bluuebunny/crossref_metadata_2025 huggingface dataset:
MRP4 and MRP5 in the ciliary epithelium of the eye and their potential role in regulating extracellular cAMP levels
Authors: Hotchkiss Adam G , Pasumarthi Kishore , Coca‐Prados Miguel , Pelis Ryan M
Published: June 2021

The purpose was to determine if the ciliary epithelium expresses multidrug resistance‐associated proteins (MRPs) that transport cyclic nucleotides, and if their activity can influence extracellular cAMP levels. MRP4 and MRP5 were detected in human ciliary body by microarray. mRNAs (RT‐PCR) for both transporters were also detected in a human ciliary epithelial (CE) cell line. Activity for MRP4/5 in CE cells was assessed using monochlorobimaneglutathione. The intracellular accumulation of monochlorobimaneglutathione increased dose‐dependently in the presence of the MRP inhibitors: indomethacin, probenecid, ibuprofen and furosemide. Treatment of CE cells with isoproterenol (100 μM) and IBMX (500 μM) increased intracellular cAMP levels 4.8‐fold (4.1 to 19.7 pmoles/106 cells) and extracellular cAMP 5.8‐fold (2.7 to 15.6 nM). The β‐adrenergic stimulation of extracellular cAMP levels was reduced 27–46% by ibuprofen, furosemide and probenecid (500 μM each), whereas indomethacin (500 μM) nearly prevented it completely (94%). MRP4/MRP5 activity may play an important role in aqueous humor dynamics through modulation of the extracellular cAMP‐adenosine signaling pathway. Funding: CIHR, NSERC and NIH.
Cab39 modulates SPAK/OSR1 activation of NKCC1
Authors: Ponce‐Coria José , Rios Kerri , Gagnon Kenneth B. , Delpire Eric
Published: June 2021

Calcium binding protein 39 (Cab39) is a scaffolding protein that stabilizes a heterotrimeric protein complex that modulates the activity of the adenosine mono‐phosphate kinase which regulates cellular energy homeostasis. Cab39 also modulates SPAK (Ste20 related Proline Alanine rich Kinase) and OSR1 (Oxidative stress responsive kinase 1), two kinases involved in activating the Na‐K‐2Cl cotransporter‐1 (NKCC1). In this study, we demonstrate that mouse Cab39 can substitute for WNK4 phosphorylation of a key serine residue (S383) in the SPAK‐T243E mutant, and even substitute for WNK4 phosphorylation of another key activating residue (threonine 243) when two wild‐type SPAK molecules are tethered together. Western blot analysis of frog oocyte lysates shows an intense signal for an endogenous Cab39. This observation raises the question of why the mouse isoform is necessary to observe the Cab39 effect on SPAK/OSR1 activation of NKCC1. To address this problem, we cloned the cDNA encoding the Xenopus Cab39 isoform and tested its effect on SPAK/OSR1 activation of NKCC1 in frog oocytes. Using site‐directed mutagenesis, we further examined residues that might be involved in the Cab39‐SPAK‐NKCC1 regulatory pathway.
Characterization of the Organic Anion Transporter 2 (OAT2) and its interaction with cytostatics
Authors: Yohannes Hagos , Marad Venkata , Flörl Saskia , Kühne Annett
Published: June 2021

The ability of a compound to exert its cytostatic effect is largely governed by transport proteins that are involved in the uptake as well as efflux of the compound from a cell. A lot of focus has been on the efflux transport systems which drive the compound out of the cell whereas relatively little is known about influx transporter systems. The knowledge about influx transporter proteins becomes all the more important in the case of tumor cells wherein, different expression patterns of influx transporters has been observed among patients. In such a scenario, administering a cytostatic to a patient with a tumor which has the expression of corresponding influx transporter supressed might lead to a drastic effect with the compound taking its toll on the surrounding tissue. We are interested in one such influx transporter, the Organic Anion Transporter 2 (SLC22A7) protein. In the current study, we performed experiments on the interaction of OAT2 stably transfected HEK cells with several cytostatics which are routinely being used in cancer chemotherapy. We found that OAT2 is strongly inhibited by the cytostatics bendamustine, irinotecan and paclitaxel. By Dixon plot analysis, we calculated the Ki values for these inhibitors to be 43.3 μM, 26.4 μM and 10.4 μM respectively. Further experiments are focused on finding out whether they are transported by OAT2 and the expression of this protein in various hepatocarcinoma cell lines.
Endothelial dysfunction in rats which exhibit depression‐like symptoms following exposure to chronic mild stress
Authors: Matchkov Vladimir V. , Bouzinova Elena V. , Wiborg Ove , Aalkjaer Christian
Published: June 2021

Major depression is associated with cardiovascular disease, but the reason for this is unclear. In the rodent chronic mild stress (CMS) model of depression only some rats develop depression‐like symptoms (‘anhedonia’ evidenced by reduced sugar intake), while others are stress‐resistant (‘resilient’). Using isometric myograph, we have here tested different components of endothelium‐dependent relaxation in arteries from rats after 8 wks of CMS. Mesenteric small arteries from anhedonic, resilient and non‐stressed rats were compared. Relaxations to acetylcholine were not different between the groups under control conditions. The inhibition eNOS with L‐NAME had greater effect on relaxation in the anhedonic group. This was associated with increased eNOS protein expression in arteries from anhedonic rats. The COX1 inhibitor, SC560 indicated that COX1 activity had contractile action. The COX2 inhibitor, NS398 revealed that relaxation by a COX2 product was significantly elevated in arteries from anhedonic rats. No difference in the expression of COX1 and COX2 proteins was found. Finally, a significant reduction of the EDHF‐like relaxation (TRAM34 and apamin sensitive relaxation to acetylcholine) was seen in anhedonic rats. This was associated with a significant reduction in the expression of intermittent‐conduction K+ channel mRNA, while small‐conduction K+ channels were not affected. Our results indicate that depression is associated with changes in endothelial function in resistance arteries. These changes might be important for the cardiovascular events associated with major depression.
Hyperglycemia induced gene expression changes in HUVEC and HMEC
Authors: Patel Hemang , Kavdia Mahendra
Published: June 2021

