bluuebunny/crossref_metadata_2025 huggingface dataset:
Corporate Stakeholders
Authors: Drobak John N.
Published: June 2021

Chapter 5 echoes the growing sentiment that corporations need to take into account other interests besides that of their shareholders. It traces the origins of the idea that corporations exist solely to increase the wealth of their shareholders and explains how this belief in shareholder primacy came to be accepted as a truism by many scholars, judges, and commentators. When Milton Friedman originally popularized this idea in 1962, he wrote that corporations should serve shareholder interest “within the rules of the game.” These days the rules of the game are influenced tremendously by business lobbying. The chapter explains how the political influence of labor waned and was replaced by business influence in the 1970s. Since that time, Congress has done very little to protect labor because business interests have become extremely powerful lobbyists and substantial donors to political campaigns.
The Effectiveness of Human Rights
Authors: de Búrca Gráinne
Published: June 2021

This chapter surveys existing theories of the effectiveness of human rights, and notes that several prominent accounts have adopted either a ‘top down’ or a ‘bottom up’ theory of effectiveness, emphasizing either external intervention or grassroots mobilization as the primary motor of change. The experimentalist theory advanced in this chapter and throughout the book, however, argues that the effectiveness of much human rights law and advocacy comes neither primarily from top-down intervention nor primarily from bottom-up action but through the iterative interaction between multiple actors, norms and institutions situated at different levels within and outside the state. Building on an emerging scholarship from political scientists, anthropologists, and human rights practitioners, the chapter advances an experimentalist account of international human rights law and advocacy, and introduces the three case studies of human rights campaigns which will be discussed in subsequent chapters. The experimentalist account emphasizes the crucial importance of social mobilization and civil society activism, but argues that the interaction of domestic activism with international accountability institutions is particularly effective in promoting human rights.
Essays in Ancient Epistemology
Authors: Fine Gail
Published: June 2021

This volume brings together thirteen of my essays on ancient epistemology, published between 2000 and 2020, along with a new, synoptic introduction. The essays focus on Plato, Aristotle, and the Pyrrhonian sceptics, though some attention is also given to the Cyrenaics and Descartes. Some essays compare these philosophers to one another, and/or to more recent discussions of these topics. One central theme is cognitive conditions and their contents. For example, is epistêmê knowledge as it is conceived of nowadays? Are doxa and dogma belief as it is conceived of nowadays? I also ask whether Plato and/or Aristotle is committed to the Two Worlds Theory; and whether Pyrrhonian skeptics take anything to be subjective and whether they are external world skeptics.
Criminalizing Villainy
Authors: Patterson Jonathan
Published: June 2021

In juridical theory, a villainy implied a malicious offence in word or deed that took on a legal dimension if it was particularly grievous, and thus warranted judicial redress. Chapter 1 gives an overview of the juridical concepts and categories of crime that were associated with ‘villainous’ or ‘foul’ allegations known as vilains cas. From the late fifteenth century, the notion of vilain cas expanded to include the very worst crimes (those dubbed vilains et énormes). Chapter 1 ends with two case studies of vilains cas, showing how in practice, punishing a villainy and even identifying a villain in law was far from straightforward.
The Villain
Authors: Patterson Jonathan
Published: June 2021

This chapter begins to expose villainy’s ambivalent flow between juridical theory and pre-modern literature. In literary-cultural discourse, the villain was the vilain: in other words, it was a commonplace to assert that the low-born, originally called vilains, would exhibit vile and potentially criminal standards of behaviour. This stereotypical alignment of low morality and low status divided French jurist opinion, not least because it entailed profoundly conflicting ideologies of nobility. The chapter ends with a reading of Giovan Battista Nenna’s Il Nennio (1542), a fiction with quasi-legal elements that deftly exposes nobility and villainy as a problematic binary in transcultural debates on vera nobilitas (true nobility).
The Testator’s Villainy
Authors: Patterson Jonathan
Published: June 2021

The poetry and persona of François Villon have been subject to transcultural reinvention since the fifteenth century (and especially since the nineteenth century, among Anglo-American poets). The legend of Villon goes back to an impecunious poet–criminal who disappeared in 1463, and who both encourages and resists a historicizing interpretation of villainy through his works. This chapter reassesses the singularity of the testator persona in Le Testament Villon (c.1461–2) in his complex vilifying manoeuvres. Some of these have distinct parallels in the early sixteenth-century works of the English poet John Skelton. Yet Villon idiosyncratically instantiates poetic discourse as a para-legal means of attesting to the rigours of torture, where the villain-poet is a direct (if unreliable) witness of his own subjection to judicial punishment.
A French History of ‘Most Shamefull Villanie’
Authors: Patterson Jonathan
Published: June 2021

By the 1580s, fragments of individual suffering in France’s Wars of Religion were being pieced together to form a larger picture within English cultural memory. A significant contribution came from Anne Dowriche’s The French Historie (1589), ostensibly based on the testimonies of Huguenot exiles. The French Historie reverses the villain–hero pairing of Chantelouve’s tragedy (Chapter 11): Charles IX becomes a consummate dissembler, while Coligny (in keeping with Protestant polemical discourse) becomes a blessed martyr. However, Dowriche’s underlying concern is to promote a selective kind of epistemic vigilance (in Relevance Theory, an ‘alertness to error’) in her readers: they must not be blind to the presence of ‘a strange Italian weede’—the villainy of Machiavelli and Catherine de’ Medici—proliferating like a rhizome across European culture.
The Nature and Significance of Culpability
Authors: Brink David O.
Published: June 2021

