistry or Molecular Biology Majors must understand to complete their major coursework. The Allied Fields working group created a survey to validate six Essential Concepts and Underlying Theories from Physics, Chemistry, and Mathematics identified from participant feedback at various workshops. One hundred twenty participants from the various regional workshops responded to the survey and 68% of the respondents answered yes to the question. “We have identified the following as the Core Concepts and Underlying Theories from Physics, Chemistry and Mathematics that biochemistry majors or molecular biology majors need to understand after they complete their major courses: 1. Mechanical Concepts from Physics 2. Energy and Thermodynamic Concepts from Physics 3. Critical Concepts of Structure from Chemistry 4. Critical Concepts of Reactions from Chemistry 5. Essential Mathematics. In your opinion, is the above list complete?” Respondents also delineated sub‐categories they felt should be included in these broad categories. The outcomes from the survey will help inform biochemistry and molecular biology educators when considering the ASBMB recommended curriculum for Biochemistry or Molecular Biology Majors. Supported by NSF Grant: 0957205: RCN‐UBE to EB
Micronutrient intake adequacy associated with shifting livelihoods in pastoralist communities of Samburu, Kenya
Authors: Iannotti Lora , Lesorogol Carolyn
Published: June 2021

Pastoralist communities have traditionally depended on animal foods in their diets. Environmental and political pressures have led millions to agriculture and other income diversification strategies. This longitudinal study of two pastoralist communities with differing land adjudication policies, compared the probability of micronutrient intake adequacies in relation to livelihood transitions. Three waves of social, economic, and dietary intake data using 24‐hour recalls were collected in 2000, 2005, and 2010 (n=197). The pastoralist diet derived an average 49% of energy from maize followed by sugar (13%) and milk (11%), and showed high probability of inadequacies for vitamins A, B12, and C. Panel regression modeling revealed income, education of household head, cattle and chicken ownership, and beans and rice consumption were positive determinants of dietary diversity (overall adj R2=0.33; p<0.001). Livestock ownership predicted nutrient adequacy for vitamin A, B12, and zinc, while income predicted vitamin C adequacy. No differences by community, except for folate and vitamin C adequacy, were found. Future strategies to improve nutrition among pastoralists should: uphold livestock production and associated milk and meat consumption; improve access to fruits, vegetables, and legumes; and offer general poverty alleviation services. The research was supported by the National Science Foundation.
Vitamin B12 Supplementation of Bangladeshi Women in Pregnancy and Lactation: Effects on Maternal and Infant Status, Breast Milk and Immunity
Authors: Siddiqua T , Islam S , Ahsan K , Rashid M , Hampel D , Shahab‐Ferdows S , Allen LH , Raqib R
Published: June 2021

Vitamin B12 deficiency is highly prevalent in Bangladesh and other countries with low animal source food intake. Poor B12 status is associated with several adverse outcomes in pregnancy and infancy, and low breast milk B12; however no B12 supplementation trials have been carried out in pregnant women. In a pilot study, women (n=69, 18–35 y, Hb <115 g/L, 11–14 wk pregnant) were randomized to 250 ug B12/d or placebo through 4 mo postpartum. Both groups also received 400 ug folic acid + 60 mg Fe. Women were immunized with HINI vaccine at 26–28 wk gestation. There were no sig. differences between groups at baseline, when 26% women were B12 deficient (<150 pM), 41% had marginal status (150–221 pM), 26% elevated MMA and 61% tHcy (>;8uM). Supplementation increased plasma B12 and lowered MMA in cord blood and in mothers and infants at 4 mo; it also reduced tHcy in cord blood and infants (all P<0.001) but not in mothers of whom 12% remained elevated (>;12 uM) after supplementation. Hematology, iron status indicators and H1N1 antibody titer did not respond to B12 supplementation. Median B12 in colostrum of the suppl.group increased to 778 vs. 320 pM in the placebo group (P<0.001), and 235 vs. 170 pM at 4 mo (P<0.05) in breast milk. Maternal plasma B12 at baseline remained correlated (r=0.446) with concentrations at 4 mo in spite of supplementation. Maternal and infant B12 status sig. improved by 250 ug/d supplements through pregnancy and lactation.

Grant Funding Source: Nestle Foundation, SIDA, and USDA, ARS WHNRC
Consumption of carotenoid‐rich diet improves plasma inflammatory markers
Authors: Whigham Leah , Jahns Lisa , Claycombe Kate , Johnson LuAnn K
Published: June 2021

Multiple studies have investigated the effects of carotenoids on a limited selection of cytokines. However, inflammation is a complex system involving numerous interacting cytokines. In this study, a broad array of 30 cytokines was measured after consumption of low‐ and high‐carotenoid diets. Nine men and 19 women (age 32.3 ± 13.1 y, BMI 23.9 ± 3.2 kg/m2) consumed their usual diet while excluding high‐carotenoids foods (6 wk, depletion) followed by a provided diet based on the Dietary Guidelines (8 wk, repletion; 62 mg carotenoids/d). Plasma was collected after an overnight fast at baseline, end of depletion, and end of repletion. Cytokine concentrations (Milliplex Human Cytokine/Chemokine kits) at each time point were ranked within each subject and analyzed using repeated measures ANOVA followed by Tukey contrasts. Compared to baseline, levels of the pro‐inflammatory cytokines tumor necrosis factor (TNF)‐α, macrophage inflammatory protein (MIP)‐1α, and MIP‐1β were no different after depletion, but levels were decreased after carotenoid repletion (p < 0.05). Interferon‐α, which can act to inhibit TNF‐α secretion, increased after repletion. There was no effect of diet on the other 26 cytokines. These data support an anti‐inflammatory role of dietary carotenoids in healthy volunteers. Study supported by USDA‐ARS Grand Forks Human Nutrition Research Center.
Health professionals’ perceptions of maternal obesity and how it relates to breastfeeding
Authors: Dieterich Christine M , Rasmussen Kathleen M , Devine Carol M
Published: June 2021