Hyperglycemic condition causes endothelial dysfunction through increased oxidative stress and downstream changes in enzymatic activity and gene expression. Research studies are examining the effect of hyperglycemia on enzymatic activities and gene expression changes in endothelial cells from individual sources of vascular system. One of the crucial hypotheses is to understand the effects of hyperglycemia in endothelial cells from different areas of vasculature. In this study, we investigated and compared the effects of high glucose exposure on HUVEC and HMEC using comparative gene expression analysis approach. Gene expression levels of pro‐oxidant enzymes such as NOS and NOX family, ROS neutralizing enzymes such as SOD, catalase (CAT) and Peroxidase (PRDX) and, cell survival signaling pathway intermediates such as ASK1 and Nrf‐2 were compared in both endothelial cell types upon exposure to high glucose. Significant up‐regulation in gene expression levels of NOS and NOX family enzymes, SOD, CAT, ASK1 and Nrf‐2 were observed in HMEC cells under high glucose exposure. On the contrary, HUVEC showed up‐regulation in ASK1 and a down‐regulation in SOD and CAT enzymes. The results are significant to understand differential effects of hyperglycemia in HUVEC and HMEC endothelial cells. Supported by NIH R01 HL084337.
Bringing Augmented Reality to a Metabolic Pathways Puzzle
Authors: Galembeck Eduardo , Magrini Márcio L , Anderson Trevor
Published: June 2021

During several years of using a paper puzzle to introduce students to the principles of metabolic pathways, we noticed that both the number of instructors and the time they spend with the students, plays a major role in the success of the activity. An insufficient number of instructors does not permit adequate contact with all the students, which both frustrates and discourages them. To bring this activity to larger audiences, while involving only a few instructors, we added a technological tool called augmented reality to the paper puzzle, turning it into a game. One of the game goals is still to assembly the puzzle, but by using an app that runs in a tablet, the students can now check their achievements as they progress through the activity. The app also includes answers to some of the frequently asked questions, while the augmented reality permits the addition of more information to the cards, via virtual flashcards. The virtual flashcards displayed on the tablet screens show information such as the molecular 3D structure, and clues for assembling the puzzle. The abovementioned technological improvement to the paper puzzle has enabled its use in larger classrooms with fewer instructors, but we still recommend also using the paper cards with students playing in groups as a powerful way of promoting student learning about metabolic pathways.

This research was partially supported by FAPESP and CNPq, Brazil.
From anatomy to acoustics, a study of the sound generation process in baleen whales (mysticetes)
Authors: Cazau Dorian , Adam Olivier , Laitman Jeffrey , Reidenberg Joy
Published: June 2021

The mechanism of baleen whale (mysticete) sound production is a topic of much interest, particularly as they make the loudest natural sounds and transmit them across the longest distances. Prior studies on mysticete sound generation have explored either acoustics or anatomy, but unfortunately have done so in isolation of the other discipline. This study links the two disciplines to derive a comprehensive understanding of the sound production process. Anatomical data (vocal fold measures, respiratory tract volumes, air flow paths) are used to derive initially an acoustic model that explains how sounds are made and transmitted. The model is then tested by comparisons to parameters (frequency, amplitude, duration) derived from hydrophone recordings of live whales. The model defines four mutually exclusive components: air sources (providing initial energy), vibrators generating harmonic frequencies (vocal folds, corniculate flaps), air flow paths (between lungs, trachea, laryngeal sac, nasal cavities), and radiating areas (transmitting sound energy to water). This model explains, for the first time, the acoustic function of each part of the mysticete sound generating system.

Grant Funding Source: AAA
Botulinum toxin injection for hemiplegic shoulder pain: Do we know enough yet?
Authors: Lee Roberts Shannon , Warden Julia , Chang Youjin , Samarakoon Amila , Baker Ross , Boulias Chris , Ismail Farooq , Agur Anne
Published: June 2021

In patients with spastic hemiplegia, botulinum toxin A (BoNTA) injection into the subscapularis (SB) has been shown to decrease shoulder pain and increase range of motion (Unlu et al., 2010; Yelnik et al., 2007). The intramuscular innervation pattern of SB has not been well studied, but could provide insight into neuromuscular partitioning, enabling the development of an optimal injection approach that enhances the efficacy of BoNTA. The purpose of this study is to document the extra‐ and intramuscular innervation patterns of SB throughout the muscle volume to identify neuromuscular partitions. Forty‐five formalin embalmed cadaveric specimens were used in this study. Extraand intramuscular innervation patterns of SB were documented using digitization and 3D modelling (n=7), and dissection and photography (n=38). Innervation patterns throughout the muscle volume were analyzed. The number of extramuscular branches (2–5) were correlated with the four identified intramuscular innervation patterns, partitioning the muscle into two parts (superior and inferior; n=11) or three parts (superior, middle and inferior; n=34). Due to the presence of neuromuscular partitions, injection in multiple locations may be required to achieve maximal effect. Future studies are needed to determine whether the number of partitions injected correlates with clinical outcomes.
Prototype 3‐D Model of the Musculotendinous Architecture of Infraspinatus
Authors: Hermenegildo Jason Aldeia , Ko Dominic M , Li Zhi , Johnson Marjorie , Merrifield Peter , Agur Anne M
Published: June 2021