Narrow culpability is the elemental sense of mens rea, which provides the mental or subjective dimension of criminal wrongdoing. Broad culpability is the responsibility condition in virtue of which the agent’s wrongdoing is blameworthy and without which she would be excused. Inclusive culpability is the combination of wrongdoing and responsibility that together make the agent blameworthy and deserving of blame and punishment. Each kind of culpability plays an important role in a broadly retributive rationale for the criminal law that predicates blame and punishment on the fair opportunity to avoid wrongdoing. Different kinds of culpability align with attributability and accountability. Strict liability crimes that permit liability without culpability are rejected.
Countering Public Misinformation
Authors: Lepoutre Maxime
Published: June 2021

This chapter examines how we should counteract the dangers posed by misinformation (such as fake news and conspiracy theories) in public discourse. Legal norms suppressing misinformation are an even less promising response here than with hate speech. Yet speech-based responses also face grave difficulties: the damage wrought by misinformation is often ‘sticky’, or difficult to reverse. The chapter offers two recommendations for counterspeech that targets misinformation. The first draws on the negative/positive distinction introduced in Chapter 3: unlike negative counterspeech, positive counterspeech can rebut misinformation without triggering the properties that render it sticky. This insight casts doubt on the popular practice of online fact-checking. Second, the chapter recommends adopting a diachronic conception of counterspeech: verbally responding to misinformation should be a continuous process, which pre-empts as well as follows misinformation. Only if it takes this diachronic form can counterspeech keep the most resilient types of misinformation, like conspiracy theories, from taking root.
Introduction
Authors: Lyman R. Lee
Published: June 2021

Graphs of quantitative data are analytical tools that facilitate visual thinking. In many disciplines, the use of graphs was preceded by tables summarizing quantitative data. Graphs known by North American archaeologists as “battleship curves” are temporal frequency distributions of relative abundances of specimens in each of several artifact types. They are unimodal frequency distributions known as spindle graphs. In the early 1950s, it was suggested that the idea of spindle graphs was borrowed by archaeologists from paleontology. Archaeologists occasionally used bar graphs and line graphs to diagram change in artifact inventories in the early twentieth century. The questions addressed in this volume are: (i) did North American archaeologists borrow the idea of spindle graphs from paleontology, and (ii) what was the frequency of use by North American archaeologists of each of the various graph types to diagram culture change during the early and middle twentieth century?
Observations on Graphing Prehistory
Authors: Lyman R. Lee
Published: June 2021

Despite years of graphing culture change using different types and styles of diagram, there is minimal discussion of graph grammar—how to construct an effective and efficient graph, and how to decipher a graph of change. Part of the difficulty attending graph decipherment resided in (and continues to reside in) unclear distinction of transformational change from variational change. Models reflecting the former tend to be commonsensical and are similar to Petrie’s classic sequence dating graphs. The difficulty of graph decipherment is exacerbated by parsing temporally continuous variation into discontinuous spatio-temporally bounded units known as artifact types, cultures, phases, periods, stages, etc. These units are reified and (implicitly) conceived as real entities to be discovered for want of a well-developed theory of change and an attendant ontology of how continuously variable phenomena should be parsed into types for analysis. Archaeologists did perfect models of diffusion—the movement of culture traits (ideas or norms manifest as artifact types) across space over time—and built models of how it should be reflected in the archaeological record. A majority of introductory archaeology textbooks published since 1965 typically present graphs of culture change in the form of a spindle graph but with minimal discussion of graph grammar. Texts on regional or continental prehistory typically summarize culture change in spatio-temporal rectangle diagrams, which for pedagogical reasons may be reasonable. A few spindle graphs have been published in other disciplines and, like archaeological spindles, display temporally shifting frequencies.
Social Proximity, Information, and Incentives in Local Bank Lending
Authors: Wang Junbo , Wang Yun , Wu Chunchi , Yang Xiaoguang , Zhao Lin
Published: June 2021

Exploring the role of social proximity in bank lending using a proprietary data set, we uncover evidence of a significant social proximity effect on private information acquisition. Local loan officers have better information to identify higher-quality borrowers through improved screening. This advantage is greater when firms have higher information asymmetry and lower ratings. Results suggest that sociocultural resources drive locals’ informational advantages and improve their lending performance. Importantly, we find that the social proximity advantage interacts with loan officer incentives. Strong evidence suggests that loan officers can be incentivized to use their social proximity advantage to make better loans. (JEL G12, G13)

Authors have furnished an Internet Appendix, which is available on the Oxford University Press Web site next to the link to the final published paper online.
Nitrogen-influenced competition between the genders of Salix rehderiana
Authors: Song Haifeng , Chen Yao , Cai Zeyu , Wu Xinxin , Zhang Sheng
Published: June 2021

Male and female willow plants show spatial segregation of genders along the environmental gradients. The skewed gender ratio of willows is related not only to altitude, but also to nutrient status and sexual competition, which can affect their growth and defense by altering secondary metabolite production. The relationship between metabolites and nutrients in the two genders of Salix rehderiana was explored in the Gongga Mountain. We found that the gender ratio was altered with a change in soil nitrogen (N) status; in the high N habitats, secondary metabolites accumulated in males. Furthermore, a pot experiment was conducted to test the effect of N supply on gender competition in S. rehderiana. Sufficient N supply stimulated females to produce amino acids and carbon (C)-containing secondary metabolites for maintaining their C-N balance, but extra N for males was used for growth to occupy more space. Nitrogen supply induced foliar nutrient imbalances and growth of opportunistic species, allowing them to outcompete neighbors. Better C allocation and storage in male than female willows would benefit intersexual competitiveness of males if environment N increases. Competition between the genders has a significant correlation with skewed gender ratio, spatial separation and resource utilization. Female willows would suffer fiercer competition for space by males with the increased soil N, which would result in the gender ratio alteration. Therefore, gender ratio of willows is likely to convert to gender balance from female-biased with long-term N deposition in the future.
From mom to prof: “Make an effort”
Authors: Finn Olivera J.
Published: June 2021