Obese women breastfeed for a shorter time than normal‐weight women, but it is unknown how health professionals (HPs) understand maternal obesity and perceive its effects on breastfeeding. We explored these issues using qualitative methods. HPs who care for pregnant or breastfeeding women were recruited in central New York. Interviews with 21 HPs were audio‐recorded, transcribed and verified for accuracy. Content analysis was used to identify themes. HPs identified “what is obese” based on BMI and weight cutoffs, as charted in problem list, and by appearance, which often reflected extreme obesity. HPs described obesity either as posing difficulties for BF or as being unrelated to BF. Benefits of obesity for BF were not mentioned. Whether or not HPs described maternal obesity as a factor affecting breastfeeding, nearly all discussed ways that obese women might need extra help or “tweaking” with breastfeeding. HPs regarded physical difficulties with positioning the infant as most prominent; and they speculated about psychosocial differences that could affect breastfeeding. These findings suggest HPs recognize challenges of BF among obese women with differing levels of saliency and could benefit from education about caring for this population. Funded by NIH (5T32HD007331) and USDA (Hatch 399449).
Why Do Women Stop After Successfully Breastfeeding for Three Months?
Authors: Schwager Haley Nicole , Kennedy Tay , Aubuchon‐Endsley Nicki , Thomas David G.
Published: June 2021

Although breastfeeding (BF) initiation rates are up, duration of BF remains less than ideal. The objective of this study was to determine when and why a sample of 121 women who had participated in a previous study and were predominately BF when their infants were 3 months old stopped BF. The sample was predominately white, well‐educated and married. The on‐line survey (47% response) asked women to indicate the degree to which 21 factors influenced their decision to stop BF. The survey included open ended questions asking what physicians and communities could do to support BF. Participants were also asked where they received most of their support during their BF experience. Age of infant was the major reason for stopping BF. Two major themes from the qualitative questions were that women wanted access to private spaces to BF their infant outside the home and that husbands were critical supports for BF success. Regional differences in barriers to BF duration need to be understood to meet public health goals. Supported by National Research Initiative Grant 2008–35200‐18779 from USDA National Institute for Food and Agriculture.

Grant Funding Source: USDA
Differential regulation of gene expression by α‐Tocopherol and α‐Tocopheryl‐phosphate in pre‐adipocytes and adipocytes
Authors: Lirangi Melania , Meydani Mohsen , Zingg Jean‐Marc , Azzi Angelo
Published: June 2021

Adipocytes play an important role in lipid and energy homeostasis and dysregulation of these processes contributes to obesity and type 2 diabetes mellitus. In addition, adipocytes influence inflammation and play a major role in preventing lipotoxicity by removing excess fatty acids from the circulation and storing them as triglycerides and thus reducing exposure of other cells to their potentially harmful effects. Although the many aspects of metabolic disorders associated with lipotoxicity and obesity cannot be fully clarified with cultured cells, the NIH3T3‐L1 preadipocytes/adipocytes differentiation model has been useful to study the molecular processes involved. In this model, several vitamins and phytochemicals have been shown to influence proliferation and differentiation. Here, we report by using quantitative RT‐PCR that in NIH3T3‐L1 pre‐adipocytes, α‐tocopheryl phosphate (αTP) (but not α‐tocopherol (αT)) activates a set of genes [TRB3 (Tribbles Homolog 3), Sestrin‐2, and Insulin Induced Gene 1 (INSIG)] potentially preventing lipids accumulation. In contrast, in differentiated adipocytes, αTP inhibits transcriptional expression of the same genes, potentially facilitating lipid uptake and storage. Thus, in proliferating pre‐adipocytes αTP foils lipid accumulation, whereas in adipocytes it enhances lipid accumulation. Supported by USDA Contract #58–1950‐0–014.
Quercetin protects osteoblast development in the presence of oxidative stress in fetal rat calvaria cells
Authors: La Stephanie , Messer Jonathan G. , Hopkins Robin G. , Kipp Deborah E.
Published: June 2021

Studies examining quercetin (QRC) effects on bone health are limited. The purpose of this study was to examine the extent that QRC impacts osteoblast cell development in the presence of oxidative stress, using osteoblast‐like cells isolated from fetal rat calvaria. At confluence, cells were pre‐treated for 12 hours with QRC aglycone (0 or 20 μM). Cells were then incubated for 4 days with QRC aglycone continued at the same dose as pre‐treatment (0, 20 μM) with or without hydrogen peroxide (H2O2; 0, 300 μM), a known inducer of oxidative stress. There were four groups: untreated Control, QRC alone, H2O2 alone, and QRC/H2O2. Expression of genes related to osteoblast phenotype development (runt‐related transcription factor 2, Runx2; alkaline phosphatase, ALP; type I collagen, COLL; bone sialoprotein, BSP; and osteocalcin, OCN) were analyzed using real‐time quantitative PCR. Additionally, alkaline phosphatase staining was used as an indicator of cells with osteoblastic potential. Results for Controls and QRC treated cells were similar. H2O2 alone suppressed ALP, BSP, and OCN gene expression (p < 0.05) and alkaline phosphatase staining. QRC, in the presence of H2O2, partially prevented the H2O2‐ induced suppression (p < 0.05). These results suggest that QRC is partially protective of osteoblast development in the presence of oxidative stress.

Grant Funding Source: USDA Agricultural Research Services