Muscle architecture, the arrangement of fibre bundles within the muscle volume, has important functional implications. Previous studies of infraspinatus (IS) muscle architecture have focused on dissection and photography of the superficial muscle layers rather than a volumetric analysis. The aim of this study was to develop a methodology to quantify the architectural parameters of the fibre bundles throughout the volume of IS. As a prototype, one formalin‐embalmed cadaveric specimen was used. The IS was exposed and each fibre bundle was meticulously dissected and digitized from end to end. The digitized data was imported and a 3D model of the fibre bundle architecture, as in situ, was constructed in Autodesk® Maya®2012. Architectural parameters including fibre bundle length (FBL), pennation angle (PA), and physiological cross sectional area (PCSA) were computed. Based on the architectural parameters, muscular partitioning was determined. This technique successfully captured IS architecture throughout its volume. The IS was found to consist of 2 architecturally distinct regions, superior and inferior. The average measures for the superior and inferior regions were respectively: FBL 115.3mm; PA 22.2°; PCSA 180.3mm2 and FBL 84.9 mm; PA 15.9°; PCSA 550.5 mm2. The results suggest that there is muscular partitioning of the IS. This methodology will form the basis of a continuing study to understand detailed IS architecture at the fibre bundle level.
Study of the anatomic variations of the pyelocaliceal system
Authors: Vergamini Lucas Bonachi , Menegazzo Gian Luigi Martins , Barros Mirna Duarte , Pinto Antonio Cardoso
Published: June 2021

Introduction and Purpose

Many are the anatomical variations of the pyelocaliceal system, which influence the arterial irrigation and venous drainage. This study adopted a classification given by Sampaio in “Anatomia Renal para Urologia”. The system is divided in: Type A‐I has the mesorenal region drained by chalices from the superior or inferior caliceal group. Type A‐II has the mesorenal region drained by crossed chalices from both superior and inferior caliceal groups. Type B‐I has the mesorenal region drained by an independent caliceal group. Type B‐II has small chalices that drain the mesorenal region directly to the renal pelvis. The study aims to describe the structure of the pyelocaliceal system and its variations. Compare findings with those found in literature.

Methods

Kidneys obtained from adults that belong to the Morphology Department of Faculdade de Ciênicas Médicas da Santa Casa de São Paulo were injected with resin through the ureter and later corroded by acid. Kidneys obtained from someone who had nefropathy were excluded.

Results

52 kidneys were obtained. 39.6% were type A‐I. 12.5% were type A‐II. 18.8% type B‐I and 29.2% type B‐II.

Conclusion

Results were similar to the ones found in Brazilian population studies. Many are the anatomic variations of the pyelocaliceal system and they must be considered especially involving surgical procedures.
Regeneration of mental nerve of rats with the use of mesenchymal stem cells obtained from dental pulp of human primary teeth
Authors: Sasaki Robson Tetsuo , Saez Daniel Martinez , Monteiro Babyla G , Almeida Sonia Y , Kerkis Irina , Silva Marcelo C
Published: June 2021

The aim of this study was to evaluate the regeneration of the mental nerve after injury by compression and treatment with mesenchymal stem cells obtained from human dental pulp of primary teeth of children with age ranging 5–7 years old. Thirty‐six male Wistar rats were used in this experiment and the rats had their right side mental nerve injured by compression and left side nerve used as positive controls. The right side mental nerves were then distributed in two study groups: control group with compression and no treatment (n=18) and experimental group that underwent injury by compression and treatment with mesenchymal stem cells (n=18). Animals were euthanized at different times of study: 1, 3, 7, 14, 21 and 42 days after lesion and they had their mental nerves and trigeminal ganglion removed for analysis with transmission electron microscopy. The images of neural fibers were obtained for the measurement of external circunference of mielinic fibers and thickness of mielinic layer. The nerves after 14 days of treatment showed similar morphological aspects to the intact nerve. We can conclude that the use of mesenchymal stem cells for mental nerve regeneration was effective in a single aplication after two weeks of the treatment.
Differentiation of porcine adipose‐derived stem cells into glucose responsive insulin‐secreting pancreatic islet‐like clusters
Authors: Liu Hui‐Yu , Lin Yuan‐Yu , Chen Yu‐Jen , Wu Cheng‐Yu , Chen Chih‐chien , Ding shih‐torng
Published: June 2021

While its ease of isolation, abundant source and multipotency have made the adipose‐derived stem cell (ADSC) a promising system for cell therapy and regenerative medicine, the approach on utilizing porcine ADSC to generate insulin‐secreting cells has not been explored. In our current study, subcutaneous adipose tissue on the back were dissected from 9‐day‐old male piglets (Y × L) purchased from a local pig farm and processed as previously described to isolate the stromal‐vascular fraction (SVF). Cells were then cultured in differentiation medium based on serum‐free DMEM/F12 as modified from Timper et al.. Our results showed that under light microscope, the morphology of pancreatic islet‐like clusters were observed at day 3 of culture, the function of insulin production identified by immunocytochemical analysis with confocal microscope and the expression of pancreatic β‐cell differentiation‐associated genes, including Pdx1, Pax4, Islet1, glucokinase, glucagon and Glut2, by RT‐PCR. Moreover, upon glucose challenge, the clusters secreted significantly higher amount of insulin as measured by ELISA on day 9 in a dose‐dependent manner compared to the basal level (5.5 mM glucose). Our present study, therefore, demonstrates the success of isolating and differentiating porcine ADSC into glucose responsive insulin‐secreting clusters. Their potentials in ameliorating type I diabetes in mouse and pig models are currently under investigation.
Associations between localized variation in brain anatomy and social behavior in healthy human subjects
Authors: Loyet Mackenzie , Schoenemann P. Thomas , Avants Brian B , Gee James C
Published: June 2021