This presentation will discuss challenges that women, and increasingly also the “new” men, face in combining the goals for a rewarding life in science and a rewarding family life. The path to success on both fronts is not always straight and often quite difficult, but always exhilarating. Personal examples will be provided to illustrate certain choices and priorities one can make at crucial steps in career development to achieve success. Responsibilities of the society and employers in academia, government or industry to support that effort will be discussed and specific suggestions made. The conclusion will be made that even under still highly unfavorable conditions, many women are successfully combining their professional and personal lives to the benefit of the society and to great personal satisfaction. They are also reaching positions of leadership and authority that should empower them to force societal changes that will make the same road they traveled a little less bumpy for the next generation.
Can a b/delta fusion protein replace individual b and delta subunits of ATP synthase in Escherichia coli?
Authors: Gajadeera Chathurada Sugeeshvarie , Weber Joachim
Published: June 2021

ATP synthase is a ubiquitous membrane protein that plays a key role in biological energy metabolism. In Escherichia coli, ATP synthase contains eight different subunits, with a stoichiometry of α3β3γδεab2c10. The stator stalk which is composed of two copies of subunit b and one copy of subunit delta (δ) is the least well understood part of ATP synthase. To get a clearer picture of the role of individual b subunits, construction of a fusion protein between one of the b subunits and δ was the main intention of the project. Such a fusion protein does exist in mycobacteria.

A b/δ fusion protein in E. coli ATP synthase was functional and could replace the individual b and δ subunits. If no additional b subunit is present, the fusion protein forms a homodimer, as dimerization of b or the b segment of the fusion protein is necessary to obtain a functional enzyme. Deletion of the N‐terminal transmembrane region in the strain that carries only the bδ fusion protein failed to give ATP synthase, whereas active ATP synthase was seen in the strain that carries both bδ and a second b subunit, showing that not both transmembrane helices are required for activity. Finally, the generation of a C‐terminal truncation in the second b, b’, gave active ATP synthase, showing that, as seen in mycobacteria, also in E. coli the C‐ terminal helix of the second b subunit is expendable. This data suggest that the b subunit is responsible for binding to F1, the b’ subunit for binding to Fo.
Structural model of a2‐subunit N‐terminus and its binding interface for cytohesin‐2: Implication for regulation of V‐ATPase function
Authors: Merkulova Maria , Hosokawa Hiroyuki , Bakulina Anastasia , Dip Phat V. , Thaker Youg R. , Khatri Ashok , Ausiello Dennis A. , Grüber Gerhard , Marshansky Vladimir
Published: June 2021

We have previously discovered an important interaction between N‐terminal cytosolic tail of the V‐ATPase a2‐subunit (a2N1–402) and cytohesin‐2. We also identified six V‐ATPase derived peptides involved in interaction with cytohesin‐2. However, their localization and binding interfaces remained unknown. Here, we used a combination of homology modeling and NMR analysis to generate the structural model of complete a2N1–402. According to our model all six peptides are clustered in two binding sites, the proximal and distal lobe sub‐domains of a2N1–402. Moreover, our data indicate that the proximal lobe sub‐domain is the major interacting site with the Sec7 domain of cytohesin‐2. In particular, we showed that two V‐ATPase derived peptides a2N1–17 and a2N35–49 are forming a specific Sec‐domain interaction interface and are involved in regulation of its enzymatic GEF‐activity. The distal lobe sub‐domain of a2N1–402 most likely interacts with another cytohesin‐2 via its PH‐domain. Moreover, our model also suggests that both cytoheins‐2 molecules are binding in a close proximity with two G/E‐peripheral stalks of V‐ATPase and may modulate their interaction with a2N1–402. Thus, we hypothesize that recruitment of cytohesin‐2 to V‐ATPase during its function as a pH‐sensing receptor may regulate function of V‐ATPase. Supported by grants NIH DK038452, BADERC DK057521–08 to VM and by A*STAR BMRC09/1/22/19/609 to GG.
Develop Spatially‐ Interactive Multienzyme Complex on Selfassembled DNA Nanostructures
Authors: Fu Jinglin
Published: June 2021

Cellular activities are directed by complex, multi‐step synthetic pathways. Many of the enzymes in these pathways are spatially organized to improve both the speed and specificity. This abstracts presents a DNA scaffold‐directed strategy to assemble multienzyme systems with precisely controlled orientations and positions. It has increased our understanding of the enzymology for biological metabolic pathways and also provided the tools and design principles for creating efficient man‐made catalytic complexes.