Understanding the relationship between brain structure and function is critical for interpreting evolutionary changes in the human brain. It has been suggested that social environments play a key role in this process and brain size has been shown to correlate with social group size across primate species (Dunbar, 1998). Studies have indicated a disproportionate elaboration in areas of the prefrontal cortex in hominin evolution (Schoenemann, 2005). The prefrontal cortex is known to be relevant to social decision making, which suggests that selection for social ability may have played a significant role in prefrontal elaboration. Thus, in order for selection on social abilities to result in evolutionary changes in brain morphology, there must have been genetic correlations within modern humans between brain anatomy and social ability. To assess this possibility, associations between localized brain anatomy and several behavioral measures related to social facility were determined on a sample of 36 female sibling pairs (72 subjects total). Voxel‐based morphometric methods were used to quantify brain morphology in MRI scans, and self‐reported degrees of social interaction/engagement were used as a proxy for sociality. A within‐family analysis was used to control for possible confounding variables, such as socioeconomic status. Our results show both positive and negative associations distributed across various regions of the brain. There are positive correlations in the splenium of the corpus callosum, the anteromedial prefrontal cortex, and the left cerebellum. Negative correlations are seen in areas of the orbital frontal and anterior cingulate, though they are modest.

Grant Funding Source: Indiana University
Models of conformational selection of DNA polymerase X in the presence of oxoG lesions help interpret kinetics data
Authors: Barbati Zachary Raymond , Arora Karunesh , Bogdanovic Jasmina , Schlick Tamar , Benitez Benedetta Sampoli
Published: June 2021

DNA polymerase X (pol X) from the African swine fever virus (ASFV) is a 174‐amino‐acid repair polymerase that participates in a viral Base Excision Repair (BER) pathway. Pol X is a member of the X‐family of polymerases, which includes the homologous human DNA polymerase β (pol β). In contrast to pol β, pol X lacks the fingers subdomain and exhibits substantially lower fidelity. Molecular dynamic studies of polX/DNA complexes suggest a hybrid conformational selection, whereby polX/DNA complexes sample low‐energy conformations, which are stabilized by the correct incoming dNTP. Here we present molecular dynamic simulations of pol X/DNA complexes in which the template base opposite to the incoming dNTP contains the damaged base 7,8‐ Dihydro‐8‐oxoguanine (oxoG). OxoG is a common DNA adduct produced by the oxidation of DNA by reactive oxygen species. Kinetics data reveals that pol X accommodates A opposite to oxoG with efficiency even higher than the non‐damaged G:C base pair. The goal of this molecular dynamic study is to help interpret, at an atomistic level, previously published kinetics data. The simulations presented in this study reveal that pol X accommodates the oxoGsyn:A mispair by sampling closed active conformations that mirror those observed in traditional Watson‐Crick ternary complexes. Comparisons with other polX/DNA systems with oxoG on the templating strand will also be presented.
Stimulation of the BLM‐hDNA2 pathway of DNA double‐strand break end resection by TopoIIIα/RMI1/RMI2
Authors: Daley James Matthew , Xue Xiaoyu , Chiba Tamara , Sung Patrick
Published: June 2021

Homologous recombination (HR) is an important mechanism by which highly toxic DNA double‐strand breaks (DSBs) are accurately repaired. HR begins with nucleolytic resection of the 5′ ends of the DSB, yielding long single‐stranded 3′ ssDNA tails. Multiple partially redundant pathways together carry out this process. Resection appears to be initiated by the DSB end‐binding Mre11‐Rad50‐Nbs1 complex together with CtIP. These proteins catalyze limited resection near the break site, which is followed by long‐range resection via two redundant pathways. In the first, the BLM helicase unwinds the DNA duplex to generate ssDNA strands, followed by cleavage of the 5′ strand by the nuclease DNA2. The second pathway is catalyzed by EXO1, an exonuclease also involved in DNA mismatch repair and processing of damaged or stalled DNA replication forks.

To delineate the mechanism of DNA end resection in human cells, we have reconstituted the resection system using highly purified human proteins. Our initial efforts have focused on characterizing the role of Topo IIIα/RMI1/2 (TR) in resection. These proteins form a complex with BLM which performs double Holliday junction dissolution. Topo IIIα is a type I topoisomerase, and the RMI proteins are OB‐fold containing proteins thought to play a structural and stimulatory role in the complex. We have found that TR stimulates resection by BLM/DNA2, but not EXO1. TR stimulates unwinding of double‐stranded DNA by BLM in a manner that is independent of the topoisomerase activity of Topo IIIα. Topo IIIα also promotes 5′ – 3′ resection by preferentially inhibiting the 3′ flap endonuclease activity of DNA2 relative to its 5′ flap endonuclease activity, similar to a previously described role of RPA in this pathway. Atomic force microscopy has revealed that Topo IIIα localizes to the ends of DSBs. These data suggest that TR stimulates resection by recruiting BLM to the break ends and modifying the nuclease activity of DNA2.
Dissect Conformational Distribution and Drug‐Induced Population Shift of Prokaryotic rRNA A‐Site
Authors: Qi Xin , Lu Jia , Zhao Liang , Xia Amy , Xia Tianbing
Published: June 2021

Accurate decoding at the ribosomal RNA A‐site is essential in protein synthesis. Previous studies showed that the dynamic behavior of the A‐site would play an important role in the recognition and function of the element. In this study, we aim to understand the mechanism of molecular recognition of prokaryotic A‐site by aminoglycosides. We have employed time‐resolved fluorescence spectroscopy to investigate the complex nature of the conformational equilibrium of the A‐site, and the effects of drug binding. The time‐resolve dynamics probing revealed that in the drug‐free state, the A‐site samples at least four distinct conformations. Changes in the fluorescence decay profiles induced by drug binding showed that population distribution shifts in a drug‐specific manner. Furthermore, time‐resolved anisotropy measurements indicated that motions of A‐site on nanosecond and picosecond timescales are differentially affected by the drug binding. Taken together, these results underscore the importance of understanding the detailed dynamic picture of molecular recognition and can potentially facilitate development of anti‐microbial drugs targeting such dynamic RNA elements. This work was supported in part by funding from UF and a grant from the Robert A. Welch Foundation (AT‐1645).
SUMO‐binding sites in Nrf2
Authors: Walters Treniqka , Arinze Ifeanyi J
Published: June 2021

The transcription factor Nrf2 influences transcription by binding to the antioxidant response element(s) (AREs) in target gene promoters, enabling oxidatively stressed cells to mount a response to oxidative stress in order to restore redox homeostasis. Post‐translational modifications (PTMs) that mediate release of Nrf2 from its inhibitory protein Keap1 have been extensively studied but PTMs that impact its biology after this release are just beginning to emerge. In this regard, only phosphorylation and acetylation have been previously reported. We recently found that Nrf2 can also be sumoylated. Here, we used three SUMO site‐predicting algorithms (SeeSUMO, SUMOsp and SUMOplot) to identify putative SUMO‐binding sites in Nrf2. Of the eight high probability sites identified by these algorithms, we found, using site‐directed mutagenesis and Co‐IP assay, that K525 is a SUMO‐targeted site. This site binds SUMO‐2/3. The impact of sumoylation on the transactivation potential and degradation of Nrf2 are shown here.

Supported by NIH grants SC1CA143985 and 5T32 GM07628–34.
Chorangiosis in Placental Malaria
Authors: McMichael Ashley B , Moore Julie M. , Burton Samantha , Okafor Uzoma
Published: June 2021

Chorangiosis is a placental change that could result in fetal, maternal, and placental disorders, and has been found to be correlated with high fetal morbidity and mortality. In this study, we investigate the possible correlation between chorangiosis and Plasmodium falciparium induced malaria in pregnant women.

Eight placentas were chosen from malaria‐infected Kenyan women. The placental tissues were cut into three sections, fixed, paraffin‐embedded, sectioned and stained. Images from each section were examined to count villi (placental structures housing the fetal blood circulation) and the number of blood vessels (vascular channels) in each.

Chorangiosis was diagnosed in those specimens in which each of the three sections have, on average, ≥10 vascular channels in each villus. Although the assessments are not complete, these preliminary results suggest that whereas uninfected placentas are negative for chorangiosis, placental malaria appears to be associated with an increased risk for chorangiosis.

The Plasmodium falciparium induced malaria interactions with maternal, placental, and fetal factors, posed an increased oxidative stress which may possibly contribute to this arising pathologic change
Modification of enzymes by protein folding memory
Authors: Satomura Atsushi , Nagayama Mitsuru , Miura Natsuko , Kuroda Kouichi , Ueda Mitsuyoshi
Published: June 2021

Propeptides, which are N‐terminal or C‐terminal domain of most proteases and lipases, are cleaved off in the protein maturation steps. Some propeptides are known to function as an intramolecular chaperone to facilitate the folding of their active region. Protein folding memory means a phenomenon in which a mutated propeptide alters the conformation of the enzymatically active region without mutations in the coding region and thereby the activity and thermal stability are altered. However, there are few examples showing the modification of enzymes by protein folding memory.

In this study, we attempted to modify carboxypeptidase Y (CPY) by introducing mutations into its propeptide. CPY with the mutated propeptide showed the higher activity and thermal stability than that with wild type propeptide [1]. This result indicated that two CPYs with the identical amino acid sequences were folded into different structures by the different propeptides. Furthermore, we recently have investigated protein folding memory using Rhizopus oryzae lipase (ROL) as the second model. ROL whose propeptide was replaced by the propeptide of ROL‐related lipase showed the higher activity than wild type ROL. Then, the mechanism causing the higher activity was investigated.
Regio‐selective alkane hydroxylation by the isolated membrane‐bound non‐heme iron oxygenase, AlkB
Authors: Luo Wen‐I , Chang Chun‐Wei , Ramu Ravirala , Tsai Yi‐Fang , Ng Kok Yaoh , Hsu Su‐Huan , Lin Chi‐Ou , Chiang Chih‐Hsiang , Yang Chung‐Ling , Yu Steve S.‐F.
Published: June 2021