As shown in Figure 1, a two‐enzyme Glucose Oxidase (GOx) ‐ Horseradish Peroxidase (HRP) cascade was assembled on rectangular DNA origami tiles with inter‐enzyme distances ranging from 10 to 65 nm. It was observed that the rate of coupled reactions dramatically increased when the two enzymes were positioned very close together (e.g. 10‐nm in Figure 1B) so that the hydration shells of enzymes overlapped. Further, a protein bridge structure was constructed to facilitate the transfer of intermediates between enzymes (Figure 1C). This opens the door for the design of much more efficient multi‐enzyme systems which should find utility in the development of catalysts for the production of high‐value products in industry and bioenergy, as well as diagnostic applications in biomedicine.
New evidence for dimerization of the short variant of PLA2g6, and regulation of its catalytic activity by Ca2+/calmodulin and Ca2+ influx factor.
Authors: Korolev Sergey , Csutora Peter , Peter Krisztina , Koroleva Olga , Subramanya Praven , Malley Konstantin , Bolotina Victoria M
Published: June 2021

Ca2+‐independent phospholipase A2β (PLA2g6) plays important role in cellular function and is associated with a wide spectrum of pathological conditions, but its structure and mechanism of regulation remain poorly understood. Here we used analytical ultracentrifugation method to characterize oligomeric state of recombinant short (S) splice variant of PLA2g6, and an in vitro real‐time fluorescent substrate assay to assess regulation of its catalytic activity by Ca2+/calmodulin (CaM) and Ca2+ influx factor (CIF). Analysis of sedimentation equilibrium revealed that PLA2g6(S) exists as a dimer of an elongated shape, which is stable with ant without Ca2+/CaM. We found that PLA2g6 is inhibited by CaM at physiological Ca2+ concentrations, suggesting that additional factors are needed to dissociate CaM to activate enzyme. We demonstrated that CIF can be one of such physiological factors, as it displaced CaM and activates PLA2g6 at any levels of Ca2+ (up to 2mM). These studies provide first strict evidence for PLA2g6(S) dimerization, and CIF‐induced full activation of PLA2g6 under physiological Ca2+/CaM conditions.
Pyruvate Revisited as a Synergistic Secondary Activator of the Escherichia coli ADP‐glucose Pyrophosphorylase
Authors: Ballicora Miguel A. , Diez Matías Asención , Iglesias Alberto A.
Published: June 2021

Bacterial glycogen synthesis is allosterically regulated at the level of ADP‐Glc pyrophosphorylase (ADP‐Glc PPase; EC 2.7.7.27) by intracellular metabolites. In Agrobacterium tumefaciens (Atu‐) the enzyme is activated by both pyruvate (Pyr) and fructose‐6P (Fru‐6P) while in Escherichia coli (Eco‐) only fructose‐1,6‐bisP (Fru‐1,6‐bisP) was described as the main activator. Previous switching of the N‐ and C‐terminal domains of Eco‐ and Atu‐enzymes supported the hypothesis of Pyr prevalence, since both constructs were activated by it. Here, we found that Eco‐ADP‐Glc PPase is activated by Pyr with an A0.5 of 25 mM increasing affinity for substrates (ATP). The high A0.5 may seem to suggest that Pyr is a poor effector; however, it has a synergistic role in Fru‐1,6‐bisP activation. Sub‐saturated Fru‐1,6‐bisP concentrations remarkably augmented Pyr affinity (5‐fold at 10 μM Fru‐1,6‐bisP) and vice versa, and 2.5 mM Pyr duplicated the Eco‐ADP‐Glc PPase affinity for Fru‐1,6‐bisP. In addition, Pyr reversed AMP inhibition with an A0.5 value of 2.76 mM. Pyr and Fru‐1,6‐bisP were synergistic and did not compete, which indicated that both effectors bind to different sites. Results with Eco‐ and Atu‐ enzymes may sustain the idea that two binding sites may be involved in allosteric regulation of other bacterial ADP‐Glc PPases. One of these regulatory sites is related to Pyr, which behaves as a fine modulator governing enzyme activity and/or sensitivity to other allosteric effectors. Supported by grants to AAI from CONICET [PIP 2519], UNL [CAI+D Orientado and Redes], and ANPCyT [PICT’08 1754]; and to MAB from the NSF [MCB‐1024945].
Regio‐ and stereo‐selective oxidation of fluorinated substrates by recombinant cytochrome P450 BM3 variants
Authors: Yu Steve S.‐F. , Wu Li‐Lan , Chiang Chih‐Hsiang , Ramu Ravirala , Yang Chung‐Ling , Ng Kok Yaoh , Luo Wen‐I , Chan Sunney I.
Published: June 2021

We recently showed that, upon the addition of fluorine substituents on n‐octane, van der Waals interactions including dispersion between a ‐CF2‐ and an aromatic π system account for changes in the alkane hydroxylation mediated by variants of recombinant cytochrome P450 BM3. Here, for the first time, we demonstrate that P450 BM3 also recognizes fluorinated C12–C15 chain‐length fatty acids. Despite the fact that residues, Arg47 and Tyr52, exert significant controls for the sub‐terminal hydroxylation via hydrogen bonding interactions at carboxylate ends of these fatty acids, in any case, the fluorine substituent(s) at omega position plays an essential role in the regio‐selective hydroxylation occurred at omega‐3 position through unique van der Waals interactions. For instance, the catalytic turnover of fluorinated lauric acid substrates by recombinant cytochrome P450 BM3 exhibit higher activities than that of their parent substrate. In conclusion, our data suggest that hydrophobic residues in the heme pocket of P450 enzymes are involved in tuning the orientation of substrates and this further leads to the controlled oxidation of aliphatics mediated by high valent iron‐oxo species.