AlkB from Pseudomonas putida GPo1, an integral membrane nonheme iron oxygenase, is a medium chain length (C3 to C12) alkane hydroxylase. It is of vast interest due to its high turnover frequency in the conversion of alkanes to alcohols. However, detailed research is hampered by its membrane‐associated and multiconstituent nature. Herein, the active AlkB and its two redox components, AlkT (rubredoxin reductase) and AlkG (rubredoxin), were purified and tested with alkane and fluoro‐alkane substrates. Products were analyzed by GC and compared with their authentic standards. Results shows that the terminal hydroxylation occurs on substrates, suggesting controls mediated by weak electrostatic forces and the associated conformational tuning are exerted inside the substrate binding pocket. Moreover, the active AlkB was subjected to X‐ray absorption spectroscopy to reveal ligand types, the number of coordination, the geometry, and oxidation states of irons. Taken together, our results provide insights to unravel the high regio‐selective features required for alkane hydroxylation by AlkB. This work is supported by Academia Sinica and grants from National Science Council, R.O.C. (NSC 101–2113‐M‐001–007‐ MY3 and 101–2811‐M‐001–123).
Identifying the Mechanism of Reduced Flavin Transfer in Alkanesulfonate Monooxygenase System
Authors: Dayal Paritosh Vijay , Singh Harsimran , Busenlehner Laura , Ellis Holly
Published: June 2021

The two‐component alkanesulfonate monooxygenase system from bacteria are involved in the acquisition of sulfonates during sulfurlimiting conditions. In this two‐component system the FMN reductase (SsuE) provides reduced flavin to the monooxygenase enzyme (SsuD). The monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of the carbon‐sulfur bond of alkanesulfonates, leading to the formation of the corresponding aldehyde and sulfite product.

The transfer of reduced flavin from SsuE to SsuD may involve either a channeling or dissociative mechanism. Prior studies have identified stable protein‐protein interactions between SsuE and SsuD supporting a channeling mechanism for flavin transfer. The SsuD enzyme belongs to the bacterial luciferase family that includes bacterial luciferase and LadA. Members of this family have a characteristic TIM‐barrel conformation with several insertion regions. The mobile loop contained on the insertion region was shown to be involved in stabilizing loop closure to protect reaction flavin intermediates, but the role of this region in flavin transfer has not been evaluated. HDX‐MS studies were conducted to identify potential interaction sites on each enzyme. The SsuD protected sites were located on the mobile loop region, while the protected sites on SsuE were near the putative active site region. There are noticeable charged and polar residues in these regions, which may play a role in the formation of a transient stable‐structure. Variants of these polar regions were generated to evaluate protein‐protein interactions and flavin transfer were altered. The interaction sites on SsuE and SsuD have the potential to act as a dynamic region which enables the transfer of reduced flavin between the two proteins.
Structural and catalytic roles of conserved cysteine residues in human lysyl‐tRNA synthetase
Authors: Maanu Christiana A Addei , Yang David C H
Published: June 2021

Human and eukaryotic tRNA‐synthetases contain substantially more cysteine residues than their bacterial counterparts, while the other nineteen amino acids show relatively comparable compositions. The significance of the evolved additional cysteines in human and eukaryotic tRNAsynthetase is not well understood. It is not known whether these cysteines play roles in catalyzing aminoacylation, maintaining the structural integrity or other functions. Human lysyl‐tRNA synthetase has eight cysteines, while E. coli lysyl‐tRNA synthetase has one cysteine. To better understand the roles of cysteines in human lysyl‐tRNA synthetase, we mutated individual cysteine to serine and alanine using site directed mutagenesis. The structure and the catalytic properties of recombinant lysyl‐tRNA synthetase mutants were examined after expression in E. coli and purification using Ni‐NTA affinity chromatography. Circular dichroism spectra of the mutants of two cysteines suggested significantly altered secondary structure, and the mutations completely inactivated the lysylation activity. Mutations of six other cysteine residues of human lysyl‐tRNA synthetase affected the lysylation of tRNA with reduced kcat/Km with respect to lysine, ATP and tRNA. The results suggest various cysteine residues play roles in both catalysis of lysylation and maintaining the structure of human lysyl‐tRNA synthetase.
IMP cyclohydrolases in the domain Archaea
Authors: Sarisky Catherine A , Plymale Nicholas I , Hunter Caroline A , Pasier Michelle L , Smee Kevin M
Published: June 2021

There is some diversity as to how archaea effect the final step of de novo purine biosynthesis to form inosine 5′‐monophosphate (IMP) from 5‐formamidoimidazole‐4‐carboxamide ribonucleotide (FAICAR). A survey of the genomes of archaea reveals a mixture of PurH2 (with or without fusion to PurH1), the Euryarchaeal signature protein PurO, and an unidentified Crenarchaeal IMP cyclohydrolase. In this report, we present the cloning and functional characterization of three representatives of the known IMP cyclohydrolases. The locus TK0430 in Thermococcus kodakarensis encodes a PurO‐type IMP cyclohydrolase with demonstrated activity despite its position in a cluster of apparently redundant biosynthetic genes. The locus AF1811 from Archaeoglobus fulgidus encodes a PurH2‐type IMP cyclohydrolase that occurs in active form without fusion to a PurH1 protein. The locus HVO_0011 in Haloferax volcanii encodes a PurO‐type IMP cyclohydrolase active only at high salt concentrations. All three enzymes convert FAICAR to IMP in vitro, and two restore the growth on purine deficient media of an E. coli purine auxotroph lacking purH2.
Using site‐directed mutagenesis to identify the most immunogenic regions of vaccine candidate P6
Authors: Bettinger John , Newman Emily , Mangan Anthony , Pichichero Michael , Michel Lea Vacca
Published: June 2021

The P6 protein is a leading vaccine candidate for protection against Nontypable Haemophilus influenzae (NTHi) infection. NTHi bacteria are a cause of middle ear infections, sinusitis, pneumonia, and chronic bronchitis. Vaccines that contain only the most immunogenic parts of the protein, often including the antibody binding sites or “epitopes,” can reduce production costs and can sometimes enhance effectiveness. Thus, identifying the most immunogenic part of P6 might be highly attractive to pharmaceutical companies who would like to use P6, or part of P6, in their next vaccine against NTHi. In this study, the P6 epitopes against monoclonal antibodies 4G4 and 7F3 were partially identified. P6 mutant T42R (threonine to arginine at residue 42) was prepared using recombinant DNA technologies, and mutated DNA was transformed into Escherichia coli cells for efficient overexpression. An Enzyme‐Linked Immunosorbent Assay (ELISA) showed decreased binding (compared to wild‐type P6) of the P6 T42R mutant for both the 4G4 and 7F3 antibodies, suggesting that 7F3 and 4G4 interact with residue 42. Nuclear Magnetic Resonance (NMR) spectroscopy was employed to assess structure changes to P6 due to the mutation. This study was funded by the Rochester Institute of Technology and NIH NIDCD RO1 08671 (to MEP).
Mouse UDP‐glucuronosyltransferase Enzymes Capable of Metabolizing Toxic Estrogen Derivatives: Serine Residues Play Crucial Role to Substrate Turnover.
Authors: RAYCHOUDHURI AMIT , JANA SIRSENDU , BASU MOUSUMI , BASU NIKHIL K , OWENS IDA S
Published: June 2021

A comprehensive study of mouse UGT isoforms Ugt2b34 and Ugt2b36 was carried in COS‐1 cells in order to understand differences and the significance of apparent adaptations between mouse and human isozymes. Understanding the mouse UGT versus human drug metabolizing enzymes will contribute significant knowledge about our physiology/biochemistry and habitat driven by evolutionary mechanism of dug metabolism. Our study by q‐RT PCR unexpectedly revealed the prostate distribution of these isozymes apart from mouse liver. Glucuronidation assay showed that both Ugt2b34 and Ugt2b36 possess genotoxic 4‐OH estrogen metabolism ability, with Ugt2b36 being far higher turnover number. Ugt2b34 has more substrate range; it is far more effective in metabolizing phyto‐estrogens and phytochemicals. These two enzymes are also effective in metabolizing xenoestrogens like bisphenol A (BPA) and diethylstilbestrol (DES). We also found by site directed mutagenesis that serine‐174 in Ugt2b34 and ser‐172 in Ugt2b36 is essential for the enzymatic activity. Further studies showed that Ugt2b34 is readily inhibited by kinase inhibitors, while, Ugt2b36 is a robust enzyme which is not susceptible to inhibition.

This work was supported, in whole or in part, by the NICHD Intramural Research Program.
Thiolmethyltransferase activity and selectivity of heterologously expressed human enzymes.
Authors: Oduaran Erica
Published: June 2021

The detoxification of sulfide by thiolmethyltransferases (TMTases) plays a role in protecting against ulcerative colitis and may be involved in motor neuron disease. This study addresses the activity and selectivity of cloned thiolmethyltransferases.
Isolation and identification of vitamin D2 and photobyproducts
Authors: Keegan Raphael , Holick Michael F
Published: June 2021

The goal of this research was to isolate and identify vitamin D2 (D2) photobyproducts in irradiated white button mushrooms (Agaricus bisporus) and determine the kinetics of the conversion of previtamin D2 (preD2) to D2. Mushrooms and ampoules containing ergosterol were irradiated with ultraviolet radiation, and prepared for HPLC. After irradiation we detected preD2, lumisterol2 (L2), and tachysterol2 (T2) in both ampoules and mushrooms. PreD2 and D2 decreased and increased in a time dependent manner. 50% of the preD2 converted to D2 11.5 hours in irradiated mushrooms and 40 hours in the ampoules. The amount of L2 was constant over time in both irradiated mushrooms and ampoules. The amount of T2 remained constant in irradiated ampoules, while in irradiated white button mushrooms, the amount decreased to 0% within 24 hours. Thus UV irradiated white button mushrooms are capable of producing pre D2, L2, and T2. Once formed the preD2 in mushrooms is more rapidly converted to D2 compared to ampoules suggesting that there is a membrane enhance catalytic conversion of preD2 to D2 similar to what occurs in human skin. The amount of T2 rapidly decreased after UV irradiation suggesting it may be metabolized and utilized by the mushroom. Supported by The Mushroom Council.
Membrane repair protein Mitsugumin 53 as a potential therapeutic for the treatment of traumatic brain injury
Authors: Moloughney Joseph Gennaro , Alloush Jenna , Le‐Hoang Oanh , Weisleder Noah Lucas
Published: June 2021