This work was supported by Academia Sinica and grants from the National Science Council, R.O.C. (NSC 97–2113‐M‐001–006‐MY3 and 101–2113‐M‐001–007‐MY3).
Cytochrome P450 autophagy: Activation by acute hepatic heme depletion
Authors: Liu Yi , Correia Maria Almira
Published: June 2021

We have previously reported that acute hepatic heme depletion activates the heme‐regulated inhibitor (HRI) eIF2α kinase resulting in the translational arrest of de novo synthesis of hepatic CYPs 2B. We now report that such hepatic heme depletion also results in the rapid autophagic‐lysosomal degradation (ALD) of existing hepatic P450s, as documented by its reversal with ALD‐inhibitors 3‐methyladenine (3MA) and NH4Cl. This is associated with the conversion of LC3‐I to its lipidated LC3‐II species as well as the accumulation and subsequent oligomerization of p62, hepatocellular hallmarks of ALD‐activation. These effects are enhanced by 3MA/NH4Cl, but more importantly reversed by hemin, thereby revealing their strategic placement in the P450 ALD cascade triggered by hepatic heme depletion. Activation of PERK and GCN2 eIF2α kinases results in the non‐canonical activation of nuclear factor kappa B (NF‐κB), a key transcription factor involved not only in inflammation, cancer, apoptosis and immunity, but also in ALD induction. Acute heme depletion we find also activates hepatic NF‐κB, and studies in HRI−/− mice are in progress to determine whether HRI activation is similarly responsible. Irrespective of HRI involvement, our findings rationalize the impaired P450‐dependent drug clearance clinically observed in the acute heme‐deficient states known as the hepatic porphyrias. Supported by NIH grants DK26506 and DK26743.
Sickle hemoglobin disturbs normal coupling between erythrocyte O2 content, glycolysis and antioxidant capacity
Authors: Rogers Stephen Colin , Ross Jerlinda , D'Avignon Andre , Gibbons Lindsey , Hassan Mojibade , McLaughlin Dylan , Griffin Sherraine , Neumayr Tara , DeBaun Malcom , DeBaun Michael , Doctor Allan
Published: June 2021

Energy metabolism in red blood cells (RBCs) is characterized by oxygen (O2)‐responsive variation in flux through the Embden Meyerhof Pathway (EMP) or the Hexose Monophosphate Pathway (HMP). As such, generation of ATP, NADH, and 2,3‐DPG (EMP) or NADPH (HMP) shifts with RBC O2 content, due to competition between deoxy‐hemoglobin (Hb) and key EMP enzymes for binding to the cytoplasmic domain of the Band 3 membrane protein (cdB3). Enzyme inactivation by cdB3 sequestration in oxy RBCs favors HMP flux and NADPH generation (maximizing glutathione (GSH)‐based antioxidant systems). We hypothesized that sickle hemoglobin (HbS) disrupts cdB3‐based regulatory protein complex assembly, creating vulnerability to oxidative stress. In RBCs from patients with sickle cell anemia (SCA), we demonstrate constrained NADPH and GSH recycling and reduced resilience to oxidative stress. We further illustrate abnormal association of HbS to RBC membrane that interferes with sequestration/inactivation of the EMP enzyme GAPDH. These findings are confirmed by EMP/HMP 1H NMR glucose flux analysis in addition to RBC immunofluorescent imaging during O2 loading/unloading. Moreover, selective inhibition of inappropriately dispersed GAPDH rescues antioxidant capacity. Such disturbance of cdB3‐based linkage between O2 gradients and RBC metabolism suggests a novel mechanism by which hypoxia may influence SCA phenotype.
Hyperargininemia in an inducible arginase‐1 deficient mouse model
Authors: Sin Angie , Ballantyne Laurel L , Mukherjee Kamalika , St. Amand Tim , McCracken Crystal , Levandovskiy Valeriy , Schulze Andreas , Funk Colin D
Published: June 2021

We generated an inducible arginase‐1 (Arg1) deficient mouse model by crossing “floxed” Arg1 mice with the Cre‐ER‐T2 model (Arg1‐Cre). These Arg1‐Cre mice die about 2 weeks after tamoxifen administration regardless of the starting age of inducing the knockout and show signs of hyperammonemia and significantly elevated arginine levels (n=4–5; four time points from 2 weeks to 14 weeks of age). Plasma creatine and creatinine levels are normal but guanidinoacetate is increased as assessed by tandem mass spectrometry. Attempts to rescue the lethality or at least to mitigate the biochemical defects caused by loss of arginase‐1 by supplementation of ornithine in the drinking water (n=5) were unsuccessful. An arginase‐1/green fluorescent protein retains full catalytic activity and is being introduced into liver hepatocytes and induced pluripotent stem cells obtained from Arg1‐Cre mice in vitro and to animals in vivo to restore normal biochemistry and to rescue the severe phenotypic consequences of deficiency of this enzyme. Arg1‐Cre mice should prove useful to explore the biochemical defects associated with arginase‐1 deficiency and to explore genetic correction strategies.
Deficiency of sphingosine kinase 1 ameliorates hepatic steatosis and inflammation in diet‐induced obesity
Authors: Geng Tuoyu , Sutter Alton , Lewin David , Ross Jessica , Chavin Kenneth D. , Cowart L. Ashley
Published: June 2021

Obesity‐associated nonalcoholic fatty liver disease (NAFLD) is characterized by a continuum of pathological changes in the liver. Although progression from simple steatosis to the inflammatory stage of NAFLD (i.e. nonalcoholic steatohepatitis/NASH) represents a critical switch from a benign to a harmful status in the progression of NAFLD, the mechanism underlying this switch is little understood. As sphingosine kinase 1 (SK1) catalyzes the biosynthesis of sphingosine‐1‐phosphate, a lipid implicated in inflammation, we hypothesized that deficiency of SK1 might prevent the progression from simple steatosis to NASH in diet‐induced obesity. We thus administrated SK1‐deficient and wild type mice with both high fat diet (HFD) and isocaloric low fat control diet.

Results

SK1 deficiency diminished HFD‐induced pathological changes, including severe lipid accumulation and macrosteatosis, increases in serum alanine aminotransferase activity, immune cell infiltration into liver parenchyma, and induction of inflammatory cytokines. Moreover, SK1 deficiency also attenuated HFD induction of prosteatotic and proinflammatory genes in the liver.

Conclusion

SK1 is essential to progression from simple steatosis into NASH in diet‐induced obesity. Funding: a VA Merit award and the NIH COBRE in Lipidomics and Pathobiology (P20RR017677), both to LAC, and NIH Grant 1R01DK069369 to KDC.
Mesenchymal stem cell therapy to promote peripheral tissue insulin sensitivity in the high‐fat fed, myocardial infarcted mouse
Authors: Hughey Curtis , Ma Lianli , James Freyja D , Bracy Deanna P , Wasserman David H , Rottman Jeffrey N , Shearer Jane
Published: June 2021

OBJECTIVE

Identify the influence of mesenchymal stem cell (MSC) therapy on peripheral tissue glucose utilization following a myocardial infarction (MI) and dietary‐induced insulin resistance (IR).

METHODS

C57BL6 mice were high‐fat fed (HFF) for eight weeks. Mice were then segregated into three groups: HFF‐SHAM mice, HFF‐MI+PBS mice undergoing coronary artery ligation to induce a MI followed by intramyocardial injection of phospho‐buffered saline and the HFF‐MI+MSC group receiving MSCs. HFF groups were age‐matched with chow‐fed mice.

RESULTS

Five weeks post‐MI, glucose utilization was assessed in the conscious, unrestrained mouse by employing a hyperinsulinemic‐euglycemic (insulin) clamp combined with administration of [14C]‐2‐deoxyglucose. The MSC‐treated, chow‐fed mice displayed elevated glucose uptake in the soleus muscle. High‐fat feeding enhanced the MSC‐effect on insulin sensitivity. Whole body insulin responsiveness was higher in the HFF‐MI+MSC, as indicated by a 1.6‐fold increase in glucose infusion rate compared to HFF‐SHAM mice (n=8–12). Also, tissue‐specific glucose uptake (μg/mg/min) was elevated in the soleus (0.95 vs 1.46; HFF‐SHAM vs. HFF‐MI+MSC), gastrocnemius (0.19 vs 0.27), vastus lateralis (0.11 vs 0.23) and adipose tissue (0.03 vs 0.05).

CONCLUSIONS

MSC transplantation represents a novel therapy for IR induced by diet and a MI. Funded by CIHR and Killam Trusts.
Using Long‐Extension PCR Assay to Detect Large‐Scale Deletions in the Mitochondrial DNA
Authors: Pogozelski Wendy Knapp , Chiappone Sam , Evans Christian , O'Donnell Robert
Published: June 2021

Human mitochondrial DNA (mtDNA) is subject to large‐scale deletions. These deletions occur at low levels and are difficult to detect. We show efforts to develop a PCR assay to amplify large sections of the mitochondrial genome, compare the lengths of the amplicons and thus detect the presence of deletions. We also show this technique can be used to determine the effect of insults such as ionizing radiation.
Can perinatal undernutrition alter mitochondrial function and metabolism in the adult liver?
Authors: Lagranha Claudia J , Silva‐Filho Reginaldo , Freitas Cristiane , Silva Alessandra , Nascimento Luciana , Leite Ana Catarina , Maia Maria Bernadete , Fernandes Mariana P
Published: June 2021

During the critical period of development, the body is likely to be influenced by external factors, such as undernutrition, and may induce biochemical changes in tissues. Thus our aim was evaluate the effects of the perinatal undernutrition in liver mitochondrial function and metabolism. We evaluate mitochondrial respiratory rates parameters (phosphorylation (State 3), resting (State 4) and uncoupled state), ATP synthesis, mitochondrial membrane potential, mitochondrial swelling, metabolic enzymes (phosphofructokinase, glucose 6‐phosphate dehydrogenase, citrate synthase and β‐hydroxyacyl‐CoA dehydrogenase) and oxidative stress (malondialdehyde, SOD, catalase and glutathione S‐transferase). Were used male Wistar rats, which were submitted to undernutrition during gestation and lactation with a diet containing 8% and 17% casein. After lactation (21 days), pups began receiving Labina. At 100 days of age, the rats were sacrificed and the liver removed for analysis. Our results showed that perinatal undernutrition induce changes in mitochondrial bioenergetics, metabolism and oxidative stress parameters in adult liver. These results suggest that perinatal undernutrition favoring a significant mitochondrial modulation, which might be associated with disease in adulthood. Supported by: FACEPE
Mammalian Ste20‐like protein kinase 3 mediates the bioenergetics of mitochondria
Authors: Yuan Chiun‐Jye , Chen Chih‐Yen
Published: June 2021