Traumatic brain injury (TBI) is an injury to the brain generated from an external mechanical force causing temporary or permanent impairment of physical and cognitive functions. The Centers for Disease Control and Prevention (CDC) estimates that TBI is a factor in a third of all injury‐related deaths in the United States, accounting for approximately 50,000 deaths annually. Research has shown that injury to neuronal cell membranes is a significant contributing factor in the progression of TBI, therefore targeting membrane repair represents a novel therapeutic strategy for the treatment of traumatic brain injuries. Mitsugumin 53 (MG53) is a TRIM family protein and an essential component of the cell membrane repair machinery. Our research group has shown that MG53 can increase the membrane resealing capability of neuronal cells following mechanical injury in vitro and that recombinant human MG53 (rhMG53) can be delivered to the injury site and minimize damage in the brain following TBI in vivo. Histological examination of brain tissue isolated from mice pretreated with rhMG53 and subjected to TBI display a significant decrease in cleaved caspase‐3 and Fluoro‐Jade C staining at the injury site, suggesting reduction in cell death due to enhanced membrane repair. In addition, motor coordination was improved in mice pretreated with rhMG53 compared to saline control as measured by the rotarod performance test after TBI. Our results show that rhMG53 can mitigate neuronal death and damage brought on by TBI and provide a protective effect in an in vivo mouse model. Research was funded by the National Football League (NFL) Charities.
The Next Generation of Nucleic Acid Microarrays: Alternative, Multi‐stranded, Plasmid and Helical Transitional DNA and RNA Microarrays
Authors: Verma Varun , Gagna Claude , Chagin Olga , Chhatlani Roshan , Ally Irshad , Thomas Elizabeth , Yang Xiao Feng , Lambert W. Clark
Published: June 2021

Nucleic acids are dynamic biological entities, namely, alternative (e.g., Z‐RNA, Z‐DNA, A‐DNA) and multi‐stranded (e.g., triplex) DNAs. DNA microarrays [i.e., based on double‐stranded (ds‐) B‐DNA] are used for gene expression studies. The next generation of nucleic acid microarrays, namely, alternative, multistranded, plasmid and helical transitional DNA and RNA microarrays can be used to characterize nucleic acid structure, and DNA‐protein, RNA‐protein, nucleic acid‐protein‐protein, and nucleic acid‐protein‐ drug complexes for, unaltered ds‐, triple‐stranded and four‐stranded nucleic acids. It can be employed to discover drugs that bind to intact, nondenatured alternative and multi‐stranded DNAs and RNAs. Sequence‐ and helical‐specific DNA‐binding proteins can be characterized and studied with protein and/or drug interactions under different environmental conditions and different substrate surfaces. Unlike conventional microarrays, which are based on hybridization of two single‐stranded DNAs, the novel microarrays take advantage of nucleic acids that are intact, unaltered and non‐denatured. Nucleic acids can be used as partially free‐floating or fully immobilized, allowing for different approaches towards studying structure and function. This technology will allow for new ways to study nucleic acids and enhance drug discovery & development for curing diseases.
Inhibition of hepatitis B virus gene expression and replication by arylnaphthalene lignan analogues HE‐145–111
Authors: Yeh Sheau Farn , Lin Chih Hsiu
Published: June 2021

We have previously reported a natural product, Heliothanxin (HE‐145) from Taiwania cryptomerioides Hayata to suppress HBV gene expression and replication in HCC cells. A series of helioxanthin analogues were synthesized and evaluated for their anti‐hepatitis B virus activity. Among them, compound HE‐145–111 is one of the most effective anti‐HBV agents. We found that HE‐145–111 suppressed not only wild type but also lamivudine‐resistant mutant HBV virus production by human hepatoma cells. To understand the molecular mechanism of HE‐145–111 on HBV gene expression, the effects of HE‐145–111 on four viral promoter activities using luciferase as a reporter were examined. It was found that HE‐145–111 selectively suppresses core promoter (CP). The suppressive effects of HE‐145–111 CP activity is liver‐specific because no suppressive activity of HE‐145–111 was observed when CP activity was assayed in non‐liver cells such as HeLa or 293T. Furthermore, we identified that the nt1656–1708 which contains hepatocyte nuclear factor 4£\ (HNF4£\) binding sequence as a response element of HE‐145–111 in HBV core promoter using serial deletion mutants of core promoter analysis. HE‐145–111 may represent a novel class of anti‐HBV drug which suppresses the HBV core promoter activity may act through modulating hepatic transcriptional machinery to suppress HBV viral gene expression and virus production.
Protein Scaffolds For Light‐Activated Delivery Of Toxic Iron To Cancer Cells
Authors: Boice Emily N , Kurtz Donald M.
Published: June 2021

An iron storage protein called bacterioferritin (Bfr) has been adapted for a novel approach to cancer therapy. Tumor‐targeting peptides (TTPs) have been fused to the protein and photosensitizers, like zinc protoporphyrin (ZnPPIX), have been substituted into the native heme binding sites. After binding to cancer cell receptors, photo‐excitation would trigger release of iron to generate a flux of toxic hydroxyl radicals that would overwhelm a cell's defenses.

The gene encoding the TTP‐Bfr was synthesized and inserted into an E. coli expression plasmid and was readily expressed with ZnPPIX and purified by standard methods. The protein was then loaded by anaerobic incubation of iron. Bfr solutions were irradiated for various time points. Ferrozine was added to quantitate the iron release. These procedures reproducibly resulted in soluble fully loaded [Fe~1,000ZnPPIX12TTP24‐Bfr]. After 3 hours irradiation, ~30% of the iron was released. At longer irradiation times, 65–70% of the iron was released. No iron was released from dark identical control solutions. We have established that [Fe~1,000ZnPPIX12TTP24‐Bfr] releases significant amounts of iron upon irradiation. Current work is aimed at assessing binding of this protein to melanoma cells, and assessing inhibition of cell growth or apoptosis upon irradiation. Results of these investigations could provide proof of principle for a new approach to cancer therapy.