Mst3 (mammalian Ste20‐like serine/threonine protein kinase 3), a key participant in the stress‐ and drug‐induced apoptosis, was recently shown recently to be present both in cytoplasm and in intermembrane space of mitochondria in different cell lines. These results suggest that the mitochondrial localization of Mst3 is a general phenomenon. Interestingly, the selective knockdown of Mst3 by RNA interference resulted in a significant reduction of the protein level of respiratory transport chain complexes I and III. Further studies showed that HeLa stable clone with selective knockdown of Mst3, HeLa(siMst3), exhibited an increased dependence of glucose for ATP synthesis. Compared with controls, the HeLa(siMst3) stable clone was less viable in a glucose‐depriving cultural medium. The mitochondrial‐targeted recombinant Mst3 and its mutants were constructed and expressed in the cells to demonstrate that Mst3 play a role in controlling the bioenergetics of mitochondria. These results indicate that the biological functions of Mst3 are spatially controlled in cells.
C3′/C4′‐Stereochemical Effects of Digitoxigenin a‐L‐/a‐D‐Glycoside in Cancer Cytotoxicity
Authors: McKenna Sean , Hinds John
Published: June 2021

Digitoxin, used for the treatment of congestive heart failure, has more recently been recognized for its potential application in oncology. The mechanism of action occurs via an increase of Ca2+ influx as a result of inhibition of the extracellular Na+/K+‐ATPase pump. In contrast to the cardiotonic effects, less detail is known for the mechanism of antitumor activity. Several studies suggest that in cancer cells, a cardiac glycoside‐bound Na+/K+‐ATPase complex signals via Src‐tyrosine kinase and 1,4,5‐ triphosphoinositol (IP3), ultimately leading to apoptosis. This signaling pathway is believed to occur through specific isoforms of the Na+/K+‐ATPase pump.

Using our lead digitoxin analogue (a‐L‐rhamno 1), we conducted a systematic SAR study, focusing on the stereochemistry and functionality of the C3′‐ and C4′‐stereocenters. We examined the cytotoxicity of digitoxin analouges against four different cancer cell lines: NCI‐H460, A549, MCF‐7, and MDA‐MB‐231. MTT assays were performed from nM to M concentrations. This study helps define limits of digitoxin monosaccharide carbohydrate substitution on cytotoxicity and points towards further analogue design. In contrast to substitution at the C‐2,4 and 5‐positions of the carbohydrate, this study shows the potential for C‐3 substitution for Na/K‐ATP‐ase isoform selectivity without loss of activity.
Specific interaction of the TCERG1 FF4–6 tandem repeat domains with RNA polymerase II requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 of the CTD
Authors: LIU JIANGXIN , Fan Shilong , Lee Chul‐Jin , Greenleaf Arno L , Zhou Pei
Published: June 2021

The human transcription elongation regulator TCERG1 physically couples transcription elongation and splicing events by interacting with splicing factors through its N‐terminal WW domains and the hyperphosphorylated C‐terminal domain (CTD) of RNA polymerase II (RNAPII) through its C‐terminal FF domains. Here, we report biochemical and structural characterization of the C‐terminal three FF domains (FF4–6) of TCERG1, revealing a rigid integral domain structure of the tandem FF domains that interacts with the hyperphosphorylated CTD (PCTD). Using peptide column binding assays and NMR titrations, we show that binding of FF4–6 toward the PCTD requires simultaneous phosphorylation at Ser2, Ser5 and Ser7 positions within the Y1S2P3T4S5P6S7 heptad repeats. Such a sequence‐specific PCTD recognition is achieved through CTD‐docking sites on FF4 and FF5 of TCERG1, but not FF6. In vivo yeast cell extract pull down assay demonstrates the essential role of Ser7P. Our study presents the first example of a nuclear factor requiring all three phosphoSer marks within the heptad repeat of the CTD for high‐affinity binding and provides a molecular interpretation for the biochemical connection between the Ser7P enrichment and co‐transcriptional splicing events. This research was supported by NIH grants GM079376 (to P.Z.) and GM040505 (A.L.G.).
Homogeneous, Sulfated Oligosaccharides from Glycosaminoglycans Activate Promatrilysin (proMMP‐7) Largely by Co‐localization
Authors: Fulcher Yan G , Zhang Fuming , Linhardt Robert J , Gari Raghavendar Reddy Sanganna , Prior Stephen H , Jeong Young J , King Gavin M , Van Doren Steven R
Published: June 2021

Human matrix metalloproteinase‐7 (MMP‐7 or matrilysin) and glycosaminolycans (GAGs) are two important players in extracellular matrix (ECM), participating in innate immunity, aging and cancer. MMP‐7 is over‐expressed in tumor cells and secreted into the ECM as a proenzyme (proMMP‐7). It has been found that MMP‐7 forms complexes with glycoproteins CD44 and heparin‐binding EGF via GAG carbohydrate chains. Recent in vitro studies showed that some GAGs regulate MMP‐7 activities by accelerating proMMP‐7 autolytic cleavage of the pro‐domain (Ra et al., 2009, JBC). We have found recombinant proMMP‐7 to be activated by homogeneous oligosaccharides from heparin in a saccharide length‐dependent manner. 6‐mer, 8‐mer, and 12‐mer heparin oligosaccharides accelerate proMMP‐7 activation at a similar rate while 16‐mer and 20‐mer oligosaccharides accelerate proMMP‐7 activation several‐fold more dramatically. ProMMP‐7 free or bound to the 6‐mer appear to be monomeric by atomic force microscopy. By contrast, proMMP‐7 and the 20‐mer formed large, elongated assemblies. Interface mapping by NMR spectroscopy suggests GAG binding sites in the catalytic and pro domains and potential modes of binding. Our results suggest GAGs regulate proMMP‐7 activation by drawing these protease molecules together. Supported by NIH grant R01GM57289.
Structure based drug design of inhibitors of the CD23 and immunoglobulin E interaction
Authors: Pang Marie Oy Yee , McDonnell James , Beavil Andrew
Published: June 2021

The binding of immunoglobulin E (IgE) to its low affinity receptor, CD23, has been implicated in a multitude of mechanisms underlying allergic asthma. The aim of this study is to develop inhibitors of the CD23‐IgE interaction, which may be a promising therapeutic approach for allergy. A computational approach was adopted to design a 5000 compound library with favourable physicochemical properties and attributes of protein‐protein inhibitors. Subsequently, a novel competitive screening assay based on time‐resolved fluorescence resonance energy transfer (TR‐FRET) was developed, optimised and utilised to screen the compounds. The assay was robust over the screening period and replicates of the compounds were reproducible. Setting a “hit” threshold of >;35% inhibition led to a “hit” rate of 1.8% (n=92). Validation of “hits” by TR‐FRET titrations revealed two compounds with low micromolar Ki values. Surface plasmon resonance titrations confirmed specific binding of these compounds to IgE. Structural studies involving crystallography and nuclear magnetic resonance have been used to identify binding sites. The work presented in this study may potentially lead to the identification and development of inhibitors of the CD23‐IgE interaction to treat allergic asthma.
Saccharomyces cerevisiae Env7 is a novel serine/threonine kinase 16 (STK16)‐related protein kinase and negatively regulates organelle fusion at the lysosomal vacuole
Authors: Gharakhanian Editte , Manandhar Surya , Ricarte Florante , Cocca Stephanie
Published: June 2021

Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report that yeast Env7 is a novel palmitoylated protein kinase ortholog and negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirm localization of tagged Env7 at vacuolar membrane and implicate membrane association via palmitoylation of its N‐terminal Cys13–15. in vitro kinase assays establish Env7 as a protein kinase. Site‐directed mutagenesis of Env7 APE motif Glu269to alanine results in an unstable kinase‐dead allele which is stabilized and redistributed to detergent resistant fraction by interruption of the proteasome system in vivo. Palmitoylation‐deficient Env7C13–15S is also kinase‐dead and mislocalized to the cytoplasm. Microscopic studies establish that env7蜐 is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3 as the two do not share a common substrate, and ENV7 is not a suppressor of yck3蜐. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of human STK16, a Golgi protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.
When fed a high‐fat diet, GPAT4 ‐/‐ mice have an increased metabolic rate and impaired growth and weight gain
Authors: Cooper Daniel , Coleman Rosalind A
Published: June 2021

Glycerol 3‐phosphate acyltransferase (GPAT) is the initial and rate‐limiting enzyme in the synthesis of triacylglycerol (TAG) and catalyzes the addition of an fatty acyl‐CoA to the sn‐1 position of glycerol 3‐phosphate. Four mammalian GPAT isoforms exist, each a product of a separate gene. To determine the mechanism whereby mice with a targeted knockout of GPAT4 (GPAT4−/−) are resistant to obesity, we characterized growth and weight gain from birth. Mice lacking GPAT4 were normal in size at birth. Compared to controls at 21 days of age, body length (nose to rump) and weight of GPAT4−/− mice were 10% and 25% lower, respectively. Mice fed a low‐fat diet until 8 weeks of age grew normally. When changed to a high‐fat diet from 2 – 3 months of age, GPAT4−/− mice gained 50% less weight and grew 20% less. Food intake, absorption and physical activity were normal. Compared to controls, the metabolic rate of GPAT4−/− mice fed a low‐fat or high‐fat diet was 4–6% or 12% higher, respectively. Compared to controls, isolated primary brown adipocytes from GPAT4−/− mice oxidized fatty acid at a 45% higher rate. In brown adipose tissue (BAT) of GPAT4−/− mice fed a low‐fat diet, UCP1 expression was unchanged. Compared to BAT in controls on a high‐fat diet, UCP1 expression in GPAT4−/− BAT was 2.5‐fold higher. These results suggest that GPAT4 in the BAT may serve to regulate metabolic rate during high‐fat feeding. Supported by DK56598, 5T32HD057824.
Identification of TMEM184A in vascular endothelial and smooth muscle cells
Authors: Slee Joshua B , Lowe‐Krentz Linda J
Published: June 2021

Transmembrane protein 184A (TMEM184A or Sdmg1) is a predominantly uncharacterized protein, predicted to have seven transmembrane regions, and is conserved throughout eukaryotic evolution. Although highly conserved, there is very limited data about its function aside from a few studies documenting roles in germ cell sex differentiation, membrane trafficking in SK11 Sertoli cells, and expression in secretory exocrine cells. Given the importance of membrane trafficking in all cells, especially cells of the vasculature, we sought to determine the presence and localization of TMEM184A in vascular cells. Western blotting of vascular endothelial and smooth muscle cells with an antibody specific for the C‐terminal region of TMEM184A demonstrated the presence of a protein of the approximate molecular weight of TMEM184A in both cell types. Immunofluorescent staining with antibodies specific for both the C‐terminal and N‐terminal region of TMEM184A in fixed and permeabilized cells shows specific detection in the vicinity of the nuclei. Similar experiments done in fixed but not permeabilized cells demonstrate specific detection of the N‐terminal and C‐terminal region of TMEM184A on the cell surface of the both cell types. These results suggest that TMEM184A is expressed in cells of the vasculature and is likely to be important outside of somatic cells, germ cells, and exocrine tissues. Funded by NIH grant HL54269